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: Primary culture of adult normal human dermal fibroblasts (NHDF, Promocell, Heidelberg, Germany) were grown in fibroblast growth medium 2 (FGM2, PromoCell).

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: Primary culture of adult normal human dermal fibroblasts (NHDF, Promocell, Heidelberg, Germany) were grown in fibroblast growth medium 2 (FGM2, PromoCell).

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: Primary culture of adult normal human dermal fibroblasts (NHDF, Promocell, Heidelberg, Germany) were grown in fibroblast growth medium 2 (FGM2, PromoCell).

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: Primary culture of adult normal human dermal fibroblasts (NHDF, Promocell, Heidelberg, Germany) were grown in fibroblast growth medium 2 (FGM2, PromoCell).

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: Primary culture of adult normal human dermal fibroblasts (NHDF, Promocell, Heidelberg, Germany) were grown in fibroblast growth medium 2 (FGM2, PromoCell).

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: Primary culture of adult normal human dermal fibroblasts (NHDF, Promocell, Heidelberg, Germany) were grown in fibroblast growth medium 2 (FGM2, PromoCell).

Techniques: Staining, Control, Comparison