Introduction:

Interferon alpha (IFNα) gene therapy with Nadofaragene firadenovec is emerging as a promising therapeutic option for patients with BCG-unresponsive non-muscle invasive bladder cancer (NMIBC). While results with monotherapy are encouraging, not all patients achieve durable responses. We hypothesized that combination therapy with gemcitabine, a nucleoside analog, and interferon gene therapy might have added benefit for patients with NMIBC as has been seen in other cancers. Herein, we sought to examine the efficacy and mechanisms of cell killing for combination therapy with either murine IFNα or IFNβ with Gemcitabine in 3 different murine bladder cancer (BLCA) cell lines.

Methods:

MB49, BBN975 and UPPL1541 murine BLCA cells were grown in-vitro and treated with Gemcitabine (Gem), IFNα, and IFNβ or combinations of Gem + IFNα or Gem + IFNβ. CellTiter-Glo® Luminescent Cell Viability Assay was used to determine the half maximal inhibitory concentration (IC₅₀) for each drug and combination. Cell cycle analysis and Annexin V staining were performed at IC₅₀ dosage to assess for difference in anti-tumor activity. GraphPad Prism 9 software was used for statistical analyses.

Results:

All cell lines showed a significant decrease of the IC₅₀ for both combination therapies compared to Gemcitabine alone (2 to 3-fold decrease). BBN and UPPL cell lines were more sensitive to combination therapy with IFNβ, while MB49 was more sensitive to combination with IFNα. Cell cycle analysis showed increase in subG0G1, decrease in G0G1, and increase in S-phase population after IFNα and Gem therapy of MB49 compared to either treatment alone, while therapy of UPPL and BBN with IFNβ and Gem led to increase in both subG0G1 and G0G1, and decrease in S-phase population. Finally, Annexin V staining was increased in all 3 cell lines with IFNα or -β combination therapy, with increase in early and late apoptosis compared to Gemcitabine, IFNα or IFNβ alone.

Conclusion:

Combination therapy with IFNα or -β and Gemcitabine led to superior cell cycle arrest and apoptosis in murine BLCA cell lines. The observed differences between the effects of IFNα and IFNβ merit further investigation to optimize intravesical gene therapy. Future in-vitro and in-vivo experiments using human and murine BLCA models are ongoing to direct future clinical development of intravesical gene therapy.

Funding: Research is supported in part by A.I. Virtanen Institute for Molecular Sciences (Kuopio, Finland) and MD Anderson CCSG program (P30 016672).