Single-Cell RNA Sequencing Reveals T-Cell Metabolic Activation and SGPL1 as a Candidate Predictor of PD-1 Resistance in Urothelial Carcinoma

Background/Objectives: Immune checkpoint blockade targeting the PD-1/PD-L1 axis is a standard treatment for urothelial carcinoma (UC), but 20–30% of patients develop resistance. The underlying mechanisms, particularly regarding tumor microenvironment metabolic reprogramming, remain unclear. Methods: We performed single-cell RNA sequencing on pre-treatment tumors from eight pembrolizumab-treated UC patients (three responders, five non-responders). Bioinformatic analyses included cell clustering, pathway enrichment, and cell–cell communication, validated using public datasets. Functional experiments involved SGPL1 knockdown in MB49 cells and a syngeneic PD-1-resistant murine model (MB49-R5). Results: Responders exhibited a higher proportion of infiltrating T-cells. Unexpectedly, CD4+ subsets, rather than CD8+ cells, were numerically enriched in responders. However, all T-cell subsets in responders demonstrated enhanced oxidative phosphorylation-dominant metabolic activity. Enhanced macrophage–T-cell crosstalk was observed, with macrophages exhibiting M1-like polarization driven by CD4+ T-cell-derived IFN-γ and CD40L signaling. In contrast, non-responders displayed dominant tumor-derived MIF–CD74 signaling and M2 polarization. Integration of public datasets identified SGPL1 as a key metabolic regulator associated with poor prognosis and reduced immune activation. Functional experiments demonstrated that SGPL1 knockdown inhibited tumor proliferation and restored anti-PD-1 sensitivity, accompanied by increased T-cell infiltration. Conclusions: T-cell metabolic activation drives anti-PD-1 responses in UC, and CD4+ T-cell-mediated M1 polarization and tumor-intrinsic SGPL1 crucially shape therapeutic outcomes, highlighting SGPL1 as a candidate metabolic target to overcome PD-1 resistance.