Abstract A81: Hyaluronan-dependent skewing of tumor-associated macrophage (TAM) phenotype in a murine pancreatic tumor model is associated with increased tumor hypoxia
Authors: Kimbler Trevor , Cowell Jessica , Lee Jisook , Thompson Benjamin J. 
Published: June 2021

Abstract
Hyaluronan (HA) is a naturally occurring glycosaminoglycan that accumulates in the tumor microenvironment of several cancer types, including pancreatic ductal adenocarcinoma (PDA), where it is an independent negative predictor of survival. An ongoing phase 3 study is evaluating pegvorhyaluronidase alfa (PEGPH20; PVHA), a first-in-class biologic that enzymatically degrades HA in the tumor microenvironment, in combination with gemcitabine and nab-paclitaxel in metastatic PDA. Recently, we reported that HA-accumulation can promote tumor hypoxia; however, the effects this may have on tumor-infiltrating immune cells are unknown. Transduction of syngeneic murine PDA cells (Pan02) with hyaluronan synthase 3 (HAS3) generates tumors (Pan02-HAS3) containing high levels of HA, and a higher frequency of TAMs with an immunosuppressive phenotype (CD206-high MHCII-low), relative to tumors generated by empty vector-transduced cells (Pan02-EV). Given the association of this TAM phenotype with tumor hypoxia, we assessed the relationship between tumor hypoxia and the TAM phenotypes of Pan02-HAS3 and Pan02-EV tumors. To confirm whether the effects seen in Pan02-HAS3 tumors require HA synthesis, we also generated tumors expressing an enzymatically inactive HAS3 mutant (Pan02-HAS3 D215A). Tumor-bearing mice were injected with pimonidazole hydrochloride 2 hours prior to harvest and dissociation of tumor tissue; subsequent flow cytometric analysis of tumor-infiltrating immune cells included an antibody against pimonidazole hydrochloride adducts (HP-1) to detect cells residing in hypoxic zones. A greater mean percentage of TAMs isolated from Pan02-HAS3 peritibial tumors had high levels of HP-1 staining (HP-1+) compared with TAMs isolated from either Pan02-EV or Pan02-HAS3 D215A tumors (18.2%, 4.2% and 3.9%, respectively). Furthermore, HP-1+ cells were enriched among CD206-high MHCII-low TAMs compared with their MHCII-high counterparts in Pan02-HAS3 tumors (38.7% vs 11.9%). Likewise, more HP-1+ cells were seen among dendritic cells (DCs) isolated from Pan02-HAS3 tumors compared with either Pan02-EV or Pan02-HAS3 D215A tumors (6.9%, 1.3% and 0.8%, respectively). Similar findings were obtained in orthotopic tumors. In contrast, no consistent differences in the percentages of HP-1+ CD8 T cells or NK cells were observed across the three tumor types in this study. Finally, exposure of cultured macrophages to hypoxia recapitulated some of the changes in gene expression observed in TAMs sorted from Pan02-HAS3 tumors, including increased expression of Arg1 and Nos2. These data suggest that tumor HA accumulation may promote an immunosuppressive TAM phenotype by creating tumor hypoxic zones that are subsequently infiltrated by TAMs, but not T or NK cells.
Citation Format: Trevor Kimbler, Jessica Cowell, Jisook Lee, Benjamin J. Thompson. Hyaluronan-dependent skewing of tumor-associated macrophage (TAM) phenotype in a murine pancreatic tumor model is associated with increased tumor hypoxia [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A81.