Journal: Nature Communications

Article Title: Periprostatic adipocytes act as a driving force for prostate cancer progression in obesity

doi: 10.1038/ncomms10230

Figure Lengend Snippet: ( a ) Graphic representation of chemokines identified by proteomic analyses of Ad-CM and their corresponding receptors. ( b ) In vitro migration of Du-145 and PC-3 cells towards medium without serum in the presence or absence of CCL7 recombinant protein (1–100 ng ml −1 ). Data are shown as mean±s.e.m. ( n =3). The statistical differences between the mean percentages of migrating cells in experiments performed in the presence versus in the absence of CCL7 were evaluated with Student's t -tests. ( c ) In vitro migration of Du-145 and PC-3 cells towards Ad-CM in the presence of m/pAbs directed against CCR3 or CCL7, or control IgG (10 μg ml −1 ). Bar plots represent the percentage of migrating cells relative to the migration of untreated cells (set to 100%). Data are shown as mean±s.e.m ( n =3). The statistical significance of differences between means of migrating cells (in %) in treated versus untreated cells was evaluated with Student's t -tests. ( d ) CCL7 secretion by murine visceral adipose tissue (mu-VAT, three samples) or human periprostatic adipose tissue (hu-PPAT, five samples). Data are shown as mean±s.e.m. ( e ) In vitro migration of PC-3 cells towards mu-VAT and hu-PPAT conditioned medium (AT-CM) in the presence or absence of CCR3 antagonist (UCB35625, 200 nM), blocking m/pAbs directed against CCR3 or CCL7, or control IgG (10 μg ml −1 ). Bar plots represent the percentage of migrating cells relative to the migration of untreated cells (set to 100%). Data are shown as mean±s.e.m. ( n =3). The statistical significance of differences between means of migrating cells (in %) in treated versus untreated cells was evaluated with Student's t -tests. ( f ) Graphic representation of the site of the staged biopsies performed in human prostatectomy specimens ( n =3) is shown (left panel) and the expression of CCL7 in these biopsies is shown (right panel). The mean expression of CCL7 in staged prostate biopsies was compared with the mean expression in PPAT. Statistical analysis: * statistically significant by Student's t -test P <0.05, ** P <0.01, *** P <0.001, NS, not significant. n stands for the number of replicated independent experiments. n stands for the number of replicated independent experiments.

Article Snippet: The inhibitor targeting CCR1 and CCR3, UCB35625 was purchased from Tocris (Bristol, UK) and used at final concentrations from 1 to 200 nM.

Techniques: In Vitro, Migration, Recombinant, Control, Blocking Assay, Expressing

Journal: Nature Communications

Article Title: Periprostatic adipocytes act as a driving force for prostate cancer progression in obesity

doi: 10.1038/ncomms10230

Figure Lengend Snippet: ( a ) mRNA were extracted from murine visceral adipose tissue (mu-VAT) of age-matched C57BL/6 mice either lean or obese (after 12 weeks of high-fat diet). Expression of CCL7 mRNA was evaluated by RT-qPCR. A panel of genes whose expression has been shown to decrease (Adiponectin, ADPN) or increase (tumor necrosis factor-α (TNF-α), Leptin) in obesity was used as a control to validate the samples. Data are shown as mean±s.e.m. ( n =5). ( b ) Secretion of CCL7 in mu-VAT-CM obtained from lean or obese C57BL/6 mice (three animals per group). Data are shown as mean±s.e.m. ( n =3). ( c ) In vitro migration of PC-3 cells towards mu-VAT-CM obtained from lean or obese animals (three animals per group) treated or not with CCR3 inhibitor (UCB35625 200 nM), blocking m/pAbs directed against CCR3 or CCL7, or control isotype (all used at 10 μg ml −1 ). Data are shown as mean±s.e.m. ( n =3). The statistical significance of differences between means of migrating cells (in %) in treated versus untreated cells was evaluated with Student's t -tests. ( d ) Graphic representation of the cellular components of adipose tissue. ( e ) CCL7 secretion by primary adipocytes or SVF cells isolated from mu-VAT of C57BL/6 lean or obese mice (six animals per group). Data are shown as mean±s.e.m. ( n =3). ( f ) In vitro migration of PC-3 cells towards mu-VAT adipocyte-CM. Adipocytes were isolated from the mu-VAT of lean or obese C57BL/6 mice (six animals per group) in the presence or absence of blocking m/pAbs directed against CCR3 or CCL7 or control IgG (10 μg ml −1 ). Data are shown as mean±s.e.m. ( n =3). The statistical significance of differences between means of migrating cells (in %) in treated versus untreated cells was evaluated with Student's t -tests. The differences between the percentages of migrating cells towards Ad-CM isolated from mu-VAT from obese versus lean mice are also shown. Statistical analysis: * statistically significant by Student's t -test P <0.05, ** P <0.01, *** P <0.001, NS, not significant. n stands for the number of replicated independent experiments.

