WO1993008301A1 - Improved method - Google Patents

Abstract

The present invention relates to a method for determining the effect of a drug or a treatments, comprising introducing, in a non-human vertebrate recipient which may be an immunodeficient, e.g. a nude mouse, cells from a donor vertebrate, e.g. a human, which mediate the disease, the donor cell or cells of the recipient being modified, e.g. marked with a genetic marker such as lacZ gene, administering the drug or the treatment to the non-human recipient vertebrate, and determining the effect of the drug or the treatment. When the genetic marker is lacZ gene, the detection is performed by staining with the chromogenic substrate X-gal resulting in a blue staining of the labelled cell. In a further aspect, the invention relates to a method for controlling the progression of cancer cells in a human or animal patient, comprising administering to the patient fibroblasts which have been transformed with a genetic construct expressing a gene product inhibiting the progression of cancer cells.

Description

AN IMPROVED DRUGSCREENING METHOD

The present invention relates to improvements in the fields of expe¬ rimental animals, drug screening and drug development, and to related developments, including new means and a new method for treating can- cer.

The environment in which the present invention was made is cancer research, in particular research related to the invasion of cancer cells into other tissue.

Dissemination of cancer involves escape of cells from the primary tumour, degradation of the normal tissue and migration, intravasa- tion, homing, extravasation, and colonization in an environment potentially different from the original location. Although cancer cells are known to produce proteolytic enzymes, adhesion molecules and integrins which may all contribute to the invasive and metastatic phenotype, the mechanisms involved in the metastatic process are not fully understood.

Human cancer cell invasion studies have mainly been confined to in vitro conditions (Albini et al., 1987, and Mignatti et al. , 1986). A major limitation of in vitro models for this type of study, however, is the lack of the many host factors which participate in the physio¬ logical and pathological mechanisms contributing to the malignant phenotype. Important interactions that occur between the cancer cells and the surrounding non-malignant tissue include the interaction of the cancer cells with extracellular matrix and stromal cells, i.e. fibroblasts, endothelial cells, parenchymal cells and other host cells capable of non-immune host reactions (Hakim et al., 1989; Basset et al., 1990; Grøndahl-Hansen et al. , 1991). Thus, the cancer cells exist in a competitive microenvironment with surrounding normal cells as well as non-malignant tumour-infiltrating cells, and the ability of the cancer cells to dominate and evade host non-immune and immune responses determines their invasive/metastatic capacity.

Only a few experimental models have been available for in vivo inves¬ tigations of human cancer cell invasion and metastasis (Dagg et al. , 1956, Kozlowski et al. , 1984). One model is the athymic nude mouse (Fidler, 1986, and Neulat-Duga et al. , 1984). However, most studies in this model have been based on injections of tumour cells intra¬ venously (iv) , and this approach involves only the later steps of the metastatic process. Subcutaneously (sc) inoculated or serially trans¬ planted tumours demonstrating local invasion and spontaneous metasta¬ tic spread are a more adequate model for the invasive and metastatic behaviour of cancer cells. However, not only has there been a limited success in establishing human tumour cell lines that reproducibly spread in these animals after sc inoculation, but difficulties in identifying small metastases throughout the mouse have also been a significant problem (Ossowski and Reich, 1980) . A number of human melanoma cell lines have been found to metastasize in nude mice (Ishikawa et al. , 1988, Wilson et al. , 1988), and the expression of melanin in some of these melanoma cell lines facilitated the identi¬ fication of metastasis in the nude mouse.

Another attempt has been orthotopic implantation of tumour cells such as described in W090/04017. This patent application relates to an animal model for human neoplastic disease having neoplastic dis- ease obtained from a human organ, implanted into the corresponding organ of the animal, said animal having sufficient immunodeficiency to allow the implanted tissue to grow and metastasize. Tumour imaging is carried out by injecting the animal with a labeled anti- umour antibody such as an antibody labeled with a radioactive isotope, allowing the antibody time to localize within the tumour, and then scanning the animal using a radiation detector.

Recently, Lin et al. (1990) demonstrated that metastases in mice injected with ras-transformed mouse 3T3 cells transfected with the E. coli lacZ gene were easily detected following X-gal staining. The la.cZ gene codes for ?-D-galactosidase, the activity of which can be detected by staining with the chromogenic substrate 5-bromo-4-chloro- 3-indolyl-?-D-galactopyranoside (X-gal) , which gives a dark blue reaction product. According to the present invention, materials and techniques are provided which make it possible to perform detections, determinations and investigations which were previously either not possible or had only been performed in less realistic environments, both in connec- tion with cancer drug and cancer treatment development, and in con¬ nection with the development and testing of drugs and treatment principles in relation to other diseases. Thus, in its widest per¬ spective, the invention leads the way to new drugs and treatments and new drug and treatment principles, and as an example, one novel cancer therapy according to the invention is also disclosed herein.

In one of its aspects, the invention relates to a method for detect¬ ing, in a non-human recipient vertebrate, a cell which has the geno¬ type of a donor vertebrate, and which is identical to or derived from a cell transferred from a donor of the vertebrate genotype into the non-human recipient vertebrate, the donor being a vertebrate of the donor vertebrate genotype or a cell line comprising cells from such vertebrate, the method comprising using, as the donor cell, a cell labelled with a genetic marker which, either directly or latently, permits or facilitates distinction between the donor cell genotype and cells of the recipient and, when the marker is one which latently permits distinction, developing the latent distinction, with the proviso that when the donor cell genotype is a murine genotype, then either 1) the donor is a mouse and not a cell culture, or 2) the donor cell is from an immunodeficient mouse.

While the above-cited work by Lin et al. relates to the in vitro modification of human EJ Ha-ras oncogene-transfected BALB/c 3T3 mouse cells which could then be transferred into athymic nude mice, the gist of this aspect of the present invention is to provide testing and screening materials and methods which are much closer to the natural human environment. Thus, as far as cancer is concerned, the invention makes it possible to establish or mimic a broad range of the factors which influence or are influenced by the growing cancer cells in the tissues in which the cancer cells operate, in that the invention makes it possible to directly expose human cancer cells to realistic environments with respect to the factors influencing their growth, and at same time monitor them extremely sensitively and accurately. As will appear from the following, these advantages are critical and decisive for a number of important new developments.

Thus, as appears, e.g., from Example 2 herein, X-gal staining of human cancer cells in whole mouse organs allowed visual detection of even very small metastatic foci, and did not stain normal mouse tissue. Furthermore, lacZ transduction was found not to alter the human neoplastic cells' phenotype, including invasive and metastatic growth mode in immune-deficient mice. Thus, lacZ transduction of human cancer cells is a highly specific and sensitive method for quantitative detection of invasive and metastatic human cancer cells in the nude mouse. The model has been found to be useful not only for examining interactions between cancer cells and host tissue in rela¬ tion to the invasive and malignant phenotype, but also for quantita- tively evaluating the effect of drugs which may interfere with biolo¬ gical events involved in the invasive and metastatic process.

By the term "genetic marker" is meant a DNA sequence encoding a. polypeptide or RNA species, the presence of which is detectable and thus revealing the presence of the genetic marker DNA sequence. In its broadest concept, the term genetic marker also includes the gene product e.g. RNA, polypeptide or glycoprotein.

The genetic marker used according to the present invention is normal¬ ly a gene encoding a product which in itself is visually distinguish¬ able from non-marked cells, or which is capable of being made visual- ly distinguishable from the non-marked cells. Genes which encode enzymes, antigens or other biologically active proteins which can be monitored easily by biochemical techniques are preferred. For most purposes, it is preferred that the gene product be a coloured or fluorescent product or a product which can be converted into a colou- red or fluorescent product. A suitable and presently preferred gene is a lacZ gene, in particular a bacterial lacZ gene, such as an E. coli lacZ gene. The gene product of this gene is not directly visual¬ ly distinguishable, but rather latently distinguishable in the sense of the use of the term "latently" herein, which means that the gene product must be subjected to a conversion treatment in order to develop the visual distinction. Thus, in accordance with this, the conversion of the lacZ gene product into a coloured product is per¬ formed by staining with the chromogenic substrate 5-bromo-4-chloro-3- indolyl- -D-galactopyranoside (X-gal) resulting in a blue staining of the labelled cell. Alternative staining procedures like red gal or FDG may be used. Other examples of suitable genes are selected from the group consisting of a Drosophila alcohol-dehydrogenase gene, and a placental alkaline phosphatase gene, both of which give rise to latently distinguishable gene products, and a gene encoding melanine, which is directly distinguishable or a gene encoding luciferase or peroxidase.

In further important embodiments of the invention the donor cells comprises a genetic marker and, furthermore, a second transgene which is different from the genetic marker, such as a gene encoding a polypeptide, such as a growth factor (e.g. TGF-3 or mammostatin) or a growth factor receptor (e.g. EGF receptor, PDGF receptor or IGF-1 receptor), a cytokine (e.g. IL-1, IL-2 or TNF-α) , or an oncogene product. Evidently, also embodiments which contain a third, a fourth or further transgenes, are within the scope of this invention.

In important aspects of the invention, the non-human recipient verte¬ brate is immunodeficient, thus allowing cells of another genotype to be introduced into the recipient without causing immunological reac¬ tions against the donor cells. In particular, the recipient is prefe¬ rably a vertebrate which is deficient with respect to thymus such as a nude mouse. The immunodeficiency may be obtained, e.g., by chemical treatment or whole body irradiation in a manner known per se . A number of immunodeficient experimental animals, in particular mam¬ mals, which are useful as recipients in accordance with the present invention are commercially available, such as rodents, in particular rats and mice. Other interesting recipient mammals are, e.g, rabbits, monkeys, cows, horses, and pigs.

In most of the important embodiments of the method of the invention, the donor cell genotype is mammalian, although it is, of course, within the scope of the invention to develop drugs and treatments for use, e.g., in poultry breeding or the breeding of other vertebrates such as fish.

The most important aspects of the present invention are the ones in which the donor cell genotype is human. On the other hand, as will appear from the discussion below, there are a number of cases where it becomes important to use donor cells of other genotypes, in parti¬ cular of the same genotype as the recipient, which, for many of the most important embodiments means donor cells which are of a cell of the rodent genotype, in particular of rat or a mouse genotype, in particular donor cells from a thymus-deficient rodent, such as a nude mouse.

While, in all of the above-mentioned embodiments, the donor cells are the ones which are labelled with a genetic marker, another important aspect of the invention is the use of a recipient vertebrate, cells of which have been labelled with a genetic marker, i.e. an animal transgenie for a genetic marker. Thus, the invention also relates to a method for detecting, in a non-human recipient vertebrate, a cell whicTi has the genotype of a donor vertebrate, and which is identical to or derived from a cell transferred from a donor of the vertebrate genotype into the non-human recipient vertebrate, the donor being a vertebrate of the donor vertebrate genotype or a cell line comprising cells from such vertebrate, the method comprising using, as the recipient vertebrate, a vertebrate cells of which are marked with a genetic marker which, either directly or latently, permits distinc- tion between the donor cell genotype and cells of the recipient and, when the marker is one which latently permits distinction, developing the latent distinction.

While it is possible to label specific cell types of the recipient vertebrate, it will, for most purposes, normally be preferred to label substantially all cells of the recipient vertebrate with the genetic marker. This is obtained when the genetic marker is part of a transcription unit which is capable of functioning in substantially all cells of the vertebrate in such a way that the genetic marker is expressed in substantially all cells of the vertebrate. Accordingly, in one aspect the present invention relates to an ex¬ pression system or transcription unit comprising a genetic marker, the system or unit comprising a 5'-flanking sequence capable of mediating expression of said genetic marker.

A transgenic animal contains one or more transgenes within its geno¬ me. A transgene is a DNA sequence which has been introduced into the genome of the animal and is generally integrated at a locus of a genome, wherein the' transgenic DNA sequence is not otherwise normally found at that locus in that genome. Transgenes may comprise heterolo- gous DNA sequences (sequences normally not found in the genome of species in question) or homologous DNA sequences (sequences derived from the genome of the species in question) . Homologous recombina¬ tion, i.e. the exchange of an endogenous gene with a variant of the gene, is also within the definition of the transgene as used herein. Heterologous polypeptides are thus polypeptides which are not normal¬ ly produced by the transgenic animal. Transgenic animals have been reported. For example, U.S. Patent No. 4,736,866 discloses a trans¬ genic mouse containing a c-myc oncogene. Other reports of transgenic animals include PCT Publication No. WO 82/04443 (rabbit 9-globin gene DNA fragment injected into the pronucleus of a mouse zygote) ; EPO Publication No. 0 264166 (Hepatitis B surface antigen and Tissue Plasminogen Activator genes under control of the whey acid protein promoter for mammary tissue specific expression); EPO Publication No. 0 247 494 (transgenic mice containing heterologous DNA encoding various forms of insulin); PCT Publication No. WO 88/00239 (tissue specific expression of DNA encoding factor IX under control of a whey protein promoter); PCT Publication No. WO 88/01648 (transgenic mammal having mammary secretory cells incorporating a recombinant expression system comprising a mammary lactogen-inducible regulatory region and a structural region encoding a heterologous protein) ; EPO Publication No. 0 279 582 (tissue specific expression of chloramphenicol acetyl- transferase under control of rat 3-casein promoter in transgenic mice) ; and WO 91/08216 (production of recombinant polypeptides by bovine species and transgenic methods) .

In the present context, the term "gene" is used to indicate a DNA sequence which is involved in producing a polypeptide chain or RNA species and which includes regions preceding and following the coding region (5'-upstream and 3'-downstream sequences) as well as interven¬ ing sequences, introns, which are placed between individual coding segments, exons, or in the 5'-upstream or 3'-downstream region. The 5'-upstream region comprises a regulatory sequence which controls the expression of the gene, typically a promoter. The 3'-downstream region comprises sequences which are involved in termination of transcription of the gene and optionally sequences responsible for polyadenylation of the transcript and the 3' untranslated region.

The above mentioned regulatory or expression regulation sequences in addition to controlling transcription also contribute to RNA stabi¬ lity and processing, at least to the extent they are also transcri¬ bed.

In general, the 5' regulatory sequence includes the transcribed por- tion of the endogenous gene upstream from the translation initiation sequence (the 5' untranslated region or 5' UTR) and those flanking sequences upstream therefrom which comprise a functional promoter. As used herein, a "functional promoter" includes those necessary un- transcribed DNA sequences which direct the binding of RNA polymerase to the endogenous gene to promote transcription. Such sequences typically comprise a TATA sequence or box located generally about 25 to 30 nucleotides from the transcription initiation site. The TATA box is also sometimes referred to as the proximal signal. In many instances, the promoter in this sense further comprises one or more distal signals located upstream from the proximal signal (TATA box) which are necessary to initiate transcription. Such promoter sequen¬ ces are generally contained within the first 100 to 200 nucleotides located upstream from the transcription initiation site, but may extend up to 500 to 600 nucleotides or more from the transcription initiation site. Such promoter sequences alone or in combination with the 5' untranslated region are referred to herein as "proximal 5' expression regulation sequences".

