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Background In 2011, we first demonstrated the presence of tertiary lymphoid structures (TLSs) in human colorectal cancer linked to improved immunotherapy response, which was extended to melanoma. 1 2 However, the mechanisms underlying TLS formation and maintenance remain unclear. Given the selective occurrence of TLSs in certain solid tumors, it is likely tumor cell-intrinsic mechanisms contribute to their assembly. A key upstream mechanism promoting tumor cell antigenicity is the activation of the STING pathway, which we previously showed to be heterogeneously regulated in melanoma but can be therapeutically targeted to prime antitumor T cell responses.3 4 Intact STING activation in melanoma cells also induces T cell-homing chemokines, including CXCL10 and CCL5—present in our earlier reported 12-chemokine (12-CK) gene expression signature (GES) predicting the presence of tumor-localized TLSs.2 Building on these findings, we hypothesized that melanoma cell-intrinsic STING signaling is a significant determinant in TLS formation.Methods We assessed the correlation between STING expression and chemokines in the 12-CK GES across melanomas using cBioPortal datasets. We also determined the correlation between STING promoter methylation levels and the 12-CK GES score using the TCGA PanCancer Atlas. Using Yumm1.7 mouse melanoma models in STING-deficient hosts, we evaluated TLS formation following STING agonist ADU-S100 and 5-aza-2’-deoxycytidine (5AZADC) treatment by immunohistochemistry and multiplex immunofluorescence.Results We identified a strong correlation between STING-expressing tumors and each of the twelve chemokines in melanoma samples, particularly with secondary lymphoid organ-associated chemokines CCL19 (r = 0.61), CCL21 (r = 0.42), and CXCL13 (r = 0.60). Using TCGA PanCancer datasets, we observed a positive correlation between the expression of STING and the 12-CK GES score (r=0.37). In contrast, the methylation levels of STING were inversely correlated with the 12-CK GES score (r= -0.41). In STING-deficient mice, therapeutic activation of STING using ADU-S100 and 5AZADC in Yumm1.7 melanoma cells led to significantly enhanced tumor control (p < 0.0001) and the formation of tumor-localized lymphoid structures, with CD19+ B cell follicles, CD3+ T cells in marginal zones, and CXCL13 expression—closely resembling TLSs observed in human melanoma.Conclusions We show that epigenetic state of STING not only shapes tumor antigenicity but is also associated with the 12-CK GES and TLS presence in human melanoma. In mice, restoring STING signaling in melanoma cells through combination therapy with ADU-S100 and 5AZADC promotes the formation of tumor-localized TLSs. Considering the prognostic value of TLSs, targeting epigenetic suppression of STING to induce TLSs may guide more effective immunotherapy-based interventions.Acknowledgements This work has been supported by the Moffitt Vivarium, Flow Cytometry, Analytical Microscopy, Tissue Core Histology, Advanced Analytical and Digital Laboratory; all comprehensive cancer center facilities designated by the National Cancer Institute (P30-CA076292). This work was funded by the NCI-NIH (P30 CA076292, and P50 CA168536), CJG Fund, Chris Sullivan Fund, V Foundation, the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the Melanoma Research Foundation (MRF) Career Development Award, and Moffitt Cancer Center Melanoma and Skin Cancer Center of Excellence Award.References Coppola D, Nebozhyn M, Khalil F, Dai H, Yeatman T, Loboda A, Mulé JJ. Unique ectopic lymph node-like structures present in human primary colorectal carcinoma are identified by immune gene array profiling. Am J Pathol. 2011;179(1):37.Messina JL, Fenstermacher DA, Eschrich S, Qu X, Berglund AE, Lloyd MC, Schell MJ, Sondak VK, Weber JS, Mulé JJ. 12-Chemokine gene signature identifies lymph node-like structures in melanoma: potential for patient selection for immunotherapy?. Sci Rep. 2012;2(1):765.Falahat R, Berglund A, Putney RM, Perez-Villarroel P, Aoyama S, Pilon- Thomas S, Barber GN, Mulé JJ. Epigenetic reprogramming of tumor cell-intrinsic STING function sculpts antigenicity and T cell recognition of melanoma. PNAS. 2021;118(15).Falahat R, Berglund A, Perez-Villarroel P, Putney RM, Hamaidi I, Kim S, Pilon-Thomas S, Barber GN, Mulé JJ. Epigenetic state determines the in vivo efficacy of STING agonist therapy. Nat Commun. 2023;14(1):1573.