Journal: bioRxiv

Article Title: The novel compensatory reciprocal interplay between neutrophils and monocytes drives cancer progression

doi: 10.1101/2022.07.21.500690

Figure Lengend Snippet: ( A ) Correlations between CCL7, CCL8, and CCL13 expression levels and patient survival were analyzed using an IDH-WT cohort from TCGA. High and low expression were defined as +/- 0.5 STDEV from the mean of all samples (n = 260). Both Log-rank and Mantel Cox (MC) tests were applied. P = 0.1868 by MC for CCL13 . ( B ) Normalized protein expressions of MCPs examined by Olink proteomic assay. Two-tailed Student’s t -test. (C) Expression distribution of MCP family members in human GBM tissue as determined in tandem by laser capture microdissection and RNA-seq queried from the IVY Gap database (n=34). (D) Schematic illustration of CRISPR/Cas9-mediated deletion of the MCP genes. (E) Serum MCP levels were measured by ELISA following LPS treatment. CCL5 was used as an internal control. Two-tailed Student’s t -test. (F) Flow cytometry quantification of Ly6c Hi monocytes in the bone marrow of healthy adult mice. (G) Multiplex flow cytometry analysis was used to enumerate blood cells in the circulation. (H) Analysis of blood cells in healthy adult mice. Two-tailed Student’s t -test. (I) Schematic illustration of orthotopic transplantation of primary tumors. ( J ) Kaplan Meier-survival curves of PDGFB -driven tumors generated in WT and qMCP -/- mice. ( K ) flow cytometric quantification of myeloid cells in tumors at humane endpoint. Student’s t -test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant. MS = median survival.

Article Snippet: ELISAs were performed for CCL2 (R&D, DY479), CCL7 (Boster Bio, EK0683), CCL8 (R&D, DY790), CCL11 (R&D, MME00), CCL12 (R&D, MCC120) and CCL5 (R&D, DY478-05) on cell lysates and cell supernatants according to the manufacturer’s instructions.

Techniques: Expressing, Proteomic Assay, Two Tailed Test, Laser Capture Microdissection, RNA Sequencing, CRISPR, Enzyme-linked Immunosorbent Assay, Control, Flow Cytometry, Multiplex Assay, Transplantation Assay, Generated

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Chemotherapy induces breast cancer stemness in association with dysregulated monocytosis

doi: 10.1158/1078-0432.CCR-17-2545

Figure Lengend Snippet: (A) Twenty pairs of pre- and post-NT sera from TNBC (n=8; black) or HER2+ BC (n=12; blue) patients were analyzed for the activity to stimulate the ALDEFLUORbright population of BC cells. MDA-MB-231 cells were cultured for 48 h in base medium supplemented with 10% human serum before ALDEFLUOR assays by flow cytometry. Wilcoxon test was performed. (B) ELISA to determine the levels of human CCL2/7/8 in the 20 pairs of sera. Wilcoxon tests were performed. (C) ALDEFLUOR assays using 6 pairs of sera (3 cases for each BC subtype) were performed as in A except that CCL2/7/8 neutralizing antibodies (NAb; 30 ng/ml; alone or all 3 combined) or control IgG were added during serum treatment. (D) ALDEFLUOR assays of MDA-MB-231 cells treated with patient sera in the presence of a CCR2 inhibitor (MK-0812; 600 nM) or vehicle. (E) NSG mice with or without MDA-MB-231 xenograft tumors and tumor-free BALB/c mice received 3 weekly injections with doxorubicin (DOXO; 4 mg/kg) or docetaxel (DTX; 25 mg/kg) (n=3). Six days later, serum was collected to treat MDA-MB-231 cells for ALDEFLUOR assays as in A. (F) ELISA to determine the serum levels of mouse CCL2/7/8. (G) ALDEFLUOR assays of MDA-MB-231 cells treated with mouse sera in the presence of the CCR2 inhibitor MK-0812 or vehicle. (H) Sera from chemotherapy-treated patients and mice induce tumorigenicity. MDA-MB-231 cells pre-treated with human (case T1) or mouse (tumor-free BALB/c) sera for 48 h were injected into the mammary fat pad of NSG mice (n=10) at the indicated numbers. Tumor incidence after 4 weeks was shown. The estimated TIC frequency was estimated by ELDA. *P<0.05, **P<0.01, ***P<0.001 (compared to the corresponding IgG group in C or as indicated).

