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Supplementary Figure 2 shows In vitro scratch migration assay.

Description of western blot methodology for supplemental Figure and sources of gene expression data.

Supplementary Figure S2 shows that combination treatment with OS2966 and oHSV does not enhance oHSV replication in intracranial glioma-bearing mice in vivo.

Supplementary Table S1 shows sequences of primers used in this study.

Supplementary Figure 3 shows Full scans of the western blotting shown in the figure 6.

Supplemental Figure 1: The ability of 34.5ENVE to infect and replicate in DB-7 and Met-1 BC cells

Supplementary Figure S1 shows that enhanced virus replication by combination treatment with oHSV and OS2966 significantly increases tumor necrosis in mammary fat-pad injected MDA468 tumor bearing mice.

Supplementary Fig. S1. Therapeutic efficacy of RV and HDACi against cancer B cells; Supplementary Fig. S2. HDACi increase JAM-1 expression without affecting cell viability; Supplementary Fig. S3. RV+HDACi display synergistic anti-myeloma activity; Supplementary Fig. S4. Combination treatment of MCL cells with RV and HDAC inhibitors results in increased cell death, compared to single agent treatments.

Supplementary Figure S4 shows that oHSV infection increases ITGB1 expression.

Anti-VEGF treatments such as bevacizumab have demonstrated convincing therapeutic advantage in patients with glioblastoma. However, bevacizumab has also been reported to induce invasiveness of glioma. In this study, we examined the effects of rapid antiangiogenesis mediated by oncolytic virus (RAMBO), an oncolytic herpes simplex virus-1 expressing vasculostatin, on bevacizumab-induced glioma invasion. The effect of the combination of RAMBO and bevacizumab in vitro was assessed by cytotoxicity, migration, and invasion assays. For in vivo experiments, glioma cells were stereotactically inoculated into the brain of mice. RAMBO was intratumorally injected 7 days after tumor inoculation, and bevacizumab was administered intraperitoneally twice a week. RAMBO significantly decreased both the migration and invasion of glioma cells treated with bevacizumab. In mice treated with bevacizumab and RAMBO combination, the survival time was significantly longer and the depth of tumor invasion was significantly smaller than those treated with bevacizumab monotherapy. Interestingly, RAMBO decreased the expression of cysteine-rich protein 61 and phosphorylation of AKT, which were increased by bevacizumab. These results suggest that RAMBO suppresses bevacizumab-induced glioma invasion, which could be a promising approach to glioma therapy.

Supplementary Figure S3 shows that Combination treatment with OS2966 and oHSV does not enhance mice survival rate in intracranial glioma-bearing mice in vivo.

Supplementary Figure 1 shows Evaluation of glioma cells infected by oncolytic virus and conditioned medium.

Integrin β1 receptor, expressed on the surface of tumor cells and macrophages in the tumor microenvironment (TME), has been implicated in both tumor progression and resistance to multiple modalities of therapy. OS2966 is the first clinical-ready humanized monoclonal antibody to block integrin β1 and was recently orphan designated by the FDA Office of Orphan Products Development. Here, we tested therapeutic potential of OS2966-mediated integrin β1 blockade to enhance the efficacy of oncolytic herpes simplex virus-1 (oHSV) through evaluation of virus replication, tumor cell killing efficiency, effect on the antiviral signaling pathway, co-culture assays of oHSV-infected cells with macrophages, and in vivo bioluminescence imaging on mammary fat pad triple-negative breast cancer xenograft and subcutaneous and intracranial glioma xenografts. OS2966 treatment decreased interferon signaling and proinflammatory cytokine induction in oHSV-treated tumor cells and inhibited migration of macrophages, resulting in enhanced oHSV replication and cytotoxicity. OS2966 treatment also significantly enhanced oHSV replication and oHSV-mediated antitumor efficacy in orthotopic xenograft models, including triple-negative breast cancer and glioblastoma. The results demonstrated the synergistic potential of the combinatory treatment approach with OS2966 to improve antitumor efficacy of conventional oHSV therapy.

