Overexpression Of Human Aspartate (Asparaginyl) β-hydroxylase In Exosomes For Diagnosis Of NSCLC

Background and Objective: Primary lung cancer is a malignant tumor that seriously endangers human health in the world.Patients with advanced lung cancer have low 5-year survival rate and poor prognosis.There is an urgent need for safe and efficient early screening strategies.At present,the total sensitivity of CYFRA21-1 or SCC,a widely used tumor marker of lung cancer,is only 35%(27%-44%).It is an urgent problem to find tumor markers with good specificity and high sensitivity.Liquid biopsy is a relatively noninvasive biopsy technique,which mainly detects CTC,ct DNA and exosomes.Because exosomes are more stable in biological properties,richer in specimen forms and higher in detection abundance than the other two,it has attracted much attention in recent years.This study intends to study the expression of ASPH in NSCLC cell line and supernatant exosomes,lung cancer tumor tissues and patients’ alveolar lavage fluid exosomes from the perspectives of cell level and clinical tumor patients,compare the expression differences between cells and exosomes,and analyze the sensitivity and specificity of ASPH in the diagnosis of NSCLC lung cancer by ROC curve.To explore whether the expression of ASPH in exosomes can be used as a biomarker for the diagnosis of NSCLC and whether the expression of ASPH in BAL exosomes of NSCLC patients can be used as a new means of liquid biopsy of NSCLC.Methods: Immunofluorescence and RT-qPCR were used to detect differences of ASPH m RNA expression between NSCLC cell lines(A-549,NCI-H1299,NCI-H1650,HCC-827)and normal bronchial cells NL-20.Then the exosomes were extracted from the cell culture supernatant of NSCLC cells.Firstly,flow cytometry was used to detect the exosomes surface markers CD63 and CD81,and then the morphology,size and distribution of exosomes were observed by transmission electron microscope.Finally,the expression of ASPH in exosomes of NSCLC cell lines was detected by RT qPCR.The data of 90 patients with pathologically confirmed non-small cell lung cancer from January 2015 to June 2019 were collected,The clinicopathological data of 90 patients with non-small cell lung cancer(NSCLC)confirmed by pathology from January 2015 to June 2019 in the Department of Pathology were collected.There were 62 males and 28 females with a median age of 56 years.According to the 7th AJCC cancer staging manual,20 patients were in stage I,40 in stage II,26 in stage III and 4 in stage IV;46 cases were adenocarcinoma and 44 cases were squamous cell carcinoma.According to the degree of histological differentiation,30 cases were well differentiated,42 cases were moderately differentiated and 18 cases were poorly differentiated;34 cases had no lymph node metastasis and 56 cases had lymph node metastasis.At the same time,the matched normal tissues more than 5 cm away from the cancer and 20 cases of benign lung diseases underwent surgical resection at the same time were taken as controls.Among them,7 cases were tuberculoma,6 cases were inflammatory pseudotumor,4 cases were hamartoma,2 cases were bronchial adenoma and 1 case was sclerosing pulmonary cell tumor.All patients with lung cancer were newly diagnosed and initially treated.They did not receive radiotherapy,chemotherapy or targeted drug therapy before operation,and had complete clinical data and follow-up data.All specimens were fixed with 10% neutral formalin,routinely embedded in paraffin,4 μm thick serial sections,and HE-stained paraffin sections were reviewed and diagnosed by two associate senior pathologists.Immunohistochemical staining was used to detect the expression of ASPH in 90 cases of NSCLC.All 90 patients who underwent surgical resection received bronchoalveolar lavage before surgery.20 patients with benign lung disease underwent the same bronchoalveolar lavage process.The expression of ASPH in BAL exosomes of NSCLC patients and patients with benign lung disease was detected by RT qPCR.In addition,the BAL of 27 NSCLC patients with different histological types and 15 patients with benign lung disease were collected