Oncolytic HSV Therapy Modulates Vesicular Trafficking Inducing Cisplatin Sensitivity and Antitumor Immunity

Abstract

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.

Fig. 1:. RAMBO increases cisplatin accumulation in cisplatin-resistant ovarian cancer cells.

A: Representative histogram (left) and quantification (right) of percentage of GFP⁺ infected cisplatin resistant ovarian cancer cells. TR127 cells infected with RAMBO (MOI=0.1) for 1hr and then treated with CP (2μM) for 16hr. Data shown are mean percentage of GFP⁺ RAMBO infected TR127 cells (± s.d) analyzed by flow cytometry (n=3/group). B: Quantification of intracellular CP retained inside platin resistant OC cells treated with/without RAMBO. OVCAR8, TR127, TR182, OVTOKO and ID8 were treated with/without RAMBO (MOI = 0.1) for one hour and then incubated with 2.5 μM Texas-red labeled CP. Twenty-four hours post-infection CP accumulation in cells was analyzed by flow cytometry. Data shown are mean fluorescence intensity (MFI) of Texas-red labeled CP (± s.d., n=3/group) C: Confocal immuno-fluorescent images of TR127 cells treated with RAMBO (MOI=0.5) (bottom) or untreated (top) for 1hr and then treated with 2.5 μM Texas-red labeled CP (red) for 24hr. Cells were stained with DAPI and F-actin to image nuclei (blue) and F-actin (white) respectively. Images on the right are magnifications of the inset on the left. D: Gene Ontology terms and KEGG pathway enrichment analysis of differentially expressed genes in RAMBO infected versus uninfected OVTOKO cells. Pathways significantly changed in infected cells are shown color coded for fold change. For a pathway, the size and color represent the number of down-regulated genes in that pathway and the False Discovery Rate (FDR), respectively. Briefly, RNA harvested from OVTOKO cells treated with RAMBO (MOI=1) for 16hr was analyzed by RNAseq. The value shown is the average of three samples for each group. E–F: Heatmaps showing changes in gene expression within each panel of the Gene Ontology terms: extracellular exosome (E) and lysosome GO pathways (F). G: Confocal immuno-fluorescent images of OVTOKO cells treated with Texas-red labeled CP and stained with lysotracker (white) and DAPI (blue). OVTOKO cells were plated in poly-lysine coated chamber slides and infected with RAMBO (MOI=1) for 16hr and then treated with Texas-red labeled CP (2 μM) for another 4hr. Infected cells become GFP+ve (bottom). H: Western blot analysis of efflux proteins ATP11B and ATP7B expression in TR127 and TR182 cells treated with RAMBO and/or CP. I: Quantification of Texas-red labeled CP accumulation in TR127 cells knocked down for either ATP7B, ATP11B, or both was analyzed by flow cytometry (n=3/group). J: Quantification of Texas-red labeled CP accumulation in OVTOKO cells overexpressing (OE) both ATP11B and ATP7B, or control (Ctl). Cells were analyzed by flow cytometry. Error bars are s.d., *p value < 0.05, M=Mock, CP=Cisplatin, R=RAMBO.

Fig. 2:. Effect of RAMBO on DNA damage and STING activation in cisplatin resistant ovarian cancer cells.

A: Representative histograms from flow cytometry analysis of cisplatin-specific DNA adducts in CP treated TR127 and TR182 cells also treated either with RAMBO (solid line) or without (dotted line). Isotype control is shaded in grey. Right panel shows the quantification of cisplatin-specific DNA adducts in TR127 and TR182 cells treated with/without RAMBO (MOI=0.1) and with/without CP (2 μM) for 16hr. (shaded = isotype, dash line = CP, solid line = RAMBO+CP, n=3/group). B: Western blot analysis of p-Chk2 and p-H2X in TR182 (left) and TR127 (right) cells treated with RAMBO (MOI=0.1) and/or CP (2 μM) and harvested 0, 1, 16, or 30 hours post treatment. C: Immunofluorescence staining of micronuclei (extranuclear γH2AX staining) in OVTOKO control or cells treated with RAMBO and/or CP. p-H2AX (red) was used as a marker for damaged DNA double strand breaks, tumor cell nuclei is stained with DAPI (blue). p-H2AX staining outside the nucleus marks micronuclei containing damaged DNA. D: Heatmaps of differentially expressed genes in the different signaling pathways derived from an RNAseq analysis of OVTOKO cells treated with/without RAMBO and with/without CP. E: GSEA plot for cGAS-STING signaling and type I interferon signaling in OVTOKO cells treated with RAMBO and CP relative to untreated cells (top), or CP alone (bottom) treated cells. F: Quantification of cGAMP in the culture supernatant of OVTOKO cells treated with RAMBO (MOI=1) and/or CP (5 μM) for 16 hr as measured by ELISA (n=3/group)). G: Quantification of IFNβ mRNA in OVTOKO cells treated with RAMBO (MOI=1) and/or CP (5 μM) for 16 hr analyzed by qRT-PCR (n=3/group). Error bars are s.d, *p value < 0.05, M=Mock, CP=Cisplatin, R=RAMBO, RCP=RAMBO + CP.

