murine ccl2 protein levels  (R&D Systems)

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: Inflammatory monocytes associate with survival in LUSC. a Kaplan–Meier plots of overall survival in lung squamous carcinoma (LUSC) patients split by median (left, p < 0.0001) and quartile (right, p = 0.0006) expression levels of CD14. P -values are obtained with the log-rank test; FDR were calculated according to Benjamini and Hochberg. b Proportion of patients by mRNA subtype that have CD14 expression levels above (red) or below (black) the median CD14 expression level. Binomial tests for proportions were performed. (Black asterisks: significant enrichment below the median, red asterisks: Significant enrichment above the median). c Kaplan–Meier plots of overall survival in LUSC by expression levels of CCL2 ( p = 0.001), CCL3 ( p = 0.018) and CSF1 ( p = 0.015) expression. d Pearson’s correlations of CCL2, CCL3, and CSF1 chemokines versus CD14 (gene expression). e Dynamic range of mRNA expression of CD14 and CCL2 for each LUSC mRNA subtype. P -values were obtained with analysis of variance. The purple shading for CD14 expression represents samples in the ‘IM-rich subset’ (above the median CD14 expression level). f Dynamic range of mRNA expression of CCL3 and CSF1 by LUSC mRNA subtype. g Representative multiplex IHC for CD14 (green), CCR2 (red) and pan-cytokeratin (light blue) in a LUSC tumor sample, and enumeration of CD14+/CCR2+ cells in CK+ and CK- regions. #1-3 represent CK- regions, #4 represents a CK + region. Scale bar 100 μm. h Representative dual CD14+/CCR2+ cells (white arrows) in CK- (#1-3) and CK+ (#4) regions. Note: CK + region shown in white channel to more easily appreciate green and red. Scale bar 25 μm. i Enumeration of CD14+/CCR2+ cells in CK- and CK+ regions by mRNA subtype. Classical ( n = 14), Basal ( n = 9), Primitive ( n = 6) and Secretory ( n = 12). p -values were obtained with Student’s t-test in comparison to the Classical subtype. * P < 0.05, ** P < 0.01, *** P < 0.0001

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Expressing, Gene Expression, Multiplex Assay, Comparison

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: TNFα activation of NFκB promotes CCL2-mediated IM recruitment. a Microarray expression data (left) comparing murine bronchial epithelial cells (MBECs), parental KLN205 and the LN4K1 sub-clone. Top upstream pathways (right) from Ingenuity Pathway Analysis (IPA) are shown for the differentially regulated genes shown in brackets. b An upstream network visualization from IPA of all over-expressed genes (all nodes) in the upper portion of the heat map shown in Fig. with significant log-rank (survival analysis) p -value ( < 0.05). TNFα and NFκB (blue nodes) were amongst the top upstream regulators known to have direct roles (black lines) in promoting CCL2, CCL3 and CSF1 chemokines (the degree of redness corresponds with increasing statistical significance). c Relative expression of TNFα. d CCL2, CCL3 and CSF1 by qPCR. Data are averages ± s.e.m. P -values were obtained with Student’s t-test in comparison with KLN205. e Relative levels of CCL2 as measured by ELISA from secreted media of cells growing in vitro or, f , from plasma of tumor-bearing mice. Data are averages ± s.e.m. g Relative mRNA expression of CCL2 and p65 in LN4K1 cells following treatment with control or p65 siRNA with or without exogenous TNFα (100 ng/mL). h Relative expression of CCL2 mRNA (top) and phospho-p65 and p65 protein (bottom) following treatment with DMSO or an IKKβ inhibitor (Compound A, 5 μM) for 5 h. i Relative IM counts in the bone marrow, blood, spleen from healthy DBA2 mice versus those with LN4K1 tumors. j Relative IM, TAM and TReg counts from the lungs of healthy versus LN4K1-bearing mice. IMs were also assessed in age-matched DBA2 mice following HBSS ‘Mock’ injection. P -values obtained with one-sided Student’s t-test, n = 5 mice/group for f , i , and j . * P < 0.05, ** P ≤0.01, *** P ≤0.001

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Activation Assay, Microarray, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, In Vitro, Clinical Proteomics, Control, Injection

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: CCL2-mediated IM recruitment is critical for LUSC metastasis. a Relative expression of CCL2 for KLN205 and sub-clones. b Quantification of luciferase signal. c Representative images obtained 10 days after cell injection of (i) KLN205-Scr ORF, (ii) KLN205-CCL2 ORF, (iii) LN4K1-Cntrl shR, (iv) LN4K1-CCL2 shR#1 and (v) LN4K1-CCL2 shR#2. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 10 mice/group. d Survival plots of mice following tail vein injection of KLN205 cell lines. The black arrow indicates tissue harvest, n = 10 mice/group. e Number of IMs per lung lobe, n = 12 lobes/group. f Survival plots of mice following tail vein injection of LN4K1 cell lines. The black arrow indicates tissue harvest, n = 10 mice/group. g Number of IMs per lung lobe, n = 12 lobes/group. h Schematic (left) and quantification of luciferase signal (right) of mice treated with vehicle or PF-04136309 to assess effects on established metastases. i FACS plots and ( j ) quantification of percent IMs in the blood and ( k ) right lung of LN4K1-bearing mice. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 5 mice/group. l , FACS analysis of percent immune infiltrates for TAMs (gated on F480), DCs (gated on SiglecF-/CD11c), CD4, CD8, Tregs (gated on TCRb + ) and NK cells (gated on SiglecF-/B220-/TCRb-). m Schematic (left) and quantification of luciferase signal (right) of mice treated at the time of cell injection to assess effects on preventing metastasis. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 10 mice/group. n.s. = non-significant, * P ≤0.05, *** P < 0.001. For panel b , * FDR < 0.05, ** FDR < 0.01

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Expressing, Clone Assay, Luciferase, Injection

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: Schematic of the ‘IM-rich subset’ of lung squamous carcinoma. TNFα activation of the canonical NFκB leads to LUSC cell secretion of chemo-attractant CCL2, which stimulates the bone marrow to release inflammatory monocytes (IMs) into circulation. The IMs bring large payloads of FXIIIA into the tumor microenvironment, leading to cross-linked fibrin, LUSC invadopodia formation and progression

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Activation Assay