WO1996018101A1

WO1996018101A1 - Histologic method of tissue analysis

Info

Publication number: WO1996018101A1
Application number: PCT/US1994/014211
Authority: WO
Inventors: Thomas M. Donndelinger
Original assignee: CELLULAR DIMORPHISM INSTITUTE
Current assignee: CELLULAR DIMORPHISM INSTITUTE
Priority date: 1994-12-09
Filing date: 1994-12-09
Publication date: 1996-06-13
Anticipated expiration: 1997-06-09

Abstract

A histologic method of analyzing tissue is disclosed. The method includes preparing the tissue under conditions effective to differentially stain two dimorphic cell types making up the tissue, and analyzing the relative numbers and spatial distribution of the two cell types. The method may be used, for example, to distinguish normal and neoplastic epithelial-cell tissue. Also disclosed is a method of identifying growth factors that regulate the growth of the two cell types.

Description

HISTOLOGIC METHOD OF TISSUE ANALYSIS

Field of the Invention

The present invention relates to a histologic method of tissue analysis, in particular, analysis of epithelial-cell tissue.

References

Aaronson, S.A., Science, 254 : 1146 (1991). Bast, R.C., et al., CANCER Supplement , 71(4) : 1597 (1993).

Bodenstein, L. , Cell Differentiation, 19: 19 (1986) .

Ennis, B.W. et al., Cancer Investigation , 9(5) : 553 (1991).

Smolle, J. , Analyt Cellular Path, 6::149 (1994) .

Background of the Invention Histological examination of cells in a tissue, such as epithelial-cell tissue, may be carried out by light microscopy, for observing structures with a resolution in the low micron range, or by electron microscopy, for observing structure with a resolution as low as 5-20 A. Light microscopy offers the advantages of simple preparative methods, the ability to view the tissue in a living, or at least hydrated state, and the availability of a variety of colored or fluorescent stains that can be used to stain specific cellular structures. Electron microscopy offers the advantage of near-atomic resolution, and has allowed a variety of subcellular and macromolecular structures to be identified.

Although both light and electron microscopy offer different views of the cell, neither has led, heretofore, to the observation that multicellular tissues, as exemplified by epithelial-cell tissues, are composed of two distinct, i.e., dimorphic cell types which have characteristic spatial arrangements and relative cell numbers. In theory, these two cell types, by producing growth factors that regulate the other's growth, would have the ability to form complex "emergent" structures, such as complex tissue morphologies, by employing simple neighborhood rules (Smolle, Bodenstein) . By studying the binding of growth factors to these two cell types, it is possible to identify the cellular mediators that act on each cell type. Among the growth factors, or inhibitors for the growth factor, which may be identified are epidermal growth factor (EGF) , transforming growth factor-alpha (TGF-α) , platelet derived growth factor (PDGF) , fibroblast growth factor (FGF) insulin-like growth factor (IGF) , and transforming growth factor-beta (TGF-6) which all play a role in cell growth (Aaronson, Ennis, Bast) .

Information about the nature and levels of growth factors, and their cell targets in a tissue, in turn, can be used to identify inhibitors of cell mediators, for use in blocking cell growth in neoplasms, for example, or for disrupting developmental patterns, e.g., in larval-stage insects.

. Summary of the Invention The invention includes, in one aspect, a histologic method for analyzing a tissue. The method includes first preparing a tissue under conditions effective to differentially stain two dimorphic cell types in the tissue. One cell type is characterized by pale staining with hematoxylin-hydroquinone/eosin-phloxine stain, and round nucleus with central-tending nucleolus; the second cell types is characterized by dark staining with he atoxylin-hydroquinone/eosin- phloxine stain, and dark staining nucleus, with irregular cell contours.

The relative numbers and spatial distribution of the two cell types in the tissue are then analysed.

The method may be used, for example to distinguish normal from neoplastic epithelial-cell tissue, where normal tissue is characterized by an unequal ratio of the two cell types, with one of the two cell types, e.g., dark cells, predominating at a ratio typically of 3:1 to 8:1, and neoplastic tissue is characterized by a more equal ratio of the two cells types, that is, closer to a 1:1 ratio.