Figure S1

OV-αmCD47-G2b treatments promote macrophage M1 polarization and NK cell activation in ID8 immunocompetent ovarian cancer metastasis mouse model.

Supplemental Figure S3. Representative fluorescent images of uninfected and infected co-cultures 12 hours post infection.

Supplemental Figure S2. Species specificity of murine TNFα ELISA kit for murine and not human TNFα

Purpose:Here we investigated the impact of oncolytic herpes simplex virus (HSV) treatment on cisplatin sensitivity of platinum-resistant ovarian cancer, and the impact of the combination on immunotherapy.Experimental Design:Therapeutic efficacy of the combination was assessed in platinum-resistant human and murine ovarian cancer peritoneal metastatic mouse models (n = 9–10/group). RNA sequencing along with flow cytometry of splenocytes from treated mice was employed to examine the effect of antitumor immune response (n = 3/group). Anti-PD-1 antibody was performed to evaluate impact on checkpoint inhibition in vivo.Results:Gene Ontology pathway analysis uncovered disruption of cellular extracellular vesicle (EV)-related pathways in infected cells (FDR = 2.97E-57). Mechanistically, we identified reduced expression of transporters expressed on EV implicated in cisplatin efflux. The increased cisplatin retention led to increased cisplatin–DNA adducts, which resulted in micronuclei and the subsequent activation of cGAS–STING pathway with a significant activation of innate immune cells and translated to an increase in antitumor immunity and efficacy. In mice bearing platinum-resistant ovarian cancer, we also observed a feedback induction of PD-L1 on tumor cells, which sensitized combination-treated mice to anti-PD-1 immune checkpoint therapy.Conclusions:To our knowledge, this is the first report to show HSV-induced cisplatin retention in infected cells. The consequential increased damaged DNA was then expelled from cells as micronuclei which resulted in induction of inflammatory responses and education of antitumor immunity. The combination therapy also created an environment that sensitized tumors to immune checkpoint therapy.

Co-culture model of endogenous Vstat120 impact on microglia response to oHSV-infected glioma.

Figure S3

Supplementary Figure Legend

PDF file, 77K.

Supplemental Figure S4. oHSV treatment increases secreted TNFα.

Supplementary Figure and Table Legends. Supplementary legends for figures and tables characterizing patient ascites samples.

PDF file - 198KB, Supplementary Figure S1. Combinatorial treatment with bortezomib and oHSV1716 induce synergistic cell killing against S462TY malignant peripheral nerve sheath tumor (MPNST) cell. Supplementary Figure S2. Induction of ER stress and unfolded protein response (UPR) in CAL27 head and neck cancer cells treated with bortezomib.

The effect of αCD47-G1 on the expression of the genes markering macrophage polarization in vitro.

OV-Q1, OV-αCD47-G1 and OV-αCD47-G4 treatments increase immune cell infiltration in the ID8-hCD47 immunocompetent ovarian cancer metastasis mouse model.

Supplementary Materials and Methods

Figure S1

Supplemental Figure S1. Percoll gradient isolated CD11b+CD45hi cells are predominantly infiltrating macrophages

