Journal: Cancer Biology & Therapy

Article Title: ENPP3 drives ccRCC progression by cGAMP hydrolysis and STING–IFN suppression

doi: 10.1080/15384047.2026.2632995

Figure Lengend Snippet: ENPP3 is aberrantly high in ccRCC. (A) Box plot showing the distribution of ENPP3 expression in normal kidney tissue ( n = 72) compared to ccRCC tissue ( n = 523). (B) Kaplan–Meier overall survival curves for ccRCC patients stratified by ENPP3 expression levels. (C) Scatter plot showing the relative ENPP3 expression in normal kidney tissue ( n = 100) and ccRCC tissue ( n = 100). (D) Upper panel: Western blot analysis of ENPP3 protein expression in three representative and paired ccRCC tumor and adjacent normal tissue samples. Lower panel: qPCR quantification of ENPP3 mRNA levels in a panel of ccRCC cell lines (Caki-2, ACHN, 786-O, A498, 769-P, RCC4, and SNU-333) and the non-malignant HEK293 control. (E) Representative IHC staining of ENPP3 in normal (left) and ccRCC tissue (right).

Article Snippet: ENPP3-knockdown (shENPP3) or control (shNT) RCC4 xenografts ( n = 3/group) were harvested at day 18; where indicated mice received three i.p. injections of 250 μg neutralizing (WT) STING (TP308418, Origene).

Techniques: Expressing, Western Blot, Control, Immunohistochemistry

Journal: Cancer Biology & Therapy

Article Title: ENPP3 drives ccRCC progression by cGAMP hydrolysis and STING–IFN suppression

doi: 10.1080/15384047.2026.2632995

Figure Lengend Snippet: ENPP3 overexpression promotes tumor growth, and its inhibition enhances the efficacy of immune checkpoint blockade therapy. (A) Tumor growth curves of ACHN cells stably transfected with NC or ENPP3 in a xenograft model. (B) Tumor growth curves of Caki-2 cells stably transfected with NC or ENPP3 in a xenograft model. (C) Tumor growth curves of RCC4 cells stably transfected with shNT or shENPP3 in a xenograft model. (D) Tumor growth curves of SNU-333 cells stably transfected with shNT or shENPP3 in a xenograft model. (E) Pooled individual tumor growth curves for each SNU-333 xenografts ( n = 5 mice per arm) treated with control IgG, anti-PD-L1, anti-ENPP3, or the combination; the same individual data are displayed separately by treatment in the four right-hand panels.

Article Snippet: ENPP3-knockdown (shENPP3) or control (shNT) RCC4 xenografts ( n = 3/group) were harvested at day 18; where indicated mice received three i.p. injections of 250 μg neutralizing (WT) STING (TP308418, Origene).

Techniques: Over Expression, Inhibition, Stable Transfection, Transfection, Control

Journal: Cancer Biology & Therapy

Article Title: ENPP3 drives ccRCC progression by cGAMP hydrolysis and STING–IFN suppression

doi: 10.1080/15384047.2026.2632995

Figure Lengend Snippet: ENPP3 modulates the extracellular cGAMP and immune landscape in ccRCC. (A) Measurement of extracellular and intracellular cGAMP in ACHN and Caki-2 cells transfected with NC or ENPP3. (B) Measurement of extracellular and intracellular cGAMP in RCC4 and SNU-333 cells transfected with shNT or shENPP3. (C) Tumor volume in the RCC4 (ENPP3-intact or knockdown) xenograft model in combination with neutralizing STING administration. (D) Quantification of macrophage infiltration in RCC4 (ENPP3-intact or knockdown) xenograft tumors in combination with neutralizing STING administration. (E) The mean intensity of pIRF3 in M1-like macrophage in RCC4 (ENPP3-intact or knockdown) xenograft tumors in combination with neutralizing STING administration. (F) Quantification of MHC-II hi CD103 + in CD11b − CD11c + cDCs in RCC4 (ENPP3-intact or knockdown) xenograft tumors in combination with neutralizing STING administration. (G) Quantification of CTL activation (CD69 + ) in RCC4 (ENPP3-intact or knockdown) xenograft tumors in combination with neutralizing STING administration. (H) Quantification of CTL activation (CD25 + ) in RCC4 (ENPP3-intact or knockdown) xenograft tumors in combination with neutralizing STING administration. (I) The mean intensity of FOXP3 of CD3 hi CD4 + CD8 − CD45 + population in RCC4 (ENPP3-intact or knockdown) xenograft tumors in combination with neutralizing STING administration.

