Journal: eLife

Article Title: FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy

doi: 10.7554/eLife.47327

Figure Lengend Snippet: ( A ) Paraffin-embedded IHC serial section staining (H and E, Pax8, pY397 FAK, and active β-catenin) of peritoneal ascites cells (tumorspheres) from initial (patient 1014086) biopsy. ( B ) OVCAR3 lysates from 2D adherent, suspended (1 hr), and cells in anchorage-independent serum-free (PromoCell) conditions facilitating tumorsphere formation were analyzed by total FAK and pY397 FAK immunoblotting. ( C ) Representative images of OVCAR3 tumorsphere formation at Day 1, Day 2, and Day 3. Scale is 2 mm. ( D ) OVCAR3 cells as tumorspheres (Day 3) treated with DMSO or CP (1 µM) for 1 hr and protein lysates blotted for pY397 FAK, total FAK, pY142 β-catenin, and total β-catenin.

Article Snippet: Proliferating cells were seeded in 3D tumorsphere growth conditions (PromoCell), DMSO or GSK3β inhibitor (CHIR99021, Sigma, 3 µM) added after 24 hr, and cells evaluated by flow cytometry (BD FACSCelesta) after 5 days.

Techniques: Staining, Western Blot

Journal: eLife

Article Title: FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy

doi: 10.7554/eLife.47327

Figure Lengend Snippet: Quantification of OVCAR3 and KMF tumorsphere formation (panel A), ALDEFLUOR activity (panel B), and tumorsphere viability (panel C) in the presence of DMSO (control), CP (1 µM), FAKi (VS-4718, 1 µM) or CP plus FAKi for 5 days. Values are means (± SEM, *p<0.05, **p<0.01, ***p<0.001 unpaired T-test) of three independent experiments. Panel C, values are means (± SEM, ***p<0.001, one-way ANOVA) from three independent experiments.

Article Snippet: Proliferating cells were seeded in 3D tumorsphere growth conditions (PromoCell), DMSO or GSK3β inhibitor (CHIR99021, Sigma, 3 µM) added after 24 hr, and cells evaluated by flow cytometry (BD FACSCelesta) after 5 days.

Techniques: Activity Assay

Journal: eLife

Article Title: FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy

doi: 10.7554/eLife.47327

Figure Lengend Snippet: ( A ) Growth of KMF cells in 2D culture + /- FAKi (VS-4718, 1 µM) over 72 hr. ( B ) Cell viability (trypan blue exclusion) of KMF cells grown in 3D (PromoCell) in the presence of DMSO, cisplatin (CP, 1 µM), or FAKi (1 µM). ( A and B ) Values are means + /- SD. NS, not significant. ( C ) Representative images of KMF cell tumorsphere formation (PromoCell, 5 days) in DMSO, cisplatin (CP, 1 µM), FAKi (1 µM), or CP plus FAKi (1 µM each). Scale is 1 mm. ( D ) KMF cells grown in 2D or 3D as tumorspheres for 3 days, treated with DMSO or FAKi (VS-4718, 1.0 µM), stained with propidium iodide, and analyzed by flow cytometry. Percentage of cells in G0, G0/G1, S, and G2/M phase of the cell cycle was determined using FlowJo. ( E ) Percent of KMF tumorsphere cell staining for annexin V or 7-AAD (7-aminoactinomycin D) treated with DMSO (control, ( C ), FAKi (VS-4718, 1 µM 24 hr), staurosporine (1 µM, 24 hr), or heat shock (100°C, 15 min) by flow cytometry. ( D and E ) Values are means from two independent experiments. ( F ) KMF cells grown as 3D tumorspheres for 3 days were treated with DMSO, FAKi (VS-4718, 1.0 µM), cisplatin (CP, 1 µM), or CP plus FAKi (1 µM each) for 2 hr and lysates evaluated by FAK and pY397 FAK immunoblotting.

Article Snippet: Proliferating cells were seeded in 3D tumorsphere growth conditions (PromoCell), DMSO or GSK3β inhibitor (CHIR99021, Sigma, 3 µM) added after 24 hr, and cells evaluated by flow cytometry (BD FACSCelesta) after 5 days.

Techniques: Staining, Flow Cytometry, Western Blot

Journal: eLife

Article Title: FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy

doi: 10.7554/eLife.47327

Figure Lengend Snippet: ( A ) Growth of OVCAR3 cells in 2D culture + /- FAKi (VS-4718, 1 µM) over 72 hr. ( B ) Cell viability (trypan blue exclusion) of OVCAR3 cells grown in 3D (PromoCell) in the presence of DMSO, cisplatin (CP, 1 µM), or FAKi (1 µM). ( A and B ) Values are means + /- SD. NS, not significant. ( C ) Representative images of OVCAR3 tumorsphere formation (PromoCell, 5 days) in DMSO, cisplatin (CP, 1 µM), FAKi (1 µM), or CP plus FAKi (1 µM each). Scale is 1 mm. ( D ) OVCAR3 cells grown in 2D or 3D as tumorspheres for 3, treated with DMSO or FAKi (VS-4718, 1 µM), stained with propidium iodide, and analyzed by flow cytometry. Percentage of cells in G0, G0/G1, S, and G2/M phase of the cell cycle was determined using FlowJo. ( E ) Percent of OVCAR3 tumorsphere cell 7-AAD (7-aminoactinomycin D) staining when treated with DMSO (control, ( C ), FAKi (VS-4718, 1 µM 24 hr) or heat shock (100°C, 15 min) by flow cytometry. ( D and E ) Values are means from two independent experiments.

