Journal: Oncogene

Article Title: Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC

doi: 10.1038/s41388-019-0728-3

Figure Lengend Snippet: Effect of SPARC on homing of ovarian cancer (OvCa) cells to omental adipocytes. a In vivo homing of ID8-GFP cells to SP +/+ and SP –/– omenta in the presence or absence of prior injection of 5 µg/ml SPARC. Bars represent means ± Standard error of the mean (SEM) of fluorescence intensity of adherent cells to omenta harvested at the indicated time points. * P < 0.05 comparing SP+/+ and SP–/–. ** P < 0.05 comparing cells with and without SPARC treatment. ns, non-significant. b Schema of the in vitro homing/chemotaxis of ID8 cells towards SP +/+ and SP –/– omental adipocytes. Bars represent means ± SEM of fluorescence intensity of ID8 cells that migrated through trans-wells towards adipocytes. Complete growth media were used as controls for migration ( n = 4). * P < 0.05 comparing ID8 migration towards control media, SP+/+, SP–/– adipocytes in absence of SPARC. ** P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. c Schema of the adhesion assay of GFP-OvCa cells over-layed on top of adipocytes for 120 min (left). Bars represent means ± SEM of fluorescence intensity of adherent cells ( n = 6/experimental condition). * p < 0.05 comparing ID8 migration towards control media, SP+/+,SP–/– adipocytes in absence of SPARC. ** P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. Photomicrographs of fluorescent adherent cells (top, 5×). d Schema of adipocyte-induced OvCa invasiveness through trans-well inserts towards omental adipocytes in the bottom chamber. Bars represent the means ± SEM of migrated cells counted in five random fields/insert, ( n = 3). * P < 0.05 comparing ID8 migration towards control media, SP+/+, SP–/– adipocytes in absence of SPARC. ** P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. Student’s t -test. e Western blots showing the expression of SPARC after overexpression and knockdown in primary human omental adipocytes (hAdi). f Schema of the in vitro homing assay with GFP-labeled human OvCa cell lines. Bars represent means ± SEM of fold change of OvCa cells that migrated through trans-wells towards genetically engineered hAdi compared with cells migrated to control media (without adipocytes) considered as 1. ( n = 4/experimental condition. Experiments were repeated three times). * p < 0.05 Student’s t -test with multiple comparisons

Article Snippet: Adipocytes were maintained in adipocytes maintenance medium (ADMM, Zen Bio, Raleigh, NC) supplemented with 10% fetal bovine serum (FBS), antibiotic–antimycotic solution.

Techniques: In Vivo, Injection, Fluorescence, In Vitro, Chemotaxis Assay, Migration, Control, Cell Adhesion Assay, Western Blot, Expressing, Over Expression, Knockdown, Labeling

Journal: Oncogene

Article Title: Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC

doi: 10.1038/s41388-019-0728-3

Figure Lengend Snippet: Effect of SPARC on omental adipocyte-induced OvCa cell proliferation. a Schema of the effect of human and murine omental adipocytes on GFP-OvCa cells proliferation. b Line graphs representing means ± SEM of changes in the proliferation (GFP fluorescence) of ID8-GFP cells over-layed on top of SP +/+ and SP –/– adipocytes in the presence or absence of 5 µg/ml SPARC, over 72 h. c Means ± SEM of changes of tumours volumes after ID8 SC injection either alone or with SP +/+ and SP –/– adipocytes (1:2) in athymic nude mice. * p < 0.05, between experimental conditions starting at week 3 post-injection ( n = 8/group; two-way ANOVA with Tukey’s multiple comparison test). d Means ± SEM of changes in the proliferation (GFP fluorescence) of GFP-labeled human OvCa cells on top of genetically engineered human adipocytes over 72 h. ( n = 6/experimental condition). * P < 0.05 comparing hAdi-SP and hAdi-shSP with their corresponding vector control at the indicated time points, two-way ANOVA. e Changes in the proliferation (GFP fluorescence) of OvCa cells over-layed on top of adipocytes in presence or absence of 5 µg/ml SPARC over 72h. ( n = 6/experimental condition). * p < 0.05, ** P < 0.001, *** p < 0.0001, comparing proliferation of GFP-cells on uncoated UC, wells (black), matrigel (blue) and adipocytes (red) in presence and absence of SPARC, two-way ANOVA with Tuckey's multiple comparison test

Article Snippet: Adipocytes were maintained in adipocytes maintenance medium (ADMM, Zen Bio, Raleigh, NC) supplemented with 10% fetal bovine serum (FBS), antibiotic–antimycotic solution.

