WO2013003234A1 - Tumor extract - Google Patents

Tumor extract Download PDF

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Publication number
WO2013003234A1
WO2013003234A1 PCT/US2012/043824 US2012043824W WO2013003234A1 WO 2013003234 A1 WO2013003234 A1 WO 2013003234A1 US 2012043824 W US2012043824 W US 2012043824W WO 2013003234 A1 WO2013003234 A1 WO 2013003234A1
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WO
WIPO (PCT)
Prior art keywords
tumor
matrix
cells
basement membrane
reflective
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Application number
PCT/US2012/043824
Other languages
French (fr)
Inventor
Jay George
Hynda Kleinman
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Trevigen, Inc.
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 Trevigen, Inc. filed Critical Trevigen, Inc.
Publication of WO2013003234A1 publication Critical patent/WO2013003234A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • breast cancer biopsies have been used to generate cell lines and also injected directly into rodent models.
  • the cell lines can also be injected into rodent models for tumor growth.
  • the problem is that while the tumor may grow, it does not spread to the typical organs that breast cancer most commonly metastasizes, such as bone and brain, and it lacks the stromal elements normally found in human breast cancer. This lack of appropriate models may explain the failure of many drugs which appear to reduce or eliminate the cancer in rodent models but do not work well or at all in humans with cancer.
  • a basement membrane extract described 25 years ago has been found to increase the take and rate of tumor growth in vivo with both biopsy material and with cell lines.
  • This matrix contains many biologically active molecules including laminins, collagen IV, proteoglycans, growth factors and proteases. This matrix has been used to assess in vitro tumor cell behavior including cell adhesion, growth, invasion, migration, and
  • the matrix is derived from a murine tumor of unknown tissue origin found in a wild mouse almost 70 years ago. Despite the widespread use of this murine tumor extract in many in vitro assays and in vivo tumor models, the development of effective drugs for blocking cancer growth and progression is still suffering from poor translation to the cancer patient.
  • the current matrix from this tumor contains proteins known to be present in the basement membrane matrix.
  • the basement membrane matrix underlies epithelial and endothelial cells and surrounds fat, muscle, and nerves.
  • the amount and type of the matrix protein vary with the tissue of origin and even with the developmental stage of the tissue. Thus there are many different levels of components in tissue-specific basement membrane matrices. Since tumors are generally of epithelial origin, this matrix represents the matrix that most tumor cells would contact and it would be expected that tumors depending on their origin would have a tumor-specific extracellular matrix.
  • stromal compartment matrix An additional variable in the matrix of tumors which has not been considered in the generation of human tumor models is stromal compartment matrix.
  • the level of stromal cells and stromal extracellular matrix vary in different tumors but as much as 50% of mammary tumors can be composed of stromal cells and matrix.
  • the stromal matrix in non malignant tissue is generally found in the skin and in fibrotic disorders such as scleroderma, liver fibrosis, pulmonary fibrosis, etc. This stromal matrix contains mainly collagen I.
  • the stromal matrix is important for tumor growth and for resistance to chemotheraputic drugs. Drugs that block collagen I synthesis reduce tumor growth.
  • the tumor microenvironment is very unusual relative to other tissues because of the rapidly growing tissue. Because it is generally growing faster than surrounding tissue, it has a unique vasculature that is rapidly made to feed the growing tissue.
  • the tumor tissue is generally lacking in oxygen (hypoxia condition) and has lower glucose because it is used so rapidly for growth.
  • the tumor microenvironment is acidic (approximately pH 6.8) whereas the surrounding normal tissue is close to neutral (7.2) pH.
  • the present invention relates to a tumor matrix comprising extracellular matrix components from a basement membrane, stroma, and tumor, that promotes tumor cell growth in vitro and in vivo reflective of the tumor type.
  • the present invention relates to a method for the preparation of the tumor matrix described above, which comprises (a) growing a basement membrane tumor in the presence of stromal cells and tumor cells, and (b) isolating the resulting tumor, and (c) preparing the matrix from the isolated tumor.
  • the present invention relates to a tumor matrix which enhances the physiological growth of tumor cells, tumor cell lines, and biopsy specimens both in vitro and in vivo where physiological growth is defined as appropriate growth rate and appropriate morphology in vitro consistent with the tumor of origin and appropriate growth rate, histology, and metastatic spread consistent with the tumor of origin.
