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WO1996018101A1 - Procede histologique d'analyse tissulaire - Google Patents

Procede histologique d'analyse tissulaire Download PDF

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Publication number
WO1996018101A1
WO1996018101A1 PCT/US1994/014211 US9414211W WO9618101A1 WO 1996018101 A1 WO1996018101 A1 WO 1996018101A1 US 9414211 W US9414211 W US 9414211W WO 9618101 A1 WO9618101 A1 WO 9618101A1
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WO
WIPO (PCT)
Prior art keywords
tissue
cell
stain
staining
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1994/014211
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English (en)
Inventor
Thomas M. Donndelinger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CELLULAR DIMORPHISM INSTITUTE
Original Assignee
CELLULAR DIMORPHISM INSTITUTE
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CELLULAR DIMORPHISM INSTITUTE filed Critical CELLULAR DIMORPHISM INSTITUTE
Priority to PCT/US1994/014211 priority Critical patent/WO1996018101A1/fr
Publication of WO1996018101A1 publication Critical patent/WO1996018101A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Definitions

  • the present invention relates to a histologic method of tissue analysis, in particular, analysis of epithelial-cell tissue.
  • Histological examination of cells in a 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.
  • EGF epidermal growth factor
  • TGF- ⁇ transforming growth factor-alpha
  • PDGF platelet derived growth factor
  • FGF fibroblast growth factor
  • IGF insulin-like growth factor
  • TGF-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 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.
  • 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.
  • 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 ⁇ m; 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.
  • 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 ⁇ m; 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.
  • 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 examined to determine which of the two cell types the reporter-labeled growth factor is bound to.
  • 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
  • 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.
  • Epithelia 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.
  • Parentacrine 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.
  • 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.
  • fixatives are glutaraldehyde and formaldehyde.
  • 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 % piccolyteTM 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 PolyfinTM, 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 ( ⁇ m) , preferably 1 ⁇ m +/- 0.5 ⁇ m. Sectioning is by conventional microtome. This tissue section thickness is important for visualizing the dimorphic cell structures of the tissue being analysed.
  • 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.
  • a chromatin stain is a hematoxylin/hydroquinone, such as the hematoxylin/hydroquinone stain described in the Example.
  • the staining step involves several dehydration steps with xylene and 1 PC-7 S94/14211
  • 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.
  • the eosin stain is used to stain acidic cytoplasmic and nuclear proteins.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • dark cells predominate in this region of the tissue.
  • 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.
  • 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.
  • the chemical mediators released by the cells may include a variety of intercellular mediators, such as peptide hormones.
  • An important class of cell mediators include growth factors, such as epidermal growth factor (EGF) , transforming growth factor -alpha and -beta (TGF- ⁇ 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.
  • 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- ⁇ , PDGF, TGF- ⁇ , 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.
  • agents such as antibodies or specific growth factor inhibitors, which can be used to disrupt intercellular communication between light and dark cells.
  • 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.
  • 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.
  • 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.
  • Phloxine B 0.5% - 10 ml (Sigma Chem, St. Louis)

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Abstract

L'invention concerne un procédé histologique d'analyse tissulaire. Ce procédé consiste à préparer le tissu dans des conditions efficaces pour colorer différentiellement deux types cellulaires dimorphiques constituant le tissu, et à analyser les nombres relatifs et la répartition spatiale des deux types cellulaires. Ce procédé peut être utilisé, par exemple, pour distinguer un tissu cellulaire normal et un tissu cellulaire épithélial néoplasique. L'invention concerne d'autre part un procédé pour identifier des facteurs de croissance régulant la croissance des deux types cellulaires.
PCT/US1994/014211 1994-12-09 1994-12-09 Procede histologique d'analyse tissulaire Ceased WO1996018101A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US1994/014211 WO1996018101A1 (fr) 1994-12-09 1994-12-09 Procede histologique d'analyse tissulaire

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1994/014211 WO1996018101A1 (fr) 1994-12-09 1994-12-09 Procede histologique d'analyse tissulaire

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0822403A1 (fr) * 1996-08-02 1998-02-04 Milestone S.r.l. Procédé pour le traitement d'échantillons organiques
WO2002005794A3 (fr) * 2000-07-13 2002-08-01 Univ Bristol Inhibition du canal chlorure ctfr (regulateur transmembranaire de la fibrose kystique)
WO2002005793A3 (fr) * 2000-07-13 2002-09-06 Univ Bristol Activation du canal a chlorure cftr (regulateur de la permeabilite transmembranaire de la fibrose kystique)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BREAST CANCER RESEARCH AND TREATMENT, Volume 15, issued 1990, OSBORNE et al., "Autocrine and Paracrine Growth Regulation of Breast Cancer: Clinical Implications", pages 3-11. *
CANCER SURVEYS, Volume 7, Number 4, issued 1988, GREIG et al., "Growth Factors as Novel Therapeutic Targets in Neoplastic Disease", pages 653-674. *
INTERNATIONAL JOURNAL OF GYNECOLOGICAL PATHOLOGY, Volume 7, Number 3, issued 1988, SAHIN et al., "An Immunohistochemical Study of Primary Ovarian Sarcoma: An Evaluation of Nine Tumors", pages 268-279. *
ONCOLOGY RESEARCH, Volume 4, Number 10, issued 1992, NICOLSON, "Paracrine/Autocrine Growth Mechanisms in Tumor Metastasis", pages 389-399. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0822403A1 (fr) * 1996-08-02 1998-02-04 Milestone S.r.l. Procédé pour le traitement d'échantillons organiques
WO1998005938A1 (fr) * 1996-08-02 1998-02-12 Milestone S.R.L. Methode pour traiter des echantillons organiques
WO2002005794A3 (fr) * 2000-07-13 2002-08-01 Univ Bristol Inhibition du canal chlorure ctfr (regulateur transmembranaire de la fibrose kystique)
WO2002005793A3 (fr) * 2000-07-13 2002-09-06 Univ Bristol Activation du canal a chlorure cftr (regulateur de la permeabilite transmembranaire de la fibrose kystique)
US7304090B2 (en) 2000-07-13 2007-12-04 The University Of Bristol Inhibition of the cystic fibrosis transmembrane conductance regulator chloride channel

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