normal human prostate stromal fibroblast cell line prsc  (Lonza)

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Representative images and quantitation of MAOB IHC staining in the stroma of hormone-naïve (HNPC, n = 38) and castration-resistant (CRPC, n = 16) PCs. Scale bars, 50 μm. ( B ) Representative images and corresponding Pearson’s correlation analysis of stromal MAOB and adjacent epithelial CHGA expression from the CRPC cohort ( n = 16) in (A). Scale bars, 100 μm. ( C ) Western blot of MAOB in PrSC and PCF2 cells upon ENZ treatment (20 μM) at indicated times. ( D and E ) Kaplan-Meier recurrence-free (D, n = 195) and cancer-specific (E, n = 161) survival curves of PC patients from the NYU cohort with either low or high stromal MAOB protein levels. ( F ) Kaplan-Meier recurrence-free survival curves of BC patients from GSE9014 with either low or high MAOB mRNA levels in the tumor stroma. Statistical analysis was performed using unpaired Student’s t test in (A) and log-rank test in (D) to (F). Data represent means ± SEM. ** P < 0.01.

Article Snippet: The normal human prostate stromal fibroblast cell line PrSC was obtained from Lonza.

Techniques: Quantitation Assay, Immunohistochemistry, Expressing, Western Blot

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Western blot of MAOB in control and MAOB-OE/MAOB-KD PrSC fibroblasts. ( B ) Quantitation of PC cells in 2D coculture with control and MAOB-manipulated PrSC cells by Luc assays ( n = 3). ( C ) Representative fluorescence images and quantitation of green fluorescent protein (GFP)–tagged PC-3 or red fluorescent protein (RFP)–tagged C4-2 cells in 2D coculture with control and MAOB-manipulated PrSC cells by fluorescence microscopy ( n = 3). Scale bars, 100 μm. ( D ) Quantitation of PC cells incubated with CM from control and MAOB-manipulated PrSC cells for 3 to 5 days ( n = 3). ( E ) Quantitation of EpCAM + cancer epithelium in 3D cocultures of PC-3 cells with control and MAOB-manipulated PrSC cells by flow cytometry ( n = 3). ( F ) Representative images and quantitation of invasive PC cells in transwell-based coculture with control and MAOB-manipulated PrSC cells ( n = 3). Scale bars, 200 μm. ( G ) Representative fluorescence images and quantitation of LuCaP 147CR and LuCaP 93 PC PDX–derived organoids after 14-day incubation with CM from control and MAOB-manipulated primary fibroblasts ( n = 3). Scale bars, 50 or 800 μm. ( H ) BLI-based growth curves of subrenal capsule xenografts combining Luc-tagged C4-2 cells with control or MAOB-KD PrSC cells in male SCID mice ( n = 5). ( I ) Representative anatomical images of tumor-grown mouse kidneys from each group. Green dashed circles denote size of tumor outgrowth from renal capsules. ( J ) Tumor weights from each group ( n = 5). ( K ) Representative images of hematoxylin and eosin (H&E) and IHC staining of stromal (Str) MAOB and tumor (T) Ki-67, AR, and SYP and their quantitation in tumor samples from each group ( n = 5). Scale bars, 10 μm. Statistical analysis was performed using unpaired Student’s t test for comparisons between two groups and one-way analysis of variance (ANOVA) with Dunnett’s test for comparisons between three groups in (B) to (F), (G), (J), and (K), and two-way ANOVA with Sidak’s test in (H). Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: The normal human prostate stromal fibroblast cell line PrSC was obtained from Lonza.

Techniques: Western Blot, Control, Quantitation Assay, Fluorescence, Microscopy, Incubation, Flow Cytometry, Derivative Assay, Capsules, Immunohistochemistry

