Spatial Heterogeneity of Immune Drivers Coordinates the Organisation of Antitumor Immunity in Pancreatic Cancer, Affecting Patient Outcome

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is considered low immunogenic with “cold” tumor microenvironment (TME) and is mostly unresponsive to immune checkpoint blockade therapies. Aims We aim to decipher the impact of intratumoral heterogeneity of immune determinants on antitumor response. Methods 130PDAC specimens were classified according to overall survival in long-term survivors (LTSs, n=29, OS≥60 months) and short-term survivors (STSs, n=101, OS60 months). Spatial compartment masks were defined by immunofluorescence imaging (tumor cells: Pancytokeratin+CD45-, leukocytes: CD45+Pancytokeratin-, stroma cells: PanCK-CD45-). 4regions of interest from tumor center (TC) and the invasive front (IF) were examined by transcriptomic and proteomic analysis using the Nanostring platform for immune pathway targets. Results of NGS and morphological features were integrated. 20samples underwent immunophenotypic analysis by multiplex immunofluorescence. Results LTSs displayed mostly homogeneous morphology with extended glandular differentiation and immunogenic TME both at TC and IF, with increasing gradient towards the IF. There was higher presence of immune checkpoint-associated and immunogenic genes and proteins at the IF as compared to the TC, including CD40, CD3, CD8, CD4, GZMB and PD-L1. In contrast, STSs were characterized by morphologic heterogeneity, including areas with reduced glandular differentiation and high tumor budding, and a mostly immunosuppressive TME with negative gradient towards the IF. Moreover, there was reduced gene expression and protein abundance at the IF as compared to the TC, including CD3, CD8, CXCL10, GZMB, IFNG, HLA-DR and CD40. Conclusion LTSs display a highly immunogenic TME, underscoring their effective antitumor immunity, especially at the area of IF compared with STSs. A significant intratumoral heterogeneity between TC and IF exists with regards to immune determinants in both, LTSs and STSs, which might explain the different antitumor immune responses, affecting patient outcome. The differential expression of immune drivers may help selecting patients for combination therapies to improve antitumor immunity and harness the responsiveness to immune checkpoint inhibitors in PDAC.