| The complement system is an important part of innate im-munity,which consists of a series of serine proteases encoded by the same ancestral gene as the coagulation pro-tein.In recent years,the role of the complement system has shifted from a blood-based antimicrobial infection to a wide range of immune regulation and tissue homeostasis regulation.In addition to participating in innate im-munity,many complement proteins facilitate cross-talk be-tween the immune cells and tumor cells in the tumor microenvironment?TME?.Complement C3 is a central component in complement activation,activation of C3 results in the generation of C3a,which is a prominent tumor-promoting factor in TME.Numerous studies have shown that myeloid cells?including monocytes,macrophages,DCs?and T cells express C3aR.In addition,activated astrocytes,endothelial cells,epithelial cells,smooth muscle cells,renal tubular epithelial cellsare also regulated by the C3a-C3aR axis.Previously,we and others demonstrated that C3aR signaling promotes tumor growth by promoting immune inhibition.However,the role of C3a-C3a R signaling in breast cancer metas-tasis remains to be explored.Published studies suggested that C3a-C3aR signaling contributes to the formation of pul-monary fibrosis and renal fibrosis,characterized by activation of fibroblasts,which reveal the potential of C3a-C3aR axis in promoting metastasis via regulating fibroblasts in TME.A growing body of evidence suggests that tumor me-tastasis is not only dependent on tumor cells themselves,but is also regulated by the TME.Carcinoma associated fibroblasts?CAFs?are the largest populations of tumor cells which accumulate in TME?e.g.breast cancer,hepatocellular carcinoma?and promote cancer metastasis through multiple pathways.Manipulating the function of CAFs is a promising strategy to treat cancer.However,whether and how C3a-C3aR signaling is involved in the regulation of CAFs remain largely unknown.In the current study,we demonstrated that C3a promotes tumor cell metastasis by modulating CAFs.C3a binds to its cognate receptor C3aR to activate PI3K/AKT signaling,which resulted in CAFs activation.More-over,in human invasive breast cancers,C3expression is positively correlated with expression of CAFs activation markers and functional effectors.Genetic or pharmacological blockade of C3aR signaling effectively inhibits lung metastasis of breast cancer.Our data demonstrated that targeting C3aR might be an effective strategy in tumor metastasis control.Methods:1.Effect of complement C3a/C3aR signal deficiency on lung metastasis of breast cancer?1?4T1 cells were orthotopicaly injected?a mouse breast cancer cell line?,which closely mimics stage IV of human breast cancer,into mammary fat pad of Balb/c C3aR+/+mice and C3aR-/-mice,respectively?2?Tumor sizes were monitored,tumor growth curve of the two groups of mice was drawn?3?Mice were sacrifice 16 or 28 days after tumor inoculation and tumor were removed and weighed.The weight of tumors from the WT and C3aR-/-mice was compared.?4?28 days after inoculation,the lungs of mice were perfused and the number of metastatic nodules in the lung was compared.?5?Cell suspension was obtained by tumor digestion and stained by FACs.The percentage of Ki67 of tumor cells was compared between the two groups.?6?Frozen sections of mammary tissue from WT/C3aR-/-mice were stained with E-Cadherin mouse monoclonal antibodies?AF0138,Beyo-time,1:50 diluted?and Vimentin Mouse Monoclonalantibodies by tissue immunofluorescence staining.2.Complement C3a/C3aR signaling regulates CAF function?1?4T1 cells were orthotopically injected into WT or C3aR-/-mice.Mice were sacrificed 16 days post tumor inoculation and tumors were harvested.RNA-sequencing was conducted.Gene ontology enrichment analysis of WT and C3aR-/-4T1 tumors.?2?16 days after inoculation,CAF cells were isolated from tumor tissue of each mice by FACs as PDGFR+F4/80-.?3?Cell