In addition to such proximal 5' expression regulation sequences, it is preferred that additional 5' flanking sequences (referred to herein as "distal 5' expression regulation sequences") also be inclu- ded in the transgene. Such distal 5' expression regulation sequences are believed to contain one or more enhancer and/or other sequences which facilitate expression of the endogenous gene and as a conse¬ quence facilitate the expression of the genetic marker when operably linked to the distal and proximal 5' expression regulation sequences. These 5' expression regulation sequences regulate the spatial and temporal distribution of gene expression. The amount of distal 5' expression regulation sequences depends upon the endogenous gene from which the expression regulation sequences are derived.

In addition, it is preferred that 3' expression regulation sequences also be included to supplement tissue or cell-type specific expres¬ sion. Such 3' expression regulation sequences include 3' proximal and 3' distal expression regulation sequences from an appropriate endoge¬ nous gene. The 3' proximal expression regulation sequences include transcribed but untranslated DNA positioned downstream from the translation stop signal in the inserted genetic marker (also refer¬ red to as the 3' untranslated region or 3' UTR) . Such sequences generally terminate at a polyadenylation sequence (either from the endogenous gene or from other sources such as SV40) and sequences that may affect RNA stability. Generally, 3' UTR's comprise about 100 to 1000 nucleotides or more downstream from the translation stop signal in the gene from which the 3' regulation sequence is derived. Distal 3' expression regulation sequences include flanking DNA se¬ quences downstream from the proximal 3' expression regulation se- quence. Some of these distal sequences are transcribed, but do not form part of the mRNA while other sequences in this 3' distal expres¬ sion regulation sequence are not transcribed at all. Such distal 3' expression regulation sequences are believed to contain enhancer and/or other sequences which enhance expression.

Although the use of both 5' and 3' expression regulation sequences are preferred, in some embodiments of the invention, endogenous 3' regulation sequences are not used. In such cases, the 3' proximal expression regulation sequences normally associated with the genetic marker are used to direct polyadenylation. As with the 5' expression regulation sequences, the optimal amount of 3' expression regulation sequence may be readily determined by varying the amount of 3' flank- ing sequence to obtain maximal expression of the genetic marker poly¬ peptide. In general, the distal 3' regulation sequence, be it from an endogenous gene or a heterologous gene, will not extend into the adjacent gene from which it is derived and will exclude any sequences which adversely effect the level of transgene expression.

When the term "genetic marker" is used, it should be understood that this term relates to a gene encoding a polypeptide, glycoprotein or RNA the presence of which can be detected as well as to any modifica¬ tions or analogues of said gene which do not have any significant adverse effect on the expression or activity of the polypeptide or RNA to be detected. Such modifications or analogues may be obtained by e.g. substitution, addition, insertion or deletion of the DNA sequence encoding the genetic marker.

When "substitution" is performed, one or more nucleotides in the full nucleotide sequence are replaced with one or more different nucleo¬ tides* when "addition" is performed, one or more nucleotides are added at either end of the full nucleotide sequence; when "insertion" is performed one or more nucleotides within the full nucleotide sequence is inserted; and when "deletion" is performed one or more nucleotides are deleted from the full nucleotide sequence whether at either end of the sequence or at any suitable point within it.

A modified genetic marker DNA sequence may be obtained by well-known methods, e.g., by use of site-directed mutagenesis or chemical syn¬ thesis of desired sequence of DNA as described in textbooks in the field. A subsequence of the genetic marker DNA sequence which com¬ prises a sufficient part of the genetic marker to ensure a signifi¬ cant polypeptide or RNA production, the presence of which is to be detected, is also within the scope of the present invention. When reference is made to a genetic marker DNA sequence this should thus be understood to include "analogues", "subsequences" and "modi¬ fied sequences" as defined above.

Because of the evident and important advantages of having a recipient which will not generate an immune response, confer above, it is pre¬ ferred that the non-human recipient vertebrate is immunodeficient, and the preferred types of recipient for this aspect of the invention are generally the same as mentioned above for the "opposite" aspect.

One advantage of the last-mentioned aspect of the present invention i.e. using a transgenic animal as a recipient is that the distinction between the donor cells and the recipient cells is obtained without the necessity of manipulating with the donor cells.

In addition, the lacZ transgenic animals may be crossed into other transgenic animals, resulting in animals expressing more than one transgene, such as p53, ras, TGF-β , u-PA, etc.

In the most important embodiments, the donor cells will, also in this aspect be of mammalian genotype, in particular human, or alternative¬ ly, of the same species or genotype as the recipient, such as will be further elaborated in the following.

As indicated further above, the above-discussed methods for the very sensitive and precise detection of donor cells, such as human cells, in tissue of recipient non-human vertebrates, constitute important new tools in the development of therapeutic or prophylactic princip¬ les relating to a large number of diseases or adverse conditions. In the following, these important exploitations of the invention will be described, both on a general level and in connection with a number of specific examples. Thus, in its broad principle, this aspect of the invention relates to a method for determining the effect of a drug or a treatment with respect to preventing, diminishing, control¬ ling or inhibiting a disease mediated by cells, comprising introdu- cing, in a non-human vertebrate recipient, cells from a donor verte¬ brate which mediate the disease, the donor cells or cells of the recipient being modified to either directly or latently permit dis¬ tinction between on the one hand cells which are identical to the donor or cells derived therefrom, and on the other hand cells of the non-human recipient vertebrate, administering the drug or applying the treatment to the non-human recipient vertebrate, and determining the effect of the drug or the treatment on the basis of detection or investigation, in the recipient vertebrate, of cells identical to or derived from the donor cells, utilizing, in the detection or investi- gation, the distinction obtained through modification of the donor cells or of cells of the recipient, whereby, when the modification is one which latently permits distinction, the latent distinction is developed prior to or in connection with the detection or investiga- tion.

Within the scope of the invention is also a method as described above, wherein the donor vertebrate cells further comprises a second, third or further transgenes which is different from the genetic marker.

As will appear from the above, this method is defined as encompassing both the embodiment where the donor cell is genetically labelled and the recipient cells are not_r and the reverse embodiment, in accor¬ dance with the explanation of these two principles above. (Evidently, it is also possible - and within the scope of the present invention and the above definitions - to have both the donor cells and the recipient cells labelled with different genetic markers allowing, and even enhancing, distinction therebetween.)

In accordance with the above discussion, it is generally preferred that the recipient vertebrate is immunodeficient, and the examples and preferences expressed above will of course, also apply in this exploitation of the invented principle.

The above-mentioned general drug or treatment screening or testing method can be further enhanced and elaborated on in a number of ways, utilizing the basic principles of the invention. Thus, e.g., after the recipient has been subjected to the drug or the treatment, the cells identical to or derived from the donor cells can be further investigated by transferring such cells to another non-human recipi¬ ent vertebrate and determining any effect of the drug or treatment conferred to the cells identical to or derived from the donor cells and manifesting itself after the transfer to the other non-human recipient vertebrate. In a number of embodiments, this other non- human recipient vertebrate is immunodeficient. Also in connection with this investigation, it is, of course, normal¬ ly a great advantage that the donor cells or cells of the recipient are modified to either directly or latently permit distinction bet¬ ween on the one hand cells which are identical to the donor or cells derived therefrom, and on the other hand cells of the other non-human recipient vertebrate, the determination of any effect of the drug or the treatment being performed on the basis of detection or investiga¬ tion, in the other recipient vertebrate, of cells identical to or derived from the donor cells, utilizing, in the detection or inves- tigation, the distinction obtained through modification of the donor cells or of cells of the recipient, whereby, when the modification is one which latently permits distinction, the latent distinction is developed prior to or in connection with the detection or investiga¬ tion. In the case where the original donor cells were genetically labelled, this establishment of distinguishability is obtained in¬ herently when using a recipient vertebrate the cells of which are not labelled, because the donor cells or their derivatives will retain their labelling. In the case where not the donor cells, but rather the first recipient vertebrate, was modified by labelling of its cells, the distinction is obtainable by using, as the second recipi¬ ent non-human vertebrate, one which has not been modified by genetic labelling of its cells as explained above.

In specific embodiments of the invention, the cells of the non-human recipient vertebrate which have been interacting with the donor cells are investigated during or after the drug administration or the treatment. The investigation of the cells of the non-human recipient vertebrate which have been interacting with the donor cells may involve transferring such cells of the non-human recipient vertebra¬ te, after the first non-human recipient, has been subjected to the drug or the treatment, to another non-human recipient vertebrate, which may be immunodeficient, and determining any effect of the drug or treatment conferred to the cells from the first recipient ver¬ tebrate and manifesting itself after the transfer to the other non- human recipient vertebrate.

A specific embodiment of the invention relates to a method wherein the cells transferred from the first non-human recipient vertebrate or cells of the second recipient vertebrate are modified to either directly or latently permit distinction between on the one hand cells which are identical to or derived from the cells from the first non- human recipient vertebrate, and on the other hand cells of the other non-human recipient vertebrate, the determination of any effect of the drug or the treatment being performed on the basis of detection or investigation, in the other non-human recipient vertebrate, of cells identical to or derived from the cells from the first non-human recipient vertebrate, utilizing, in the detection or investigation, the distinction obtained through modification of the cells from the first non-human recipient vertebrate or of cells of the second non- human recipient vertebrate, whereby, when the modification is one which latently permits distinction, the latent distinction is deve¬ loped prior to or in connection with the detection or investigation.

In the a number of the embodiments enlisted above, the non-human recipient vertebrate is a mammal, such as a rodent, in particular a rat or a mouse. The recipient rodent may be a thymus-deficient ro¬ dent such as a nude mouse.

Within the scope of the present invention is a method of producing a transgenic non-human vertebrate cells of which are marked with a genetic marker which, either directly or latently, permits distinc¬ tion between cells having the genotype of the non-human vertebrate and cells which are donated to the non-human vertebrate said method comprising chromosomally incorporating genetic marker into the genome of a non-human mammal.

This method may be performed by injecting a transcription unit com¬ prising said genetic marker into a fertilized egg or a cell of an embryo of a vertebrate so as to incorporate the transcription unit into the germline of the vertebrate and developing the resulting injected fertilized egg or embryo into an adult vertebrate.

The invention thus also relates to a transgenic non-human vertebrate whose germ cells and somatic cells contain a genetic marker which, either directly or latently, permits distinction between cells having the genotype of the non-human vertebrate and cells which are donated to the non-human vertebrate, as a result of chromosomal incorporation of the genetic marker into the non-human vertebrate genome, or into the genome of an ancestor of said non-human vertebrate. A specific and important embodiment of the invention is a transgenic non-human vertebrate such as a mouse, rat or rabbit, wherein the genetic marker is lacZ gene.

An interesting embodiment of the invention is, analogous to what is described above, a method of producing a transgenic non-human ver¬ tebrate cells of which are marked with a genetic marker and a second, third, fourth or further transgene which is different from the gene¬ tic marker said method comprising chromosomally incorporating the genetic marker and/or the second transgene into the genome of a non- human mammal. These second, third, fourth or further transgene genes may be selected from genes encoding polypeptides, such as a growth factors or growth factor receptors, cytokines or oncogene products.

The transgenic non-human vertebrate of the invention are produced by introducing a "transgene" into an embryonal target cell of the animal of choice. In one aspect of the invention, a transgene is a DNA sequence which is capable of producing a desirable phenotype when contained in the genome of cells of a transgenic non-human vertebra¬ te. In specific embodiments, the transgene comprises a genetic marker encoding a detectable polypeptide or RNA. In such cases, the trans¬ gene is capable of being expressed to produce the detectable poly¬ peptide.

The incorporation of the transcription unit into the germline of the vertebrate may be performed using any suitable technique, e.g. as described in Hogan B. , Constantini, F. and Lacy, E. Manipulating the Mouse Embryo. A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1986 or in W091/08216.

Methods of introducing transgenes or overlapping transgene fragments into embryonal target cells include microinjection of the transgene into the pronuclei of fertilized oocytes or nuclei of ES cells of the non-human animal, or by transfection of ES cells, e.g. after electro- poration. Such methods for murine species are well known to those skilled in the art. Alternatively, the transgene may be introduced into an animal by infection of zygotes with a retrovirus containing the transgene (Jaenisch, R. (1976), Proc. Natl . Acad. Sci. USA, 73 , 1260-1264). Embryos are thereafter transferred to an appropriate female by standard methods to permit the birth of a transgenic or . chimeric animal depending upon the stage of development when the transgene is introduced. As is well known, mosaic animals can be bred to form true germline transgenic animals.

Transgene integration can be detected by taking an appropriate tissue biopsy such as from the ear or tail of the putative transgenic ani¬ mal. About one to two centimeters of tail or about five to ten square millimeters of ear are obtained followed by southern blotting with a probe for the transgene according to the method of Hogan et al. (1986) Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory.

Normally, not all of the injected eggs will develop into transgenic vertebrate. Transgenic founder animals can be identified. From a statistically point of view about half of the vertebrate will be males. One the basis of the identified transgenic individuals - male and female - progeny can be established and stable lines of trans- genie animals established.

While the transgenic non-human vertebrate of the invention in its broadest aspect is not restricted to any particular type of vertebra¬ te, the vertebrate will normally be selected from the group consist¬ ing of mice, rats and rabbits which are especially interesting due to the fact that these animals are conventionally use in the laboratory and the manipulation of these animals is simple.

Also progeny of a transgenic vertebrate as defined above, having a genetic marker integrated within its genome is within the scope of the present invention.

Another important further elaboration of the basic method is to sub¬ ject the cells of the non-human recipient vertebrate which have been interacting with the donor cells, to investigation during or after the drug administration or the treatment. As one interesting example, the cells of the non-human recipient vertebrate which have been interacting with the donor cells can be transferred to another non- human recipient vertebrate, and any effect of the drug or treatment conferred to the cells from the first recipient vertebrate and mani- festing itself after the transfer to the other non-human recipient .vertebrate can be investigated. Also in this case, the cells trans¬ ferred from the first non-human recipient vertebrate or cells of the second recipient vertebrate are preferably modified to either direct¬ ly or latently permit distinction between on the one hand cells which are identical to or derived from the cells from the first non-human recipient vertebrate, and on the other hand cells of the other non- human recipient vertebrate, the determination of any effect of the drug or the treatment being performed on the basis of detection or investigation, in the other non-human recipient vertebrate, of cells identical to or derived from the cells from the first non-human reci¬ pient vertebrate, utilizing, in the detection or investigation, the distinction obtained through modification of the cells from the first non-human recipient vertebrate or of cells of the second non-human recipient vertebrate, whereby, when the modification is one which latently permits distinction, the latent distinction is developed prior to or in connection with the detection or investigation.

