Significance This study provides an insight into stem cell-based oncolytic virus therapies for advanced melanoma tumors that have metastasized into the brain by developing clinically relevant mouse tumor models and testing the fate and efficacy of oncolytic herpes simplex virus-armed mesenchymal stem cells in such models. This study therefore overcomes the hurdles of systemic delivery of oncolytic viruses and provides a clinically applicable therapeutic platform to target melanoma brain metastasis.

Abstract The recent Food and Drug Administration approval of immunogenic oncolytic virus (OV) has opened a new era in the treatment of advanced melanoma; however, approximately 50% of patients with melanoma develop brain metastasis, and currently there are no beneficial treatment options for such patients. To model the progression of metastases seen in patients and to overcome the hurdles of systemic delivery of OV, we developed melanoma brain metastasis models in immunocompromised and immunocompetent mice, and tested the fate and efficacy of oncolytic herpes simplex virus (oHSV)-armed mesenchymal stem cells (MSCs). Using brain-seeking patient-derived melanoma cells and real-time in vivo imaging, we show a widespread distribution of micrometastases and macrometastases in the brain, recapitulating the progression of multifoci metastases seen in patients. We armed MSCs with different oHSV variants (MSC-oHSV) and found that intracarotid administration of MSC-oHSV, but not of purified oHSV alone, effectively tracks metastatic tumor lesions and significantly prolongs the survival of brain tumor-bearing mice. In a syngeneic model of melanoma brain metastasis, a combination of MSC-oHSV and PD-L1 blockade increases IFNγ-producing CD8+ tumor-infiltrating T lymphocytes and results in a profound extension of the median survival of treated animals. This study thus demonstrates the utility of MSCs as OV carriers to disseminated brain lesions, and provides a clinically applicable therapeutic platform to target melanoma brain metastasis.

Melanoma, the most aggressive type of skin cancer, accounts for a large proportion of skin cancer-related deaths (1). Among all cancer types, melanoma has a particularly high propensity to metastasize to the brain, occurring in >50% of all patients with advanced disease. More than 90% of melanoma brain metastases lead to death, and the median survival is 17–22 wk after detection (2⇓–4).

Current therapeutic options of chemotherapy, surgery, and radiation have very limited efficacy for patients with melanoma brain metastasis (5⇓–7). These patients either have multiple metastatic lesions or diagnostically challenging asymptomatic lesions, making surgery an inadequate therapeutic option by itself. In addition, the blood-brain barrier (BBB) limits central nervous system (CNS) penetration of systemic therapies, and the negative side effects of radiotherapy (8) pose challenges for the success of existing therapies, contributing to the failure to improve overall patient survival. As such, there is an urgent need for new therapies for melanoma brain metastasis.

The development and characterization of preclinical tumor models that authentically recapitulate the clinical disease settings are critical for developing and testing new therapies. Most previous studies have used either subcutaneous (s.c.) injection or intracranial injection of established melanoma lines in mice (9⇓–11), which do not mimic the actual clinical settings of melanoma brain metastasis, such as initial adhesion of tumor cells to brain capillaries, extravasation, continuation of perivascular position, vessel co-option, micrometastatic growth, and macrometastatic growth (12). In addition, long-established melanoma lines often fail to recapitulate the key aspects of human malignancy and thus poorly predict the clinical efficacy of tested therapeutic agents (13).

In this study, we created in vivo imageable mouse models of melanoma brain metastasis by internal carotid artery (ICA) injection of patient-derived primary melanoma and brain-seeking melanoma lines [either BRAF mutant or wild type (WT)], as well as the syngeneic mouse model of melanoma brain metastasis using a BRAF mutant line isolated from BrafV600E/wtCdkn2A−/−Pten−/− mice.

Oncolytic viruses (OVs) that selectively replicate in tumor cells are an emerging modality of cancer treatment that shows promising results in both preclinical studies and clinical trials (14, 15). Among these OVs, oncolytic herpes simplex viruses (oHSV) have shown promising therapeutic efficacy in treating advanced melanoma (16, 17). Recently, the US Food and Drug Administration approved talimogene laherparepvec (T-VEC) for the treatment of melanoma lesions in the skin and lymph nodes (17). Although induction of an antitumor immune response is implicated in activity for distant uninjected lesions, T-VEC has not been shown to improve overall patient survival of stage IVM1b and IVM1c disease that has metastatic lesions to the brain, bone, liver, lungs, or other internal organs (18). The unavailability of appropriate clinically translatable mouse models of melanoma brain metastasis and issues related to oHSV delivery via the bloodstream (19), such as virus neutralization, sequestration, and inefficient extravasation, pose major barriers to the development of oHSV-based therapies for melanoma brain metastasis.