Techniques: Fluorescence, Injection, Comparison, Labeling, Plasmid Preparation, Control

Journal: Oncogene

Article Title: Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC

doi: 10.1038/s41388-019-0728-3

Figure Lengend Snippet: Adipocyte-SPARC suppresses the reciprocal transcriptional activity of pro-inflammatory/adipogenic factors. a Schematic illustration of the experimental design and the trans-well assays for the single and co-cultures. b The levels of adipokines in SP +/+ and SP –/– omental adipocytes and ID8 cells in single and in co-cultures for 24 h was determined by qRT-PCR. c Transcriptional activity of cEBP, NFκB, and AP-1 in primary SP +/+ and SP –/– omental adipocytes and ID8 cells in single and in co-cultures was determined by measuring the luciferase reporter activity in each cell type. Results were normalized to fold change of DNA content of each cell type measured before and after the experiment as determined by CyQuant assay. Bars represent mean ± SEM from one of three experiments, performed in triplicate. * p < 0.05 Student’s t- test comparing SP +/+ and SP –/– adipocytes; # p < 0.05 Student’s t- test comparing adipocytes in single to co-cultures with ID8 cells; and ** p < 0.05 Student’s t- test comparing ID8 cells in single to co-culture with SP +/+ and SP –/– adipocytes

Article Snippet: Adipocytes were maintained in adipocytes maintenance medium (ADMM, Zen Bio, Raleigh, NC) supplemented with 10% fetal bovine serum (FBS), antibiotic–antimycotic solution.

Techniques: Activity Assay, Quantitative RT-PCR, Luciferase, CyQUANT Assay, Co-Culture Assay

Journal: Oncogene

Article Title: Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC

doi: 10.1038/s41388-019-0728-3

Figure Lengend Snippet: SPARC suppresses pro-inflammatory/adipogenic factors in ID8 intraperitoneal tumour. a Immunoblots of total and phosphorylated cJun, p65NFκB, in lysates from ID8 intraperitoneal tumours growing in SP –/– and SP +/+ mice. Tubulin was used as a loading control. b Immunostaining of cEBPβ, NFκB, and cJun in tumours dissected from SP +/+ and SP –/– (magnification, ×200). c Box plots of the expression scores of the nuclear transcription factors in tumour cells and adipocytes of both genotypes. p < 0.05, Mann–Whitney test

Article Snippet: Adipocytes were maintained in adipocytes maintenance medium (ADMM, Zen Bio, Raleigh, NC) supplemented with 10% fetal bovine serum (FBS), antibiotic–antimycotic solution.

Techniques: Western Blot, Control, Immunostaining, Expressing, MANN-WHITNEY

Journal: Oncogene

Article Title: Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC

doi: 10.1038/s41388-019-0728-3

Figure Lengend Snippet: Correlation of tumour SPARC and cEBPβ, NFkB, and AP-1. a Photomicrograph of stage III HGSC specimens (CHTN) showing distinctive compartmentalization of SPARC in the cancerous vs stromal compartments and the expression of cEBPβ, NFkB, and cJun. b Scatter plots of the expression scores of SPARC, cEBPβ, NFkB, and cJun. p < 0.05, Mann–Whitney test. c Spearman’s correlation of the expression scores of tumour-SPARC and nuclear transcription factors. d Immunofluorescence staining of the expression of omental metastases of HGSC specimens, showing the expression of cEBPβ, RelA, cJun, and SPARC in the tumour (T)–adipocyte (A) interface. e Spearman’s correlation of the nuclear expression of tumour and adipocyte cEBPβ, RelA, and cJun with tumour and adipocyte SPARC. Scale bars 100 µm

Article Snippet: Adipocytes were maintained in adipocytes maintenance medium (ADMM, Zen Bio, Raleigh, NC) supplemented with 10% fetal bovine serum (FBS), antibiotic–antimycotic solution.

