Tumor-infiltrating lymphocytes demonstrate potent anti-tumor efficacy and synergize with PD-1 blockade in bladder cancer

Abstract Background Bladder cancer (BCa) is one of the most prevalent urological malignancies globally with substantial clinical challenges characterized by high recurrence rates and limited treatment options for advanced disease. Although immune checkpoint inhibitors (ICIs) provide clinical benefit, their efficacy is restricted, with objective response rates of only 20–25%. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has shown remarkable success in other solid tumors but remains largely unexplored in BCa. The emergence of patient-derived organoids (PDO) offers a physiologically relevant ex vivo platform for functionally evaluating TIL by preserving tumor heterogeneity. Methods TIL were isolated from BCa patient specimens and robustly expanded ex vivo using interleukin-2 (IL-2) and a rapid expansion protocol (REP). Expansion efficiency was quantified, and TIL phenotypes were characterized via flow cytometry. Transcriptional and clonal dynamics were profiled using single-cell RNA sequencing (scRNA-seq) coupled with T-cell receptor (TCR) sequencing. The anti-tumor efficacy of TIL, both as monotherapy and in combination with the PD-1 inhibitor Nivolumab, was assessed against BCa cell lines and autologous PDO through viability (ATP), cytokine release (IFN-γ, ELISA), and apoptosis (caspase 3/7) assays. In vivo validation was performed in a 5637 cell-line-derived xenograft (CDX) mouse model. Results TIL were successfully expanded from 33 of 48 BCa samples with high yields of viable cells. Expanded TIL were predominantly CD8 + cytotoxic T lymphocytes. Integrated scRNA and TCR-seq analysis revealed that REP enriched for cytotoxic effector clones and reduced regulatory T-cell populations. TIL mediated potent tumor-killing efficacy against BCa cell lines and autologous PDO in vitro and significantly suppressed tumor growth in vivo. Critically, combining TIL with Nivolumab synergistically enhanced tumor cell death in PDO and resulted in near-complete tumor suppression in the CDX model, significantly outperforming TIL monotherapy. Conclusions TIL isolated from bladder cancer patients can be robustly expanded in vitro into a population enriched with CD8⁺ T cells, which exhibited potent anti-tumor activity across both in vitro and in vivo models. Notably, combining TIL with PD-1 blockade significantly enhanced efficacy, establishing TIL-based adoptive cell therapy as a viable immunotherapeutic strategy for bladder cancer and provide a compelling rationale for the clinical translation of TIL combined with Nivolumab.