  • the tumor matrix of the present invention contains components present in tumor tissue-specific basement membrane and stromal matrices and preferably should be acidic and have low levels of glucose. These components may include but are not limited to laminin, collagen IV, heparan sulfate proteoglycan and various growth factors.
  • This matrix can be obtained by growing a basement membrane tumor in the presence of stromal cells and tumor cells in a suitable animal.
  • the stromal cells and tumor cells are human cells, but cells from other suitable animals may be used, for example, primate cells.
  • tumor cells are intended to include tumor derived cell lines, transformed cell lines, tumor cells, etc.
  • the basement membrane tumor is of animal origin, preferably murine origin, and the suitable animal is preferably a rodent.
  • the basement membrane tumor may originate in other animals, such as pig, rabbit, etc.
  • the basement membrane tumor is an Engelbreth-Holm- Swarm (EHS) mouse tumor.
  • EHS Engelbreth-Holm- Swarm
  • the ratios of the three different cells types in the mixture will vary depending on the tumor cell type. Optimal ratios may readily be determined by those of skill in the art, for example by observing the physiological growth rate or the tumor-appropriate histology of the tumor. An illustrative ratio would be about 5:1 :10. This mixture of cells should grow well in rodents and yield tumors within a few weeks.
  • the tumor is then harvested and the matrix prepared according to methods known in the art, including those described in Kleinman, et ah, Biochemistry, volume 25, page 312, 1986, the content of which is incorporated herein by reference (the process involves general washes of the tumor followed by extraction with urea or a similarly functioning material) for the basement membrane tumor except that the final medium is preferably low in glucose (less than or equal to about 7 mM) and preferably has an acidic pH (less than or equal to about pH 7.0), although glucose in an amount of greater than about 7 mM and a basic pH could also be acceptable, depending on the materials used.
  • the amount of basement membrane tumor, stromal cells, and tumor should be such that the composition of the resulting tumor mainly reflects the composition of the tumor tissue of origin and is enriched in proteins from the tumor source, e.g., human proteins. Any amount of enrichment could be suitable, depending on the materials used. Preferably, such enrichment may be by about 10% or greater; about 20% or greater; about 30% or greater; about 40% or greater; or by more than about 50%.
  • This matrix will be a mixture of stromal and basement membrane components. This resulting material will promote tumor growth in vivo and in vitro in a manner comparable to the tumor of origin.
  • the material may be derived from any desired tumor type.
  • Non-limiting examples include breast, lung, liver, kidney, brain, prostate, and colon cancer cells.
  • the compositions of the present invention will preferably gel at a protein concentration of about 4 mg/ml or greater.
  • a highly metastatic breast cancer should grow quickly on this material and be invasive in vitro and when coinjected in vivo, it should promote a comparable rapid growth and spread, and be histologically comparable to an invasive tumor with regard to stromal matrix and cells.
  • a slow growing breast cancer should grow slowly in vitro and be less invasive in the matrix and grow at a slower rate in vivo, be less or non metastatic, and maintain the histology of the primary tumor in terms of stroma and cells.
  • the proposed matrix of the present invention will differ from known basement membrane extract in the following ways.
  • the invention may be further illustrated by reference to the following non- limiting example.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present invention relates to a tumor matrix comprising extracellular matrix components from a basement membrane, stroma, and tumor, that promotes tumor cell growth in vitro and in vivo reflective of the tumor type. Also disclosed are methods for the preparation of the tumor matrix.

Description

TUMOR EXTRACT
BACKGROUND OF THE INVENTION
Animal models for human tumors have been difficult to generate and when obtained such models do not mimic the human tumor in composition or behavior well. For example, breast cancer biopsies have been used to generate cell lines and also injected directly into rodent models. The cell lines can also be injected into rodent models for tumor growth. The problem is that while the tumor may grow, it does not spread to the typical organs that breast cancer most commonly metastasizes, such as bone and brain, and it lacks the stromal elements normally found in human breast cancer. This lack of appropriate models may explain the failure of many drugs which appear to reduce or eliminate the cancer in rodent models but do not work well or at all in humans with cancer.
