Single-cell profiling unveils the immuno-favorable tumor microenvironment remodeling after successful neoadjuvant therapy for advanced gallbladder cancer.

[BACKGROUND] Neoadjuvant treatment has demonstrated clinical benefits in advanced gallbladder cancer (GBC). However, the overall response rate remains suboptimal, and the underlying mechanisms driving treatment efficacy are not fully understood.

[MATERIALS AND METHODS] This study aimed to evaluate the therapeutic effects of neoadjuvant chemo-immunotherapy (NAT) combining gemcitabine, nab-paclitaxel, and anti-PD-1 immunotherapy in advanced GBC, and to investigate the associated tumor microenvironment (TME) alterations. Single-cell RNA sequencing and multiplex immunohistochemistry were utilized to analyze the cellular composition of the TME in patients who successfully underwent downstaging.

[RESULTS] NAT significantly reshaped the tumor-immune landscape, characterized by an expansion of follicular helper T (Tfh) cells and the formation of tertiary lymphoid structures (TLSs) in 50% of treated tumors. Inflammatory cancer-associated fibroblasts (CAFs) increased and exhibited upregulation of CCL19 and CXCL12 , potentially promoting Tfh cell recruitment and TLS formation. Additionally, NAT led to an expansion of GZMB + cytotoxic CD8 + T cells with an exhausted phenotype in GBC, but not in adjacent normal tissues. The treatment also increased the number and effector functions of natural killer cells while reducing tumor-promoting macrophages and angiogenesis-related CAFs. Furthermore, NAT decreased the cancer stem cell-like subpopulation while increasing a cancer cell subset with enhanced antigen-presenting capacity.

[CONCLUSION] This study suggested the potential efficacy of NAT in advanced GBC and revealed alterations in the TME following treatment. The findings provided insights into the mechanisms underlying NAT responses and offered valuable directions for optimizing therapeutic strategies in GBC.