The secretome of hypoxic mammary cancer-associated fibroblasts unveils pro-angiogenic factors

Hypoxia correlates with abnormal angiogenesis, increased risk of metastasis and poor prognosis of breast cancer patients. The structural and functional abnormalities of the tumour vasculature impair the delivery and efficacy of existing cancer therapies, aid the metastatic dissemination and fuel an aggressive tumour milieu. Cancer-associated fibroblasts (CAFs) are an abundant stromal component in breast tumours; yet, their role in angiogenesis under hypoxic conditions is poorly characterised. Here, using a 3D co-culture system, we have shown that endothelial cells (ECs) co-cultured with CAFs under hypoxia or exposed to the conditioned medium (CM) of hypoxic CAFs sprouted significantly more than the normoxic counterpart. To identify the secreted factors responsible for this phenotype, we have used SILAC-based MS secretomics of mammary CAF cell lines. More than 1,000 proteins were quantified in the soluble and extracellular vesicles fractions of the CAF secretome. We found that targets of HIF1α and TGFβ were highly induced by the low oxygen environment. Moreover, a variety of secreted proteins known to influence angiogenesis were regulated by hypoxia. Strikingly, we have identified two novel hypoxia-induced secreted proteins, a leucine-rich repeat containing (LRRC) protein and an antisense protein (AS). The up-regulation of LRRC by hypoxia occurred at transcriptional level in a HIF1α-dependent manner only in fibroblasts but not in cancer or endothelial cells. In agreement with this finding, in vivo LRRC is expressed specifically in the stroma of murine and human breast tumours. The increased levels of the protein AS2 induced by hypoxia was not a result of transcriptional regulation, and in vivo AS2 was found in both cancer and stromal cells. Strikingly, ECs treated with the CM of hypoxic CAFs silenced for LRRC or AS2 significantly decreased their sprouting and migration ability. We then measured in an unbiased fashion the global changes induced in the secretome of hypoxic CAFs upon silencing of LRRC and AS2. Notably, the knockdown of LRRC in hypoxic CAFs decreased the secretion of IL-6, CTSH and SOD3, which have been reported to induce angiogenesis. The silencing of AS2 in hypoxic CAFs decreased the levels of secreted VEGFA, the master pro-angiogenic growth factor. We have identified novel stroma-derived secreted proteins that enhance angiogenesis in hypoxic conditions. Our findings can provide new avenues to normalise the dysfunctional vasculature found in advanced breast tumours