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) GSEA plot of “response to wounding” gene signature enriched in MAOB-OE PrSC cells versus controls. ( B ) Western blot of αSMA in control and MAOB-OE/MAOB-KD PrSC cells. ( C ) Representative αSMA IHC staining of C4-2 tumors co-inoculated with control or MAOB-KD fibroblasts in mice. Scale bars, 100 μm. ( D ) Enzyme-linked immunosorbent assay (ELISA) of TGFβ1 secretion in the culture media of control and MAOB-manipulated PrSC cells ( n = 3). ( E ) Western blot of p-Smad2 and p-Smad3 in control and MAOB-manipulated PrSC cells. ( F ) GSEA plots of “TGFβ1 targets up” gene signature enriched in MAOB-manipulated PrSC cells versus controls. ( G ) Quantitation of intracellular ROS levels in control and MAOB-KD PrSC cells ( n = 3). ( H ) GSEA plots of two ROS-related gene sets enriched in MAOB-KD PrSC cells versus controls. ( I ) Western blot of αSMA in control and MAOB-manipulated PrSC cells upon NAC (5 mM, 48 hours) or H 2 O 2 treatment (40 μM, 24 hours). ( J ) qPCR of indicated reactive stromal markers in control and MAOB-manipulated PrSC cells upon NAC (5 mM, 48 hours) or H 2 O 2 treatment (40 μM, 24 hours) ( n = 3). ( K and L ) Determination of collagen levels deposited (K) and collagen-based cell contraction (L) in control and MAOB-KD PrSC cells under H 2 O 2 treatment (40 μM, 24 hours) ( n = 3). ( M ) Quantitation of PC-3 cells in coculture with control and MAOB-OE PrSC cells pretreated with NAC (5 mM, 24 hours) ( n = 3). Statistical analysis was performed using unpaired Student’s t test for comparisons between two groups and one-way ANOVA with Dunnett’s test for comparisons between three groups in (D) and (G); one-way ANOVA with Tukey’s test in (J), (K), and (M); and two-way ANOVA with Tukey’s test in (L). Data represent means ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: The normal human prostate stromal fibroblast cell line PrSC was obtained from Lonza.

Techniques: Western Blot, Control, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Quantitation Assay

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) GSEA plots of “chemokine signaling pathway” and “chemotaxis” gene sets enriched in MAOB-OE PrSC cells compared with controls. ( B ) ELISA of CXCL12 secretion in the culture media of control and MAOB-KD PrSC cells ( n = 3). ( C ) Representative IHC images and corresponding Pearson’s correlation analysis of stroma-expressed MAOB and CXCL12 protein levels in a PC TMA ( n = 37). Scale bars, 100 μm. ( D ) Pearson’s correlation analysis of MAOB and CXCL12 mRNA levels in patient-derived cultured prostatic stromal cells (left, n = 20) and laser-capture microdissected breast tumor stroma (right, n = 53) from GSE34312 and GSE9014 datasets, respectively. Statistical analysis was performed using one-way ANOVA with Dunnett’s test in (B). Data represent means ± SEM. ** P < 0.01.

Article Snippet: The normal human prostate stromal fibroblast cell line PrSC was obtained from Lonza.

Techniques: Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Control, Derivative Assay, Cell Culture

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Western blot of Twist1 in control and MAOB-manipulated PrSC cells upon NAC (5 mM, 48 hours) or H 2 O 2 (40 μM, 24 hours) treatment. ( B ) ELISA of CXCL12 secretion in culture media of control and MAOB-OE PrSC cells treated with TWIST1 siRNA or NAC (5 mM, 48 hours) ( n = 3). ( C ) qPCR of CXCL12 in indicated PrSC cells upon NAC treatment (5 mM, 48 hours) or Twist1/ TWIST1 siRNA expression ( n = 3). ( D and E ) Determination of CXCL12 mRNA (D) and 0.7-kb promoter activity (E) in PrSC cells upon Twist1 expression and/or TGFβ1 treatment (10 ng/ml, 12 hours) ( n = 3). ( F ) Schematic diagrams of WT and mutated CXCL12 E-box/SBE-Luc constructs and determination of their activities in PrSC cells upon Twist1 expression and/or TGFβ1 treatment (10 ng/ml, 12 hours) ( n = 3). ( G ) Representative proximity ligation assay staining and quantitation of indicated Twist1-Smad interactions by per-nucleus fluorescence intensity in control and MAOB-OE PrSC cells. Smad antibody incubation alone served as negative control. Numbers of nuclei included for comparisons between groups are denoted. Scale bars, 50 μm. ( H ) Co-IP assays of indicated Twist1-Smad interactions in PrSC cells with coexpression of Twist1 and individual Smads. Immunoglobulin G (IgG) was used in IP as negative control. Ten percent input was blotted as positive control. ( I ) ChIP analysis of chromatin from control and MAOB-OE PrSC cells precipitated with anti-Twist1, anti-Smad4, or a control IgG, followed by qPCR probing the E-box/SBE-centric CXCL12 promoter region ( n = 3). ( J ) ChIP analysis of chromatin from PrSC cells precipitated with anti-Smad4 antibody and then reprecipitated with anti-Twist1 or a control IgG (re-ChIP), followed by qPCR probing the E-box/SBE-encompassing CXCL12 promoter sequence ( n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s test. Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: The normal human prostate stromal fibroblast cell line PrSC was obtained from Lonza.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Expressing, Activity Assay, Construct, Proximity Ligation Assay, Staining, Quantitation Assay, Fluorescence, Incubation, Negative Control, Co-Immunoprecipitation Assay, Positive Control, Sequencing

