Establishment Of TRPC5 (EVs)-based TRFIA To Detect Chemoresistance In Breast Cancer

Breast cancer is a serious threat to women's health.Chemotherapy is one of the main treatment of breast cancer,but chemoresistance has become a major dilemma.At present,the diagnosis of chemoresistance can not be early judgment and dynamically monitored,so that eventually resulted in ineffective or excessive treatment.Therefore,real-time in vitro diagnosis of drug resistance at this stage is the key to breast cancer drug therapy.Drug-resistant human breast cancer cells possessed numerous transient receptor potential channel 5?TRPC5?-containing extracellular vesicles?EVs?on the cell surface.Suppressing TRPC5 expression diminished the formation of EVs.Incubation of drug-sensitive recipient cells with EVs endowed recipients with drug-resistant properties.It may be worthwhile to further explore the potential of using TRPC5-containing EVs as a diagnostic biomarker for chemotherapy resistance breast cancer.Therefore,we hope to establish the quantitative detection method of TRPC5?EVs?.In this paper,we established the enrichment method of EVs in the cell membrane of chemoresistance breast cancer cells.The MUC1 antibody was used to coat beads to enrich the EVs,and the related parameters were optimized.The method was as follows.The supernatant was collected at 1000 rpm and centrifuged at room temperature for 5 min.The supernatant was incubated with the beads coated with MUC1 antibody for 80 min at 25 o C or 60 min at 37 oC.The reaction system was placed on the magnetic frame,standing 10-15 s,until the magnetic beads and the reaction solution is completely separated.Suck the liquid carefully,then added PBS and clean the beads once gently.Using immunofluorescence staining and laser confocal microscopy,we screened for specific binding effects of extracellular TRPC5 antibody.We selected time-resolved fluorescence immunoassay for the construction of indirect competition TRPC5?EVs?-TRFIA.The optimal detection conditions were as followed.The concentration of TRPC5 peptide was 0.19 ?g/m L.The dilution ratio of anti-TRPC5 antibody was 1: 1000.The dilution ratio of Eu³⁺-IgG?goat anti-rabbit?was 1: 400.The reaction temperature and time were as followed,at 37oC,90 min-80 min;at 25oC,120 min-100 min.The immunoassay method was evaluated in accuracy,sensitivity,stability and recovery rate.The sensitivity was 1 ng/m L and the range was 4-430 ng/mL.The mean value of ED80,ED50 and ED20 were?4.68±0.13?ng/mL,?321.66±2.77?ng/m L and?714.42±4.07?ng/m L.The deviation of the three effects points was small,which indicated that the detection method had better stability.The average recoveries of different sample were in 85%-115%,which met the requirements of immunoassay method,indicating that the detection method had good accuracy.Collecting blood samples from breast cancer chemotherapy resistance,breast cancer chemotherapy tolerance and healthy women,we analyzed these samples using the TRPC5?EVs?-TRFIA method.We found that the result between healthy women and chemosensitivity patients had no significant difference?p>0.6?.There was a significant difference between the test results of chemoresistant and chemosensitivity patients?p<0.0001?,and the results are related to the results of EVs experiments in samples separated by high speed centrifuge?R2=0.69,p<0.0001?.It is worth further study.