Article Snippet: ENPP3-knockdown (shENPP3) or control (shNT) RCC4 xenografts ( n = 3/group) were harvested at day 18; where indicated mice received three i.p. injections of 250 μg neutralizing (WT) STING (TP308418, Origene).

Techniques: Transfection, Knockdown, Activation Assay

Journal: Cancer Biology & Therapy

Article Title: ENPP3 drives ccRCC progression by cGAMP hydrolysis and STING–IFN suppression

doi: 10.1080/15384047.2026.2632995

Figure Lengend Snippet: ENPP3 deficiency induces a local type I IFN response in ccRCC. (A) qRT-PCR analysis of Ifnb1 and ISGs in TAMs isolated from RCC4 xenografts treated with shNT or shENPP3. (B) qRT-PCR analysis of Usp18, Oas3, and Ifit1 in PBMCs. (C) Measurement of IFNβ protein levels in RCC4 xenografts treated with shNT or shENPP3. (D–H) Quantitative ELISA analysis of CCL3, CCL4, CCL5, CCL7, and CCL12 in RCC4 xenografts treated with shNT or shENPP3. (I) Pooled individual tumor growth curves for each SNU-333 xenografts ( n = 5 mice per arm) treated with control IgG, anti-ENPP3, anti-IFNAR1, or the combination; the same individual data are displayed separately by treatment in the four right-hand panels.

Article Snippet: ENPP3-knockdown (shENPP3) or control (shNT) RCC4 xenografts ( n = 3/group) were harvested at day 18; where indicated mice received three i.p. injections of 250 μg neutralizing (WT) STING (TP308418, Origene).

Techniques: Quantitative RT-PCR, Isolation, Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: 3D vascularized microtumors unveil aberrant ccRCC vasculature and differential sensitivity to targeted treatments

doi: 10.1101/2025.03.27.645644

Figure Lengend Snippet: A, B and D : Spheroids of 500 RCC4-VHL or RCC4 cells embedded in collagen I hydrogel and cultured for 0 or 6 hours in complete DMEM ( A ) or for 3 days in complete DMEM or fibroblast-conditioned medium (NHDF-CM) ( B and D ), growth factor-depleted ECGM2 (ECGM2-Δ5) or supplemented with EGF (ECGM2-Δ5+EGF), VEGF-depleted ECGM2 (ECGM2-ΔVEGF) ( B ), stained for F-actin by phalloidin (red for RCC4-VHL, green for RCC4) and for nuclei by DAPI (blue). Images are displayed as z-stack projection ( A and B ) and optical section localized at the spheroid center determined by the highest diameter size ( D ). Scale bar: 100 µm ( A ) or 200 µm ( B and D ). C : Area ( a ), perimeter ( b ) and circularity index ( c ) of spheroids cultured in conditions described in panels B and D. Quantifications were performed on z-stack projections. E : Lumen area (left panel) and lumen area over total spheroid area (right panel) of RCC4-VHL spheroids cultured in conditions described in panels B and D. Quantifications were performed on central optical section. Graphs represent the mean of 3 independent experiments +/- SEM ( C and E ). Statistical analyses were performed using Kruskal-Wallis ( C ) or Welch’s t ( E ) tests. ns p>0.05, * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

Article Snippet: Brightfield images of RCC4 or RCC4-VHL spheroids in hanging drops were acquired by a Leica DMi1 microscope with an MC170HD camera (10x/0.3 objective).

