Abstract P1-06-10: Characterization of novel activated human mammary fibroblast lines and their protumorigenic effect on human breast cancer xenotransplants in mice

Abstract

Abstract Human breast cancer is typically characterized by an extensive stromal compartment enriched in fibroblasts. In contrast, xenotransplants of human breast cancer cell lines are typically epithelial-rich and characterized by sparse in-growth of murine fibroblast stroma. Stromal fibroblasts are important for structural integrity of normal tissue and constitute a major element of the stromal microenvironment of invasive cancer. Cancer associated fibroblasts (CAFs) frequently undergo activation as they co-evolve with cancer cells, serving to promote tumor growth and angiogenesis through secretion of multiple paracrine factors. Presence of activated CAFs in solid malignancies is generally associated with higher grade tumors and poor prognosis. Activated CAFs are resistant to apoptosis and display a myofibroblastic phenotype, including expression of a-smooth muscle actin (α-SMA). To more accurately model human tumor-stroma interactions in human breast cancer xenograft lines in mice, we aimed to develop immortalized activated human mammary fibroblasts for admixture xenografting with human breast cancer cells. Human mammary fibroblasts (HMFs) were isolated from multiple surgically excised tissues of reduction mammoplasties or mastectomies. Five isolated primary HMF lines were screened for α-SMA expression. HMF1 expressed the highest levels of α-SMA and was immortalized by stable lentiviral-delivered hTERT. Quantitative real-time PCR, anchorage-independent growth assay, and in vivo studies were used to further characterize selected HMFs. hTERT-HMF1 and HMF2, despite lower level of α-SMA expression in HMF2, displayed significantly elevated levels of mRNA of proteins commonly associated with activated fibroblasts, including SDF-α, SDF-β, HGF, IL-6, VEGF and podoplanin, as well as promoted a 10-fold increase in anchorage-independent growth of MCF7 breast cancer cells in vitro. To evaluate the ability of these HMF lines to promote tumor growth in vivo, MCF7 human breast cancer cells were orthotopically injected into murine mammary fat pads in the presence or absence of hTERT-HMF1 or HMF2. Both HMF lines facilitated in vivo MCF7-xenograft growth and induced histological changes, including higher grade and greater stromal development, when compared to pure MCF7 cell xenografts. IHC for SMA and collagen confirmed the presence of activated fibroblasts in all tumors; however HMF/MCF7 xenografts showed a distinct distribution of fibroblast stroma throughout the tumor in contrast to sparser stroma between larger epithelial aggregates in tumors grafted from MCF7 cells alone. hTERT-HFM1 cells promoted tumor growth in vivo more robustly than HFM2 cells. By 60 days post xenografting, hTERT-HMF1/MCF7 tumors were significantly larger (∼4-fold) than control MCF7 tumors. In addition, admixture with hTERT-HMF1 cells promoted increased tumor angiogenesis and cancer cell proliferation as measured by Ki67 expression. hTERT-HMF1 represents a novel mammary myofibroblast line that may be useful for improved preclinical xenotransplant modeling and tumor drug response testing of human breast cancer in vivo. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-10.