Multiplex microscopy reveals unique spatiotemporal effects of cancer immunotherapies

Abstract

Abstract Pancreatic ductal adenocarcinoma (PDA) is an aggressive and heterogeneous cancer that is often refractory to current treatments. To address this issue, we employ a mouse PDA-derived tumor clone library where individual clones elicit a spectrum of unique immune cell infiltration profiles in the tumor microenvironment (TME). We found that a combination of anti-PD1, anti-CTLA-4, and anti-CD40 results in tumor regression only in tumor clones with a high number of infiltrating T cells. The aim of our study is to gain mechanistic understanding into this therapeutic combination. To that end, we leverage quantitative high multiplex microscopy, enabling us to decipher the complexities of cellular behaviors, interactions, and phenotypes in an intact TME. We found that our therapy distinctively alters the TME of both responsive and non-responsive tumors. Unique to non-responsive tumors, we found discrete myeloid clusters scattered through the TME that increase in frequency and size post therapy. Additionally, we witnessed prominent cellular movement within myeloid clusters using 2-photon intravital imaging. In contrast, in the TME of responsive tumors our therapy severely depleted, reprogrammed, and restricted regulatory T cells to the tumor periphery within 48 hours post anti-CD40 administration. We found this to be ADCC–independent, but anti-CD40–dependent, with the mechanistic factors driving the fate of these cells under detailed investigation. Combined, these data reveal mechanistic insight into anti-CD40 combination therapies and provide a platform for investigating the factors driving the formation and maintenance of an immunosuppressive TME. This work was supported by (1) Intramural Research Program of NIAID, NIH, (2) Postdoctoral Research Associate Training (PRAT) Fellowship of NIGMS, NIH, (3) Parker Institute for Cancer Immunotherapy, and (4) Bench to Bedside Award.