The above-described general drug or treatment development, screening or testing methods are extremely advantageous in connection with a wide range of, e.g human diseases mediated by cells, including auto- immune diseases such as various types of diabetes, conditions related to the functions of the thyroid gland, the pancreas, the adrenal gland, the intestine, and arthritis, encephalitis and the autoimmune diseases like systemic lupus erythematosus, multiple sclerosis, haematological diseases. In addition, diseases causing obesity are examples of cell-mediated diseases which can be advantageously ex¬ amined.

Examples of the types of substances which may be used in the treat¬ ment and may thus be developed as drugs, are chemical compounds, antibodies or antigens, amino acids, proteins such as a growth fac- tors, lymphokines, interferons, interleukines, insulin, hormones, leukotrienes, anti-sense RNA etc. By using this method of the invention, the time needed for the deve¬ lopment of drugs and/or treatments can be very drastically reduced as compared to the clinical examination and trial which, until the pre¬ sent invention, was the only possible development scenario.

One of the most important uses of the drug or treatment development method described herein is for the assessment of the usefulness of drug or a treatment for preventing, diminishing, controlling or inhibiting the progression of metastases in a donor vertebrate, in particular a human. This embodiment comprises introducing, into an immunodeficient non-human recipient vertebrate in which cancer cells of the donor vertebrate genotype are capable of invading and meta- stasizing, cancer cells of the donor vertebrate genotype, the donor cancer cells or cells of the recipient being modified to either di¬ rectly or latently permit distinction between on the one hand cells which are identical to or derived from the donor cells, and on the other hand cells of the non-human recipient vertebrate, administering the drug or applying the treatment to the non-human recipient verte¬ brate, and determining the effect of the candidate drug on the pro¬ gression of metastases in the animal, utilizing, in the detection or investigation, the distinction obtained through the modification of the donor cells or of cells of the recipient, whereby, when the modification is one which latently permits distinction, the latent distinction is developed prior to or in connection with the detection or investigation.

A preferred embodiment of the method described above is a method, wherein the modification of the donor cells or the cells of the recipient comprises labelling the cells with a genetic marker which, either directly or latently, permits or facilitates distinction between the donor cell genotype and cells of the recipient vertebra- te. In a specific embodiment of the method described above, the modified donor or recipient cells further comprises a second, third, fourth or further transgene which is different from the genetic marker. The genetic marker is a gene encoding a product which in itself is visually distinguishable from non-marked cells, or which is capable of being made visually distinguishable from the non-marked cells, the gene product can thus be a coloured or fluorescent product or a product which can be converted into a coloured or fluorescent pro¬ duct. In a specific embodiment of the method, the genetic marker is a lacZ gene, and the conversion of the gene product thereof into a coloured product is performed by staining with the chromogenic sub¬ strate 5-bromo-4-chloro-3-indolyl-_/S-D-galactopyranoside (X-gal) resulting in a blue staining of the labelled cell.

By far the most important embodiment of this aspect is where the donor cells are human cells such as human cancer cells.

In order to obtain the advantages of the acceleration offered by the method of the invention in connection with adjuvant treatment, pri- mary tumours may be removed from the non-human recipient vertebrate before the drug is administered to or the treatment applied to the non-human recipient vertebrate, such as described in greater detail in Example 5.

An example of the use of the method for drug screening is described in Example 6, in which the inhibition of the invasive and metastatic process of human cancer cells introduced into nude mice using mono¬ clonal antibodies directed against urokinase type plasminogen ac¬ tivator, u-PA, is tested.

Example 5 illustrates a case where the very high sensitivity and selectivity of the method of the invention is utilized in that the donor cancer cells have been marked with a lacZ gene in such a manner that it is expressed by the cancer cells, and removal of the primary tumours is performed substantially at a time where metastasizing in the non-human recipient vertebrate has just begun to be visually detectable by staining with X-gal and metastasing is still undetec- table in a reference non-human vertebrate animal the cells of which are not labelled and to which non-labelled cancer cells of otherwise the same kind as the donor cancer cells have been transferred. Prior to the present invention, detection of micrometastases required very detailed histological examination, but, even such very detailed examination was not sensitive enough to always secure detection of micrometastases which were in fact present.

Human cancer cells which are particularly useful as donor cells in this method of the invention are cells selected from the group consisting of the human cell lines MDA-MB-231, MDA-MB-435, and OVCAR- 3. Cells of these kinds are suitably used in combination with an im¬ munodeficient recipient mammal, in particular a nude mouse, in which the cells are capable of metastasizing.

Thus, a particularly valuable type of recipient immunodeficient mammal is a mammal in which at least one of the cell lines MDA-MB- 231, MDA-MB-435, or 0VCAR-3 is capable of metastasizing. A very advantageous immunodeficient mammal is a mammal, in particular a thymus-deficient or nude mouse, in which the cell lines MDA-MB-231, MDA-MB-435, and OVCAR-3 are capable of metastasizing. Examples of such mice are the nu/nu META/Bom mouse available from Bomholtgaard, Gammel Ry, Denmark, the SCID mouse available from Bomholtgaard, Gammel Ry, Denmark, and the Bar mouse available from the Bartholin Institute, Kommunehospitalet, Denmark, and the use of the specific mouse strains and other strains having the same capability as reci¬ pients or donors in connection with the above-described drug and treatment screening methods constitutes valuable embodiments of the invention. The Bar mouse is produced by mating a male nu/nu META/Bom mouse with a female C3H+/+ and then mating the female offsprings with the male nu/nu META/Bom mouse.

A further, important, aspect of the invention is a mammal in which at least one of the cell lines MDA-MB-231, MDA-MB-435, or OVCAR-3 is capable of metastasizing, and which mammal has been modified by labelling cells of the mammal, preferably ^"substantially all cells of the mammal, with a genetic marker as defined above. A very advan¬ tageous mammal of this kind is a mammal, in particular a thymus- deficient or nude mouse, in which the cell lines MDA-MB-231, MDA-MB- 435, and OVCAR-3 are capable of metastasizing. Thus, examples of such mice are the nu/nu META/Bom mouse available from Bomholtgaard, Gammel Ry, Denmark, the SCID mouse available from Bomholtgaard, Gammel Ry, Denmark, and the BAR mouse available from the Bartholin Institute, Kommunehospitalet, Denmark, whenever they have been modified with a genetic marker as defined above.

The nu/nu META/Bom mouse has a chromosomal analysis substantially as shown in Table 1 below. The references referred to in Table 1 are the following:

1. Krog, H.-H.: Biochemical Genetics, 14, 319-324, 1976

2. Wilcox, F.H. et al.: Biochemical Genetics, 17, 1093-1107, 1979 3. Birdsall, A. et al.: Biochemical Genetics, 4, 655-658, 1970

4. Kozak, L.P. and Erdelsky, K.J.: Cell Physiol., 85, 438-448, 1975

5. Ruddle, L.P. et al.: Genetics 58, 599-606, 1968

6. Carter, N.D. and Parr, C.W. : Nature 216, 522, 1967

7. Ritte, F.H. et al. : Biochemical Genetics, 20, 475-481, 1982 8. Henderson, N. : J.Exp.Zool. , 158, 263-274, 1965

9. Womack, J.E. et al.: Biochemical Genetics, 13, 511-518, 1975

10. Khlebodarova, T.M. and Serov, O.L. : Biochemical Genetics, 18, 1027-1039.1, 1980

11. Shows, T.D. et al.: Biochemical Genetics, 4, 707-718, 1970 12. Wilcox, F.H.: Biochemical Genetics, 13, 243-245, 1975

13. Lewis, W.H.P. and Truslove, G.M. : Biochemical Genetics, 3, 493- 498, 1969

14. Shreffler, D.C. : Linkage of the mouse transferring locus. J. Hered. 54, 127-129, 1963 15. Shows, T.B. et al.: Biochemical Genetics, 3, 25-35, 1969

16. Roderick, T.H. and Guidi, J.N.: Strain distribution of polymor¬ phic variants. In Lyon M F, and Searle, A.G.: Genetic Variants and Strains of the Laboratory Mouse. Gustav Fischer Verlag, Stuttgart, N.Y., 2nd ed. 76-77, 1989 17. Nomura, T. : In Sordat, B. (ed.): Immune-deficient Animals. 4th Int. Workshop on Immune-deficient Animals in Exp.Res. Karger, Basel, 1984, 160-171 18. Nomura, T. , Esaki, K. and Tomita, T. (ed.): ICLAS Manual for Genetic Monotoring of Inbred Mice. p. 29, University of Tokyo Press, 1984 Table 1

nu/nu and i/i

Markers (chromosomes) BALB/cA nu/nu-META/BOM Reference

Another, also very interesting aspect of the invention is a cancer cell which is labelled with a genetic marker in the form of a gene encoding a product which in itself is visually distinguishable from non-marked cells, or which is capable of being made visually distin¬ guishable from the non-marked cells, in particular a gene the product of which is a coloured or fluorescent product or a product which can be converted into a coloured or fluorescent product, such as explain¬ ed above. Example 1 herein describes the construction of such a human cancer cell wherein the genetic marker is a lacZ gene, the conversion of the gene product of which into a coloured product can be performed by staining with the chromogenic substrate 5-bromo-4-chloro-3-indoyl-3- D-galactopyranoside (X-gal) resulting in a blue staining of the labelled cell.

Important specific embodiment of such labelled cancer cells are labelled cancer cells of one of the cell lines MDA-MB-231, MDA-MB- 435, MDA-MB-435, and OVCAR-3.

As indicated above, one further aspect of the invention is a new method for cancer therapy. This method is a method for controlling the progression of cancer cells in a human or animal patient, com¬ prising administering, locally or systemically, to the patient, tumour-infiltrating fibroblast cells which have been modified with a genetic construct expressing a gene product inhibiting the progres¬ sion of cancer cells. This aspect of the invention is based on the finding, according to the invention, that non-malignant fibroblasts which colonize cancer tumours are able to proliferate substantially proportionally to the proliferation of the cancer cells, which means that in a growing tumour, the proportion of non-malignant fibroblasts will be substantially constant, and that, thus, the non-malignant fibroblasts will be present throughout the growing tumour.

The utilization of the screening or developing method of the present invention in connection with the development of this gene therapy is a good example of the value of the screening of developing method for arriving, within a relatively short time, at a stage where the therapy can be carried into clinical trial:

In order to arrive at conclusive experiments the result of which will, firstly, indicate the potential efficiency of a gene which is a candidate for this gene therapy, and, secondly, serve as the indis¬ pensable pre-trials which will make it possible to obtain permission to enter into the clinical trial stage, fibroblasts which are capable of finding and infiltrating a malignant tumour of cancer cells of a human genotype in an immunodeficient non-human recipient vertebrate into which cancer cells of the said mammal genotype have been intro¬ duced, are modified so that they contain, and are capable of expres¬ sing, a gene which is a candidate for the gene therapy. It is then tested, using the screening method according to the invention, whether the fibroblasts containing the gene are capable of control¬ ling the progression of the cancer cells of the said mammal in the recipient vertebrate. One of the starting conditions for this experi¬ ment, that is, that the fibroblasts in question will be capable of localizing the cancer cells, can suitably be tested, using the dis¬ tinction method and the modified non-human recipient vertebrate according to the invention, in the following manner:

First, non-labelled human cancer cells are introduced into a non- human recipient, such as a nude mouse e.g. a nu/nu META/Bom mouse, the cells of which are labelled with, e.g. lacZ. After growth of tumours in the recipients, the tumours with the infiltrating fibro¬ blasts are stained, which will reveal presence of host cells in the tumour. From other tumours, the tumour-infiltrating fibroblasts are isolated and introduced into another non-human recipient, such as a nude mouse, the cells of which have not been labelled, together with or subsequent to the introduction of human cancer cells corresponding to the cells which were introduced into the first recipient, and it is investigated, utilizing staining of the gene product of the gene¬ tic marker, whether the fibroblasts are capable of localizing the cancer cells and proliferating together with the cancer cells.

Once this has been established, a next step could be to introduce the candidate gene in the fibroblasts in such a way that it is expressed when the fibroblasts have been transferred to a recipient.

In the above-mentioned gene therapy method, particularly preferred fibroblasts for use in a patient are fibroblasts of a tissue type which is compatible with the tissue type of the patient, most prefe¬ rably fibroblasts from the patient.

One aspect of the gene therapy according to the invention are fibro¬ blasts which are capable of finding and infiltrating a malignant tumour in a mammal and which contain, and are capable of expressing, a gene producing a gene product which is capable of controlling the progression of cancer cells in the mammal.

With reference to the screening model, such fibroblasts of the inven- tion can be defined as fibroblasts which are capable of finding and infiltrating a malignant tumour of a mammal genotype, in particular human genotype, in an immunodeficient non-human recipient vertebrate into which cancer cells of the said mammal genotype have been intro¬ duced, the fibroblasts containing 1) a gene which, when it is expres- sed in the immunodeficient recipient vertebrate by fibroblasts which are capable of finding and colonizing, in the recipient vertebrate, colonies of cancer cells of the said mammal genotype, is capable of controlling the progression of the cancer cells of the said mammal, and 2) a promoter securing expression of the gene product when the fibroblasts have been transferred to the said mammal.

One strategy for exploiting the gene therapy method would be to keep the gene-expressing fibroblasts in cell cultures, ready for use in gene therapy, which therapy is preferably instituted briefly after removal of a malignant tumour from a patient. In this strategy, a collection of cell cultures should be maintained, comprising the modified fibroblasts of a multitude of human tissue types. Another strategy is to isolate, immediately after removal of a tumour from a patient, fibroblasts from the tumour, modifying the fibroblasts with a gene which has been found to be able to control the growth of the cancer cells in question, and reintroducing the so modified fibro¬ blasts into the patient. Within the scope of the present invention is thus a method of treating a patient in need thereof with the above- mentioned fibroblasts.

Test systems for carcinogenesis and mutagenesis are known and descri- bed e.g. in W091/15579 which relates to mutagenesis testing using transgenic non-human mammals having a genome characterized by the presence of an excisably-integrated lambda phage containing a target gene system comprising LacI and LacZ operatively linked to prokaryo- tic expression signals. The carcinogenesis testing system according to the present invention uses a different approach as described below:

The invention also relates to a method for determining the carcino¬ genicity of a substance or a treatment on cells of a vertebrate donor genotype, comprising either

1) exposing the cells of the vertebrate donor genotype to the substance or the treatment followed by introduction of the cells into a non-human vertebrate recipient, or

2) introducing the cells of the vertebrate donor genotype into a non-human recipient vertebrate, followed by exposure of the recipient to the substance or the treatment,

the donor cells being modified by labelling with a genetic marker to either directly or latently permit distinction between on the one hand cells which are identical to the donor or cells derived there¬ from, and on the other hand cells of the non-human recipient verte- brate, and determining any capability of the substance or the treat¬ ment to convert the donor cells into cancer cells by examining any invasive and metastatic activity of the donor cells, either in vitro, in the recipient, or after transfer of the donor cells to another recipient, on the basis of detection of any metastasis utilizing the distinction obtained through the modification of the donor cells.

