Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: C3a-C3aR signaling promotes breast cancer lung metastasis via modulating carcinoma associated fibroblasts

doi: 10.1186/s13046-019-1515-2

Figure Lengend Snippet: Lung metastasis were reduced in C3aR deficiency breast tumor-bearing mice. a-f 4 T1 cells were orthotopically injected into WT ( n = 7) or C3aR −/− ( n = 6) mice. Mice were anesthetized and sacrificed 28 days post tumor inoculation. a Lung metastases burden in the WT and C3aR −/− tumor-bearing mice, and scans of H&E stained sections of the lungs of WT and C3aR −/− mice with breast tumors. b Quantification of lung metastases. c-d Expression of E-Cadherin and Vimentin in the primary tumor tissue detected by immune-fluorescence, and western blot assay ( e-f ). (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: The primary antibody against mouse C3aR (14D4, Hycult biotech, 1:50 diluted) and rabbit anti-mouse α smooth muscle actin (E184, Abcam, 1:500 diluted) were incubated at 4 °C overnight.

Techniques: Injection, Staining, Expressing, Fluorescence, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: C3a-C3aR signaling promotes breast cancer lung metastasis via modulating carcinoma associated fibroblasts

doi: 10.1186/s13046-019-1515-2

Figure Lengend Snippet: The reduced lung metastasis of breast cancer in C3aR−/− mice is associated with the altered function of CAFs. a-b 4 T1 cells were orthotopically injected into WT or C3aR −/− mice. Mice were sacrificed 16 days post tumor inoculation and the tumors were harvested. RNA-sequencing was conducted. a Gene ontology enrichment analysis of WT and C3aR −/− 4 T1 tumors. Enrichment scatter plot in which the abscissa is the GeneRatio (the ratio of the number of differential genes on the GO pathway to the total number of differential genes). b Heat map of mRNA expression for differential extracellular matrix related genes. c qPCR analysis of mRNA levels of CAF markers ( ACTA-2, PDGFRα ) and functional cytokines ( TGFβ, HGF, CXCL12, and VEGF-A ) of CAFs, isolated from WT or C3aR −/− tumors (* P < 0.05,** P < 0.01)

Article Snippet: The primary antibody against mouse C3aR (14D4, Hycult biotech, 1:50 diluted) and rabbit anti-mouse α smooth muscle actin (E184, Abcam, 1:500 diluted) were incubated at 4 °C overnight.

Techniques: Injection, RNA Sequencing Assay, Expressing, Functional Assay, Isolation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: C3a-C3aR signaling promotes breast cancer lung metastasis via modulating carcinoma associated fibroblasts

doi: 10.1186/s13046-019-1515-2

Figure Lengend Snippet: C3aR signaling promotes the pro-meatastatic function of CAF. a CAFs were sorted by Flow cytometry as PDGFRa + F4/80 − cells of 4 T1 tumor tissues from WT or C3aR −/− mice. Immunofluorescence analysis of C3aR expression in WT and C3aR −/− CAF. b The migratory properties of 4 T1 cells cultured with WT and C3aR −/− CAFs detected in scratch assays (* P < 0.05). c-d The migration and invasive capability of 4 T1/EO771 tumor cells co-cultured with WT CAFs and C3aR −/− CAFs (* P < 0.05). CAF obtained from 4 T1 tumor-bearing WT mice were stimulated with rmC3a(0.5μg/ml) for 24 h, the expression of α-SMA was analyzed by immunofluorescence ( e ) and western blotting assay ( f ). The software ImageJ was used to qualify the signal intensities of the western blot, and the ratio of α-SMA/β-actin is presented. g Quantitative PCR analysis of mRNA level of CAF markers ( PDGFRA, FAP, ACTA2 ) and functional cytokines ( TGF-β1, HGF, VEGFA ) in treated or untreated CAFs was performed. h-i 4 T1 cells were co-injected with CAFs derived from WT or C3aR −/− mice in the mammary fat pad. The number of 4 T1 lung metastasis tumor burden was calculated after 28 days. Data are expressed as the mean ± SEM. (* p < 0.05,** p < 0.01,*** p < 0.001)

Article Snippet: The primary antibody against mouse C3aR (14D4, Hycult biotech, 1:50 diluted) and rabbit anti-mouse α smooth muscle actin (E184, Abcam, 1:500 diluted) were incubated at 4 °C overnight.

