Fig 2: CMP5 effect on BLV ENV-mediated syncytia formation. A BLV-producing cell line FLK-BLV was co-cultured with a reporter cell line CC81-GREMG and treated with Milli-Q water (CMP5−) or CMP5 at the indicated concentrations (CMP5+) for 48 h. (A,B). Fluorescent syncytia in nine fields of view in each well were automatically scanned by EVOS2 fluorescence microscopy with a 4× objective. Fluorescent syncytia were recognized by enhanced green fluorescent protein (EGFP) expression and gated by their area and intensity. Red arrows indicate positions of fluorescent syncytia. Hoechst 33342 was used to stain the nucleus. Each picture is a representation of nine pictures acquired for each well. (B) Data analysis of (A) shows syncytia count using HCS Studio Cell Analysis software. Error bars represent the standard deviation of three independent experiments, the p value was calculated by ANOVA (0.00031), and Dunnett’s multiple comparisons test showed a significant decrease in the syncytia count at 10 µM (* p = 0.017) and 20 µM (*** p = 0.0002). (C) Effects of CMP5 on the total cell count taken from gating and counting of Hoechst 33342 staining using HCS Studio Cell Analysis software (Thermo Fisher Scientific). (D) CMP5 effect on cell viability according to WST-1 assay.

Fig 3: Cell populations proportions retained in 786-O and ACHN tumoroids compared to their original tumor tissue.A Immunofluorescent images of 786-O whole mounted tumoroids and their counterpart tissues. Tissue sections are 8 µm thick. Each column represent a different co-labeling : (1) FAP & vimentin, (2) CAIX & Ki67, (3) CD31 & E-cadherin, (4) collagen Pan & CD44, (5) CD68 & actin. Images were obtained using the Cell In Sight CX7 High throughput automated microscope (ThermoFisher) using 20x objective (NA 0.45) for tumoroids and 10x objective (NA 0.4) for tissue slices. Tumoroid scale bar = 50 µm; Tissue scale bar = 100 µm. B Percentage of cells expressing the different markers in tumoroids and tissues in a grouped analysis. 786-O tumoroids and ACHN tumoroids were respectively grouped to obtain global proportions. Quantification of different populations was made by the Thermo Fisher’s HCS studio software using the spot detector bioapplication. Reference levels for positive cells to each marker was the same for all samples. Quantification was made on the following number of replicates : Tumoroids 786-O = 27; Tumoroids ACHN = 20–28; Tissues = 4. Statistical analysis was made using student t-test, p < *0.05, ****0.0001. C Correlation and linear regression of the percentage of cells that are positive for a markers in tumoroids compared to their respective tissue they are derived from. X-axis correspond to the percentage of positive cells for a marker present in the tissue while Y-axis correspond to the mean of the percentage of positive cells for 6 to 8 tumoroids per tumor. Correlations are significant for FAP, vimentin, CAIX, CD68 and CD44 (p < 0.05).

Fig 4: Characterization and drug sensitivity of the human ccRCC tumoroids.A Human tumoroid and its counterpart tumoral tissue from patient NJ115 stained with HE. Scale bar = 40 µm. B, C Tumoroids (B) and tissue slices (C) treated with KU-CX (10 µM each) or Sunitinib (10 µM) or DMSO as control. Mortality was assessed as in Fig. 5. D Immunofluorescent images of NJ115 whole mounted tumoroids and its counterpart tissue. Tissue sections are 8 µm thick. Each column represent a different co-labeling: (1) FAP & vimentin, (2) CAIX & Ki67, (3) CD31 & E-cadherin, (4) collagen Pan & CD44, (5) CD68 & actin. Images were obtained using the Cell In Sight CX7 High throughput automated microscope (ThermoFisher) using 20x objective (NA 0.45) for tumoroids and 10x objective (NA 0.4) for tissue slices. Tumoroid scale bar = 50 µm; Tissue scale bar = 100 µm. E Percentage of cells expressing the different markers in tumoroids and tissue for the human tumor NJ115. Quantification of different populations was made by the Thermo Fisher’s HCS studio software using the spot detector bioapplication. Reference levels for positive cells to each marker was the same for all samples. Quantification was made on the following number of replicates: Tumoroids = 6–8; Tissues = 2. Statistical analysis was made using student t-test, *p < *0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.