Fig. 2

Combination of EwS-specific expression cassette and targeted gene delivery confers strong therapeutic effects in vivo. a mRNA log2 expression intensities of GPR64, FAT4, LECT1, and CD99 from publicly available microarray data of EwS (n = 50) and normal tissues (n = 928, comprising 70 different tissue types). Data are presented as boxplots with the horizontal line representing the median, the box the interquartile range (IQR) and the whiskers 1.5×IQR of the expression intensity. b Validation of surface expression of GD2, GPR64, CD99, FAT4 and LECT1 by antibody staining and flow cytometry. Isotype controls for both antibody host species were included separately. Dots indicate mean fluorescent intensity (MFI) for 4 independent experiments. Mean and standard deviation per group are depicted as horizontal bars and whiskers. c IRS (immunoreactive score) of GPR64 in immunohistochemistry of primary EwS tumors and relevant normal tissues. Representative EwS samples with high, medium and low GPR64 expression are shown aside. d Flow cytometry analysis of EwS and non-EwS cell lines after transduction with GPR64-targeting, GFP-encoding lentiviruses. CD99 and isotype- targeting lentivirus was used as positive and negative control. Dots indicate percentage of GFP positive cells determined by flow cytometry of 4 biologically independent experiments. Horizontal bars and whiskers represent mean and standard deviation per group. e Resazurin-based cell viability assay of EwS and non-EwS cell lines treated with GCV (20 µM) or DMSO vehicle control 24 h after GPR64-targeted transduction with pLenti_25_LT. Readout was performed 72 h after GCV addition. CD99-targeting lentiviruses, non-targeting lentiviruses (isotype) and VSV-G pseudotyped lentiviruses were included as controls. Dots indicate cell viability relative to that of vehicle control for 4 biologically independent experiments. Mean standard deviation per group are represented by horizontal bars and whiskers. f Bioluminescence measurements (exposure time: 20 s) of NSG mice bearing subcutaneous RD-ES xenografts 14 d after a single intratumoral injection of 0.5 × 10⁶ TU of pLenti_25_LT or pLenti_CMV_LG lentiviral particles pseudotyped with 2.2. GPR64- or CD99-targeting antibodies were used to coat 2.2 pseudotyped viruses. 2.2 pseudotyped viruses without antibodies were included as negative control. g Tumor volumes of A-673 subcutaneous xenografts treated with GPR64-targeting pLenti_25_LT or pLenti_CMV_LG (mock) lentiviruses. Valganciclovir (VGCV, 0.5 mg/ml in drinking water enriched with 5% sucrose) or sucrose (5% in drinking water) was administered orally ad libidum once the tumor had reached an average diameter of 5 mm. Lentiviruses were intratumorally injected twice per week starting from day 7. Data are shown as mean tumor volume and SEM of 6–7 mice per treatment condition. P-values were determined by one-tailed Mann-Whitney test. h Relative bioluminescence (right) and bioluminescent images (left) of NSG mice after intraperitoneal tumor inoculation with Firefly luciferase-expressing A-673. 3 days after tumor injection mice were randomized and repeatedly received either GPR64-directed 2.2. pseudotyped lentivirus (pLenti_25_TK) or PBS by intraperitoneal injection. VGCV was orally administered in both groups 3 days after the first virus injection. The representative bioluminescent pictures show both groups 12 and 19 days after tumor inoculation. Dots indicate bioluminescence signal relative the mean measured on of VGCV initiation (day 6) for 6–7 mice per group. Horizontal bars indicate mean and whiskers SEM per group. P values were determined by one-tailed Mann-Whitney test. i CD8⁺ T cell count per mg of tumor tissue and absolute CD8⁺ T cell count per spleen 5 days after human T cell transfer into mice bearing subcutaneous A-673 xenografts treated with GPR64-coated lentiviral particles (pLenti_25_IX) or PBS. Where not indicated otherwise, P-values were determined with two-tailed Mann-Whitney test, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001