suspension was obtained by tumor digestion and stained by FACs.The percentage of Ki67 in PDGFR+F4/80-cells was compared between the two groups.?4?CAF cells cultured in vitro were stained with immunofluorescence to detect the expression of C3aR on the surface of CAF cells.?5?CAF cells were obtained from 4T1 tumors of WT/C3aR-/-mice by flow cytometry sorting as PDGFR+F4/80-.The expression of ACTA2,CXCL12,HGF,TGF-?1,HGF,PDGFRa,FAP were detected by qRT-PCR.?6?According to the RNA-Seq database,the correlation between the expression of C3in human breast cancer tissues and the functional molecules of CAF cells was analyzed.?7?To compare the migration and invasiveness of 4T1 tumor cells,transwell with CAF cells from WT/C3aR-/-mice.Scratch test was used to detect the movement ability of 4T1 cells under the action of two kinds of CAF cells.?8?4T1 cells alone,or 4T1 cells with CAF cells from Balb/c or C3aRKO mice were orthotopic inoculated into the breast fat pad of nude mice at a ratio of 1:10.The growth of the tumor was monitored and the growth curve was drawn.?9?28 days after inoculation,the mice were removed and weighed,and the tumor weights of the three groups were compared.The metastatic foci in lungs of mice were observed and the number of metastatic foci in lungs of the two groups was counted.3.Complement C3a/C3aR axis converts fibroblasts to CAFs via activating PI3K signaling.?1?Mammary fibroblasts isolated from BALB/C?WT?or C3aR-/-mice were sorted by flow cytometry and cultured routinely?2?Fibroblasts obtained from WT mice were stimulated by recombinant C3a for 48hours,and the expression of alpha-SMA was detected by Western blot.?3?Fibroblasts were stimulated by recombinant C3a for 48 hours,and the levels of TGF-beta in the supernatant were detected by ELISA assay.?4?Fibroblasts derived from WT or C3aR-/-were stimulated by recombinant C3a in mice.Proteins were collected at corresponding time points and the expressions of AKT and p-AKT were detected by Western Blot method.?5?Fibroblasts were stimulated by recombinant C3a in mice,or C3a with C3aRA treatment,or anti-C3aR antibody?Hycult 14D4?pre-treatement,or PI3K inhibitor,and the expression of p-AKT and AKT were detected by Western Blot method.?6?After pretreated with Ly294002,the fibroblasts were stimulated by recombinant C3a in mice.TGF-beta levels in the supernatant of the cells were detected by ELISA.4.Tumor-cell derived C3 promotes breast cancer metastasis?1?Identification of 4T1-C3KO cell lines by ELISA method.?2?4T1 or 4T1-C3KO cells were inoculated into the mammary fat pad of Balb/c mice,the growth of the tumor volume were monitored.?3?4T1 or 4T1-C3KO cells were inoculated in the third mammary fat pad of t of nude mice?T/B cell deletion?,tumor growth was monitored.?4?After orthopotic inoculation of tumor cells into nude mice,the mice were sacrificed on the 28th day and the tumor weight was compared.?5?Compared the number of lung tumor metastases in two groups of mice.?6?4T1 or 4T1-C3KO cells were cultured in vitro,and CCK-8 assay was used to detect the proliferation of two kinds of cells in vitro.5.C3aR will become a new target for breast cancer anti-metastasis therapy.?1?Balb/c mice were inoculated with 4T1 cells in situ and weighed.The mice were divided into two groups and injected with PBS,SB290157?C3aR antagonist;10mg/kg body weight,twice a week?.The growth of the tumor was observed and the weight of the tumor was compared after 28 days.?2?Balb/c mice were inoculated with 4T1 cells in situ and weighed.The mice were divided into two groups.PBS,SB290157 trifluoride salt?C3aR antagonist;10mg/kg body weight,twice a week?were injected into the lungs of Balb/c mice for 28 days.The lungs of the two groups were perfused and fixed with Indian ink.The number of lung metastasis of two groups was compared.?3?Six-week-old PyMT mice were weighed and divided into two groups,which were injected with PBS or C3aRA?10mg/kg