Previous studies from our laboratory demonstrated that therapeutic human and mouse stem cells home extensively to multiple tumor deposits in the brain (20) and act as cell carriers for onsite delivery of tumor-specific agents or OV (21) in mouse models of different brain tumor types (22). In the present study, we tested the therapeutic efficacy of MSC-loaded oHSV (MSC-oHSV) in both BRAF mutant and WT in vivo imageable mouse models of melanoma brain metastasis, and explored the combined therapeutic efficacy of PD-L1 blockade and MSC-oHSV in a syngeneic mouse model of melanoma brain metastasis.

Discussion In this study, we show that oHSV has a potent cell-killing effect in a broad spectrum of malignant melanoma lines. To explore the therapeutic efficacy of oHSV in melanoma brain metastasis, we created in vivo imageable mouse models of melanoma brain metastasis in both immunocompromised and immunocompetent mice. We demonstrate that ICA-delivered MSC-oHSV, but not purified oHSV, efficiently track metastatic tumor deposits in the brain, suppress brain tumor growth, and prolong survival in mouse models of melanoma brain metastasis. Furthermore, our studies demonstrate that the combination therapy of MSC-oHSV and anti–PD-L1 has improved therapeutic efficacy in syngeneic mouse model of melanoma brain metastasis, which is associated with an increased CD8+IFNγ+ TIL population. Melanomas are molecularly heterogeneous tumors bearing different mutations and are resistant to a number of currently used chemotherapies (25, 26). In this study, we screened a panel of seven melanoma cell lines consisting of both established and patient-derived brain metastatic melanoma lines for their responses to oHSV infection and oncolysis. Our results reveal that oHSV infection has a consistent cell-killing effect on melanoma lines regardless of their BRAF mutational status, thus strongly supporting the use of oHSV for treating melanoma brain metastasis. Our screening results also demonstrated that the yields of oHSV in melanoma lines correlate with the efficiency of oHSV-mediated cell killing, suggesting that virus replication underlies the direct oncolytic effects. We also found that the oHSV yield in the patient-derived brain metastatic melanoma cell line M15 was relatively lower than that in the other melanoma lines, which correlated with less cell death in M15 cells treated with oHSV. Compared with MeWo, M12, and MSCs, M15 melanoma cells have decreased expression of Nectin-1 receptor, a major cell surface receptor for HSV entry (SI Appendix, Fig. S12), which may contribute to less permissive entry of oHSV into M15 cells. However, our data show that oHSV achieves better infection and spread in M15 cells at higher MOI (SI Appendix, Fig. S13 A and B). Based on our previous findings that the efficacy of oHSV-mediated cell killing can be significantly increased using a proapoptotic variant of oHSV, oHSV-TRAIL, in tumor lines that are less permissive to oHSV-mediated oncolysis (27), our future studies will focus on testing the efficacy of oHSV-TRAIL in such melanoma lines. To test the therapeutic effects of oHSV in melanoma brain metastasis, we developed and extensively characterized in vivo imageable mouse models of melanoma brain metastasis that display the various features of brain metastasis observed in patients with advanced melanoma. Melanoma brain metastasis originates either directly from primary melanoma lesions or from metastatic lymph nodes and visceral lesions (13); therefore, we chose MeWo (derived from the metastatic lymph nodes in advanced melanoma) and M12 (derived from melanoma brain metastases) to mimic these two types of metastasis. These two melanoma lines are either BRAF WT or mutant (BRAFV600E), the most frequent BRAF mutation seen in melanoma patients (28). Our results indicate that ICA injection of such lines results in the formation of clinically relevant mouse models that resemble the diverse features of metastatic melanoma, including widely disseminated numerous foci in the brain, aggressive and fatal growth, different mutational status of BRAF, and pigmented and nonpigmented lesions. These mouse models provide a unique and valuable platform for testing existing and novel therapeutic approaches for melanoma brain metastasis and help us better understand the pathogenesis of melanoma brain metastasis. Previous studies typically used either intratumoral injection of oHSV into solid tumor lesions or systemic injection of high-dose oHSV (19, 29, 30). Given the multiple metastatic melanoma lesions in the brain, intratumoral injection into each single lesion is not a feasible approach. Systemic delivery of high-dose viruses carries a risk of virus-related toxicity (31). ICA delivery of oHSV has been explored previously in multiple glioblastoma and breast cancer brain metastasis models (32, 33); however, its efficiency is largely impeded by either antiviral activity present in plasma or undamaged BBB. Moreover, our studies have shown that ICA injection of purified oHSV (2 × 106 pfu) is unable to access multiple metastatic lesions in the brain. To overcome this limitation, we developed a strategy that uses MSCs as cellular carriers to shield oHSV from neutralization and achieve onsite delivery of oHSV to multiple tumor deposits in the brain. Stem cells, such as MSCs, are promising cell carriers for various antitumor viruses mainly because they can home to tumor deposits in the brain (34⇓⇓–37), can be easily isolated from patients and grown in culture, and have high metabolic activity, which is important for virus production (20, 38). Furthermore, MSCs are less immunogenic (39) and have been used in various clinical trials for different indications (40). In addition, MSCs also have been used as virus carriers in a phase 1 clinical trial in ovarian cancer patients (41). Using oHSV mutants bearing diagnostic proteins combined with bioluminescence and fluorescence imaging, our experiments reveal that MSCs act as oHSV carriers and track metastatic tumor deposits in the brain, ultimately releasing the oHSV. Our in vivo imaging data suggest that after ICA injection of MSC-oHSV-Fluc, virus replication initially occurs within infected MSCs, which releases oHSV-Fluc upon cell lysis, transfers oHSV to adjacent tumor cells, and results in subsequent virus replication in tumor cells. Comparison of the accessibilities of MSC-oHSV and purified oHSV to metastatic tumor lesions in the brain reveals that MSC-oHSV has superior tumor-tracking capability and results in a significant reduction in tumor foci and a survival advantage in mice bearing melanoma brain metastases. Importantly, ICA injection of MSC-oHSV was safe, and we did not observe any acute systemic toxicities or local adverse events, such as brain infarction. Although the mechanism of oHSV-mediated killing of MSCs remains unclear, our results indicate that it is not mediated via apoptosis due to the absence of cleaved PARP, a hallmark of cell apoptosis (SI Appendix, Fig. S14). The CNS is protected by the BBB and the blood-cerebrospinal fluid barrier, which prevent most therapeutic agents from entering into the brain. Although studies have shown increases in BBB permeability in various brain tumor models, it remains the key mitigating factor for delivering therapeutics into the CNS. Given that delivery of therapeutic agents to the tumors in the brain is a major challenge, significant efforts have been made to develop efficient delivery routes to brain tumors, which include both invasive and noninvasive administration strategies (42). In a previous study, we showed that local implantation of encapsulated MSCs loaded with oHSV have therapeutic efficacy in mouse models of resected brain tumors (21). Recent studies have shown that i.v. injected MSC-oHSV have therapeutic efficacy in treating lung metastatic tumors (43). These studies imply that i.v.-injected MSC-oHSV would be more suitable for treating both primary and metastatic tumors in the lungs as opposed to the tumors in the brain. Therefore, exploring alternate routes of administration of MSC-oHSV to tumors in the brain was critical. Immune checkpoint blockade is a major advance in recent cancer therapy, especially for the treatment of metastatic melanoma (44), which is typically immunogenic, likely due to the large numbers of UV-associated mutations (45). Two monoclonal antibodies that block PD-1/PD-L1 interactions (pembrlizumab and nivolumab) have shown objective responses in 30∼40% of patients with melanoma brain metastasis (46, 47). oHSV represents a novel approach to tumor immunotherapy and is an attractive option based on its ability to preferentially target, infect, and replicate in cancer cells. Furthermore, oHSV viral genomes can be easily attenuated to limit host pathogenicity or engineered to express immune-potentiating genes to enhance the host antitumor immune response (48). Because PD-1 is activated mostly at tumor sites or other areas of active immune response, the side effects of anti–PD-1/PD-L1 therapy tend to be less severe than those associated with anti–CTLA-4 antibodies, which potentially affect all circulating T cells in the body and thus can cause significant, albeit manageable, autoimmune side effects (49). Meanwhile, our results showed strong PD-L1 expression in melanoma brain metastasis in the syngeneic mouse model. We thus chose to use an anti–PD-L1 immune-checkpoint blocker to antagonize the immune suppression posed by metastatic melanoma cells. Our study investigated the therapeutic efficacy of MSC-oHSV in combination with anti–PD-L1 for melanoma brain metastasis. We found that CD8+IFNγ+ TIL population was associated with the survival benefits achieved by the combination therapy of MSC-oHSV plus anti–PD-L1, suggesting that cytotoxic CD8+ TIL may play a critical role in killing metastatic melanoma cells in the brain, likely via activation of IFNγ-related signaling pathways. Release of IFNγ at the tumor site could limit oHSV spread but trigger a variety of beneficial responses, such as activation of other immune cell subsets, up-regulation of MHC class I, and antiangiogenesis. The transient nature of IFNγ secretion likely would limit the detrimental impacts of IFNγ-induced inflammatory reactions in the brain. The overall cellular responses to the oHSV infection, coupled with the release of tumor antigens by virally infected dying tumor cells into the tumor microenvironment, attract innate and adaptive immune cells, including tumor-specific CD4+ and CD8+ T cells. This oHSV infection-mediated response makes virotherapy an ideal modality to combine with immune checkpoint blockers to achieve a more durable response and outcome. Our data suggest that the increased population of CD8+IFNγ+ TIL represents a beneficial antitumor immune response elicited by MSC-oHSV therapy for melanoma brain metastasis. In conclusion, we have shown that intra-arterial delivery of MSC-loaded oHSV can effectively track and kill metastatic melanoma cells in the brain, and that combination therapy with an immune checkpoint blocker boosts the therapeutic efficacy of MSC-oHSV. Thus, our study warrants clinical testing of MSC-oHSV alone or in combination with immune checkpoint blockers for patients with melanoma brain metastases. Attributed to their innate tumor tropism, stem cells carrying oHSV have been shown to target tumor lesions in the lung and prevent metastases upon i.v. injection (43). Based on previous findings and our present findings, stem cell-based oncolytic virotherapies could have the potential to be broadly applicable in targeting metastatic lesions in organs such as the liver, colon, and lung.

