Plastic Surgery Research Council

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Characterizing the Clonal Nature of Cancer Associated Fibroblasts
Deshka S. Foster, M.D., M.A., Ryan C. Ransom, B.S., Ruth E. Jones, M.D., Ankit Salhotra, B.S., Michael S. Hu, M.D., M.P.H., M.S., Michael T. Longaker, M.D., M.B.A..
Stanford University, Stanford, CA, USA.

PURPOSE: Solid tumors are composed of cancer cells along with a heterogeneous group of participatory cells. Cancer associated fibroblasts (CAFs) are an integral component of solid tumors, providing a nutritive, metabolically-active microenvironment for tumor cells to proliferate and a scaffold for fibrous tumors such as melanoma, breast and pancreas cancers to invade and ultimately metastasize. The recruitment patterns and clonal nature of CAFs, however, have yet to be fully elucidated. The aims of this project are to characterize tumor stroma in solid cancers in terms of the clonal proliferation and epigenetic phenotype of CAFs both in primary cancers and metastatic disease.
METHODS: Rainbow mice (ActinCreER; R26VT2/GK3) systemically induced with tamoxifen were used for allograft tumor models. The Rainbow system is a Cre-dependent model with a four-color (mCerulean, eGFP, mOrange, and mCherry) reporter construct in the ROSA (R26) locus. When Cre-recombination occurs, cells are genetically marked with one of the possible colors. All progeny cells are marked with the same color as the parent cell creating a fluorescent mosaic pattern upon analysis. At the time of tumor implantation, local induction with activated tamoxifen was also pursued. Tumors and surrounding tissue were harvested, whole-mounted and analyzed using confocal microscopy. In this model, tumor cells are dark but stoma cells are fluorescent allowing detailed characterization of the stroma cell niche. All experiments were conducted in the Longaker Laboratory at Stanford University School of Medicine.
RESULTS: CAFs develop as a small sub-set of local fibroblasts (rather than a systemic infiltration of a heterogeneous cell population) that are recruited by primary tumor cells and undergo extensive clonal expansion. When compared with wound healing studied in the same mouse model, tumor stroma shows increased clonality and more disorder in the proliferation of recruited fibroblasts (Figure 1). Thus far, tumor stroma has been investigated in fibrous cancer types including breast and melanoma. Results from experiments using endogenous tumor models (bred with rainbow mice) as well as metastatic disease models, along with epigenetic characterization of this cell niche, are forthcoming.
CONCLUSION: CAFs are recruited locally by cancer cells and proliferate in a highly clonal manner in the tumor microenvironment, which can be visualized in whole tissue using the rainbow mouse system.
Figure 1. Tumor stroma rainbow mouse model showing highly clonal proliferation of cancer associated fibroblasts. In this model, stroma cells appear fluorescent whereas cancer cells are dark. T indicates Tumor, the tumor edge is outlined with a dotted line, eG = eGFP, mCe = mCerulean, mOr = mOrange, mCh = mCherry.


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