In a preferred method, the tissue is prepared by embedding the tissue in a polymer-hardened paraffin matrix; sectioning the embedded tissue to produce a tissue slice having a thickness between about 0.5-2 μm; and staining the slice with a chromatin stain effective to produce differential chromatin staining between the two cell types, such that one cell type is identifiable as having lightly stained nuclei, and the other cell type, as having darkly stained nuclei.

A preferred nucleic acid stain is a hematoxylin/hydroquinone stain. The tissue may also be stained with an eosin-containing cytoplasmic stain.

In a related aspect, the invention includes a histologic method for visualizing two distinct cell types making up epithelial-cell tissue. The method includes embedding the tissue in a polymer- hardened paraffin matrix; sectioning the embedded tissue to produce a tissue slice having a thickness between about 0.5-2 μm; and staining the slice with a chromatin stain effective to differentially stain the chromatin in the two cell types, such that one cell type is identifiable as having a lightly stained nucleus, and the other cell type, as having a darkly stained nucleus.

The ability to distinguish distinct dimorphic cell types in epithelial-cell tissue allows for the identification of growth factors that regulate the control of the growth of each of the two cell types, in accordance with another aspect of the invention. The method involves obtaining a normal or neoplastic tissue sample from a selected human tissue, where the tissue is composed of a first cell type characterized by (i) pale staining with hematoxylin-hydroquinone/eosin-phloxine stain and (ii) round nucleus with central-tending nucleolus, and a second cell type characterized by (i) dark staining with hematoxylin-hydroquinone/eosin- phloxine stain and (ii) dark staining nucleus, with irregular cell contours.

The tissue is reacted with a selected reporter-labeled growth factor, under conditions effective to promote binding of the growth factor to one of the two cells types making up the tissue. Following growth factor binding to one of the two tissue cell types, the cells in the tissue are examined to determine which of the two cell types the reporter-labeled growth factor is bound to.

These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings. Brief Description of the Drawings Fig. 1 is a pictorial representation of two distinct, characteristic cells types that have been observed in normal epithelial tissue, and in epithelial-cell tumors;

Fig. 2 illustrates a epithelial-cell tissue section from colon showing the distribution of light- and dark-stained cells in the tissue;

Fig. 3 illustrates a general mechanism of paracrine cellular control in a tissue composed of a population of dimorphic cells; and

Figs. 4A-4C illustrate steps in identifying growth factors that are specific to each of the two distinct cell types in tissue composed of dimorphic cell types.

Detailed Description of the Invention I. Definitions

"Epithelial cells" refer to the cells lining the inner and outer surfaces of the body and that form coherent cell sheets called epithelia.

"Epithelial-cell tumor" is a neoplasm derived from epithelial cells and associated with or growing in epithelia. "Paracrine control" or "paracrine stimulation" refers to a mechanism where the metabolism and growth of a first cell type is influenced by a factor released from a second, adjacent cell type and the metabolism and growth of the second, adjacent cell type is influenced by a factor released from the first cell type.

"Growth factor" is a polypeptide which plays a role in cellular growth and proliferation.

"Growth factor inhibitor" refers to an agent or compound that acts to inhibit the biological effect of a growth factor, either by inhibiting binding of the growth factor to its target cell, or by inhibiting the activity of the growth factor.

A "chromatin stain" is a stain that stains chromatin by virtue of its binding to basic groups of chromatin histones. A chromatin stain will produce darker staining in chromatin rich in histones, and particularly histones, such as histone H3, that are rich in arginine and lysine, and less darkly stain chromatin having fewer histones and/or less basic histones. One preferred chromatin stain is the hematoxylin/hydroquinone stain described in the Example.

