Fig 2: Antitumor efficacy of oncolytic vectors and the combination therapy with anti PD-1 antibody in murine melanoma B16V (1 × 106 cells/flank, one mouse had 2 tumors/6 tumors per group) xenograft immunocompetent C57BL/6 model. (A) Tumor volume (mm3) measured through the study. The treatment was performed once per day on days 1–6. The mice were treated according to the scheme (Table 1) with viruses (i.t.) and anti PD-1 antibody (i.v.) (B) At the end of the study, mice were sacrificed and tumors harvested for weight assessment. (C,D) Tumor volume measurement on days 6 and 20. (E) Body weight measurements throughout the study. (F) Survival profile was calculated by Kaplan–Meier test. (H) Evaluation of CRT exposure after the treatment with oncolytic adenoviruses AdV-24-ICOSL-CD40L and AdV-D24, and in combination with anti PD-1. CRT exposure was measured in the end of the study (after mice sacrifice) with anti-calreticulin antibody staining and subsequent flow cytometry analysis (Beckman-Coulter Cytomics FC500). (G) Assessment of ATP release after the treatment. ATP concentration from the tumors was evaluated in the end of the study (after mice sacrifice) with CellTiter-Glo® Luminescent Cell Viability Assay ATP detection kit by Promega. Error bars, mean ± SEM, * = p = 0.05, *** = p = 0.0001.

Fig 3: Immunogenic cell death assessment. (A) Evaluation of calreticulin (CRT) exposure by melanoma cell lines after treatment with oncolytic adenoviruses AdV-24-ICOSL-CD40L and AdV-D24, and in combination with anti PD-1. CRT exposure was measured 48 h post-treatments with anti-calreticulin antibody staining AlexaFluor® 488 and subsequent flow cytometry analysis (Beckman-Coulter Cytomics FC500). (B) Assessment of ATP release after the treatment. ATP concentration in a supernatant was evaluated 72 h after infection with CellTiter-Glo® Luminescent Cell Viability Assay ATP detection kit by Promega. (C) Evaluation of high-mobility group box 1 (HMGB-1) release after treatment with oncolytic adenoviruses and the combination with anti PD-1. HMGB-1 level was measured from the supernatant collected 72 h after infection with ELISA kit (MBL International, Woburn, MA, USA), according to manufacturer’s dispositions. Statistical analysis was carried out with a Mann–Whitney test to compare two groups (* = p = 0.05; ** = p = 0.001, *** = p = 0.0001).

Fig 4: Evaluation of ICOSL and CD40L expression in tested melanoma cells. (A) ICOSL concentration was detected on supernatants collected 24–48 h after treatment using ELISA kit. (B) CD40L was detected from supernatants collected 24–48 h after treatment using an ELISA kit according to the manufacturer’s instructions. Statistical analysis was carried out with a Mann–Whitney test to compare two groups (ns = not significant, p > 0.05). (C) Transgene expression. The expression of ICOSL and CD40L from AdV-D24-ICOSL-CD40L was assessed by infecting A549 cells with the virus, harvesting proteins 48 h after the infection and detecting ICOSL and CD40L by Western blot. 1–4: protein extracts from A549 cells infected with AdV-D24-ICOSL-CD40L clones #1–4. N: protein extract from non-infected A549 cells. M: PageRuler Prestained Protein Ladder (ThermoScientific # 26616). ICOSL MW: 70 kDa (due to heavy protein glycosylation); CD40L MW: 30 kDa.

Fig 5: Evaluation of cell viability by MTS assay (cell cytotoxicity assay). (A) Cell viability was evaluated 72 h post-infection with AdV-24-ICOSL-CD40L and AdV-24 at the concentration of 100 VP/cell and combination with anti-PD1 (pembrolizumab). Data are expressed as the percentage of viable cells according to MTS cell viability assay protocol (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega). Statistical analysis was carried out with a Mann–Whitney test to compare two groups (p > 0.05). (B,C) Microscopic photographs visualizing the morphology and cytopathic effect (CPE) of the cells representing the investigated groups and assessed by MTS assay at 48 and 72 h post-infection (magnification 10×) (** = p = 0.001).