Enhanced antitumor effect of combination intravesical mitomycin C and bacillus Calmette-Guerin therapy in an orthotopic bladder cancer model

Abstract

Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) is currently the most successful adjuvant agent for the treatment and/or prophylaxis of non-muscle-invasive bladder cancer (NMIBC). However, NMIBCs recur in 60-70% of cases and 30% of these recurrent tumors present with a higher grade and more invasive properties. Patients that do not respond to intravesical BCG therapy are considered to be a challenge for urologists. Thus, novel conservative possibilities should be explored. To test the efficacy of a novel therapeutic approach, we examined the antitumor effect of combination therapy by intravesical administration of mitomycin C (MMC) plus BCG, infusing the two drugs simultaneously, in an orthotopic bladder cancer model. Intravesical BCG and MMC administration showed a dose-dependent survival (n=8 per group). The combination of MMC and BCG provided a significant survival advantage compared to the BCG-alone (p=0.035) and MMC-alone groups (p=0.040) (n=8 per group). The group with combined MMC/BCG exhibited a survival period similar to that achieved with an amount eight times higher that of BCG (n=10 per group). Ki-67 labeling index of cancer cells, showing tumor proliferation, was significantly lower in the combined group compared to the BCG-alone (p<0.05), MMC-alone (p<0.01) and control groups (p<0.01). No difference was detected between the combined group and the BCG-alone group with regard to CD3, T-cell infiltration and CD68 macrophage activity. The combined MMC/BCG treatment decreased the tumor appearance rate, improved the survival period and reduced the cellular proliferation rate in tumors compared to the BCG-alone treatment. The results suggest that the combined intravesical MMC/BCG treatment induced an enhanced antitumor effect against bladder tumors. The combined MMC/BCG treatment also showed a survival period similar to that achieved using a dose eight times higher of BCG-alone.

Figure 1

Kaplan-Meier analysis of the survival rates of mice according to five times intravesical instillation treatment, following MBT-2 cell implantation in an orthotopic bladder cancer model. A significant survival advantage in the combined BCG/MMC (◆) group was observed, compared to that in the BCG-alone (●) group (p=0.035) or the MMC-alone (■) group (p=0.040).

Figure 2

The mice were randomly allocated to four groups 17 days after the intravesical implantation of 1×10⁶ MBT-2 cells. A single intravesical administration of 100 μl of PBS, 50 μg of MMC in 100 μl of PBS, 100 μg of BCG in 100 μl of PBS or a combination of 50 μg of MMC and 100 μg of BCG in 100 μl of PBS were instilled into the bladder. The bladders were removed and fixed with 10% formalin 1 day after the single intravesical instillation, and the bladder tumor specimens were then immunostained with the antibodies, Ki-67, CD68 and CD3. Statistical analysis among the various treatment groups (mean ± SD, n=4, ANOVA, Bonferroni’s multiple correction) were examined. Cells labeled with Ki-67, CD68 and CD3 antibodies were expressed as the percentage to the total cell numbers per mm².