Abstract 1808: Role of natural killer (NK) cells in glycoasminoglycan-mediated anti-cancer immunity

Abstract

Abstract Background: Immune-modulating therapies have limited efficacy in colorectal cancers (CRC). This resistance to treatment is attributed to low neoantigen expression, and the inability of T cells to kill HLA-I negative tumor cells, especially cancer stem cells (CSCs). Advantageously, NK cells can preferentially kill CSCs via stress ligand recognition. We have previously shown that glycosaminoglycans (GAGs) of a specific chain length, and synthetic mimetics thereof, selectively inhibit colon CSCs. In the current study, we evaluated the immunomodulatory role of natural GAGs and synthetic GAG mimetics (SGMs) in eliciting Natural Killer effector functions that regulate the anti-tumor immune response. Methods: 8-12 wk old C57bl/6 mice were treated with SGM or natural GAG (Fucoidan, heparin hexasaccharide (HS06)) and vehicle for 2 wks. Splenocytes were expanded in IL-2-containing media with or without respective GAG/SGMs, and subjected to co-culture with syngeneic MC38 colon cancer cells. The effects of each treatment on immune cell profile and anti-cancer properties were evaluated using a) multiplex flow cytometry Cytek® immune profiling assay and b) MTT assay. In vitro, MACS-enriched human peripheral blood NK cells were stimulated ex vivo with GAG/NSGMs and analyzed for activation using flow cytometry and cytotoxicity towards primary human colon cancer-derived spheroids. PDL-1 expression and CSC marker expression in GAG/SGM-treated MC38 spheroids was analyzed using western blotting and qPCR. Results: Unbiased multiplex flow cytometry revealed a robust activation of NK cells, evidenced by increased CD335 (NKp46) expression in SGM or Fucoidan-treated mice. SGM pulsed splenocytes also showed increased cytotoxicity toward MC38 cells compared to vehicle controls ex vivo. Furthermore, our preliminary findings showed that human peripheral blood NK cells (CD45+CD3-CD56+) showed activation (increased CD69 expression) with both GAGs and SGMs but showed increased proliferation (Ki67+) upon ex vivo stimulation with GAG oligosaccharide HS06 and not polymeric GAG. Furthermore, GAG treatment significantly enhanced activated autologous NK cell-induced apoptosis in primary human tumor-derived spheroids. Mechanistically, a significant reduction in Pdl-1 protein expression was observed in MC38 spheroids concurrent with the downregulation of various CSCs maker genes, e.g., Oct4, Sox2, and c-my when treated with various SGMs in vitro. Conclusions: The work highlights the role of novel SGMs in the activation of NK cells to target CSCs and it has major implications for development of novel immunotherapy strategies in CRC. Citation Format: Pavani Pingali, Jackson Faulx, Aditi Nandi, Umesh R. Desai, Bhaumik B. Patel. Role of natural killer (NK) cells in glycoasminoglycan-mediated anti-cancer immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1808.