A basement membrane extract described 25 years ago has been found to increase the take and rate of tumor growth in vivo with both biopsy material and with cell lines. This matrix contains many biologically active molecules including laminins, collagen IV, proteoglycans, growth factors and proteases. This matrix has been used to assess in vitro tumor cell behavior including cell adhesion, growth, invasion, migration, and
morphology. It has been highly useful in improving and advancing our understanding of tumor growth and progression. The matrix is derived from a murine tumor of unknown tissue origin found in a wild mouse almost 70 years ago. Despite the widespread use of this murine tumor extract in many in vitro assays and in vivo tumor models, the development of effective drugs for blocking cancer growth and progression is still suffering from poor translation to the cancer patient.
The current matrix from this tumor contains proteins known to be present in the basement membrane matrix. The basement membrane matrix underlies epithelial and endothelial cells and surrounds fat, muscle, and nerves. The amount and type of the matrix protein vary with the tissue of origin and even with the developmental stage of the tissue. Thus there are many different levels of components in tissue-specific basement membrane matrices. Since tumors are generally of epithelial origin, this matrix represents the matrix that most tumor cells would contact and it would be expected that tumors depending on their origin would have a tumor-specific extracellular matrix.
An additional variable in the matrix of tumors which has not been considered in the generation of human tumor models is stromal compartment matrix. The level of stromal cells and stromal extracellular matrix vary in different tumors but as much as 50% of mammary tumors can be composed of stromal cells and matrix. The stromal matrix in non malignant tissue is generally found in the skin and in fibrotic disorders such as scleroderma, liver fibrosis, pulmonary fibrosis, etc. This stromal matrix contains mainly collagen I. The stromal matrix is important for tumor growth and for resistance to chemotheraputic drugs. Drugs that block collagen I synthesis reduce tumor growth.
Finally the tumor microenvironment is very unusual relative to other tissues because of the rapidly growing tissue. Because it is generally growing faster than surrounding tissue, it has a unique vasculature that is rapidly made to feed the growing tissue. The tumor tissue is generally lacking in oxygen (hypoxia condition) and has lower glucose because it is used so rapidly for growth. The tumor microenvironment is acidic (approximately pH 6.8) whereas the surrounding normal tissue is close to neutral (7.2) pH.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a tumor matrix comprising extracellular matrix components from a basement membrane, stroma, and tumor, that promotes tumor cell growth in vitro and in vivo reflective of the tumor type.
In another aspect, the present invention relates to a method for the preparation of the tumor matrix described above, which comprises (a) growing a basement membrane tumor in the presence of stromal cells and tumor cells, and (b) isolating the resulting tumor, and (c) preparing the matrix from the isolated tumor.
In another aspect, the present invention relates to a tumor matrix which enhances the physiological growth of tumor cells, tumor cell lines, and biopsy specimens both in vitro and in vivo where physiological growth is defined as appropriate growth rate and appropriate morphology in vitro consistent with the tumor of origin and appropriate growth rate, histology, and metastatic spread consistent with the tumor of origin.
DETAILED DESCRIPTION OF THE INVENTION
The tumor matrix of the present invention contains components present in tumor tissue-specific basement membrane and stromal matrices and preferably should be acidic and have low levels of glucose. These components may include but are not limited to laminin, collagen IV, heparan sulfate proteoglycan and various growth factors. This matrix can be obtained by growing a basement membrane tumor in the presence of stromal cells and tumor cells in a suitable animal. Preferably, the stromal cells and tumor cells are human cells, but cells from other suitable animals may be used, for example, primate cells. As used herein, tumor cells are intended to include tumor derived cell lines, transformed cell lines, tumor cells, etc. The basement membrane tumor is of animal origin, preferably murine origin, and the suitable animal is preferably a rodent. The basement membrane tumor may originate in other animals, such as pig, rabbit, etc. In a preferred embodiment, the basement membrane tumor is an Engelbreth-Holm- Swarm (EHS) mouse tumor. The ratios of the three different cells types in the mixture (basement membrane tumor, stromal cells, and tumor) will vary depending on the tumor cell type. Optimal ratios may readily be determined by those of skill in the art, for example by observing the physiological growth rate or the tumor-appropriate histology of the tumor. An illustrative ratio would be about 5:1 :10. This mixture of cells should grow well in rodents and yield tumors within a few weeks. The tumor is then harvested and the matrix prepared according to methods known in the art, including those described in Kleinman, et ah, Biochemistry, volume 25, page 312, 1986, the content of which is incorporated herein by reference (the process involves general washes of the tumor followed by extraction with urea or a similarly functioning material) for the basement membrane tumor except that the final medium is preferably low in glucose (less than or equal to about 7 mM) and preferably has an acidic pH (less than or equal to about pH 7.0), although glucose in an amount of greater than about 7 mM and a basic pH could also be acceptable, depending on the materials used. The amount of basement membrane tumor, stromal cells, and tumor should be such that the composition of the resulting tumor mainly reflects the composition of the tumor tissue of origin and is enriched in proteins from the tumor source, e.g., human proteins. Any amount of enrichment could be suitable, depending on the materials used. Preferably, such enrichment may be by about 10% or greater; about 20% or greater; about 30% or greater; about 40% or greater; or by more than about 50%. This matrix will be a mixture of stromal and basement membrane components. This resulting material will promote tumor growth in vivo and in vitro in a manner comparable to the tumor of origin.
