Inhibition Of PI3K/AKT/mTOR Pathway Reverses The Chemotherapy Drug Sensitivity Of Primarily Mesenchymal Phenotype Circulating Tumor Cells

Objective Chemotherapy resistance in tumor patients is the main cause of poor prognosis and death.At present,the molecular mechanism of tumor chemotherapy resistance is still not fully understood.Circulating tumor cells(CTCs)shed from the primary tumor site or tumor metastasis site and enter the blood circulation.They can transfer to distant sites along with the blood circulation and form new tumor.CTCs play an important role in tumor chemotherapy resistance,metastasis and poor prognosis of patient.The epithelial-mesenchymal transition(EMT)of CTCs plays an important role in the resistance of tumor cells to chemotherapy.Our team’s previous studies have found that CTCs have a higher level of basic autophagy,a stronger ability to repair DNA damage and a lower level of apoptosis in the blood,and stronger survival ability in the blood.Studies on the mechanism of drug resistance of triple negative breast cancer showed that PI3K/AKT/mTOR pathway was activated in EPI-Hs578 T Epirubicin-resistant cells,and they had stronger ability to repair DNA damage.This study further explored the mechanisms of influencing the susceptibility of CTCs to epirubicin and taxol,and the role of EMT biological processes in the resistance of CTCs to chemotherapy.Methods1.Isolation and identification of primary tumor cells(PTCs): 4T1 parent cells were injected into the fourth pair of fat pads on the right side of mice to establish 4T1 breast cancer in situ transplantation tumor model.PTCs were separated by collagenase digestion.The positive rate of EpCAM was detected by flow cytometry,and the isolated PTCs were identified by fat pad tumorigenesis assay and HE staining.2.Extraction and identification of circulating tumor cells(CTCs): CTCs were captured from the blood of model mice by Ficoll density gradient centrifugation;The positive rates of leukocyte marker CD45 and epithelial marker EpCAM were detected by flow cytometry,and the captured CTCs were identified by fat pad tumor formation assay and HE staining.3.Type identification of CTCs: Related indicators of Epithelial-Mesenchymal Transition(EMT)were detected,and positive rates of epithelial marker EpCAM and mesenchymal marker Vimentin of CTCs were detected by flow cytometry.The expression levels of epithelial markers E-cadherin and EpCAM and mesenchymal markers N-cadherin,Vimentin,Slug and Snail of CTCs were detected by Western Blot.The expression levels of epithelial marker E-cadherin and mesenchymal marker Vimentin of CTCs were detected by cellular immunofluorescence.4.Detection of CTCs drug resistance: MTT assay was used to detect the sensitivity of PTCs and CTCs to epirubicin and taxol.The expression levels of MRP1 and LRP in PTCs and CTCs were detected by RT-PCR.Western Blot was used to detect the expression levels of MRP1 and LRP in PTCs and CTCs.5.PI3K/AKT/mTOR signaling pathway detection: Western Blot analysis was performed to detect the expression levels of phosphorylated AKT and mTOR in PTCs and CTCs.6.Detection of relevant indicators after the effect of AKT inhibitor MK2206 on PTCs and CTCs: MTT method was used to detect the appropriate concentration of AKT inhibitor MK2206 on PTCs and CTCs.Drug sensitivity of PTCs and CTCs to epirubicin and taxol.The expression levels of phosphorylated AKT and mTOR in PTCs and CTCs were detected by Western Blot.The expression levels of epithelial markers E-cadherin and EpCAM,mesenchymal markers of N-cadherin,Vimentin,Slug and Snail;Expression levels of drug-resistant proteins LRP and MRP1.The expression levels of epithelial marker E-cadherin and mesenchymal marker Vimentin of PTCs and CTCs were detected by cellular immunofluorescence.The expression levels of drug resistance genes LRP and MRP1 in PTCs and CTCs were detected by RT-PCR.Results1.Successful isolation of primary tumor cells(PTCs): 4T1 breast cancer in situ tumor animal model was established successfully,using collagenase digestion method to isolate primary tumor cells;Flow cytometry showed that EpCAM(+)cells accounted for 99.2%.After the isolated cells were injected into the fat pad of mice mammary gland,tumor formation was confirmed by HE staining,and the captured cells were confirmed as PTCs.2.Successful extraction of circulating tumor cells(CTCs): Ficoll density gradient centrifugation was used to capture cells from the blood of model mice.Flow cytometry showed that EpCAM(+)cells accounted for 17.6%,while CD45(+)cells accounted for 0.064%,excluding the possibility of leukocyte cells.After the isolated cells were injected into the fat pad of mice mammary gland,tumor formation was confirmed by HE staining,which suggested that the isolated cells were derived from cancer cells,and the captured cells were confirmed as CTCs.3.The isolated CTCs showed primarily mesenchymal phenotype phenotype: Flow cytometry showed that EpCAM(-)Vimentin(+)cells accounted for 70.3% of CTCs.Western Blot results showed that the expression levels of epithelial markers E-cadherin and EpCAM of CTCs were lower than PTCs,while the expression levels of mesenchymal markers N-cadherin,Vimentin,Slug and Snail were higher than PTCs(P < 0.05);The results of cell immunofluorescence showed that the expression level of epithelial marker E-cadherin was lower than PTCs,and the expression level of mesenchymal marker Vimentin was higher than that of PTCs(P < 0.05).The results of the three methods showed that the extracted CTCs showed primarily mesenchymal phenotype.4.Detection of drug sensitivity: CTCs are less sensitive than PTCs(P < 0.05);The expression levels of drug resistance genes LRP and MRP1 in CTCs are higher than PTCs(P < 0.05);The expression levels of drug-resistant proteins LRP and MRP1 in CTCs were higher than PTCs(P < 0.05).5.PI3K/AKT/mTOR signaling pathway detection: phosphorylated AKT and mTOR expression levels in CTCs are higher than PTCs(P < 0.05).6.The expression levels of phosphorylated AKT and mTOR are decreased after MK2206 is treated with CTCs(P < 0.05);Western Blot analysis showed that epithelial markers E-cadherin and EpCAM increased,while mesenchymal markers N-cadherin,Vimentin,Slug and Snail decreased(P < 0.05);Cell immunofluorescence results showed that the expression level of epithelial marker E-cadherin increased,and the expression level of mesenchymal marker Vimentin decreased(P < 0.05);The expression levels of drug-resistant proteins LRP and MRP1 were decreased(P < 0.05);The expression levels of drug-resistant genes LRP and MRP1 were decreased(P <0.05);The sensitivity of CTCs to chemotherapy resistance increased(P < 0.05).Conclusion1.4T1 circulating tumor cells were isolated from the blood of 4T1 breast cancer in situ model mice and identified as primarily mesenchymal phenotype with EMT phenotype.2.The sensitivity of 4T1 primarily mesenchymal phenotype circulating tumor cells to chemotherapy drugs epirubicin and taxol decreased;3.Chemotherapy resistance in 4T1 circulating tumor cells is related to the biological behavior of EMT mediated by PI3K/AKT/mTOR signaling pathway activation.