Single-Cell Analysis of Residual Esophageal Squamous Cell Carcinoma after Neoadjuvant Immunochemotherapy Reveals TFAM-Mediated Immunoregulation in Dendritic Cells

Abstract Neoadjuvant immunochemotherapy (nICT) has emerged as a promising neoadjuvant strategy for esophageal squamous cell carcinoma (ESCC). Identification of the factors affecting responsiveness to nICT could help further improve treatment efficacy. In this study, we performed single-cell analysis on 14 patients with ESCC undergoing nICT and revealed tumor microenvironment (TME) features associated with differential treatment responses. Nonnegative matrix factorization (NMF) identified five coordinated cellular programs with distinct response associations. Specifically, the NMF3 program, mainly comprising immunosuppressive cell subsets, was enriched in the minimal or no pathologic tumor regression [tumor regression staging 3 (TRS3)] group, in which regulatory T cells (Treg) and dendritic cells (DC) exhibited a close correlation. In addition, elevated expression of mitochondrial transcription factor A (TFAM) in DCs was associated with increased Treg infiltration in the TRS3 group. A myeloid-specific Tfam knockout mouse model showed that TFAM deficiency reversed the immunosuppressive TME, inhibited tumor growth, and enhanced the response to anti–programmed cell death protein 1 therapy in ESCC. Mechanistically, TFAM deficiency in DCs activated the stimulator of IFN genes (STING)–TBK1–IRF3 pathway, thereby promoting DC maturation to enhance antitumor immunity. Overall, this study characterized the TME in residual ESCC after nICT and revealed an association between elevated TFAM expression in DCs and poor responsiveness to nICT. These findings indicate the critical role of TFAM deficiency in DCs in activating antitumor immunity, highlighting the potential of targeting TFAM to improve the efficacy of immunotherapy and optimize therapeutic strategies. Significance: TFAM mediates dendritic cell activation through the STING-TBK1-IRF3 pathway and is associated with responsiveness to neoadjuvant immunochemotherapy in esophageal squamous cell carcinoma, identifying TFAM as a putative immunotherapeutic target and biomarker.