Project Summary/Abstract: Recent success in utilizing patients’ own immune cells to combat metastatic cancers highlights the ability of the immune system in providing sustained remission for patients with late-stage cancers like melanoma. However, the role of the immune system in eliminating premalignant cells and preventing their progression to invasive cancers remains uncertain. To determine the benefit of activating the immune system to prevent cancer development and recurrence, we study the immune pathways that lead to effective immune activation against early phases of skin cancer development. Non-melanoma Skin cancers (NMSCs) are collectively the most commonly diagnosed cancers in the United States. cutaneous squamous cell carcinoma (SCC) is associated with severe morbidities including disfigurement, ulceration and infection, and a mortality rate similar to that of melanoma. Importantly, the SCC precursors can be clinically identified and treated. Based on compelling preclinical data, we have performed a randomized double-blind clinical trial in which we find topical calcipotriol together with 5-fluorouracil (5-FU) to promote a robust adaptive immune activation against skin cancer precursors, actinic keratoses, and their complete clearance. Importantly, this immunotherapy led to a significantly lower risk of SCC compared to 5-FU monotherapy group in 3 years post-trial. In order to determine the precise mechanism of calcipotriol plus 5-FU action against early premalignant cells in the skin, and to fully harness its potential to prevent skin cancer, we aim to (1) determine the signals that direct the immune cells to the premalignant cells treated with calcipotriol plus 5-FU combination, (2) investigate the mechanism by which these activated immune cells eliminate the targeted tumor cells, and (3) identify the immune cells and factors that mediate the long-term protective effects of calcipotriol plus 5-FU against SCC. The outcomes of the proposed research will establish a fundamental role for immune activation in combating the early stages of skin cancer development and provide the rationale for the use of calcipotriol plus 5-FU combination in skin cancer immunotherapy and immunoprevention.