Article Snippet: The inhibitor targeting CCR1 and CCR3, UCB35625 was purchased from Tocris (Bristol, UK) and used at final concentrations from 1 to 200 nM.

Techniques: Expressing, Quantitative RT-PCR, Control, In Vitro, Migration, Blocking Assay, Isolation

Journal: Nature Communications

Article Title: Periprostatic adipocytes act as a driving force for prostate cancer progression in obesity

doi: 10.1038/ncomms10230

Figure Lengend Snippet: ( a ) One representative experiment out of three showing CCR1 and CCR3 expression detected by flow cytometry in the murine prostate cancer cell line TRAMP-C1P3. Solid histogram: control isotypes; open histogram: CCR1 or CCR3. ( b ) In vitro migration of TRAMP-C1P3 towards medium without serum, supplemented or not with CCL7 recombinant protein (1–100 ng ml −1 ). Data are shown as mean±s.e.m. ( n =3). The statistical differences between the mean percentages of migrating cells in experiments performed in the presence versus in the absence of CCL7 were evaluated with Student's t -tests. ( c ) In vitro migration of TRAMP-C1P3 cells towards Ad-CM as a chemoattractant treated or not with UCB35625 (200 nM), anti-CCR3, anti-CCL7 Abs, or control isotype (10 μg ml −1 ). Data are shown as mean±s.e.m. ( n =3). Data are expressed as the percentage of migrating cells relative to the migration of untreated cells (set to 100%). The statistical significance of differences between means of migrating cells in treated versus untreated cells was evaluated with Student's t -tests. ( d ) Mean fluorescence intensity of CCR3 expression determined by flow cytometry in TRAMP-C1P3 cells (WT), transfected with control shRNA (shCtrl) or with one of three different shRNA sequences targeting the CCR3 receptor (shm4, m5 and m6CCR3). Data are shown as mean±s.e.m. ( n =3). The statistical significance of differences between means of fluorescence in control or CCR3 invalidated cells versus WT cells was evaluated with Student's t -tests. ( e ) In vitro migration of TRAMP-C1P3 cells transfected with either shCtrl or three different shRNA sequences targeting the CCR3 receptor (shm4, m5 and m6CCR3) towards Ad-CM. As indicated, cells were treated with the CCR3 antagonist UCB35625. Data are shown as mean±s.e.m. ( n =3). ( f ) I n vitro migration of TRAMP-C1P3 cells transfected with either shCtrl or shm6CCR3 towards CM of mu-VAT adipocytes obtained from lean or obese mice. As indicated, cells were treated with Abs against CCR3 or CCL7, or control isotype (10 μg ml −1 ). Data are shown as mean±s.e.m. ( n =3). Statistical analysis: * statistically significant by Student's t -test P <0.05, ** P <0.01, *** P <0.001, NS, not significant. n stands for the number of replicated independent experiments.

Article Snippet: The inhibitor targeting CCR1 and CCR3, UCB35625 was purchased from Tocris (Bristol, UK) and used at final concentrations from 1 to 200 nM.

Techniques: Expressing, Flow Cytometry, Control, In Vitro, Migration, Recombinant, Fluorescence, Transfection, shRNA