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 ) IHC staining of paraffin-embedded serial initial tumor biopsy sections (patient 3000276) with H and E, Pax8, pY397 FAK, and Ki67. Scale is 200 µm. Inset (green box) region is shown at 40X (below). Scale is 60 µm. ( B and C ) FAK pY397 staining intensity of paired patient ovarian tumor samples from initial biopsies (panel B) and after surgical removal following neoadjuvant chemotherapy (panel C) within Pax8-positive (tumor) and Pax8-negative (stroma) regions. Dot plots are quantified staining from 14 paired patient samples (Aperio software) and bars show mean ± SEM (analyzed 11 regions per sample, ***p<0.001, unpaired T-test). ( D ) Increased FAK pY397 staining within Pax8-positive regions post-chemotherapy (*p<0.05, paired T-test). Lines are connecting paired patient tumor samples collected prior to and after neoadjuvant chemotherapy.

Article Snippet: Antibody , anti-Pax8 (rabbit polyclonal) , Proteintech Group , Cat# 10336–1-AP; RRID: AB_2236705 , WB (1:1000).

Techniques: Immunohistochemistry, Staining, Software

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: Antibody , anti-Pax8 (rabbit polyclonal) , Proteintech Group , Cat# 10336–1-AP; RRID: AB_2236705 , WB (1:1000).

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:

Article Snippet: Antibody , anti-Pax8 (rabbit polyclonal) , Proteintech Group , Cat# 10336–1-AP; RRID: AB_2236705 , WB (1:1000).

Techniques: Transfection, Protease Inhibitor, Recombinant, Proliferation Assay, Reporter Assay, SYBR Green Assay, ALDH Detection Assay, Isolation, Plasmid Preparation, Clone Assay, Expressing

Journal: The EMBO Journal

Article Title: Stable expansion of high‐grade serous ovarian cancer organoids requires a low‐Wnt environment

doi: 10.15252/embj.2019104013

Figure Lengend Snippet: Summary of cancer patient data with TNM and FIGO classifications showing advanced stage of disease at the time of surgery. Graphic representation of the standard experimental procedure for tumor patient material. Samples were obtained at the time of primary debulking surgery from the high purity tumor deposits in peritoneum/omentum. In vitro niche dependency of HGSOC tumor cells. Phase‐contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth, while they do not grow at all in Wnt3a‐supplemented medium. Also, inhibition of BMP signaling through Noggin has strong negative effect on the initial growth. Scale bar: 500 μm. E—EGF, F—FGF10, N—Noggin, R—R‐spondin1, B—Basic medium, P—Passage. Cancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines. Scale bar: 20 μm. HE staining of organoids and respective tissue confirms high similarity in cellular structure and tissue organization. Scale bar: 100 μm. HGSOC organoids show differential response to carboplatin treatment, confirming patient‐specific sensitivity of the cultures. Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors. Data represent mean ± SD of technical triplicates.

Article Snippet: Rabbit anti‐Pax8, IHC (1:50) , Proteintech , 10336‐1‐AP.

Techniques: In Vitro, Isolation, Inhibition, Staining, Viability Assay

Journal: The EMBO Journal

Article Title: Stable expansion of high‐grade serous ovarian cancer organoids requires a low‐Wnt environment

doi: 10.15252/embj.2019104013

Figure Lengend Snippet: Diagnosis of ovarian cancer in tissue fragments used for organoid generation was confirmed by experienced pathologists to be of HGSOC phenotype, being marked by strong EpCAM, Pax8, and p16 expression. Depicted are four examples of the pathology stainings. Scale bar: 100 μm. Phase‐contrast images of the establishment of HGSOC organoid culture (OC1) showing strong negative effect of low‐BMP (presence of Noggin) and high‐Wnt signaling, achieved here by the addition of GSK3‐β inhibitor CHIR (3 μM) (lower panel). While isolates seeded in EFR medium gave rise to expanding organoids (upper panel), only small aggregates formed in the presence of CHIR (lower left) and entered growth arrest with prominent cell debris indicative of cell death (lower right). This culture could not be further maintained. Scale bar: 500 μm. Three HGSOC organoid lines were tested for their response to the drug carboplatin showing slightly different responses as determined by cell viability assay. Data represent mean ± SD of technical triplicates.

Article Snippet: Rabbit anti‐Pax8, IHC (1:50) , Proteintech , 10336‐1‐AP.

Techniques: Expressing, Viability Assay

Journal: The EMBO Journal

Article Title: Stable expansion of high‐grade serous ovarian cancer organoids requires a low‐Wnt environment

doi: 10.15252/embj.2019104013

Figure Lengend Snippet:

Article Snippet: Rabbit anti‐Pax8, IHC (1:50) , Proteintech , 10336‐1‐AP.

Techniques: Recombinant, Plasmid Preparation, Microscopy, Western Blot, Software, Viability Assay, Labeling, Microarray, Ligation, SYBR Green Assay, Spectrophotometry, Real-time Polymerase Chain Reaction