human prostate stromal cell line wpmy  (ATCC)

Journal: Journal of Nanobiotechnology

Article Title: Epithelial cells derived exosomal miR-203a-3p facilitates stromal inflammation of type IIIA chronic prostatitis/chronic pelvic pain syndrome by targeting DUSP5 and increasing MCP-1 generation

doi: 10.1186/s12951-024-02513-5

Figure Lengend Snippet: A scheme visually represents how exosomal miR-203a-3p, originating from inflammatory prostate epithelial cells, activates the ERK1/2-MCP-1 signaling pathways. This activation occurs by decreasing the expression of DUSP5, which further promotes inflammation in stromal cell

Article Snippet: The human prostate epithelial cell line (PEC: RWPE-1 cell) and human prostate stromal cell line (PSC: WPMY-1 cell) were purchased from the American Type Culture Collection (ATCC, USA) and tested for no mycoplasma contamination.

Techniques: Protein-Protein interactions, Activation Assay, Expressing

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

Journal: Oncogene

Article Title: Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance

doi: 10.1038/s41388-022-02476-7

Figure Lengend Snippet: a Transcriptome-wide profiling of gene expression changes in primary normal human prostate stromal cell line (PSC27) by microarray. Cell lysates were collected for analysis 7 days after treatment. CTRL control. H 2 O 2 hydrogen peroxide. BLEO bleomycin. RAD radiation. Red highlighted, EREG. Agilent microarray data adapted from Sun et al. with permission from Nature Medicine , copyright 2012, Springer Nature . b Representative immunofluorescence staining images (γH2AX and p-53BP1 co-staining, left) and comparative statistics (right) of DNA damage response (DDR) in PSC27 cells treated by DOX (doxorubicin), MIT (mitoxantrone), BLEO (bleomycin), DTX (docetaxel), PTX (paclitaxel) and VBL (vinblastine). DDA DNA-damaging agents (DDAs). NDDA non-DNA-damaging agents. DDR were classified into four sub-categories including 0 foci, 1–3 foci, 4–10 foci and >10 foci per cell. Scale bars, 15 μm. c SA-β-Gal staining of PSC27 cells treated by various agents used in b . Cells were stained 7 days after in vitro treatments. Scale bars, 30 μm. Right, comparative statistics. d BrdU staining of stromal cells treated by different agents as indicated in b and c . Scale bars, 15 μm. Right, comparative statistics. e Quantitative RT-PCR of EREG expression after treatment of PSC27 cells by various agents. Cell lysates were collected for measurement 7 days after treatment. Signals normalized to CTRL. f Immunoblot analysis of EREG expression in stromal cells 7 days after treatments performed as indicated. IC intracellular samples. CM conditioned media. GAPDH, loading control. g Time course expression assessment of a subset of EREG and other typical SASP factors (CXCL8, CSF2, WNT16B, IL6 and MMP3) after drug treatment of stromal cells in vitro. Numeric numbers indicate the individual days after treatment. h Immunoblot measurement of EREG expression at the protein level in the time course described in g . i Comparative appraisal of EREG transcript expression in stromal cells (PSC27) versus cancer epithelial cells (PC3, DU145, LNCaP and M12). Signals normalized to untreated sample per cell line. j Immunoblot assessment of EREG expression in protein lysates of stromal and epithelial cells after bleomycin treatment as performed in i . Data are representative of three independent experiments. ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. p values were calculated by Student’s t test ( c – e , g ) and two-way ANOVA ( b , i ). ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary normal human prostate stromal cell line PSC27, breast stromal cell line HBF1203 and lung stromal cell line HFL1 (ATCC) were maintained in stromal complete medium as described [ ].