Purpose: Novel therapeutic regimens are needed to improve dismal outcomes associated with late-stage ovarian cancer. Oncolytic viruses are currently being tested in patients with ovarian cancer. Here, we tested the therapeutic efficacy of combining doxorubicin with 34.5ENVE, an oncolytic herpes simplex virus transcriptionally driven by a modified stem cell–specific nestin promoter, and encoding for antiangiogenic Vasculostatin-120 (VStat120) for use against progressive ovarian cancer.Experimental Design: Antitumor efficacy of 34.5ENVE was assessed in ovarian cancer cell lines, mouse ascites–derived tumor cells, and primary patient ascites–derived tumor cells by standard MTT assay. The ability of conditioned medium derived from 34.5ENVE-infected ovarian cancer cells to inhibit endothelial cell migration was measured by a Transwell chamber assay. Scope of cytotoxic interactions between 34.5ENVE and doxorubicin were evaluated using Chou–Talalay synergy analysis. Viral replication, herpes simplex virus receptor expression, and apoptosis were evaluated. Efficacy of oncolytic viral therapy in combination with doxorubicin was evaluated in vivo in the murine xenograft model of human ovarian cancer.Results: Treatment with 34.5ENVE reduced cell viability of ovarian cancer cell lines, and mouse ascites–derived and patient ascites–derived ovarian tumor cells. Conditioned media from tumor cells infected with 34.5ENVE reduced endothelial cell migration. When combined with doxorubicin, 34.5ENVE killed synergistically with a significant increase in caspase-3/7 activation, and an increase in sub-G1 population of cells. The combination of doxorubicin and 34.5ENVE significantly prolonged survival in nude mice bearing intraperitoneal ovarian cancer tumors.Conclusions: This study indicates significant antitumor efficacy of 34.5ENVE alone, and in combination with doxorubicin against disseminated peritoneal ovarian cancer. Clin Cancer Res; 20(24); 6479–94. ©2014 AACR.

Purpose: The inhibitory role of secreted chondroitin sulfate proteoglycans on oncolytic viral (OV) therapy was examined. Chondroitinase ABC (Chase-ABC) is a bacterial enzyme that can remove chondroitin sulfate glycosaminoglycans from proteoglycans without any deleterious effects in vivo. We examined the effect of Chase-ABC on OV spread and efficacy.Experimental Design: Three-dimensional glioma spheroids placed on cultured brain slices were utilized to evaluate OV spread. Replication-conditional OV-expressing Chase-ABC (OV-Chase) was engineered using HSQuik technology and tested for spread and efficacy in glioma spheroids. Subcutaneous and intracranial glioma xenografts were utilized to compare antitumor efficacy of OV-Chase, rHsvQ (control), and PBS. Titration of viral particles was performed from OV-treated subcutaneous tumors. Glioma invasion was assessed in collagen-embedded glioma spheroids in vitro and in intracranial tumors. All statistical tests were two sided.Results: Treatment with Chase-ABC in cultured glioma cells significantly enhanced OV spread in glioma spheroids grown on brain slices (P < 0.0001). Inoculation of subcutaneous glioma xenografts with Chase-expressing OV significantly increased viral titer (>10 times, P = 0.0008), inhibited tumor growth, and significantly increased overall animal survival (P < 0.006) compared with treatment with parental rHsvQ virus. Single OV-Chase administration in intracranial xenografts also resulted in longer median survival of animals than rHsvQ treatment (32 vs. 21 days, P < 0.018). Glioma cell migration and invasion were not increased by OV-Chase treatment.Conclusions: We conclude that degradation of glioma extracellular matrix with OV-expressing bacterial Chase-ABC enhanced OV spread and antitumor efficacy. Clin Cancer Res; 17(6); 1362–72. ©2010 AACR.

Supplemental Figure S6. TNFα increases cell proliferation and is not cytotoxic to uninfected GB cells.

OV-Q1, OV-αmCD47-G2b and OV-αmCD47-G3 treatments increase immune cell infiltration in the ID8 immunocompetent ovarian cancer metastasis mouse model.

The supplementary figures provide data supporting the combination drug screening process that uncovered synergy between PDI and HDAC inhibitors, and further supplements the findings in the main figures demonstrating the genomic, biochemical, and cellular characterization of the molecular mechanisms responsible for that synergy.

Schematic map of OV-αCD47 used in this study.

Primers used in this study.

PDF file - 1614K, Methylated CpG sites in the Nestin promoter region of rQNestin34.5, immediately upstream of the gamma1 34.5 gene.