The donor cells of particular interest are human cells, in particular leukocytes, bone marrow cells or keratinocytes. The other parameters of this method, such as the choice of the recipient and the kind of genetic marker, are preferably the same as described above.

An example of such a carcinogenicity test according to the invention is given in Example 14.

The invention also relates to the use of a drug, the effect of which with respect to preventing, diminishing, controlling or inhibiting a disease has been established using the methods according to the invention for the preparation of a pharmaceutical composition for preventing, diminishing, controlling or inhibiting the disease, and to the use of a drug, the effect of which with respect to preventing, diminishing, controlling or inhibiting the progression of metastases has been established using the methods according to any the inven¬ tion, for the preparation of a pharmaceutical composition for preven- ting, diminishing, controlling or inhibiting the progression of metastases.

EXAMPLE 1

Labelling of human cancer cell lines with the lacZ gene

A number of human cancer cell lines have been described as being invasive and metastatic in immune incompetent animals. However, it is difficult to assess metastatic spread of a subcutaneously injected or inoculated cell line, since an exact detection of all microfoci of human tumour cells in the animals by usual histological procedures would require extensive sectioning of the whole animal. To overcome this problem, human cancer cells is transduced or transfected with a genetic marker allowing subsequent identification of the human cancer cells following inoculation into immunedeficient mice. The following example describes the labelling of the human cancer cells.

Cell lines

The MCF-7 and MDA-MB-231 human breast cancer cell lines were origi- nally obtained from ATCC, Maryland, USA, and the MDA-MB-435 human breast cancer cell line was kindly provided by Dr. Janet Price, MD Anderson Hospital, Houston, Texas, USA. The cells were routinely propagated in DMEM (Flow Laboratories, Scotland) with 10% Fetal Calf Serum (D10) or IMEM without phenol red and supplemented with 5% charcoal stripped fetal calf serum.

Retroviral transduction

Viral stocks of the BAG vector (Fig. 1) (Price et al. , 1987) packaged in PA317 cells (Miller et al., 1986) were used to transduce the breast cancer cell lines. The human breast cancer cells were plated one day before infection at a 1:10 split ratio. To infect the cells, the culture medium was replaced with 5 ml of viral supernatant con¬ taining 4 μg/ml of polyprene and incubated for 2 hours at 37°C in a 5% CO2 incubator. An additional 5 ml of D10 was then added and the cells were returned to the incubator. The medium was replaced with fresh D10 the next day. The resulting cell lines were named MDA-MB- 231 BAG and MDA-MB-435 BAG, respectively. Transfection using lipofection mediated gene transfer

MCF-7, MDA-MB-231 and MDA-MB-435 human breast cancer cells were transfected using the lipofection mediated gene transfer method (Chang et al. , 1989) with a vector containing the lacZ gene and a neomycin-resistance gene under a RSV promotor (Fig. 2) . 5 x 10-¹ cells were seeded in 25 cm^ culture flasks, grown overnight, and transfect¬ ed with 10 μg DNA in IMEM supplemented with 5% charcoal stripped serum. After 18 h, the DNA-containing medium was replaced by fresh IMEM containing 5% charcoal stripped serum. The following day, cells were split 1:10 and selection with G418 was initiated. Resistant colonies were isolated by means of cloning cylinders and tested for lacZ expression according to the method described below. Positive cell clones were expanded and used for further studies. The resulting cell lines were named MCF-7 pRSVIacZ, MDA-MB-231 pRSVIacZ and MDA- MB-435 pRSVIacZ, respectively.

In a similar manner, the cells can be genetically labelled by transfection methods such as calcium phosphate precipitation, DEAE- dextran, popyprene, protoplast fusion, electroporation, lipofection, lipid-mediated transfection, laser-mediated transfection and scrape- loading or by transduction methods by use of retrovirus, SV40, BPV or vaccinia virus, and similarly, other antibiotics, e.g. hygromycin, can be used for the selection of transfectants.

Selection procedure

To increase the number of cells expressing the lacZ gene, the trans- duced or transfected cell populations were grown in medium containing G418. The selected cells, however, did not all express ?-galactosi- dase as determined by X-gal staining. Therefore, cells were subjected to fluorescein-di-9-D-galactopyranoside (FDG)-FACS selection, as described by Nolan, et al. (1988). In this procedure, the release of fluorescein from a non-fluorescent substrate within cells expressing the lacZ gene product, 3-galactosidase, allows cell separation in a flow cytometer. A confluent 100 mm dish was trypsinized and the single cell suspension was adjusted to 1-5 x 10' cells/ml in D10. 100 μl of the cell suspension was incubated at 37^C in a Falcon 2058 tube. Uptake of the substrate FDG by the cells was accomplished by hypotonic shock with the addition of 100 μl of 2mM FDG in dtt^O. Following a 1 minute incubation at 37°C, the substrate was trapped within the cells by the addition of 1.8 ml ice cold D10. The cells were incubated on ice for 1 hour and sorted on a Becton Dickinson dual laser FACStar Plus flow cytometer set to a 488 nm wavelength.

In a similar manner other malignant and non-malignant human cells may be labelled.

X-gal staining of cells in culture

Cells in culture were placed on glass slides and allowed to grow for two days. They were then fixed with 0.5% (vol/vol) glutaraldehyde in PBS for 5 minutes, washed three times with PBS, and incubated over¬ night at 37^CC in X-gal staining solution (1 mg X-gal /ml; 35 mM potassium ferricyanide; 35mM potassium ferrocyanide; 2 mM MgCl in PBS) .

Results

lacZ transduction

MDA-MB-231 and MDA-MB-435 cells were infected with the BAG vector (Fig. 1). These cells do not stain with X-gal unless they have been infected with the BAG vector as it is shown in Fig. 3 for MDA-MB-435 cells. Because of a low viral titre (5 x 10^^" cfu/ml) , only about 1% of either cell line was initially found to stain positive with X-gal. Following G418 selection, approximately 60 - 70% of the G418- resistant cells expressed the lacZ gene as based upon X-gal staining. In order to enrich for lacZ-expressing cells, both breast cancer cell lines were subjected to FDG-FACS selection. This enriched both cell populations to >95% X-gal positivity. In order to determine the stability of the lacZ transduction, cells were passaged without G418 15 times following FDG-FACS selection and stained with X-gal. As seen in fig. 3B, MDA-MB-435 BAG cells showed only a slight loss in the relative number of lacZ expressing cells. However, heterogeneity in expression level was noted. Similar results were obtained with the other three cell lines.

EXAMPLE 2

Introduction and subsequent detection of lacZ expressing human cancer cells in nu/nu META/Bom mice

In this example the inoculation of lacZ expressing human cancer cells into nu/nu META/Bom mice with the subsequent extirpation of the pri¬ mary tumours and various organs from the host is described, aiming at identifying the human cancer cells following metastatic spread in the animals (see Brunner et al. , 1992).

Nude mice

6-8 weeks-old intact female nu/nu-META/Bom (Bomholtgaard, Denmark) mice were used. The mice were kept in sterile laminar flow clean benches at 25^βC and 50% humidity. At the end of the experiment, the mice were killed by cervical dislocation.

Inoculation of lacZ expressing human breast cancer cells into nu/nu META/Bom mice

2 x 10° tumour cells of the human breast cancer cell lines described in example 1 were inoculated subcutaneously, bilaterally into the flanks of nu/nu META/Bom mice. The cells were placed below.the tho¬ racic wall. The mice were sacrificed 6 or 8 weeks after cell inocu¬ lation.

X-gal staining of tumours and whole organs

Primary tumours and whole organs (liver, spleen, pancreas, intestine and lungs) were dissected from the animals and placed in a mixture of 2% (vol/vol) paraformaldehyde and 0.2% (vol/vol) glutaraldehyde in PBS for 2-3 hours at 4°C. After fixation, the tissue blocks were rinsed 3 times with PBS and then incubated for 24 hours at 4°C in 1 mg X-gal /ml; 35 mM potassium ferricyanide; 35 mM potassium ferro- cyanide; 2 mM MgCl₂; 0.02% (vol/vol) Nonidet P-40; 0.01% (wt/vol) sodium deoxycholate in PBS. The tissue blocks were then rinsed, first with 3% (vol/vol) dimethyl sulfate in PBS and then with PBS only. Until photographs were taken, the tissues were stored at 4^CC in 0.02% sodium azide in PBS. Photos were taken through an inverted dissecting microscope.

Frozen sections

Primary tumours and host organs were fixed at 4°C for 2 hours in 2% paraformaldehyde in 0.1 M PBS pH=7.4, rinsed with 0.1 M PBS and dehydrated in 0.1 M PBS with 7% sucrose for 2 hours and 15% sucrose for additional 2 hours, and finally processed for cryo-sections. The cryo-sections were then stained with X-gal following the procedure used for the cell lines and described in Example 1.

Histology

After paraffin embedding and haematoxylin-eosin (HE) staining, se¬ lected areas from primary tumours and from secondary foci identified by X-gal staining were processed for routine histological examina¬ tion.

Results

Detection of human tumour cells in vivo

The purpose of transducing the human tumour cells with the lacZ gene was to be able to find the cells after their dissemination in the nude mouse. Primary subcutaneous tumours of each transduced cell line demonstrated highly specific X-gal staining, whereas tumours from non-transduced cells did not stain blue. This is shown for MDA-MB-231 and MDA-MB-231 BAG tumours in Figure 4A and 4B, respectively. Identi¬ cal results were seen following staining of MDA-MB-435 and MDA-MB-435 BAG primary xenotransplants. Both BAG lines were serially passaged in nude mice, and they retained lacZ expression after at least four pas- sages. In cryo-sections of the primary tumours, the X-gal staining was also confined to the BAG-transduced human cancer cells (Fig. 5). Both untransduced and transduced tumours was locally invasive, pene¬ trating the peritoneal wall of the animals. X-gal staining of mouse liver, spleen, pancreas, intestine and lungs from mice with trans¬ duced sc tumours of either tumour line demonstrated blue staining of secondary tumour formation within organs in the peritoneal cavity (Fig. 5) . Untransduced and lacZ transduced secondary tumours were locally invasive in various intraperitoneal organs: MDA-MB-231 BAG preferentially spread to pancreas and the hepatic portal tract, whereas secondary MDA-MB-435 BAG tumours most often were on the omentum of the intestine, and in the spleen, pancreas, and hepatic portal tract. Lung metastases were found constantly in mice with either tumour (Fig. 5). Histologic examination of the blue stained areas in lungs confirmed the presence of micrometastases (Fig. 5) . No positive X-gal staining were found in mice inoculated with uninfected tumour cells or in organs without tumour metastases, with the exception of weakly positive staining of the gastro-intestinal tract in some animals. The percentage of mice with intraperitoneal spread and lung metastases following either 6 or 8 weeks of subcutaneous tumour growth, is shown in Table 2.

TABLE 2

INTRAPERITONEAL SPREAD LUNG METASTASES

17/18

5/5

18/19

5/5

In a similar manner other lacZ expressing malignant or non-malignant human cells may be introduced into immunedeficient animals intra¬ venously, intraperitoneally, intracranially or into various organs. Comparative studies between non- ransduced and transduced tumour cells

The aim of the study is to obtain a model for studying the invasive and metastatic phenotype of human cancer cells. Determination of possible effect of a retroviral transduction on the transformed phenotype of the tumour cells was to be performed. First, it was observed that the transduction altered neither the tumourigenicity nor the lag period or growth rate of the tumours. Second, comparison betwen in vitro and in vivo invasion of the cells before and after transduction was performed. Since the invasion process of tumour cells includes infiltration of the basement membrane, we studied the cells' ability to cross an artificial basement membrane (Matrigel) (Albini et al.,1987). In this assay, no significant activity diffe¬ rence was found between non-transduced and transduced cells. By X-gal staining of cells which had crossed the Matrigel (cells located on the lower part of the polycarbonate filter) , lacZ expressing cells were shown to be capable of crossing the Matrigel. In nude mice, transduction did not interfere with the cells' ability to locally invade the peritoneal wall or to invade into the peritoneal cavity and into internal organs. However, a reliable in vivo comparison between the invasive and metastatic activity of wild-type cells ver¬ sus transduced cells were not possible, since micrometastases from wild-type tumours could not be readily identified. With this reserva¬ tion, transduced and non-transduced cells seem to behave similarly in both in vitro and in vivo invasion assays, indicating that the retro¬ viral integration of the lacZ gene did not significantly change the invasive behavior of the cells.

EXAMPLE 3

In vivo examination of the adherence properties of labelled human cancer cells

Adherence properties of cancer cells are of importance in the process of metastasis. Far from all cancer cells that enter the blood stream establish metastases, and furthermore, cancer cells of different ori- gin (different histological type) tend to have characteristic pat¬ terns as to where secondary tumours, i.e. metastases, occur. This indicates that specific mechanisms/properties of recognizing certain structures and adhering to these are involved in tissue specific metastatic spread (Frandsen et al. , 1992).

Intravenous injection of lacZ transduced human cancer cells (accord¬ ing to Example 1) into the tail vein of immunedeficient mice provides a means of investigating in vivo adhesion capability as well as capa¬ bility of those cells to form experimental metastases. This model might be used 1) for studying the specificity for various organs of different tumour cells and 2) as a quick in vivo screening for the effect of experimental anti-metastatic adjuvant therapy (ct. Example 5).

Cell lines

3 lacZ transduced (Example 1) human breast cancer cell lines MDA-MB- 231 BAG, MDA-MB-435 BAG and MCF-7 BAG were used.

Mice

6-8 weeks old intact nu/nu-META/Bom female mice were used. One day prior to cell inoculation an Estrogen deposit pill was administered to all mice, subcutaneously.

Cell harvesting

The cells were harvested non-enzymatically by scraping using a rubber policeman. After centrifugation and resuspension the cells were counted for viability using Trypan-Blue, and kept on ice until in- jected. Each mouse was injected in the tail vein with 50.000 cells per 0,1 ml medium. Surplus of cell suspension was incubated as a viability control for the cells. Experimental design

At intervals following cell injection (5 min; 1 hour; 4 hours; 24 hours; 1,2,4 and 6 weeks) 3 mice were sacrificed and the lungs were processed for X-gal staining according to Example 2. Fresh or frozen tumour tissue was included as a positive staining control.

