Project Summary Immune therapy in cancer has made great strides in the past decade. However, not all tumor types benefit from current therapies. Additionally overcoming resistance to both targeted and immune therapy for second- or third- line therapies remains a critical challenge for all cancers. Developing experimental models to advance new immune therapy strategies has faced impediments, hindering progress. Most studies require clinical trials for even minor advancements in approaches, protocols, timing, dosage, and sequential treatments. Our laboratory has developed a new experimental model of immune cell-tumor interactions that will fill an important gap in immune oncology investigations because it is: i) autologous, as immune and tumor cells are derived from the same patients; and ii) reproducible, as tumor cells and iPS (induced pluripotent stem) cells have unlimited growth capacities. The source of melanoma cells are the patients’ lesions, which are maintained as PDX (patient- derived xenografts), organoids, or cell lines. iPS cells are conveniently derived from peripheral blood mononuclear cells or normal fibroblasts. Three cell types, monocytes, pre-T cells and thymic epithelial cells, are independently developed from the same iPS cultures. The monocytes are loaded with tumor antigens (tumor cells, extracts, proteins, or peptides), acting as antigen presenting cells. When the three cell types are placed together as thymic organoids, the T cells mature and appear stable and can be expanded. The first aim proposes to investigate T cell receptor diversity and achieving tumor specificity. The second aim characterizes tumor cell-T cell interactions and how the system can be used to overcome resistance to both checkpoint inhibitors and targeted therapies. We plan to develop melanoma cell-iPS cell pairs from patients resistant to checkpoint inhibitors including anti-PD1 and/or CTLA4 and those who are resistant to both targeted and immune checkpoint therapies. We will then develop complex organoids with stromal and immune cells to mimic tumor-T cell interactions within an intact microenvironment. Finally, we will transfer the in vitro model to an in vivo setting using our extensive experience in humanizing mice. Furthering our on-going collaboration with NCI’s Patient-Derived Models Repository (PDMR) will enable us to expand the technology and conduct critical research aimed at developing new approaches to overcome therapy resistance. Moreover, this collaboration will allow for sharing our new models with the scientific community and extending this innovative approach from melanoma to other malignancies. 1