ABSTRACT – Project 1 Project 1 will explore how photodynamic therapy (PDT) can be used for immunological priming in new combination treatments for skin cancer. Nonmelanoma skin cancers (NMSC), comprising squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), are the most common of all human cancers, representing a significant healthcare burden (>3 million U.S. cases/year), with increased lethality for certain patient subsets, e.g. organ transplant recipients. We showed previously that aminolevulinate (ALA)-based PDT can be effective for NMSC patients, and that 5-fluorouracil (5FU) and Vitamin D (VitD) further improve treatment outcomes; yet tumor clearance rates remain poor for large and advanced NMSC. To ameliorate this situation, we propose to exploit the concept of photodynamic priming (PDP), a natural consequence of PDT. PDT triggers immunogenic cell death, enhances influx of tumor infiltrating lymphocytes (TILs), and induces an adaptive anti-tumor immune response, sensitizing the tumor microenvironment to the effects of immune checkpoint inhibition. Our preliminary data show that a low-intensity PDT regimen can clear tumors in a murine SCC model, accompanied by induction of damage-associated molecular patterns (DAMPs) and recruitment of neutrophils, macrophages and TILs, along with altered expression of immune checkpoint molecules such as PD-1. These findings set the stage for a further combination approach using PDT and immune checkpoint inhibitors (ICI). In Aim 1 (preclinical) we will evaluate in murine SCC and BCC models the therapeutic response to various combinations of PDT, 5FU, VitD, and ICI agents, and measure specific timing of TIL recruitment, checkpoint molecule expression, and tumor clearance. In Aim 2 (clinical studies) we will determine the nature and timing of immune responses in BCC and SCC patients, post-PDT. Tumors will be subjected to PDT (guided by Core C dosimetry to measure photosensitizer levels); then after 1-to-14 days the tumor will be surgically excised and analyzed for checkpoint molecule expression and intratumoral immune cell infiltration using the histological immunoscore (IS; Project 3). Systemic T-cell activation in circulating blood will be measured in collaboration with Project 2. Another arm of these studies will test effects of 5FU or VitD pretreatment of NMSC tumors upon the magnitude of anti-tumoral immune responses. Aim 3 will feature exploratory studies to validate the IS approach for measuring TIL recruitment in human SCC tumor models in vitro. In collaboration with Project 3 and Core B, 3D patient-derived tumor-immune organoids (PDIOs), consisting of stratified SCC cultures admixed with PBMCs (lymphocytes) will be tested for differential immune cell activation following PDT using cutting-edge hyperspectral optical cytometry techniques developed in Core B that can image multiple immune markers simultaneously. Relevance and Impact: Overall, this Project will benefit patients by providing the ba...