Results

Adhesion capability was quantitated by X-gal staining lungs from animals injected intravenously with lacZ transduced human breast cancer cells. Fig. 6 shows the results from an experiment where MDA- MB-231 BAG cells were injected into the tail vein of nu/nu META/Bom mice. Lungs were removed at 5 min, 24 hours and 2 weeks following cell injection. The lungs were stained with X-gal according to the method described in Example 2. Few minutes after cell injection, a large number of the lacZ expressing human cancer cells were trapped in the capillaries of the lungs (Fig. 6A) , but most of these cells were cleared from the lung tissue following another 24 hours (Figure 6B) . After two weeks a small number of tumour nodules (experimental metastasis) consisting of several tumour cells had been established in the lungs (Figure 6C) .

Distinct differences have been found between the three cell lines: MDA-MB-231 BAG having the best in vivo adhesion properties followed by MCF-7 BAG. MDA-MB-435 BAG was found to be almost non-adhering. These conclusions are based on the findings of time periods shorter than 24 hours, i.e. thus not including differences in capabilities of forming experimental metastases.

In a similar manner other genetically labelled human cancer cells may be introduced into the veins of nude mice. The model may be used in screening of potential anti-metastatic compounds. EXAMPLE 4

Inhibition of the invasive and metastatic process in mice using monoclonal antibodies directed against urokinase

The significance of the enzymatic activity of urokinase type plasmi- nogen activator (u-PA) in the invasive and metastatic process of cancer cells was examined as described below and the results of this experiment substantiates that this model will be valuable for testing the anti-invasive and anti-metastatic effect of substances inhibiting activity of proteolytic enzymes in cancer, as for example interaction of uPA binding to its receptor (uPAR) , inhibition of proteolytic ac¬ tivity of metalloproteinases, inhibition of the proteolytic activity of cathepsins etc.

Materials and methods

Cell lines

The human breast cancer cell line MDA-MB-231 was routinely propagated in DMEM with 5% Fetal Calf Serum (D5). Cells for nude mouse experi¬ ments were harvested using a cell scraper instead of using enzymes. The cell lines were tested and found free from Mycoplasma contamina¬ tion.

Mice

Nude female mice 6-8 weeks-old nu/nu META/Bom (Bomholtgaard, Denmark) were used. The mice were kept in laminar flow clean benches and all equipment used was autoclaved.

Retroviral transduction

Retroviral transduction with the lacZ gene of the human cancer cells was performed as described in Example 1. Selection procedure

Selection of transduced cells expressing the lacZ gene was performed as described in Example 1.

Cell inoculation

A total number of 2 x 10⁶ MDA-MD-231-BAG tumour cells were inoculated subcutaneously into each flank of the animals. The tumour cells were always placed inferior to the thoracic wall. One day before cell inoculation 250 μg of antibodies were injected into the mice. Based on pharmacokinetic studies of the antibody used, a second injection of 250 μg antibody was given on day 21 after cell inoculation. The mice were sacrificed 6 weeks after cell inoculation.

Antibodies

u-PA-antibodies clone 5 (Nielsen et al. , 1986) were used to inhibit the enzymatic activity of uPA, and an IgGl mouse monoclonal antibody directed against a barley protein was used as an irrelevant control antibody.

Examination of the transduced human breast cancer cell line

The transduced cell line was examined for the presence of mRNA coding for both u-PA and u-PAR by Nothem blotting.

Total RNA was extracted from MDA-MB-231 BAG xenografts as previously described (Chirgwin et al. , 1979) . 25 μg of RNA was size fractionated by electrophoresis in a 1.2% agarose gel containing 2.2 M formal¬ dehyde. The RNA was blotted onto a nitrocellulose filter and hybrid!- zed with ³²P-labeled cDNA probes at 58°C for 18 hours in standard hybridization buffer (Chomczynski et al. , 1987) . The cDNA for u-PA comprising pHUK8 (Verde et al., 1984) and the cDNA for the u-PAR com¬ prising p-u-PAR-1 (Roldan et al., 1990) were used. Following hybridization, filters were washed with three changes of 0.1 x SSC for a total of 1 hour at 65°C. Autoradiography was performed at -80^CC using two Chronex Quanta III intensifying screens. X-gal staining of cells in culture

X-gal staining of cells in culture was performed as described in Example 1.

Tumour growth

Tumours were measured twice weekly in two dimensions and tumour growth curves were constructed on the basis of a transformed Gompertz function.

Evaluation of local invasion

Six weeks after cell inoculation, all animals were sacrificed by cervical dislocation. Primary subcutaneous tumours located at the site of cell inoculation were excised from the direction of the intraperitoneal cavity towards the skin, thus leaving the peritoneal muscle wall at one site and the skin on the other site of each tu¬ mour. Each tumour was divided into two by an incision perpendicular to the longest diameter of the tumour. Another incision was made close to the border of the tumour. Histological sections were made from the site of incision. Local invasion was defined as tumour cells placed between the peritoneal muscle fibers. No attempt was made to relate the treatment to invasion of the skin. In cases where the tumour cells were located close to the muscle fibers but without splitting the muscle, additional sections were made to exclude inva¬ sion.

Evaluation of metastases

At autopsy, liver, diaphragm, spleen, pancreas, intestine and lungs were excised from each animal. The organs were processed with X-gal according to the method described in Example 2. To confirm the pre¬ sence of human tumour cell metastases, blue areas from various organs were fixed in 4% formalin and processed for routine histology. If an organ presented one or more blue areas it was registered as positive for metastatic lesion. Alternatively, an ELISA for 3-D-galac osidase as described by Fugiwara et al. could be used as an objective measurement of metastatic spread. Frozen sections

Examination of the frozen tissue sections was performed as described in Example 2.

Inhibition of cell-surface plasminogen activation by anti-u-PA anti¬ bodies

The capacity of MDA-MB-231 BAG cells to activate plasminogen was determined by a modification of the method previously described for use with suspension-growing cells (Ellis et al. , 1990) . Briefly, the MDA-MB-231 BAG cells were grown to confluency in 24 well Costar trays maintained in serum-free DMEM (Flow Laboratories, Scotland) in the presence of the plasmin inhibitor aprotinin (10 μg/ml) (Bayer, Ger- many). Prior to assay the cells were washed 3 times i Hepes-buffered DMEM, followed by incubation for 15 minutes at room temperature in the presence or absence of 10 μg/ml of a monoclonal antibody to u-PA (clone 5). After 2 subsequent washes the cells were incubated with plasminogen (20 μg/ml) and the specific fluorogenic plasmin specific substrate H-D-Val-Leu-Lys-AMC (0.2 mM) in PBS containing 0.2% BSA (200 μl in each of 12 wells per incubation) at 37°C was added. At timed intervals 150 μl of the cell supernatant was removed, diluted with 150 μl 0.05 M Tris pH 7.4, 0.1 NaCl and the fluorescence inten¬ sity measured using micro-cuvettes in a Perkin Elmer LS-5 lumines- cence spectrometer with excitation and emission wavelengths of 380 and 480 nm, respectively. Plasmin concentrations were then determined by calculation of substrate hydrolysis (dF/dt) over each time inter¬ val, and comparison with calibration curves constructed using active site-titrated plasmin (Ellis et al., 1990).

Pharmacokinetics of anti-u-PA-antibodies

Seven groups each of 5 nude mice were injected intraperitoneally with a single dose of 100 μg anti-u-PA-antibodies clone 5. At 0, 1, 2, 4, 8, 16 and 32 days after this injection a group of 5 mice were bleed and serum was pooled. Furthermore, in the experiment with tumour- bearing animals, serum was obtained from all anti-uPA-antibody-treat¬ ed animals day 14 after antibody injection. Nunc Immunoplates (Flat bottom Maxisorb, Nunc A/S, Denmark) were coated over-night at 4°C with 1 μg/ml of u-PA (UKIDAN Leo) in 0.1 M Na₂C0₃, pH 9.8, Excess u- PA was washed away and additional protein binding sites were blocked by a one hour incubation with 1% skimmed milk powder in PBS. After washing, the plates were incubated for 1 hour at 37°C with standard dilutions of anti-u-PA antibody clone 5 in mouse serum or sample to be tested. The plates were washed again and then incubated for an¬ other hour with biotinylated rabbit anti-mouse IgG (Dako) . After an additional wash the plates were incubated with peroxidase-coupled avidin (Dako) for one hour and the peroxidase reaction was developed using OPD-tablets and H2O2 in 0.1 M citrate-phosphate buffer, pH 5.0. The reaction was stopped by adding 100 μl 1 M H2SO and the absor¬ bance was read at 490 nm. All the samples were run in duplicates.

Results

lacZ transduction and selection of cells

The results of examination of the lacZ transduction and selection of cells is described in Example 1.

Results of the examination of the transduced human breast cancer cell line

MDA-MB-231 BAG tumours expressed aboundant mRNA for both u-PA and u-PAR and the cross-linking experiment using l²⁵ι ^TF clearly demon¬ strated the presence of u-PAR protein in the detergent phase of the cell lysate.

Detection of human tumour cells in vivo

Detection of human tumour cells in vivo was performed as described in Example 2. Inhibition of the invasive and metastatic process in mice using anti- u-PA antibodies

MDA-MB-231 BAG tumours in mice not injected with anti-u-PA antibodies were growing invasively into the peritoneal muscle layer of the mice. The cancer cells expanded into the muscle fibers, and often tumour cells were located at the peritoneal side of the muscles. The irrelevant antibody had no effect on local tumour invasion. The anti-u-PA antibodies inhibited to some extent local spread of tumour cells.

As seen from Table 3, 5 out of 8 untreated control mice had metasta¬ tic spread of the cancer cells to intraperitoneal organs. Most com¬ monly, tumour cells were found in the portal tract of the liver and in pancreas. The irrelevant antibody had no effect on the metastatic formation to the intraperitoneal cavity (Table 3). Injection with anti-u-PA antibodies significantly inhibited the extension of tumour cells to intraperitoneal organs (Table 3) . There was also a signifi¬ cant reduction in the number of animals with metastatic deposits to the lungs in the group of mice injected with anti-u-PA antibodies as compared to untrated control mice and mice treated with the irrele- vant antibody.

Metastatic spread of the human cancer cells was confirmed by the presence of cancer cells in the blue spots, as determined by conven¬ tional histologic examinations. In a similar manner, other potential anti-invasive, anti-metastatic and/or anti-angiogenic compounds can be tested in the described model (see Example 5) .

TABLE 3

intraperitoneal lung diss. metastases

Control 5/8 8/8

Barley antib. 5/10 9/10

anti-u-PA antib. 0/8 1/8

Effect of anti-u-PA antibodies on tumour growth

Examination of the effect of the anti-u-PA antibodies on tumour growth showed that neither anti-u-PA antibodies nor irrelevant anti¬ bodies caused significant changes in tumour growth (Figure 7)

Pharmacokinetics

A single intraperitoneal injection of 100 μg anti-u-PA antibody resulted in a serum concentration of the antibody of approximately 25 ng/ml. The serum concentration only declined slowly with an esti¬ mated

°f about 14 days (Figure 8)

EXAMPLE 5

The Adjuvant Model

Inhibition of micrometastases

Development of better strategies for adjuvant therapy of breast cancer has so far been limited to clinical trials, which means a minimum observation period of 5 to 10 years. An experimental model that mimics the clinical events of breast can¬ cer, in terms of allowing invasion and metastatic spread of cancer cells, might be used in research aiming at inhibiting the early deve¬ lopment of micrometastases. The model described below is based on the use of genetically labelled human breast cancer cells introduced sub- . cutaneously into female nu/nu-META/Bom mice.

Cell lines

3 lacZ transduced human breast cancer cell lines, MDA-MB-231 BAG, MDA-MB-435 BAG and MCF-7 BAG were used. Transduction was performed as described in Example 1.

Mice

6-8 weeks old intact nu/nu-META/Bom female mice were used.

Cell harvest

Cell harvest was performed non-enzymatically as described in Example 3, but resuspension of the cells was performed at a concentration of 1-2 x 10⁶ cells pr. 0,2 ml.

Cell introduction

Cells were introduced bilaterally into 35 female nu/nu META/Bom mice according to the method described in Example 2.

Preliminary experiments

In order to establish the time of non-detectable cancer cell dissemi¬ nation a number of experiments have been performed (see Table 4) . TABLE 4

2 weeks 4 weeks 6 weeks 9 weeks

Tumour + + +

.extirpation

Sacrificed + + + +

The mice were divided into 7 groups of 5 mice each.

After 2 weeks the subcutaneous tumours were extirpated from one group of mice, following which the animals lived until 9 weeks after the cancer cell introduction. At the same time a similar group of 5 mice were sacrificed and examined for lung metastases according to the method described in Example 2.

The entire procedure was repeated at 4 and 6 weeks, respectively.

After 9 weeks the experiment was terminated and all previously opera¬ ted mice plus the 5 non-operated mice were sacrificed and examined for lung metastases according to the method described in Example 2. The results will be used as a guide for the design of future thera- peutic experiments as to when tumours can be excised with "non- detectable" lung metastases i.e. metastases which are only visible when the tumour cells are labelled with the genetic marker, and thus prior to "visible" lung lesions.

For each time interval the tumours were measured in two dimensions prior to other procedures in order to calculate the average tumour burden for statistical comparison between the two groups.

.Results

The preliminary experiments indicate for both MDA-MB-231 BAG and MDA- MB-435 BAG that "non-detectable" cancer cell dissemination to the lungs takes place in the interval of 4-6 weeks after cell inocula¬ tion. For MCF-7 BAG the interval has not yet been established. Therapeutic experiments

The model will be used for experiments studying the effect of con¬ ventional and experimental adjuvant therapy:

1) Anti-proliferative therapy, such as conventional chemotherapy (Cyclophosphamid, Methotrexat, 5-Fluoro-Uracil and Adriamycin) and endocrine therapy (Tamoxifen) and combinations hereof.

2) Experimental adjuvant therapy aiming at inhibiting the development of micro-metastases.

The processes involved in invasion and metastasis can be divided into 3 main parts: A) dissolution of the extracellular matrix and basement membranes; B) cancer cell migration; and C) attachment either by cell-to-cell or cell-to-substrate interaction.

The experimental adjuvant therapy will be directed against each of these mechanisms:

A) Inhibiting invasion by blocking extracellular proteolysis (Ossow- ski et al., 1991). Monoclonal antibodies directed against the cataly¬ tic part of the urokinase-type plasminogen activator^' (u-PA) will be used in order to prevent invasion and metastases.

In a similar manner antibodies inhibiting binding of uPA to the uPAR (Rønne et al., 1991) might be used.

B) Modulation of growth factors of significance for cell-migration (Brύnner et al., 1992). Experiments will include monoclonal anti¬ bodies directed against growth factors and/or growth factor recep¬ tors. One example is the type-1 Insulin-like growth factor receptor, since IGF-1 has been shown to influence cancer cell migration (Stracke et al., 1989).

C) Blocking cellular adhesion molecules, and thereby preventing the cells from adhering to endothelial cells of capillaries (Bretti et al., 1989; Humphries et al., 1988; Saiki et al., 1989). For these experiments monoclonal antibodies against E-Cadherin and N-CAM will be used.

