Abstract:Background Accumulating evidence suggests that cancer-associated fibroblast (CAF) autophagy is crucial in tumor development and may be a therapeutic target for pancreatic ductal adenocarcinoma (PDAC). However, the role of CAFs autophagy during immune surveillance and cancer immunotherapy is unclear. MethodsIn vivo efficacy and mechanistic studies, using ATG5f/f α-SMAcreERT2 mice, KPC-genetically engineered mouse model (GEMM) mice, C57BL/6 mice and nude mice with KPC cell‑derived orthotopic tumors, employed immune checkpoint blockades (ICBs) and gemcitabine. Mass cytometry (CyTOF), immunohistochemistry (IHC), and flow cytometry analyzed local and systemic alterations in the immunophenotype. The regulatory effects of CAFs autophagy on tumour PD-L1 and its downstream signaling were analyzed by RNA-seq, 4D mass spectrometer (MS), IP-MS, western blotting, GST-pulldown, immunofluorescence (IF), IHC, and flow cytometry. Results The present study revealed that the inhibition of CAF autophagy suppresses in vivo tumor development in immune-deficient xenografts. This deletion compromises anti-tumor immunity and anti-tumor efficacy both in vitro and in vivo by upregulating PD-L1 levels in an immune-competent mouse model. A deletion in CAF autophagy reduced the production of interleukin 6, disrupting high desmoplastic TME and decreasing USP14 expression at the transcription level in pancreatic cancer cells. We further identify USP14 as the post-translational factor responsible for downregulating PD-L1 expression by removing K63 linked-ubiquitination at the K280 residue. Finally, chloroquine diphosphate-loaded mesenchymal stem cell (MSC)-liposome, by accurately targeting CAFs, inhibited CAF autophagy, improving the efficacy of immunochemotherapy to combat pancreatic cancer. Conclusions Targeting CAFs autophagy disrupt the desmoplastic and immunosuppressive TME to enhance the efficacy of immunochemotherapy by inhibiting adaptive immune resistance.