Introduction: Intravesical immunotherapy with mycobacterium bovis baccillus Calmette-Guerin (BCG) has been the standard of care for high grade non-muscle invasive bladder tumors for nearly 40 years. While BCG has been successful at lowering recurrence rates and preventing progression, it does not engage adaptive memory and is associated with recurrence rates as high as 50%. Thus the search for treatment strategies that promote a durable anti-tumor memory response is warranted.
One such strategy developed by our group is the intravesical administration of a simple coformulation of the TH1 polarizing cytokine interleukin-12 (IL-12) and the biopolymer chitosan (chitosan/IL-12). Chitosan is not bioactive but is cationic and viscous in solution and acts as a delivery vehicle for IL-12. It is presumed to enhance three aspects of delivery. 1) Chitosan's positive charge interacts with the urothelium's tight junctions to transiently increase its permeability. 2) Mucoadhesive forces between the urothelium's mucosa and chitosan prolong the contact of IL-12 with the urothelium. 3) The viscous nature of chitosan limits expulsion of IL-12 during voiding of the bladder.
Our group has recently demonstrated that four weekly intravesical administrations of chitosan/IL-12 can 1) eliminate established orthotopic in >90% of mice, 2) provide protective local immunity, and 3) promote durable systemic immunity. The purpose of this study is to investigate the mechanisms of intravesical chitosan/IL-12 immunotherapy. Specifically, we look at the role of natural killer cells (NK), CD8+ cytotoxic T-cells, and CD4+ T-helper cells in chitosan/IL-12 therapy of orthotopic tumors as well as their importance for maintaining systemic immunity to those bladder tumors.
Methods: Orthotopic bladder tumors were generated via intravesical implantation of 75,000 MB49 cells into female C57BL/6J mice. Briefly, anesthetized mice were catheterized with a 24G x 3/4” Teflon catheter and the cells injected after a 10 minute wash with Poly-L-Lysine. Upon onset of hematuria, usually one week post implantation, mice received four twice-weekly intravesical administrations of either saline or chitosan/IL-12 immunotherapy (1 µg IL-12 in 100µl of 10 mg/ml chitosan). For immune subset depletion studies, four daily administrations of 100 µg anti-CD4 (GK1.5), anti-CD8 (2.43), or anti-NK1.1 (PK136) antibodies were given prior to implantation. Depletion was maintained for the duration of the study with 100 µg antibody given twice per week. For systemic rechallenge studies, mice were injected with 300,000 MB49 in the right flank.
Results: Mice were depleted of NK, CD8+, or CD4+ lymphocytes prior to tumor inoculation to investigate the importance of immune cell types on the efficacy of intravesical chitosan/IL-12. Mice depleted of CD8+ T-cells (n=9) failed to eliminate orthotopic tumors, but did extend survival over the saline treated control. Mice depleted of CD4+ or NK+ immune cells elicited a more mixed result with 4/8 and 3/8 mice respectively surviving long term, free of tumors. Mice that were not depleted, but were given chitosan/IL-12 therapy eliminated their tumors in 7/9 instances.
In a separate study to investigate the importance of T-cell subtypes in maintaining systemic immunity, mice previously cured of orthotopic disease were depleted of CD8+ or CD4+ T-cells and then rechallenged subcutaneously. Eight out of 10 CD8-depleted mice rejected the tumors, while only 1/9 CD4-depleted mice rejected the rechallenge. All naïve mice grew subcutaneous tumors (n=9). All mice previously cured of orthotopic disease and not depleted rejected the subcutaneous rechallenge (n=4).
Conclusions: The elimination of established orthotopic tumors in response to chitosan/IL-12 therapy involves multiple immune cells types. As expected CD8+ T-cells appear to be the primary effectors with CD4+ and NK+ cells playing lesser, but still important roles. Interestingly, the relative importance of CD8+ and CD4+ T-cells seems to reverse for the systemic memory response to tumor rechallenge; almost all subcutaneously rechallenged mice depleted of CD4 grew tumors, but nearly all CD8 depleted mice rejected tumors. Further mechanistic studies are warranted to explain and build upon these results.
Citation Format: Sean Smith, Jack Baltz, Bhanu Koppolu, Sruthi Ravindranathan, Khue Nguyen, David Zaharoff. Effector cells in chitosan/interleukin-12 immunotherapy of bladder tumors in mice. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B020.
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