Introduction:

Although Bacillus Calmette-Guérin (BCG) is widely used to treat bladder cancer, its precise mechanism, particularly in sustaining a long-lasting immune response, is still not fully understood. Tumor immune microenvironment (TIME) refers to the complex and dynamic environment surrounding a tumor, which includes a variety of immune cells, signaling molecules, and cellular structural components. Tumor-infiltrating lymphocytes (TIL) have been postulated to have prognostic value in various cancers, including melanoma, colon cancer, ovarian cancer, and bladder cancer, where presence of TIL signifies favorable oncologic outcomes. TILs also play an important role in TIME, mediating its response to tumorigenesis and immunotherapeutic drugs. Neutrophils are drivers of inflammation, and a key player in TIME, they are involved in the initiation, development and progression of bladder cancer. This study features mice treated with BCG, we demonstrate that TIL and neutrophil count in bladder stroma correlates with tumor stage and response to treatment.

Methods:

Specific pathogen-free C57BL/6 male and female mice were obtained from Taconic Laboratory. BBN was acquired from TCI America and administered at 0.05% in water, provided ad libitum and replenished weekly 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. Following this, mice received regular water until study completion. Tumors were evaluated via ultrasonography starting at week 14. Upon detecting intravesical tumors, intravesical BCG (TICE) treatment (200 µl of 1x10^6 CFU/ml) was given weekly for up to 6 weeks, with BCG retained in the bladder for at least 1 hour under anesthesia. Bladders were collected for histopathological analysis, and hematoxylin and eosin (H&E) stains were obtained. (Figure 1A)

Results:

Tumor-infiltrating lymphocytes showed significant association with tumor stage and treatment response to BCG. In BCG-treated mice, mean TIL counts for stages T0-T4 were 96±13.2 (T0), 11.1±15.1 (T1), 15±4.2 (T2), and 4 .1 (T4) (Figure 1B). Neutrophil count also showed a significant association with tumor stage. In the BCG-treated cohort, mean neutrophil counts for stages T0-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) (Figure 1D). 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. The ROC analysis for predicting poor response (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% (Figure 1E).

Conclusion:

In conclusion, this study demonstrates that in a murine bladder cancer model treated with BCG, both the neutrophil count and the neutrophil to lymphocyte ratio (NLR) are positively correlated with tumor stage. Using the NLR to predict poor response to BCG (defined as ≥ T2), the ROC curve yielded an AUC of 0.72, with a sensitivity of 81.25% and a specificity of 87.50%. Additionally, tumor-infiltrating lymphocyte (TIL) counts were significantly associated with the response to BCG treatment. Further validation in human studies is underway.

Funding: BCAN Young Investigator Award