II. Histological Method

The invention includes in one aspect, a histological method for analyzing tissue. The procedure allows for visualization of the two distinct cell types, also referred to herein as dimorphic cell types, which appear to be found in intimate relationship in all tissues in higher organisms. The dimorphic cell types, and their relative distribution in tissue, will be described in Section III below. The histologic method involves first fixing the tissue in a suitable fixative. A preferred fixative is a formalin/ Zn sulfate fixative, at a Zn⁺² concentration preferably between 5% and 10%. An initial formalin solution (5%) containing Zn sulfate (5-10%) fixative insures sufficient crosslinking, without agglutination of the nuclear network, and consequent loss of histological detail. Other suitable fixatives are glutaraldehyde and formaldehyde. After a second fixation, e.g., in formalin (10%) with Zn sulfate (5-10%), the tissue is dehydrated, typically by means of a series of alcohol and xylene dehydration steps, such as shown in the Example below. The dehydrated tissue is then placed in paraffin, for solvent exchange, and finally fixed in a paraffin block, preferably one containing a resin, such as 2 % piccolyte™ elastomer resin (Hercules, Wilmington, DE) . The resin increases the hardness of the paraffine matrix. The resin is a conventional resin, such as epoxy or an olefinic resin used in commercial polymer-hardened paraffin compositions for use in tissue preparation, and is present typically at a weight percentage of 1-5%. A suitable type of paraffin/resin material, employed in the Example, is Polyfin™, available from Polyscience (Warrington, PA) . The paraffin or paraffin/resin block is cured according to standard methods or manufacturer's instructions.

The tissue is sectioned to a thickness between 0.5 and 2 microns (μm) , preferably 1 μm +/- 0.5 μm. Sectioning is by conventional microtome. This tissue section thickness is important for visualizing the dimorphic cell structures of the tissue being analysed.

According to another important feature of the invention, the tissue is now stained with a chromatin stain under conditions effective to enhance the staining of the chromatin (nuclear DNA/histone complex) , such that one cell type is identifiable as having a lightly stained nucleus, and the other cell type, as having a darkly stained nucleus in the slice. One preferred stain is a hematoxylin/hydroquinone, such as the hematoxylin/hydroquinone stain described in the Example. As seen in the exemplary staining procedure given in the Example, the staining step involves several dehydration steps with xylene and 1 PC-7 S94/14211

8

100% alcohol, a quick emersion in 1% acetic acid, then distilled water, followed by a 4 minute incubation in hematoxylin (Richard Allen Medical) containing 0.3 g hydroquinone/750 ml at room temp (about 20-^24oC) .

The tissue section is then further treated with clarifier, a bluing agent, and an eosine- phloxine stain, as detailed in the Example. The clarifier serves to remove background color. The purpose of the bluing agent is to develop the blue color of hematoxylin. And the eosin stain is used to stain acidic cytoplasmic and nuclear proteins.

After staining, the tissue slice is dehydrated by further alcohol and xylene treatment. All of the washing and staining procedures disclosed in the Example are carried out at room temperature, for the times indicated. The fixed and stained tissue section is then analysed by light microscopy to determine the relative numbers and spatial distribution of the two dimorphic cell types identifiable in sections prepared according to the above. The nature of the two cell types is considered in the section below.

III. Dimorphic Cell Types

According to one feature of the invention, it has been discovered that tissue prepared, sectioned, and stained as above show two readily distinguishable cell types, referred to herein as dimorphic cell types because of their different, distinctive morphologies.

A. Light and Dark Cell Types Fig. 1 is a graphic representation of the two different cell types, as seen in an epithelial- cell tissue, a light cell 10 and an adjacent dark cell 12.

The light cell is characterized by a round nucleus 14 with lightly peripherally clumped chromatin, centrally finely divided chromatin and, generally, a central-tending nucleolus 16. The light cell tends to have fewer organelles and less cytoskeletal elements within the cytoplasm than the dark cell, giving a paler appearance upon staining. Mitotic figures are identified in this cell line and they tend to be substrate oriented in the two-cell population.

The darker cell 12 has more cytoplasmic organelles and cytoskeletal elements relative to the light cell. The nucleus 18 absorbs the stain more deeply, presumably because of a greater density of histones and/or histones rich in Arg and Lys. The darker cell tends to have an irregular contour with a smudged appearance. When secretion is a function of the cell pair, those products are usually observed within the cytoplasm of the dark cell. That is, the dark cell seems to be preferentially programmed for a secretory protein function. Mitotic figures are identified within this cell line and when secretory products are expressed, these cells tend to be located toward the exterior.

