Journal: Frontiers in Immunology

Article Title: The role of the C5a-C5aR pathway in iron metabolism and gastric cancer progression

doi: 10.3389/fimmu.2024.1522181

Figure Lengend Snippet: C5a-C5aR pathway activation in gastric cancer. (A) Quantification of C5AR1 mRNA expression in tumor tissues and normal gastric tissues in the STAD cohort and GTEx. Data were analyzed using the GEPIA2 database. (B) The assessment of overall survival (OS) in patients correlated with varying levels of C5aR1 expression was performed using the online Kaplan-Meier plotter tool. (C) C5aR1 expression in GC and paired adjacent normal tissues were determined by IHC (representative panels scale bar, 50 μm), and statistically analyzed the percentage of positive areas (n=3). (D) Protein levels of C5aR1 in GC and paired adjacent normal tissues were examined using western blot analysis results were normalized to the internal control β-actin, and the relative protein expression levels were statistically analyzed (n=4). Statistical analyses were executed using GraphPad Prism 9 software, with comparisons made via unpaired two-tailed Student’s t-test. Data are presented as mean ± standard deviation (SD). Significance levels were set at p<0.05 (denoted by *), p<0.01 (denoted by **).

Article Snippet: Membranes were incubated with a monoclonal rabbit anti-human/mouse C5aR antibody (1:1000, Proteintech, Cat#21316-1-AP, China), LCN2 antibody (1:5000, Proteintech, Cat#26991-1-AP, China), GRP78 antibody (1:1000, Beyotime Biotechnology, Cat#AF0171, China), CHOP antibody (1:1000, Beyotime Biotechnology, Cat#AF6684, China), β-actin antibody (1:1000, Beyotime Biotechnology, Cat#AF5003, China), or α-tubulin antibody (1:1000, Beyotime Biotechnology, Cat#AF5012 China) and the respective HRP-conjugated secondary antibodies (1:1000; Beyotime Biotechnology, Cat#A0208, China) were used to detect the target proteins.

Techniques: Activation Assay, Expressing, Western Blot, Control, Software, Two Tailed Test, Standard Deviation

Journal: Frontiers in Immunology

Article Title: The role of the C5a-C5aR pathway in iron metabolism and gastric cancer progression

doi: 10.3389/fimmu.2024.1522181

Figure Lengend Snippet: C5a-C5aR pathway enhanced gastric cancer progression by facilitating iron transfer from macrophages to cancer cells. THP-1 cells were cultured in 6-well plates, with 1×10 6 cells per well, pre-treated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 24h. Then, in certain experiments, cells were pretreated with 10 nM C5aR antagonist (C5aRA) or 10μM LCN2 inhibitor, ZINC00640089 for 3 hours, followed by stimulation with 100 ng/mL recombinant human C5a protein. After a 6-hours incubation, the treatment medium was discarded and replaced with fresh 1640 medium. Subsequently, distinct THP-1 macrophage culture supernatants were collected and utilized to cultivate gastric cancer cells for assessing cancer cells viability or iron content. (A) Cell viability of gastric cancer cells including BGC-823 cells, HGC-27 cells, AGS cells culturing in different culture medium (Control, C5a or C5a+C5aRA) was examined using CCK8 assay (n=4). (B) Intracellular iron content in gastric cancer cells was tested by Iron Content Assay Kit (n=3~5). (C) Cell viability of gastric cancer cells including BGC-823 cells, HGC-27 cells, AGS cells culturing in different culture medium (Control, C5a or C5a+ZINC00640089) was examined using CCK8 assay (n=4). Statistical analyses were executed using GraphPad Prism 9 software, with comparisons made via one-way ANOVA test. Data are presented as mean ± standard deviation (SD). Significance levels were set at p<0.05 (denoted by *), p<0.01 (denoted by **), p<0.001 (denoted by ***), and p<0.0001 (denoted by ****).

Article Snippet: Membranes were incubated with a monoclonal rabbit anti-human/mouse C5aR antibody (1:1000, Proteintech, Cat#21316-1-AP, China), LCN2 antibody (1:5000, Proteintech, Cat#26991-1-AP, China), GRP78 antibody (1:1000, Beyotime Biotechnology, Cat#AF0171, China), CHOP antibody (1:1000, Beyotime Biotechnology, Cat#AF6684, China), β-actin antibody (1:1000, Beyotime Biotechnology, Cat#AF5003, China), or α-tubulin antibody (1:1000, Beyotime Biotechnology, Cat#AF5012 China) and the respective HRP-conjugated secondary antibodies (1:1000; Beyotime Biotechnology, Cat#A0208, China) were used to detect the target proteins.

