Natural killer T cell immunotherapy combined with oncolytic vesicular stomatitis virus or reovirus treatments differentially increases survival in mouse models of ovarian and breast cancer metastasis

Abstract

Background: Oncolytic 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.

Methods: Using 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.

Results: VSV 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.

Conclusion: Taken 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.

Keywords: cytotoxicity; dendritic cells; immunologic; immunotherapy; natural killer T-Cells; oncolytic virotherapy.

Figure 1

Combining natural killer T (NKT) cell activation therapy with oncolytic virus therapy to target ID8 ovarian cancer. (A) Schematic of treatments in the ID8 ovarian model. (B) Survival was assessed following treatment with unloaded dendritic cells (DCs), α-galactosylceramide (α-GalCer)-loaded DCs, reovirus, UV-inactivated reovirus, or α-GalCer-loaded DCs in combination with reovirus (n=7–10 per group). (C) Survival was assessed following treatment with unloaded DCs, α-GalCer-loaded DCs, vesicular stomatitis virus (VSV), UV-inactivated VSV, or α-GalCer-loaded DCs in combination with VSV (n=13–26 per group). *p<0.05 compared with unloaded DCs; †p<0.05 compared with single treatments; ‡p<0.05 compared with reverse order treatment.

Figure 2

Combining natural killer T (NKT) cell activation therapy with oncolytic virus therapy to target metastatic 4T1 mammary carcinoma. (A) Schematic of treatments in the post-surgical 4T1 metastasis model. (B) Survival was assessed following treatment with unloaded dendritic cells (DCs), α-galactosylceramide (α-GalCer)-loaded DCs, reovirus, UV-inactivated reovirus, or α-GalCer-loaded DCs in combination with reovirus (n=10 per group). (C) Survival was assessed following treatment with unloaded DCs, α-GalCer-loaded DCs, vesicular stomatitis virus (VSV), UV-inactivated VSV, or α-GalCer-loaded DCs in combination with VSV (n=9–10 per group). (D, E) Number of 4T1 CFUs present in lung cell suspensions isolated at 28 days post-4T1 injection following treatments incorporating (D) reovirus or (E) VSV (n=9–10 per group). *p<0.01 compared with unloaded DC; †p<0.01 compared with virus alone; ‡p<0.01 compared with α-GalCer-loaded DCs alone.

Figure 3

Tumor growth in survivors following rechallenge with 4T1. Tumor resected mice that survived to day 120 after treatment with α-galactosylceramide (α-GalCer)-loaded dendritic cells (DCs), vesicular stomatitis virus (VSV), combination of VSV and α-GalCer-loaded DCs (figure 2) were rechallenged in the contralateral mammary fat pad with 2×0⁵ 4T1 cells. (A) Tumor volume was compared with tumors grown in naïve mice (n=4–7 per group, except n=1 for VSV group). (B) Metastatic burden at day 25 was compared with naïve tumor-bearing mice (n=4–7 per group, except n=1 for VSV group). *p<0.05 compared with naïve control.

Figure 4

Killing of 4T1 and ID8 cells by VSV∆M51 and reovirus. (A) 4T1 and (B) ID8 cells were infected with vesicular stomatitis virus (VSV) or reovirus at a multiplicity of infection of 1 in vitro. After 24 hours, cells were collected and stained for Annexin V and 7AAD (n=3 per group). *p<0.05 compared with media; † p<0.05 compared with reovirus.

Figure 5

Induction of antigen presentation machinery by 4T1 and ID8 cells infected with VSV∆M51 or reovirus. (A, B) Mean fluorescent intensity (MFI) of major histocompatibility complex (MHC) I, MHC II and CD1d surface expression on cultured (A) 4T1 and (B) ID8 cells 24, 48 and 72 hours after vesicular stomatitis virus (VSV) or reovirus infection at multiplicity of infection (MOI) of 1. UV-inactivated viruses were used as controls (n=4 per group). (C, D) Quantitative PCR examining the expression of tap1 and tap2 in (C) 4T1 and (D) ID8 cells treated with VSV or reovirus for 24 hours. UV-inactivated viruses were used as controls. qPCR was analyzed using the 2^−ΔΔCT quantification technique relative to the validated housekeeping gene GAPDH (n=3–6 per group). *p<0.05 compared with media; † p<0.05 compared with UV-inactivated virus; ‡p<0.05 compared with reovirus.

Figure 6

Cytokine and chemokine mRNA expression in 4T1 cells infected with VSV∆M51 or reovirus. 4T1 cells were treated with reovirus, vesicular stomatitis virus (VSV), or UV-inactivated viruses for 24 hours. qPCR was analyzed using the 2^−ΔΔCT quantification technique relative to the validated housekeeping gene GAPDH (n=3–6 per group). *p<0.05 compared with media; † p<0.05 compared with UV-inactivated virus; ‡<0.05 compared with reovirus treatment.

Figure 7

Virus-induced markers of immunogenic cell death in 4T1 and ID8 cells. Flow cytometric expression of surface calreticulin (CLR) was measured on (A) 4T1 and (D) ID8 cells 8 hours after infection with vesicular stomatitis virus (VSV) or reovirus (n=3 per group). ATP release from (B) 4T1 and (E) ID8 cells was measured by ELISA 24 hours after indicated virus treatment (n=4 per group). HMGB1 release from (C) 4T1 and (F) ID8 cells was measured by ELISA 24 hours following the indicated virus treatments (n=4 per group). *p<0.05 compared with media; †p<0.05 compared with UV-inactivated virus; ‡p<0.05 compared with reovirus treatment.