Abstract A104: Matrix metalloproteinase-2 and Toll-Like receptors modulating immune responses in the tumor microenvironment
Authors: Muniz-Bongers Luciana Rebiero , Saxena Mansi , Bhardwaj Nina 
Published: February 2019

Successful antitumor immunotherapies improve survival in many malignancies but many patients still do not respond or relapse after treatment. This may result from an immunosuppressive tumor microenvironment (TME), composed of many of cells and extracellular matrix proteins such as matrix metalloproteinases (MMPs) that contribute towards tumor growth and metastasis while dampening anti-tumor immunity. We have previously shown that MMP2 is produced in the melanoma TME and modulates human dendritic cell (DC) function to skew T-cells towards a deleterious TH2 phenotype. Briefly, MMP-2 inhibits IL-12 and triggers Toll-like receptor-2 (TLR2) stimulation, leading to NFκB activation to promote OX40L expression and inflammatory cytokine production. We aimed to investigate the mechanisms underlying MMP2-TLR2 interaction in the TME and how they promote immune suppression and melanoma tumor growth. Like human cells, mouse primary and immortalized bone marrow (BM) macrophages and DCs respond to MMP2 and secrete proinflammatory cytokines like TNFα and IL-6. In mice, however, MMP-2 signaling depends on TLR2 and TLR4 with TLR2-/-/TLR4-/- and single KO cells demonstrating compromised TNFα signaling. This response requires MyD88. Moreover, mouse primary splenic and lung DCs respond to MMP-2, and the lack of TLR2/4 abrogates the response, indicating TLR-MMP2 signaling is also systemically significant. Accordingly, in vivo MMP2 injections induced cytokine secretion in the serum of WT but not TLR2-/-, TLR4-/- or dKO mice. To study MMP2 in the TME, we used B16 melanoma tumors. We characterized B16 mRNA and protein expression profiles in cellular compartments and compared with that of whole tumors. We detected MMP2 expression in cultured cells and whole tumors. To identify the source of MMP2 in the TME, we sorted tumor cells and stromal cells and confirmed that both express MMP2 mRNA. Regarding TLR signaling, tumor cells lacked TLR2 and CD14 expression and thus, did not signal via TLR2/TLR4. Following subcutaneous injections of B16 tumors in mice, we observed that TLR2-/-/TLR4-/- mice had significant slower tumor growth kinetics and smaller tumors but similar cellular infiltration in the tumor when compared to WT mice. Next, we modulated TLR and MMP2 signaling in the TME. First we assessed the contributions of the hematopoietic or stromal compartment TLRs into MMP2 signaling using BM chimeras. Our results revealed differences in tumor development due to lack of TLR signaling in hematopoietic cells, as WT mice that received TLR2-/-/TLR4-/- BM have significant slower tumor kinetics and smaller tumors than WT BM recipients. Then, in order to modulate the levels of MMP2 in the TME, we knocked out and overexpressed MMP2 in tumor cells and compared tumor growth in WT mice. Our pilot experiments suggest that the lack of MMP2 in tumor cells significantly delays tumor development and size in WT mice when compared with MMP2-sufficient tumor cells. Co-immunoprecipitation studies confirmed that MMP2 binds with both TLR2 and TLR4. Currently, we are working on pinpointing the exact MMP-2 domain responsible for binding TLRs. Preliminary results thus far indicate the SP-Pro domains of MMP2 is responsible for binding to TLR2 and TLR4 as MMP2 that lacks these domains does not bind TLRs and SP-Pro domains alone are enough for binding. Our data suggests that MMP2, expressed in the TME, serves as a strong promoter of tumor growth and metastasis through stimulation of TLR2 and TLR4 on hematopoietic cells in vivo. Previous work targeting TLRs and endogenous alarmins has showed benefits for tumorigenesis control. Therefore, a deeper knowledge of how MMP-2 interacts with its signaling receptors will provide opportunities to specifically target MMP-2 TLR axis for controlling immune suppression in the TME.

Citation Format: Luciana Rebiero Muniz-Bongers, Mansi Saxena, Nina Bhardwaj. Matrix metalloproteinase-2 and Toll-Like receptors modulating immune responses in the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A104.