Materials and Methods Detailed information on the materials and methods used in this study is provided in SI Appendix. All of the animal studies were approved by Massachusetts General Hospital's institutional review board. Cell Lines. MeWo, SK-Mel-2, SK-Mel-28, MALME-3M, and YUMM1.1 melanoma cells were cultured in DMEM (MeWo, MALME-3M, and YUMM1.1) or RPMI (SK-Mel-2 and SK-Mel-28) supplemented with 10% FBS and 1% penicillin-streptomycin. TXM-13 cells were kindly provided by I. J. Fidler and cultured in TXM medium (MEM supplemented with 10% FBS, 1% vitamin, 1% sodium pyruvate, 1% nonessential amino acid, and 1% penicillin-streptomycin). M12 and M15 patient-derived melanoma brain metastatic lines (kindly provided by J. Sarkaria, Mayo Clinic, Rochester) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Human and mouse MSCs were cultured in NutriStem XF Medium and MesenCult MSC Basal Medium, respectively. Normal human astrocytes were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Engineered Viral Vectors, Viral Packaging, and Transduction of Tumor Cells. The following lentiviral constructs were used in this study: Pico2-Fluc-mCherry and Pico2-Fluc-GFP. Lentiviral packaging was performed by transfection of 293T cells as described previously (50). MeWo and M12 cells were transduced at an MOI of 5 in medium containing protamine sulfate (10 µg/mL). All cells were visualized by fluorescence microscopy for GFP or mCherry expression to confirm transduction. oHSV-mCherry and oHSV-Fluc were previously generated by cloning mCherry or Fluc cDNA under the HSV IE4/5 immediate early promoter or CMV promoter, respectively, using site-specific recombination between the G47delta BAC and the shuttle plasmid (27, 51). All of the recombinant oHSVs express Escherichia coli lacZ driven by endogenous ICP6 promoter. Statistical Analysis. Data were analyzed using the Student t test when comparing two groups and ANOVA when comparing more than two groups. Data were plotted as mean ± SEM, and differences were considered significant at P < 0.05. Survival curves were compared using the log-rank test. Analyses were done using GraphPad Prism 5.01.

Acknowledgments We thank Mark Schroeder and Jan Sarkaria for providing patient-derived M12 and M15 tumor lines, Yohei Kitamura and Deepak Bhere for helping with the in vivo M12 tumor model, Jennifer Lo for assisting with the melanoma cell culture, and Ravi Mylvaganam for FACS analysis. This work was supported by Department of Defense Idea Award CA140744 (to K.S.) and National Institutes of Health Grants R01 CA204720 (to K.S.), P01 CA163222 (to D.F.).