Journal: Cancer Communications

Article Title: The Fibrillin‐1/VEGFR2/STAT2 signaling axis promotes chemoresistance via modulating glycolysis and angiogenesis in ovarian cancer organoids and cells

doi: 10.1002/cac2.12274

Figure Lengend Snippet: FBN1 knockout inhibits glycolysis and angiogenesis, leading to increased cisplatin sensitivity in cisplatin‐resistant ovarian cancer organoids and cells. ( A ) Glucose uptake, lactate, ATP and NADPH production in cisplatin‐resistant ovarian cancer organoids and cell line. Data are presented as mean ± SD of triplicate measurements repeated three times. ( B ‐ C ) ECAR ( B ) and OCR ( C ) in cisplatin‐resistant ovarian cancer organoids and cells. ( D ) Effect of FBN knockout on HUVEC tube formation. HUVEC cells were treated with supernatant obtained from OVCA433‐CisR/FBN1‐KO1, OVCA433‐CisR/FBN1‐KO2, or the corresponding control cells. ( E ) Cell viability assay of organoids treated with 5 μg/L cisplatin and/or 20 μmol/L apatinib in different time intervals. ( F ) IC50 values of cisplatin for FBN1‐knockout and control ovarian cancer cells treated with different concentrations of cisplatin for 48 h with or without 20 μmol/L apatinib; IC50 values of apatinib for the cells treated with various concentrations of apatinib for 48 h with or without 2.5 μg/mL cisplatin. ( G ) Relative colony formation efficiency of cisplatin‐resistant ovarian cancer organoids and cells treated without drugs or with 2.5 μg/mL cisplatin and/or 20 μmol/L apatinib for 7 days. ( H ) Cell viability assay of organoids treated with 5 μg/L cisplatin alone or in combination with 2.5 mmol/L 2‐DG in different time intervals. ( I ) IC50 values of cisplatin for ovarian cancer cells treated with different concentrations of cisplatin with or without 5 mmol/L 2‐DG for 48 h. ( J ) Relative colony formation efficiency of cisplatin‐resistant ovarian cancer organoids and cells treated with 2.5 μg/mL cisplatin alone or in combination with 2.5 mmol/L 2‐DG for 7 days. **, P < 0.01. Abbreviations: FBN1, fibrillin‐1; SD, standard deviation; CR: cisplatin‐resistant; KO, knockout; NC, negative control; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; HUVECs, human umbilical vein endothelial cells. IC50, half maximal inhibitory concentration; 2‐DG, 2‐deoxy‐D‐glucose

Article Snippet: Shortly, 24‐well plate coated Matrigel was prepared in advance, and then 2 × 10 4 human umbilical vein endothelial cells (HUVECs, ATCC) were inoculated in the plate with 200 μL DMEM medium.

Techniques: Knock-Out, Control, Viability Assay, Standard Deviation, Negative Control, Concentration Assay

Journal: Cancer Communications

Article Title: The Fibrillin‐1/VEGFR2/STAT2 signaling axis promotes chemoresistance via modulating glycolysis and angiogenesis in ovarian cancer organoids and cells

