Association between neutrophil and tumor-infiltrating lymphocyte count and bladder cancer stage and treatment response in BCG-treated mice models.
Authors: Wang Betty , Nandwana Devika , Campbell Rebecca , Weight Christopher , Almassi Nima , Haywood Samuel , Abouassaly Robert , Abbosh Philip , Bukavina Laura 
Published: February 2025

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Background:
Although Bacillus Calmette-Guérin (BCG) is widely used to treat bladder cancer, its mechanism is still not fully understood. Tumor-infiltrating lymphocytes (TIL) have been postulated to have prognostic value in bladder cancer, where presence of TIL signifies favorable outcomes. TILs play an important role in the tumor immune microenvironment, mediating its response to tumorigenesis and immunotherapeutic drugs. Neutrophils are also key players in the initiation, development and progression of bladder cancer. This study features BCG-treated mice, demonstrating that TIL and neutrophil count in bladder stroma correlates with tumor stage and response to treatment.
Methods:
Specific pathogen-free C57BL/6 mice were obtained. BBN acquired from TCI America was administered at 0.05% in water, provided ad libitum to half of the cages, while the other half received regular water. BBN administration began at 8-10 weeks of age and continued for 12 weeks. Subsequently, mice received regular water until study completion. Tumors were evaluated via ultrasonography starting at week 14. Upon detecting intravesical tumors, intravesical BCG treatment was given weekly, up to 6 weeks. Bladders were harvested for histopathological analysis, H&E stains were obtained. BCG-treated bladder tumors collected from 85 patients via transurethral resection were similarly analyzed.
Results:
TILs showed significant association with tumor stage and BCG-treatment response. In BCG-treated mice, mean TIL counts for stages T0 to T4 were 96±13.2 (T0), 11.1±15.1 (T1), 15±4.2 (T2), and 4.1 (T4). Neutrophil count also showed a significant association with tumor stage progression. In BCG-treated mice, the mean neutrophil counts for stages T0 to T4 were 30.25±11.6 (T0), 37.7±18 (T1), 46±34 (T2), 48±25.5 (T3), and 75.5±44 (T4) (p<0.01). We further analyzed the neutrophil to lymphocyte Ratio (NLR) to predict tumor response. Using an optimal threshold of 1.93, NLR was calculated as the ratio of neutrophils to total lymphocytes in the stroma. ROC analysis for predicting poor response to BCG (defined as ≥ T2) yielded an AUC of 0.72, demonstrating a reasonably good predictive ability. Sensitivity was 81.25%, specificity was 87.50%, and overall accuracy was 83.33%. Tumor-infiltrating neutrophil (TIN) count in humans showed a significant association to tumor recurrence, with an OR of 1.25 (p=0.03).
Conclusions:
In conclusion, this study demonstrates that in a BCG-treated murine bladder cancer model, both the neutrophil count and the NLR are positively correlated with tumor stage. Using NLR to predict poor response to BCG, ROC curve yielded an AUC of 0.72, with a sensitivity of 81.25% and specificity of 87.50%. Additionally, TIL counts were significantly associated with response to BCG treatment. Validation in humans showed a significant association between TIN count and tumor recurrence.