Fig 2: PD-L1 inhibitors secreted from VV-iPDL1/GM-infected cells bind to PD-L1 on tumor cells and immune cells.a MC38 tumor cells were infected with VV-RFP, VV-iPDL1/GM at an MOI = 0.5, or PBS for 24 h. The percentage of IgG Fc+ population representing iPDL1 (soluble PD-1-IgG Fc)-bound VV-infected (RFP+) or uninfected (RFP−) PD-L1-expressing tumor cells was measured by flow cytometry. b MC38 tumor cells that were stimulated with IFN-γ (20 ng/mL) for 48 h were infected with the indicated VVs. PD-L1 expression of infected (RFP+) or uninfected (RFP−) cells was determined by flow cytometry. c–g MC38 cells were subcutaneously inoculated into the left (1 × 106) and right (5 × 105) flanks of C57BL/6 mice. When left flank tumor sizes reached ~100 mm3 (counted as day 0), the tumors of the left flank were intratumorally injected with 50 μL of PBS, VV-RFP, VV-GM, or VV-iPDL1/GM (5 × 107 pfu per tumor), or 200 μg of anti-PD-L1 antibody (clone 10F.9G2) intravenously on days 0 and 3. Two days post-second VV treatment, VV-treated (primary tumor) and untreated, distant tumors were collected, weighed and digested with collagenase type I and DNase. Tumor cell suspensions were blocked with anti-CD16/32 antibody and then stained with antibodies against CD45, CD3, CD8, CD4, CD11c, CD11b, Gr-1, FoxP3, PD-L1, and IgG Fc to assess PD-L1 expression or IgG Fc+ frequency on infected (RFP+) or uninfected (RFP−) tumor cells from the treated primary tumors c or untreated distant tumors (CD45− cells) d, and PD-L1 expression on infiltrating immune cells from treated e or untreated distant tumors f, IgG Fc+ frequency on infiltrating immune cells g. Infiltrating immune cells include cytotoxic T cells (CD45+CD3+CD8+), DCs (CD45+CD11c+), myeloid-derived suppressor cell MDSCs (CD45+CD11c−CD11b+Gr-1+), and Treg (CD45+CD3+CD4+FoxP3+); n = 5 mice. Significant differences are indicated as **P < 0.01, ***P < 0.001, or ****P < 0.0001 determined by two-tailed Student’s t-test.

Fig 3: (A) High, middle and low uptake of tumor in mice by 18F-RGD PET/CT imaging; (B) Changes of tumoral PD-L1 expression score (6-step scoring system, ‘Cologne Score’) of mice with days; (C) Changes of SUVmean and SUVmax of mice with days. P < 0.05 is considered statistically significant.

Fig 4: (A) ROC curve of 18F-RGD PET/CT parameters to reflect tumoral PD-L1 expression status (relatively high or low expression) in tumor-bearing mice: AUC of SUVmax and SUVmean were 0.871 (95% CI, 0.743-0.998) and 0.848 (95% CI, 0.699-0.997), respectively; (B) ROC curve of 18F-RGD PET/CT parameters to reflect tumoral PD-L1 expression status (relatively high or low expression) in NSCLC patients: AUC of SUVmax, SUVmean, SUVpeak and GTV were 0.881 (95% CI, 0.692-1.000), 0.774 (95% CI, 0.499-1.000), 0.786 (95% CI, 0.525-1.000) and 0.762 (95% CI, 0.490-1.000) respectively.

Fig 5: (A) Low, middle and high uptakes of 18F-RGD PET/CT scanning for NSCLC patients; (B) Relatively low and high expression of PD-1 and PD-L1 (×400) in NSCLC patients; (C) Scatter diagrams of correlations between SUVmax, SUVmean, SUVpeak and PD-L1 expression in NSCLC patients. P < 0.05 is considered statistically significant.