Intralesional Injection of Rose Bengal Induces a Systemic Tumor-Specific Immune Response in Murine Models of Melanoma and Breast Cancer Paul Toomey 1,3. , Krithika Kodumudi 1. , Amy Weber 1 , Lisa Kuhn 1 , Ellen Moore 1 , Amod A. Sarnaik 1,2,4" , Shari Pilon-Thomas 1,2,4 * " 1 H. Lee Moffitt Cancer Center and Research Institute, Immunology Program, Tampa, Florida, United States of America, 2 H. Lee Moffitt Cancer Center and Research Institute, Cutaneous Oncology Program, Tampa, Floria, United States of America, 3 University of South Florida, College of Medicine, Tampa, Florida, United States of America, 4 University of South Florida, Department of Oncologic Sciences, Tampa, Florida, United States of America Abstract Intralesional (IL) injection of PV-10 has shown to induce regression of both injected and non-injected lesions in patients with melanoma. To determine an underlying immune mechanism, the murine B16 melanoma model and the MT-901 breast cancer model were utilized. In BALB/c mice bearing MT-901 breast cancer, injection of PV-10 led to regression of injected and untreated contralateral subcutaneous lesions. In a murine model of melanoma, B16 cells were injected into C57BL/ 6 mice to establish one subcutaneous tumor and multiple lung lesions. Treatment of the subcutaneous lesion with a single injection of IL PV-10 led to regression of the injected lesion as well as the distant B16 melanoma lung metastases. Anti- tumor immune responses were measured in splenocytes collected from mice treated with IL PBS or PV-10. Splenocytes isolated from tumor bearing mice treated with IL PV-10 demonstrated enhanced tumor-specific IFN-gamma production compared to splenocytes from PBS-treated mice in both models. In addition, a significant increase in lysis of B16 cells by T cells isolated after PV-10 treatment was observed. Transfer of T cells isolated from tumor-bearing mice treated with IL PV- 10 led to tumor regression in mice bearing B16 melanoma. These studies establish that IL PV-10 therapy induces tumor- specific T cell-mediated immunity in multiple histologic subtypes and support the concept of combining IL PV10 with immunotherapy for advanced malignancies. Citation: Toomey P, Kodumudi K, Weber A, Kuhn L, Moore E, et al. (2013) Intralesional Injection of Rose Bengal Induces a Systemic Tumor-Specific Immune Response in Murine Models of Melanoma and Breast Cancer. PLoS ONE 8(7): e68561. doi:10.1371/journal.pone.0068561 Editor: Fabrizio Mattei, Istituto Superiore di Sanita `, Italy Received March 26, 2013; Accepted May 30, 2013; Published July 17, 2013 Copyright: ß 2013 Toomey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a sponsored research agreement from Provectus Inc. to the H Lee Moffitt Cancer Center and Research Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors would like to declare that Provectus, Inc. provided funding for this project. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. Provectus, Inc. had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. * E-mail: shari.pilon-thomas@moffitt.org . These authors contributed equally to this work. " These authors also contributed equally to this work. Introduction Intralesional (IL) injection of anti-tumor therapeutic agents has been used for the treatment of cutaneous malignancies. IL therapy can control local disease and avoid potential surgical complications and systemic toxicity. While many of these agents lead to regression of injected lesions, systemic responses are rarely observed thus limiting this strategy for patients with metastatic disease. Immunotherapeutic strategies incorporating IL therapy to induce anti-tumor immune responses are currently being explored as a potential means to induce both local and systemic tumor regressions. IL injection of agents that can increase the presen- tation of tumor-specific antigens, induce infiltration of immune cells, or enhance ongoing immunity may lead to the induction of robust systemic anti-tumor immunity. Intratumoral injection of dendritic cells (DCs) has led to increased lymphocyte infiltration and systemic anti-tumor effects in patients with advanced melanoma [1,2]. Injection of adjuvants such as BCG or TLR agonists has been shown to enhance tumor-specific immunity [3– 6]. Intratumoral injection of immune-enhancing cytokines such as IL-2 or GM-CSF has also been shown to enhance anti-tumor immunity in melanoma patients. [6,7]. Rose Bengal is a water-soluble xanthene dye that had been previously used in liver function studies and is still in use by ophthalmologists [8,9]. PV-10 is a 10% solution of Rose Bengal formulated for IL injection. IL PV-10 therapy has been shown to induce regression of both injected melanoma lesions and un- injected bystander lesions in patients with melanoma [10]. PV-10 has been documented to induce cell death in melanocytes without affecting normal dermal fibroblasts [11]. Intralesional injection of PV-10 followed by radiotherapy has shown promising results in three patients with metastatic melanoma [12]. Relatively little is known about the mechanism by which PV-10 can induce resolution of bystander lesions. However, an immune-mediated process is likely as responses in untreated lesions occur only when there is response in injected lesions, and regression of bystander PLOS ONE | www.plosone.org 1 July 2013 | Volume 8 | Issue 7 | e68561