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Fig. 7 | Journal of Experimental & Clinical Cancer Research

Fig. 7

From: Therapeutic targeting of P2X4 receptor and mitochondrial metabolism in clear cell renal carcinoma models

Fig. 7

P2XR4 expression and mitochondrial respiration in ccRCC organoids. A Representative hematoxylin–eosin (H&E) staining of different clear cell carcinomas PDOs sections together with the bright-field microscopy images of corresponding PDOs (inset). Scale bars = 50 μm (section) and inset scale bar = 1 mm. B Representative immunofluorescence images of PDOs stained with typical markers of clear cell renal cancers, e.g., CD10 (green) and carbonic anhydrase 9 (red) Ki67 (pink). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars = 100 μm or 20 μm. C Phase contrast images of organoids from different patients treated with different doses of 5-BDBD as indicated, or vehicle for 4 days, Scale bars = 100 μm. D Quantification of PDOs diameters from different patients (PDOs 1–5) treated for 4 days with the indicated dose of 5-BDBD. Data are reported as mean of 20 replicates ± SD determined by two-tailed Mann–Whitney test. **p < 0.01. E Immunofluorescence of PDOs incubated with DMSO or 5 μM, 10 μM or 50 μM 5-BDBD for different days. Nuclei are indicated by DAPI staining (blue), live cells by calcein-AM (green), and dead cells by Caspase 3/7 (red). Scale bars = 200 μm. F and G Dose response curve in PDOs from patients 1 to 10 treated for 4 days with different doses of 5-BDBD as indicated. Data are reported the means of 20 biological replicates for each dose. H IC50 (micromol/L) of individual organoids (n = 20) from different patients. Data reported are the mean of 20 biological replicates. I Area under the curve (AUC) of individual organoids from different patients. The means of twenty replicates is indicated. Significance was calculated by two-way ANOVA **p < 0.001. J Oxygen consumption rate (OCR) in PDOs 1–10 or parental A-498 cells measured by Seahorse XF96 analyzer. Data are representative of 4 biological replicates. The selected cut-off of OCR is indicated by line. K Basal extracellular acidification rate (ECAR) in PDOs and parental A-498 cells. Data are representative of 4 biological replicates. L PDOs diameters after different time of treatment with 5-BDBD. Red lines represent PDOs with high mitochondrial activity, blue lines are PDOs with low mitochondrial activity. Data are reported as mean of 20 replicates and ± SD. Significance was determined by unpaired Student’s t-test. **, p < 0.01

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