Fig. 3:. Combination RAMBO with cisplatin treatment increases innate immunity.

A–B: Quantification of viable TR127 and TR182 cells treated with RAMBO and/or CP with PBMCs (black bars) or without (grey) overlaid. Cell tracker labelled TR127 cells (A) or TR182 cell (B) were infected with RAMBO (MOI 0.04) for 1 hr and then treated with or without CP (5 μM) and overlaid with or without PBMCs (Effector: Target=10:1) for another 96 hrs. Data shown are mean viable tumor cells (annexin and PI double negative) ± s.d quantified by flow cytometry (n=3/group). C: Quantification of IFNγ secretion in PBMCs co-cultured with TR127 or TR182 cells treated with RAMBO and/or CP. Data shown are mean IFNγ ± s.d released into conditioned medium (CM) (n=3/group). D: Quantification of gene expression of NKG2D ligands: ULBP1, ULBP2, ULBP3, MICA, and MICB relative to GAPDH by qRT-PCR from OVTOKO cells treated with RAMBO (MOI=0.2) and/or CP (2 μM) for 16 hours (n=3/group). E: Representative dot blots and quantification of NKG2D expression (activation marker) on CD56⁺ NK cells following co-cultured with OVTOKO cells by flow cytometry. OVTOKO cells were treated with RAMBO (MOI=0.5) and/or CP (5 μM) for 16hrs and then overlaid with NK cells (Effector :Target = 2:1) isolated from human donor-derived PBMCs for 48hr. (F) Mean IFNγ secretion in the supernatants from OVTOKO and NK cell co-cultures quantified by ELISA (n=3/group). Error bars are s.d, *p value < 0.05, CP=Cisplatin.

Fig. 4:. Combination of RAMBO with cisplatin treatment increases anti-tumor activity in TR182-Luc/ NSG model.

A: Treatment schematic TR182-luc xenograft establishment and therapy in NSG mice. 6-week old female NSG mice were inoculated with 5 × 10⁵ TR182-Luc-GFP tumor cells intraperitoneally. 5 days later tumor-bearing mice were treated with 5 × 10⁵ pfu RAMBO with or without CP (2mg/Kg) given on the indicated days. B–C: IVIS imaging and quantification TR182-Luc tumors in NSG mice after treatment with RAMBO and/or CP. Tumor growth of individual mice was monitored by IVIS imaging on day-1, day 18 and day 25 after RAMBO treatment (n=9/group, dpi = days post infection). D–E: Quantification of tumor nodules (D) and tumor weights (E) in TR182-Luc implanted mice twenty-eight days following RAMBO and/or CP treatment. F: Representative dot plots and quantification of GFP⁺ TR182 tumor cells and CD45⁺ leukocytes in tumor nodules harvested from TR182-Luc bearing mice twenty-eight days following RAMBO and/or CP treatment. Data shown are mean percentage of GFP⁺ TR182-Luc cells ± s.d. (n=3/group). G: H&E and Ki67 IHC staining of tumor nodules harvested from TR182-Luc-bearing mice 28 days following RAMBO and/or CP treatment. H: Quantification of tumor associated macrophage phenotype markers CD206 and CD86 expressed in tumor nodules harvested from TR182-Luc bearing mice 28 days following RAMBO and/or CP treatment. Tumor nodules were harvested and single cell suspension were prepared for flow cytometry analysis of %CD86⁺ and %CD206⁺ cells gated on F4/80 (n=3). I: Quantification of cisplatin by flameless atomic absorption spectrophotometry in TR182 tumor-bearing NSG mice 24 hrs after CP treatment (9 different tumor nodules from three mice/group). J: Quantification of cGAMP by ELISA in TR182 tumor lysates from tumor-bearing mice 24 hrs after RAMBO+CP treatment (n=3/group). Error bars denote s.d, *p value < 0.05, **p value < 0.01, Mock=7/group, CP=6/group, RAMBO=9/group, RAMBO+CP=9/group, CP=Cisplatin.

Fig. 5:. Combination of RAMBO with cisplatin treatment increases anti-tumor activity in ID8-Luc C57/BL6 model.

