High-dose vitamin C improves BCG immunotherapy's efficacy in a murine ectopic model of bladder cancer

Management of non-muscle-invasive bladder cancer (NMBIC) typically involves transurethral resection of bladder tumor (TURBT) followed by intravesical Bacillus Calmette-Guérin (BCG) immunotherapy. However, 30-50 % of patients may not respond to BCG or experience recurrence. High-dose vitamin C (VitC) has shown promise in improving the outcome of immunotherapies such as immune checkpoint blockade. However, evidence of its ability to augment BCG immunotherapy in bladder cancer remains lacking. Tumor-bearing male C57BL/6 mice were divided into four treatment groups receiving placebo, BCG, VitC, and BCG/VitC, respectively. After 22 days, tumors were collected and subjected to histopathological evaluation using H&E staining. Expression of CD4, CD8, NK1.1, F4/80, CD80, CD163, and Ki67 in the tumor microenvironment was assessed by immunohistochemistry (IHC). mRNA levels of Th1 cytokines (IL-2, IL-12, IFNG, TNFA), Th2 cytokines (IL-4, IL-5, IL-6, IL-10), and NOS2 were determined using qPCR. Serum levels of IL-12 and TNF-α were quantified using enzyme-linked immunosorbent assay (ELISA). The results demonstrated that VitC remarkably improves the efficiency of BCG immunotherapy. The combination of BCG and VitC resulted in greater inhibition of cell proliferation, reduced tumor sizes, and increased infiltration of inflammatory cells compared to BCG monotherapy, indicating a synergistic effect on the antitumor immune responses. Additionally, BCG/VitC treatment led to a predominance of M1 macrophages and a substantial increase in the infiltration of natural killer cells, as well as T-lymphocytes. Furthermore, mice receiving combination therapy exhibited the highest levels of intratumoral Th1 cytokines while decreasing Th2 cytokines. In conclusion, VitC appears to play a critical role in enhancing BCG-mediated immune responses against NMBIC.