Cytotoxic lymphocytes can inhibit growth of tumors by seeking out and killing malignant cells. Recent retrospective studies across many different human cancers have shown that the presence of cytotoxic CD8 T cells within the tumor core is associated with better prognosis. Still, in most cancers, the immunosuppressive tumor microenvironment inhibits the capacity of tumor infiltrating lymphocytes (TILs) to lyse their targets. Recently, it was shown that such immunosuppression is mediated not only by functional paralysis of T cells but also by physical barriers that limit TIL proximity to cancer cells. This immunosuppressive milieu created by tumors remains one of the principal obstacles limiting host immunity to cancer and success of current therapeutic approaches. The objective in this proposal is to determine how collagen fibers influence retention of TIL in specific tumor compartments and impair their functional potential. The central hypothesis is that stromal matrix restrains anti-tumor immunity by preventing cytotoxic lymphocytes from penetrating compartments of the tumor that are rich in cancer cells. This hypothesis is based on imaging studies demonstrating that T cells are excluded from the core of human and mouse tumors by a collagen-rich cortical barrier and functional studies showing that fibroblast-derived collagen can potently suppress T cell activation. The rationale for the proposed research is that defining mechanisms by which TILs are rendered ineffective has the potential to translate into new immunotherapeutics. Two specific aims have been proposed that will z test the hypothesis: 1) Address the contribution of tumor matrix architecture in the compartmentalization and function of tumor-infiltrating CD8 T cells in vivo; and 2) Demonstrate therapeutic efficacy of a nanoparticle- based drug delivery system for localized disruption of tumor collagen matrix. In the first aim the impact of stromal matrix on localization and activation of TILs will be evaluated. Pharmacologic and genetic approaches will be exploited to examine the consequences of tumor matrix disruption on TIL compartmentalization and effector differentiation. In the second aim pharmacokinetics, biodistribution, pharmacodynamics, and therapeutic efficacy of a novel nanoparticle formulation targeting tumor matrix will be evaluated in a mouse model of triple negative human breast cancer. The approaches are innovative, making use of cutting edge imaging technologies and nanocarriers to enable in depth characterization of the relationship between TIL compartmentalization and matrix architecture. The long-term goal of this research is to create efficacious immunotherapeutics for treatment of cancer. The proposed studies are significant because understanding how TILs lose tumoricidal potential in the tumor microenvironment could enable development of next generation cancer therapies that interfere with immunosuppressive mechanisms. Unleashing the host's powerful armament of cytotoxic TILs has the potential to extend and improve the quality of life for cancer patients.