Article Snippet: The CCL7 mouse ELISA kit was purchased from Cusabio (College Park, MD).

Techniques: Activity Assay, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, Injection

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Chemotherapy induces breast cancer stemness in association with dysregulated monocytosis

doi: 10.1158/1078-0432.CCR-17-2545

Figure Lengend Snippet: Serum CSC-stimulating activity and MCP levels in stratified patients. *

Article Snippet: The CCL7 mouse ELISA kit was purchased from Cusabio (College Park, MD).

Techniques: Activity Assay, Polymerase Chain Reaction

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Chemotherapy induces breast cancer stemness in association with dysregulated monocytosis

doi: 10.1158/1078-0432.CCR-17-2545

Figure Lengend Snippet: (A) Clodronate liposomes or control were injected into BALB/c mice through the tail vein every 2 days starting at 2 days prior to the first chemotherapy treatment (n=3). At day 6 after 3 weeks of chemotherapy, the number and percentage (out of total white blood cells) of monocytes were analyzed by a complete blood count (left). The CD11b+CD115+ monocyte population in the BM was analyzed by flow cytometry (right). (B) ELISA to determine the serum levels of mouse CCL2/7/8 (n=3). (C) ALDEFLUOR assays of MDA-MB-231 cells treated with indicated mouse sera (n=3). (D) Chemotherapy induces expansion of monocytes in the rebound phase. BALB/c mice received 4 times of treatment with DOXO or DTX, or PBS on days 1, 7, 14, and 21 (blue arrowheads). At each indicated time point, complete blood count was conducted to determine the numbers and percentages of various cell populations (n=3). Total BM cell count was also shown. WBC: white blood cells; LY: lymphocytes; MO: monocytes; NE: neutrophils; RBC: red blood cells; PLT: platelets. On days 7 and 14, blood was collected before treatment. (E) BM cells were collected at the indicated time from C57BL/6 mice that have received 1–4 times of treatment with DOXO or DTX, or PBS (treatments given on days 1, 7, 14, and 21). Flow cytometry was performed to determine the population of indicated cell types (n=3). HSC: hematopoietic stem cells; CMP: common myeloid progenitors; GMP: granulocyte-monocyte progenitors; MEP: megakaryocyte-erythrocyte progenitors. (F) Representative flow cytometry plots on day 28 from all three groups in E. *P<0.05, **P<0.01, ***P<0.001 (compared to the corresponding PBS group in D & E or as indicated).

Article Snippet: The CCL7 mouse ELISA kit was purchased from Cusabio (College Park, MD).

Techniques: Liposomes, Control, Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Counting

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Chemotherapy induces breast cancer stemness in association with dysregulated monocytosis

