Classifications

• • 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
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5082—Supracellular entities, e.g. tissue, organisms
  • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
  • A01K2267/0331—Animal model for proliferative diseases
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/475—Assays involving growth factors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/475—Assays involving growth factors
  • G01N2333/50—Fibroblast growth factors [FGF]

Definitions

• the present disclosure relates to animal models of cancer. Specifically, the present disclosure relates to a breast cancer xenograft model comprises a human breast cancer cell line engineered to express vascular endothelial growth factor (VEGF) on induction by an inducer and applications of said animal models in treatment of breast cancer growth, metastasis and other characteristics associated with breast cancer.
• VEGF vascular endothelial growth factor
• Antiestrogen therapies are effective in both premenopausal and postmenopausal breast cancer patients and in the metastatic, adjuvant, and chemopreventive settings. Compared to chemotherapy, the antiestrogenic drugs are well tolerated with low toxicity and relative mild side effects. Currently, the most widely used antiestrogen drug is tamoxifen, which is administered orally as the citrate salt.
• tamoxifen resistance was mainly caused by either 1) ER-independent alternative signaling pathways to support tumor cells to grow, or by 2) ER-mediated mechanism such as switch of tamoxifen action from an ER antagonist to an ER agonist.
• VEGF vascular endothelial growth factor
• VEGF family comprises several members.
• VEGF-A commonly referred to in the art as VEGF, stimulates endothelial cell proliferation and migration in culture and angiogenesis in vivo (Klagsbrun et al., Cytokine.GrowthFactor.Rev., 7: 259-270, 1996).
• VEGF is a key regulator of endothelial cell functions required for both physiological and pathological angiogenesis.
• Other members of the VEGF family include VEGF-B, -C, -D, -E, and placenta growth factor (P1GF).
• VEGFR-1 membrane tyrosine kinases
• VEGFR-2 Flk-1 or KDR
• VEGFR-3 Flt4
• neuropilin-1 neuropilin-1
• VEGF occurs in a number of isoforms, including polypeptides of 121, 145, 165, 189, and 206 amino acids (amino acid length calculated without signal peptide sequences), which are produced by the alternate splicing of a single gene containing eight exons (Neufeld et al., FASEB J., 13: 9-22, 1999).
• VEGF- ⁇ 2 ⁇ and VEGF-i ⁇ s are the isoforms most commonly secreted by tumor cells, it is the VEGF- ⁇ 65 isoform that acts most strongly on endothelial cells, leading to the formation of new capillaries (Veikkola et al, Cancer Res., 60: 203-212, 2000; Ferrara et al., Nat.Rev.Cancer, 2: 795-803, 2002).
• a variety of studies have shown the importance of VEGF as a prognostic indicator of the severity of breast cancer (Foekens et al., Cancer Res., 61: 5407-5414, 2001).
• Metastases rather than primary tumors, are responsible for most cancer deaths.
• the development of effective therapies is partially hampered by the lack of suitable animal models (Chambers et al., Nat.Rev.Cancer, 2: 563-572, 2002; Couzin, J. Science, 299: 1002-1006, 2003).
• Dissemination of cancer cells involves different mechanisms, such as direct invasion of surrounding tissues, spread via the blood vascular system (hematogenous metastasis), and spreads via the lymphatic system (lymphatic metastasis).
• VEGF increases potential of metastasis through neoangiogenesis
• Angiogenesis is essential for tumor growth, and is implicated in the initial progression from a dormant microscopic metastases into clinically detectable metastatic lesion (Cristofanilli et al., Nat.Rev.Drug Discov., 1: 415-426, 2002).
• Breast cancer can spread to almost any area of the body. The most common metastasis sites are lymph node, lung, bone, and liver.
• VEGF vascular endothelial growth factor-1
• C9V cell lines MCF-7- derived human breast cancer cell lines
• Dox doxycycline
• Both C9V and MCF-7 parental cell lines are estrogen receptor positive, exhibit estrogen growth dependence, tamoxifen sensitivity, and minimal metastatic potential when introduced subcutaneously into nude mouse.
• Addition of Dox in mouse drinking water induces VEGF expression in C9V xenografts and demonstrates that induction of VEGF to clinical relevant levels is sufficient to promote ER-positive, tamoxifen-responsive and non-metastatic breast tumors to become tamoxifen resistant and metastatic through a mechanism by which VEGF facilities the remodeling tumor-microenvironment.
• VEGF expressing tumor cells are able to induce a desmoplastic response, a feature of host stromal response common to human breast cancers but seldom seen in xenograft models. It is observed that VEGF expression increases colonization of disseminated tumor cells at the distant organ lungs, suggesting a new role of VEGF in addition to its angiogenic effect. When VEGF overexpression was turned off at early stage in the mouse, tumor growth became static and the metastasis incidence was reduced.