Techniques: Flow Cytometry, Immunofluorescence, Expressing, Cell Culture, Migration, Western Blot, Software, Real-time Polymerase Chain Reaction, Functional Assay, Injection, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: C3a-C3aR signaling promotes breast cancer lung metastasis via modulating carcinoma associated fibroblasts

doi: 10.1186/s13046-019-1515-2

Figure Lengend Snippet: C3aR signaling shifts CAF function via PI3K activation. a Dynamic up-regulation of phosphorylated AKT (Ser473) in CAFs stimulated with rmC3a at different time points. b Dynamic phosphorylated AKT (Ser473) expression of C3aR −/− CAFs treated with rmC3a at different time points. c rmC3a-induced phosphorylated AKT (Ser473) expression of WT CAFs after pretreatment of C3aR antagonist (SB290157), PI3K inhibitor (LY294002), or C3aR antibodies (14D4). d α-SMA expression of C3a-induced CAFs blocked with C3aR antagonist, PI3K inhibitor, or C3aR antibodies. (* p < 0.05) ( e ) Transwell assay demonstrated that rmC3a treated CAF facilitate migration capacity of 4 T1 cells, and can be inhibited by C3aR antagonist and PI3K inhibitor. (** p < 0.01,*** p < 0.001,**** p < 0.0001) ( f )TGF-ß1 secretion of CAF stimulated by rmC3a after blockade with C3aR antagonist or PI3K inhibitor (LY294002), compared with medium only. (* p < 0.05)

Article Snippet: The primary antibody against mouse C3aR (14D4, Hycult biotech, 1:50 diluted) and rabbit anti-mouse α smooth muscle actin (E184, Abcam, 1:500 diluted) were incubated at 4 °C overnight.

Techniques: Activation Assay, Expressing, Transwell Assay, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: C3a-C3aR signaling promotes breast cancer lung metastasis via modulating carcinoma associated fibroblasts

doi: 10.1186/s13046-019-1515-2

Figure Lengend Snippet: Targeting C3aR Inhibits Lung Metastasis of Breast Cancer. a 4 T1 cells were inoculated into the third mammary fat pad of mice on day 0. C3aRA or PBS were injected i.p.10 mg/kg bodyweight from day 1, twice a week. b 28 days after tumor inoculation, lung metastasis burden in the C3aRA treated ( n = 7) or PBS injected ( n = 6) tumor-bearing mice. c-d Vimentin and E-Cadherin expression were detected by immunofluorescence assay. Data shown as three repeated experiments. e 4–5-week-old female MMTV-PyMT mice were intraperitoneally injected with (10 mg/Kg body weight) C3aR antagonist twice a week. f The experimental mice were euthanized at 16 weeks of age, the lungs were inflated with India ink, and the white nodules visible in the lungs represented the metastases burden. (* P < 0.05). g Kaplan–Meier curve for survival of human triple negative breast cancer patients from the TCGA-BRAC data sets. Data were obtained from the PROGgeneV2 web application and C3aR1 and PDGFA gene co-expression was divided into high and low at the median expression. n represents the number of patients at day 0. P -values less than 0.05 were considered significant

Article Snippet: The primary antibody against mouse C3aR (14D4, Hycult biotech, 1:50 diluted) and rabbit anti-mouse α smooth muscle actin (E184, Abcam, 1:500 diluted) were incubated at 4 °C overnight.

Techniques: Injection, Expressing, Immunofluorescence