Detection Of HER2 Positive Circulating Tumor Cell By Using Anti-HER2-renilla Luciferase Fusion Protein

BackgroundBreast cancer(BC)is the most common cancer in woman all over the world,and it is the second reason of cancer-related death in woman.Approximately 20%breast cancer patients over-expressed human epithelial cell growth factor receptor 2(HER2),HER2 is a 185kDa tyrosine kinase receptor that is encoded by a proto-oncogene located on chromosome 17q21,and it always correlated with disease recurrence,rapid disease progression and poor prognosis.Although target drugs may ameliorate disease progression and improve prognosis,high recurrence and acquired resistance still remain.Patients are selected for HER2 directed therapy based on immunohistochemical(IHC)detection of primary tumor HER2 over-expression as well as on gene-based fluorescence in situ hybridization(FISH).However,breast cancer is a heterogeneous disease,with a hallmark of its genetic instability.Although several studies showed that there has a highly concordance between HER2 status of primary and metastatic site in the same breast cancer,however,after multiple therapies,HER2 status may difference between CTCs and the corresponding primary tumor cells.Therefore,it is inaccurate to make individualized treatment plan based on the primary tumor phenotype.Compared with the existing tumor detection methods,the liquid biopsy is non-invasiveness and can be detected for multiple times and its rapid reaction ability shows significant advantages.The main detection markers include:circulating tumor cells(CTCs),circulating tumor DNA(ctDNA)and exosomes(exosomes),which come from the tumor tissue,exist in the blood,can predicted the tumor progression and drug resistance and other information to guide the individual accurate treatment.Among them,CTCs test is an important tool of "liquid biopsy" and is the hot point of translational medicine research at present.A large number of clinical studies suggest that CTCs has important clinical potential.Circulating tumor cells(CTCs)are cancer cells that fall off the tumor and enter the circulatory system,where they can be detected by simple blood tests,is a non-invasive detection method.Compared with the traditional diagnostic method such as imaging,endoscopy and pathological biopsy diagnosis,CTCs detection has obvious advantages:(1)CTCs are more sensitive than conventional to detect changes in tumors,and can monitor the prognosis,recurrence and metastasis of cancer patients;(2)CTCs detection is a non-invasive diagnostic tool,only a small amount of peripheral blood from the patient can be taken for CTCs detection.Multiple tests can be performed at different stages of disease development and at different times.(3)CTCs can be found in many malignant tumors such as lung cancer,pancreatic cancer,prostate cancer,bladder cancer,and colorectal cancer,etc.However,the proportion of patients with detectable CTCs in the blood varies according to the type of cancer.Therefore,circulating tumor cells can be used as biomarkers for cancer,indicating whether there are tumors at the far end,as well as their genetic conditions and drug susceptibility.CTCs in the peripheral blood of patients with early or metastatic breast cancer can be used as an easily detectable biomarker to monitor tumor progression in real time and can be used for early metastatic detection of breast cancer,then guide treatment decisions.However,there are a large number of white and red cells in the blood.The number of CTCs in 10 ml of blood may be range from several to dozens.Therefore,more sensitive detection methods were needed to detect such rare cells.Exosomes are endosome-derived micro-vesicles that are small and have a specific physical structure.Tumor cell-derived exosomes contain specific antigens and have potential value for immunodetection.Accordingly,we use a bioluminescent protein sequence from the sea pansy,Renilla-reniformis,conjugated to a single chain variable fragment(scFv)of the HER2 antibody,this chimeric anti-HER2-Renilla luciferase fusion protein(anti-HER2-Rluc)targeted HER2 cell surface antigens on CTCs and emitted light.ObjectiveThe aim of this study was to establish a new detection method for HER2 positive circulating tumor cells(anti-HER2-Renal luciferase fusion protein(anti-HER2-Rluc)detection method),which may provide a valuable reference for physicians to improve diagnosis and guide individualized treatment decisions.Methods1.Construction of anti-HER2-Renilla luciferase plasmid,transfection,and measurement of anti-HER2-Renilla luciferase activity in an LB942 luminometer.2.Cell experiments(1)Binding assay to MDA-MB-231 and SK-BR-3:various concentration of cells incubated with anti-HER2-Renillaluciferase,Luminometry was detected.(2)Specificity of anti-HER2-Renilla luciferase fusion protein:SK-BR-3 cells were incubated with HER2 antibody at different dilution concentrations,then cells were incubated with anti-HER2-Rluc,Luminometry was detected.(3)Sensitivity of the anti-HER2-Renilla luciferase fusion protein:various concentration of SK-BR-3 cells was mixed with a fixed number of MDA-MB-231 cells,then cells were incubated with anti-HER2-Rluc,Luminometry was detected.3.Optical imaging of tumor-bearing mice(1)Establishment of mouse tumor model,inject anti-HER2-Rluc via the tail vein.After 2,4,6,and 8 h,mice were injected via the tail vein with coelenterazine and immediately imaged for bioluminescence.(2)At the end of the experiment,the whole blood of the mouse was used to detection of HER2 positive cells.4.Breast cancer patient peripheral blood HER2 positive CTCs detection:isolation of PBMCs with Ficoll density gradient centrifugation,after incubated with anti-HER2-Rluc,Luminometry was detected.5.Screening of CTCs:(1)A fixed number of MDA-MB-231 cells(1 x 105)were added to different amounts of SK-BR-3 cells and then use protein A/G-coated plate to screen HER2 positive cells,the light signal was detected.(2)Breast cancer patients blood were used to do the same experiment.6.Exosome isolation and detection in culture medium:cell culture medium was collected and incubated withHER2-Luc protein.Thermo Fisher Total Exosome Isolation reagent was used to isolate exosomes,the bioluminescence was measured.7.Statistical analysesofdifferent groupwere performed by one-way ANOVA followed by Student's t-test.Each experiment was conducted in triplicate,P<0.05 was considered significant.Results1.HER2 positive cells can be effectively and specifically detected byanti-HER2-Rluceven in a large population of negative background cells.2.Although anti-HER2-Rluc cannot identify HER2 positive tumor cells in mouse breast cancer xenografts model,it can detect HER2 positive CTCs intumor inoculated mouse blood.3.Anti-HER2-Rluc can effectively detected HER2 positive CTCs in patient blood.4.Protein A/G-coated plate screening method could effectively catch HER2 positive CTCs.5.Anti-HER2-Rluc could effectively detected exosomes shed from HER2 positive cell.ConclusionHER2 positive circulating tumor cells and exosomes can be effectively detected by anti-HER2-renilla luciferase fusion protein.