• Another object of the disclosure is to provide human ER-positive breast cancer cell lines that expresses VEGF in a Dox-regulated manner to clinically relevant levels.
• Another object of the disclosure is to provide an animal model to further investigate the role of VEGF in breast cancer development and progression, induction of tamoxifen resistance, colonization of disseminated cancer cells in secondary sites distant from the primary tumor, and induction of metastasis.
• a further object of the disclosure is to provide an animal model to study the mechanism of action of VEGF in breast cancer development and progression, induction of tamoxifen resistance, colonization of disseminated cancer cells in secondary sites distant from the primary tumor, and induction of metastasis.
• a further object of the disclosure is to provide an animal model to study the mechanism of action of VEGF in tumor stromal bidirectional interaction, tumor microenvironment remodeling, and colonization of disseminated cancer cell in distant organ in breast cancer progression, anti-estrogen hormonal resistance, and development of metastases.
• An additional object of the disclosure is to provide an animal model to identify inhibitors of VEGF action to inhibit the role of VEGF in breast cancer development, progression, induction of tamoxifen resistance and induction of metastatic potential.
• a further object of the disclosure is to provide an animal model for preclinical efficacy studies of potential therapeutic agents, new treatment of multi-drug therapy targeting both estrogen and VEGF signaling pathways in settings of metastatic and adjuvant therapy.
• FIGS. 1A and IB are diagrams of the DNA constructs that have been stably transfected into MCF-7 cells to produce human VEGFi ⁇ s-
• FIG. 1 (a) illustrates the bicistronic tet-transactivator construct (pBTE) and FIG. 1(b) illustrates the tet-responsive element construct (pTRE-hVEGF).
• the MCF-7 cells stably integrated with these constructs are produced as described.
• FIG. 2 shows Western Blotting analysis of secreted VEGF protein in conditioned media from representative MCF-7-derived cell lines.
• MCF-7 Non-transfected MCF-7 cells
• MCF-7 cells transfected only with the pBTE construct designated C9
• a cell line constitutively expressing VEGF designated MLV-165-16
• human recombinant 165 amino acid fonn VEGF PeproTch, Rocky Hill, NJ
• C9V4 and C9V18 cell lines transfected with both the pTRE- hVEGF and pBTE constructs were analyzed as indicated in the presence and absence of 0.2 ug/ml Dox.
• the high molecular weight band could reflect VEGF post-translational modification. shows the absence of an autocrine effect of VEGF on cell proliferation in vitro.
• C9V cells were stripped of estrogen, cultured in estrogen-free medium as described, and treated as indicated. Cell number was measured on day 3 by cell counting.
• the treatment conditions as indicated are: VEGF: 20ng/ml; FGF: FGFa, 20ng/ml; Tarn: 4-hydroxytamoxifen, 10 "7 M, and E2 : 17-beta estradiol, 10 "8 M.
• FIGS. 4A and 4B show the effect of VEGF expression on tumor growth (as measured by tumor volume) and the correlation of tumor volume with cytosolic VEGF concentration.
• the VEGF expressing tumors were produced in nude mice as described.
• FIG. 4A shows that the addition of Dox in mouse drinking water to induce VEGF expression increased tumor growth rate in a dose- dependent manner over a 50-day period.
• FIG. 4B shows that the average tumor volume in each mouse group was directly correlated with the tumor VEGF content determined by ELISA and dose of Dox in the mouse drinking water.
• FIGS. 5A-5D show Dox-regulated VEGF expression was associated with an increased percentage of nuclei staining positive for a cell proliferation marker Ki-67 in tumor sections, indicating that Dox- regulated.
• the VEGF expressing tumors were produced in nude mice as described. VEGF expression stimulated the proliferation of tumor cells in vivo. Dox-regulated VEGF expression increases proliferation of tumor cells in both tamoxifen-treated mice (FIGS. 5 A and 5B) and estrogen- supplemented mice (FIGS. 5C and 5D).
• FIGS. 6A and 6B show Dox-regulated VEGF expression increased tumor microvessel density characterized by immunohistochemical staining with a mouse endothelial cell-specific marker PECAM-1.
• the VEGF expressing tumors were produced in nude mice as described.
• FIG. 6A shows immunohistochemical staining in the presence of estrogen with no induction of VEGF expression while FIG. 6B shows immunohistochemical staining in the presence of estrogen in the presence of Dox-induced VEGF expression.
• FIG. 7 shows that the effect of VEGF on the growth rate of breast tumors is reversible.
