Experiments In Vivo Of The Impact Of Hypoxia On Circulating Tumor Cells In Gastric Adenocarcinoma

Objective:The first part of this experiment is attempting to estimate the specificity and sensitivity of the CTCs enrich/identify system which were based on density gradient centrifugation and CK19immunofluorescence; We investigate the relationship between hypoxic microenvironment and the presence of circulating tumor cells in gastric adenocarcinoma in the second part of this experiment.Methods:1、The enrichment efficiency,the specificity and sensitivity were evaluated by the spike recovery experiment,that is the human poorly differentiated gastric adenocarcinoma BGC-823cells were cultrued regularly, using sterile PBS to adjust cell concentration to100/ml by microscope,then add100ul cell suspension to10ml blood of healthy volunteers (per blood contains10BGC-823cells),CTCs were enriched by density gradient centrifugation for mononuclear cells, and then the cells were identified and counted by immunofluorescence with anti-cytokeratinl9antibody, the CTCs from the peripheral bloods of healthy volunteers and gastric cancer patients were enriched and identified by the method above,the specificity and sensitivity of the method in CTCs detection were evaluated by comparing the difference of CTCs number between healthy volunteers and gastric cancer patients.2、The HIF-la expression level of BGC-823cells dealed with different concentration of CoCl2(0.100、200、300、400umol/L) for4hours were detected through Western Blot.3、60BALB/C mice were randomized divided into three groups:hypoxia group, normoxia group, and contronl group(normal saline),20mice of each group.BGC-823cells dealed with culture medium contain200umol/L CoC12for4h as hypoxia group,BGC-823cells dealed with serum-free medium for4h as normoxia group,and then injected the cells into corresponding mice through the tail vein,puncturing the angular vein for collecting1ml blood sample to EDTA anticoagulant tubes after successfully injected for3days,enriching and identifying CTCs by the method above.Results: 1、The spike experiment showed that the average enrichment efficiency was75%;It revealed that there were none of CTCs found in bloods from10healthy volunteers, while among20gastric cancer patients,0CTCs were detected in40%patients, and more than1CTCs were detected in60%patients.2、BGC-823cells dealed with culture medium contain200umol/L CoCl2for4h had the highest HIF-la expression level,there was significant difference compared with cells dealed with culture medium contain other different concentration of CoCl2(P<0.05).3、The presence of circulating tumor cells significantly related to hypoxia,the CTCs number of mics of hypoxia group was higher than mics of normoxia group(4.70±1.86vs1.15±0.67, P=0.000).Conclusion:1、The enrichment and identification method used could well recover spiked cells in peripheral blood and have significant specificity and sensitivity in CTCs clinical detection.2、Hypoxic microenvironment could enhance development of CTCs in gastric adenocarcinoma.