Techniques: Cell Culture, Recombinant, Incubation, Control, CCK-8 Assay, Software, Standard Deviation

Journal: Frontiers in Immunology

Article Title: The role of the C5a-C5aR pathway in iron metabolism and gastric cancer progression

doi: 10.3389/fimmu.2024.1522181

Figure Lengend Snippet: C5a-C5aR pathway promoted macrophages polarization to M2 phenotype in vitro . THP-1 cells were cultured in 6-well plates, with 1×10 6 cells per well, pre-treated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 24h. While RAW264.7 cells were cultured in 6-well plates, with 5×10 5 cells per well. Then both cell types were stimulated with 100 ng/mL C5a for 48 hours. In certain experiments, cells were pretreated with 10 nM C5aRA for 3 hours prior to C5a stimulation. After the 48-hour incubation period, cell samples were harvested for quantitative RT-PCR analysis. (A) Relative gene expression of CCR7 and CD206 in THP-1 cell treated with different group (Control, C5a or C5a+C5aRA) were examined by QRT-PCR (n=3). (B) Relative gene expression of CCL3 and CD206 in RAW264.7 cell treated with different group (Control, C5a or C5a+C5aRA) were examined by QRT-PCR (n=3). Statistical analyses were executed with one-way ANOVA test. Data are presented as mean ± standard deviation (SD). Significance levels were set at p<0.01 (denoted by **), p<0.001 (denoted by ***), and p<0.0001 (denoted by ****).

Article Snippet: Membranes were incubated with a monoclonal rabbit anti-human/mouse C5aR antibody (1:1000, Proteintech, Cat#21316-1-AP, China), LCN2 antibody (1:5000, Proteintech, Cat#26991-1-AP, China), GRP78 antibody (1:1000, Beyotime Biotechnology, Cat#AF0171, China), CHOP antibody (1:1000, Beyotime Biotechnology, Cat#AF6684, China), β-actin antibody (1:1000, Beyotime Biotechnology, Cat#AF5003, China), or α-tubulin antibody (1:1000, Beyotime Biotechnology, Cat#AF5012 China) and the respective HRP-conjugated secondary antibodies (1:1000; Beyotime Biotechnology, Cat#A0208, China) were used to detect the target proteins.

Techniques: In Vitro, Cell Culture, Incubation, Quantitative RT-PCR, Expressing, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: The role of the C5a-C5aR pathway in iron metabolism and gastric cancer progression

doi: 10.3389/fimmu.2024.1522181

Figure Lengend Snippet: C5a-C5aR promoted LCN2 expression via activation of ER stress in macrophages. PMA pre-treated THP-1 cells and RAW264.7 cells were stimulated with 100 ng/mL C5a for 48 hours. In certain experiments, cells were pretreated with 10 nM C5aRA or 250 ng/mL ER stress antagonist, 4-Phenylbutyric acid (4-PBA). After the 48-hour incubation period, cell samples were harvested for subsequent western blotting and quantitative RT-PCR analysis to detect LCN2 and ER stress related markers (CHOP, GRP78 and activating transcription factor 4, ATF4). (A) Relative gene expression of LCN2, ATF4, CHOP and GRP78 in THP-1 cell treated with different group (Control, C5a or C5a+C5aRA) were examined by QRT-PCR (n=3). (B) Protein levels of GRP78, CHOP and LCN2 in THP-1 cell treated with different group (Control, C5a or C5a+C5aRA) were examined using western blot analysis. (C) Relative gene expression of ATF4, CHOP, GRP78 and LCN2 in THP-1 cell treated with different group (Control, C5a or C5a+4-PBA) were examined by QRT-PCR (n=3). (D) Protein levels of GRP78, CHOP and LCN2 in THP-1 cell (Control, C5a or C5a+4-PBA) were examined using western blot analysis. (E) Relative gene expression of ATF4, CHOP, GRP78 and LCN2 in RAW264.7 cell treated with different group (Control, C5a or C5a+C5aRA) were examined by QRT-PCR (n=3). (F) Protein levels of GRP78, CHOP and LCN2 in RAW264.7 cell treated with different group (Control, C5a or C5a+C5aRA) were examined using western blot analysis. (G) Relative gene expression of ATF4, CHOP, GRP78 and LCN2 in RAW264.7 cell treated with different group (Control, C5a or C5a+4-PBA) were examined by QRT-PCR (n=3). (H) Protein levels of GRP78, CHOP and LCN2 in RAW264.7 cell treated with different group (Control, C5a or C5a+4-PBA) were examined using western blot analysis. Statistical analyses were executed with one-way ANOVA test. Data are presented as mean ± standard deviation (SD). Significance levels were set at p<0.05 (denoted by *), p<0.01 (denoted by **), p<0.001 (denoted by ***), and p<0.0001 (denoted by ****).