• the VEGF expressing tumors were produced in nude mice as described. The inoculated mice were divided into three groups (all groups receiving estrogen). Group 1 received 0.2 mg/ml Dox throughout the experiment, group 2 received 0.2 mg/ml Dox for 11 days only, while group 3 received sucrose (negative control) throughout the experiment. The tumor growth rate of the mice receiving Dox for only 11 days decreased dramatically upon the Dox removal, suggesting that inhibition of VEGF at an early stage may stabilize disease progression.
• FIGS. 8 A and 8B show that VEGF expression induces tamoxifen resistance. The VEGF expressing tumors were produced in nude mice as described.
• FIG. 8A shows results from mice where the C9V4 cell line was used to establish the xenograft
• FIG. 8B shows results from mice where the C9V18 cell line was used to establish the xenograft.
• FIGS. 9A-9D show VEGF expression induced primary tumor metastasis.
• Parental C9V4 MCF-7 cells were Lac Z-tagged.
• the VEGF expressing tumors were produced in nude mice as described.
• X-gal staining of the lungs showed macrometastatic deposits of tumor cells in the mice that received Dox and either estrogen (FIGS. 9A and 9C) or estrogen followed by tamoxifen (FIGS. 9B and 9D).
• X-gal staining is shown in FIGS. 9 A and 9B and H&E histological staining of the lung tissue samples is shown in FIG. 9C and 9D.
• FIG. 10A-10D show VEGF expression increases colonization of disseminated tumor cells in distant organ, a new role of VEGF in addition to its angiogenic effect.
• C9V cells were directly introduced into the circulation by tail vein injection and the patterns of disseminated tumor cells in mouse lungs were examined by immunostaining with antibody against cytokeratin, an epithelial cell marker.
• FIG. 12A and 12C show immunostaining with cytokeratin of lung sections obtained 2 hours after tail vein injection in animal treated receiving no Dox (FIG. 12A) and in an animal receiving Dox (FIG. 12C).
• FIG. 12B and 12D show immunostaining with cytokeratin of lung sections obtained 4 weeks after tail vein injection in animal treated receiving no Dox (FIG.
• tumor cells were detected in the lungs of animals regardless of Dox treatment 2 hours after tail vein injection, 4 weeks after tail vein injection, tumor cells were only detected in the lungs of animals receiving Dox treatment to induce VEGF expression.
• FIG. 11 shows VEGF expression induced macroscopic metastatic lesions. 10 weeks after tail vein injection, several macroscopic metastatic lesions were observed in lungs from mice that were received Dox in their drinking water. Mixed pattern of single tumor cells, various sizes of tumor cell colonies, blood vessels, blood lakes, and stromal cells were observed in the macroscopic metastatic lesion by cytokeratin immunohistological staining (tumor appears dark surrounded by lighter staining stromal cells; arrows point to blood lakes in the lesions).
• FIG. 12 shows VEGF increased chemotactic mobility of human fibroblast cells. Cell migration transwell assay with human foreskin fibroblast showed VEGF dramatically increased chemotactic mobility of fibroblast toward VEGF concentrations as compared to control and 10% FBS.
• FIGS. 13A-13C show VEGF expression increased desmoplastic response in the primary tumors from tamoxifen-treated mice. Immunohistological staining of the epithelial cell marker, cytokeratin, shows an increased desmoplastic response in primary tumors in the mice treated with Dox.
• FIG. 13A shows the desmoplastic response in animals not treated with Dox to induce VEGF expression.
• FIG. 13B shows the desmoplastic response in animals treated with Dox to induce VEGF expression.
• VEGF induction increased level of desmoplastic response from 10% of stromal cell per view field to about 40% in tamoxifen treated mice (FIG. 13C).
• FIG. 14 shows VEGF expression increased bFGF content in the primary tumors from tamoxifen- treated mice.
• bFGF content were determined by ELISA assay with tumor lysates prepared from tamoxifen treated tumors.
• bFGF was significantly increased in tumors from mice received Dox in their drinking water. This may contributed by high level of stromal content in tumor with VEGF induction. This assay was done by using bFGF ELISA kit from R&D Systems, Minneapolis, MN as per manufacturer's instructions.
• VEGF levels in breast cancer are associated with resistance to anti-estrogen therapy, increased tumor cell proliferation and tumor metastasis.
• an inducible human VEGF gene expression system was developed.
• C9V cell lines Several ER-positive MCF-7-derived cell lines, designated as C9V cell lines, were developed using an improved tet-inducible gene expression system that expressed VEGF in a Dox- regulated manner (Qu et al., Gene, 2004, 327, 61-73). With these cell lines, a xenograft mouse model of breast cancer was developed to study the effects of VEGF expression and/or over-expression.
• VEGF includes VEGF-A and derivatives thereof.
• derivatives includes, but is not limited to, “fragments,” “degenerate variants,” “variants,” “mutants,” “analogs” and “chemical derivatives.”
