rm9 cell line  (ATCC)

Journal: Journal of Leukocyte Biology

Article Title: TLR9 expression and secretion of LIF by prostate cancer cells stimulates accumulation and activity of polymorphonuclear MDSCs

doi: 10.1189/jlb.3MA1016-451RR

Figure Lengend Snippet: C57BL/6 and FVB mice were subcutaneously engrafted with RM9-Tlr9ON (A and B) and Myc-CaP-Tlr9ON (C and D) cancer cells, respectively. After tumors were established, 2 mg of tetracycline or PBS as a vehicle was injected intravenously every other day. The induction of Tlr9 expression in CD45− tumor cells was confirmed by real-time qPCR: (A) RM9-Tlr9ON and (C) Myc-CaP-Tlr9ON. Growth kinetics of RM9-Tlr9ON (B) and Myc-CaP-Tlr9ON tumors (D) were determined by caliper measurements. (E–G) Tumor infiltration by PMN-MDSCs (CD45+CD11b+Ly6GHILy6CLO), M-MDSCs (CD45+CD11b+Ly6GLOLy6CHI), and TAMs (CD45+CD11b+Ly6G−F4/80+) was analyzed with flow cytometry. The representative flow cytometry analysis (E) and frequency of tumor-infiltrated PMN-MDSCs, M-MDSCs, and TAMs from PBS- or tetracycline-treated RM9-Tlr9ON (F) and Myc-CaP-Tlr9ON (G) tumors are shown. (H) RM9-Tlr9ON tumor-bearing mice treated with PBS or tetracycline were injected intratumorally with either 40 μg of anti-Gr1 antibody or control rat IgG. Tumor growth kinetics was monitored (n ≥ 5 per group); data are means ± sem. *P < 0.05; **P < 0.01.

Article Snippet: Cells The RM9 cell line was kindly provided by Dr. T. Thompson (University of Texas MD Anderson Cancer Center, Houston, TX, USA) and Myc-CaP cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Injection, Expressing, Flow Cytometry, Control

Journal: Journal of Leukocyte Biology

Article Title: TLR9 expression and secretion of LIF by prostate cancer cells stimulates accumulation and activity of polymorphonuclear MDSCs

doi: 10.1189/jlb.3MA1016-451RR

Figure Lengend Snippet: (A) Tumor-infiltrating PMN- and M-MDSCs were sorted from RM9-Tlr9ON tumor-bearing mice after treatment with PBS (n = 3) or tetracycline (n = 3). The mRNA expression levels of S100a8, S100a9, Cebpb, and Stat3 were assessed with real-time qPCR. (B) Levels of tyrosine-pSTAT3, arginase-1 (Arg-1), and IL-10 were analyzed in tumor-infiltrating PMN- and M-MDSCs from PBS- or tetracycline-treated Myc-CaP-Tlr9ON tumors by flow cytometry (n = 6/group). (C) PMN- and M-MDSCs were sorted from spleens of PBS- or tetracycline-treated mice bearing RM9-Tlr9ON tumors (n = 3/group). CFSE-labeled CD3+ T cells were activated with CD3/CD28 microbeads and cocultured for 3 d with PMN- or M-MDSCs (MDSC:T cell ratio = 1:2). Cells were harvested and T cell proliferation was analyzed by CFSE dilution assay and flow cytometry. Data are means ± sem. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Cells The RM9 cell line was kindly provided by Dr. T. Thompson (University of Texas MD Anderson Cancer Center, Houston, TX, USA) and Myc-CaP cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Expressing, Flow Cytometry, Labeling, Dilution Assay

Journal: Journal of Leukocyte Biology

Article Title: TLR9 expression and secretion of LIF by prostate cancer cells stimulates accumulation and activity of polymorphonuclear MDSCs