The material may be derived from any desired tumor type. Non-limiting examples include breast, lung, liver, kidney, brain, prostate, and colon cancer cells. The compositions of the present invention will preferably gel at a protein concentration of about 4 mg/ml or greater.
For example, a highly metastatic breast cancer should grow quickly on this material and be invasive in vitro and when coinjected in vivo, it should promote a comparable rapid growth and spread, and be histologically comparable to an invasive tumor with regard to stromal matrix and cells. In contrast, a slow growing breast cancer should grow slowly in vitro and be less invasive in the matrix and grow at a slower rate in vivo, be less or non metastatic, and maintain the histology of the primary tumor in terms of stroma and cells. The proposed matrix of the present invention will differ from known basement membrane extract in the following ways.
1. It will be generated with a mixture of three cells types (EHS murine or other basement membrane tumor, stromal cells, and tumor) as opposed to one cell type (EHS or other basement membrane tumor) for the basement membrane extract.
2. It will be enriched in, e.g., human proteins as opposed to the all murine protein basement membrane extract.
3. It will contain a mixture of stromal, basement membrane, and tumor-specific proteins as opposed to all basement membrane proteins found in the basement membrane extract. 4. It will contain low glucose (preferably about 7 mM or less) as opposed to the higher glucose (25 mM or greater) of the basement membrane extract.
5. It will be low pH (pH of about 7 or less) as opposed to the neutral pH (7.2-7.4) of the basement membrane extract.
6. It will show growth when cultured in vitro reflective of the tumor type and malignant potential as opposed to the rapid growth for all tumor cells observed with the basement membrane extract.
7. It will show growth when coinjected with tumor cells in vivo reflective of the tumor type and malignant potential as opposed to the rapid growth for all tumor cells observed with basement membrane extract.
8. It will maintain the tumor stroma when coinjected with tumor cells in vivo reflective of the tumor type as opposed to the lack of stroma observed with the basement membrane extract. 9. It will increase organ-specific metastasis when coinjected in vivo orthotopically as opposed to the basement membrane extract which does not encourage metastasis well and when it occurs it is not organ-specific to the tumor type.
The invention may be further illustrated by reference to the following non- limiting example.
Example 1 (manufacturing protocol)
1. Develop humanized tumors in athymic mice using MCF-7 breast cancer and AS27 stromal cell lines and EHS tumor tissue and expand and cell lines for injection into mice. a. Inject nude athymic mice and inject with ratios:
A B C
5xl06 2xl06 lxlO6 MCF-7
5xl05 2xl05 lxlO5 stromal cells
.12 grams .12 grams .12 grams EHS tissue
2. Harvest and analyze tumor tissue. a. Harvest 3-4 gm tumors and take a small area of 4 (for hematoxylin and eosin stain, and chrome stain for collagen and vimentin by standard procedures). b. Save 1/5 of 3 tumors each for future inoculation of additional mice. c. Analyze by histology to determine tumor phenotype. d. Analyze by immunohistochemistry for vimentin and collagen I
3. Test morphology and drug sensitivity of cells grown on new breast tumor aligned basement membrane extract (BTA-BME) vs BME a. Test cells for growth and morphology b. Test cells for drug sensitivity with known drugs that work and do not work on patients with breast cancer.
