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Characterizing The Clonality Of Breast Cancer Associated Fibroblasts In Primary And Metastatic Disease
Malini Chinta, BA, Deshka Foster, MD, Alan Nguyen, Ankit Salhotra, R. Chase Ransom, R. Ellen Jones, MD, Ashley L. Titan, MD, Shamik Mascharak, Derrick C. Wan, MD, Gerlinde Wernig, MD, Jeffrey A. Norton, MD, Michael T. Longaker, MD, MBA.
Stanford University, Palo Alto, CA, USA.

PURPOSE: Fibroblasts are a predominant stromal cell type in breast cancer, and contribute to tumor growth by depositing desmoplastic stroma that supports proliferation and angiogenesis, and regulates the immune cell activities. Although many functions of cancer-associated fibroblasts (CAFs) have been demonstrated, therapeutically targeting these cells remains difficult due to remaining questions concerning their activation and heterogeneity. The aim of this project is to identify and characterize progenitor-type breast CAF phenotypes and understand how these cells proliferate in the tumor stroma.
METHODS: To characterize CAF progenitor activation and proliferation, we used the Rainbow mouse model. The Rainbow construct is a Cre-dependent model that contains a four-color reporter construct located in the Rosa-26 locus. When Cre-recombination is induced, cells are genetically marked with one of the available colors. All progeny cells are labeled with the same color as it's parent cell permitting precise lineage tracing. We bred Rainbow mice with an aSMA-CreER driver and then crossed these mice with an endogenous mouse mammary tumor virus (MMTV) breast cancer model. The mice received tamoxifen induction just prior to the time of tumor development to initiate Rainbow expression in aSMA+ cells (activated fibroblasts). Primary tumors as well as spontaneous lung metastases were harvested and analyzed using confocal microscopy. We performed bulk RNA-sequencing on florescence-activated cell sorting (FACS) isolated CAFs along with control fibroblasts from healthy littermates to characterize CAF gene expression. RESULTS: Using the Rainbow mouse model (Rosa26VT2/GK3), we identified clonal proliferation of breast CAFs in the tumor stroma suggesting the presence of progenitor-type fibroblasts that are activated in the context of neoplasia. We saw similar clonal proliferation of activated CAFs in spontaneous lung metastases. (Figure 1A-B). On bulk RNA-seq, significant differential gene expression was observed between tumor and control CAFs. CAFs demonstrated upregulation of multiple genes related to fibrosis including aSMA and osteopontin (SPP1) (Figure 1C). CAF clones appear to co-express aSMA and SPP1. CONCLUSION: Activated CAFs proliferate clonally in both primary and metastatic mouse breast cancer, suggesting that these CAFs may derive from tissue-resident, progenitor-type fibroblasts which are activated with response to neoplasia. Breast cancer CAFs upregulate aSMA and SPP1 at the transcript level, suggesting a role in the recruitment and activation of these fibroblasts. Future directions for this project include further gene expression and protein analyses to characterize mouse breast CAF activation and heterogeneity, as well as exploration of our results in human disease.


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