BRCA mutational status shapes the stromal microenvironment of pancreatic cancer linking CLU+ CAF expression with HSF1 signaling (CAF)

Cancer-associated fibroblasts (CAFs) give rise to desmoplastic stroma, which supports tumor progression and metastasis, and comprises up to 90% of the tumor mass in pancreatic cancer. Recent work by us and others has shown that CAFs are transcriptionally rewired by adjacent cancer cells to form heterogeneous subtypes. Whether this rewiring is differentially affected by different driver mutations in cancer cells is largely unknown. Here we address this question by dissecting and comparing the stromal landscape of BRCA-mutated and BRCA Wild-type (WT) pancreatic ductal adenocarcinoma (PDAC). We comprehensively analyze PDAC samples from a cohort of 42 patients by laser-capture microdissection, RNA-sequencing and multiplexed immunofluorescence, revealing different CAF subtype compositions in germline BRCA-mutated vs. BRCA-WT tumors. In particular, we detect an increase in a subset of Clusterin (CLU)-positive CAFs in BRCA-mutated tumors. Using cancer organoids, co-cultures and in-vivo models we show that loss of BRCA function in cancer cells leads to a transcriptional shift of pancreatic stellate cells from myofibroblastic into immune-regulatory CLU+ CAFs. This process is mediated through activation of heat-shock factor 1 (HSF1), the transcriptional regulator of Clu. Our findings unravel a new dimension of stromal heterogeneity, influenced by germline mutations in cancer cells, with direct translational implications for clinical research. Overall design: CAFs were collected for bulk RNA-seq as follows: 2-3 weeks following KPC shControl or KPC shBrca2 orthotropic injection into the pancreas the mice were sacrificed, PDAC tumors were excised, digested into single cells and suspended in MACS buffer. The cell suspension was depleted of CD45+ and EpCAM+ cells using manganic beads. The CAF-enriched flow through was then stained for FACS sorting with the following antibodies: CD45-FITC, CD31-FITC, EpCAM-FITC, PDPN-APC, and Ghost Dye-violet 450 for detection of live cells. To collect CAFs, the following gating strategy was applied: CD45-/CD31-/EpCAM-/PDPN+