Establishment And Clinical Application Of A New Circulating Tumor Cells Detection Method Based On Aggregation-Induced Emission Probe

Background&ObjectiveCirculating tumor cells(CTCs)can provide important information about tumor early diagnosis,dynamic observation,curative effect judgment,medication guidance and recurrence monitoring,and have important value in research and clinical application.However,the identification of CTCs still lacks ideal markers.Studies have shown that tumor cells and normal cells are significantly different in mitochondrias,and in our previous work,we have found a novel aggregation-induced emission(AIE)molecule TPN,which can target mitochondrias of live HeLa cells with fluorescence imaging,and it is expected to provide ideas for developing a new CTC rapid identification method based on mitochondrial difference.In this research,the aim of the first part is to establish a complete and effective CTC detection platform.And in the second and the third part,the aim is to investigate the detection effect of TPN probe in tumor cell lines from all aspects,explore the identification effect of TPN in CTCs from whole blood samples of tumor patients and tumor cells from pleural effusion by collecting clinical samples.Methods1.CTCs were enriched by ClearCell,and then identified by immunofluorescence analysis of CK and CD45.single CTCs were picked by CellCelector,and single CTC genome amplification was performed with multiple dilacement amplification(MDA)and multiple annealing and looping based amplification cycles(MALBAC)and single CTC transcriptome amplification was also performed.And comprehensive evaluation and optimization of the CTC detection platform were carried out.2.Fluorescent microscopy and flow cytometry analysis were performed to compare fluorescence difference of 6 tumor cell lines and white blood cells.With a small amount of tumor cells spiked recovery experiments,the recovery rate of TPN detecting tumor cell lines was explored.And the effect of TPN on the activity of tumor cells was explored by determining the cell death using PI.Single A549 cell labeled and unlabeled with TPN was picked for single cell transcriptome amplification and sequencing,and the sequencing results were compared.3.27 whole blood samples of lung cancer patients,16 whole blood samples of liver cancer patients,10 whole blood samples of healthy donors and 11 pleural effusion of lung cancer patients were collected.CTCs and tumor cells were identified by TPN.Results1.The establishment of the CTC detection platformThe average recovery rate of ClearCell enrichment was 64.0%,and with ClearCell enrichment,CTCs could be successfully found in the blood of cancer patients.And the success rate of single cell picking with CellCelector is close to 100%.At the same time,the coverage rate of single cell genome amplification was excellent,and the concentration,total amount and integrity of single cell transcriptome amplification product was satisfying.2.The detection effect of TPN for tumor cell linesFluorescence microscopy and flow cytometry analysis showed that the fluorescence intensity of 6 tumor cell lines after TPN labeling was significantly higher than that of white blood cells.And the average recovery rate of TPN detecting H1975 and HepG2 cells were over 90%.In addition,the survival rate of 6 tumor cell lines after TPN labeling reached 90%.The results of transcriptome sequencing of single A549 cell labeled and unlabeled TPN revealed that the TPN did not affect the transcriptome of single A549 cell.3.The identification effect of TPN for tumor cells in clinical samplesTPN could identify TPN-positive,CD45-negative suspected CTCs in clinical samples,and the TPN-positive CTC number of lung cancer and liver cancer patient samples was significantly higher than that of healthy donor samples.At the same time,TPN could identify TPN-positive,CD45-negative tumor cells in pleural effusion.ConclusionWe have successfully established a complete and effective CTC detection platform which will ultimately enable the acquisition and analysis of genetic information from single CTC.And we also confirmed the detection effect of TPN in tumor cell lines and clinical whole blood and pleural effusion samples.The new fluorescent molecule TPN is expected to be used in detection and clinical application of CTCs and benign and malignant identification of pleural effusion and it has broad clinical application prospects.