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

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: Pancreatic stellate cells (PSCs) were cultured in matrigel growth factor reduced (GFR) with growth medium for 4 days. The media was then replaced to KPC organoid-shBRCA2 or KPC organoid -shControl conditioned medium (CM), or with PSC own conditioned medium as control for 4 days. The cells were harvested and RNA-sequencing was performed.

癌症相关成纤维细胞（Cancer-associated fibroblasts, CAFs）可形成促结缔组织增生性间质，该间质能够支持肿瘤进展与转移，在胰腺癌中其占肿瘤组织总质量的比例最高可达90%。我们及其他研究团队近期的工作表明，邻近的肿瘤细胞可使CAFs发生转录重编程，进而形成具有异质性的亚型。目前，肿瘤细胞的不同驱动突变是否会对这种重编程产生差异性影响，在很大程度上仍不明确。本研究通过剖析并对比BRCA突变型与BRCA野生型（WT）胰腺导管腺癌（pancreatic ductal adenocarcinoma, PDAC）的间质景观，解答了这一科学问题。我们对包含42名患者的队列的PDAC样本进行了全面分析，采用激光捕获显微切割、RNA测序（RNA-sequencing）与多重免疫荧光技术，发现生殖系BRCA突变型肿瘤与BRCA野生型肿瘤的CAFs亚型组成存在差异。具体而言，我们在BRCA突变型肿瘤中检测到簇集素（Clusterin, CLU）阳性CAFs亚群的占比升高。通过使用癌症类器官、共培养体系与体内模型，我们证实肿瘤细胞中BRCA功能缺失会促使胰腺星状细胞（pancreatic stellate cells, PSCs）从肌成纤维细胞表型向免疫调节型CLU阳性CAFs发生转录方向的转变。这一过程由Clu的转录调控因子热休克因子1（heat-shock factor 1, HSF1）的激活所介导。本研究揭示了受肿瘤细胞生殖系突变影响的间质异质性新维度，其研究结果对临床转化研究具有直接的指导意义。

实验整体设计如下：将胰腺星状细胞（PSCs）在生长因子减少型基质胶（growth factor reduced, GFR）中配合生长培养基培养4天，随后更换为KPC类器官-shBRCA2或KPC类器官-sh对照的条件培养基（conditioned medium, CM），同时以PSC自身的条件培养基作为对照，继续培养4天后收集细胞并进行RNA测序。