Techniques: Gene Expression, Microarray, Control, Immunofluorescence, Staining, In Vitro, BrdU Staining, Quantitative RT-PCR, Expressing, Western Blot

Journal: Oncogene

Article Title: Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance

doi: 10.1038/s41388-022-02476-7

Figure Lengend Snippet: a Schematic of putative NF-κB binding sites in the proximal region of EREG promoter. A set of reporter constructs was generated by sequential cloning of the promoter fragments into a pGL4.22 vector (pGL-EREG-P01 to P05) that expresses firefly luciferase. Numeric numbers on the top denote the core site of each putative NF-κB binding motif, while numbers at the left mark the length of each segmental promoter clone. TSS transcription start site. Lower-left inlet, consensus binding motif of the NF-κB subunit p65. b Assessment of luciferase activities upon exposure of 293F cells pre-transfected with the individual EREG promoter constructs to TNF-α at 40 ng/ml in culture. The empty vector was used as a negative control, while a construct NAT11-Luc2CP encoding multiple copies of typical NF-κB binding sequences and an optimized IL-2 minimal promoter served as a positive control. Signals were presented as relative ratios of firefly/renilla luciferase activities. c Luciferase activity assay with lysates of PSC27 cells pre-transfected with each of the constructs used in b prior to treatment by 50 μg/ml bleomycin (BLEO) in culture. d Chromatin immunoprecipitation (ChIP) was performed to identify potential NF-κB binding sites in the proximal promoter of EREG. Left, EREG-p2/p3/p4/p5 denotes four representative genomic sites in EREG promoter region, while selective NF-κB binding sites from IL6 and CXCL8 served as positive controls. e EREG and MMP3 transcript expression in PSC27 cells exposed to BLEO, MIT (mitoxantrone) or DOX (doxorubicin), with or without the NF-κB inhibitor BAY (Bay 11-7982, 5 μM). Signals were normalized to untreated cells, with MMP3 expression analyzed as positive control. f The reporter construct pGL-EREG-P05 was transiently transfected into PSC27 cells before treatment by BLEO. BAY (5 μM), BA (betulinic acid, 10 μM), T-5224 (10 μM) were applied with BLEO as small molecule inhibitors against NF-κB, C/EBP family and AP-1, respectively. SR (SR 11302, 3 μM), a positive control inhibitor against AP-1. Cells were lysed 7 days after treatment, with lysates subject to luciferase activity assay. g PSC27 cells were treated in the same conditions as described in f , with lysates collected for total RNA preparation and subject to quantitative RT-PCR analysis. Expression of EREG (left), IL6 (mid) or CXCL8 (right) was compared between CTLR (untreated), Mock (PBS-treated), BAY, BA, T-5224 and SR treatment groups. Cells were damaged by BLEO (50 μg/ml) or VBL (vinblastine, 20 nM) treatment. h Immunoblot analysis of DDR signaling (ATM), p38MAPK activation, cellular senescence (p16, p21) and NF-κB activation (p65) in PSC27 cells treated by various chemotherapeutic agents as indicated. GAPDH, loading control. i Immunoblot analysis Expression assay of p65 nuclear translocation in PSC27 cells treated by VBL, PTX, BLEO or MIT, individually. C cytoplasmic, N Nuclear. Histone H3, loading control for nuclear proteins. Note, the relative signal intensities (RSI, presented as percentage) of p65 were quantified as the virtual intensity of an individual sample after scanning, and calculated in relative to that of the strongest signal (BLEO, C for the p65 blot). j Presentation of p65-specific ChIP-seq tracks of the gene locus of several SASP hallmarks and senescence-associated factors. Illustrations were prepared from datasets deposited in the GEO (accession number GSE141992), with raw data available at publicly released sources . Data are representative of three independent experiments. All p values were calculated by Student’s t tests. ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary normal human prostate stromal cell line PSC27, breast stromal cell line HBF1203 and lung stromal cell line HFL1 (ATCC) were maintained in stromal complete medium as described [ ].