Techniques: Expressing, MANN-WHITNEY, Immunofluorescence, Staining

Journal: Oncogene

Article Title: Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC

doi: 10.1038/s41388-019-0728-3

Figure Lengend Snippet: Effect of SPARC on metabolic programming of adipocytes and OvCa cells. a Schema of the co-culture (upper), bars represent means ± SEM of FFA release in media of co-cultures for 48h. * p < 0.05, Student’s t -test. b WB showing increased lipases in adipocytes in mono- and co-cultures with ID8 cells. c Schema showing co-cultures of ID8 and adipocytes. d Photomicrographs of FA uptake by ID8 cells visualized by Bodipy staining of in co-cultures (upper, 40×; scale bar 50 µm) and electron microscopy (lower, 6800×; scale bar, 500 nm). e Quantification of Bodipy fluorescence intensity in the in ID8 cells in mono- and co-cultures with adipocytes. The number and size of lipid droplets (lower) was quantified in 10 cells/condition. Plots represent means ± SEM of the fluorescent intensity quantified in 10 fields/experimental condition ( n = 3 experiments), and unpaired P < 0.05, t -test. f Effect of SPARC on FA uptake by human OvCa cells. p < 0.05, one-way ANOVA. g Immunofluorescence showing the effect of SPARC (5 µg/ml) on FABP4 and CD36 expression in SKOV3 and OVCAR3 cells. Scale bars, 10 µm. h Bars report means ± SEM of fluorescence intensity of FABP4 and CD36 quantified in 10 fields/experimental condition, Student’s t -test. i mRNA expression of enzymes involved in FA oxidation of ID8 in mono- and co-culture with SP +/+ and SP –/– adipocytes. Bars report means ± SEM of a representative of three independent experiments each in triplicate. * p < 0.05. Student’s t -test. j Bars depict mean ± SEM of the changes in the transcript levels ( n = 3/genotype). p- values are determined by unpaired Student’s t -test

Article Snippet: Adipocytes were maintained in adipocytes maintenance medium (ADMM, Zen Bio, Raleigh, NC) supplemented with 10% fetal bovine serum (FBS), antibiotic–antimycotic solution.

Techniques: Co-Culture Assay, Staining, Electron Microscopy, Fluorescence, Immunofluorescence, Expressing

Journal: Oncogene

Article Title: Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC

doi: 10.1038/s41388-019-0728-3

Figure Lengend Snippet: SPARC exerts anti-adipogenic effect on omental adipocytes and 3T3-L1 cells. a Oil Red O (ORO) staining of differentiating SP +/+ and SP –/– pre-adipocytes (×10 magnification). b SP –/– adipocytes exhibit increased size and number of fat droplets day 10 (D10) post-differentiation as shown by ORO (×20 magnification) and electron microscopy (×6800). c Plots indicate mean ± SEM of the number (upper) and size (lower) of lipid droplets quantified in EM images (10 cells/experimental condition). P values are determined by Student's t-test. d Western blots showing the kinetics of expression of adipogenic transcription factors during differentiation of SP +/+ and SP –/– pre-adipocytes. e The expression of SPARC protein during differentiation of SP +/+ adipocytes. f Effect of SPARC (5 µg/ml in PBS–0.4% BSA) on the expression of the adipogenic transcription factors during differentiation of SP +/+ adipocytes. Tubulin was used as loading control. g Confluent 3T3-L1 pre-adipocytes (D0) were differentiated either in the presence or absence of 5 μg/ml rSPARC up to day 10. The cells were harvested at days 0, 3, 7, and 10. Intracellular lipids were stained with ORO. h Bars (right) represent means ± SEM of the quantification of ORO-stained cells solubilized with 100% isopropanol for 5min and measuring the absorbance A 492 nm. * p < 0.0001, two-way ANOVA with Sidak post-hoc test. h Images of Bodipy fluorescent staining of intracellular lipids in differentiating 3T3-L1 pre-adipocytes in the presence or absence of 5 μg/ml rSPARC. i Western blots showing the expression of SPARC and the adipogenic differentiation markers in differentiating 3T3-L1 cells in the presence or absence of SPARC

Article Snippet: Adipocytes were maintained in adipocytes maintenance medium (ADMM, Zen Bio, Raleigh, NC) supplemented with 10% fetal bovine serum (FBS), antibiotic–antimycotic solution.

Techniques: Staining, Electron Microscopy, Western Blot, Expressing, Control