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Background Recombinant human adenovirus type 5 (H101) may remodel the immune tumor microenvironment (TME) via viral mimicry and immunogenic cell death. The combination treatment of H101 with PD-1 inhibitors may overcome immunotherapy resistance. This study comprehensively characterizes the dynamic mechanisms of TME changes through spatial transcriptomics.Methods This prospective single-arm study enrolled 10 patients with PD-1-resistant advanced melanoma. All patients completed at least two cycles of intratumoral H101 administration combined with intravenous anti-PD-1 therapy. Spatial transcriptomics (Xenium Prime 5K Human Pan Tissue & Pathways Panel) was employed to dynamically monitor molecular changes in the TME before and after treatment. Cell type annotation was performed based on predefined marker gene lists, and baseline cellular subtype characteristics were compared between treatment responders and non-responders.Results This study successfully enrolled 10 patients with PD-1-resistant advanced melanoma. Following H101 combined with anti-PD-1 treatment, tumor response assessment revealed complete response (CR) 0, partial response (PR) 2, stable disease (SD) 4, and progressive disease (PD) 4, achieving a disease control rate of 60% (6/10). Baseline TME analysis demonstrated that treatment responders (n=6) had significantly higher proportions of monocytes (5.83% vs 2.41%, p<0.05) and melanoma cells (72.85% vs 58.92%, p<0.01) compared to non-responders (n=4), while non-responders showed significantly elevated proportions of endothelial cells (9.33% vs 4.17%), fibroblasts (8.25% vs 3.84%), and B cells (0.63% vs 0.28%) (all p<0.05). Spatial transcriptomic dynamic monitoring revealed significant immune reprogramming in the TME following H101 treatment: CD3+/CD4+/CD8+ T cell infiltration increased 2.3-fold compared to baseline (p<0.001), with a notable increase in regulatory T cells (Tregs) that exhibited distinct transcriptional signatures from conventional suppressive Tregs, suggesting the existence of functionally reprogrammed subtypes; chemokines CXCL9 and CXCL10 expression were upregulated 4.2-fold and 3.8-fold, respectively, compared to pre-treatment levels (both p<0.001); CD68+ macrophage density increased 1.9-fold (p<0.01), however, classical M1/M2 polarization markers (iNOS, Arg1, IL-10) showed no significant changes, indicating that oncolytic virus induced a novel macrophage activation state.Conclusions This study systematically elucidates for the first time the unique immune reprogramming patterns induced by H101 combined with anti-PD-1 therapy through spatial transcriptomic analysis, including the differentiation of functionally reprogrammed Treg subtypes, chemokine-mediated effector immune cell recruitment, and the formation of non-classical macrophage polarization states. These findings provide novel molecular mechanistic insights into how oncolytic viruses reshape the immunosuppressive tumor microenvironment and establish an important theoretical foundation for personalized precision treatment strategies in PD-1 inhibitor-resistant melanoma.