| Despite considerable advancements in diagnosis and treatment of solid tumors,distant metastases remain the main cause of cancer-related mortality.Circulating tumor cells(CTCs)can be detected in the blood of patients with a variety of solid tumor.The higher counts of CTCs in blood of patients resulted in the poorer prognosis and shorter survival of the patients.CTCs can be used as "liquid biopsy" for prognosis and prediction for disease recurrence.Though CTCs can be the source of metastasis-initiating cells,little is known about whether CTCs could influence tumor metastasis through other mechanisms.Objective:Our purpose is to investigate whether CTCs could promote tumor metastasis,and how CTCs promote metastasis.Methods:(1)We established an CTCs animal model by using an non-metastatic tumor cell,B16F0;and an metastasis animal model by using an metastatic tumor cell,B16F1.(2)Mice were injected with CFSE-labeled B16F1 cells 12 h after non-labeled B16F0 cells injection via tail vein.The mice were sacrificed 5 h(for analysis of tumor cell arrest)or 24 h(for analysis of extravasation)after B16F1 cells injection.Tumor cells in frozen sections were visualized by fluorescence microscopy and counted.(3)Serum levels of IL-6 and IL-1β from different mouse models were detected by ELISA.(4)The infiltration of neutrophils in lung from different mouse models was detected by immunohistochemical staining.(5)Mice received the i.v.injection of B16F1 cells with or without B16F0 cells.Neutrophils were depleted in vivo by i.p.injection of anti-Ly6G antibody.The metastatic nodules on the surface of lung were counted.(6)Neutrophils were isolated from the bone marrow(BM)or peritoneal cavity(PC)of different mouse model.The neutrophils were used for co-inoculation with B16F1 cells to naive mice in the right hind thigh.Tumors were dissected and weighted on d12 after tumor cell inoculation.(7)To investigate the function of CTCs on tumor metastasis and neutrophil function,we expressed an anti-inflammatory cytokine,IL-37 in our models.(8)Neutrophils were isolated from the bone marrow(BM)of cir-B16F0 mice without further stimulation,or isolated from the peritoneal cavity(PC)of cir-B16F0 mice and stimulated with T-sMs(0.5 mg/ml)for 12 h.The expression of pro-tumor genes,Mmp9,Bv8,Argl and Nos2,and anti-tumor genes,Trail and Rab27a was detected by real-time RT-PCR.(9)Neutrophils were isolated from the peritoneal cavity of cir-B16F0 mice with or without further stimulation with T-sMs(0.5 mg/ml)for 30 min.The release of MPO from neutrophils was detected after stimulation with T-sMs,and the CD63,which is associated with the primary granules and transferred to the surface of neutrophils during degranulation,was detected by flow cytometry.The phosphorylation of Akt and p38 MAPK was detected by Western blot after stimulation with T-sMs.(10)The expression of Csf3(g-csf)and 116 genes in lung and spleen tissues of cir-B16F0-mice was detected by real-time RT-PCR.The serum levels of G-CSF,IL-6 and IL-1β in cir-B16F0-mice were detected by ELISA.(11)Cir-B16F0-mice were untreated or treated with anti-IL-1β antibody to neutralize IL-1β in vivo.The expression of Csf3(g-csf)and 116 genes in lung and spleen tissues was detected by real-time RT-PCR.(12)HMGB1 and HSP70 in the serum of cir-B16F0 mice were detected by Western blot.Cir-B16F0-mice were untreated or treated by i.v.injection of psTLR2 and/or psTLR4 plasmids.S100A8,S100A9 and SAA3 proteins in lung tissues were detected by Western blot.(13)Cir-B16F0-mice were untreated or treated by i.v.injection of psTLR2 and/or psTLR4 plasmids.Serum levels of G-CSF,IL-6,and IL-1β were detected by ELISA.Metastatic nodules on the surface of lung were counted.Results:(1)CTCs(circulating B16F0 cells)promoted the metastatic colonization of disseminated carcinoma cells(B1671),thus promoted tumor metastasis.(2)CTCs can elevate the serum level of pro-inflammatory cytokines,IL-6 and IL-1β,and thus induced a systemic inflammatory response.(3)CTCs promote the infiltration of neutrophils in lung.(4)CTCs could not promote lung metastasis of B16F1 cells when neutrophils were depleted in vivo.(5)CTCs promote the expression of Mmp9,Bv8,Argl and Nos2,and suppress the expression of TRAIL and Rab27a in neutrophils.(6)CTCs increased the responsiveness of neutrophils to the stimuli in tumor milieu.(7)CTCs attenuated neutrophil degranulation and the phosphorylation of p38 MAPK and Akt in neutrophils.(8)The expression of IL-37 in vivo can effectively suppress the CTC-induced inflammatory response and the influence of CTC on neutrophil function.However,IL-37 cannot directly influence the proliferation and colonization of B16F1 cells.Meanwhile,IL-37 also cannot directly regulate the function of CTC on neutrophil function and responsiveness.(9)The expression of Csf3(g-csf)and 116 genes in lung and spleen tissues of cir-B16F0 mice were higher than naive mice.Consistently,the serum levels of G-CSF and IL-6 were also elevated in cir-B16F0 mice.(10)The increased expression of Csf3(g-csf)and 116 in lung and spleen tissues of cir-B16F0-mice could be significantly suppressed by blocking IL-1β in vivo.(11)The protein levels of HMGB1 and HSP70,the representatives of TLR2/4 ligands which have been implicated in promoting the production of proinflammatory cytokines,were elevated in the serum of cir-B16F0 mice.Moreover,the expression of S100A8,S100A9 and SAA3,which are also the ligands for TLR2/4 and have been implicated in promoting G-CSF,IL-6 and IL-1β expression,in lung tissues were increased in cir-B16F0-mice.(12)The expression of soluble TLR2(sTLR2)and soluble TLR4(sTLR4)in vivo could effectively suppress not only the promotion effect of CTC on the production of G-CSF,IL-6 and IL-1β,but also the promotion effect of CTC on tumor metastasis.Conclusion:CTCs promote the metastatic colonization of disseminated carcinoma cells by initiating systemic inflammatory responses,promoting neutrophil infiltration in pre-metastatic organs,and inducing the conversion of neutrophil function to pro-metastasis,which consequently promoting tumor metastasis. |
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