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Quantitation of C4-2 and PC-3 PC cells in monoculture and coculture with indicated PrSC fibroblasts upon anti-CXCL12 (0.1 μg/ml) antibody treatment ( n = 3). ( B ) Quantitation of PC cells in coculture with indicated PrSC cells treated with rCXCL12 protein (50 ng/ml; n = 3). ( C ) Western blot of CXCR4 and CXCR7 in control and CXCR4-KD/CXCR7-KD PC cells. ( D and E ) Quantitation of control and CXCR4-KD/CXCR7-KD PC cells in coculture with indicated PrSC cells treated without (D) or with (E) rCXCL12 (50 ng/ml; n = 3). ( F ) Quantitation of PC cells in coculture with indicated PrSC cells treated with 10 nM AMD3100 ( n = 3). ( G ) Representative images and quantitation of PC-3 cell migration and invasion in coculture with indicated PrSC cells upon treatment with anti-CXCL12 antibody (0.1 μg/ml) or 10 nM AMD3100 ( n = 3). Scale bars, 200 μm. ( H ) Representative images and quantitation of PC cell migration in coculture with indicated PrSC cells treated with rCXCL12 (50 ng/ml; n = 3). Scale bars, 100 μm. ( I ) Phospho-antibody array analysis of PC-3 cells treated with indicated PrSC cell CM. All phosphoprotein levels were normalized to their total forms from a single array with six replicate spots, with significantly activated phosphoproteins (fold change > 1.5, P < 0.05) denoted. ( J ) Western blot of p-Src and p-JNK in control and CXCR4-KD PC-3 cells treated with indicated PrSC cell CM. ( K ) Western blot of p-Src and p-JNK in PC-3 cells treated with indicated PrSC cell CM plus rCXCL12 (50 ng/ml). ( L ) Quantitation of PC cells in coculture with indicated PrSC cells following pretreatment with 40 nM Src inhibitor 1 or 10 μM SP600125 for 24 hours ( n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s test. Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: The normal human prostate stromal fibroblast cell line PrSC was obtained from Lonza.

Techniques: Quantitation Assay, Western Blot, Control, Migration, Ab Array

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Quantitation of C4-2 and PC-3 cell proliferation in monoculture and coculture with control and MAOB-OE PrSC cells upon selegiline treatment (10 nM, 72 hours) ( n = 3). ( B ) BLI-based growth curves of Luc-tagged PC-3 tumors grown in the prostates of male NSG mice treated with selegiline at various doses (0.5, 2, and 10 mg/kg) or saline as a vehicle ( n = 5 per group). ( C ) BLI images of mice from each group at the end point. ( D ) Determination of tumor weights ( n = 5). ( E ) Determination of MAOA and MAOB enzymatic activities in mouse liver tissue from each group at the end point ( n = 3). ( F ) Representative images of H&E and IHC staining of tumor-expressed Ki-67, p-Src, and p-JNK and stroma-expressed αSMA and CXCL12 and their quantitation in tumor samples from each group ( n = 5). Scale bars, 100 μm. ( G ) Mouse body weights determined weekly ( n = 5). ( H ) Representative H&E images of mouse liver and kidney tissue from each group. Scale bars, 100 μm. ( I to L ) ELISA of ALT (I), AST (J), BUN (K), and creatinine (L) in mouse sera at the end point ( n = 5). ( M ) Schematic depicting stromal-derived MAOB activation of paracrine CXCL12-CXCR4/Src/JNK signaling through interplay between ROS-dependent Twist1 (via a HIF1α/VEGF-A/AKT/FOXO1 pathway) and TGFβ1/Smads to promote stromal-epithelial interactions for PC growth and progression. Statistical analysis was performed using one-way ANOVA with Tukey’s test in (A), (D) to (F), and (I) to (L) and two-way ANOVA with Tukey’s test in (B) and (G). Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: The normal human prostate stromal fibroblast cell line PrSC was obtained from Lonza.

Techniques: Quantitation Assay, Control, Saline, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Derivative Assay, Activation Assay