doi: 10.1002/cac2.12274

Figure Lengend Snippet: FBN1 combines directly with VEGFR2 in ovarian cancer cells. ( A ) Co‐IP assay of interactions between FBN1 and VEGFR2 proteins in cisplatin‐resistant ovarian cancer organoids and OVCA433‐CisR cells. ( B‐C ) Interactions between FBN1 and VEGFR2 confirmed by FRET‐FLIM upon transient co‐expression in OVCA433‐CisR cells. **, P < 0.01. ( D ) Western blotting assay for determining the relationship between FBN1 and VEGFR2, p‐VEGFR2, downstream molecules of VEGFR2‐mediated signaling in ovarian cancer cells. ( E ) Immunofluorescence assay for determining the relationship between FBN1 and p‐VEGFR2 (Tyr1054) in cisplatin‐resistant ovarian cancer organoids and cell lines. Green signals, FBN1; red signals, p‐VEGFR2 (Tyr1054); blue signals, DAPI. ( F ) Western blotting assay of p‐AKT1 (Ser473) in cisplatin‐resistant ovarian cancer organoids and OVCA433‐CisR cells. ( G ) Co‐IP assay demonstrated an interaction between VEGFR2 domains 2 & 3 and FBN1 protein. Binding of VEGFA and the extracellular domains 2 & 3) of VEGFR2 was used as the positive control. ( H ) Mutant codons and amino acids of D2 and D3 of VEGFR2. ( I ) Western blotting assay of p‐AKT1 (Ser473) in cisplatin‐resistant ovarian cancer organoids and OVCA433‐CisR cells. Abbreviations: FBN1, fibrillin‐1; CR, cisplatin‐resistant; KO, knockout; NC, negative control; SDS‐PAGE, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis; AKT, protein kinase B; Co‐IP, co‐Immunoprecipitation; VEGFR2, vascular endothelial growth factor receptor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; FRET‐FLIM, Fӧrster resonance energy transfer‐fluorescence lifetime imaging. FE, FRET efficiency

Article Snippet: Shortly, 24‐well plate coated Matrigel was prepared in advance, and then 2 × 10 4 human umbilical vein endothelial cells (HUVECs, ATCC) were inoculated in the plate with 200 μL DMEM medium.

Techniques: Co-Immunoprecipitation Assay, Expressing, Western Blot, Immunofluorescence, Protein Binding, Positive Control, Mutagenesis, Knock-Out, Negative Control, SDS Page, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Förster Resonance Energy Transfer, Fluorescence, Imaging

Journal: Cancer Communications

Article Title: The Fibrillin‐1/VEGFR2/STAT2 signaling axis promotes chemoresistance via modulating glycolysis and angiogenesis in ovarian cancer organoids and cells

doi: 10.1002/cac2.12274

Figure Lengend Snippet: STAT2 is the downstream target molecule of the FBN1/VEGFR2 signaling axis. ( A ) GSEA analysis was performed using FBN1‐knockout and control cisplatin‐resistant ovarian cancer organoids (CR‐organoids/FBN1‐KO1 and CR‐organoids/NC). The signature was defined by genes showing significant expression changes. ( B ) Effects of FBN1 knockout on mRNA levels of STAT family members. ( C ) FBN1 knockout greatly altered the distribution and expression of STAT2 and p‐STAT2 (Tyr690) in cytoplasm and nucleus of OVCA433‐CisR ovarian cancer cells. ( D ) Immunofluorescence assay to determine the association between p‐STAT2 (Tyr690) and FBN1 in cisplatin‐resistant ovarian cancer organoids and cells. ( E ) Western blotting assay to detect expression of STAT2 and p‐STAT2 (Tyr690) in cisplatin‐resistant ovarian cancer organoids and OVCA433‐CisR cells. The protein quantification was analyzed by Image J software. ( F ) mRNA expression of glycolysis and angiogenesis‐associated genes in FBN1‐knockout and control cisplatin‐resistant ovarian cancer organoids and OVCA433‐CisR cells. ( G ) Effect of FBN1 knockout, VEGFR2 overexpression, and STAT2 knockdown on HUVEC tube formation. HUVEC cells were treated with supernatant obtained from groups of NC, FBN1 KO‐1, FBN1 KO‐1+VEGFR2 OE, and FBN1 KO‐1+VEGFR2 OE+shSTAT2 in OVCA433‐CisR cells. Abbreviations: FBN1, fibrillin‐1; CR, cisplatin‐resistant; KO, knockout; NC, negative control; OE, overexpression; STAT, signal transducer and activator of transcription; VEGFR2, vascular endothelial growth factor receptor 2

Article Snippet: Shortly, 24‐well plate coated Matrigel was prepared in advance, and then 2 × 10 4 human umbilical vein endothelial cells (HUVECs, ATCC) were inoculated in the plate with 200 μL DMEM medium.