EXAMPLE 6

Neo-adjuvant therapy

It is often debated whether or not, or to what extent, surgical procedures accelerates cancer cell spread by introducing the cancer cells into the circulation.

It would therefore be of interest to develop a model in which this question can be addressed. With this model, the effect of neo-ad¬ juvant therapy, which comprises treatment prior to removal of a tumour, can be studied in details.

The adjuvant model (Example 5) will be used for experiments of neo- adjuvant therapy by administering therapy prior to tumourectomy. As an end-point for evaluating the effect of the applied neo-adjuvant therapy, the frequency of lung metastases as visualised by staining of the product encoded by the genetic marker in treated animals compared to untreated animals will be used.

In a similar manner, animals which recieve adjuvant therapy as de- scribed in Example 5, could be randomized prior to the adjuvant therapy into two groups, one of which recieves neo-adjuvant therapy.

EXAMPLE 7

Establishment of a lacZ transgenic nu/nu META/Bom mouse.

As a tool in the study of tumour/stroma cell interactions a lacZ expressing transgenic nu/nu META/Bom mouse is established. The lacZ gene is attempted expressed ubiquitously, but also tissue/cell specific expression is pursued. By inoculating human cancer cells subcutaneously into lacZ transgenic nu/nu META/Bom mice, the stromal component of the xenotransplanted human tumours is -D-galactosidase expressing whereas the human cancer cells will not express the lacZ gene. Thereby, the different cell types can be distinguished from each other, either by X-gal staining or by FACS as described in Example 1.

Construction of lacZ transgenic nu/nu META/Bom mice

A plasmid is prepared in which the lacZ gene is brought into such a context that it is efficiently transcribed and translated in mammalian cells. In particular, the enhancer/promoter combination is chosen to give the desired specificity of expression, e.g. in vir¬ tually all cells of transgenic animals. Examples of enhancer/promo¬ ters with very general expression are derived from the eEF-1 alpha gene, the HMG gene, the UBI gene, and the early genes of CMV. Each of these includes enhancer, promoter, an intron, the lacZ gene with a nuclear targeting sequence, a polyadenylation site, and a MAR ele¬ ment. Introns increase transgene expression (Palmiter et al. , 1991), the MAR element (matrix attachment region) is optional but has been reported to increase consistent expression of transgenes (McKnight et al., 1992; Phi-Van et al., 1990; Stief et al. , 1989).

The EF enhancer/promoter is derived from the eukaryotic translation elongation factor 1 alpha (eEF-1 alpha) , which normally directs the synthesis of one of the most abundant proteins in eukaryotic cells (Mizushima and Nagata, 1990). It is active in all cells that synthe- size protein. The derivative is active in transfection experiments with many cell types.

The HMG enhancer/promoter belongs to the housekeeping gene for murine hydroxy-methylglutaryl-coenzyme A reductase, and it has also been used for ubiquitous expression (Mehtali et al., 1990).

The UBI enhancer/promoter normally drives the synthesis of ubiquitin, a protein present in all cells (Wiborg et al. , 1985). The CMV enhancer/promoter belongs to the early transcription unit of cytomegalovirus. Like the parent virus, it is active in many cell types, and can be used for ubiquitous expression (Schmidt et al., 1990).

Examples of suitable vectors are shown in Figures 9-12.

To facilitate detection of the expression of the lacZ gene in mamma¬ lian cells, the sequence contains a nuclear localization signal so that the θ-galactosidase is targeted to the nucleus. Cells containing this transgene and developed with the chromogenic substrate X-gal have the blue stain concentrated to the nucleus and are therefore more easily detected than if the stain were dispersed over the whole cell volume.

A linear fragment of DNA prepared to contain the lacZ gene and its control sequences, but not irrelevant plasmid sequences, is micro- injected by standard techniques into a pronucleus of mouse one-cell zygotes. These are then implanted into the oviduct of recipient female mice which have been made receptive by mating with sterile males. The one-cell zygotes used for microinjection are derived from crosses between male nu/nu META/Bom mouse and female +/+ META/Bom mice.

Progeny is tested for lacZ expression by X-gal staining in important cell types in tail biopsies. After breeding to preserve transgene and nu character, the tissue specificity is further characterized in descendants of the transgenic founders.

In a similar manner, lacZ transgenic animals is produced from other immunedeficient animals e.g. nu/nu BALB/cABom, nu/nu-C57BL/6JBom, nu/nu-SJL/NBom, Bom:NMRI-nu, FOX CHASE SCID, nu/nu-FOX CHASE SCID, hr/hr-FOX CHASE SCID, NIH-III (bg/nu/xid) and BAR mice, and for DNA constructs under different gene control, suitable for several purposes. Testing of off -spring

With the aim of isolating off-spring with lacZ expression, fibroblast and endothelial cells from nude off-springs of the transgenic animals is stained with X-gal as described in Examples 1 and 2. Animals with a positive X-gal staining reaction is further tested for lacZ expression in other cell types using X-gal staining as described in Examples 1 and 2. Animals with high expression of β- D-galactosidase is used for establishment of a lacZ transgenic strain of nu/nu META/Bom mice.

In a similar manner lacZ transgenic animals may be produced from other immunedeficient animals .

EXAMPLE 8

Use of the immunedeficient transgenic mouse for examination of the development of autoimmune diseases in vivo and for identification of substances suitable for treatment of autoimmune diseases

The experimental scheme described below is used for examination of the development of various autoimmune diseases. The scheme comprises the transfer of cells from a human donor, having or being in the risk of having an autoimmune disease, to a transgenic lacZ nu/nu META/Bom mouse prepared as described in Example 7. Due to the possibility of identifying the human donor cells in the immunedeficient transgenic mouse, the cells of which can be visualized by X-gal staining, in particular the experimental scheme may be used for localization of cells causing or participating in the development of an autoimmune disease and for examination of the interaction between the cells causing an autoimmune disease and the surrounding cells. In addition, the capability of the mouse cells from the transgenic immunedeficient mouse having been in contact with the human donor cells to induce or transfer a disease to other mouse cells may be examined by transfer- ring cells from the immunedeficient transgenic mouse to another immunedeficient non- ransgenic mouse. Furthermore, the experimental scheme may be used to identify substan¬ ces which can be used to inhibit or counteract the progress of the disease or which can be used for prophylaxis and thereby as drugs to prevent, control and/or combatting the autoimmune disease. Thus, the scheme opens widespread possibilities for examining human cells in vivo and for examining the effect of various substances on the human cells in vivo .

Experimental scheme

The experimental scheme comprises the following steps:

1) Cells from a human patient having an autoimmune disease or possessing the potential to develop an autoimmune disease are transferred to a lacZ transgenic nu/nu META/Bom mouse produced as described in Example 7.

2) After a period of time, the duration of which should be established individually for the various diseases to be examined and which should allow the cells to be established in the lacZ transgenic nu/nu META/Bom mouse, the localiza¬ tion of the human donor cells is identified by X-gal stain¬ ing of tissues considered likely to harbour the human donor cells. X-gal staining of organs and cells is described in Examples 1 and 2. Any interaction between the donor cells and the cells from the lacZ transgenic nu/nu META/Bom mouse, e .g. in the form of infiltration, necrosis, prolife¬ ration etc. may be determined in the same manner.

3) In order to determine the nature of the interaction between the human donor cells and the cells from the lacZ trans¬ genic nu/nu META/Bom mouse such as the capability of the mouse cells to provoke the onset of the disease or to transfer the disease to other cells, cells from the lacZ transgenic nu/nu META/Bom mouse is transferred to other non-transgenic immunedeficient mice. Thereby, the donor cells may be identified in the recipient mice using visua¬ lization with X-gal staining. This differentiation allows localization and examination of the interaction between the donor and cells from recipient mice such as described above under 2) .

4) The effect of a substance on the development of the human donor cell, the interaction between the human donor cells and the cells from the lacZ transgenic nu/nu META/Bom mouse, the pro¬ gress of the disease and the potential as a prophylactic treat¬ ment is examined by treating the mouse with the substance in question using various treatment schemes, various treatment ways and various doses of the substance In question. The treat¬ ment may be locally or generally applied and may be combined with e.g. surgical treatment in some instances. Due to the possibility of visualizing the human donor cells, the direct effect of the treatment on the human donor cells and the pos- sible Interaction between the human donor cells and the cells from the lacZ transgenic nu/nu META/Bom mouse can be determined. In addition the above outlined experiment for determination of the effect of a substance on the human donor cells may also be performed on the additional transfer of cells from an lacZ transgenic nu/nu META/Bom mouse to a recipient mouse as described above under 3) .

Autoimmune diseases which may be examined using the above outlined experimental scheme comprises various types of diabetes such as diabetes mellitus in which pathological processes In the islets of pancreas takes place before clinical manifestation of diabetes mel¬ litus (type 1)). Furthermore, conditions related to the functions of the thyroid gland, In particular hyper- or hypofunctioning of the gland, e.g. thyroiditis, the pancreas, the adrenal gland, in parti¬ cular hypofunction, the intestine, and arthritis, encephalitis and the autoimmune diseases like systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, sarcoidosis, primary biliary cirrhosis, haematolocial diseases like autoimmune hemolytic anemia, immune thrombocytopenia purpura and together with autoimmune glomerulone- phritis may also be examined. In addition, diseases causing obesity may be examined. The substance which may be. used in the treatment may be a chemical compound, antibodies or antigens, amino acids, proteins such as a growth factors, lymphokines, interferons, interleukines, insulin, hormones, leukotrienes, anti-sense RNA etc.

In a similar manner the human cells may be genetically engineered with a gene the product of which allows visuable detection. The gene could be the lacZ gene or other genes the product of which can be visualized by specific staining procedures.

In addition, the lacZ transgenic animals may be crossed into other transgenic animals resulting in animals expressing both genes.

EXAMPLE 9

Examination of the neurological development using brain tissue from lacZ transgenic nu/nu META/Bom new-born (or embryos) mouse introduced into a nu/nu META/Bom mouse

Donor tissue

Brain tissue is excised from new-borne or embryos of the lacZ trans¬ genic mouse described in Example 7.

Tissue transplantation

Whole tissue samples or single cell suspensions of the removed brain tissue are inoculated into the brain of 4-6 week old nu/nu META/Bom mice.

Evaluation

2-3 months following brain tissue inoculation, the animals are killed and the brain is removed. In order to identify the brain transplant, the recipient brain is stained for ?-galactosidase production accord¬ ing to the methods described in Example 2. The interaction between the donor and recipient cells can now be studied using histology, immune histochemistry, in situ hybridization or similar methods.

In a similar manner, other tissues and organs from lacZ transgenic nu/nu META/Bom mice can be excised and subsequently inoculated into -various organs or sites in conventional nu/nu META/Bom mice.

EXAMPLE 10

Isolation of tumour infiltrating fibroblasts from the transgenic mouse

Recent studies have indicated that the non-malignant cell population, e.g. fibroblasts and endothelial cells, in epithelial cancers con¬ tribute to the malignant phenotype (Basset et al., 1990; Pyke et al., 1991). Furthermore, significant genotypic differences between "tu¬ mour-derived" fibroblasts and "non-tumour-derived" fibroblast, have been reported (Hakim, 1989; Cullen et al., 1991), indicating that fibroblast in tumours may represent a subgroup of fibroblasts. In addition, it has been observed that each of the human breast cancer xenografts has a characteristic and constant fraction of diploid cells during tumour growth, suggesting that the stromal compartment of the tumours proliferates at a rate proportional with the epithe- lial human breast cancer cells.

Finally, using the Boyden chamber assay (Albini et al. , 1987; Frand- sen et al., 1992) , it has been shown that TIF (tumour infiltrating fibroblasts) are active in degrading matrigel (basement membrane material).

In this example, the isolation of tumour infiltrating fibroblast from xenotransplanted human breast cancer cells growing in lacZ transgenic nu/nu META/Bom mice will be described. Tumour cell inoculation

As described in Example 2, human breast cancer cells (MDA-MB-231 or MCF-7) are inoculated subcutaneously into nu/nu META/Bom mice.

Isolation of tumour infiltrating fibroblasts

6-8 weeks following tumour cell inoculation, the primary subcutaneous tumour is removed and transferred to a sterile petri dish containing tissue culture medium. The tumour is disaggregated mechanically and/or enzymatically (Engelholm et al. , 1985) into a single cell suspension. Based on expression of lacZ, or another marker, the fibroblasts can be isolated from the single cell suspension using FACS.

Alternatively, the tumour infiltrating fibroblasts may be separated from the human tumour cells by differences in growth requirements in vi tro .

Results

As seen in Fig. 9A, the first passage in vitro of an MCF-7 human breast cancer xenograft resulted in the outgrowth of epithelial tumour cells and fibroblasts. However, as seen in Fig. 9B, already in the second in vitro passage, the tumour infiltrating fibroblast overgrew the tumour cells, resulting in a pure fibroblast culture. The fibroblasts have now been serially passaged for more than 10 passages without any sign of epithelial tumour cells.

In a similar manner, other human cancer cell lines may be inoculated into the lacZ transgenic nu/nu META/Bom mouse with subsequent isola- tion of tumour infiltrating fibroblasts. EXAMPLE 11

Co-inoculation of tumour infiltrating fibroblasts from lacZ trans¬ genic nu/nu META/Bom mice and human breast cancer cells into nu/nu META/Bom mice

A prerequisite for using tumour Infiltrating fibroblasts as a carrier in gene therapy (see Example 10) is that the tumour infiltrating fibroblasts are able to repopulate a human xenograft, either by being inoculated simultaneously with the human cancer cells or by being in¬ jected Intravenously into a mouse carrying a human tumour xenograft.

Cell inoculation

10-* tumour infiltrating fibroblasts from lacZ transgenic mice or fibroblasts which in vitro are transfected or transduced with lacZ are mixed with 10° human tumour cells and inoculated subcutaneously into a nu/nu META/Bom mouse. 6-8 weeks later, the tumour is removed. In order to evaluate the fate of the tumour infiltrating fibroblast, the tumour is stained for ?-galactosidase production according to the procedure described in Example 2.

In a similar manner tumour infiltrating fibroblast from lacZ trans¬ genic nu/nu META/Bom mice might be introduced by intravenous injec- tion into nu/nu META/Bom mice carrying human breast cancer xenografts with subsequent extirpation and X-gal staining of the primary human breast cancer.

EXAMPLE 12

Gene therapy utilizing tumour Infiltrating fibroblasts as carrier of a gene whose product is inhibitory to either proliferation, invasion or metastatic spread of the human cancer cells

In order to inhibit either tumour growth or tumour invasion and metastasis it would be desirable to apply a local therapy, thereby reducing potential side effects on normal cells. One possibility would be to direct genetically engineered cells expressing a factor which is inhibitory to the cancer cells, to the tumour or to metas¬ tases. Data from the literature suggest that tumour infiltrating lymphocytes may be candidate cells for such a therapy strategy. Another possibility would be to genetically engineer tumour infil¬ trating fibroblasts.