body weight twice a week?.The number of lung metastasis of the two groups was compared at 16 weeks.?4?ProGgene V2 was used to analyze the effect of high expression of C3AR1 and PDGFA on prognosis,survival and metastasis of breast cancer.Tumor sizes were measured three times/week and tumor volume was calculated.Tumor volume was calculated by the following formula:volume=0.5×?width?2×length.For the co-implantation assay,a total of5×104 4T1 cells mixed with 2.5×105 CAFs and co-injected into the third mammary fat pad of4-6-week-old female Balb/c nude mice.Results1.C3aR deficiency reduced metastasis of breast cancer to the lungs?1?The 4T1 tumor growth and size in C3aR-/-mice were similar with those in WT mice.?2?The proliferation of 4T1 cells was not affected by C3a/C3aR deficiency in host mice?3?Complement C3a/C3aR signal enhanced the EMT of tumor cell?4?The number of lung metastasis decreased significantly in C3aR-/-mice compared with the WT mice.2.Involvement of CAFs in C3aR mediated breast cancer lung metastasis?1?Tumor tissue from C3a R-/-mice displayed significant differences in gene expression profiles compared with that of WT tumors.genes associated with extracellular matrix components were significantly downregulated in tumors isolated from C3aR-/-mice than those from WT mice?2?The percentage of CAF cells?PDGFR+F4/80-?in Living cells in tumor tissues of C3aR-/-was similar with that in wild type mice.Complement C3a/C3aR signal deficiency does not affect the ability of CAF cells to promote tumor growth.?3?CAF cells isolated from 4T1 orthotopic tumor,expressed C3aR on the surface.?4?Compared with WT mice,the expression of CAF cell phenotype?acta2,FAP,PDGFRa?and functional molecule?CXCL12,HGF,TGF-??in C3aRKO mice decreased significantly.?5?In the human breast cancer RNA-Seq database?WWW.cbioportal.org?,the expression of C3 is positively correlated with the expression of acta2,FAP,PDGFRa.?6?The CAF cells of C3aR-/-mice lose the function of promoting tumor cells?migration,invasion??7?Complement C3a/C3aR signal deficiency attentuent the ability of CAF cells to promote lung metastasis of 4T1 tumor cells.3.PI3K-AKT signaling is involved in C3aR signaling-driven CAFs activation?1?Mouse recombinant C3a protein promotes the expression of marker in fibroblasts.?2?Mouse recombinant C3a protein promotes the expression of functional related molecules in fibroblasts.?3?AKT can be phosphorylated after stimulation with recombinant C3a in mice.?4?Blocking C3aR signal,the phosphorylation of AKT induced by recombinant C3a was inhibited.?5?After blocking the PI3K-AKT signal,the functional molecules expressed in fibroblasts were down regulated.4.4T1 tumor cell derived C3 promotes breast cancer metastasis?1?C3 knockout 4T1 tumor cells inhibit tumor growth in immunocompetent mice.?2?C3 knockout 4T1 tumor cells slightly inhibited tumor growth in immunodeficient nude mice.?3?C3 knockout of 4T1 tumor cells significantly reduced the number of lung metastases in mice.?4?C3 knockout 4T1 tumor cells did not affect the proliferation ability of mice in vitro.5.Pharmacological inhibition of C3aR signaling inhibits breast cancer metastasis?1?C3aRA?C3aR antagonist?significantly inhibited the number of lung metastasis in Balb/c 4T1-bearing mice?2?C3aRA?C3aR antagonist?significantly inhibited the number of metastases foci in lungs of PYMT breast cancer mice.?3?High expression of C3aR1 and PDGFA in breast cancer patients was more prone to metastasis with shorter survival.Conclusions1.C3a-C3aR signaling in CAFs facilitates the metastasis of breast cancer2.C3a-C3aR signaling augments pro-metastatic cytokine secretion and extracellular matrix components expression of CAFs via the activation of PI3K-AKT signaling.3.Genetic or pharmacological blockade of C3aR signaling effectively inhibited lung metastasis of breast cancer in mouse models. |
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