| Background:Malignant tumors are a large group of diseases threatening human health.Metastasis is one of the main malignant biological characteristics of tumors.About 90%of the deaths of cancer patients are caused by metastasis of tumors.Hematogenous metastasis is the main way for distant metastasis of tumors.In recent years,cancer stem like cells（CSCs）have been considered to be the key to determine the recurrence and metastasis of tumors because of their malignant biological characteristics such as self-renewal,anti-apoptosis,proliferation and differentiation,invasion and drug resistance.It has been found that cancer cells in the blood circulation can detect stem cell phenotypes,so cancer stem cells may be the key to mediate the hematogenous metastasis of tumors.Adhesion and exosmosis of tumor cells to vascular endothelial cells of target organs in blood circulation is one of the key steps in the process of blood metastasis.Studies have shown that PMNs and hypercoagulable state of blood circulation can promote the adhesion and exosmosis of tumor cells to vascular endothelial cells.This study focuses on the adhesion and exosmosis of tumor stem cells to vascular endothelial cells.To explore the mechanism of Fuzheng Qudu Recipe in preventing and treating hematogenous metastasis of tumors from the perspectives of recruitment of PMNs,vascular endothelial adhesion,vascular extravasation and target organ implantation.To explore the role and mechanism of breast cancer stem cells in mediating hematogenous metastasis,and the intervention and molecular mechanism of Fuzheng Qudu recipe,so as to provide a new scientific basis for targeting and regulating cancer stem cells to prevent and treat hematogenous metastasis.Methods:Tumor Spheres Formation method was used to isolate and enrich breast cancer stem cells on the basis of 4T1 breast cancer cell line,and established a stable co-culture system of breast cancer stem cells.4T1 breast cancer cells and neutrophils.Parallel plate flow chamber was used to establish a parallel plate flow experimental system to simulate the hypercoagulable state of blood in cancer patients in vitro.The levels of vascular endothelial adhesion and exosmosis between breast cancer stem cells and 4T1 breast cancer cells mediated by PMNs in the flow system were compared,and the intervention effect of Fuzheng Qudu recipe was also studied.The level of IL-8 secretion in cell supernatant was detected by ELISA,and the expression levels of ICAM-1,alpha-vbeta-3 and beta-2 integrin on membrane surface of breast cancer stem cells,4T1 breast cancer cells and PMNs were detected by flow cytometry.The mechanism of vascular endothelial adhesion and exosmosis of breast cancer stem cells mediated by neutrophils in the flow system and the intervention mechanism of Fuzheng Qudu recipe were clarified.The effects of PMNs on the migration of breast cancer stem cells and 4T1 breast cancer were compared by Transwell Migration Experiment system.The expression level of t-PA fibrinolytic system related molecules was detected and its mechanism was explored.By inoculating 4T1 breast cancer cells into tail vein of BALB/c mice to simulate the process of blood metastasis of breast cancer,the model of lung metastasis induced by tail vein inoculation was established to study the effect and mechanism of Fuzheng Qudu Recipe on preventing and treating blood metastasis.The effect and mechanism of Fuzheng Qudu Recipe on regulating blood metastasis of breast cancer stem cells were verified in vivo.Results:（1）CSLCs showed stronger vascular adhesion and extravasation ability of 4T1 cells under the action of PMNs in simulated hypercoagulable flow system（adhesion:843.0±1.16 vs 152.0±2.31,P<0.01;extravasation:666.3±8.65 vs 627.0±8.39,P<0.05）.（2）The high vascular adhesion and extravasation ability of CSLCs under the action of PMNs in simulated hypercoagulable flow system may be related to the high expression of ICAM-1（80.25%±4.45%vs 55.35%±5.81%,P<0.05）,alpha v beta 3（15.28%±0.85%vs 0.54%±0.11%,P<0.01）on the surface of CSLCs membrane,and the secretion of higher level of IL-8（78.72%±4.24 vs 9.705%±0.47pg/ml,P<0.01）.（3）PMNs can promote the migration of 4T1 cells（174±7.239 vs 284.8±8.139 P<0.0001）.Its mechanism may be related to activating t-PA fibrinolytic system and promoting the expression of t-PA related catabolic enzymes（P<0.01）.（4）In Fn-simulated hypercoagulable state,PMNs-mediated vascular adhesion and extravasation increased（574.3±8.09 vs 666.3±8.65,P<0.01）,and migration increased（137± 15.37 vs 218.6±9.158,P<0.05）.（5）CSLCs have stronger t-PA fibrinolytic system activation than 4T1 cells（P<0.01）,which may have stronger t-PA system-mediated effects.