J. A. Perone1, T. Tamesa1,2, M. Tsutsui2, R. E. Alvarado1, S. Moore1, P. Dolber1, I. Pinchuk1, K. Olino1, D. Tyler1 1University Of Texas Medical Branch,Galveston, TX, USA 2Durham Veterans Affairs Medical Center,Durham, NC, USA
Introduction:
Advanced melanoma remains a highly lethal disease with poor long-term survival in the majority of patients. Although systemic immunotherapy has improved outcomes, there is much room for improvement. One strategy to help augment systemic immunotherapy has been regional therapeutics. Regional therapies offer variable immunogenic effects on the microenvironment, usually with minimal toxicity, that could synergize with systemic treatments to improve antitumor responses. To better define the spectrum of immune effects produced by regional therapy, we picked four different chemotherapeutic agents to explore the range of immune mediated events that occur in the context of variable mechanisms of cell death.
Methods:
B16 F10.9-OVA melanoma cells were injected intradermally in the left leg of C57BL/6 female mice to create a solitary leg tumor. At tumor volumes of ~50mm3 animals were randomized and isolated limb infusion (ILI) was performed using the maximally tolerated dose of saline, melphalan, doxorubicin, temozolomide, or oxaliplatin. Following survival studies, immunologic evaluation was performed by harvesting Inguinal (ILN) and popliteal lymph nodes (PLN) on days 3 and 10 post ILI and immunologic evaluation was performed utilizing RT-PCR and flow cytometry, staining for CD4, CD8, FoxP3, Tbet, PD-1, and PD-L1.
Results:
While regional chemotherapy was exceptionally effective at killing tumor, we were surprised to see the induction of immunosuppressive profiles in the microenvironment at days 3 and 10. Increased expression of PD-L1 on CD4+ and CD8+ Tcells was observed not only in inguinal nodes but also in popliteal nodes and tumors on day 10 following treatment with either melphalan or doxorubicin (Figure 1). Immunosuppression associated with temozolomide and oxaliplatin was primarily manifested as an increase in tregs(CD4+FoxP3+) in both the ILN and PLN between days 3 and 10, as well as an increase in exhausted Tcells (CD8+PD1+) and suppressor Tcells (CD8+FoxP3+) (Figure 2). PCR analysis of tumors treated with doxorubicin also showed similar increase in tregs compared to naïve animals.
Conclusion:
Regional chemotherapy used in ILI creates an immunosuppressive environment around treated tumors that varies depending on the chemotherapeutic agent utilized. Characterization of the immunosuppressive profile induced by regional treatment allows development of novel strategies, such as combining systemic anti-PD1 treatment with melphalan-based ILI, to augment both regional and systemic anti tumor response.