The relative numbers of light and dark cells in a tissue is dependent on the type of tissue, and the region of the tissue from which the section is obtained. For normal (non-cancerous) epithelial-cell tissue, one of the two cell types, e.g., the dark cell type, predominate, in a ratio typically between 3:1 to 8:1 one cell type to the other. The ratio appears to be important for normal tissue functioning, since epithelial cell tissue which has become neoplastic shows ratios of the two cell types closer to 1:1, indicating that some control between the cells responsible for maintaining the integrity of the tissue has been lost. This change in relative cells number between normal and neoplastic tissue may be used, in accordance with the invention, to monitor the neoplastic state of tissue, e.g., in a tumor biopsy. other tissue types appear to have ratios closer to 1:1 in the normal state. For example, dimorphic patterns of bone marrow cells, and even circulating blood cells, in which the ratio of the two cells types is close to 1:1.

Fig. 2 shows a photomicrograph of a section 19 of colon epithelial tissue prepared, sectioned and stained in accordance with the invention. The section shows a lumen bordered by light and dark cells, such as indicated at 21, 23, respectively. As seen, dark cells predominate in this region of the tissue. Also as seen, the cells are arranged spatially so that each cell type is either directly adjacent or spaced by only one or a few cells from the second cell type, such that cellular mediators produced by each cell type are readily transmitted, through the intercellular space, to the other cell type. This cell pattern or motif appears to hold both for normal epithelial-cell structures, and for epithelial- cell neoplasms.

B. Model of Paracrine Cellular Control The discovery of this motif in epithelial tissue where two cell types, a light cell and a dark cell, are arranged together in a distinct relationship has lead to the recognition, in accordance with the invention, that control of cellular growth, including the transformation of healthy tissue to neoplastic tissue, is regulated by a paracrine feedforward mechanism in which growth regulators produced by each distinct cell type ar involved in regulating the growth and metabolism of the other cell type.

In the proposed feed forward mechanism, a first cell type secretes a local chemical mediator which acts on a second, adjacent cell type in the immediate environment. The second cell type in turn releases a second chemical mediator, either on its own initiation or in response to the first chemical mediator secreted by the first cell type. The second chemical mediator interacts with the first cell type. In this way, through secretion of chemical mediators, two-way communication between the two cell types is accomplished. Communication between the two cell types, in a paracrine fashion, allows for controlled, coordinated growth. The chemical mediators released by the cells may include a variety of intercellular mediators, such as peptide hormones. An important class of cell mediators, for purposes of the present invention, include growth factors, such as epidermal growth factor (EGF) , transforming growth factor -alpha and -beta (TGF-α and TGF-6) , platelet derived growth factor (PDGF) , fibroblast growth factor (FGF) and insulin-like growth factor (IGF). Fig. 3 illustrates paracrine control between the two cells types identified in the present invention, where the chemical mediator released by the cells is a growth factor. Seen here is a light cell 20, as described above, with specific cellular surface receptors 22. The light cell secretes a growth factor 24, which interacts or binds with the surface receptors 26 of the dark cell 28. The dark cell releases a growth factor 30 which interacts with the surface receptors 22 of the light cell. The two cells communicate through the release and binding of the growth factors influencing the development and proliferation of each cell type.

The paracrine control described in relation to Fig. 3 is a feed forward loop, meaning that each of the two cell types has the potential to influence the growth of the other cell type in a positive way. It can be appreciated that this type of control has the potential for unregulated growth, in which abnormal growth in one cell type leads to a corresponding abnormal stimulation in the growth of the other cell type.

In support of this feed forward model, the general pattern and changed relative number of the two distinct cell types that have been observed in normal epithelial tissue appears to be preserved in epithelial-cell neoplasms, indicating that both cell types are required for tumor-proliferative growth.

IV. Identification of Cell-Specific Mediators The ability to stain an epithelial tissue in such a way that allows for the identification of two dimorphic cells, as described in Section III above, allows the chemical mediators involved in the paracrine control between the two cells to be identified. An important class of chemical mediators is growth factors, to the extent they are important in the proliferation of cells. This section will describe methods for identifying which growth factors are associated with the two cells types of the invention.

