Accelerated Preclinical Testing Using Transplanted Tumors from Genetically Engineered Mouse Breast Cancer Models

Center for Cancer Research, National Cancer Institute, Frederick, Maryland.
Clinical Cancer Research (Impact Factor: 8.72). 04/2007; 13(7):2168-77. DOI: 10.1158/1078-0432.CCR-06-0918
Source: PubMed


The use of genetically engineered mouse (GEM) models for preclinical testing of anticancer therapies is hampered by variable tumor latency, incomplete penetrance, and complicated breeding schemes. Here, we describe and validate a transplantation strategy that circumvents some of these difficulties.
Tumor fragments from tumor-bearing MMTV-PyMT or cell suspensions from MMTV-PyMT, -Her2/neu, -wnt1, -wnt1/p53(+/-), BRCA1/p53(+/-), and C3(1)T-Ag mice were transplanted into the mammary fat pad or s.c. into naïve syngeneic or immunosuppressed mice. Tumor development was monitored and tissues were processed for histopathology and gene expression profiling. Metastasis was scored 60 days after the removal of transplanted tumors.
PyMT tumor fragments and cell suspensions from anterior glands grew faster than posterior tumors in serial passages regardless of the site of implantation. Microarray analysis revealed genetic differences between these tumors. The transplantation was reproducible using anterior tumors from multiple GEM, and tumor growth rate correlated with the number of transplanted cells. Similar morphologic appearances were observed in original and transplanted tumors. Metastasis developed in >90% of mice transplanted with PyMT, 40% with BRCA1/p53(+/-) and wnt1/p53(+/-), and 15% with Her2/neu tumors. Expansion of PyMT and wnt1 tumors by serial transplantation for two passages did not lead to significant changes in gene expression. PyMT-transplanted tumors and anterior tumors of transgenic mice showed similar sensitivities to cyclophosphamide and paclitaxel.
Transplantation of GEM tumors can provide a large cohort of mice bearing mammary tumors at the same stage of tumor development and with defined frequency of metastasis in a well-characterized molecular and genetic background.

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    • "We have now created an improved model system in which Hif1a is efficiently deleted in the mammary tumor epithelium via ex vivo viral transduction with Cre recombinase prior to the injection of mammary tumor cells to the cleared fat pads of recipient mice. Validating the use of a transplantation paradigm in lieu of intact transgenic mice, previous studies have shown that when isolated PyMT tumor cells are serially regenerated as tumors in recipient mice the morphology and gene expression profiles are similar to the tumors found in the transgenic mouse [28]. "
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    ABSTRACT: Overexpression of the oxygen-responsive transcription factor hypoxia-inducible factor 1α (HIF-1α) correlates with poor prognosis in breast cancer patients. The mouse mammary tumor virus polyoma virus middle T (MMTV-PyMT) mouse is a widely utilized preclinical mouse model that resembles human luminal breast cancer and is highly metastatic. Prior studies in which the PyMT model was used demonstrated that HIF-1α is essential to promoting carcinoma onset and lung metastasis, although no differences in primary tumor end point size were observed. Using a refined model system, we investigated whether HIF-1α is directly implicated in the regulation of tumor-initiating cells (TICs) in breast cancer. Mammary tumor epithelial cells were created from MMTV-PyMT mice harboring conditional alleles of Hif1a, followed by transduction ex vivo with either adenovirus β-galactosidase or adenovirus Cre to generate wild-type (WT) and HIF-1α-null (KO) cells, respectively. The impact of HIF-1α deletion on tumor-initiating potential was investigated using tumorsphere assays, limiting dilution transplantation and gene expression analysis. Efficient deletion of HIF-1α reduced primary tumor growth and suppressed lung metastases, prolonging survival. Loss of HIF-1α led to reduced expression of markers of the basal lineage (K5/K14) in cells and tumors and of multiple genes involved in the epithelial-to-mesenchymal transition. HIF-1α also enhanced tumorsphere formation at normoxia and hypoxia. Decreased expression of several genes in the Notch pathway as well as Vegf and Prominin-1 (CD133)was observed in response to Hif1a deletion. Immunohistochemistry confirmed that CD133 expression was reduced in KO cells and in tumorspheres. Tumorsphere formation was enhanced in CD133hi versus CD133neg cells sorted from PyMT tumors. Limiting dilution transplantation of WT and KO tumor cells into immunocompetent recipients revealed > 30-fold enrichment of TICs in WT cells. These results demonstrate that HIF-1α plays a key role in promoting primary mammary tumor growth and metastasis, in part through regulation of TICs. HIF-1α regulates expression of several members of the Notch pathway, CD133 and markers of the basal lineage in mammary tumors. Our results suggest that CD133, which has not been profiled extensively in breast cancer, may be a useful marker of TICs in the PyMT mouse model. These data reveal for the first time that HIF-1α directly regulates breast TIC activity in vivo.
    Breast cancer research: BCR 01/2012; 14(1):R6. DOI:10.1186/bcr3087 · 5.49 Impact Factor
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    • "First, if GEM tumors are harvested and fragments successfully implanted subcutaneously (SC) or orthotopically into syngeneic, immune competent mice, enough animals can be amassed to perform traditional xenograft-like screens with genetically desirable murine tumors (allografts). Varticovski et al., (2007) provide an example of this approach, harvesting MMTV-PyMT breast tumors from a few mice and passing them as fragments or cell suspensions into numerous host animals for subsequent drug studies. DNA microarrays were used to verify only slight changes in gene expression after two serial passages from the original tumor. "

    Drug Discovery and Development - Present and Future, 12/2011; , ISBN: 978-953-307-615-7
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    • "Preclinical studies focusing on tumor response rather than prevention can expedite experiment duration by using mammary transplants. This adapted method of preclinical testing distributes dissociated primary tumor cells into large numbers of immunocompromised or syngeneic hosts at the same time, resulting in a tumor-staged and highly reproducible experimental model (Martinez et al. 2006; Varticovski et al. 2007). Studies in our lab have used this methodology to screen drug candidates in C3(1)Tag mammary transplants prior to testing in the GEM model. "
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    ABSTRACT: Cross-species genomic analyses have proven useful for identifying common genomic alterations that occur in human cancers and mouse models designed to recapitulate human tumor development. High-throughput molecular analyses provide a valuable tool for identifying particular animal models that may represent aspects of specific subtypes of human cancers. Corresponding alterations in gene copy number and expression in tumors from mouse and human suggest that these conserved changes may be mechanistically essential for cancer development and progression, and therefore, they may be critical targets for therapeutic intervention. Using a cross-species analysis approach, mouse models in which the functions of p53, Rb, and BRCA1 have been disrupted demonstrate molecular features of human, triple-negative (ER-, PR-, and ERBB2-), basal-type breast cancer. Using mouse tumor models based on the targeted abrogation of p53 and Rb function, we identified a large, integrated genetic network that correlates to poor outcome in several human epithelial cancers. This gene signature is highly enriched for genes involved in DNA replication and repair, chromosome maintenance, cell cycle regulation, and apoptosis. Current studies are determining whether inactivation of specific members within this signature, using drugs or siRNA, will identify potentially important new targets to inhibit triple-negative, basal-type breast cancer for which no targeted therapies currently exist.
    Toxicologic Pathology 02/2010; 38(1):88-95. DOI:10.1177/0192623309357074 · 2.14 Impact Factor
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