Article Snippet: Proliferating cells were seeded in 3D tumorsphere growth conditions (PromoCell), DMSO or GSK3β inhibitor (CHIR99021, Sigma, 3 µM) added after 24 hr, and cells evaluated by flow cytometry (BD FACSCelesta) after 5 days.

Techniques: Staining, Flow Cytometry

Journal: eLife

Article Title: FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy

doi: 10.7554/eLife.47327

Figure Lengend Snippet: ( A ) Human A2780, A2780-CP70, OVCAR10, and OVCAR10-CP tumorsphere lysates immunoblotted for FAK pY397, total FAK, and actin. ( B ) Growth of A2780, OVCAR10, A2780-CP70, or OVCAR10-CP cells as tumorspheres in the presence of FAKi (VS-4718, 0.1 to 1.0 µM) for 4 days. Values are means (± SEM, ***p<0.001, one-way ANOVA) from two independent experiments. ( C ) A2780-CP70 or OVCAR10-CP cells grown as tumorspheres (3 days) were treated with DMSO or FAKi (VS-4718, 1 µM) for 24 hr, stained with propidium iodide, and analyzed by flow cytometry. Shown is percent of cells in G0/G1, S, or G2/M phase of the cell cycle. ( D ) Quantitation of A2780-CP70 and OVCAR10-CP colony formation in methylcellulose (21 days) with DMSO (control) or FAKi (VS-4718, 1 µM). Values are means (± SEM, *p<0.05, ***p<0.001, unpaired T-test) from two independent experiments. ( E ) A2780-CP70 and OVCAR10-CP tumorsphere ALDEFLUOR activity treated with DMSO or FAKi (VS-4718, 1 µM) for 24 hr. Values are means (± SEM, **p<0.01, ***p<0.001, one-way ANOVA compared to DMSO) for three independent experiments. ( F ) Quantitation of A2780-CP70 and OVCAR10-CP methylcellulose colony formation (21 days). Values are means (± SEM, *p<0.05, ***p<0.001, unpaired T-test) from two independent experiments. ( G and H ) Representative OVCAR10-CP methylcellulose colony formation (21 days) (panel G) and colony size (panel H) in the presence of DMSO (control), CP (1 µM), FAKi (1 µM), or CP plus FAKi. Scale is 2.5 mm. Values are means (± SEM, *p<0.05, ***p<0.001, one-way ANOVA) from two independent experiments. ( I ) A2780-CP70 and OVCAR10-CP tumorsphere cytotoxicity (annexin V) in the presence of DMSO (control), CP (1 µM), FAKi (1 µM), or CP plus FAKi. Values are means (± SEM, **p<0.01, one-way ANOVA) from three independent experiments.

Article Snippet: Proliferating cells were seeded in 3D tumorsphere growth conditions (PromoCell), DMSO or GSK3β inhibitor (CHIR99021, Sigma, 3 µM) added after 24 hr, and cells evaluated by flow cytometry (BD FACSCelesta) after 5 days.

Techniques: Staining, Flow Cytometry, Quantitation Assay, Activity Assay

Journal: eLife

Article Title: FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy

doi: 10.7554/eLife.47327

Figure Lengend Snippet: ( A ) Representative images of A2780 and A2780-CP70 methylcellulose colony formation (21 days) + /- FAKi (1 µM VS-4718). Scale is 2 mm. ( B ) Quantitation of A2780 and A2780-CP70 colony formation from panel A. Values are means + /- SD from two independent experiments (NS, not significant and ***p<0.001). ( C ) A2780 and A2780-CP70 cells grown as 3D tumorspheres for 3 days, treated with DMSO or FAKi (VS-4718, 1 µM), stained with propidium iodide, and analyzed by flow cytometry. Percentage of cells in G0, G0/G1, S, and G2-M phase of the cell cycle was determined using FlowJo. ( D ) Quantitation of cyclin D1 protein levels by immunoblotting of cell lysates from tumorsphere experiment in panel F. Values are normalized to DMSO-treated control and are means + /- SD from three independent experiments (*p<0.05 and **p<0.01). ( E ) Representative flow cytometry histograms of A2780-CP70 spheroids treated with DMSO (gray) 1 µM VS-4718, 5 µM cisplatin, VS-4718 plus cisplatin, or 1 µM staurosporine for 18 hr (orange) and analyzed for annexin V binding.