Fig. 4A shows four cells in a normal or neoplastic tissue sample which contains the two cell types described in the invention. The growth factors associated with each of the two cells, the light cell and the dark cell, can be identified as follows. The tissue, in an unfixed state, is exposed to selected reporter-labeled growth factors, designated gf-F* in Fig. 4B, under conditions effective to allow binding of the labeled growth factor to the cells making up the tissue. After a designated amount of time, the excess labeled growth factor is washed away. Labeled growth factor is shown at 40 bound to two of the four cells in Fig. 4B, where the growth factor is typically fluorescent labeled or radiolabeled. The tissue is then fixed, which fixes bound growth factor to its receptor, embedded in paraffin, sectioned, and stained as above, to differentially stain light and dark cell types. The stained section is examined by light microscopy, to identify the light or dark cell type having bound, labeled growth factor. This may be done by viewing the section by a both light and fluorescent microscopy, where the growth factor is fluorescent labeled, or by correlating the light microscopic pattern with a radiomicrograph pattern, where the growth factor is radiolabeled. In the illustration shown in Fig. 4C, growth factor is bound to the dark cells in the sample. This procedure of identifying the growth factors specific to the light cell and to the dark cell is repeated with each of the growth factors that may play a role in cellular proliferation and, particularly, in uncontrolled cellular growth. These growth factors include EGF, TGF-α, PDGF, TGF-β, IGF and FGF. More generally, the invention provides a method of identifying growth factors that regulate the control of the growth of dimorphic cell types in an epithelial-cell tumor. The method includes obtaining a normal or neoplastic tissue sample from a selected human tissue, the tissue sample being composed of a first cell type characterized by (i) pale staining with hematoxylin- hydroquinone/eosin-phloxine stain and (ii) round nucleus with central-tending nucleolus, and a second cell type characterized by (i) dark staining with hematoxylin-hydroquinone/eosin- phloxine stain and (ii) dark staining nucleus, with irregular cell contours. The tissue is reacted with a selected reporter-labeled growth factor, under conditions effective to promote binding of the growth factor to one of the two cells types making up the tissue. The cells in the tissue are then examined to determine which of the two cell types the reporter-labeled growth factor is bound to.

The identification of growth factors that are responsible for cell mediation between the two cell types in tissue may be used, for example, to identify or produce agents, such as antibodies or specific growth factor inhibitors, which can be used to disrupt intercellular communication between light and dark cells. For example, in tumor tissue, an imbalance of one or more growth factors may be corrected by addition to the tumor region of an inhibitor of that factor, as a means of treating the tumor.

Alternatively, since interaction between dimorphic cell types would be especially sensitive to perturbation during embryonic development of tissue, inhibitors or activators of a growth factor should be effective in disrupting embryonic tissue growth, e.g., in larval-stage insects, as an insect-specific type of insecticide.

The following example is intended to illustrate, but in no way limit the invention.

Example Preparing Thin Sections Epithelial tissue samples were processed by successive treatment in the following solutions. 5% zinc formalin¹ 30 min.

10% zinc formalin¹ 1 hour, 30 min. 95% alcohol 45 min. 95% alcohol 45 min. 95% alcohol 45 min. 100% alcohol 45 min. 100% alcohol 45 min. 100% alcohol 45 min. xylene 1 hour xylene 1 hour paraffin² 30 min. paraffin² 30 min. paraffin² 30 min.

Sections were cut at 0.5 to 1.0 μm with disposable blades. ¹Formalin concentrate purchased from Anatech Ltd. , Battle Creek, Michigan

²Paraffin: Polyfin purchased from Polyscience, Inc., Warrington, PA

Staining Procedure

Sectioned tissue was stained by successive treatment with the following solutions. xylene 2.5 min. xylene 2.5 min. 100% alcohol 1 min. 30 sec.

100% alcohol 1 min. 30 sec.