Article Snippet: Membranes were incubated with a monoclonal rabbit anti-human/mouse C5aR antibody (1:1000, Proteintech, Cat#21316-1-AP, China), LCN2 antibody (1:5000, Proteintech, Cat#26991-1-AP, China), GRP78 antibody (1:1000, Beyotime Biotechnology, Cat#AF0171, China), CHOP antibody (1:1000, Beyotime Biotechnology, Cat#AF6684, China), β-actin antibody (1:1000, Beyotime Biotechnology, Cat#AF5003, China), or α-tubulin antibody (1:1000, Beyotime Biotechnology, Cat#AF5012 China) and the respective HRP-conjugated secondary antibodies (1:1000; Beyotime Biotechnology, Cat#A0208, China) were used to detect the target proteins.

Techniques: Expressing, Activation Assay, Incubation, Western Blot, Quantitative RT-PCR, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: The role of the C5a-C5aR pathway in iron metabolism and gastric cancer progression

doi: 10.3389/fimmu.2024.1522181

Figure Lengend Snippet: C5aRA inhibited gastric cancer progression in BGC-823 xenograft mouse model. BALB/C female nude mice were 6 weeks old at the experiment’s onset. They were randomly assigned to two groups (n=6) and received subcutaneous transplantation of BGC-823 cells (~2×10 6 BGC-823 cells into the subcutaneous on back). After tumor formation within three days, for C5aRA group, mice were treated with the C5aR antagonist (C5aRA) (1 mg/kg, intravenously via the tail), every two days, with tumor volume being monitored. While for control group, mice were treated with 0.9% NaCl. (A, B) Tumor size, weight and volume of BGC-823 xenograft mice treated with or without C5aRA. (C) Body weight of BALB/C nude mice treated with or without C5aRA. Statistical analyses were executed with unpaired two-tailed Student’s t-test. Data are presented as mean ± standard deviation (SD). Significance levels were set at p<0.001 (denoted by ***), and p<0.0001 (denoted by ****).

Article Snippet: Membranes were incubated with a monoclonal rabbit anti-human/mouse C5aR antibody (1:1000, Proteintech, Cat#21316-1-AP, China), LCN2 antibody (1:5000, Proteintech, Cat#26991-1-AP, China), GRP78 antibody (1:1000, Beyotime Biotechnology, Cat#AF0171, China), CHOP antibody (1:1000, Beyotime Biotechnology, Cat#AF6684, China), β-actin antibody (1:1000, Beyotime Biotechnology, Cat#AF5003, China), or α-tubulin antibody (1:1000, Beyotime Biotechnology, Cat#AF5012 China) and the respective HRP-conjugated secondary antibodies (1:1000; Beyotime Biotechnology, Cat#A0208, China) were used to detect the target proteins.

Techniques: Transplantation Assay, Control, Two Tailed Test, Standard Deviation

Journal: Frontiers in Immunology

Article Title: The role of the C5a-C5aR pathway in iron metabolism and gastric cancer progression

doi: 10.3389/fimmu.2024.1522181

Figure Lengend Snippet: The mechanism by which C5a-C5aR pathway accelerated the progression of gastric cancer through enhancing iron transfer from macrophages to cancer cells. The C5a-C5aR pathway enhances macrophage polarization towards the M2 phenotype while simultaneously increasing the expression of the iron-transporter protein LCN2 through ER stress activation. Consequently, gastric cancer cells acquire sufficient iron from M2 macrophages within the tumor microenvironment to sustain their proliferative capacity.

Article Snippet: Membranes were incubated with a monoclonal rabbit anti-human/mouse C5aR antibody (1:1000, Proteintech, Cat#21316-1-AP, China), LCN2 antibody (1:5000, Proteintech, Cat#26991-1-AP, China), GRP78 antibody (1:1000, Beyotime Biotechnology, Cat#AF0171, China), CHOP antibody (1:1000, Beyotime Biotechnology, Cat#AF6684, China), β-actin antibody (1:1000, Beyotime Biotechnology, Cat#AF5003, China), or α-tubulin antibody (1:1000, Beyotime Biotechnology, Cat#AF5012 China) and the respective HRP-conjugated secondary antibodies (1:1000; Beyotime Biotechnology, Cat#A0208, China) were used to detect the target proteins.

