Supplementary Figure 5 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

A. Cytolytic effect of VLV-GFP in cancer cells is mediated by the induction of pro-apoptotic genes. Treatment of cancer cells with VLV-GFP is associated with a significant increase on TRAIL and FAS mRNA expression. *=p>0.005; **= p>0.001 B. In vivo induction of pro-apoptotic genes. Mice bearing mCherry-TKO tumors were treated 2X with VLV-GFP. The expression of TRAIL and FAS was determined 24h after the last treatment. Note the significant increase on TRAIL and FAS mRNA expression following VLV-GFP treatment. ***= p>0.0001</p

Supplementary Table S1 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

Primers used to clone CARG-2020, VLV-12 and VLV-GFP</p

Supplementary Figure 4 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

A. TKO mouse ovarian cancer cells were treated with increasing concentrations of CARG-2020 or VLV-GFP and effect on cell growth and cell death is quantified in real time using Cytation5/Biospa by measuring mCherry confluence and CelltoxTM fluorescence; B. Representative fluorescence images showing PBS Control and CARG-2020-treated cultures. red, mCherry; green, CelltoxTM; C. Human ovarian cancer cells, clone OCSC1-F2 and the hTERT-immortalized human endometrial stromal cells were treated with increasing concentrations of CARG-2020 for 72h and cell death was quantified using CelltoxTM fluorescence. Note that CARG-2020 induces cell death only in cancer cells and not in normal cells.</p

Supplementary Table S2 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

Primers used for qPCR reactions</p

Supplementary Figure 3 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

A. Diagram of CARG-2020 vector and GFP control vector; B. TKO mouse ovarian cancer cells were treated in vitro with PBS Control or 1x105 PFU/cell of CARG-2020 for 24h. IL12 mRNA was quantified by qPCR and secreted IL-12p40 and p70 protein were quantified using xMAP technology; C. Expression of IL-12 and IL-17R ECD were detected by western blot analysis in total cell lysate. D. Secretion of IL-17 RA-ECD was detected by western blot analysis in the supernatant of cells treated with CARG-2020. Control cells were treated with VLV-GFP vector. Representative images of 3 independent experiments. E. Expression of PDL-1 following treatment with CARG-2020. Note the time dependent decrease on PDL-1 expression on cells treated with CARG-2020. Control=VLV-GFP. Representative imagesof 3 independent experiments.</p

Supplementary Figure 6 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

A. Kaplan Meier curve comparing overall survival in mice treated with PBS Control, CARG-2020, VLV-IL-12, VLV-17R ECD, VLV-shRNA PD-L1, and VLV-GFP; p<0.0001. B. CARG-2020 but not VLV-IL-12 modulates the innate immune system. Peritoneal lavage from VLV-IL-12 and CARG-2020-treated mice (n=6) were analyzed by flow cytometry for macrophages using CD11b and CD206. VLV-IL-12 treatment is not able to increase the number of CD11blow/CD206- macrophages compared to CARG2020. Representative dot plots shown. Gating strategy is shown in Supp. Fig. 1. C. Decrease on Ly6C+/Ly6G+ MSC is observed only in the CARG2020 treated group but not in those treated with VLV-IL-12 group. Representative dot plots shown. Gating strategy is shown in Supp. Fig. 1.</p

Supplementary Figure 2 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

Diagram showing major immune phenotypes in each phase of ovarian tumor progression.</p

Supplementary Figure 1 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

Flow cytometry gating strategy CD8 T cells, Macrophages and MDSC.</p

Supplementary Figure 7 from Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory

A. Mean tumor burden over time of nude mice bearing OCSC1-F2 human ovarian tumors. mCherry fluorescent ROI was used to provide a measure of tumor burden. Data are presented as mean ± SEM. Treatments began on day 6 and continued through to day 12. B. Survival analysis using Kaplan-Meier.  </p