• fragment is meant to refer to any polypeptide subset of VEGF amino acid sequence, incorporating 5 or more sequential or contiguous amino acids of VEGF (such as but not limited to VEGF 16 s) or any nucleic acid coding for said polypeptide.
• VEGF an analog of VEGF encompasses the various isoforms and splice variants of VEGF, including, but not limited to, VEGF ⁇ 21 , VEGF ⁇ 45 , VEGF ⁇ 65 , VEGF ⁇ 89 , and VEGF 206 .
• nucleic acid sequences which contain alternative codons which code for the eventual translation of the identical amino acid in VEGF or a fragment thereof For purposes of this specification, a nucleic acid sequence bearing one or more alternative codons will be defined as a "degenerate variation.”
• a variant is defined as a VEGF molecule containing a conservative amino acid changes, such as, but not limited to, substitution of valine for leucine or asparagine for glutamine may not cause a change in functionality of the polypeptide, as well as the nucleic acid coding for said variants.
• mutants either in the translated VEGF polypeptide ("mutants") and nucleic acid coding for said mutant polypeptides.
• the mutants may be isolated from cell lines of tissue or may be introduced via recombinant mechanisms.
• the mutants may or may not substantially alter the ultimate physical properties of the expressed VEGF polypeptide. Examples of altered properties of mutants include, but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
• chemical derivative refers to VEGF that contains additional chemical moieties or amino acid that are not normally a part of the base VEGF molecule, but do not substantially alter its structure.
• a molecule is "substantially similar" to VEGF in terms of structure if both molecules have at least 75% identity between their respective amino acid sequences.
• VEGF is VEGF 105 .
• the amino acid sequence of VEGF 1 65 is shown in SEQ ID NO: 1.
• the amino acid sequence of VEGF 206 is shown in SEQ ID NO: 2.
• MCF-7 cells are human breast cancer cells derived from the pleural effusion of a 69 year old female. MCF-7 cells are ER-positive, estrogen responsive, antiestrogen sensitive and exhibit minimal invasiveness when introduced subcutaneously into nude mice. Two nucleic acid vectors were used to introduce VEGF, in this embodiment VEGF 165 : 1) a bicistronic Tet-transactivator expression vector (pBTE); and 2) a Tet-responsive element vector (pTRE-hVEGF) controlling the expression of VEGF (FIG. 1). Vector pBTE is constructed as described in the method section.
• pBTE incorporates a number of modifications resulting in decreased VP16 toxicity, reduced basal transcription activity, and increased Dox sensitivity in human cells 24 .
• the tet-transactivator is expressed as part of a bicistronic message that includes a downstream internal ribosome entry site linked to a second set of coding sequences conferring resistance to blasticidin thereby allowing selection of cells that maintain the tet-transactivator expression.
• These vectors were integrated through stable transfection into chromosomal DNA in ER-positive, lac-Z tagged MCF-7 cells, resulting in MCF-7-derived cell lines (including but not limited to such cell lines designated C9V) which express or over-express VEGF in a Dox-regulated manner.
• the clones expressing VEGF were selected with the antibiotic blasticidin and by hVEGF ELISA with conditioned media from the cell culture treated with and without Dox as described. Western blotting of the conditioned media with an anti-VEGF antibody showed that the secreted VEGF was tightly controlled by Dox (FIG. 2).
• Human, recombinant VEGF 165 was used as a control, along with the parental MCF-7 cells (designated MCF-7) and a cell line constitutively expressing VEGF (designated MLV-165-16).
• Two representative clones, C9V4 and C9V18 showed increased VEGF expression when exposed to Dox. The high molecular band could reflect the VEGF post- translational modification.
• VEGF had no effect on cell proliferation or tamoxifen sensitivity in vitro. This is consistent with the observation that MCF-7 and C9V cells do not express VEGF receptors Rl and R2 proteins. C9V cells grew at a similar rate in the presence and absence of VEGF in culture medium and remained equally sensitive to 4-hydroxytamoxifen (FIG. 3). Therefore, VEGF does not appear have an autocrine effect on C9V cell proliferation. FGF and estrogen did stimulate C9V cell growth (FIG. 3).
• the instant specification describes in detail one method to prepare human breast cancer cell line modified to increase the expression VEGF to clinically relevant levels.
• the cell line is the MCF-7 cell line.
• other breast cancer cell lines may also be used, such as T47D or ZR-75-1. Modifications to the method provided as would be obvious to one of skill in the art are included in the scope of the disclosure.
• methods other than the one described may be used to produce cell lines modified to increase the expression of VEGF to clinically relevant levels, such as transient transfection. These alternate methods are also considered within the scope of the disclosure.
• VEGF Expression stimulates breast tumor growth in a manner that is additive to the mitogenic stimulus provided by estrogen VEGF expression stimulates breast cancer growth in a manner that is additive to the mitogenic stimulus provided by estrogen.