Background: Bortezomib is an FDA-approved proteasome inhibitor, and oncolytic herpes simplex virus-1 (oHSV) is a promising therapeutic approach for cancer. We tested the impact of combining bortezomib with oHSV for antitumor efficacy.Experimental Design: The synergistic interaction between oHSV and bortezomib was calculated using Chou–Talalay analysis. Viral replication was evaluated using plaque assay and immune fluorescence. Western blot assays were used to evaluate induction of estrogen receptor (ER) stress and unfolded protein response (UPR). Inhibitors targeting Hsp90 were utilized to investigate the mechanism of cell killing. Antitumor efficacy in vivo was evaluated using subcutaneous and intracranial tumor xenografts of glioma and head and neck cancer. Survival was analyzed by Kaplan–Meier curves and two-sided log-rank test.Results: Combination treatment with bortezomib and oHSV (34.5ENVE), displayed strong synergistic interaction in ovarian cancer, head and neck cancer, glioma, and malignant peripheral nerve sheath tumor (MPNST) cells. Bortezomib treatment induced ER stress, evident by strong induction of Grp78, CHOP, PERK, and IRE1α (Western blot analysis) and the UPR (induction of hsp40, 70, and 90). Bortezomib treatment of cells at both sublethal and lethal doses increased viral replication (P < 0.001), but inhibition of Hsp90 ablated this response, reducing viral replication and synergistic cell killing. The combination of bortezomib and 34.5ENVE significantly enhanced antitumor efficacy in multiple different tumor models in vivo.Conclusions: The dramatic synergy of bortezomib and 34.5ENVE is mediated by bortezomib-induced UPR and warrants future clinical testing in patients. Clin Cancer Res; 20(14); 3787–98. ©2014 AACR.

The effect of αCD47-G1 or αCD47-G4 on the expression of NK cell markers.

Ewing sarcoma subcutaneous tumors stained for CD68+ macrophages, with hematoxylin counterstain.

PDF file - 7683K, HSV Antigen immunohistochemistry.

OV-αCD47-G1 treatments promote macrophage M1 polarization and NK cell activation in ID8-hCD47 immunocompetent ovarian cancer metastasis mouse model.

Purpose:Oncolytic herpes simplex virus-1 (oHSV) infection of brain tumors activates NOTCH, however the consequences of NOTCH on oHSV-induced immunotherapy is largely unknown. Here we evaluated the impact of NOTCH blockade on virus-induced immunotherapy.Experimental Design:RNA sequencing (RNA-seq), TCGA data analysis, flow cytometry, Luminex- and ELISA-based assays, brain tumor animal models, and serum analysis of patients with recurrent glioblastoma (GBM) treated with oHSV was used to evaluate the effect of NOTCH signaling on virus-induced immunotherapy.Results:TCGA data analysis of patients with grade IV glioma and oHSV treatment of experimental brain tumors in mice showed that NOTCH signaling significantly correlated with a higher myeloid cell infiltration. Immunofluorescence staining and RNA-seq uncovered a significant induction of Jag1 (NOTCH ligand) expression in infiltrating myeloid cells upon oHSV infection. Jag1-expressing macrophages further spread NOTCH activation in the tumor microenvironment (TME). NOTCH-activated macrophages increased the secretion of CCL2, which further amplified myeloid-derived suppressor cells. CCL2 and IL10 induction was also observed in serum of patients with recurrent GBM treated with oHSV (rQnestin34.5; NCT03152318). Pharmacologic blockade of NOTCH signaling rescued the oHSV-induced immunosuppressive TME and activated a CD8-dependent antitumor memory response, resulting in a therapeutic benefit.Conclusions:NOTCH-induced immunosuppressive myeloid cell recruitment limited antitumor immunity. Translationally, these findings support the use of NOTCH inhibition in conjunction with oHSV therapy.

Supplementary Data

Supplementary Methods and Supplementary Figure Legends

OV-αCD47-G1 improves the therapeutic efficacy against ovarian cancer in a xenograft model.

List of genes differentially induced ({greater than or equal to} 1.5 fold) in macrophages cultured with rHSVQ1 (Q) vs. RAMBO (R) infected glioma cells (UI, uninfected).

Supplementary Figure from NOTCH-Induced MDSC Recruitment after oHSV Virotherapy in CNS Cancer Models Modulates Antitumor Immunotherapy

PDF file, 157K, Ex vivo serum rescue assay.