100% alcohol 1 min. 30 sec. -

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750ml distilled water plus 7.5ml concentrated acetic acid 30 sec. distilled water 1 min. hematoxylin¹ 4 min., 30 sec. distilled water 1 min. clarifier² 30 sec. distilled water 1 min. bluing³ 1 min. distilled water 1 min. 95% alcohol 1 min. eosin-phloxine⁴ 3 min.

100% alcohol 1 min.

100% alcohol 1 min. xylene 2 min. xylene 2 min.

The following reagents were obtained from the following sources or prepared as follows:

1. hematoxylin - Richard Allen Medical (Richland, MI) I, 750 ml + 0.3 grams hydroquinone- Aldrich

(Milwaukee, WI)

2. clarifier - Richard Allen

3. bluing - 10-15 ml concentrated ammonium hydroxide + 750 ml distilled water 4. eosin - Eosin Y (Ricca Chem, Arlington, TX) 1% w/v alcohol solution) - 100 ml

Phloxine B 0.5% - 10 ml (Sigma Chem, St. Louis)

95% alcohol - 780 ml acetic acid - 4 ml

Although the invention has been described with reference to specific embodiments and features, it will be appreciated that various changes and modifications can be made without departing from the invention.

Claims

IT IS CLAIMED:

1. A histologic method for analyzing a tissue, comprising (a) preparing a tissue under conditions effective to differentially stain two dimorphic cell types in the tissue, one cell type being characterized by:

(i) pale staining with hematoxylin- hydroquinone/eosin-phloxine stain, and

(ii) round nucleus with central-tending nucleolus, and the second cell type being characterized by: (i) dark staining with hematoxylin- hydroquinone/eosin-phloxine stain, and

(ii) dark staining nucleus, with irregular cell contours, and

(b) examining the relative numbers and spatial distribution of the two cell types in the tissue.

2. The method of claim 1, for use in analyzing epithelial cell tissue.

3. The method of claim 2, for use in distinguishing normal from neoplastic tissue, where normal tissue is characterized by an unequal ratio of the two cells types, with one of the two predominating at a ratio between 3:1 to 8:1, and neoplastic tissue is characterized by a more equal ratio of the two cell types.

4. The method of claim 1, wherein said preparing includes the steps of: embedding the tissue in a polymer-hardened paraffin matrix; sectioning the embedded tissue to produce a tissue slice having a thickness between about 0.5- 2 μm; staining the slice with a chromatin stain effective to produce differential chromatin staining in the two different cell types.

5. The method of claim 4, wherein the stain is hematoxylin/hydroquinone stain.

6. The method of claim 4, which further includes staining the tissue with an eosin- containing cytoplasmic stain.

7. A method for visualizing two distinct cell types making up epithelial-cell tissue, comprising embedding the tissue in a polymer-hardened paraffin matrix; sectioning the embedded tissue to produce a tissue slice having a thickness between about 0.5- 2 μm; staining the slice with a chromatin stain effective to produce differential chromatin staining of the two cells types, such that one cell type is identifiable as having a lightly stained nucleus, and the other cell type, as having a darkly stained nucleus in the slice.

8. The method of claim 7, wherein the chromatin stain is a hematoxylin/hydroquinone stain.

9. The method of claim 7, which further includes staining the slice with an eosin- containing cytoplasmic stain.

10. A method of identifying growth factors that regulate the control of the growth of dimorphic cell types in an epithelial-cell tumor, comprising: obtaining a normal or neoplastic tissue sample from a selected human tissue, the tissue sample being composed of a first cell type characterized by (i) pale staining with hematoxylin-hydroquinone/eosin-phloxine stain and (ii) round nucleus with central-tending nucleolus, and a second cell type characterized by (i) dark staining with hematoxylin-hydroquinone/eosin- phloxine stain and (ii) dark staining nucleus, with irregular cell contours, reacting the tissue with a selected reporter- labeled growth factor, under conditions effective to promote binding of the growth factor to one of the two cells types making up the tissue, and examining the cells in the tissue to determine which of the two cell types the reporter-labeled growth factor is bound to.

11. The method of claim 10, wherein said examining includes staining the tissue sample with a chromatin stain effective to differentially stain the two cells types, and examining the cells by light microscopy.