SUMMARY While significant progress has been made in the past decade with immunotherapy for metastatic melanoma and renal cell carcinoma (RCC), not all patients respond, and others develop resistance over time. Hundreds of novel regimens are being developed to overcome primary and acquired resistance. Some combinations increase the infiltration of cytotoxic T cells in the tumor to kill cancer cells, while others modulate tumor-associated macrophages (TAMs) to enhance the T cell activity in the tumor microenvironment. However, toxicity to patients is an important limitation to immunotherapy regimens. Non-invasive imaging techniques are needed to determine response to combination therapy to save potential non-responders from unnecessary toxicity. Current imaging techniques do not assess the dynamic changes in the levels of T cells and TAMs at the same time. The overall hypothesis is that imaging the infiltration of cytotoxic T cells and TAMs concurrently can better determine response to immunotherapy regimens. The goal of this proposal is to develop non-invasive imaging probes for single photon emission computed tomography (SPECT) imaging of T cells and TAMs concurrently to determine response to combination therapy. We anticipate that SPECT will accurately recapitulate pathology and may ultimately replace on-treatment biopsies in patients who might have pseudoprogression. SPECT may also complement tissue-based analyses to further interrogate the tumor microenvironment through digital spatial profiling (DSP), an in situ profiling method that preserves spatial information. DSP can quantify multiple biomarkers simultaneously in immune cells responsible for the tumor’s sensitivity to combination therapy. In our preliminary work we developed the tools to implement the tracking of CD8+ T cells and CD68+ TAMs in vivo with radiolabeled antibodies. We will test our SPECT agents in established murine tumor models of melanoma and RCC in mice with intact immune systems. We will characterize their binding properties to CD8+ T cells and CD68+ TAMs in vitro (Aim 1). We will image tumoral T cell and TAM infiltration in vivo by SPECT imaging in syngeneic tumor models of melanoma and RCC and assess activation status of immune cells by DSP after immunotherapy in murine and patient tissues (Aim 2). These studies will lay the foundations for future clinical trials of dual-isotope SPECT in immunotherapy-treated patients.