Journal: bioRxiv

Article Title: Interplay between PML NBs and HIRA for H3.3 deposition on transcriptionally active interferon-stimulated genes

doi: 10.1101/2021.11.30.470516

Figure Lengend Snippet: A. (left) Fluorescence microscopy visualization of HIRA (green) and PML (red) in BJ cells treated with a control siRNA targeting luciferase (siLuc) or with an siRNA against DAXX (siDAXX) for 48h. Ruxolitinib (Ruxo) was added at 2μM in the last 24h. Cell nuclei are visualized by DAPI staining (grey). Scale bars represent 10μm. (right) Histogram shows quantitative analysis of cells with HIRA localization at PML NBs. p-values (Student t-test): *<0,05; ns: non significant. Numbers represent the mean of 3 independent experiments (±SD). B. Histogram shows quantitative analysis of PML protein levels from western blot analysis presented in Sup. Figure 3C. p-values (Student t-test): ns: non significant. Numbers represent the mean of 3 independent experiments (±SD). C. (left) Fluorescence microscopy visualization of HIRA (green) and H3.3-HA (red) in BJ eH3.3i cells treated with doxycyclin (Dox) and IFNβ for 24h. Arrowheads indicate nuclei with high levels of nucleoplasmic H3.3-HA preventing HIRA accumulation in PML NBs despite IFNβ treatment. Scale bars represent 10μm. (right) Histogram shows quantitative analysis of cells with HIRA localization at PML NBs in BJ eH3.3i treated with Dox and IFNβ as indicated for 24h. p-values (Student t-test): *<0,05; **<0,01; ns: non significant. Numbers represent the mean of 3 independent experiments (±SD). D. Fluorescence microscopy visualization of HIRA (green) and H3.3-HA (red) in BJ eH3.3i cells treated with doxycyclin and with or without IFNβ for 24h. High exposure indicates a lane where H3.3-HA signal was specifically increased in order to show H3.3-HA localization in PML NBs without saturating the signal in cells with higher expression. Scale bars represent 10μm. E. Quantification of nuclear H3.3-HA intensity levels in BJ eH3.3i cells treated as in C. Mean H3.3-HA intensity levels were calculated on a pool of n=121 nuclei from 3 independent experiments (total). Nuclei were then separated on basis of accumulation of HIRA in PML NBs (nuclei with HIRA in PML NBs, n=58) or without it (nuclei with no HIRA in PML NBs, n=63) and mean H3.3-HA intensity was plotted for each category. Bars represent median with interquartile range. p-values (Mann-Whitney u-test): ****<0,0001. F. Fluorescence microscopy visualization of HIRA (green) and H3.3-HA (red) in IMR90 eH3.3i cells treated as in C. Scale bars represent 10μm. Arrows indicate nuclei showing accumulation of HIRA in PML NBs together with H3.3-HA, while arrowheads indicate nuclei with high levels of nucleoplasmic H3.3-HA preventing HIRA accumulation in PML NBs despite IFNβ treatment.

Article Snippet: Human BJ primary foreskin fibroblasts (ATCC, CRL-2522), Human IMR90 fetal lung fibroblasts (ATCC, CCL-186), human HEK 293T embryonic kidney cells (Intercell, AG) and mouse MEFs embryonic fibroblasts Pml -/- (from Dr. Lallemand-Breitenbach) were cultivated in DMEM medium (Sigma-Aldrich, D6429) containing 10% of fetal calf serum (FCS) (Sigma-Aldrich, F7524), 1% of penicillin/streptomycin (Sigma-Aldrich, P4458), at 37°C under 5% CO2 and humid atmosphere.

Techniques: Fluorescence, Microscopy, Control, Luciferase, Staining, Western Blot, Expressing, MANN-WHITNEY

Journal: medRxiv

Article Title: A 3D ovarian cancer metastasis model using a decellularized peritoneal matrix to study therapy response