• C9V cells were inoculated into mammary fat pat of ovariectomized nude mice. The inoculated mice were divided into three groups: 1) group 1 received 0.05 mg/ml Dox in their drinking water over the entire time course of the experiment; 2) group 2 received 0.2 mg/ml Dox in their drinking water over the entire time course of the experiment; and 3) group 3 received an appropriate concentration of sucrose over the entire time course of the experiment (negative control).
• mice All three groups of mice were supplemented with estrogen pellets to allow for maximum C9V cell proliferation. Tumor volume was monitored twice a week (FIG. 4A). At the end of the 60-day time course, tumors were excised and their cell lysates were prepared for the determination of cytosolic VEGF content by ELISA assay (FIG. 4B). The mean tumor volumes and the amount of cytosolic VEGF in the xenografts from each group of mice correlated to Dox concentration supplied in the drinking water of the mice (FIGS. 4 A and 4B). Continued administration of Dox via the drinking water to estrogen-supplemented mice resulted in significantly increased tumor volumes and increase cytosolic tumor VEGF content.
• the percentage of Ki-67 positive cells is increased when Dox is administered in conjunction with estrogen, indicating the VEGF stimulated growth proceeds by a mechanism additive to estrogen growth stimulation (FIG. 5D).
• Administration of the anti-estrogen tamoxifen decreased the number of tumor cells staining positive for Ki-67 as compared to estrogen administration (compare FIG. 5A to FIG. 5C).
• the percentage of Ki-67 positive cells is increased when Dox is administered in conjunction with tamoxifen (FIG. 5D) again indicating that VEGF stimulated growth proceeds by a mechanism independent of estrogen growth stimulation.
• the level of VEGF induced by Dox is clinically relevant, ranging from 0.6 ng to 20 ng of VEGF per mg of total cytosolic proteins (FIG. 4B).
• a clinical study including 845 breast cancer patients showed the median level of VEGF expression was 0.22 ng/ g of total cytosolic protein.
• Patients who received tamoxifen for their advanced disease and with a VEGF level above the median were correlated with significantly shorter post-relapse survival time.
• the survival rate of patients with tumor VEGF level in a range of 1.73 ng/mg to 542 ng/mg was significantly lower than the rate of patients with the VEGF level in a range of 0 to 0.22 ng/mg and 0.22 ng/ l to 1.73 ng/mg.
• the tumor VEGF expression of the xenograft mouse in this study is within the range that is associated with clinically aggressive or acquired tamoxifen resistant breast cancer.
• the term "clinically relevant” means a concentration of VEGF expressed in a modified, human cell line (such as MCF-7 cells modified to express VEGF as described herein) that is within the range of VEGF concentrations statistically associated with advanced breast cancer tumors (such as those tumors that have acquired tamoxifen resistance and tumors that have acquired the ability to metastasize).
• the concentration of VEGF expressed in the modified human cell line is in the range of about 0.2 ng to 500 ng VEGF per mg of total cytosolic protein.
• the concentration of VEGF expressed in the modified human cell line is in the range of about 0.5 ng to 250 ng VEGF per mg of total cytosolic protein.
• the concentration of VEGF expressed in the modified human cell line is in the range of about 1.7 ng to 100 ng VEGF per mg of total cytosolic protein.
• the instant disclosure describes modifying the human cell line by integrating the nucleic acid constructs that encode the VEGF protein into the genome of the parental cell line.
• modifications to increase the expression of VEGF to clinically relevant levels should be considered within the scope of this disclosure.
• the use of other breast cancer cell lines should also be considered within the scope of this disclosure. Therefore, the xenograft mouse model described is an accurate in vivo model of the role of VEGF in breast cancer development and progression and may be used for further study of the VEGF effect on human breast cancer progression and to isolate compounds that inhibit such progression and development. This model may also be used for preclinical efficacy study of breast cancer therapeutics.
• tumor microvessel density was characterized by immunohistochemical staining with a mouse endothelial cell-specific marker PECAM-1 (FIGS. 6A and 6B). Increased microvessel density was observed in xenograft tissue when VEGF expression was induced by Dox (FIG. 6B) as compared to control xenograft tissue when Dox was not present (FIG. 6A) demonstrating the angiogenic effect of VEGF. This is consistent with the clinical studies in human primary breast cancer that elevated cytosolic VEGF levels are associated with increased microvessel density.
• C9V cells were inoculated into mammary fat pat of ovariectomized nude mice.
• the inoculated mice were divided into three groups: 1) group 1 received 0.2 mg/ml Dox in their drinking water during the entire 60 day period of the experiment; 2) group 2 received 0.2 mg/ml Dox in their drinking water for the first 11 days of the experiment; and 3) group 3 received an appropriate concentration of sucrose (negative control). All three groups of mice were supplemented with estrogen pellets to allow for maximum C9V cell proliferation (FIG. 7).
