ROCK2-regulated LIF-STAT3 drives immunosuppression in pancreatic cancer

Background: Immunosuppression is a key characteristic of pancreatic ductal adenocarcinoma (PDAC), contributing to metastasis and poor survival. Our studies have identified tumor cell intrinsic Rho-associated coiled-coil containing protein kinase-2 (ROCK2) as a key regulator of extracellular matrix remodeling. In this study, we investigated how ROCK2 regulates immunosuppression in PDAC by modulating Leukemia inhibitory factor (LIF) and its effects on STAT3. Methods: TCGA PDAC patient dataset was used to compare the ROCK2 and LIF expression in normal and PDAC tissues. CIBERSORT analysis of the PDAC dataset estimated the proportion of tumor infiltrating immune cell subsets. Genomic editing using the CRISPR/Cas9-system in LSL-Kras G12D/+; Trp53 R172H/+ ; Pdx1 Cre/+ (KPC) cells was performed to generate KPC Rock2 knockout (Rock2 KO ) cells. Cytokine array was performed on Rock2 EV and Rock2 KO conditioned media and ELISA was used to validate the results. Flow cytometry was used to profile LIF receptor (LIFR) expression in different cell types from KPC orthotopic tumors. Bone marrow-derived macrophages from C57BL/6 mice were treated with recombinant LIF (rLIF) and LIFR inhibitor (EC359), then analyzed for polarization by flow cytometry. KPC orthotopic tumors were generated, and Immune cell profiling was performed to evaluate alterations in immune cell subsets following treatment with EC359. Findings from ROCK2 and ROCK2-regulated LIF-STAT3 targeting, both in vitro and in vivo were validated using Immunoblotting and immunohistochemistry. Results: Analysis of the TCGA dataset revealed that Human PDAC tissues have increased expression and correlation of ROCK2 and LIF. Further analysis showed that macrophages constitute a substantial proportion of the immune cell infiltrate. Cytokine array and ELISA-based studies revealed decreased LIF secretion with ROCK2 knockout, providing evidence for ROCK2 dependent regulation of LIF. KPC orthotopic tumors demonstrated higher LIFR expression in tumor-associated macrophages (TAMs) and EC359 treatment reduced ARG1 and PD-L1 expression on these cells. Additionally, EC359 treatment led to a significant increase in the activated effector and effector memory T cell populations. Furthermore, rLIF treatment increased pSTAT3 levels in macrophages, while EC359 lowered its expression, highlighting the role of ROCK2-regulated LIF-LIFR in STAT3 activation. Conclusion: These findings demonstrate that tumor cell-intrinsic ROCK2 regulates LIF-STAT3, which mediates immunosuppression and can serve as a potential therapeutic target for PDAC.