Article Snippet: Proliferating cells were seeded in 3D tumorsphere growth conditions (PromoCell), DMSO or GSK3β inhibitor (CHIR99021, Sigma, 3 µM) added after 24 hr, and cells evaluated by flow cytometry (BD FACSCelesta) after 5 days.

Techniques: Quantitation Assay, Staining, Flow Cytometry, Western Blot, Binding Assay

Journal: eLife

Article Title: FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy

doi: 10.7554/eLife.47327

Figure Lengend Snippet: ( A ) Immunoblotting of KMF and CRISPR-mediated FAK KO clones KT3 and KT13 cell lysates for FAK, Pyk2, β-catenin, and actin. ( B ) KMF and FAK KO KT13 cell viability treated with DMSO (control) or CP (1 µM) after 72 hr as measured by Alamar Blue. Values are means (± SEM, *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA with Fisher’s LSD multiple comparison test) for three independent experiments. ( C ) β-catenin transcriptional reporter activity (TOPFlash) in transfected KMF and KT13 FAK KO cells + /- GSK3β inhibitor. Values are arbitrary units (***p<0.001, unpaired T-test, two independent experiments). ( D ) Immunoblotting for pY397 FAK, FAK, and actin in lysates of KMF, FAK KO, GFP-FAK-WT, and GFP-FAK-R454 re-expressing cells. ( E ) Top 10 proteomic differences (fold-change) detected by mass spectroscopy of membrane associated proteins in KT13 FAK KO, GFP-FAK-WT, and GFP-FAK R454 re-expressing cells. ( F ) Immunoblotting for β-catenin and actin in lysates of KT13 FAK KO cells stably expressing GFP-FAK-WT, GFP-FAK-R454, or β-catenin (ΔGSK). ( Gand H ) XTT metabolic activity (panel G) or tumorsphere formation (panel H) of KMF, KT13 FAK KO, or the indicated reconstituted cells in PromoCell after 5 days. Values are means (± SEM, *p<0.05, ***p<0.001, one-way ANOVA with a Tukey’s multiple comparisons test) from 2 (panel G) or 3 (panel H) independent experiments. 10.7554/eLife.47327.027 Figure 8—source data 1. KMF FAK KO clone KT13 exome sequencing variants. 10.7554/eLife.47327.028 Figure 8—source data 2. Summary of mass spectrometry-detected proteomic changes between KMF FAK KO, FAK-WT, and FAK kinase-inactive (K454R) re-expressing cells grown as tumorspheres.

Article Snippet: Proliferating cells were seeded in 3D tumorsphere growth conditions (PromoCell), DMSO or GSK3β inhibitor (CHIR99021, Sigma, 3 µM) added after 24 hr, and cells evaluated by flow cytometry (BD FACSCelesta) after 5 days.

Techniques: Western Blot, CRISPR, Clone Assay, Activity Assay, Transfection, Expressing, Mass Spectrometry, Stable Transfection, Sequencing

Journal: eLife

Article Title: FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy

doi: 10.7554/eLife.47327

Figure Lengend Snippet: ( A ) Immunoblotting for FAK and actin in OVCAR3 lysates and identification of FAK KO clones (AB21 and AB28 are starred). ( B ) Growth of OVCAR3 parental, AB21, and AB28 cells in 2D culture (black bars) or enumeration of colonies in methylcellulose after 14 days (red bars). Values are means as percent of OVCAR3 control (± SEM, ***p<0.001, one-way ANOVA or NS, not significant) from two independent experiments. ( C ) Cell viability treated with DMSO (control) or CP (1 µM) after 72 hr as measured by AlamarBlue. Values are means (± SEM, *p<0.05, ***p<0.001) for three independent experiments. ( D ) Immunoblotting for FAK, active β-catenin, β-catenin, Myc, and actin in the indicated cell lysates from 3D conditions. ( E ) Tumorsphere formation (5 days). ( F ) ALDEFLUOR activity. ( E and F ) Values are means (± SEM, n = 2, *p<0.05, ***p<0.001, one-way ANOVA with a Tukey’s multiple comparisons test) from three (panel E) or four (panel F) independent experiments. ( G ) Immunoblotting for ALDH-1A2, ALDH-1B1, or actin in the indicated cell lysates. ( H ) Cytotoxicity (percent Annexin V negative) of OVCAR3 (black circles), OVCAR3 FAK KO (red squares), and OVCAR3 FAK KO + GFP-FAK-WT (green triangles) cells in PromoCell treated with increasing CP concentrations for 5 days. Values are means (± SEM, ***p<0.001, two-way ANOVA with a Bonferroni’s multiple comparisons test) from three independent experiments. EC50 values were determined independently and calculated using Prism (Graphpad).

Article Snippet: Proliferating cells were seeded in 3D tumorsphere growth conditions (PromoCell), DMSO or GSK3β inhibitor (CHIR99021, Sigma, 3 µM) added after 24 hr, and cells evaluated by flow cytometry (BD FACSCelesta) after 5 days.

Techniques: Western Blot, Clone Assay, Activity Assay