Abstract:

Objective: To identify the cytokine genes influencing the formation of tertiary lymphoid structures (TLS) through CRISPR-Cas9 library screening, and to discover potential key regulatory molecules, providing new targets for enhancing the efficacy of bladder cancer immunotherapy. Methods: Based on a mouse whole-genome library, 44 chemokine-related genes were identified, and an single-guide RNA (sgRNA) library targeting these genes was designed and constructed, with three sgRNAs assigned to each gene. Using a lentiviral packaging system, the library plasmids were used to transfect HEK293T cells to generate a lentiviral library, which was then used to infect the mouse bladder cancer cell line MB49. Purinomycin selection was performed to obtain the MB49-mCherry cell line stably over-expressing chemokines. The cells were inoculated into the peritoneal cavity of C57BL/6 mice to establish a bladder cancer xenograft model, and tumor growth was monitored. Three weeks later, tumor tissue was excised, genomic DNA was extracted for high-throughput sequencing, and sgRNA enrichment was analyzed to screen for differentially expressed cytokine genes. Concurrently, immunohistochemical staining was performed to detect TLS markers CD20 and CD3, and the number, distribution, and maturity of TLS were assessed. The selected candidate genes were validated individually in vivo to further confirm their impact on TLS formation. Results: We successfully constructed a cytokine gene library containing 132 sgRNAs, covering 44 chemokine genes. Following lentiviral infection, we obtained the MB49-mCherry cell line, which stably expressed the library, and isolated dead Cas9-positive monoclonal cell lines via flow cytometry to ensure the homogeneity and reproducibility of subsequent experiments. Intratumoral tumor experiments in mice revealed that the number of TLS cells in the experimental group was significantly higher than in the control group, primarily distributed at the tumor margins. High-throughput sequencing results showed that, compared with the control group, in the experimental group, Cxcl16 sgDNA was significantly enriched, while Ccl20 and Cx3lc1 sgDNA levels decreased compared with baseline (P < 0.05). Further validation of the individual roles of each factor via intraperitoneal injection revealed that the number of TLSs in tumors decreased in the group treated with the CX3CL1 chemokine, suggesting that CX3CL1 might negatively regulate TLS formation. Immunohistochemical results showed that in the CX3CL1-treated group, the aggregation of CD20-positive B cells and CD3-positive T cells in the tumor tissue was reduced, and the TLS structure was incomplete. Conclusion: Through CRISPR-Cas9 library screening combined with in vivo validation, this study successfully identified CX3CL1 as a potential negative regulator of TLS formation in bladder cancer. High CX3CL1 expression was associated with a reduction in TLS numbers, suggesting that it might exert an inhibitory role in the immune microenvironment of bladder cancer. This finding provides new clues and research directions for understanding the molecular mechanisms of TLS formation in bladder cancer. However, whether CX3CL1 can serve as an immunotherapeutic target remains to be further validated through clinical specimen analysis, multidimensional mechanistic investigation, and immunotherapy response correlation studies.

Key words: Urinary bladder neoplasms, CRISPR-Cas systems, Tertiary lymphoid structures, Gene library, Chemokines