Techniques: Knock-Out, Control, Expressing, Immunofluorescence, Western Blot, Software, Over Expression, Knockdown, Negative Control

Journal: Cancer Communications

Article Title: The Fibrillin‐1/VEGFR2/STAT2 signaling axis promotes chemoresistance via modulating glycolysis and angiogenesis in ovarian cancer organoids and cells

doi: 10.1002/cac2.12274

Figure Lengend Snippet: FBN1 knockout inhibits progression of ovarian cancer and sensitizes response to cisplatin in vivo. ( A ) Images of tumors generated by FBN1‐knockout and control cisplatin‐resistant ovarian cancer organoids with or without cisplatin and/or apatinib. ( B ) Growth curves of xenograft tumors in mice. ( C ) Average tumor weights in nude mice. ( D ) Representative images of PET‐CT used for detection of glucose uptake. Each group contained 5 mice. ( E ) Average SUVmax values of nude mice bearing tumors. ( F ) Effect of FBN1 morpholino in zebrafish model was tested by qRT‐PCR. ( G ) Zebrafish model treated with or without cisplatin (0.2 mmol/L) and/or apatinib (40 μmol/L). ( H ) VEGFA mRNA in zebrafish models. ( I ) Immunofluorescence of FBN1 and specific angiogenesis marker CD31 in the xenograft tumors of FBN1 knockout and control groups with cisplatin treatment. ( J ) qRT‐PCR analysis of the indicated genes in FBN1‐knockout group and the control in nude mouse tumor tissues without drug treatment. Error bars, 95% CIs. *, P < 0.05, **, P < 0.01. ( K ) Heatmap showing that FBN1‐affected genes are involved in glycolysis and angiogenesis with cisplatin treatment. Abbreviations: FBN1, fibrillin‐1; CR, cisplatin‐resistant; KO, knockout; NC, negative control; OE, overexpression; VEGFA, vascular endothelial growth factor A; qRT‐PCR, quantitative real‐time PCR; CD31, platelet endothelial cell adhesion molecule‐1

Article Snippet: Shortly, 24‐well plate coated Matrigel was prepared in advance, and then 2 × 10 4 human umbilical vein endothelial cells (HUVECs, ATCC) were inoculated in the plate with 200 μL DMEM medium.

Techniques: Knock-Out, In Vivo, Generated, Control, Positron Emission Tomography-Computed Tomography, Quantitative RT-PCR, Immunofluorescence, Marker, Negative Control, Over Expression, Real-time Polymerase Chain Reaction

Journal: Cancer Communications

Article Title: The Fibrillin‐1/VEGFR2/STAT2 signaling axis promotes chemoresistance via modulating glycolysis and angiogenesis in ovarian cancer organoids and cells

doi: 10.1002/cac2.12274

Figure Lengend Snippet: Immunohistochemical staining and immunofluorescence of FBN1, p‐VEGFR2, and p‐STAT2. ( A ) Representative images of immunohistochemistry of FBN1, p‐VEGFR2 (Tyr1054), and p‐STAT2 (Tyr690) in ovarian cancer tissues. ( B ) Human serum VEGFA concentration in 50 pairs of ovarian cancer patients measured by ELISA kit. ( C‐D ) Association between SUVmax of PET/CT technology and FBN1 expression in the lesions of adnexal carcinomas from 100 ovarian cancer patients. ( E ) The relationship between SUVmax of PET/CT image and overall survival of 100 ovarian cancer patients. ( F ) mRNA expression levels of genes associated with glycolysis and angiogenesis assessed via qRT‐PCR in 45 pairs of ovarian cancer samples with high or low FBN1 expression. ( G ) Representative images of immunofluorescence of FBN1, p‐VEGFR2 (Tyr1054), and p‐STAT2 (Tyr641) in ovarian cancer patients’ tissues. Green signals, FBN1 or p‐VEGFR2 (Tyr1054); red signals, p‐VEGFR2 (Tyr1054) or p‐STAT2 (Tyr641); blue signals, DAPI. (H) Schematic model on the proposed role of the FBN1/VEGFR2/STAT2 signaling axis in modulating glycolysis, angiogenesis, and cisplatin sensitivity. Abbreviations: FBN1, fibrillin‐1; ELISA, enzyme‐linked immunosorbent assay; SUVmax, maximum of standardized uptake value; PET‐CT, positron emission tomography‐computed tomography; VEGFA, vascular endothelial growth factor A; STAT2, signal transducer and activator of transcription 2; VEGFR2, vascular endothelial growth factor receptor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride

