Evaluating the role of cancer-associated fibroblasts in T cell–mediated proinflammatory reprogramming of tumor associated macrophages in the prostate cancer tumor microenvironment.

Abstract

248 Background: Tumor-associated macrophages (TAMs) have been implicated as a critical mediator of T cell exclusion and inhibition within the prostate TME. However, using a microscale cell culture platform, known as Stacks, we have previously demonstrated that while prostate tumor cells induce an immunosuppressive phenotype in TAMs, activated T cells can reprogram these macrophages towards a proinflammatory phenotype that augments tumor-directed T cell responses. To investigate how other cell populations within the TME impact T cell mediated macrophage reprogramming, we expanded our models to include cancer-associated fibroblasts (CAFs), which have been previously shown to have immunosuppressive effects that regulate macrophage function in the TME. Methods: Primary T cells and autologous monocyte-derived macrophages (MDMs) were derived from the peripheral blood samples of patients receiving care at the University of Wisconsin Carbone Cancer Center and with William S Middleton Memorial Veterans Hospital in Madison, WI. MDMs were cultured with prostate tumor (22Rv1) and CAF (hPrCSC-44) cells in mono-, co-, and tri-culture models for 72h in the Stacks microfluidic cell culture platform. Activated autologous T cells were then added to each condition for an additional 24h of culture. Models were analyzed for T cell migration and surface marker expression by confocal microscopy, secreted cytokine and chemokine profiles by protein bead array, and gene expression by qRT-PCR. Results: We found that MDM-mediated augmentation of CD4 + and CD8+ T cell migration within the prostate TME models was abrogated in the presence of CAFs. Analysis of gene expression further demonstrated that the presence of CAFs in our quad culture models resulted in a decrease in expression of T cell recruitment chemokines by the MDMs, including CCL2, CCL5, CXCL9, CXCL10, and CXCL11, compared to MDM tri-culture with tumor cells and T cells. MDMs also expressed higher levels of IL-10 mRNA, a potent immune suppressive factor, when CAFs were present in the TME. Conclusions: Using microscale cell culture technologies, we provide the first direct evidence that CAFs inhibit T cell mediated proinflammatory reprogramming of MDMs in the prostate TME. Furthermore, we demonstrate that this inhibition of MDM reprogramming is associated with a decrease in tumor-directed T cell migration compared to MDM, tumor cell, and T cell tri-cultures. This data suggests that CAFs, through their effects on MDMs/TAMs, may be playing a vital role in the inhibition of tumor-directed T cell responses. The pathways that are mediating this effect therefore represent valuable therapeutic targets to augment immunotherapeutic efficacy in prostate cancer treatment.