doi: 10.1189/jlb.3MA1016-451RR

Figure Lengend Snippet: (A) mRNA expression levels of various IL-6-type cytokines including Lif, Il6, Il11, Osm, and Cntf were measured in mouse TLR9+ RM9 prostate cancer cells with real-time qPCR. (B) The gene expression levels for LIF, IL-6, IL-11, OSM, and CNTF were determined with TCGA datasets from 490 patients with prostate cancer. (C) Total RNA was extracted from CD45− tumor cells from PBS- or tetracycline-treated mice bearing RM9-Tlr9ON (left) or MycCAP-Tlr9ON (right) tumors, and the mRNA level of LIF was measured with real-time qPCR (n ≥ 5). Data are means ± sem. (D) Protein level of LIF was measured in CD45− tumor cells from PBS- or tetracycline-treated mice bearing RM9-Tlr9ON. (E) Pearson correlation analysis between Tlr9 and Lif expression. Each dot represents results from CD45− tumor cells isolated from a single Myc-CAP-Tlr9ON tumor-bearing mouse after PBS (black) or tetracycline treatment (red). (F) The association of NF-κB and Stat3 at the putative NF-κB-binding site were examined by ChIP assay followed by RT-qPCR using primers encompassing −200 upstream from the transcription start site. Transcription factor binding sites were predicted at the Lif promoter region (n = 3) with MotifMap. Data are means ± sem. (G–I) PC3 (TLR9+) and LNCAP (TLR9−) prostate cancer cells were genetically modified to alter basal levels of TLR9, creating PC3-mock/PC3-shTLR9 and LNCaP-mock/LNCaP-hTLR9, as described elsewhere [13]. Shown is the effect of LIF expression on the mRNA levels by qPCR (G), protein levels by Western blot analysis (H), and the concentration of LIF in cell culture supernatant by ELISA (I). Data are means ± sem (n = 3). (J) The correlation between TLR9 and LIF expression (r = 0.32; P = 8.88 × 10−15), as well as between TLR9 and IL-6 expression (r = 0.22; P = 2.17 × 10−14), as assessed with a TCGA dataset from 490 patients with prostate cancer. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Cells The RM9 cell line was kindly provided by Dr. T. Thompson (University of Texas MD Anderson Cancer Center, Houston, TX, USA) and Myc-CaP cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Expressing, Gene Expression, Isolation, Binding Assay, Quantitative RT-PCR, Genetically Modified, Western Blot, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Leukocyte Biology

Article Title: TLR9 expression and secretion of LIF by prostate cancer cells stimulates accumulation and activity of polymorphonuclear MDSCs

doi: 10.1189/jlb.3MA1016-451RR

Figure Lengend Snippet: (A) Mice with subcutaneously engrafted RM9-Tlr9ON tumors were injected intravenously with 2 mg tetracycline to induce Tlr9 expression. When tumor size reached 50 mm3, LIF-neutralizing antibody (α-LIF, 10 mg) or control IgG (10 mg) was injected intratumorally every other day. Kinetics of tumor growth and tumor weight at the end of the experiment. (B) The percentage of tumor-associated MDSCs in mice treated with LIF-neutralizing antibodies or control IgG, as assessed with flow cytometry. Representative contour plots and bar graphs are shown. (C) Intracellular levels of arginase-1 (Arg-1), IL-10, and tyrosine-pSTAT3 in tumor-infiltrated PMN- and M-MDSCs following LIF neutralization or IgG treatment as measured cytofluorimetrically. Shown are means ± sem (n ≥ 5/group). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Cells The RM9 cell line was kindly provided by Dr. T. Thompson (University of Texas MD Anderson Cancer Center, Houston, TX, USA) and Myc-CaP cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Injection, Expressing, Control, Flow Cytometry, Neutralization

Journal: Journal of Leukocyte Biology

Article Title: TLR9 expression and secretion of LIF by prostate cancer cells stimulates accumulation and activity of polymorphonuclear MDSCs

doi: 10.1189/jlb.3MA1016-451RR

Figure Lengend Snippet: (A) The biodistribution of fluorescence-labeled CpG-STAT3dODNCy3 after single intratumoral injection. CpG-STAT3dODNCy3 (5 mg/kg) was injected into RM9 tumors. The uptake by tumor-infiltrating T cells, TAMs, and M- and PMN-MDSCs was evaluated using flow cytometry (n = 4). (B) Local treatment using CpG-STAT3dODN but not control CpG-scrODN (5 mg/kg, every other day; n = 5) results in RM9 tumor growth inhibition in mice. Data are means ± sem. *P < 0.05; **P < 0.01.

Article Snippet: Cells The RM9 cell line was kindly provided by Dr. T. Thompson (University of Texas MD Anderson Cancer Center, Houston, TX, USA) and Myc-CaP cells were purchased from ATCC (Manassas, VA, USA).

Techniques: Fluorescence, Labeling, Injection, Flow Cytometry, Control, Inhibition