Adipose stromal cell-derived cancer-associated fibroblasts promote pancreatic adenocarcinoma through SFRP4 signaling

Progression of pancreatic ductal adenocarcinoma (PDAC) and other carcinomas relies on cancer-associated fibroblasts (CAFs). A subset of CAFs is derived from adipose stromal cells (ASCs) recruited by tumors and the ASC-CAF conversion has been associated with invasiveness and poor prognosis. To explore the underlying molecular mechanisms, we used a model based on primary ASC derived from human visceral adipose tissue co-cultured with human PDAC cell line Capan-1. To investigate cancer progression in vivo, we also used mice orthotopically grafted with mouse KPC cells. Genomic analysis revealed that Capan-1 co-culture induces Wnt and TGFß signaling and extracellular matrix (ECM) gene expression in ASC. We investigated the function of two markers of the fibroblastic transition highly induced by cancer cells: a long non-coding RNA LINC01614 and a Wnt signaling modulator SFRP4. By using ASC with either SFRP4 or LINC01614 knocked out (ko), we showed that both genes are required for Wnt / TGFß signaling and ECM induction in ASCs by Capan1. Analysis of changes in Capan-1 genes that rely on LINC01614 and SFRP4 expression in ASC also identified the Wnt and TGF pathways. SFRP4 ko in ASCs suppressed both migration and invasion of Capan-1 cells. We show that tumors in SFRP4 ko mice have less desmoplasia, less epithelial dedifferentiation, reduced growth rate, and reduced progression to metastasis. We conclude that SFRP4 promotes cancer progression in pancreatic cancer and is a promising therapeutic target. Overall design: Visceral adipose tissue biopsies from a single patient were digested and the stromal vascular fraction was isolated and given the nomenclature vASC. These cells were then engineered to express red fluorescent protein (RFP) via transduction of a lentiviral contruct that also contained a puromycin resistance gene to allow for targeted selection of RFP+ cells. Capan-1 cells were engineered to express green fluorescent protein (GFP) via transduction of a lentiviral contruct that also contained a puromycin resistance gene to select for GFP+ cells. Seperate cohorts of the vASC_RFP cells were further engineered for knockdown of the LINC01614 and SFRP4 genes using CRISPR/Cas9 targeted mutagenesis via lentiviral transduction. The CRISPR/Cas9 construct targeting these genes also contained a blasticidin resistance gene to allow for selection of positively transduced cells. vASC_RFP, vASC_LINC01614_RFP, and vASC_SFRP4_RFP cells were cultured alone or in coculture with Capan-1_GFP cells in replicates for seven days. Cells were then trypsinized, made into single cell suspensions, and sorted (RFP vs GFP) using FACS. Total RNA-sequencing was performed on sorted cells to analyze changes in gene expressions.