doi: 10.1101/2024.12.27.24319681

Figure Lengend Snippet: 3D cell cultures are established with the porcine dECM scaffolds. a Schematic illustration of the setup for cell culture. Scaffolds were mounted in a Transwell®-like setup and cells were seeded in the inner chamber. After three days, the scaffolds were flipped, allowing the cells to come in direct contact with the imaging surface, enabling confocal imaging. Scaffolds were kept in this reversed position in the culture plate between imaging. b Confocal images and representative images of histological staining of cell cultures on pSIS and pPerMa. Each row represents a cell line (COV318, OV90, and Caov3). COV318 and OV90 cells were seeded in a number of 25,000 cells/scaffold, while Caov3 cells were seeded at 400,000 cells/scaffold for monoculture and 200,000 cells/scaffold for coculture. For cocultures, BJ fibroblasts were seeded in a number of 12,500 cells/scaffold (COV318), 6,250 cells/scaffold (OV90), and 25,000 cells/scaffold (Caov3). Confocal images are 3D renderings of z-stacks, visualized in the x/y plane (left, quadratic images) and the x/z plane (right, rectangular images). Magenta represents tissue autofluorescence (ex = 561 nm, em = 575-625 nm), green represents GFP+ EOC cell lines (ex = 488 nm, em = 500-550 nm), and white represents miRFP670+ BJ fibroblasts (ex = 637 nm, em = 663-738 nm). Scale bars = 100 µm. Histological stainings were performed on a cross-section of the scaffolds, corresponding to the x/z plane in the confocal images. The monocultures are stained with EpCAM. Cocultures are stained with EpCAM (top) and hematoxylin & eosin (H&E; bottom). Scale bars = 50 µm. NA = not applicable, too few cells to stain. The experiment was performed two times with three replicates for each group. c Graphs showing the cell growth of COV318, OV90, and Caov3 cells on pSIS (monoculture) and pPerMa (monoculture and coculture with fibroblasts). The cell volume was estimated using the surfaces function in the IMARIS image analysis software. Data represent mean ± SD.

Article Snippet: Human EOC cell lines Caov3 (cat# HTB-75, RRID: CVCL_0201), OV90 (cat# CRL-11732, RRID: CVCL_3768), and OVCAR3 (cat # HTB-161, RRID: CVCL_0465) were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA), and human EOC cell line COV318 (cat# 07071903, RRID: CVCL_2419) from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA).

Techniques: Cell Culture, Imaging, Staining, Software

Journal: medRxiv

Article Title: A 3D ovarian cancer metastasis model using a decellularized peritoneal matrix to study therapy response

doi: 10.1101/2024.12.27.24319681

Figure Lengend Snippet: Representative confocal images of COV318 ( a ), OV90 ( b ), and Caov3 ( c ) on pSIS (top) and pPerMa (bottom) on days 3-21. COV318 and OV90 cells were seeded in a number of 25,000 cells/scaffold, while Caov3 cells were seeded at 400,000 cells/scaffold. The confocal images are 3D renderings of z-stacks, visualized in the x/y plane. Magenta represents tissue autofluorescence (ex = 561 nm, em = 575-625 nm) and green represents GFP+ EOC cell lines (ex = 488 nm, em = 500-550 nm). Scale bars = 100 µm.

Article Snippet: Human EOC cell lines Caov3 (cat# HTB-75, RRID: CVCL_0201), OV90 (cat# CRL-11732, RRID: CVCL_3768), and OVCAR3 (cat # HTB-161, RRID: CVCL_0465) were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA), and human EOC cell line COV318 (cat# 07071903, RRID: CVCL_2419) from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA).

Techniques:

Journal: medRxiv

Article Title: A 3D ovarian cancer metastasis model using a decellularized peritoneal matrix to study therapy response

doi: 10.1101/2024.12.27.24319681

Figure Lengend Snippet: Representative confocal images of BJ fibroblasts in coculture with COV318 ( a ), OV90 ( b ), and Caov3 ( c ) on days 3-21. COV318 and OV90 cells were seeded in a number of 25,000 cells/scaffold and Caov3 cells at 200,000 cells/scaffold. For cocultures, BJ fibroblasts were seeded in a number of 12,500 cells/scaffold (COV318), 6,250 cells/scaffold (OV90), and 25,000 cells/scaffold (Caov3). The confocal images are 3D renderings of z-stacks, visualized in the x/y plane. Magenta represents tissue autofluorescence (ex = 561 nm, em = 575-625 nm), green represents GFP+ EOC cell lines (ex = 488 nm, em = 500-550 nm), and white represents miRFP670+ BJ fibroblasts (ex = 637 nm, em = 663-738 nm). Scale bars = 100 µm. NA = not applicable (only imaged on days 14 and 21).

Article Snippet: Human EOC cell lines Caov3 (cat# HTB-75, RRID: CVCL_0201), OV90 (cat# CRL-11732, RRID: CVCL_3768), and OVCAR3 (cat # HTB-161, RRID: CVCL_0465) were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA), and human EOC cell line COV318 (cat# 07071903, RRID: CVCL_2419) from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA).

