Modulating Cholangiocarcinoma's Tumor Microenvironment Enhances Response To Immune Checkpoint Blockade

Abstract

Presenter: Laurence Diggs MD | National Institutes of Health Background: Intrahepatic cholangiocarcinoma (iCCA) carries a very poor prognosis due to a typically late clinical presentation and a poor response to current therapeutics. ICCA’s tumor microenvironment (TME) has been shown to be immunosuppressive. While these tumors commonly express PD-1 and PD-L1, the response to immune checkpoint inhibitors (ICI) is poor. We hypothesized that modulating the TME by stimulating antigen-presenting cells (APCs) can improve response to ICIs. Specifically, stimulating CD40, a known receptor on all APCs, can drive increased effector cell cytotoxicity when combined with ICI. Methods: We treated tumor bearing C57BL/6 mice with a CD40 agonistic antibody, an anti-PD1 antibody, and the combination (CD40/PD1) to compare treatment response. We used three different mouse models including subcutaneous and orthotopic injections of SB1, an established iCCA cell line as well as hydrodynamic tail vein injection of the YAP and AKT plasmids. Mice were then treated for three weeks with a CD40 agonist (2.5ug/g) weekly, an anti-PD-1 (10ug/g) weekly or the combination starting on post-tumor inoculation day five. Tumors were measured, weighed and evaluated histologically for differences between treated and untreated mice. Tumors were histologically proven iCCA (CK7 +, CK19+, HNF4α-). Immunohistochemical (IHC) staining characterized the TME including lymphocytes (CD4&CD8 T cells, B cells), myeloid cells (macrophages, dendritic cells), as well as PD-1 and CD-40 expression. Flow cytometric analysis of lymphoid and myeloid cell populations including activation status was performed. qPCR allowed for the identification of the cytokine mediators of treatment response. Finally, CD4, and CD8 T cell depletion was undertaken to identify effector cells. Results: In subcutaneous, orthotopic, and plasmid models, mice treated with anti-PD-1 alone did not demonstrate significantly (p>0.05) lower tumor burden. Mice did exhibit a response to monotherapy with a CD40 agonist (p<0.05). When these agents were combined (CD40/PD-1) however, a significantly greater reduction in tumor burden was seen (p<0.05) (Figure 1). IHC confirmed iCCA with increased areas of necrosis and calcification in CD40/PD-1 in the liver tumor tissue of treated mice. Flow cytometry demonstrated increased CD4+ and CD8+ T cells presence and activation as well as increased activation of myeloid cells in CD40/PD-1 treated mice. qPCR revealed increased presence of IL-2, TNF, iNOS, and Granzyme B within the tumor tissue and lymphocytes of mice treated with CD40/PD-1. T cell depletion demonstrated reduced efficacy of treatment with CD8 depletion and complete loss of efficacy of treatment with CD4 depletion (p<0.05). Conclusion: In Conclusion, modulation of the TME by CD40-mediated activation of APCs in iCCA appears to activate T cells and significantly enhances response to anti-PD-1 therapy. This process appears to be mediated both by CD4+ and CD8+ T cells. These findings offer a potential alternative to current cytotoxic chemotherapy regimens.