• VEGF Expression Induces Anti-estrogen Resistance Clinical studies have revealed that a high level of tumor VEGF is correlated with decreased effectiveness of anti-estrogen therapy, such as tamoxifen therapy, and relapse after the treatment.
• anti-estrogen therapy such as tamoxifen therapy
• tumor growth in anti-estrogen treated xenograft mice in which tumor VEGF expression is induced by Dox was examined. Tumors were initially grown by injecting C9V cells into the mammary fat pad in ovariectomized nude mice.
• mice were supplemented with estrogen as described. When the size of tumors reached 150-200 mm 3 after about 10 days, the estrogen pellets were replaced with tamoxifen pellets. .
• the mice were divided into two groups at this point, one group was provided with sucrose-containing drinking water (negative control) and the other group was provided with Dox in the drinking water.
• Dox-induced VEGF expression allowed for continued tumor growth C9V xenograft mice (FIGS. 8A and 8B). In contrast, the tumors became static and eventually regressed in the tamoxifen -treated mice that did not received Dox in their drinking water (FIGS. 8 A and 8B).
• VEGF vascular endothelial growth factor
• VEGF induced lung metastasis was observed in several xenograft mouse experiments with two cell lines C9V4 and C9V18.
• Experimental metastasis assay with tumor cell tail vein injection also demonstrated VEGF induced metastasis.
• the lung metastasis incidence in mice experiments is summarized in Table 1. 6 of nine 9 total estrogen-supplemented mice and 4 of 9 total tamoxifen-treated mice presented with lung metastases when the mice were received VEGF induction. In contrast, no metastasis was found in lung from any mouse that did not receive Dox. In addition, removal of Dox from the drinking water not only reduced tumor growth rate, but also significantly reduced the incidence of lung metastases.
• FIG. 9A is representative images of X-gal stained lung metastases in tumor-bearing mice.
• H&E histological staining (FIG. 9B) of the tumor samples confirmed lung macroscopic metastasis indicated by X-gal staining.
• Figures on the left side represent C9V xenograft mice exposed to Dox to induce VEGF expression and estrogen.
• Figures on the right side represent C9V xenograft mice exposed to Dox to induce VEGF expression and 4-hydroxytamoxifen.
• VEGF Expression Increases Colonization of Disseminated Tumor Cells and Increases Fibroblast Chemotactic Mobility
• the metastatic process consist of a series of steps in which tumor cells escape from a primary tumor into the blood circulation, disseminate to secondary sites, and arrest in and invade into a new organ. Sequential steps, colonization to form microscopic metastases in the target organ environment and proliferation to develop macroscopic metastatic lesions, are limiting steps of metastasis development.
• VEGF play a key role in metastasis development tumor cells were directly introduced into the circulation by tail vein injection and the patterns of disseminated tumor cells in mouse lungs were examined by immunostaining with antibody against cytokeratin, an epithelial cell marker.
• the desmoplastic response is a stromal reaction (predominately fibroblastic) to carcinoma, often observed with breast carcinoma.
• desmoplasia can be observed both at the primary tumor site and at sites of tumor metastasis.
• the stroma comprise the majority of the tumor mass, in some cases accounting for over 90% of the tumor mass (Elenbaas et al., Exp. Cell Research, 264, 169-184, 2001).
• desmoplasia is rarely observed in animal cancer models, including both transgenic and xenograft models.
• the desmoplastic response is characterized by the presence of a dense collagenous stroma, which can be responsible for the clinical presentation of a tumor as a 'lump 1 .
• Evidence suggests that the collagen and other material observed in the desmoplastic response are synthesized by myofibroblasts present in the interstitium.
• myofibroblasts activation and collagen synthesis have been proposed such as cytokine mechanisms and microvascular injury (with features analogous to wound healing), and paracrine activation of myofibroblasts by growth factors released by tumor cells.
• fibroblast stromal cells surround each colony of VEGF-expressing tumor cells suggests that VEGF may increase chemotactic mobility of fibroblastic stromal cells toward to tumor cells. Therefore, human foreskin fibroblast cells were used to assess whether VEGF affect fibroblast chemotactic mobility in vitro.
• the results from cell migration trans-well assay showed VEGF indeed dramatically increased chemotactic mobility of fibroblast (FIG. 12). This result suggests that tumor released VEGF attracts and interacts with fibroblast stromal cell to induce a remodeling of the tumor microenvironment; and this new environment is suitable for tumor cell survival and proliferation.
• the interaction of VEGF with other types of stromal cell may play equally important roles in the metastatic progression.