Techniques:

Journal: medRxiv

Article Title: A 3D ovarian cancer metastasis model using a decellularized peritoneal matrix to study therapy response

doi: 10.1101/2024.12.27.24319681

Figure Lengend Snippet: Carboplatin sensitivity for OV90 cells. a Dose-response curve for OV90 cells in 2D culture, treated with different doses of carboplatin. Y axis represents the absorbance of formazan in treated cells relative to control (%). X axis represents doses of carboplatin in moles/liter (M). The EC50 drug dose was determined to be 45.60 µM. b,c Response to carboplatin for OV90 cells in 2D culture, evaluated with Annexin V/PI staining and flow cytometry. b Example gating shown for one sample treated with 45.6 µM carboplatin. c Graph showing the percentage of dead cells for the different doses of carboplatin and untreated, calculated as the sum of Q1-Q3. Data represent mean ± SD. d Representative confocal images of OV90 cells cultured on pPerMa and treated with carboplatin at different doses (10 µM, 50 µM, and 100 µM). The confocal images are 3D renderings of z-stacks, visualized in the x/y plane. Magenta represents live cells stained with CytoCalcein Violet (ex = 405 nm, em = 425-475 nm), green represents apoptotic cells stained with Apopxin Green (ex = 488 nm, em = 500-550 nm), and white represents necrotic cells stained with 7-AAD (ex = 637 nm, em = 663-738 nm). e Graphs showing the surface volume (left graph) and cell count (three right graphs) of the markers for live, apoptotic, and necrotic cells for scaffolds treated with increasing doses of Carboplatin. Data represent mean ± SD.

Article Snippet: Human EOC cell lines Caov3 (cat# HTB-75, RRID: CVCL_0201), OV90 (cat# CRL-11732, RRID: CVCL_3768), and OVCAR3 (cat # HTB-161, RRID: CVCL_0465) were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA), and human EOC cell line COV318 (cat# 07071903, RRID: CVCL_2419) from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA).

Techniques: Control, Staining, Flow Cytometry, Cell Culture, Cell Counting

Journal: medRxiv

Article Title: A 3D ovarian cancer metastasis model using a decellularized peritoneal matrix to study therapy response

doi: 10.1101/2024.12.27.24319681

Figure Lengend Snippet: CAR T cell sensitivity is assessed in the 3D cell cultures using confocal microscopy. a Schematic illustration of the setup for treatment with CAR T or Mock T cells. The EOC cell lines OV90 and OVCAR3 were seeded on PerMa. OV90 cells were seeded at 25,000 cells/scaffold and OVCAR3 cells at 100,000 cells/scaffold. After three days, the scaffolds were flipped and imaged with confocal microscopy. After four more days, the scaffolds were imaged again and flipped back to the original position with the cells in the inner chamber. The next day, CAR T or Mock T cells were seeded on the scaffolds. After 24 hours, the scaffolds were flipped, stained with 7-AAD, and imaged with confocal microscopy. b,d Representative confocal images of OV90 cells treated with CAR T cells targeting TAG72 or Mock T cells (b), and OVCAR3 cells treated with CAR T cells targeting Muc16 or Mock T cells (d). CAR T and Mock T cells were derived from three different donors. The experiment was performed with three replicates in each group. Magenta represents 7-AAD (ex = 561 nm, em = 575-625 nm), green represents GFP+ EOC cell lines (ex = 488 nm, em = 500-550 nm), and white represents CellTraceTM Violet stained T cells (ex = 405 nm, em = 425-475 nm). The T cells that were adhered to the scaffold and/or tumor cells, did not detach when the scaffolds were flipped and can be seen in white on the confocal images. Scale bars = 100 µm. NA = not applicable. c,e Graphs showing the estimated cell volume of OV90 cells (c) and OVCAR3 cells (e) untreated and treated with Mock T cells or CAR T cells (left). Graphs showing the change in estimated cell volume from the day before treatment to 24 hours after treatment for Mock T cells (middle) and CAR T cells (right). The cell volume was estimated using the surfaces function in the IMARIS image analysis software. Data represent mean ± SD, * P < 0.05, ** P < 0.01. Different symbols in the graphs represent donor 1 (circle), donor 2 (square), and donor 3 (triangle).

Article Snippet: Human EOC cell lines Caov3 (cat# HTB-75, RRID: CVCL_0201), OV90 (cat# CRL-11732, RRID: CVCL_3768), and OVCAR3 (cat # HTB-161, RRID: CVCL_0465) were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA), and human EOC cell line COV318 (cat# 07071903, RRID: CVCL_2419) from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA).

Techniques: Confocal Microscopy, Staining, Derivative Assay, Software