Transfection of tumour infiltrating fibroblasts derived from a lacZ transgenic nu/nu META/Bom mouse

Tumour infiltrating fibroblasts are isolated from a human breast car¬ cinoma xenograft grown in a nu/nu META/Bom mouse or a lacZ transgenic nu-nu/META/Bom mouse or another immune deficient animal as described in Example 10. The fibroblasts are transfected or transduced (see Ex¬ ample 1) with a vector encoding a gene product which is inhibitory to either tumour cell proliferation or tumour cell invasion and meta- stasis. The gene product could be TGF-3, interleukin 1, 2, or 6, the amino terminal fragment of urokinase type plasminogen activator (u-PA), PAI-1, PAI-2, TIMP-1, TIMP-2 etc.

Introduction of transfected tumour infiltrating fibroblast into xenotransplanted human breast cancer cells

Two different routes of administration will be tested:

A: Co-inoculation subcutaneously into a nu/nu META/Bom mouse of transfected tumour infiltrating fibroblasts together with the human cancer cells as described in Example 11.

B: Intravenous injection of transfected tumour infiltrating fibro- blasts into a nu/nu META/Bom mouse which is carrying a human breast cancer xenograft under the skin.

Evaluation

In order to evaluate the effect of the applied gene therapy, tumour growth and invasion/metastasis will be recorded and compared to the same parameters in a control experiment where the tumour infiltrating fibroblasts are untransfected. Furthermore, the primary tumours are stained with X-gal in order to test for the presence of lacZ expres¬ sing fibroblasts.

EXAMPLE 13

Human breast cancer-derived ascites tumours grown in nude mice as in vivo models for the screening of anti-proliferative and anti- invasive/anti-metastatic drugs

Many drugs with an anti-proliferative activity against human cancer are available. However, development of relapse of the disease with subsequent cross-resistance to other groups of anti-cancer compounds represent one of the most serious problems in the clinical management of patients with various types of cancer, including breast cancer.

Precllnical drug screening programs most often include sensitivity testing of human cancer cells in vitro and murlne ascites tumours grown in vivo in syngenic mice. A more appropriate assay system would be the in vivo testing of human cancer cells. However, human tumours grown as solid tumours in nude mice often have a slow growth rate resulting in time-consuming and expensive assay conditions.

A more rational assay would be one similar to the murine ascites tumour systems, where the human cancer cells are inoculated intra- perltoneally into nude mice with subsequent application of therapy. The end-point should be survival time of the mice in relation to that of a control group consisting of nude mice injected in a similar manner with the human ascites tumour cells but without treatment.

If the treatment in question is directed against tumour cell invasion and metastasis, the human cancer cells could be genetically labelled with a gene whose product would facilitate identification and quanti¬ fication of metastasis in the animals of the human cancer. Ascites cell lines

It has been found that invasively growing human breast cancer and ovarian cell lines, such as MDA-MB-231, MDA-MB-435 and OVCAR-3, when inoculated subcutaneously into nu/nu META/Bom mice grow invasively into the peritoneal wall and into the peritoneal cavity and sub¬ sequently produce ascites containing tumour cells. From nu/nu META/Bom mice carrying each of such tumours, tumour cells were isolated from the peritoneal cavity and maintained in vitro as described in Example 1. These cell lines, reproducibly produce ascites tumours when injected intraperitoneally into nu/nu META/Bom mice. Two of these cell lines were transduced with the lacZ gene according to the method described in Example 1. Ascites-tumour forming cell lines obtainable in this manner, are most useful for in vitro drug testing.

In vitro drug testing

In order to characterize the sensitivity patterns of the ascites tumour forming cell lines derived from MDA-MB-231 and MDA-MB-435, respectively, they were examined using an in vitro clonogenic assay described by Roed et al. (1987). Briefly, the cell lines were tested against anti-proliferative drugs conventionally used in clinical treatment of cancer. Triplicates of 2 x 10^ cells of each cell line were seeded in soft agar and continuously exposed to different doses of adriamycin (ADR), BCNU, cisplatin (CIS), VP-16, vincristine (VCR), ARA-C. Cellular dose-survival curves were constructed from the number of colonies produced by treated tumour cells and untreated control cells, and the dose-survival curves for each drug were applied in the calculation of the dose killing 50% of the exposed cells (LD50) .

In vivo cell inoculation

5 x 10° lacZ labelled MDA-MB-231-derived ascites-tumour forming cells and 5 x 10° lacZ labelled MDA-MB-435 -derived ascites-tumour forming cells were injected intraperitoneally in the animals. X-gal staining of ascites

Smears of ascites fluid were produced on glass slides from the intra¬ peritoneal cavity. The slides were dried and subsequently fixed and stained as described for cells In culture (see Example 1) .

X-gal staining of organs

The method described in Example 2 was used.

Administration of anti- cancer drugs

The drug in question is administered intraperitoneally at the same time as the tumour cells. Different treatment schedules and routes of administration is used.

Evaluation of in vivo therapy

Measurements such as increase in life span, body weight, or formation of metastasis is used as end-points in the evaluation of treatment effect.

Results

In vitro sensitivity

The in vitro sensitivity patterns of MDA-MB-231-derived ascites- tumour forming cells and MDA-MB-435-derived ascites-tumour forming cells are summarized In Table 5 below. The cell lines showed a diffe- rential sensitivity to the six drugs tested. The MDA-MB-231-derived cells were more sensitive than the MDA-MB-435-derived cells to ADR, BCNU and VP-16, whereas the cell lines were similar in sensitivity to the other drugs. The LD50 values of the individual drugs are within the ranges found for other human cancer cells (Jensen et al. , 1992). TABLE 5

MDA-MB-231-derived MDA-MB-435-derived LD₅₀ LD₅₀

ADR 0.007 μg/ml 0.016 μg/ml

BCNU 0.825 μg/ml 2.993 μg/ml

CIS 0.146 μg/ml 0.176 μg/ml

VP-16 0.105 μM 0.135 μM

VCR 0.001 μM 0.001 μM

ARA-C 20.82 nM 22.48 nM

Ascites tumour production

Both cell lines produced ascites when injected intraperitoneally. Staining with X-gal of ascites demonstrated highly specific staining of the human tumour cells only.

In vivo evaluation

The life span of mice injected with either of the cell lines was approximately 3 weeks. X-gal staining of mouse organs showed heavy tumour cell infiltration in almost all intraperitoneal organs as well as tumour cell spread to the lungs.

EXAMPLE 14

Testing of carcinogenicity of various substances

Below, a test for determination of the carcinogenicity of various substances is outlined. The test may be performed in vivo on human cells whereby a much more biologically correct and informative test on the actual carcinogenicity of a substance on human cells being placed in a biologically active environment is provided as compared to known in vitro tests. The test is performed as follows:

1) Human cells labelled with the lacZ-gene as described in Example 1 are transferred to nu/nu META/Bom mice prior to exposure to the substance to be tested or after exposure which may take place in vitro by exposing the cell culture for the substance in question.

2) The mouse and thus the human cells are exposed to the sub¬ stance to be tested. Exposure of the mouse may be performed using various routes and should optimally be In line with the normal exposure of humans to the substance to be tested.

3) The human cells are Identified using visualization of the lacZ-gene with X-gal staining (Examples 1 and 2) and the human cells are examined for the occurrence of characte- ristics known to be present in cancer cells such as cyto- logical and morphological characteristics for malignant cells. Also various DNA and RNA analysis using techniques like, Southern Blot and Northern Blot may be used to exa¬ mine the cells for malignancy criterias, e.g., gene ampli- fIcations, gene deletions etc.

4) Human cells isolated from the mice after exposure may be grown in vitro In culture and examined for their malignant potential using various in vitro based test systems, e.g., in vitro invasion assays, soft agar colony formation etc.

5) Transfer of the human cells either directly from the mice as described under 3) or from the cell culture established as described under 4) to other nu/nu META/Bom mice may be used to examine the capability of cells exposed to a car¬ cinogenic substance to form tumours and possibly to invade and metastasize. The human cells used in the above described test may be leukocytes, in particular lymphocytes, cells from the bone marrow and keratino- cytes or other cell types.

EXAMPLE 15

Identification of substances capable of inhibiting, preventing or controlling diseases caused by human microbiological pathogens using lacZ-labelled human cells in vivo in an immunedeficient mouse

A test for identification of substances which may be used in the treatment of human pathogenic organisms such as bacteria, vira and unicellular parasites is described. The test is performed as follows below:

1) Human cells transformed with the lacZ-gene are infected with the pathogen for which a substance capable of combat¬ ting the pathogen is sought. The infection is performed using a technique normally used when infecting cells with the pathogen in question.

2) The infected lacZ transformed human cells are transferred to an immunedeficient mouse, such as a nu/nu META/Bom mouse using a method suitable for the cell in question.

3) The mouse and thus the infected human cells are exposed to the substance to be tested in various doses and using various exposure duration schemes.

4) The effect of the substance on the pathogen and on the human cells are examined after identification of the human cells by visualization using X-gal staining.

The pathogens for which substance capable of combatting the pathogens are sought may be bacteria such as Mycobacterium tuberculosis , Myco- bacterium lepra, Pneumocystis carinae , vira such as retrovira, in particular HIV, herpes vira, cancer-inducing vira etc., unicellular parasites such as Plasmodium falciparum, Toxoplasma gondii, flagel¬ lates such as Giardia sp., Trypanosoma sp., Leishmania sp., Trichomo - nas sp. and amoebae such as Entamoeba sp. and fungi.

LEGEND TO FIGURES

Fig. 1 Schematic illustration of the BAG vector (Price et al. , 1987). The BAG vector consists of the lacZ gene under the transcrip¬ tional control of the Moloney murine leukemia Long Terminal Repeat (LTR) promotor and the SV40 promoted neo® gene.

Fig. 2 Illustration of the construction of the vector pRSVIacZ used for transfection in Example 1 (9.0 Kb). The lipofection mediated gene transfer method with a vector containing the lacZ gene and a neomycin-resistance gene under a RSV promotor was used.

Fig. 3 X-gal staining of tumour cells grown in vitro . Cells were fixed and processed for X-gal staining (see Example 1) . A: MDA-MB-435 cells (non-transduced) B: lacZ transduced MDA-MB-435 BAG at passage 22 after transduction.

Fig. 4 X-gal staining of tumour cells grown in vivo . Tumours were fixed and processed for X-gal staining (see Example 2) ; A: Subcutaneous MDA-MB-231 xenograft (non-transduced) ; B: Subcutaneous lacZ transduced MDA-MB-231 BAG xenograft passage 2 in nude mice.

Fig. 5

A: Cryosection of a primary MDA-MB-435 BAG tumour. Tumour tissue was processed for cryo-sectioning and stained with X-gal (see Example 2). Only the tumour cells stained positive with X-gal. TT: tumour tissue; MT: mouse tissue. B - D: Macroscopic appearance of secondary MDA-MB-435 BAG tumours. Whole organ staining with X-gal. B: liver;

C: spleen and pancreas; D: intestine. 3E - G: Metastatic spread of MDA-MB-435 BAG tumour cells to mouse lung.

E: Macroscopic appearance of lung metastases; F: Single lung metastasis (arrow) ; G: Histological section of the lung metastasis (arrow) seen in 3F.

H.E. staining, 40 X. H: X-gal staining of ascites from a mouse inoculated sc with

MDA-MB-435 BAG tumour cells.

Fig. 6 MDA-MB-231 BAG: A few minutes after intravenous injection, a large number of tumour cells are trapped in the capillaries of the lungs (Figure 6A) , but most of these cells are cleared from the lung tissue following another 24 hours (Figure 6B). After 2 weeks a small number of tumour nodules (experimental metastases) consisting of several tumour cells is been established in the lungs (Figure 6C) .

Fig. 7 Macroscopic growth of untreated control tumours, tumours in mice treated with an irrelevant antibody and tumours exposed to anti- u-PA antibodies.

Fig. 8 Serum concentration of anti-u-PA antibodies in nude mice following injection of 100 μg anti-u-PA antibodies from clone 5.

Fig. 9 Schematic Illustration of the EF-lacZ-MAR vector (11515 bp)

Fig. 10. Schematic illustration of the HMG-IacZ-MAR vector (14.50 Kb)

Fig. 11. Schematic illustration of the UBI -lacZ-MAR vector (11664 bp)

Fig. 12 Schematic illustration of the CMV-lacZ-MAR vector (10094 bp)

Fig. 13 MCF-7 human breast cancer xenografts established in tissue culture. A: After 1 passage in culture both tumour cells and fibroblasts are present B: After second in vitro passage only fibroblasts are seen. ^β7

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Claims

1. A method for detecting, in a non-human recipient vertebrate, a cell which has the genotype of a donor vertebrate, and which is identical to or derived from a cell transferred from a donor of the vertebrate genotype into the non-human recipient vertebrate, the donor being a vertebrate of the donor vertebrate genotype or a cell line comprising cells from such vertebrate, the method comprising using, as the donor cell, a cell labelled with a genetic marker which, either directly or latently, permits or facilitates distinc- tion between the donor cell genotype and cells of the recipient and, when the marker is one which latently permits distinction, developing the latent distinction, with the proviso that when the donor cell genotype is a murine genotype, then either 1) the donor is a mouse and not a cell culture, or 2) the donor cell is from an immunodeficient mouse.

2. A method according to claim 1 wherein the donor vertebrate cells further comprises a second transgene which is different from the genetic marker.

3. A method for detecting, in a non-human recipient vertebrate, a cell which has the genotype of a donor vertebrate, and which is identical to or derived from a cell transferred from a donor of the vertebrate genotype into the non-human recipient vertebrate, the donor being a vertebrate of the donor vertebrate genotype or a cell line comprising cells from such vertebrate, the method comprising using, as the recipient vertebrate, a vertebrate cells of which are marked with a genetic marker which, either directly or latently, permits distinction between the donor cell genotype and cells of the recipient and, when the marker is one which latently permits distinc¬ tion, developing the latent distinction.

4. A method according to claim 3, wherein substantially all cells of the recipient vertebrate are labelled with the genetic marker.

5. A method according to claim 3 or 4 wherein the recipient verte¬ brate cells further comprises a second transgene which is different from the genetic marker.

6. A method according to any of claims 1-5, wherein the genetic marker is linked to a promoter and a transcription terminator which are capable of functioning in substantially all cells of the ver¬ tebrate in such a way that the genetic marker is expressed in sub¬ stantially all cells of the vertebrate.

