<p>Spread sheet including a single tab per Figure panel conveying each graph's statistical analyses. Note that tabs are named accordingly. The tabs include the comprehensive statistical analyses that were performed for each experiment in which statistical significance was given. Each tab contains the full statistical output for its labeled experiment. The statistical test performed for each experiment is identified within the corresponding tab. Statistical tests were carried out using the software, Prism, version 7.05.</p>
<div><p>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). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEV). The tumor-supportive CAFs possess a distinct phenotypic profile, compared with normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α<sub>5</sub>β<sub>1</sub> localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1<sup>+</sup> CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Furthermore, we show that NetG1 expression in CAFs is required for the prosurvival properties of sEVs. In addition, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α<sub>5</sub>β<sub>1</sub> being enriched in exosomes. Finally, we found that NetG1 and Integrin α<sub>5</sub>β<sub>1</sub> were detected in sEVs collected from plasma of patients with PDAC, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor–stroma interactions and pathogenic stroma detection.</p>Significance:<p>Results from this study identified two unique types of tumor-supporting CAF EVs, with evidence of these being detected in patients. Thus, this study facilitates a novel avenue to further dissect the subtleties of the tumor–stroma interactions responsible for PDAC homeostasis and progression, as well as the possibility of establishing future means to detect and monitor dynamic stroma staging.</p></div>
<p>Supplementary Figure 1. Confirmation that CAFs produce ECMs distinct from NLFs. Supplementary Figure 2. Direct co-culture of CAFs support PDAC cells during nutrient-deprivation. Supplementary Figure 3. Additional CAF cell lines generate sEVs that rescue PDAC cells from nutrient deprivation-induced apoptosis. Supplementary Figure 4. NetG1+ CAF-sEVs support PDAC cell survival in a NGL-1 dependent manner. Supplementary Figure 5. NetG1 expression in CAFs is necessary for sEV-mediated survival of nutrient-deprived PDAC cells. Supplementary Figure 6. NetG1 ablation does not affect the uptake of sEV cargo in PDAC cells. Supplementary Figure 7. sEV supernatant contains DNPs enriched with sub-exosome sized EVs. Supplementary Figure 8. Enriched gene ontology clusters from proteomic and metabolomic analysis comparing sEV and DNP fractions. Supplementary Figure 9. EV cargo requires transfer in intact vesicles to provide tumor-supportive effect.</p>
<p>Spreadsheet including the metabolomic analyses corresponding to the experiments indicated by the naming of the assorted tabs. Specific analysis parameters for each experiment are provided within the respective main figure legends.</p>
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