Epithelial-mesenchymal Transition Typing And Glucose Metabolism Characteristics Of Circulating Tumor Cells

Backgrounds:Metastasis is the leading cause of poor prognosis and high mortality in malignant tumors.It is also a major challenge for clinical treatment of tumors.Epithelial-Mesenchymal Transition(EMT)refers to the phenomenon that epithelial cells lose their original polarity and convert into interstitial cells that can move freely between extracellular matrices under specific physiological or pathological conditions.Tumor cells become more aggressive through EMT to promote tumor metastasis.Metabolism Reprogramming is an important feature of tumor cells,which is manifested as mitochondrial oxidative breathing disorders,high dependence on aerobic glycolysis,and enhanced pentose phosphate pathway and glutamine metabolism.Thus the tumor cell can intake and consumpt large amounts of glucose to ensure the energy consumption and macromolecule synthesis during cell growth and metastasis.Circulating Tumor Cells(CTCs)refer to tumor cells derived from the primary tumor or metastatic tumor,and released to the peripheral blood circulation due to spontaneous or therapeutic factors.CTCs can spread to the distant organs or tissue through the blood system and form a new metastases by colonization.The level of CTCs in peripheral blood is closely related to the progression of disease,metastasis and recurrence.And the CTCs counting and molecular characterization are of great significance for the diagnosis of clinical stage,treatment response and prognosis,recurrence and metastasis monitoring.Therefore,the aim of this study is to analyze the CTCs characteristics of EMT phenotype and metabolic function in peripheral blood of cancer patients.The research contents include the following three parts:i)the clinical analysis of circulating tumor cells EMT classification technology(Chapter 2);ii)the screening and validation of differentially expressed glucose metabolism genes related to tumor metastasis(Chapter 3);and iii)the analysis of circulating tumor cells metabolic characteristics in peripheral blood and its clinical significance(Chapter 4)(Chapter 1 is the introduction part).METHODS:A new Canpatrol(?)CTCs typing platform was used to analyze the peripheral blood CTCs in cancer patients.In the second chapter,the results of CTCs typing in the blood samples of 551 tumor patients from Nanfang Hospital during December 1,2014 to December 31,2016 were analyzed,and the information of gender,age,tumor stage tumor-specific serum markers expression were collected to analyze the correlation between CTCs typing and these clinical pathological features.In the third chapter,the gene expression profiles of high metastatic and low metastatic prostate cancer cell lines PC-3M 1E8 and PC-3M 2B4 were compared by the Functional Microarray technique.Meanwhile the expression of array identified genes in other prostate cancer cells with different metastatic potentials(LNCAP,PC-3 and DU145 cells)was also detected by fluorescence quantitative PCR and Western blot to screen the glucose metabolism genes related to tumor metastasis.In the fourth chapter,the Canpatrol(?)CTCs typing platform was used to detect the expression of tumor metastasis-related glucose metabolism gene in peripheral blood CTCs of patients with common cancers,such as hepatocellular carcinoma,lung cancer and cervical cancer.Besides,the joint markers for metabolism active CTCs detection were determined to analyze the metabolic characteristics of CTCs in peripheral blood from patients with prostate cancer and breast cancer.RESULTS:In the second chapter,we analyzed the blood test results of 551 cancer patients,mainly including hepatocellular carcinoma(195 cases),nasopharyngeal carcinoma(38 cases),lung cancer(114 cases),colorectal cancer(74 cases),osteosarcoma(35 cases),renal carcinoma(18 cases)and other tumors(77 cases).Charging CTCs positive by CTCs count?3/5mL peripheral blood,the positive rate of CTCs was 61%to 80%in these patients(hepatocellular carcinoma 75%,lung cancer 61%,nasopharyngeal carcinoma 63%,colorectal cancer 68%,osteosarcoma 80%,renal cell carcinoma 72%and other tumors 64%).The median CTCs count in CTCs-positive patients ranged from 5 to 10/5mL,totally ranging from 3 to 86 cells/5mL.In addition,in 380 cases of CTCs positive samples,the numbers for CTCs detected in three types was 202 cases of E type,338 cases of H type and 235 cases