Investigating the combination of natural killer immunotherapy with radiation therapy for muscle invasive bladder cancer

Background: Muscle-invasive bladder cancer (MIBC) is commonly treated with radical cystectomy (RC) or radiation therapy (RT). However, both approaches have significant limitations: RC is associated with a major impact on quality of life and a high risk of metastasis, while RT, despite preserving the bladder, has a recurrence rate of about 30%, largely due to the presence of radio-resistant tumors. This study explores the potential of combining adoptive Natural Killer (NK) cell immunotherapy with RT to enhance efficacy and offer a more effective bladder-preserving alternative. Methods: Three human bladder cancer cell lines (UM-UC3, UM-UC1, RT4) were treated with RT (2 × 3 Gy), NK cells (5 × 10⁶ cells IV, twice weekly for two weeks), or the combination. Cell viability was assessed using a colorimetric Kit-8 assay at effector-to-target ratios (1:1, 2:1, 5:1). In vivo efficacy was tested in a subcutaneous mouse model. Clinical relevance was examined using NanoString GeoMx spatial transcriptomic data from MIBC patients treated with RT (responders n = 107; non-responders n = 35). NK infiltration and NK-associated gene signatures were analyzed via Singscore and xCell deconvolution. Results: RT significantly reduced viability in UM-UC3 and RT4 cells (p<0.01), and the combination of RT+NK further enhanced cytotoxicity (p<0.001). In UM-UC1, NK cells alone markedly decreased viability (p<0.001), with no additional reduction observed when combined with RT. In vivo, combination therapy produced a sustained reduction in tumor growth compared to RT (p=0.0117) or NK therapy alone (p=0.0004), demonstrating enhanced therapeutic efficacy. Clinically, responders exhibited higher NK infiltration (p<0.01), elevated NK anti-tumor scores (p<0.01), and increased expression of MICA, NKG2C, and HLA-E, molecules involved in NK-cell activation and target-cell recognition (all p<0.05). Conclusion: Overall, our results suggest that NK cells can play an important role in improving the response to RT in MIBC. We observed that combining NK cell therapy with RT led to better tumor control in preclinical models, and clinical data supported this by showing that patients who responded to RT had higher NK cell infiltration and expression of key immune markers. While further studies are needed to confirm these findings, this work adds to growing evidence that NK cells could play a role in modulating treatment response. Exploring this further may help identify patients who are more likely to benefit from combination strategies and guide future efforts toward more personalized approaches in MIBC.