Gut Microbiota inhibit the potency of MSLN-CAR-T cells against pancreatic carcinoma by modulating Cholesterol Metabolism

Background Pancreatic cancer is one of the gastrointestinal malignant tumors with the highest mortality rate in the world, and CAR-T immunotherapy has shown poor therapeutic efficacy for pancreatic cancer despite its promising applications. Methods In this study, we first collected fecal samples from 9 pancreatic cancer patients and 20 healthy individuals for 16s-rRNA high-throughput sequencing to compare the compositional differences between the two groups. Secondly, the effect on CART phenotype was detected by co-culturing gut microbial supernatants with primary CAR-T cells. Finally, the influence mechanism was initially explored by metabolomics and RNA interference replication experiments. Results The difference in α and β diversity between the gut microbiota of PC patients and healthy controls was significant; LEfSe analysis showed that the abundance of genera such as Streptococcus and Klebsiella was significantly higher in the PC group, in which lipid-associated regulated metabolites were also significantly increased. Co-culture of gut flora supernatants with pancreatic cancer cell lines showed that PC gut microbial metabolites were able to reduce the migration ability of CAR-T cells and increase the proportion of Treg subtypes, as well as inhibit the killing effect of CAR-T (E/T ratio=1:1), while the transcription of genes related to the synthesis, transport, and secretion of cholesterol metabolism of CAR-T was up-regulated. In the RNA interference response experiments, we inhibited ACAT-1, a key enzyme for cholesterol metabolism in CD8+ T cells, and found that the CAR-T killing effect was reversibly enhanced (E/T ratio=1:1). Conclusion The gut microbiota of PC patients differed significantly from that of healthy individuals. PC gut microbiota could inhibit the killing effect of CAR-T by regulating cholesterol metabolism and could be reversed by inhibiting ACAT-1. This provides a possible basis for enhancing the therapeutic effect of CART by changing the structure of CAR-T or regulating the gut microbiota of PC patients in the future.