Techniques: Cell Culture, Staining

Journal: bioRxiv

Article Title: 3D vascularized microtumors unveil aberrant ccRCC vasculature and differential sensitivity to targeted treatments

doi: 10.1101/2025.03.27.645644

Figure Lengend Snippet: A: Capillaries of HUVEC seeded in collagen I hydrogel and cultured for 5 days in conditioned medium (CM) from fibroblast (NHDF), RCC4, RCC4-VHL cells or 1:1 mixture of NHDF-CM/RCC4-CM or of NHDF-CM/RCC4-VHL-CM, stained for F-actin by phalloidin (green) and for nuclei by DAPI (blue). Images are displayed as maximum intensity projections of the capillary network (260 µm z-stacks). Scale bar: 200 µm. B: 3D quantification of total capillary length, capillary number, branch point number and connectivity index. Results were normalized to control condition (NHDF-CM). C: Capillaries formed and grown for 5 days in NHDF-CM were further treated for 4 days by the various cell-derived CM described in A. Images were performed as in A. Scale bar: 200 µm. D: 3D quantifications were performed as in B. Results were normalized to control condition in NHDF-CM. Graphs represent the mean of 3 independent experiments +/- SEM. Statistical analyses were performed using Brown-Forsythe and Welch ANOVA tests and represented with regard to the values of NHDF-CM (B and D). ns p>0.05, * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

Article Snippet: Brightfield images of RCC4 or RCC4-VHL spheroids in hanging drops were acquired by a Leica DMi1 microscope with an MC170HD camera (10x/0.3 objective).

Techniques: Cell Culture, Staining, Control, Derivative Assay

Journal: bioRxiv

Article Title: 3D vascularized microtumors unveil aberrant ccRCC vasculature and differential sensitivity to targeted treatments

doi: 10.1101/2025.03.27.645644

Figure Lengend Snippet: A : Schematic representation of 3D co-culture model. Spheroids of fluorescent RCC4 or RCC4-VHL were co-embedded in collagen I hydrogel with HUVEC suspension and cultured for 4 to 7 days in fibroblast-conditioned medium (NHDF-CM). B-E : Spheroids of fluorescent RCC4 (green) or RCC4-VHL (red) and capillaries after 4 days of culture. Images of vascularized microtumors immunostained for CD31 (white) and stained by DAPI for nuclei (blue) are displayed as z-stack projections ( B ) and optical sections at 3 spheroid z-levels ( C ). The endothelial pond is identified by a red triangle in the mid-plane section of RCC4 spheroid. Scale bar: 100 μm. 3D quantification of total capillary length, capillary number, branch point number and connectivity index of the capillary network formed in RCC4 (green) or RCC4-VHL (red) spheroid ( D ). Graphs represent the mean of 3 independent experiments +/- SEM. Statistical analyses were performed using Mann-Whitney test. ns p>0.05, ** p<0.01, *** p<0.001. Mid-plane section of RCC4 (green) spheroid reveals an endothelial pond immunostained for collagen IV (white), CD31 (red), and stained by DAPI for nuclei (blue) ( E ). Magnifications of yellow-(area 1) and blue-dotted (area 2) frames display the tumor-endothelial cell interactions at the pond periphery and fragmented nuclei (blue asterisk) of endothelial cells in the pond cavity, respectively. Scale bar: 20 μm.

Article Snippet: Brightfield images of RCC4 or RCC4-VHL spheroids in hanging drops were acquired by a Leica DMi1 microscope with an MC170HD camera (10x/0.3 objective).

Techniques: Co-Culture Assay, Suspension, Cell Culture, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: 3D vascularized microtumors unveil aberrant ccRCC vasculature and differential sensitivity to targeted treatments

doi: 10.1101/2025.03.27.645644

Figure Lengend Snippet: A and B: Time-lapse images of vascularized spheroids of RCC4-VHL (A) or RCC4 (B). Fluorescent spheroids (green) were co-embedded with fluorescent HUVEC (red) in collagen I hydrogel and cultured for 6 days in fibroblast-conditioned medium. Images of the spheroid were acquired every 10 to 12 hours and are displayed as maximal z-stack projections. Scale bar: 50 µm. C: Formation and remodeling processes of the endothelial pond in the invading RCC4 spheroid. Images were acquired between 44 and 140 hours of culture and are displayed for optical z-mid-plane sections. Scale bar: 100 µm.

Article Snippet: Brightfield images of RCC4 or RCC4-VHL spheroids in hanging drops were acquired by a Leica DMi1 microscope with an MC170HD camera (10x/0.3 objective).

