Journal: Oncotarget

Article Title: Chemokines CCL2, 3, 14 stimulate macrophage bone marrow homing, proliferation, and polarization in multiple myeloma

doi:

Figure Lengend Snippet: A. qPCR analysis of expression of different MO chemokines in MM patient BM cells. The value indicates the relative expression to GAPDH. One representative sample of 4 patient samples analyzed is shown. B. Levels of CCL3, CCL14, and CCL2, measured by ELISA, in BM plasma of healthy donors (CTR) and patients with MM or MGUS. The numbers of CTR, MGUS and MM patients used for measuring CCL2 are 4, 10, and 23, respectively; for CCL14 are 7, 10, and 11, respectively; and for CCL3 are 7, 10, and 13, respectively. C. Immunohistochemistry analysis of CCL3, CCL14, and CCL2 expression in BM biopsies of 2 healthy donors (CTR1 and CTR2) and 2 representatives (MM1 and MM2) out of five MM patients. D. Linear regression analysis of the relationship between the percentage of BM MΦs and concentration of chemokines CCL14 ( n = 20) and CCL3 ( n = 18) in BM plasma in MM patients. * p < 0.05.

Article Snippet: Neutralizing antibodies to human CCL3 (MAB270), CCL14 (MAB3241) and CCL2 (MAB679), and mouse CCL3 (AB-450-NA), CCL2 (AB-479-NA) were purchased from R&D Systems.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Immunohistochemistry, Concentration Assay

Journal: Oncotarget

Article Title: Chemokines CCL2, 3, 14 stimulate macrophage bone marrow homing, proliferation, and polarization in multiple myeloma

doi:

Figure Lengend Snippet: A. qPCR analysis of CCL3, CCL14, and CCL2 expression in CD138 + primary MM cells and CD138 − non-malignant cells in BM aspirates from 3 different MM patients (MM1 to MM3). The y-axis indicates the fold change relative to GAPDH value. B. Concentration of chemokines secreted by 4 human MM cell lines. MM cells (2 ×10 5 cell per well) were cultured in vitro for 24 hours; then the chemokine expression in culture supernatant was analyzed by ELISA. C. BMSCs were cocultured with ARP-1 or MM.1S MM cells for 48 hours. CCL3, CCL14, and CCL2 expression in BMSCs was analyzed by qPCR. D. BMSCs were cultured in transwells with MM cells ARP-1 for 48 hours, followed by removal of MM cells and cultured alone in fresh medium for 24 hours. BMSCs from the same donors were cultured alone in parallel as controls. Chemokine concentrations were determined by ELISA using culture supernatants collected at the end of culture. * p < 0.05, ** p < 0.01.

Article Snippet: Neutralizing antibodies to human CCL3 (MAB270), CCL14 (MAB3241) and CCL2 (MAB679), and mouse CCL3 (AB-450-NA), CCL2 (AB-479-NA) were purchased from R&D Systems.

Techniques: Expressing, Concentration Assay, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Chemokines CCL2, 3, 14 stimulate macrophage bone marrow homing, proliferation, and polarization in multiple myeloma

doi:

Figure Lengend Snippet: A. nMΦ or mMΦ were generated as described in the Methods. The cells were then cultured in vitro in the presence of each of the chemokine neutralizing antibodies (αCCL3, αCCL14 and αCCL2, at a final concentration of 10 μg/ml each), and their combination (MIX). An equal amount of IgG was used as control. Cell proliferation at different time points was examined by MTS. B. nMΦs were cultured for 3 days in the presence of each of the recombinant chemokines (final concentration of 1 μg/ml) or their combination (MIX). mMΦs cultured in medium alone were used as a positive control. Cell proliferation at different time points was determined by MTS assay. C. Western blot showing the levels of p27Kip1 and cyclin D1 in nMΦs and mMΦs from 2 healthy donors (Do1 and Do2). D. Western blot showing the levels of p27Kip1 and cyclin D1 in nMΦs and mMΦs. mMΦs were generated in coculture with MM cells in transwells in the absence (CTR) or presence of control IgG or a combination of the chemokine neutralizing antibodies (MIX). E. Western blot showing the levels of different kinases, IL-6, c-myc, cyclin D1, and p27Kip1 in nMΦs in overnight cultures in medium or with addition of each of the chemokines CCL2, CCL14, or CCL3 individually or all three combined (MIX). mMΦs were used as a positive control. F. Proliferative response of mMΦs in medium alone (CTR) or in the presence of the PI3K-Akt inhibitor LY294002 (LY; 50 μM) or Erk1/2 inhibitor U0126 (UO; 2 μM). * p < 0.05, ** p < 0.01.