Techniques: Expressing, Activation Assay

Journal: Frontiers in Immunology

Article Title: The role of the C5a-C5aR pathway in iron metabolism and gastric cancer progression

doi: 10.3389/fimmu.2024.1522181

Figure Lengend Snippet: Schematic summarizing the proposed mechanism of the C5a-C5aR pathway’s role in gastric cancer progression.

Article Snippet: Membranes were incubated with a monoclonal rabbit anti-human/mouse C5aR antibody (1:1000, Proteintech, Cat#21316-1-AP, China), LCN2 antibody (1:5000, Proteintech, Cat#26991-1-AP, China), GRP78 antibody (1:1000, Beyotime Biotechnology, Cat#AF0171, China), CHOP antibody (1:1000, Beyotime Biotechnology, Cat#AF6684, China), β-actin antibody (1:1000, Beyotime Biotechnology, Cat#AF5003, China), or α-tubulin antibody (1:1000, Beyotime Biotechnology, Cat#AF5012 China) and the respective HRP-conjugated secondary antibodies (1:1000; Beyotime Biotechnology, Cat#A0208, China) were used to detect the target proteins.

Techniques:

Journal: iScience

Article Title: C5aR1-positive adipocytes mediate non-shivering thermogenesis in neonatal mice

doi: 10.1016/j.isci.2024.111261

Figure Lengend Snippet:

Article Snippet: PE anti-mouse CD88 (C5aR) Antibody , Biolegend , RRID: AB_2243735.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Sequencing, Software

Journal: Frontiers in Immunology

Article Title: A novel hollow iron nanoparticle system loading PEG-Fe 3 O 4 with C5a receptor antagonist for breast cancer treatment

doi: 10.3389/fimmu.2024.1466180

Figure Lengend Snippet: C5a-C5aR pathway activation in human breast cancer and its role in ferroptosis resistance and breast cancer progression in 4T-1 xenograft mouse model. (A) , the expression of C5b-9 and C5aR in BC tumoral tissues (T) and BC-adjacent non-tumoral tissues (N) of patients with different clinical stages were evaluated by immunohistochemistry. Statistical analyses of the difference of C5b-9 and C5aR expression between different groups were presented in the right. (B) Immunoblotting of NRF2 in 4T-1 cells after treatment with 480 ng/mL C5a or C5a plus 10 nM C5aRA for 48h. (C-E) , Relative cell viability (C) , ROS level (D) , NRF2 and GPX4 expression analysis (E) of 4T-1 cells after with different treatment. 1) control group; 2) the 4T-1 cells were treated with 2ug/mL Sorafenib for 48 hours; 3) the 4T-1 cells were stimulated with 2ug/mL Sorafenib and 480 ng/mL C5a; 4) before stimulation with 480 ng/mL C5a and 2ug/mL Sorafenib, these cells were pre-treated with 10 nM C5aRA for 1 hour. Mice were implanted with 1.0 × 10 6 4T-1 cells in the mammary fat pad. The tumor growth was measured by representative tumor images (F) , tumor weight (G) , and tumor size (H) (n=5). (I-K) , Immunohistochemistry and statistical analyses of the expression of GPX4 and NRF2 in tumor tissues. Statistical differences are assessed by student’s t-test. Data were mean ± s.e.m. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.001.

Article Snippet: C5aR-KO mice (BALB/C background) were purchased from Jackson Laboratory (Ellsworth, Maine, US).

Techniques: Activation Assay, Expressing, Immunohistochemistry, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: A novel hollow iron nanoparticle system loading PEG-Fe 3 O 4 with C5a receptor antagonist for breast cancer treatment

doi: 10.3389/fimmu.2024.1466180

Figure Lengend Snippet: C5a-C5aR pathway promoted macrophage polarization to M2 phenotype. (A-D) , RAW264.7 and THP-1 cells were pre-incubated with 10 nM C5aRA for 60 min before exposure to 480 ng/mL C5a for 48h and then the expressions of M1 associated gene (iNOS, CCR7) and M2 associated gene (Arg1, CCL17) were measured by RT-qPCR. The expression level of mRNA was normalized to β-actin (n=3). (E, F) , Flow cytometry analysis of IA-IE and CD206 expression in tumor-associated macrophages, representative flow cytometry diagram (E) and the ratio of M1/M2 macrophages (F) . Statistical differences are assessed by student’s t-test. Data were mean ± s.e.m. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: C5aR-KO mice (BALB/C background) were purchased from Jackson Laboratory (Ellsworth, Maine, US).

Techniques: Incubation, Quantitative RT-PCR, Expressing, Flow Cytometry