Fig 1.
Schematic procedure for 3D co-culture of HUVECs and OVCAR8 in Matrigel sandwich.
(A) HUVECs seeded on polymerised Matrigel; (B) After 4 hours, HUVECs started to form tubules; (C) OVCAR8 cell suspension in medium containing 10% Matrigel was added; (D) 24 hours later, Matrigel sandwich structure formed and co-culture stabilised, ready for longer term culture or further drug testing.
Fig 2.
Morphology characterisation of different in vitro models used in the study.
(A) Representative pictures of (A) HUVECs in mono-culture, (B) OVCAR8 in mono-culture, (C) 3D co-culture of both cell types in the Matrigel sandwich, and (D) on-top co-culture of both cell types on top of the Matrigel at day 1 (24 hours after seeding on day 0), day 3, day 5, day 7 and day 10. It is noticeable that the tubule structures of HUVECs mono-culture started to degrade at day 3.
Fig 3.
Immunostaining characterisation of 3D Matrigel sandwich models used in the study.
3D structure analysis by multiphoton microscopy (MPM). Green: GFP-expressing OVCAR8; Red: vWF staining of HUVECs by TRITC; Blue: Cell nuclei marked by DAPI. Scale bar: 200μm. (A) HUVECs 3D mono-culture in the Matrigel sandwich, at day 2 (from day 3 most tubule structure degraded); (B) OVCAR8 3D mono-culture in the Matrigel sandwich at day 10; (C) Co-culture of HUVECs and OVCAR8 in the Matrigel sandwich at day 10. (A)–(C): (i) original fluorescence photo; (ii) 3D reconstruction by Imaris; (iii) Side view of the 3D reconstruction showing the thickness. In the 3D reconstruction figure the grid unit measurement is 20μm.
Fig 4.
Image processing procedure used for bright field images.
(A) The original bright field images of 3D mono-culture of OVCAR8 cells, on-top co-culture of OVCAR8 cells and HUVECs, and 3D co-culture of OVCAR8 cells and HUVECs at day 10. (B) Background corrected images of each original image. (C) Different contour lines detected in each background corrected image. (D) Segmentation of spheroids (in black colour) and tubules (in grey colour) in each image.
Fig 5.
Semi-automated image analysis comparing morphologies of different in vitro models.
(A) Comparison of 3D on-top and 3D sandwich co-culture of OVCAR8 and HUVECs with regard to: (i) Total spheroid areas in μm2 and (ii) Mean tubule areas. (B) Comparison of 3D mono-culture of OVCAR8 and 3D co-culture of HUVECs and OVCAR8 in terms of: (i) Total spheroid areas in μm2 and (ii) Mean spheroid areas in μm2. (C) Angiogenesis parameter comparisons for 3D on-top and 3D sandwich co-culture of OVCAR8 and HUVECs over 10 days for: (i) Total tubule areas and (ii) Branching points. Results are presented as (mean ± SD), and are considered significantly different when p < 0.05 based on Student’s t-test (marked as ‘*’ in the figures showing significant difference between the compared models on the same day). Note that since HUVECs mono-culture lost their tubule network at day 2 and eventually lost signs of living cells over longer term culture (Fig 2(A)), only mono-culture of cancer cell OVCAR8 and co-culture of OVCAR8 and HUVECs were compared here over 10 days of culture.
Fig 6.
Apoptosis assay comparing co-culture of OVCAR8 with HUVECs and mono-culture of OVCAR8 at day 10.
(A) Fluorescence images of co-culture. It is noticeable that nearly no apoptotic signal was observed with thinner tubule structures, while more apoptosis was detected with the structures with larger dimensions. (B) Fluorescent images of mono-culture. Cleaved caspase 3 (magenta); DAPI (blue), HUVECs (red), and OVCAR8 (green). Scale bar: 100μm. (C) Zen localisation coefficient comparison between co-culture and mono-culture. No significant difference was observed.
Fig 7.
Drug response comparisons between 3D mono-cultures and co-cultures.
(A) Dose-dependent responses comparing monocultures and co-culture, measured by cell viability inhibition percentages in a Matrigel sandwich treated with: (i) Cisplatin; (ii) Paclitaxel. (B) Tubule area reduction comparing monocultures and co-culture, after treatment by: (i) Cisplatin and (ii) Paclitaxel. *p<0.05 **p<0.01 compared with control using Student’s t-test.
Fig 8.
Representative images of the co-culture model and mono-culture models in response to Cisplatin or Paclitaxel.
(A) Mono-culture of HUVECs; (B) Mono-culture of OVCAR8; (C) Co-culture of HUVECs and OVCAR8 in a Matrigel sandwich. The images at the concentrations of 0, 10nM, and 1μM are given as representatives, as most differences were observed between 10nM and 1μM. Scale bar: 200μm.