7. A method according to any of the preceding claims, wherein the non-human recipient vertebrate is immunodeficient.

8. A method according to any of the preceding claims, wherein the non-human recipient vertebrate is a mammal.

9. A method according to claim 8, wherein the non-human recipient mammal is a rodent, in particular a rat or a mouse.

10. A method according to claim 9, wherein the recipient rodent is a thymus-deficient rodent.

11. A method according to claim 10, wherein the thymus-deficient rodent is a nude mouse.

12. A method according to any of the preceding claims, wherein the donor cell genotype is mammalian.

13. A method according to claim 12, wherein the donor cell genotype is human.

14. A method according to claim 12, wherein the donor cell genotype is a rodent genotype, in particular a rat or mouse genotype.

15. A method according to claim 14, wherein the donor cell is from a immunodeficient rodent, in particular a thymus-deficient rodent.

16. A method according to claim 15, wherein the thymus-deficient rodent is a nude mouse.

17. A method according to any of the preceding claims, wherein the second transgene is a gene encoding encoding a polypeptide, such as a growth factor or a growth factor receptor, a cytokine or an oncogene product.

18. A method for determining the effect of a drug or a treatment with respect to preventing, diminishing, controlling or Inhibiting a dis¬ ease mediated by cells, comprising introducing, in a non-human verte- brate recipient, cells from a donor vertebrate which mediate the dis¬ ease, the donor cells or cells of the recipient being modified to either directly or latently permit distinction between on the one hand cells which are identical to the donor or cells derived there¬ from, and on the other hand cells of the non-human recipient verte- brate, administering the drug or applying the treatment to the non- human recipient vertebrate, and determining the effect of the drug or the treatment on the basis of detection or investigation, in the recipient vertebrate, of cells identical to or derived from the donor cells, utilizing, In the detection or investigation, the distinction obtained through modification of the donor cells or of cells of the recipient, whereby, when the modification is one which latently per¬ mits distinction, the latent distinction Is developed prior to or in connection with the detection or investigation.

19. A method according to claim 18 wherein the modified donor or recipient cells further comprises a second transgene which is dif¬ ferent from the genetic marker.

20. A method according to claim 18 or 19, wherein the recipient ver¬ tebrate is immunodeficient.

21. A method according to any of claims 18-20, wherein the modifl- cation of the donor cells or the cells of the recipient comprises labelling the cells with a genetic marker which, either directly or latently, permits or facilitates distinction between the donor cell genotype and cells of the recipient vertebrate.

22. A method according to any of the preceding claims, in which the genetic marker is a gene encoding a product which in itself is vi¬ sually distinguishable from non-marked cells, or which is capable of being made visually distinguishable from the non-marked cells.

23. A method according to claim 22, wherein the gene product is a coloured or fluorescent product or a product which can be converted into a coloured or fluorescent product.

24. A method according to claim 22, wherein the genetic marker is a lacZ gene, and the conversion of the gene product thereof into a coloured product is performed by staining with the chromogenic sub¬ strate 5-bromo-4-chloro-3-indoyl-_/S-D-galactopyranoside (X-gal) resul¬ ting in a blue staining of the labelled cell.

25. A method according to any of claims 18-24, wherein the investiga¬ tion of the cells identical to or derived from the donor cells invol- ves transferring such cells, after the recipient has been subjected to the drug or the treatment, to another non-human recipient verte¬ brate and determining any effect of the drug or treatment conferred to the cells identical to or derived from the donor cells and mani¬ festing itself after the transfer to the other non-human recipient vertebrate.

26. A method according to claim 25, wherein the other non-human recipient vertebrate is immunodeficient.

27. A method according to claim 25 or 26, wherein the donor cells or cells of the recipient are modified to either directly or latently permit distinction between on the one hand cells which are identical to the donor or cells derived therefrom, and on the other hand cells of the other non-human recipient vertebrate, the determination of any effect of the drug or the treatment being performed on the basis of detection or investigation, in the other recipient vertebrate, of cells identical to or derived from the donor cells, utilizing, in the detection or investigation, the distinction obtained through modifi¬ cation of the donor cells or of cells of the recipient, whereby, when the modification is one which latently permits distinction, the latent distinction is developed prior to or in connection with the detection or Investigation.

28. A method according to claim 27, wherein the cells transferred^' from the first non-human recipient vertebrate or cells of the second recipient vertebrate are modified to either directly or latently permit distinction between on the one hand cells which are identical to or derived from the cells from the first non-human recipient vertebrate, and on the other hand cells of the other non-human reci- pient vertebrate, the determination of any effect of the drug or the treatment being performed on the basis of detection or investigation, in the other non-human recipient vertebrate, of cells identical to or derived from the cells from the first non-human recipient vertebrate, utilizing, in the detection or investigation, the distinction ob- tained through modification of the cells from the first non-human recipient vertebrate or of cells of the second non-human recipient vertebrate, whereby, when the modification is one which latently permits distinction, the latent distinction is developed prior to or in connection with the detection or investigation.

29. A method according to any of claims 18-28 for the assessment of the usefulness of drug or a treatment for preventing, diminishing, controlling or Inhibiting the progression of metastases in a donor vertebrate, in particular a human, comprising introducing, into an immunodeficient non-human recipient vertebrate in which cancer cells of the donor vertebrate genotype are capable of invading and meta¬ stasizing, cancer cells of the donor vertebrate genotype, the donor cancer cells or cells of the recipient being modified to either directly or latently permit distinction between on the one hand cells which are identical to or derived from the donor cells, and on the other hand cells of the non-human recipient vertebrate, administering the drug or applying the treatment to the non-human recipient verte¬ brate, and determining the effect of the candidate drug on the pro¬ gression of metastases in the animal, utilizing, in the detection or investigation, the distinction obtained through the modification of the donor cells or of cells of the recipient, whereby, when the modification is one which latently permits distinction, the latent distinction is developed prior to or in connection with the detection or investigation.

30. A method according to claim 29, wherein the modified donor or recipient cells further comprise a second transgene which is dif- ferent from the genetic marker.

31. A method according to claim 29 or 30, wherein primary tumours are removed from the non-human recipient vertebrate before or after the drug is administered to or the treatment applied to the non-human recipient vertebrate.

32. A method according to claim 31, in which the donor cancer cells have been marked with lacZ in such a manner that it is expressed by the cancer cells, and removal of the primary tumours is performed substantially at a time where metastasizing in the non-human recipi¬ ent vertebrate has just begun to be visually detectable by staining with X-gal and metastazing is still undetectable in a reference non- human vertebrate animal the cells of which are not labelled and to which non-labelled cancer cells of otherwise the same kind as the donor cancer cells have been transferred.

33. A method according to any of claims 29-32, wherein the cancer cells are selected from the group consisting of the human cell lines MDA-MB-231 MDA-MB-435 and OVCAR-3, and the immunodeficient recipient vertebrate is a mammal, in particular a nude mouse, in which at least one of the cell lines MDA-MB-231, MDA-MB-435 or OVCAR-3 is capable of metastasizing.

34. A method according to claim 33, wherein the immunodeficient mammal is a mammal, in particular a nude mouse, in which at least one of the cell lines MDA-MB-231, MDA-MB-435 or OVCAR-3 is capable of metastasizing.

35. A method according to claim 33 or 34, wherein the immunodeficient mammal is a thymus-deficient mouse.

36. A method according to claim 35, wherein the thymus-deficient mouse has a chromosomal analysis substantially as shown in Table 1 herein.

37. A human cell which is labelled with a genetic marker in the form of a gene encoding a product which in itself Is visually distinguish¬ able from non-marked cells, or which is capable of being made visual¬ ly distinguishable from the non-marked cells.

38. A human cell according to claim 37 which is a cancer cell.

39. A human cell according to claim 37 which further comprises a second transgene which is different from the genetic marker.

40. Human cancer cells according to claim 39 which are selected from the group consisting of the cell lines MDA-MB-231, MDA-MB-435 and 0VCAR-3.

41. The use of human cancer cells as defined in any of claims 37-41 as a tool in an experimental model for the assessment of the capabi¬ lity of a drug candidate of preventing or inhibiting human cancer cell progression.

42. A method for determining the effect of a drug or a treatment with respect to preventing, diminishing, controlling or inhibiting a dis- ease mediated by cells, comprising introducing intraperitoneally, in a non-human vertebrate recipient, cells from a a cell line as claimed in claim 40, administering the drug or applying the treatment to the non-human recipient vertebrate, and determining the effect of the drug or the treatment on the basis of the determination of the sur- vival (increased life span (ILS)) or weight of the non-human recipient vertebrate.

43. A method for controlling the progression of cancer cells in a human or animal patient, comprising administering, locally or sys- temically, to the patient, fibroblasts which have been transformed with a genetic construct expressing a gene product inhibiting the progression of cancer cells.

44. A method according to claim 43, wherein the fibroblasts are tumour-infiltrating fibroblast cells which are capable of finding cancer cells in the patient.

45. A method according to claim 43 or 44, wherein the fibroblasts are fibroblasts of a tissue type which is compatible with the tissue type of the patient.

46. A method according to claim 45, wherein the fibroblasts are fibroblasts from the patient.

47. A method according to any of claims 42-45, wherein the gene pro- duct is one which is capable of controlling the progression of cancer cells of the patient genotype in a recipient vertebrate into which the cancer cells have been transferred.

47. A method according to claim 46, wherein the gene product is one which is capable of controlling the progression of the cancer cells of the patient genotype when the gene product is expressed by tumour- infiltrating fibroblasts of the recipient vertebrate genotype.

48. A method according to claim 46, wherein the gene product is one which is capable of controlling the progression of the cancer cells of the patient genotype when the gene product is expressed in the recipient immunodeficient vertebrate by tumour-infiltrating fibro¬ blasts of the donor genotype.

49. Fibroblasts which are capable of finding and infiltrating a malignant tumour in a mammal and which contain, and are capable of expressing, a gene producing a gene product which is capable of controlling the progression of cancer cells in the mammal.

50. Fibroblasts which are capable of finding and infiltrating a malignant tumour of a mammal genotype, in particular human genotype, in an immunodeficient non-human recipient vertebrate into which cancer cells of the said mammal genotype have been introduced, the fibroblasts containing 1) a gene which, when it is expressed in the immunodeficient recipient vertebrate by fibroblasts which are capable of finding and colonizing, in the recipient vertebrate, colonies of cancer cells of the said mammal genotype, is capable of controlling the progression of the cancer cells of the said mammal, and 2) a pro- moter securing expression of the gene product when the fibroblasts^' have been transferred to the said mammal.

51. A cell culture comprising fibroblasts according to claim 49 or 50.

52. A collection of cell cultures comprising fibroblasts according to claim 49 or 50, the collection comprising fibroblasts of a multitude of human tissue types.

53. A method of producing a transgenic non-human vertebrate cells of which are marked with a genetic marker which, either directly or latently, permits distinction between cells having the genotype of the non-human vertebrate and cells which are donated to the non-human vertebrate said method comprising chromosomally incorporating a gene¬ tic marker Into the genome of a non-human mammal.

54. A method according to claim 53 comprising injecting a transcrip¬ tion unit comprising said genetic marker into a fertilized egg or a cell of an embryo of a vertebrate so as to incorporate the transcrip¬ tion unit into the germline of the vertebrate and developing the resulting injected fertilized egg or embryo into an adult vertebrate.

55. A transgenic non-human vertebrate whose germ cells and somatic cells contain a genetic marker which, either directly or latently, permits distinction between cells having the genotype of the non- human vertebrate and cells which are donated to the non-human verte¬ brate, as a result of chromosomal incorporation of the genetic marker into the non-human vertebrate genome, or into the genome of an ances¬ tor of said non-human vertebrate.

56. A transgenic non-human vertebrate according to claim 55 in which the genetic marker is a lacZ gene.

57. A transgenic non-human vertebrate according to claim 55 or 56 which is immunodeficient.

58. A vertebrate according to claim 56 or 57 which is selected from the group consisting of mice, rats and rabbits.

59. A non-human immunodeficient mammal according to any of claims 55- 58, the genome of which further comprises a second transgene which is different from the genetic marker.

60. A non-human immunodeficient mammal produced by crossing a mammal according to any of claims 55-59 with another mammal of the same species the genome of which does not comprise a genetic marker or with another transgenic mammal of the same species the genome of which does not comprise the genetic marker.

61. A method for the assessment of the usefulness of drug or a treat¬ ment for preventing, diminishing, controlling or inhibiting a disease in a vertebrate, in particular a human, comprising introducing, into a non-human recipient vertebrate, cells of a transgenic animal as claimed in any of claims 55-60, the cells of the transgenic animal being modified to either directly or latently permit distinction between on the one hand cells which are identical to or derived from the transgenic animal, and on the other hand cells of the non-human recipient vertebrate, administering the drug or applying the treat¬ ment to the non-human recipient vertebrate, and determining the effect of the candidate drug utilizing, in the detection or inves¬ tigation, the distinction obtained through the modification of the donor cells whereby, when the modification is one which latently permits distinction, the latent distinction is developed prior to or in connection with the detection or investigation.

62. A method for determining the carcinogenicity of a substance or a treatment on cells of a vertebrate donor genotype, comprising either

1) exposing the cells of the vertebrate donor genotype to the substance or the treatment followed by introduction of the cells into a non-human vertebrate recipient, or 2) Introducing the cells of the vertebrate donor genotype into a non-human recipient vertebrate, followed by exposure of the recipient to the substance or the treatment,

the donor cells being modified by labelling with a genetic marker ^"to either directly or latently permit distinction between on the one hand cells which are identical to the donor or cells derived there¬ from, and on the other hand cells of the non-human recipient verte¬ brate, and determining any capability of the substance or the treat¬ ment to convert the donor cells into cancer cells by examining the invasive and metastatic activity of the donor cells, either in the recipient, or after transfer of the donor cells to another recipient, or in vitro , on the basis of detection of any metastasis utilizing the distinction obtained through the modification of the donor cells.

63. The use of a drug, the effect of which with respect to prevent- ing, diminishing, controlling or inhibiting a disease has been estab¬ lished using the method according to any of claims 18-28, for the preparation of a pharmaceutical composition for preventing, diminish¬ ing, controlling or inhibiting the disease.

64. The use of a drug, the effect of which with respect to prevent- ing, diminishing, controlling or inhibiting the progression of meta¬ stases has been established using the method according to any of claims 29-36, for the preparation of a pharmaceutical composition for preventing, diminishing, controlling or inhibiting the progression of metastases.

65. A method for preventing, diminishing, controlling or inhibiting a disease, comprising administering, to a patient in need thereof, an effective amount of a drug the effect of which for preventing, dimi¬ nishing, controlling or inhibiting the disease has been established using the method according to any of claims 18-28.

66. A method for preventing, diminishing, controlling or inhibiting the progression of metastases, comprising administering, to a patient in need thereof, an effective amount of a drug the effect of which for preventing, diminishing, controlling or inhibiting the progres- sion of metastasis has been established using the method according to any of claims 29-36.