While the invention has been described in detail above, the invention is not intended to be limited to the specific embodiments as described. It is evident that those skilled in the art may now make numerous uses and modifications of and departures from the specific embodiments described herein without departing from the inventive concepts.

Claims

Claims:
1. A tumor matrix comprising extracellular matrix components from a basement membrane, stroma, and a tumor, that promotes tumor cell growth in vitro and in vivo reflective of the tumor type.
2. The matrix of claim 1, wherein the matrix is derived from breast, lung, liver, kidney, brain, prostate, or colon cancer cells.
3. The matrix of claim 1, wherein the stroma and tumor are human cells, and the matrix contains more than about 50% human proteins.
4. The matrix of claim 1, wherein the matrix is able to gel at about 4 mg/ml or greater protein concentration.
5. The matrix of claim 1, wherein the matrix is capable of maintaining tumor morphology reflective of the tumor type and aggressiveness in vitro.
6. The matrix of claim 1, wherein the matrix is capable of maintaining tumor invasiveness reflective of the tumor type in vitro and in vivo.
7. The matrix of claim 1, wherein the matrix is capable of maintaining tumor growth reflective of the tumor type in vitro and in vitro.
8. The matrix of claim 1, wherein the matrix is capable of maintaining tumor metastasis reflective of the tumor type in vivo.
9. The matrix of claim 1, wherein the matrix is capable of maintaining organ specific metastasis reflective of the tumor type in vivo.
10. The matrix of claim 1, wherein the basement membrane is of murine origin.
11. The matrix of claim 1, wherein the tumor matrix contains less than about 7 raM glucose.
12. The matrix of claim 1, wherein the tumor matrix has a pH of less than about
7.0.
13. A method for the preparation of a tumor matrix, which comprises (a) growing a basement membrane tumor in the presence of stromal cells and tumor cells, and (b) isolating the resulting tumor, and (c) preparing the matrix from the isolated tumor.
14. The method of claim 13, wherein the growth takes place in a rodent.
15. The method of claim 13, wherein the ratio of basement membrane tumonstromal cell: tumor cell is about 5: 1 :10.
16. The method of claim 13, wherein the matrix is derived from breast, lung, liver, kidney, brain, prostate, or colon cancer cells.
17. The method of claim 13, wherein the basement membrane is of murine origin.
18. The method of claim 13, wherein step (c) comprises washing and extraction.
19. The method of claim 18, wherein the extraction is with a solvent comprising urea.
20. The method of claim 13, wherein the stromal cells and tumor cells are human cells.
21. The product of the process of claim 13.
PCT/US2012/043824 2011-06-28 2012-06-22 Tumor extract WO2013003234A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018083231A1 (en) * 2016-11-07 2018-05-11 Rise Research Institutes of Sweden AB Diagnostic methods

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4829000A (en) * 1985-08-30 1989-05-09 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Reconstituted basement membrane complex with biological activity
US20110097403A1 (en) * 2008-11-14 2011-04-28 Histogen, Inc. Extracellular matrix compositions for the treatment of cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4829000A (en) * 1985-08-30 1989-05-09 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Reconstituted basement membrane complex with biological activity
US20110097403A1 (en) * 2008-11-14 2011-04-28 Histogen, Inc. Extracellular matrix compositions for the treatment of cancer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BENTON, G. ET AL.: "Advancing Science and Technology Via 3D Culture on Basement Membrane Matrix", JOURNAL OF CELLULAR PHYSIOLOGY, vol. 221, no. ISS. 1, 2 June 2009 (2009-06-02), pages 18 - 25 *
BENTON, G. ET AL.: "Multiple uses of basement membrane-like matrix (BME/Matrigel) in vitro and in vivo with cancer cells", INTERNATIONAL JOURNAL OF CANCER, vol. 128, 17 November 2010 (2010-11-17), pages 1751 - 1757 *
HORNING ET AL.: "3-D Tumor Model for In Vitro Evaluation of Anticancer Drugs", MOLECULAR PHARMACEUTICS, vol. 5, no. 5, 5 August 2008 (2008-08-05), pages 849 - 862 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018083231A1 (en) * 2016-11-07 2018-05-11 Rise Research Institutes of Sweden AB Diagnostic methods
US11840732B2 (en) 2016-11-07 2023-12-12 Iscaff Pharma Ab Diagnostic methods

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