Abstract B036: Hypoxic cancer cells condition immunosuppressive macrophages via secreted non-protein mediators in pancreatic ductal adenocarcinoma
Authors: Cribb Matthew T , Fattani Sahar , Colon-Ortiz Ayeisha , Jiang Dadi , Spiotto Michael , Koong Albert 
Published: September 2024

Abstract
INTRODUCTION: Hypoxia is a defining feature of pancreatic ductal adenocarcinoma (PDAC) that contributes to the immunosuppressive microenvironment defined by increased tumor-associated macrophages (TAMs). However, there is an unmet need to define mechanisms by which hypoxia drives the immunosuppressive macrophage phenotype. We hypothesize that hypoxic cancer cells secrete non-protein metabolites to induce immunosuppressive phenotypes in macrophages. METHODS: We generated conditioned media (CM) by culturing the pancreatic adenocarcinoma cell line KPC under normoxia (21% O2) or hypoxia (0.5% O2) conditions for 48 hours. We treated bone marrow-derived macrophages (BMDMs) or the macrophage line RAW264.7 with CM. We assessed immunosuppressive gene expression using RNAseq and qRT-PCR. Proteins in CM were depleted using Sera-Mag Magnetic Carboxylate Modified Beads or denatured by boiling at 100°C for 15 minutes. We identified changes in metabolites between hypoxic and normoxic CM using HPLC. RESULTS: Compared to BMDMs treated with normoxic CM, we observed that BMDMs treated with hypoxic CM induced multiple immunosuppressive proteins (Arg1), cytokines (Ccl22, Cxcl3) and interleukins (Il1b, Il1a, Il6). Similarly, GSEA analysis demonstrated that hypoxic CM induced TNFa signaling via NfkB and IL6-JAK-STAT3 signaling. Consistent with RNA-seq analysis, qRT-PCR measured Arg1 expression 53 times higher in hypoxic CM-treated BMDMs compared to nomoxic CM (p=0.0286). Compared to normoxic CM, hypoxic CM stimulated macrophage migration, a marker for macrophage infiltration, as measured by transwell assays (p&lt;0.0001). An increase in Arg1 expression in RAW264.7 cells was still observed when treated with protein depleted or boiled hypoxic CM, excluding proteins as soluble factor(s) mediating immunosuppression. To identify non-protein mediators of immune suppression, mass spectrometry quantified lipids and metabolites enriched in HCM. Metabolomic analysis identified 40 metabolites increased in hypoxic CM including the monounsaturated or polyunsaturated fatty acids linoelaidic acid, DGLA, and arachidonic acid. Similarly, lipidomic analysis revealed that multiple PC (phosphatidylcholine) and LPC (lysophosphatidylcholine) lipid species were enriched in HCM. CONCLUSION: We observe that hypoxic tumor cells induce immunosuppressive changes in macrophages likely via secreted non-protein metabolites. The hypoxic metabolome of PDAC tumor cells may be a key driver of the accumulation of immunosuppressive tumor associated macrophages. We are currently characterizing the role of individual lipids or metabolites in macrophage polarization.
Citation Format: Matthew T Cribb, Sahar Fattani, Ayeisha Colon-Ortiz, Dadi Jiang, Michael Spiotto, Albert Koong. Hypoxic cancer cells condition immunosuppressive macrophages via secreted non-protein mediators in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B036.