NetrinG1⁺ cancer-associated fibroblasts generate unique extracellular vesicles that support the survival of pancreatic cancer cells under nutritional stress

Abstract

It is projected that, in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). This fibrous stroma, known as desmoplasia, causes the collapse of local blood vessels rendering a nutrient-deprived milieu. Hence, PDAC cells are nurtured by local CAF-secreted products, which include, among others, CAF-generated small extracellular vesicles (sEVs). It is well-accepted that upon culturing functionally tumor-promoting CAFs under pathophysiological-relevant conditions (e.g., within self-produced ECM), these cells express NetrinG1 (NetG1) and sustain endosomal pools rich in active α5β1-integrin; traits indicative of poor patient survival. We herein report that NetG1+ CAFs generate sEVs that rescue PDAC cells from nutrient-deprived induced apoptosis. Two unique sEVs, NetG1+ and α5β1-integrin+, were uncovered. The former constitutes cargo of CAF-generated exomeres, and the latter is detected in classic exosomes. Proteomic and metabolomic analyses showed that the sEV-dependent PDAC survival is, at least in part, dictated by the cargo packaged within sEVs in a NetG1-dependent manner. Indeed, despite producing a similar number of vesicles, selected key proteins and metabolites (e.g., glutamine) were incorporated within the unique sEVs. Finally, we found that NetG1 and α5β1-integrin were detected in sEVs collected from plasma of PDAC patients, while their concomitant levels were significantly lower in plasma of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF sEVs opens a new investigative avenue in tumor-stroma interactions and stroma staging detection.

Eribulin normalizes pancreatic cancer-associated fibroblasts by simulating TGFβ inhibition

“…The CDM is a pathophysiologic-relevant system that has been successfully used in numerous human cohorts [15,23,47]. The desmoplastic/CAF CDM system serves for studying drug effects on cancer regulated by the TME [30,31,48], as well as for measuring the direct influences of drugs on modulating fibroblastic cell function [22,23,29,33]. Since quiescent/normal fibroblasts are naturally tumor-suppressive [10] and because CAF elimination has been demonstrated to be advantageous to PDAC progression and deleterious to patients [7][8][9], the field has placed significant efforts on identifying potential stroma-normalizing drugs [6,12,13].…”

“…To this end, β5 KO CAFs constitute an ideal human cellular model as these cells present with a phenotype and function akin to perpetuating TGFβ inhibition [15]. In fact, β5 KO CAFs have been reported to generate CDMs with tumor-suppressive functions and traits [15, 22, 33]. Using β5 KO CAFs, we previously demonstrated that canonical TGFβ signaling is necessary for the production of tumor-supportive ECMs.…”

“…CDMs with tumor-suppressive functions and traits [15,22,33]. Using β5 KO CAFs, we previously demonstrated that canonical TGFβ signaling is necessary for the production of tumor-supportive…”

“…TGFβ-deficient CAFs, whereby TGFβ signaling was shown to be uniquely necessary to produce a tumor-supportive ECM [15,22,33,40].…”

“…Further, since pancreas resident fibroblastic cells, initially presenting with normal-like tumor-suppressive features, undergo an ex-vivo culturing stress-induced activation [32], our team previously used the CRISPR/Cas9 system to engineer β5-integrin deficient PADC CAFs (β5 KO ) [15]. These β5 KO CAFs, serve as “normalized” CAF controls [22, 33], as these cells perpetuate a phenotype that simulates TGFβ inhibition in CAFs [15] and constituted an important model used in this study. We herein report that CAF-generated ECMs protect PDAC cells by modestly hindering the effects of eribulin upon limiting tumor cell growth.…”