Targeting tumor associated macrophages through blockade of GM-CSF sensitizes cholangiocarcinoma to adaptive immunity

Abstract

Presenter: Luis Ruffolo MD | University of Rochester Background: Cholangiocarcinoma (CCA), is an aggressive malignancy with poor survival and increasing incidence. Immunotherapies offer the promise for better disease control, yet a thorough understanding of mechanisms of T cell exclusion in CCA remains undescribed. Here were report the immunosuppressive priming of the tumor microenvironment (TME) driven by CCA derived Granulocyte-Macrophage-Colony Stimulating Factor (GM-CSF) in a spontaneous model of CCA. Methods: KRAS(G12D); TP53(-/-); Alb-Cre mice (KPPC) develop spontaneous CCA. Disease was monitored with high frequency ultrasonography. Novel murine tumor cell lines were derived from syngeneic KPPC tumors and injected orthotopically. Histology and immunohistochemistry (IHC) staining was performed on mouse and human CCA for stromal and immunophenotypic markers. Murine tumor and immune compartments were processed for flow cytometry analysis. Genomic analysis of tumor and normal parenchyma was performed by quantitative real time polymerase chain reaction (qRT-PCR) and whole exome RNA sequencing (RNA seq). Therapeutic blockade of GM-CSF was achieved by administration of monoclonal antibody (mab), or appropriate control. The study was approved by UCAR and RSRB at the University of Rochester. Results: The human CCA TME is heavily desmoplastic with a predominant myeloid leukocyte infiltrate (CD11b+). CD68+ tumor associated macrophages (TAMs) are the most abundant leukocyte and their presence within the TME correlates with a worse overall survival after resection (p = 0.03). KPPC tumors closely recapitulate human CCA, and are similarly heavily infiltrated with TAMs and myeloid derived suppressor cells (MDSCs). Outside of the TME, myeloid leukocytes are elevated in the peripheral blood, spleen, and bone marrow of tumor bearing mice compared to littermate controls (P < 0.001). qRT-PCR of bulk tumor vs. normal parenchyma demonstrates increased transcription of myelopoietic cytokines, with GM-CSF being 1000 fold overexpressed compared to littermate controls (P < 0.001). Therapeutic blockade with anti-GM-CSF mAb slowed disease growth and onset of metastases, and prolonged survival in spontaneous tumor bearing mice (P = 0.015) (Figure). Flow cytometry analysis of treated and untreated murine CCA tumors demonstrated a significant decrease in TAMs and peripheral blood monocytes. Furthermore, GM-CSF blockade resulted in a reduction in expression of programmed cell death ligand 1 (PD-L1) and arginase (Arg1) by both TAMs and MDSCs within the TME. Concomitantly, CD8+ effector T cells were found to be increased in prevalence compared to untreated tumor bearing mice, and these cells exhibited increased expression of programmed cell death receptor 1 indicative of activation. RNA seq analysis of tumor educated macrophages treated with anti-GM-CSF mAb demonstrated repolarization away from an M2 phenotype, with reduction in CD206, Arg1, PD-L1, PD-L2, and IL6 (all p-values < 0.01). GM-CSF was found to be over expressed by human CCA tumors compared to normal liver, and tumors with a higher expression of GM-CSF conferred a worse overall survival in resected patients (P = 0.008). Conclusion: CCA induces myelopoiesis and recruitment of TAMs and MDSCs resulting in an immunosuppressive TME. GM-CSF is a key driver of myeloid cell production and immunosuppressive programming, and therapeutic blockade of GM-CSF potently inhibits TAMs resulting in enhanced cytotoxic T cell penetration and activation. GM-CSF represents a novel target which should be explored in advanced biliary malignancies.