Techniques: Cell Culture

Journal: bioRxiv

Article Title: 3D vascularized microtumors unveil aberrant ccRCC vasculature and differential sensitivity to targeted treatments

doi: 10.1101/2025.03.27.645644

Figure Lengend Snippet: HUVEC (A and B), RCC4 spheroids (C and D) or co-culture of HUVEC and RCC4 spheroids (E-G) were embedded in collagen I hydrogel and treated by temsirolimus (5, 10 or 25 µM), crizotinib (0.05 or 0.5 µM), sunitinib (0.05, 0.5 or 5 µM) or vehicle (0) in NHDF-conditioned medium for 2 days. A: Capillaries immunostained for CD31 (red) and stained bt DAPI for nuclei (blue). Scale bar: 100 µm. B: 3D quantification of total capillary length, branch point number and connectivity index of capillary network. Results were normalized to control condition (vehicle). C: RCC4 spheroids stained for F-actin by phalloidin (green) and for nuclei by DAPI (red). Scale bar: 100 µm. D: Quantification of area and perimeter of spheroids. Results were normalized to control condition (vehicle). E: Vascularized fluorescent RCC4 spheroids (green, mid- and bottom panels) immunostained for CD31 (red, top and bottom panels) and stained by DAPI for nuclei (blue, bottom panels). Scale bar: 100 µm. F: 3D quantification of total capillary length, branch point number and connectivity index of the capillary network in the vascularized microtumors. Results were normalized to control condition (vehicle). G: Quantification of area and perimeter of spheroids. Results were normalized to control condition (vehicle). Graphs represent the mean of 3 independent experiments +/- SEM. Statistical analyses were performed over control condition using Kruskal-Wallis test (B, D, F and G). ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, $ p<0.00001.

Article Snippet: Brightfield images of RCC4 or RCC4-VHL spheroids in hanging drops were acquired by a Leica DMi1 microscope with an MC170HD camera (10x/0.3 objective).

Techniques: Co-Culture Assay, Staining, Control

Journal: bioRxiv

Article Title: 3D vascularized microtumors unveil aberrant ccRCC vasculature and differential sensitivity to targeted treatments

doi: 10.1101/2025.03.27.645644

Figure Lengend Snippet: A : vascularization of spheroids was promoted for 5 days in the presence of NHDF-CM after which treatment with sunitinib (0.05 or 0.5 µM) was applied for 2 days. Vascularized fluorescent RCC4 spheroids (green, mid-panels) immunostained for CD31 (red) and stained by DAPI for nuclei (blue, mid- and right panels). Maximal z-stack projection of spheroid images (left and mid-panels) display the pond and connecting capillaries. Images of the spheroid-adjacent field (right panel) display capillary networks. Scale bar: 100 µm. B : Quantification of vascular areas of pond and connecting capillaries (black columns) and of adjacent tumor capillaries (white columns) were performed on z-stack projections. Results were normalized to corresponding control condition (vehicle). Graphs represent the mean of 3 independent experiments +/- SEM. Statistical analyses were performed using ANOVA test. Statistical mentions above columns proceed from comparison of treated pond and converging capillaries or of treated adjacent tumor capillaries to their corresponding control condition. ns p>0.05, **** p<0.0001.

Article Snippet: Brightfield images of RCC4 or RCC4-VHL spheroids in hanging drops were acquired by a Leica DMi1 microscope with an MC170HD camera (10x/0.3 objective).

Techniques: Staining, Control, Comparison

Journal: Molecular Oncology

Article Title: Loss of SETD2 in wild‐type VHL clear cell renal cell carcinoma sensitizes cells to STF ‐62247 and leads to DNA damage, cell cycle arrest, and cell death characteristic of pyroptosis

doi: 10.1002/1878-0261.13770

Figure Lengend Snippet: Drugs of interest in kidney cancer and their IC50 values measured in ccRCC cell lines.

Article Snippet: Both 786‐0 (RRID:CVCL_1051) and 786‐0/VHL cells were a gift from Dr. Amato J. Giaccia (Stanford University, CA, USA), whereas RCC4 (RRID:CVCL_UY81), RCC4/VHL (RRID:CVCL_2706) cells were purchased from Millipore Sigma (Oakville, ON, Canada).