Article Snippet: Shortly, 24‐well plate coated Matrigel was prepared in advance, and then 2 × 10 4 human umbilical vein endothelial cells (HUVECs, ATCC) were inoculated in the plate with 200 μL DMEM medium.

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Immunohistochemistry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Positron Emission Tomography-Computed Tomography, Expressing, Quantitative RT-PCR, Positron Emission Tomography, Computed Tomography

Journal: Marine Drugs

Article Title: A Truncated Galectin-3 Isolated from Skin Mucus of Atlantic Salmon Salmo salar Binds to and Modulates the Proteome of the Gram-Negative Bacteria Moritella viscosa

doi: 10.3390/md18020102

Figure Lengend Snippet: Differentially expressed bacterial proteins shown as spot intensities from two dimensional gels ( Y -axis) in bar graphs based on the PDQuest Advanced 2D Analysis software. Error bars show standard deviation, n = 4, * means p < 0.05, ** means p < 0.01, and *** means p < 0.001.

Article Snippet: Gels were normalized and analyzed using PDQuest Advance 2D analysis software (BioRad).

Techniques: Software, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Development and Preliminary Clinical Activity of PD-1-Guided CTLA-4 Blocking Bispecific DART Molecule

doi: 10.1016/j.xcrm.2020.100163

Figure Lengend Snippet: Cells Co-expressing PD-1 and CTLA-4 Are More Prevalent in the Tumor Microenvironment (A) In situ RNA hybridization of PD-1 and CTLA-4 probes in ovarian cancer tumor cores (N = 21) analyzed using RNAscope and quantified with HALO software. Each square represents an individual core, with red and blue circles representing the indicated frequency of PD-1 and CTLA-4 expression, respectively. The first square shows PD-1 and CTLA-4 expression in a non-malignant ovary sample. (B) In situ RNA hybridization of PD-1 (red) and CTLA-4 (blue) probes visualized by RNAscope in representative tumor microarray core or healthy tonsil samples. (C) Fraction of cells co-expressing PD-1 and CTLA-4 RNA detected by ISH in lymphoid organs from healthy donors (N = 7) or tumor samples from randomly selected patients (N = 12). Means and standard deviations (SDs) are shown. (D) Peripheral blood mononuclear cells (PBMCs) from healthy donors (N = 8) and PBMCs (N = 27) or dissociated tumor cells (DTCs) (N = 7) from patients with various cancers were stained for PD-1 and CTLA-4 expression and analyzed by flow cytometry. Box and whiskers plots depict the minimum, first quartile, median, third quartile, and maximum. Gated on viable CD45 + /CD3 + cells. (E) Representative fluorescence-activated cell sorting (FACS) images from (D) gated on viable T cells. See also Figure S1 .

Article Snippet: Normal human tissue microarray and tumor microarrays of ovarian, breast, lung, colon, rectal cancer samples were provided by Advanced Cell Diagnostic (Newark, USA).

Techniques: Expressing, In Situ, Hybridization, RNAscope, Software, Microarray, Staining, Flow Cytometry, Fluorescence, FACS