Pro-tumoral role of Granzyme B to aid in invasion and metastasis.

Abstract

Abstract The granule exocytosis pathway has long been known to be involved in potent responses to pathogens and cancers. This is executed by the action of Perforin and a family of granule-associated serine proteases known as Granzymes (Gzm). Many studies have shown that these proteases exert their effects by killing affected cells, executing the major function by Cytotoxic T cells (Tc) and Natural Killer cells expressing GzmB. Studies have found that the tumor cells ability to create a suppressive environment is dependent on Gzm expression by Regulatory T lymphocytes (Treg). Bird et al published data showing GzmB aids in the transmigration of Tcs, however the ability of GzmB to aid in the migration of non-T cells has not been investigated. We hypothesize that Granzyme B secretion may cause extracellular matrix remodeling in the tumor microenvironment, aiding in the outgrowth of the tumor cells via promoting invasion or metastasis. We test our hypothesis by measuring tumor invasion in vitro utilizing the xCelligence transmigration system. We found that culturing B16 cells with recombinant GzmB from Biolegend or Peprotech on Matrigel resulted in a 31% and 47% increase in cellular invasion, respectively. Also, the rate of invasion resulted in a 35% and 61% increase, respectively. We also tested our hypothesis in vivo with a preliminary study using Treg-specific GzmB KO mice (Foxp3creGzmBfl/fl). We depleted CD8+ T cells, injected B16 cells intravenously, and activated Tregs. We found a 48% decrease in metastatic nests in mice deficient of GzmB in Treg cells after 8 days and a 37% decrease after 16 days compared to WT. These findings shed light on the pro-migratory ability of secretory GzmB and helps us understand an unassuming role of GzmB as a pro-tumoral factor. Supported by funds through NIH (RO1 HL135325, T32 AI095190, P30 CA134274) and Maryland Department of Health's Cigarette Restitution Fund Program.