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Background Melanoma is the most common form of skin cancer and a leading cause of skin cancer-related mortality. While adoptive immunotherapy has shown promise in melanoma treatment, its efficacy is often limited by the tumor’s low immunogenicity. Here, we present a novel strategy to enhance the tumor-targeting capacity of autologous T cells through ex vivo education using small-molecule-treated melanoma cells.Methods Murine B16 melanoma cells were treated with small molecules (I-BET726, JQ1, C-170) to enhance tumor immunogenicity, then irradiated and co-cultured with mouse splenocytes in the presence of GM-CSF and CpG-A. After five days, the phenotype and function of the trained splenocytes were assessed via flow cytometry and IFN-γ ELISA. CD3 + and CD8+ T cells were isolated from trained splenocytes, and their cytotoxicity was evaluated using IncuCyte live-cell imaging. Therapeutic efficacy was tested in two melanoma mouse models. In the low tumor burden model, mice received 1×104 B16 cells followed by immediate administration of trained or untrained splenocytes. In the high tumor burden model, mice were injected with 1×105 B16 cells, and trained splenocytes were delivered on day 7. T cell trafficking was monitored in vivo using DiR dye.Results Trained splenocytes exhibited a marked increase in activated CD8 + T cells, along with an enrichment of double-negative CD3+ T cells (CD4–CD8–) (figure 1). Expression of exhaustion markers (TIM-3, LAG-3, TIGIT) were elevated in trained CD8+ T cells but diminished following cryopreservation. IFN-γ secretion and cytotoxicity assays confirmed robust anti-tumor activity, with CD8+ T cells alone demonstrating the greatest cytotoxic effect. In the low tumor burden model, all control mice (n=3) developed tumors, whereas none of the treated mice (n=3) showed tumor growth by day 19. In the high tumor burden model (n=5/group), tumor progression was significantly suppressed in the treated cohort (p=0.002) (figure 2). In vivo imaging confirmed efficient tumor homing of trained T cells via both local and systemic delivery.Conclusions This study supports the potential of ex vivo-trained, tumor-primed T cells as an effective and adaptable platform for adoptive immunotherapy in melanoma.Abstract 359 Figure 1Splenocytes trained with BET/JQ1/C-170–treated B16 cells for 5 days. Flow cytometry: CD8+ T cells, Tim3+, Lag3+, CD137+, CD39+, and KLRG1hiCD127lo subsets were increased, while PD1+ remained unchanged. Mean±SD; *p<0.05, **p<0.01, *p<0.001 (n=3)Abstract 359 Figure 2Tumor-trained splenocytes reg melanoma growth. (A) Low burden: B16 (1×104) + trained splenocytes (1×107) simultaneously lead to strong tumor reduction. (B) High burden: B16 (1×105) + splenocytes (1.5×107, Day7) reduce tumor growth at d19