Techniques:

Journal: bioRxiv

Article Title: TFE3 fusions direct an oncogenic transcriptional program that drives OXPHOS and unveils vulnerabilities in translocation renal cell carcinoma

doi: 10.1101/2024.08.09.607311

Figure Lengend Snippet: (a) OXPHOS and glycolysis gene signature scores in ccRCC or tRCC tumors from three independent studies (TCGA, Motzer et al., Elias et al. (PDX)) , , . (b) Principal component analysis (PCA) of H3K27ac ChIP-Seq data across 8 RCC cell lines (3 tRCC; 5 ccRCC; 4 ccRCC lines were profiled in a previously published study, see Extended Data Fig. S1 ). (c) Boxplot of averaged H3K27ac signal at typical or super enhancers at OXPHOS genes in 3 tRCC cell lines (UOK109, FU-UR-1, s-TFE) vs. 5 ccRCC cell lines (786-O, Caki-1, RXF393, TK10, A498). (d) Heatmap showing H3K27ac signal (quantified by ROSE2) at ETC and TCA cycle genes in tRCC vs. ccRCC cell lines. (e) GSEA showing enrichment of OXPHOS gene signature in tRCC cell lines (n=3, UOK109, FU-UR-1, s-TFE) versus ccRCC cell lines from CCLE (n=7, A498, A704, 786-O, 769-P, Caki-1, Caki-2, OS-RC-2). (f) Oxygen consumption rate (OCR) as measured by a Seahorse Bioflux analyzer after the addition of oligomycin, FCCP, or antimycin A/rotenone in a ccRCC cell line (786-O) and a tRCC cell line (s-TFE). Data are shown as mean ± s.d, n=5 biological replicates for 786-O cell line, n=6 biological replicates for s-TFE cell line. (g) Ratio of (OCR) to extracellular acidification rate (ECAR) as detected by a Seahorse Bioflux analyzer in ccRCC (n=6, 786-O, Caki-1, Caki-2, KRMC-1, A498, RCC4) and tRCC (n=3, UOK109, FU-UR-1, s-TFE) cell lines. OCR/ECAR ratio represents the basal respiration:glycolytic balance in each cell line. Data are shown as mean ± s.d, n=5-7 biological replicates per cell line. (h) Viability of ccRCC (n=6, 786-O, Caki-1, Caki-2, KRMC-1, A498, RCC4) and tRCC (n=3, UOK109, FU-UR-1, s-TFE) cell lines cultured in glucose or galactose-containing media for 6 days. Data are shown as mean ± s.d. n=3 biological replicates per cell line. (i) Viability of ccRCC (n=6, 786-O, Caki-1, Caki-2, KRMC-1, A498, RCC4) and tRCC (n=3, UOK109, FU-UR-1, s-TFE) cell lines cultured under hypoxic (2.5% O 2 ) or normoxic (20% O 2 ) conditions for 10 days. Data are shown as mean ± s.d. n=3-4 biological replicates per cell line. For panels (a), (c) and (g-i), statistical significance was determined by Mann-Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001, n.s. not significant.

Article Snippet: 786-O (ATCC, CatLog: ATCC ® CRL-1932 TM ), 293T (ATCC, CatLog: ATCC ® CRL-11268 TM ), RCC4 (Sigma: #3112702), KRMC-1(JCRB, CatLog:JCRB1010), A498(ATCC, CatLog: ATCC ® HTB-44 TM ), Caki-1(ATCC, CatLog: ATCC ® HTB-46 TM ), Caki-2 (ATCC, CatLog: ATCC ® HTB-47 TM ), UOK109 (Dr. W. Marston Linehan’s laboratory, National Cancer Institute), FU-UR-1(Dr. Masako Ishiguro’s laboratory Fukuoka University School of Medicine) and s-TFE(RIKEN, # RCB4699 were grown at 37°C in DMEM supplemented with 10% FBS, 100 U mL −1 penicillin, and 100 μg mL −1 Normocin (Thermo fisher: #NC9390718).

Techniques: ChIP-sequencing, Cell Culture, MANN-WHITNEY