Metastatic lesions are responsible for over 90% of breast cancer associated deaths. Therefore, strategies that target metastasis are of particular interest. This study examined the efficacy of natural killer T (NKT) cell activation as a postsurgical immunotherapy in a mouse model of metastatic breast cancer. Following surgical resection of orthotopic 4T1 mammary carcinoma tumors, BALB/c mice were treated with NKT cell activating glycolipid antigens (a-GalCer, a-CGalCer or OCH) or a-GalCer-loaded dendritic cells (DCs). Low doses of glycolipids transiently reduced metastasis but did not increase survival. A high dose of a-GalCer enhanced overall survival, but was associated with increased toxicity and mortality at early time points. Treatment with a-GalCer-loaded DCs limited tumor metastasis, prolonged survival, and provided curative outcomes in »45% of mice. However, survival was not increased further by additional DC treatments or co-transfer of expanded NKT cells. NKT cell activation via glycolipid-loaded DCs decreased the frequency and immunosuppressive activity of myeloid derived suppressor cells (MDSCs) in tumor-resected mice. In vitro, NKT cells were resistant to the immunosuppressive effects of MDSCs and were able to reverse the inhibitory effects of MDSCs on T cell proliferation. NKT cell activation enhanced antitumor immunity in tumor-resected mice, increasing 4T1-specific cytotoxic responses and IFNg production from natural killer (NK) cells and CD8 C T cells. Consistent with increased tumor immunity, mice surviving to day 150 were resistant to a second tumor challenge. This work provides a clear rationale for manipulating NKT cells to target metastatic disease.

Natural killer T (NKT) cells are glycolipid-reactive lymphocytes that promote cancer control. In previous studies, NKT-cell activation improved survival and antitumor immunity in a postsurgical mouse model of metastatic breast cancer. Herein, we investigated whether NKT-cell activation could be combined with chemotherapeutic agents to augment therapeutic outcomes. Gemcitabine and cyclophosphamide analogues enhanced the potential immunogenicity of 4T1 mammary carcinoma cells by increasing the expression of antigen-presenting molecules (MHC-I, MHC-II, and CD1d) and promoting exposure or release of immunogenic cell death markers (calreticulin, HMGB1, and ATP). In 4T1 primary tumor and postsurgical metastasis models, BALB/c mice were treated with cyclophosphamide or gemcitabine. NKT cells were then activated by transfer of dendritic cells loaded with the glycolipid antigen α-galactosylceramide (α-GalCer). Chemotherapeutic treatments did not impact NKT-cell activation but enhanced recruitment into primary tumors. Cyclophosphamide, gemcitabine, or α-GalCer-loaded dendritic cell monotherapies decreased tumor growth in the primary tumor model and reduced metastatic burden and prolonged survival in the metastasis model. Combining chemotherapeutics with NKT-cell activation therapy significantly enhanced survival, with surviving mice exhibiting attenuated tumor growth following a second tumor challenge. The frequency of myeloid-derived suppressor cells was reduced by gemcitabine, cyclophosphamide, or α-GalCer-loaded dendritic cell treatments; cyclophosphamide also reduced the frequency of regulatory T cells. Individual treatments increased immune cell activation, cytokine polarization, and cytotoxic responses, although these readouts were not enhanced further by combining therapies. These findings demonstrate that NKT-cell activation therapy can be combined with gemcitabine or cyclophosphamide to target tumor burden and enhance protection against tumor recurrence. .

BackgroundOncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in combination with other therapies. Current therapeutic approaches, including chemotherapeutics, come with dose-limiting toxicities. Another option is to combine oncolytic viruses with immunotherapeutic approaches.MethodsUsing experimental models of metastatic 4T1 breast cancer and ID8 ovarian peritoneal carcinomatosis, we examined natural killer T (NKT) cell-based immunotherapy in combination with recombinant oncolytic vesicular stomatitis virus (VSV) or reovirus. 4T1 mammary carcinoma cells or ID8 ovarian cancer cells were injected into syngeneic mice. Tumor-bearing mice were treated with VSV or reovirus followed by activation of NKT cells via the intravenous administration of autologous dendritic cells loaded with the glycolipid antigen α-galactosylceramide. The effects of VSV and reovirus on immunogenic cell death (ICD), cell viability and immunogenicity were tested in vitro.ResultsVSV or reovirus treatments followed by NKT cell activation mediated greater survival in the ID8 model than individual therapies. The regimen was less effective when the treatment order was reversed, delivering virus treatments after NKT cell activation. In the 4T1 model, VSV combined with NKT cell activation increased overall survival and decreased metastatic burden better than individual treatments. In contrast, reovirus was not effective on its own or in combination with NKT cell activation. In vitro, VSV killed a panel of tumor lines better than reovirus. VSV infection also elicited greater increases in mRNA transcripts for proinflammatory cytokines, chemokines, and antigen presentation machinery compared with reovirus. Oncolytic VSV also induced the key hallmarks of ICD (calreticulin mobilization, plus release of ATP and HMGB1), while reovirus only mobilized calreticulin.ConclusionTaken together, these results demonstrate that oncolytic VSV and NKT cell immunotherapy can be effectively combined to decrease tumor burden in models of metastatic breast and ovarian cancers. Oncolytic VSV and reovirus induced differential responses in our models which may relate to differences in virus activity or tumor susceptibility.