Article Snippet: Neutralizing antibodies to human CCL3 (MAB270), CCL14 (MAB3241) and CCL2 (MAB679), and mouse CCL3 (AB-450-NA), CCL2 (AB-479-NA) were purchased from R&D Systems.

Techniques: Generated, Cell Culture, In Vitro, Concentration Assay, Control, Recombinant, Positive Control, MTS Assay, Western Blot

Journal: Theranostics

Article Title: Crosstalk between macrophage-derived PGE 2 and tumor UHRF1 drives hepatocellular carcinoma progression

doi: 10.7150/thno.69494

Figure Lengend Snippet: TAMs promote HCC progression via the UHRF1 and CSF1 network. (A) Left: Immunofluorescent staining of CD68 + TAMs (red) in xenograft H22 tumors expressing nonsense control shRNA (shNC) or shUhrf1. The nuclei (blue) were stained by DAPI. Scale bar, 50 µm. Right: CD68 positive cells per field. Six randomly selected microscopic fields per sample. n = 6 samples per group, *P = 0.0007. Student's t -test. (B, C) PGE 2 secretion (B) and COX-2 expression (C) in human HCC TAMs. TAMs were incubated with the supernatants of HepG2 cells expressing control vector (HepG2 Ctrl sup.) or UHRF1 (HepG2 UHRF1 sup.) for 24 hours. n = 3, *P = 0.0031. Student's t -test. (D, E) The relevant mRNA levels in HepG2 cells stably expressing nonsense control shRNA (shNC) and shUHRF1 (D) , or expressing empty vector (Vector) and UHRF1 (E) . n = 3, *P < 0.05, versus corresponding control (shNC or Vector). Student's t -test. (F) CSF1 and CCL14 protein levels in HepG2 cells stably expressing empty vector (Vector) or UHRF1. (G) COX-2 protein levels in TAMs incubated with the supernatants from HepG2 cells or UHRF1-overexpressing HepG2 cells. HepG2 cells were treated without or with CSF1 neutralizing antibody (anti-CSF1, 2 μg/mL) for 24 hours. (H) PGE 2 secretion from human HCC TAMs. TAMs were incubated with the supernatants from HepG2 cells or UHRF1-overexpressing HepG2 cells that were treated without or with anti-CSF1 (2 µg/mL) for 48 hours. Isotype IgG as an antibody control. n = 3, *P = 0.03117. Student's t -test. (I) COX-2 protein levels in human HCC TAMs incubated with the supernatants from HepG2 cells or UHRF1-overexpressing HepG2 cells that were treated without or with siRNA against CSF1 (siCSF1) for 48 hours. (J) Left: Schematics showing transwell assays analyzing human HCC TAMs. Human TAMs were seeded in the upper chamber. HepG2 cells (HepG2 Ctrl ) or UHRF1-overexpressing HepG2 cells (HepG2 UHRF1 ) were seeded in the lower chamber. TAMs were cultured in the medium without or with anti-CSF1 (2 μg/mL). Isotype IgG as an antibody control. Right: Percentages of migrated TAMs relative to total TAMs. n = 3 with replicates. *P = 1.39 × 10 -4 , versus Control (HepG2 Ctrl in medium without anti-CSF1). One-way ANOVA with Dunnett's multiple comparisons test. (K) DNA methylation of CpG islands in the CSF1 promoter in HepG2 cells stably expressing control vector (Vector), UHRF1, shNC or shUHRF1. Closed circles indicate methylated CpGs. Open circles represent unmethylated CpGs. Percentage of DNA methylation (methylated CpGs/ total CpGs) is given at the bottom of each panel. (L) Pearson correlation between UHRF1 mRNA levels and CSF1 promoter methylation levels in human HCC tissues. 16 HCC patients. (M) ChIP assay showing DNMT1 abundance on the CSF1 promoter in HepG2 cells stably expressing shNC or shUHRF1. (N) CSF1 protein levels in HepG2 cells and shUHRF1-expressing HepG2 cells that were transfected without or with siRNA against DNMT1 (siDNMT1). (O, P) Tumor growth (O) and overall survival (P) of the mice. Mice were subcutaneously inoculated with H22 cells stably expressing shUhrf1 (H22 shUhrf1 ) or shNC (H22 shNC ). Half of the mice in each group were intraperitoneally injected with Clodronate liposomes to deplete macrophages (H22 shUhrf1 + MΦ-dep., and H22 shNC + MΦ-dep.). n = 4 per group. In O , *P = 0.0006, H22 shNC + MΦ-dep. versus H22 shNC ; P = 0.0740 (N.S.), H22 shUhrf1 + MΦ-dep. versus H22 shUhrf1 ; Student's t -test. In P , *P = 0.0169, H22 shNC + MΦ-dep. versus H22 shNC ; P = 0.1753, H22 shUhrf1 + MΦ-dep. versus H22 shUhrf1 ; Log-rank (Mantel-Cox) test. (Q) Effect of celecoxib on tumor growth. H22 cells were inoculated into the left posterior flank of the Balb/c mice. The tumor bearing mice were then treated with daily oral administration of celecoxib (CXB, 150 mg/kg, n = 6) or solvent (Control, n = 6). Left panel: once tumors started growing, their sizes were measured twice weekly and tumor volume was calculated. Right panel: photographs of isolated tumors from each group. *P = 0.042. Student's t -test. (R) UHRF1 staining analysis of tissue sections from celecoxib(CXB)-treated and solvent(Control)-treated H22 tumor bearing mice (n = 6 per group). Scale bar, 1 cm. *P = 0.022. Student's t -test. (S) Schematic model showing the interactions between TAMs and HCC cells. TAMs produce and release PGE 2 into the tumor microenvironment. (I) PGE 2 inhibits miR-520 transcription by dissociating KLF6 from the miR-520 promoter. (II) Reduced miR-520 permits UHRF1 upregulation. (III) High-level UHRF1 epigenetically suppresses KLF6 expression via H3K9 hypermethylation. (IV) Dampened KLF6 lowers miR-520, thus allowing further elevation of UHRF1 protein level. (V) Concurrently, high-level UHRF1 epigenetically promotes CSF1 expression via DNA hypomethylation. (VI) CSF1 secreted from HCC cells promotes COX-2 expression in TAMs, leading to macrophage tumor infiltration and activation. The upregulated COX-2 in TAMs stimulates additional PGE 2 production .

