PROJECT SUMMARY The recent approval of immunotherapies has revolutionized the approach to managing bladder cancer (BCa). Nevertheless, complete response is observed in only a minority of patients and there are no unambiguous biomarkers to guide treatment selection. A key factor that controls tumor growth and response to cancer immune therapy is the recruitment of immune cells to the tumor microenvironment by chemokines. The chemokine CCL2 (C-C motif ligand 2) is best known for its ability to induce the trafficking of immune cells by binding its primary receptor, CCR2. The recruitment of immunosuppressive myeloid cells by CCL2 promotes cancer in several tumor types. In the bladder, however, we show an unexpected finding that CCL2 and its receptor CCR2 are protective against cancer development and growth and the mechanism is T cell-dependent. We also show that post-translational nitration of CCL2 by bladder tumors inhibits CCL2’s ability to recruit T cells and its effects on tumor control. This may be one of the critical reasons behind the failure of chemokines in clinical trials and may explain certain paradoxes that are seen between CCL2 levels and patient outcomes in cancer. We propose delineating the effect of CCL2 nitration on the biology of BCa by using nitration-resistant recombinant CCL2 (rCCL2NR) which may restore bladder tumor T cell recruitment and facilitate anti-tumor T cell immunity. Based on convincing preliminary data, we hypothesize that nitration of CCL2 in BCa ablates the tumor protective function of CCL2/CCR2 by disrupting T cell recruitment to bladder tumors. To address this hypothesis, two specific aims are proposed. In Aim 1, we test the hypothesis that blocking nitration of CCL2 by rCCL2NR reduces BCa progression by enhancing the recruitment of CCR2+ T cells in the bladder. rCCL2NR treatment effects will be tested by intravesically instilling rCCL2NR vs rCCL2 vs control PBS weekly in the bladder. We will also delineate the specific mechanisms by which rCCL2NR exerts its tumor-protective effect and test whether rCCL2NR increases CCR2+ T cell recruitment or increase the activation and cytotoxicity of already recruited CCR2+ T cells in the bladder. rCCL2NR will be evaluated either as a single agent or in combination with standard BCa treatment strategies. Aim 2 will test whether nitrated CCL2 level in human bladder tumors is a prognostic factor and predicts treatment response to immune therapy in BCa patients. Multiplex immunofluorescence will be used to detect unmodified and nitrated CCL2 levels in BCa tissue microarray. We will also test whether intravesical human rCCL2NR treatment results in increased recruitment of T cells in a humanized murine model of BCa. The focus of this proposal is to develop an in-depth understanding of a novel cellular trafficking mechanism of immune cells in the biology of BCa and the development of a breakthrough immunotherapy strategy that will potentially improve the landscape of BCa treatment. ...