Fig 1: Antitumor efficacy of AdV5/3-D24, AdV5/3-D24-ICOSL-CD40L, and anti-PD-1 and the combinatory therapy in mesothelioma H226 xenograft immunodeficient BALB/c nude model (5×106 cells/flank, n=5 per group). (A) Prior to the start of treatment, tumors of sizes ~5 × 5 mm in diameter were randomized. Once tumors have been formed, the treatment has been initiated. Mice received 1×108 VP viruses i.t., 200 μg anti-PD-1 i.v. on days 0, 3, 6, 9, 12, and 15. The control group received PBS administered in a same scheme as treated groups. Tumor volume (mm3) was measured through the study. At the conclusion of the study, mice were sacrificed, and tumors were extracted. (B) Tumor volume (mm3) measured at the end of the study (day 32). (C) Body weight was measured throughout the study. Statistical analyses were carried out with ANOVA test. Error bars, mean ± SEM; *p ≤ 0.05.
Fig 2: Evaluation of ICOSL and CD40L expression in various mouse organs after the treatment with oncolytic adenoviruses, anti-PD-1, and their combinations in humanized mesothelioma H226 model. (A) ICOSL concentration was measured from mouse organs (liver, tumor, and spleen) and blood collected at sacrifice after the treatment with ELISA kit (RayBiotech, ELH-B7H2-1) according to manufacturer’s instructions. (B) CD40L was detected from mouse organs (liver, tumor, and spleen) and blood collected at sacrifice after the treatment with ELISA kit (RayBiotech, ELH-CD40L-1) as per the instructions laid down by the manufacturer. Statistical analyses were carried out with ANOVA test. Error bars, mean ± SM; *p ≤ 0.05, **p ≤ 0.01, ***p<0.001.
Fig 3: Transcriptomic analyses. To determine the putative levels of different immune cells in our data, we used two methods—MCP counter33 and quanTIseq. Both methods decompose bulk RNA-seq expression matrix using expression profile of genes characteristic only to specific cell types. For MCP counter, each cell type is assigned with a score, which correlates with putative number of each cell line. For quanTIseq for each sample, a fraction of each of predefined cell types is provided. We found out that only the combinatorial therapy resulted in the increased levels of CD8+ T cells in TME. Transcriptomic analyses revealed that (A) MUC16 and (B) MSLN were found among genes that expression was substantially decreased by the combinatorial therapy; **p ≤ 0.05, ***p<0.01. Putative levels of CD8+ T cells in TME analyzed with- quanTIseq (C) and MCP counter (D) using bulk RNA-seq data. The combinatorial therapy resulted in the highest levels of CD8+ T cells. (E) The application of AdV5/3-D24-ICOSL-CD40L or pembrolizumab alone did not yield increased levels of this subpopulation of T cells. Composition of TME predicted with quanTIseq using bulk RNA-seq data. ns, not significant.
Fig 4: In vitro cytotoxicity assay (MTS assay). (A) AdV5/3-D24-ICOSL-CD40L and AdV5/3-D24 at concentrations of 0.1, 1, and 10, and 100VP/cell were used to assess cell viability 96-h after infection. (B) Combinatory treatment with AdV5/3-D24-ICOSL-CD40L and AdV5/3-D24 at concentrations 100VP/cell with or without anti-PD-1 (100 µg/mL) were used to assess cell viability 96 h after infection. Statistical analyses were carried out with Mann–Whitney t-test. Error bars, mean ± SEM; **p ≤ 0.01, ***p<0.001.
Fig 5: Immunomodulatory properties of tested agents in humanized xenograft mesothelioma H226 NSG nude model. At the end of the study, mice were euthanized and tumors collected for immunological analyses from four groups: (i) control, (ii) AdV5/3-D24-ICOSL-CD40L, (iii) anti-PD-1, and (iv) the combinatory therapy (end of study). Tumor-infiltrating lymphocytes CD4+, CD8+, FoxP3, and CD8+GrB+ mean expression has been assessed in collected tumors. Samples were acquired using BD Lyric FACS Flow. Statistical analyses were carried out with ANOVA test; ns, not significant. Error bars, mean ± SEM.
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