of M type,and the overall positive rate was E type 53%,H type 89%,M-type 62%.Meanwhile,the correlation analysis between CTCs EMT classification and clinicopathological features demonstrated that CTCs count and the proportion of CTCs expressing M-type markers were related to the metastatic status,clinical stage and serum tumor markers in patients with hepatocellular carcinoma and nasopharyngeal carcinoma.In the third chapter,39 differentially expressed genes were identified between high metastatic PC-3M 1E8 cell and low metastatic PC-3M 2B4 cell by the functional microarray.By fluorescence quantitative PCR and Western blot validation and comparison of expression of these genes in LNCAP,PC-3 and DU145 cells as a secondary screening,we finally determined ten glucose metabolism genes related to cancer metastasis,including HK2(Hexokinase 2),PDP2(Pyruvate phosphatase catalyzed subunit 2),G6PD(Glucose-6-phosphate dehydrogenase),PGK1(Phosphoglycerate kinase 1),PHKA1(Phosphorylase kinase,alpha 1),PYGL(Liver glycogen phosphorylase),PDK1(Pyruvate dehydrogenase kinase 1),PKM2(M2 pyruvate kinase),ALDOA(Fructose diphosphate aldolase)and ENO1(enolase 1).Besides,the expression of these genes has a certain relevance to the expression levels of EMT markers(Vimentin,?-catenin,N-cadherin)and cancer stem cell related markers and tumor stem cell-related markers(CD44,CD133,OCT4,ALDH1).In the fourth chapter,the above-mentioned tumor metastasis-related metabolic genes were detected in peripheral blood CTCs from 64 patients(including 10 cases of hepatocellular carcinoma,21 cases of cervical cancer,8 cases of lung cancer,8 cases of nasopharyngeal carcinoma,9 cases of esophageal cancer,8 cases of renal cell carcinoma).The results showed that CTCs with high expression of these metabolic markers were detected in the blood of patients with solid tumors,with a total positive rate of 16%to 84%and the expression features in different EMT types of CTCs vary.The positive rate of CTCs with high expression of the metabolic markers in E,H and M type CTCs were 0-100%(G6PD highest,100%),21%-96%(G6PD highest,96%)and 0-86%(PYGL highest,86%).The positive rate of CTCs highly expressing PGK1,PYGL and PDK1 in M-type CTCs was higher than that in E-type CTCs,and the positive expression of HK2,G6PD,PGK1 and PYGL was closely related to CTCs EMT typing(P<0.05).Furthermore,G6PD and PGK1 were used as joint markers to detect the active metabolic status of CTCs and to analyze the metabolic characteristics of peripheral blood CTCs in patients with prostate cancer and breast cancer.It was found that the level of CTCs count in peripheral blood was higher in prostate cancer and breast cancer with distant cancer metastasis than that in patients without metastatis,and the ration of M-type CTCs was also relatively higher.Meanwhile,the positive rates of metabolism active(G6PD+PGK1 positive)CTCs in peripheral blood of patients with metastatic prostate cancer and metastatic breast cancer were higher in H and M-type CTCs than that in E-type CTCs,and the rates were to some extend correlated with the serum tPSA,fPSA levels and tumor Gleason scores of prostate cancer patients,and also correlated with the serum CA153,CEA levels and tissue ki67 expression of breast cancer patients.CONCLUSIONS:1.The expression of M-type markers in CTCs of peripheral blood from patients with hepatocellular carcinoma,lung cancer and nasopharyngeal carcinoma is very common,thus the detection of this kind of cells can improve the capture rate and detection accuracy of CTCs tests.Besides,the increased proportion of CTCs expressing M-type markers correlates with the clinical stage and metastasis of cancer patients,and analysis of EMT phenotype characteristics in peripheral blood CTCs may contribute to the clinical diagnosis of disease progression and metastasis in cancer patients.2.The glucose metabolic markers related to tumor metastasis are highly expressed in certain CTCs of patients with cancers,such as hepatocellular carcinoma,lung cancer and nasopharyngeal carcinoma,and it has a correlation with the EMT phenotype characteristics of CTCs.Meanwhile,the proportion of metabolism active CTCs has a certain relationship with the level of serum tumor markers in patients with prostate cancer and breast cancer,so the analysis of CTCs metabolic function status may provide useful information for the monitoring of disease progression and metastasis in cancer patients.