• VEGF Expression Induces A Desmoplastic Response and Increases FGF Content in Primary Tumors
• FIGS. 13A-13C show the representative images of cytokeratin staining of primary tumor samples from tamoxifen treated mice at 60 days after C9V cell inoculation without Dox treatment to induce VEGF expression, while FIG.
• FIG. 13B shows a representative image of primary tumor samples from tamoxifen treated mice at day 60 after C9V cell inoculation with Dox treatment to induce VEGF expression. Without VEGF induction, desmoplasia is observed in roughly 10% of area of tumor sections. However, with VEGF induction, desmoplasia is observed approximately 40% of rumor sections (FIG. 13C).
• FIG.14 shows the quantitation of bFGF content in tumor lysates as determined by an ELISA assay. Without Dox treatment, bFGF levels in tumor lysates were in the range of 750 pg/ g lysate protein. However, Dox-induced VEGF expression significantly increased bFGF levels in tumor lysates (bFGF levels in the range of 2500 pg/mg lysate protein).
• VEGF expression is capable of increasing the content of bFGF in C9V xenografts in nude mice.
• bFGF has been shown to be involved in cell growth, cell migration, differentiation, stromal responses and tissue organization as well as angiogenesis. Therefore, stromal cells interact with tumor cells may mediated by growth factors such as bFGF and others. It has been reported that constitutive expression of numbers of growth factors, growth factor receptors, or growth factor signaling intermediates can allow ER- positive breast cancer cell to acquire the ability to grow in the absence of estrogen or in the presence of anti-estrogen (Schiff et al, Clin.Cancer Res., 9: 447S-454S, 2003). These data suggest a mechanism by which tumor VEGF expression aids tumor cells in establishment a microenvironment which is suitable for their survival and growth in both the primary and metastatic sites.
• the MCF-7 cells expressing the constructs described, such as C9V4 and C9V18, can be stimulated to express clinically relevant levels of VEGF.
• the VEGF while not mitogenic for the MCF-7 cells themselves, provide paracrine stimulation of fibroblast, endothelial cells and other stromal cell components. These cells in turn provide the tumor cells with a variety of factors that aid the tumor cells in growth and survival. In addition, these factors aid in the alteration of the local microenvironment, such as the induction of desmoplasia and angiogenesis allowing for the growth of tumor cells in the primary tumor site and secondary sites of tumor metastasis. Furthermore, the stimulated stromal cell components may provide chemotactic factors that aid in tumor invasiveness and the metastatic process.
• the model system described provides for a clinically relevant model system to study the effects of VEGF on cancer development, particularly breast cancer development.
• the model system described provides a means to study VEGF involvement in a variety of pathways, including breast cancer growth, mechanisms of resistance of breast cancer cells to anti-estrogenic therapy, metastasis and desmoplasia.
• the xenograft model described is unique in that a clinically relevant range of VEGF is expressed and VEGF expression can be tightly controlled by the addition of the antibiotic Dox.
• the xenograft model has characteristics normally associated with breast cancer in humans, such as the desmoplastic response. Therefore, the xenograft model described herein provides advantages over models currently available in the art.
• Lipofectamine PlusTM, Zeocin, the pZeo-SV plasmid, and blasticidin were obtained from Invitrogen (Carlsbad, CA).
• the plasmids pTet-on, pTRE were obtained from Clontech (Palo Alto, CA).
• Improved Modified Eagle's Medium (IMEM) and fetal bovine serum were obtained from Life Technologies (Gaithersburg, MD).
• vector pBTE (FIG. 1): The modified tet-transactivator rtTA2S-M2 contained in plasmid pUHrt 62-1 was obtained from Dr. Wolfgang Hillen 18 . RtTA2S-M2 was subcloned into the vector pEF6-IVS-IRES-bsd generated as described below, resulting in an expression plasmid that produces a bicistronic message coding for both rtTA2S-M2 and the blasticidin resistance gene (BsdR). The pEF6-IVS-IRES-bsd vector was constructed by using pEF6 Myc/His A (Invitrogen,
• VEGF- 165 fonn The cDNA of human VEGF- 165 fonn, including N- terminal secretory signal peptide sequences, was obtained by RT-PCR with mRNAs of human umbilical vein endothelial cells (HUVEC).
• the hVEGFies was subcloned into pTRE vector (Clontech, Palo Alto, CA) at EcoRI site to generate the final vector pTRE-hVEGF. Orientation and VEGF insert were confirmed by digesting with appropriate enzymes and sequencing.
• LacZ-tagged MCF-7 cells were stably transfected with a modified tet-transactivator in bicistronic expression vector pBTE and were selected with 3 ug/ml blasticidin in the medium, designated as C9 cells.
• Tet-transactivator expressing clone C9 was re-transfected with pTRE-h EGF and a plasmid conferring zeocin resistance pZeo-SV.