by centrifugation,and the expression of ASPH was detected by immunocytochemistry,and the results were consistent with immunohistochemistry.ROC curve was used to analyze the sensitivity and specificity of ASPH in the diagnosis of NSCLC lung cancer.Results: 1.Compared with normal bronchial cells NL-20,the expression of ASPH m RNA in NSCLC cell lines A-549,NCI-H1299,NCI-H1650 and HCC-827 was significantly increased.2.Immunofluorescence revealed that ASPH protein was highly expressed in NSCLC cell lines,which was consistent with the result of RT-qPCR analysis.3.The positive rate of exosomal surface marker CD63 was 45.9%,which was significantly higher than 1.8% in the negative control group(P<0.01);the positive rate of surface marker CD81 was 74.0%,which was significantly higher than the negative control group 0.0%(P<0.01).4.TEM showed that the exosomes extracted from the cell culture supernatant of NSCLC cells were abundant and evenly distributed.The diameter of exosomes is about 40-150 nm.5.Compared with NL-20,the expression level of ASPH m RNA in the exosomes of NSCLC cell line increased significantly,especially in A-549 exosomes which increased by more than 10 times,while HCC-827 2.5 Times.6.The immunohistochemical staining of tumor tissues of patients with different histological types of non-small cell lung cancer showed deep brownish yellow granular staining in cytoplasm and cell membrane,showing a high level of ASPH immunoreactivity,while there was almost no staining or very weak staining in adjacent tissues and normal lung tissues of patients with non-small cell lung cancer.7.The positive expression rate of ASPH in NSCLC lung cancer,paracancerous tissue and benign lung disease was 68.9%,20% and 10%,respectively.The expression level of ASPH in lung cancer tissue was significantly higher than that of normal lung tissue and benign lung disease tissue,the difference was statistically significant(P < 0.01).8.The expression of ASPH was statistically significant in tumor size,lymph node metastasis and TNM stage(P < 0.05),but there was no significant difference in age,gender,smoking history,histological classification,histological type,differentiation degree and other groups(P > 0.05).9.ASPH m RNA expression in NSCLC patients(including patients with early lung cancer)BAL exosomes was high,while the expression of ASPH in adjacent tissues and normal lung tissues of NSCLC patients was very low.The expression level of BAL exosomes ASPH in NSCLC patients was significantly higher than that in non-malignant tissues and normal lung tissues(P<0.01).10.Immunocytochemical detection of BAL cells in NSCLC patients showed that ASPH was negative or low expressed in normal bronchial epithelial cells and normal alveolar epithelial cells of non cancer patients,and overexpressed in BAL cells of different histological types of NSCLC patients.In addition,the positive rate of ASPH in BAL exosomes of NSCLC was significantly higher than that in BAL cells(81.5% vs.59.3%,P < 0.01).11.ROC curve analysis: the sensitivity and specificity of ASPH at the critical value were 81% and 90%,respectively.The area value of AUC was 0.866,and within 95% confidence interval(0.768-0.964).The difference was statistically significant(P<0.001).Conclusions: 1.ASPH was overexpressed in NSCLC cell line and exosomes of NSCLC cell line.2.ASPH was overexpressed in different histological types of NSCLC patients.3.ASPH was overexpressed in BAL cells and exosomes of NSCLC patients,which was highly consistent with the immunohistochemical results of NSCLC lung cancer tissue.4.BAL exosomes ASPH detection technology can greatly improve the sensitivity of BAL and the specificity of ASPH in the early diagnosis of NSCLC.BAL exosomes may provide a new liquid biopsy method for NSCLC.Whatsmore,the method of BAL exosomes isolation in this study was easy to perform in a clinical laboratory.5.Compared with BAL cells,BAL exosomes have higher detection rate of ASPH and lower missed diagnosis rate.6.ROC curve shows that the high ASPH expression of BAL exosome can distinguish NSCLC patients and can be used for the diagnosis of non-small cell lung cancer.