The goal of this proposal is to test the novel concept that an effective treatment for malignant melanoma is represented by a combinatorial immunotherapy, which targets malignant melanoma initiating cells (MMIC) and tumor-associated pericytes, in combination with continuous low dose cyclophosphamide, i.e. metronomic chemotherapy. This strategy stems from the following lines of evidence: i) cancer stem cells (CSC) have to be targeted for a therapy to be an effective anticancer treatment, since the CSC theory postulates that only a small cell population in tumors is self-renewing, tumorigenic and drug- and radio-resistant, ii) there is an urgent need to develop an effective therapy of malignant melanoma, since its incidence continues to increase and no effective therapy is available, and iii) a combination of T cell- and antibody-based immunotherapy that targets MMIC and tumor-associated pericytes in the tumor microenvironment is expected to counteract the multiple escape mechanisms utilized by tumor cells to avoid immune recognition. The target selected for our strategy is chondroitin sulfate proteoglycan 4 (CSPG4) which will be targeted with T- and antibody-based immunotherapy. Utilizing a survival surgery- recurrence-metastasis model, the specific aims of this proposal are to test the hypothesis that combinatorial T cell- and antibody- based immunotherapy in combination with metronomic chemotherapy is effective in inhibiting recurrence and metastatic spreading in immunodeficient mice of human MMIC isolated from cell lines and from lesions, following surgical removal of the grafted primary tumor. The outlined studies, which will benefit from the available expertise in multiple relevant research areas and the availability of unique reagents, are expected to yield novel and potentially important contributions to the fields of CSC and immunotherapy of malignant melanoma.