Article Snippet: Protein extracts were probed with antibodies against human UHRF1 (sc-373750, Santa Cruz biotechnology, USA), KLF6 (sc-7158, Santa Cruz biotechnology, USA), COX-2 (A5787, ABclonal, USA), CSF1 (AF216, R&D Systems, USA), DNMT1 (sc-271729, Santa Cruz biotechnology, USA), and CCL14 (MAB3241, R&D Systems, USA) or β-actin (A5441, Sigma, USA).

Techniques: Staining, Expressing, Control, shRNA, Incubation, Plasmid Preparation, Stable Transfection, Cell Culture, DNA Methylation Assay, Methylation, Transfection, Injection, Liposomes, Solvent, Isolation, Activation Assay

Journal: Frontiers in Oncology

Article Title: AXL Overexpression in Tumor-Derived Endothelial Cells Promotes Vessel Metastasis in Patients With Hepatocellular Carcinoma

doi: 10.3389/fonc.2021.650963

Figure Lengend Snippet: AXL/SOX2/DKK-1 axis in HUVECs promotes HCC metastasis. (A) DKK-1 and CCL14 secretion was significantly downregulated in CM from HUVEC-AXL-KD compared with that from HUVEC-AXL-NC, as detected with a human cytokine antibody array. (B) DKK-1 and CCL14 expression was markedly upregulated in the CM of HUVECs overexpressing AXL (CCL14: p < 0.001; DKK-1: p = 0.003) compared with the CM of HUVEC-AXL-NC, as detected by ELISA assay. (C) AXL siRNA downregulated DKK-1 and CCL14 secretion in the CM of HUVEC-AXL-NC and HUVEC-AXL-OE cells (CCL14: p < 0.001 and p < 0.001; DKK-1: p < 0.001 and p < 0.001). (D) DKK1 siRNA (MHCC-97L: p < 0.001 and p < 0.001; HCC-LM3: p <0.001 and p < 0.001), but not CCL14 siRNA (MHCC-97L: p = 0.126 and p = 0.711; HCC-LM3: p = 0.901 and p = 0.694) could attenuate the effect of the CM from HUVEC-AXL-NC and HUVEC-AXL-OE cells on the migration of HCC-LM3 cells and MHCC-97L cells. (E) SOX2 mRNA expression was significantly increased in HUVEC-AXL-OE cells and decreased in HUVEC-AXL-KD cells compared with HUVEC-AXL-NC cells (HUVEC-AXL-KD: p < 0.001, HUVEC-AXL-OE: p < 0.001). (F) AXL overexpression could significantly increase SOX2 and DKK-1 protein expression in HUVEC-AXL-OE cells compared with HUVEC-AXL-NC cells, and SOX2 siRNA inhibited SOX2 and DKK-1 protein expression in HUVEC-AXL-OE and HUVEC-AXL-NC cells.

Article Snippet: The protein concentrations of CCL14 and DKK-1 in the supernatants were also measured using an enzyme-linked immunosorbent assay (ELISA) kit (CCL14: EK1123 Boster, Wuhan, China; DKK-1: EK0867 Boster, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Ab Array, Expressing, Enzyme-linked Immunosorbent Assay, Migration, Over Expression