doi: 10.1158/1078-0432.CCR-17-2545

Figure Lengend Snippet: (A & B) Co-cultures were set up using MDA-MB-231 or BT474 BC cells with monocytes isolated from C57BL/6 mice treated with DOXO, DTX, or PBS, or with DOXO/DTX/PBS-pretreated THP-1 cells, in the presence or absence of the CCR2 inhibitor MK-0812 (A) or CCL2/7/8 NAb (B) as described in Materials and Methods. BC cells and monocytes were seeded at a ratio of 1:1 or 1:3. ALDEFLUOR assays (for MDA-MB-231) and ESA+CD44+CD24−/low flow cytometry (for BT474) were performed using BC cells harvested after 48 h of co-culture. (C) MCP secretion by THP-1 and CAF was determined by ELISA of the conditioned media (CM) of 105 THP-1 cells or cancer-activated CAF (pre-treated with the CM from PDX265922 cancer cells) that had been treated with DOXO (125 nM), DTX (4 nM), or PBS for 48 h. (D) THP-1 cells under DOXO/DTX/PBS treatment and mouse monocytes isolated as in A were cultured in the presence or absence of a JNK inhibitor SP600125 (1 μM) for 48 h and analyzed by quantitative RT-PCR using GAPDH/Gapdh for normalization. (E) MDA-MB-231 cells were treated with CCL2/7/8 at the indicated concentrations for 48 h and analyzed by sphere formation assay or ALDEFLUOR assay. (F) PDX265922 cells derived from a primary TNBC were treated with CCL2/7/8 (1 ng/ml) or PBS for 48 h and analyzed by sphere formation assay or ALDEFLUOR assay. (G) CCL2/7/8-treated BT474 cells were analyzed by sphere formation assay or flow cytometry for the ESA+CD44+CD24−/low population. (H) Indicated BC cells were treated with CCL2/7/8 (1 ng/ml) or PBS for 24 h and analyzed by quantitative RT-PCR for indicated stemness-related genes. Data are normalized to GAPDH and compared to the PBS group. (I) Western blot analysis showing stemness-associated gene expression in BC cells treated with CCL2/7/8 or PBS for 24 h. (J) Western blot analysis of MDA-MB-231 cells treated with CCL2/7/8 or PBS in the presence or absence of a γ-secretase inhibitor (GSI) RO4929097 (10 μM). *P<0.05, **P<0.01, ***P<0.001 (compared to the control group in the first column of each group or as indicated).

Article Snippet: The CCL7 mouse ELISA kit was purchased from Cusabio (College Park, MD).

Techniques: Isolation, Flow Cytometry, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR, Tube Formation Assay, Derivative Assay, Western Blot, Gene Expression, Control

Journal: bioRxiv

Article Title: Cigarette smoking upregulates vascular expression of the novel atherosclerosis risk factor ADAMTS-7

doi: 10.1101/2025.10.28.685237

Figure Lengend Snippet: Impact of ADAMTS-7 deficiency on vascular smooth muscle cell supernatants secondary to incubation with CCL17. A . Representative cytokine profiler blots (left) and heatmap visualizing cytokine profiler results. Cytokines displaying a more than 50% difference between the conditions are highlighted in blue. CCL17 was similar between the groups (red). B . Validation of findings from A using enzyme-linked immunosorbent assays (n=6 each). Each symbol indicates one independent experiment. Data are mean and s.e.m. Unpaired t-test. Abbreviations: CCL20 , C-C motif chemokine 20; FC , fold-change; GM-CSF , Granulocyte-Macrophage Colony-Stimulating Factor; HGF, Hepatocyte Growth Factor; ICAM1 , intercellular adhesion molecule 1; IL-1β , Interleukin 1-β; MCP - 3 , Monocyte Chemotactic Protein 3; scr , scramble; TS7 , ADAMTS-7; VEGF , Vascular Endothelial Growth Factor.

Article Snippet: In cell culture supernatants, GM-CSF (DGM00), IL-1β (DLB50), CCL7 (DCC700), VEGF (DVE00), HGF (DHG00B), ICAM1 (DCD540), and CCL20 (DM3A00; all from R&D Systems) were used to quantify cytokine concentrations.

Techniques: Incubation, Biomarker Discovery

Journal: Frontiers in Immunology

Article Title: Inflammatory biomarkers and therapeutic potential of milk exosome-mediated CCL7 siRNA in murine intestinal ischemia-reperfusion injury

doi: 10.3389/fimmu.2024.1513196

Figure Lengend Snippet: Schematic overview of the study design. The workflow includes transcriptome sequencing of intestinal tissues from sham and IIRI mice, bioinformatics analyses (GO, KEGG, PPI, WGCNA, LASSO, SVM-RFE, GSEA, and IPA), validation of identified biomarkers (Ccl7, Cd14, Cxcl1, Hmox1, and Nfkbia) by qPCR and IHC, and evaluation of the therapeutic efficacy of milk exosome-mediated siCCL7 delivery in the IIRI model.