Techniques: Binding Assay, Construct, Generated, Cloning, Plasmid Preparation, Luciferase, Transfection, Negative Control, Positive Control, Activity Assay, Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR, Western Blot, Activation Assay, Control, Translocation Assay, ChIP-sequencing

Journal: Oncogene

Article Title: Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance

doi: 10.1038/s41388-022-02476-7

Figure Lengend Snippet: a Immunoblot analysis of EGFR-associated pathways in PC3 and DU145 cells treated by the CM from PSC27 cells transduced with the empty vector or EREG construct, or alongside the EGFR inhibitor AG-1478 (2 μM). Antibodies of p-EGFR (Y845), p-Akt (S473), p-mTOR (S2448), p-MEK (S217/S221) and p-ERK (T202/Y204) were applied to probe the individual molecules. Total protein per molecule and GAPDH were used as loading control. b Schematic diagram of the construct encoding the mature chain of EREG (upper) and immunoprecipitation (IP, lower) followed by immunoblot assay of EGFR and His-EREG (fusion protein) in the whole lysates of PC3 cells. PC3 was treated by the CM of PSC27 Vector and PSC27 His-EREG for 3 days. Antibodies including IgG and anti-EGFR were used for IP, with both EGFR and His-EREG in inputs analyzed. c Measurement of cellular senescence by quantification of SA-β-Gal staining positivity. Stromal cells were pre-transduced with shRNAs and treated by BLEO. Upper, statistics. Lower, representative images. Scale bar, 20 μm. d PCa cells were treated with the CM from PSC27 sublines for 3 days, and subject to cell proliferation assay. Native and shRNA-transduced PSC27 cells as indicated were treated by bleomycin (BLEO), with the conditioned media (CM) collected 7 days after drug treatment and used for PCa cell culture. The CM were collected from equal number of cells per condition, with a starting DMEM that contains 0.5% FBS to make the CM. e Migration assay of PCa cells seeded within transwells in 6-well plates, with cells cultured for 3 days in the CM from PSC27 sublines depicted in d . f Invasiveness appraisal of PCa cells across the transwell membrane upon culture with the CM from PSC27 sublines described in d . g Chemoresistance assay of PCa cells cultured with the CM from PSC27 sublines described in d . MIT (mitoxantrone) was applied at the concentration of IC50 value pre-determined per cell line. AG-1478 (2 μM), cetuximab (50 μg/ml) or EREG mAb (1 μg/ml) were applied alongside with PSC27 CM. h Dose-response curves (non-linear regression/curve fit) plotted from drug-based survival assays of PC3 cells cultured with the CM of PSC27 native or damaged by bleomycin (PSC27-BLEO), and concurrently treated by a wide range of concentrations MIT. AG-1478, cetuximab or EREG mAb (1 μg/ml) were applied with PSC27 CM. Data are representative of three independent experiments. All p values were calculated by Student’s t tests. ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary normal human prostate stromal cell line PSC27, breast stromal cell line HBF1203 and lung stromal cell line HFL1 (ATCC) were maintained in stromal complete medium as described [ ].

Techniques: Western Blot, Transduction, Plasmid Preparation, Construct, Control, Immunoprecipitation, Staining, Proliferation Assay, shRNA, Cell Culture, Migration, Membrane, Concentration Assay

