Quantitative Analysis Of Ncadherin On Circulating Tumor Cell By Mass Spectroscopy Based On Noble Metal Cluster Probes

Remote tumor metastasis is the main cause of death in patients with malignant tumors.Circulating tumor cells（CTC）originate from primary tumors or metastatic tumors,gain the ability to break away from the basement membrane and then invade and enter the blood through the tissue matrix.At present,CTC have now been adopted by the American Society of Clinical Oncology as a new tumor marker.Early detection and molecular classification of CTC in the blood through quantitative analysis plays an important role in prognostic judgment,curative effect evaluation and individualized cancer treatment.The key is how to develop highly sensitive biological probes that specifically recognize protein markers of CTC,and establish corresponding analysis methods at the molecular,cellular and clinical levels.Noble metal clusters possess unique physical,chemical and biological properties,and have wide application in life analysis.In this thesis,a peptide-gold cluster biological probe was constructed to identify the neural cadherin（N-cadherin）on the surface of CTC through peptide targeting.Combining the unique fluorescence and mass spectrometric properties of gold clusters established the optical imaging and absolute quantitative analysis methods of N-cadherin at the molecular and cellular levels.Further,the intrinsic correlation between N-cadherin expression and tumor molecular typing was explored by analyzing clinical samples.This cluster probe-based CTC biopsy technology is expected to be used in early diagnosis of tumors,providing personalized treatment and assisting prognostic judgment.1.A functional regionalized peptide was designed and synthesized.Its amino acid sequence is H₂N-CCYKKKKSWTLYTPSGQSK-COOH.Among them,the SWTLYTPSGQSK sequence specifically recognizes N-cadherin,and the CCY sequence is used as a biomimetic mineralization ligand for gold clusters.By optimizing the synthesis conditions,a peptide-gold cluster probe was successfully prepared,which emits red fluorescence,chemical properties and uniform size.The precise molecular composition of the probe(Au₁₉Peptide₃)was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF-MS）.2.By selecting human lung cancer cell A549 and breast cancer cell MCF-7 with obvious differences in the expression of N-cadherin protein as model cells,the targeting property of the polypeptide-gold cluster probe was verified.After co-cultivation with tumor cells,by combining with the unique photoluminescence properties of gold clusters,the spatial distribution of N-cadherin on tumor cell membranes was clarified by using laser confocal microscopy.By optimizing the labeling conditions,using inductively coupled plasma mass spectrometry（ICP-MS）for quantitative analysis at the population cell level,based on the precise composition of the probe,the N-cadherin expression level was accurately obtained.Furthermore,the LA-ICP-MS technology was used to quantitatively analyze N-cadherin at the single cell level.It found that the expression of N-cadherin was positively correlated with the invasion and metastasis ability of tumor cells.3.By simulating the CTC environment in vitro,a quantitative analysis method of N-cadherin mass spectrometry based on peptide-gold cluster probe was established at the single-cell level.The blood of 6 healthy people was drawn,a small amount of A549cell suspension was injected into it,and cell enrichment,nucleic acid fluorescence in situ hybridization（FISH）and polypeptide-gold cluster probe labeling were performed in sequence.The filter membrane was used to remove the smaller white blood cells,and the A549 cells are effectively trapped and enriched on the membrane.A549 cells were identified by FISH hybridization and peptide-gold cluster probe labeling.The LA-ICP-MS technology was used to accurately determine the gold element in a single cell,and the expression of N-cadherin on the surface of a single cell was obtained by calculation.4.Further,quantitative analysis of CTC in blood samples of clinical lung adenocarcinoma patients was carried out.5 m L of peripheral blood from 6 patients with lung adenocarcinoma who had not undergone surgical treatment and had a clinical stage of stage IV was collected and subjected to enrichment,FISH hybridization and peptide-gold cluster probe labeling.Fluorescence microscopy was used to locate cells by imaging and genotype analysis was performed to identify CTCs and determine cell types.The content of N-cadherin protein on the surface of a single CTC was determined by LA-ICP-MS.The finding showed that when the numbers of green mesenchymal gene signal points in CTC are higher,the expression of N-cadherin protein is higher.This result confirms the mesenchymal characteristic.This work provides a novel and simple method for in situ observation and precise quantitative analysis of CTC protein markers at the single-cell level.