A: Treatment schedule of C57BL/6 mice bearing ID8-Luc-GFP intraperitoneal metastatic tumors. 5 × 10⁶ ID8-Luc-GFP tumor cells were implanted into 6-week old female C57BL/6 mice. 15 days later tumor-bearing mice were treated with or without 5 × 10⁵ pfu RAMBO (day 15) with or without CP (2mg/Kg, at the indicated time points). B–C: IVIS imaging and quantification of luciferase in ID8-Luc-GFP tumors grown in C57BL/6 mice following RAMBO and/or CP treatment. Tumor growth was monitored by IVIS imaging on day −1, day 23, and day 49 following RAMBO and CP treatments (n=10/group, dpi=days post infection). D: Quantification of volume of ascites in ID8-luc-GFP-bearing C57BL6 mice after RAMBO and/or CP treatment on day 75 following RAMBO treatment (n=5/group). E–F: Flow cytometry analysis of splenocytes from mice 3 days following treatment with RAMBO and/or CP. E: Quantification of IFNγ secretion in CD4⁺ and CD8⁺ T cells, and F: Quantification of IFNγ secretion in NK1.1+ cells, (n=3/group). G: Percent quantification of dendritic cells expressing CD86 and MHCII in splenocytes of mice treated with RAMBO and/or CP. Three days following RAMBO treatment of ID8-Luc-GFP tumor-bearing mice, splenocytes were harvested and analyzed for %CD86⁺, and %MHCII⁺ CD11c⁺ dendritic cells (n=3/group). H: Quantification of cytosolic DNA and mitochondrial DNA in human donor PBMC-derived dendritic cells co-cultured with OVTOKO cells treated with RAMBO and/or CP. Cells were co-cultured for 48hrs, cytosolic DNA and mtDNA from dendritic cells was quantified by qRT-PCR (n=3/group). I: Gene expression quantification by qRT-PCR of cGAS from OVTOKO cells (left) in bottom and dendritic cells derived from the transwell experiment described in (H), (n=3/group). J: Quantification of IFNβ mRNA from human PBMC-derived dendritic cells knocked down for STING (siSTING) and co-cultured with OVTOKO cells treated with RAMBO and/or CP compared to control. IFNβ mRNA was quantified by qRT-PCR (n=3/group). K: Quantification of %CD44⁺CD8⁺ and CD44⁺CD4⁺ T cells co-cultured human donor PBMC-derived dendritic cells and OVTOKO cells treated with RAMBO and/or CP. T cell activation was measured by flow cytometry. L: IFNγ secretion in the supernatant of co-cultures from RAMBO and/or CP treated OVTOKO cells with human PBMC-derived dendritic cells and purified T cells was quantified by ELIS, (n=3/group). M: Quantification of PD1 expression in splenic T cells from ID8-Luc-GFP tumor-bearing mice. Three days following RAMBO treatment, splenocytes were harvested and analyzed for %PD1 expression on CD4⁺ and CD8⁺ T cells by flow cytometry (n=3). Error bars denote s.d, *p value < 0.05, **p value < 0.001, NS indicated no significant difference, CP=Cisplatin

Fig. 6:. PD1 blockade increases anti-tumor response in murine metastatic ovarian cancer ID8-Luc model treated with RAMBO and cisplatin.

A: Representative histogram and quantification of PD-L1 in ovarian cancer cells treated with RAMBO and/or CP. TR127 cells were treated with RAMBO (MOI=0.1) and/or CP (5 μM) for 24hr. PD-L1 expression was analyzed by flow cytometry (left). OVTOKO were treated with RAMBO (MOI=0.5) and/or CP (5 μM) for 24hr. PD-L1 expression was analyzed by qRT-PCR (right), (n=3). B: Schematic of establishment and treatment of ID8-Luc-GFP tumor model in C57BL/6 mice treated with RAMBO and CP, with or without an anti-PD-1 antibody. 5 × 10⁶ ID8-Luc-GFP tumor cells were inoculated intraperitoneally into 6-week old female C57BL/6 mice. 15 days later, tumor-bearing mice were treated with 5 × 10⁵ pfu RAMBO and CP (2mg/kg) with or without anti-PD1 antibody (100 μg/mouse). C–D: IVIS imaging and quantification of ID8-Luc-GFP tumor growth in C57BL/6 mice on day −1, day 12, and day 65 following treatment (n=10). E–J: Flow cytometry analysis of splenocytes from mice treated with RAMBO and CP with or without anti-PD-1 blocking antibody. E: Quantification of NK cell activation markers (NK1.1 and NKG2D). F: Quantification of %PD-1⁺ CD4⁺ and CD8⁺ T cells. G: Quantification of %Tim-3⁺ CD4⁺ and CD8⁺ T cells. H: Quantification of %CD62L⁻ CD4⁺ and CD8⁺ of T cells. I: Quantification of IFNγ secretion in CD4⁺ and CD8⁺ T cells, and J: Quantification of IFNγ secretion in NK1.1+ cells, (n=3). Error bars are s.d, *p value < 0.05, ns indicated no significant difference, CP=Cisplatin.