Article Snippet: Serum levels of CCL7 were measured using a mouse CCL7 ELISA kit (MCC070, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Sequencing, Biomarker Discovery, Drug discovery

Journal: Frontiers in Immunology

Article Title: Inflammatory biomarkers and therapeutic potential of milk exosome-mediated CCL7 siRNA in murine intestinal ischemia-reperfusion injury

doi: 10.3389/fimmu.2024.1513196

Figure Lengend Snippet: Screening of biomarkers in IIRI. (A, B) LASSO coefficient profiles and 6-fold cross-validation for optimal tuning parameter (λ) selection using LASSO. (C, D) Estimation of 5-fold cross-validation error and accuracy using support vector machine recursive feature elimination (SVM-RFE). (E) Venn diagram depicting the overlap of 5 genes identified by LASSO and SVM-RFE. (F) ROC curves for Ccl7, Cd14, Cxcl1, Hmox1, and Nfkbia. (G) PCA plot of the biomarkers distinguishing normal and IIRI samples.

Article Snippet: Serum levels of CCL7 were measured using a mouse CCL7 ELISA kit (MCC070, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Selection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Inflammatory biomarkers and therapeutic potential of milk exosome-mediated CCL7 siRNA in murine intestinal ischemia-reperfusion injury

doi: 10.3389/fimmu.2024.1513196

Figure Lengend Snippet: IPA analysis. (A) Significant classical pathway enrichment results. (B) Molecular interaction network of Ccl7, Cd14, Hmox1, and Nfkbia. (C) Regulatory relationship between biomarkers and reperfusion injury. (D) Regulatory relationship of the ‘Pathogen Induced Cytokine Storm Signaling’ pathway (highest Z-score). (E) Interaction network between biomarkers and cytokines.

Article Snippet: Serum levels of CCL7 were measured using a mouse CCL7 ELISA kit (MCC070, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques:

Journal: Frontiers in Immunology

Article Title: Inflammatory biomarkers and therapeutic potential of milk exosome-mediated CCL7 siRNA in murine intestinal ischemia-reperfusion injury

doi: 10.3389/fimmu.2024.1513196

Figure Lengend Snippet: Validation of biomarker expression. (A) Schematic representation of biomarker expression validation in mouse tissue samples by RT-qPCR. (B) Scatter plots showing individual data points of qRT-PCR validation for Ccl7, Cd14, Cxcl1, Hmox1, and Nfkbia expression in IIRI (n=10) vs. control samples (n=10), with median and interquartile range. (C, D) Quantification of immunohistochemical staining shown as scatter plots with individual values (n=4 per group) for Ccl7, Cd14, Cxcl1, Hmox1, and Nfkbia expression in IIRI vs. control samples, with mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control.

Article Snippet: Serum levels of CCL7 were measured using a mouse CCL7 ELISA kit (MCC070, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Control, Immunohistochemical staining, Staining

Journal: Frontiers in Immunology

Article Title: Inflammatory biomarkers and therapeutic potential of milk exosome-mediated CCL7 siRNA in murine intestinal ischemia-reperfusion injury

doi: 10.3389/fimmu.2024.1513196

Figure Lengend Snippet: M-Exo/siCCL7 treatment attenuates intestinal ischemia-reperfusion injury. (A) Transmission electron microscopy images depicting the morphology of milk-derived exosomes (M-Exos) before and after electroporation. (B) Serum CCL7 levels in the sham, IIRI, and M-Exo/siCCL7-treated (1×10 11 particles/mouse, once daily for 2 days) groups. (C) Representative gross anatomical photographs of jejunum before (normal pink coloration) and after (dark red/purple with visible edema) ischemia-reperfusion injury, showing macroscopic changes in intestinal appearance. (D) Representative histological images (H&E staining) of intestinal tissues from the three groups. (E) Chiu’s score for the assessment of intestinal injury severity. (F) Serum diamine oxidase activity as a marker of intestinal barrier function. (G) Serum lactate dehydrogenase levels as an indicator of tissue damage. Data are presented as mean ± SD; ** p < 0.01, *** p < 0.001.

Article Snippet: Serum levels of CCL7 were measured using a mouse CCL7 ELISA kit (MCC070, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Transmission Assay, Electron Microscopy, Derivative Assay, Electroporation, Staining, Activity Assay, Marker