• Zeocin resistant cell clones at lOOug/ml level were isolated and expanded. The conditioned media from each culture in IMEM containing 1% FBS with and without 0.5ug/ml Dox for 48 hours were collected.
• the VEGF concentration in conditioned medium was determinate using a human VEGF ELISA kit (R&D Systems, Minneapolis, MN as per manufacturer's instructions). The cell number from each culture was counted for the normalization of the ELISA VEGF values. The Dox-regulated expression of secreted VEGF in selected cell lines was further examined by western blotting of the conditioned media with a polyclonal anti-hVEGF antibody (Santa Cruz, California).
• Cell Proliferation Assay Cells used in this assay were stripped off estrogens over for one day by several medium exchanges with 5% charcoal-stripped calf serum (CCS) in phenol red free (PRF)-IMEM. Then, cells were seeded on 24-well plates with the same medium 20 thousand cells per well. The next day, three wells were counted as day 0. Different conditions as indicated were added to the cell cultures in triplets. The cell number of each well was counted at day 3.
• CCS charcoal-stripped calf serum
• PRF phenol red free
• VEGF and FGFa proteins were from R&D Systerm, VEGF: 20ng/ml; FGF-1: 20ng/ml; Tarn: 4-hydroxytamoxifen, 10 "7 M, and E2 : 17-beta estradiol, 10 "8 M.
• mice 7 - 8 weeks-old ovariectomized athymic nude mice were given three days before cell inoculation, drinking water containing either sucrose (2.5%) only, or sucrose and Dox at various concentrations.
• sucrose 2.5%) only, or sucrose and Dox at various concentrations.
• a 60-day-release 0.72 mg-17b-estradiol pellet (Innovative Research of America) was implanted in mouse one day before cell inoculation. The following day, 10 7 cells, suspended in 200 ml of 50% phenol-red free Matrigel (BD Biosciences), were inoculated into the mammary fat pat of each mouse subcutaneously. Both tumor size and animal weight were measured twice a week.
• the tumor was excised and cut into two halves, one half was stored in liquid nitrogen and the other was fixed in 10% phosphate-buffered formalin. Samples from different mouse organs were also collected and fixed for histology examination.
• the implanted estrogen pellet was replaced with a 60-day release 5 mg-tamoxifen pellet (Innovative Research of America) once tumors had reached a volume of 150 - 250 mm 3 .
• mice were randomly divided into two groups; with one group given drinking water containing 0.2 mg/ml Dox and 2.5% sucrose, and the other given drinking water containing sucrose only.
• 1X10 6 V9V cells in 100 ⁇ l PBS were injected into mouse tail vein and the lung tissues were collected at different time point.
• Lung Metastasis and Immunohistochemistry Lung samples were cut into several strips and paraffin embedded. 5-um sections of lung, lymph node, and tumor samples were H&E stained. Metastasis lesions were examined under LEICA DMLB microscope. The size of metastatic lesion in mouse lung bigger than 10 cells across over the diameter was considered as macrometastasis, smaller then that considered as micrometastasis. About 1 cm2 of lung section was examined from each mouse for metastatic lesion.
• the antibodies used in immunohistochemistry staining were polyclonal antibody against the epithelial cell marker cytokeratin (Keratin Pan Ab-1, from Neomarkers), rat monoclonal antibody against mouse PECAM-1 (Mec 13.3, BD, Bioscience PharMingen), polyclonal antibody against the cell proliferation marker Ki-67 (Santa Cruz, California).
• cytosolic preparation buffer was added to every 100-mg tissue sample. The tissue was homogenized with Polytron on ice at 50% power output. Cells in the processed samples were lysed by frizzing and thawing on dry ice 3 times and further disintegrated by sonication 3 times 2 seconds each at high power output. Samples were spun at 12000 m for 30 min under 4°C. The cytosol sample was collected from supernatant used for VEGF content determination by the VEGF ELISA. The protein concentration was determined to calculate the specific VEGF content.
• the cytosolic preparation buffer contained 10 mM Tris pH 7.4; 1.5 mM EDTA; 10 mM sodium molybdate (Sigma, St. Louise, MO); 1.0 mM 1-thioglycerol (Sigma, St. Louise, MO).
• the cell migration assay was conducted by using 24-well chemotaxis chamber with matrigel- coated 8 ⁇ m pore size insert from BD Bioscience following the manufacturer's instructions.
• the bottom chambers were loaded with DMEM cell culture medium or with 10% FBS and lOOng/ml recombinant VEGF protein (from R&D System) added.
• the upper chambers were seeded with 2X10 5 foreskin fibroblast cells. After incubation at 37°C for 4 hours, the filters were fixed, stained with 0.5% crystal violet, and the cells that had migrated through the filter were counted.

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