（6）Fuzheng Qudu Recipe has certain inhibitory effects on PMNs-mediated vascular endothelial adhesion and exosmosis in CSLCs and 4T1 cells in hypercoagulable flow system（adhesion:control group 843.0±1.16/visual field vs medium dose 38.33±2.03/visual field,high dose 9.333 ± 1.46/visual field,P<0.01;exosmosis effect:control group 666.3 ±8.65/visual field vs medium dose 500.3±8.83,high dose 402.7±22.98/field of vision P<0.01）.（7）Fuzheng Qudu Recipe inhibits PMNs-mediated vascular endothelial adhesion and exosmosis in CSLCs and 4T1 cells in hypercoagulable flow system by inhibiting IL-8 secretion（parallel plate fluid test:control group 84.43±3.2]pg/ml vs medium dose group 47.95±4.750pg/ml,high dose group 63.69±1.563pg/ml,P<0.01;Migration Experiment 78.72 ± 4.24pg/ml vs 23.93±3.45pg/ml,67.53±2.37pg/ml,P<0.01）,decreased the expression of integrin beta 2 on PMNs membrane surface（control group 26.59+1.52%vs medium dose group 21.20± 0.63%vs high dose group 18.78±0.49%）.Inhibition of 4T1 cells and CSLCs was related to PMNs adhesion and exosmosis.（8）Fuzheng Qudu Recipe could inhibit the migration of 4T1 cells and CSLCs（4T1:control group 284.8±8.14 vs middle dose group 282.6±8.01,high dose group 112.8±6.95,P<0.01;CSLCs:control group 218.6+9.158 vs middle dose group 116.6±7.075,high dose group 66.40 ± 8.334,P<0.01）,and its mechanism might be related to the up-regulation of the expression of adhesion molecules between tumor cells（control group 18.42±0.18%vs high dose group 33.42 ± 0.29%,P<0.01）,up-regulated the anti-cancer molecule PAI:（4T1:control group 0.99 ± 0.02 vs middle dose group 0.99±0.02,high dose group 3.37±0.22;CSLCs:control group 1.69±0.03 vs high dose group 2.61±0.10,P<001）TFPI（4T1:control group 2.67±0.02 vs high dose group3.83±0.15;CSLCs:control group 2.90±0.09 vs middle dose group3.97±0.13,P<0.01）The expression level was correlated.（9）Fuzheng Qudu Prescription could significantly inhibit the number of pulmonary metastatic nodules in mice with pulmonary metastasis inoculated by tail vein 7 days earlier（model group 81.2±13.7 vs middle dose 17.4±4.6,high dose 13.8±2.1,P<0.01）.（10）The inhibitory effect of Fuzheng Qudu Recipe on hematogenous metastasis in tail vein inoculated lung metastasis model mice may be related to the inhibition of IL-8 level in peripheral blood circulation of mice（31.54 ± 0.68 vs 29.05±0.48 pg/ml,P<0.05）,and the down-regulation of PMNs integrin beta 2 expression in peripheral blood of mice（model group 24120±1441 vs high dose group 16060±1511,P<0.05）.（11）The inhibitory effect of Fuzheng Qudu Recipe on hematogenous metastasis in mice with lung metastasis by tail vein inoculation may be related to the up-regulation of ICAM-1,a adhesion molecule on the surface of tumor cells(model group 37.50±4.89 vs a vβ3（model group 80.25%±4.45%vs high dose 55.35%±5.81%,P<0.01）,which can enhance the adhesion between tumor cells and stabilize tumors.Conclusion:（1）The biological characteristics of CSLCs under the action of PMNs in simulated hypercoagulable flow system have stronger vascular adhesion and extravasation ability of 4T1 cells.This biological characteristics of CSLCs may be related to the expression of higher levels of adhesion molecules ICAM-1 and alpha v beta 3 on the surface of CSLCs membrane,and the autocrine level of IL-8.（2）PMNs can promote the migration of 4T1 cells and CSLCs in co-culture system,and its mechanism may be related to the activation of t-PA system.（3）Fuzheng Qudu Recipe can significantly inhibit PMNs-mediated vascular endothelial adhesion and exosmosis in the hypercoagulable flow system of CSLCs.This effect may be related to the inhibition of CSLCs autocrine IL-8 by Fuzheng Qudu Recipe and the down-regulation of integrin beta 2 expression on PMNs cell membrane surface.（4）Fuzheng Qudu Recipe can significantly inhibit the migration of 4T1 cells and CSLCs.The mechanism may be related to the upregulation of the expression of adhesion molecule alpha v beta 3 on the surface of CSLCs by Fuzheng Qudu Recipe and the expression of tumor suppressor PAI-1 and TFPI in 4T1 cells and CSLCs.（5）Fuzheng Qudu Decoction can significantly inhibit the occurrence of lung metastasis in tail vein inoculated lung metastasis model mice.Its effect may be related to the inhibition of IL-8 level in peripheral blood circulation by Fuzheng Qudu Decoction,and the down-regulation of PMNs integrin beta 2 expression in peripheral blood of mice,thus reducing the vascular endothelial adhesion and exosmosis of PMNs-mediated tumor cells and cancer stem cells.（6）Fuzheng Qudu Prescription may also enhance the adhesion between tumor cells,stabilize the tumor body and inhibit the blood metastasis of tumor cells by up-regulating the expression levels of ICAM-1 and αvβ3 on the surface of tumor cells. |
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