Journal: Oncogene

Article Title: Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance

doi: 10.1038/s41388-022-02476-7

Figure Lengend Snippet: a Heatmap depicting differentially expressed human transcripts in PC3 cells after a 3-days culture with EREG-containing CM collected from PSC27 cells. In contrast to cancer cells cultured with control CM (CTRL), 970 and 1362 genes were upregulated and downregulated, respectively, in those treated with the CM from EREG-expressing PSC27 cells (EREG). b Graphical visualization of pathways by GO profiling. Those significantly enriched genes in the upregulated list were sorted according to their fold change in PC3 cells exposed to the CM of EREG-expressing PSC27 cells. c Venn diagram displaying the overlap of 39 transcripts upregulated in PC3 and DU145 cells upon treatment with EREG-containing CM from stromal cells (970 and 309 genes with unique annotations for PC3 and DU145, respectively). d Statistics of transcripts differentially expressed (fold change either ≥2 or ≤0.5, with p < 0.05) in PC3 and DU145 upon EREG stimulation, and classified into typical categories according to functional annotations mapped by Genecode (V27). e Heatmap showing the top 39 transcripts upregulated by both PC3 and DU145 cells, sorted according to their expression fold change in PC3. f Pie chart depicting the biological processes associated with transcripts upregulated by EREG after GO analysis of the 39 transcripts in PC3. g Quantitative RT-PCR measurement of the expression of KIF20A, MARCHF4 and SPNS2 in both PCa lines upon exposure to CM of stromal cells expressing EREG. Signals normalized to those of cells exposed to PSC27 cells transduced with vector. h Dose-response curves (non-linear regression/curve fit) plotted from drug-based survival assays of PC3 cells transduced with vector or MARCHF4 construct and treated by a range of concentrations of MIT. i Dose-response curves (non-linear regression/curve fit) plotted from drug-based survival assays of DU145 cells treated in a manner similar to that of PC3 cells. j Immunoblot assessment of protein expression of EMT-associated molecules. CD81, a downstream target of MARCHF4. β-actin, loading control. k Immunoblot profiling of apoptosis-related factors of self-cleavage activity in both PCa cell lines pre-transduced with vector or MARCHF4 construct and exposed to MIT for 72 h. β-actin, loading control. Data in g – k are representative of three independent experiments. All p values were calculated by Student’s t tests. ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary normal human prostate stromal cell line PSC27, breast stromal cell line HBF1203 and lung stromal cell line HFL1 (ATCC) were maintained in stromal complete medium as described [ ].

Techniques: Cell Culture, Control, Expressing, Functional Assay, Quantitative RT-PCR, Transduction, Plasmid Preparation, Construct, Western Blot, Activity Assay

Journal: Oncogene

Article Title: Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance

doi: 10.1038/s41388-022-02476-7

Figure Lengend Snippet: a Schematic workflow of experimental procedure for severe combined immunodeficient (SCID) mice. Two weeks after subcutaneous implantation and in vivo uptake of tissue recombinants, animals received either single or combinational agents administered as metronomic treatments composed of several cycles. b Statistical profiling of tumor end volumes. PC3 cells were xenograted alone or together with PSC27 cells to the hind flank of SCID mice. Prior to implantation, PSC27 cells were transduced with the control vector or EREG construct to make stable sublines. MIT was administered to induce tumor regression. Right, representative tumor images. c Transcript assessment of several canonical SASP factors expressed in stromal cells isolated from the tumors of SCID mice. Tissues from animals implanted with both stromal and cancer cells were subject to LCM isolation, total RNA preparation and expression assays. d Representative IHC images of EREG expression in tissues isolated from placebo or MIT-treated animals. Square regions in the upper images were zoomed into lower images. Scale bars, 100 μm. e Statistical comparison of tumor growth in animals that underwent several different treatment modalities. Mice were implanted with PC3 alone or in combination with PSC27, before treated by the chemotherapeutic drug (MIT) or combinational agents (MIT/cetuximab or MIT/EREG mAb). Tumor volumes were measured at the end of an 8-week preclinical regimen. f Representative bioluminescence images (BLI) of PC3/PSC27 tumor-bearing animals in the preclinical trial. Digital signals were proportional to in vivo luciferase activities measured by an IVIS device. g Statistical assessment of DNA-damaged and apoptotic cells in the tumor specimens analyzed in e . Values are presented as percentage of cells positively stained by IHC with antibodies against γH2AX/p-53BP1 (co-staining) or caspase 3 (cleaved). h Representative IHC images of caspase 3 (cleaved) in tumors at the end of therapeutic regimens. Biopsies of placebo-treated animals served as negative controls for MIT-treated mice. Scale bars, 50 μm. i EREG concentration assessment in circulating blood of experimental mice treated by chemotherapy and/or EREG mAb. Data were derived from human EREG-specific ELISA assays. Data are representative of three independent experiments. Animal studies were performed with ten mice per group ( n = 10). All p values were calculated by Student’s t tests. ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary normal human prostate stromal cell line PSC27, breast stromal cell line HBF1203 and lung stromal cell line HFL1 (ATCC) were maintained in stromal complete medium as described [ ].

Techniques: In Vivo, Transduction, Control, Plasmid Preparation, Construct, Isolation, Expressing, Comparison, Luciferase, Staining, Concentration Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay