murine prostate cancer cell line rm1  (ATCC)

Journal: Nature communications

Article Title: Recruitment of Mesenchymal Stem Cells Into Prostate Tumors Promotes Metastasis

doi: 10.1038/ncomms2766

Figure Lengend Snippet: ( a ) CXCR6 mRNA expression by human MSCs (P 1 and P 2 ). ( b ) Expression of CXCR6 protein by human MSCs. Controls included isotype matched controls and fibroblast-specific protein 1 (FSP1) for MSCs. Scale bars, 100μm. ( c ) CXCR6 mRNA by mMSCs. CXCR6 expression was determined in freshly isolated, non-cultured (P 0 ) or P 2 murine MSCs from CXCR6 +/+ or CXCR6 −/− mice. Human and murine osteoblasts (HOB and MC3T3-E1) were used as a negative control. ( d ) Expression of CXCR6 by murine P 2 CXCR6 +/+ or CXCR6 −/− MSCs by IHC staining. Scale bar 100μm. Data in ( a-c ) are representative of mean with standard deviation for triplicates in each of three independent experiments (Student’s t -test). ( e ) CXCL16 expression in human prostate cancer tissue microarray in . Differences noted between normal prostate ( n = 30), Gleason 4+5 ( n = 9), Gleason 6+7 ( n = 18), and Gleason 8+9 ( n = 15) (mean±s.d. Student’s t -test). Secretionof CXCL16 by human prostate cancer cell lines ( f ) and murine prostate cancer cell lines ( g ) as determined by ELISA (mean±s.d., n = 3 independent experiments, Student’s t -test). ( h ) Migration of freshly isolated, non-cultured (P 0 ) or P 2 murine MSCs from CXCR6 +/+ or CXCR6 −/− mice in response to CXCL16. The % migrated MSC was determined by hemocytometer counting (mean±s.d., n = 3 independent experiments, Student’s t -test). ( i ) CXCR6 +/+ or CXCR6 −/− mice were implanted s.c. with RM1 cells and caliper measurements of tumor growth performed over 25 days. *Significant differences between tumors grown CXCR6 +/+ and CXCR6 −/− mice (mean±s.d, for 7 animals/group, n = 3 independent experiments, P < 0.05; Student’s t -test). ( j ) % MSCs (P 0 ) present in RM1 tumors grown in CXCR6 +/+ or CXCR6 −/− mice at day 25 (mean±s.d. for 7 animals/group, n = 3 independent experiments, Student’s t -test). ( k ) SCID mice were implanted s.c. with PC3 cells mixed with MSC P0 CXCR6 +/+ or MSC P0 CXCR6 −/− cells and tumor growth was evaluated by caliper measurements over 42 days. *Significant differences between tumors grown with PC3 cells mixed with MSC P0 CXCR6 +/+ and MSC P0 CXCR6 −/− cells (mean±s.d. for n = 5 animals/group, n = 1 independent experiment, P < 0.05, Student’s t -test).

Article Snippet: The human prostate cancer cell lines PC3, LNCaP, and DU145, and murine prostate cancer cell lines RM1 and Tramp were used (American Type Culture Collection (ATCC), Rockville, MD).

Techniques: Expressing, Isolation, Cell Culture, Negative Control, Immunohistochemistry, Standard Deviation, Microarray, Enzyme-linked Immunosorbent Assay, Migration

Journal: Nature communications

Article Title: Recruitment of Mesenchymal Stem Cells Into Prostate Tumors Promotes Metastasis

doi: 10.1038/ncomms2766

Figure Lengend Snippet: ( a ) Expression of CXCL16 mRNA in the RM1 Control or RM1 shCXCL16 cells by qRT-PCR. ( b ) Secretion of CXCL16 by RM1 Control or RM1 shCXCL16 cells was determined by ELISA. ( c ) Migration of MSC CXCR6 +/+ cells was determined toward RM1 Control cells or RM1 shCXCL16 cells. Data in ( a-c ) are representative ofmean with standard deviation for triplicates in each of three independent experiments (Student’s t -test). Significance was determined using a Student’s t -test. ( d ) Experimental scheme of RM1 Control or RM1 shCXCL16 cell implantation to CXCR6 +/+ mice for examining tumor growth and MSC cell recruitment to tumors. ( e ) The tumor growth of RM1 Control or RM1 shCXCL16 cells on CXCR6 +/+ mice was evaluated by caliper measurements over 23 days. *Significant differences between tumors grown with RM1 Control and RM1 shCXCL16 cells (mean±s.d., for n = 5 animals/group, n = 2 independent experiments, P < 0.05;ANOVA). ( f ) % MSCs present in RM1 Control or RM1 shCXCL16 tumors grown in CXCR6 +/+ mice (mean±s.d., for n = 5 animals/group, n = 2 independent experiments, Student’s t -test).

Article Snippet: The human prostate cancer cell lines PC3, LNCaP, and DU145, and murine prostate cancer cell lines RM1 and Tramp were used (American Type Culture Collection (ATCC), Rockville, MD).

Techniques: Expressing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Migration, Standard Deviation

Journal: Nature communications

Article Title: Recruitment of Mesenchymal Stem Cells Into Prostate Tumors Promotes Metastasis

doi: 10.1038/ncomms2766

Figure Lengend Snippet: ( a ) Vehicle or CXCL12 treated RM1 cells, or RM1 cells co-cultured with MSCs from CXCR6 +/+ or CXCR6 −/− mice were examined by phase contrast microscopy and IHC staining for cytokeratin, E-cadherin, N-cadherin, vimentin, and α-SMA. Scale bars, 100μm. Representative images from 2 independent studies. ( b ) Western blots analysis for epithelial (E-cadherin) and mesenchymal (N-cadherin, β-catenin, snail, slug) markers. Representative images from 2 independent studies. ( c ) EMT markers in the primary tumor were examined by IHC. Colocalization of E-cadherin or N-cadherin with FSP1 was observed. More E-cadherin by prostate cancer cells (red; white arrows) was detected in close proximity to FSP1 expressing MSC cells (green; orange arrows) in tumors grown in CXCR6 −/− mice compared to tumors grown in CXCR6 +/+ mice. In contrast, more N-cadherin expressing prostate cancer cells (red; white arrows) were detected in close proximity to N-cadherin and FSP1 co-expressing CAF cells (yellow; yellow arrows) when the tumors were grown in CXCR6 +/+ mice compared to tumors grown in CXCR6 −/− mice. Blue, DAPI nuclear stain. Scale bars, 100μm. Representative images derived from n=10 mice/group). ( d ) IHC of E-cadherin or N-cadherin positive cells within benign or Gleason 4+5 prostate cancers in human prostate tissue microarrays (TMAs) (red, E-cadherin or N-cadherin, white arrows; blue, DAPI nuclear stain). Scale bars, 100μm. ( e ) Quantification of . Mean expression scores were multiplied by percent positive cells in the field. Significant differences were noted between benign ( n = 30) or Gleason 4+5 prostate ( n = 6) (mean±s.d ANOVA). ( f ) CXCR4 mRNA was determined for EMT-induced RM1 cells following CXCL12 treatment or co-culture with MSCs derived from CXCR6 +/+ or CXCR6 −/− mice (mean±s.d., n = 3 independent experiments). ( g ) More CXCR4 expressing RM1 cells (red; white arrows) were detected in close proximity to CXCR4 and FSP1 (green; orange arrows) co-expressing CAF cells (yellow; yellow arrows) when the tumors were grown in CXCR6 +/+ mice compared to tumors grown in CXCR6 −/− mice. Scale bars, 100μRepresentative images from an experiment with n=10 animals/group). ( h ) AMD3100 or anti-CXCR4 antibody prevents the development of EMT by RM1 cells following CXCL12 exposure. Scale bars, 100μm. Representative images from an experiment with n=10 animals/group.

Article Snippet: The human prostate cancer cell lines PC3, LNCaP, and DU145, and murine prostate cancer cell lines RM1 and Tramp were used (American Type Culture Collection (ATCC), Rockville, MD).

Techniques: Cell Culture, Microscopy, Immunohistochemistry, Western Blot, Expressing, Staining, Derivative Assay, Co-Culture Assay

Journal: Nature communications

Article Title: Recruitment of Mesenchymal Stem Cells Into Prostate Tumors Promotes Metastasis

doi: 10.1038/ncomms2766

Figure Lengend Snippet: (a) Migration assays were performed in Transwell® plates using 10% serum or CXCL12 as chemoattractants. Migration toward 0.5% serum was used as a negative control. (b) Blockade of CXCR4 by AMD3100 or anti-CXCR4 antibody prevents prostate cancer migration towards CXCL12 or MSCs isolated from CXCR6 +/+ , but not CXCR6 −/− animals. Data in (a,b) are representativedata from two independent studies (mean±s.d., ANOVA). Significance was determined using a Student’s t -test. RFP-labeled RM1 WT or RM1 EMT cells were incubated with vehicle or AMD3100 in vitro , and then inoculated by intra-cardiac ( i.c. ) injection into CXCR6 +/+ or CXCR6 −/− ( n = 7). Metastasis was assessed by qPCR for RFP in a number of tissues. (c,d) Number of metastatic RM1 cells following i.c. injection. *Significance between RM1 WT treated with vehicle and RM1 WT treated with AMD3100 ( P < 0.05). # Significance between RM1 WT treated with vehicle and RM1 EMT cells treated with vehicle ( P < 0.05). † Significance between RM1 EMT treated with vehicle and RM1 EMT treated with AMD3100 ( P < 0.05). Error bars represents mean±s.d., n = 2 independent experiments, P < 0.05; Student’s t -test. (e-h) RM1 cells expressing RFP were identified in the femur of CXCR6 +/+ or CXCR6 −/− mice following i.c . injection. Red arrows identify RM1 cells. White arrows identify osteoblast on the bone surface staining positive for CXCL12 expression. Scale bars, 100μm. (f,h) Quantification of Fig. 5e and Fig. 5g, respectively. The numbers of RM1 cells were quantified on the endosteal region of the 7 long bones. Endosteal regions were defined as 12 cell diameters from bone surfaces. ((Mean±s.d. ( n = 3)., ANOVA).

Article Snippet: The human prostate cancer cell lines PC3, LNCaP, and DU145, and murine prostate cancer cell lines RM1 and Tramp were used (American Type Culture Collection (ATCC), Rockville, MD).

Techniques: Migration, Negative Control, Isolation, Labeling, Incubation, In Vitro, Injection, Expressing, Staining