Heterogeneity Analysis Of Genetic Mutation And PD-L1 Expression In Circulating Lung Cancer Cells

Background:Tumor heterogeneity often leads to differences and failures in response to targeted tumor therapy(such as EGFR)and anti-PD-1 therapy.The traditional single-point biopsy of the primary tumor only represents some tumor cell clones or subclones,which lacks metastasis information.Circulating tumor cells(CTCs)are theoretically derived from multiple tumors in the body,with better representation and with more comprehensive information coverage.Tumor heterogeneity can be expressed in the CTCs genome,in-depth studies involved in single CTCs level can help the final elucidate the phenomenon of tumor heterogeneity,and analyze the dynamic and persistent mutation evolution of the genome during tumor progression.Method:1.Single CTCs separation technology and detection of lung cancer patients.Based on Cell Search principle,we optimized Ep CAM antibody and improved cell membrane breaking process.For patients with lung cancer,5ml of anticoagulated peripheral blood was used to enrich CTCs by using Ep CAM immunomagnetic beads.CTCs were identified by Ep CAM fluorescence signal amplification,CD45~+and Hoechst immunofluorescence staining,and a single CTCs was then isolated by microscopic manipulation to perform single-cell-level genome DNA extraction and amplification,followed by sequencing of 72 target genes that related lung cancer.Copy number variation(CNV)and gene mutations of each gene were analyzed to further confirm the isolated CTCs.A total of 98 patients with lung cancer were enrolled,and the efficacy of clinical detection of CTCs was also analyzed.2.Heterogeneity analysis of lung cancer tissue and single CTCs gene mutation.(1)Heterogeneity analysis of EGFR gene mutation.9 CTCs with EGFR-sensitive mutations in the tissues were involved for analyzing the heterogeneity of the mutant genes between CTCs and between CTCs and tissues.(2)Heterogeneity analysis of the clinical association-driven gene mutations performed(ALK,BRAF,ROS1,RET,MET,etc.)among CTCs.3.Heterogeneity of CTCs-PD-L1 protein expression and detection of regulatory factors.(1)31 patients with lung cancer and CTCs matching samples were inclved,by using establish CTCs protein detection method to detect and analyze the consistency of PD-L1 expression between CTCs and tissues;(2)To determine the level of cytokines in peripheral blood and analyze the correlation between cytokines and CTCs-PD-L1expression.Among the different cytokines that may regulate the expression of PD-L1in lung cancer cell lines,IFN-?is tested to be a main regulatory for PD-L1 expression,and we used experiment in vitro to verify the CD137 signaling pathway may induces T lymphocytes to secrete IFN-?,by which promoting the expression of PD-L1 in lung cancer cells.Result:Part I:Optimization of single-circulation tumor cell isolation technology in peripheral blood.Goat anti-mouse polyclonal antibody(Goat Anti-Mouse p Ab,dylight488)was selected to stain the tumor cells of mouse anti-human Ep CAM antibody immunomagnetic beads,and the secondary antibody labeling staining was used to effectively increase the fluorescence intensity and simplify the cell membrane breaking process.A staining scheme for identifying CTCs and CD45~+to exclude white blood cells based on Ep CAM~+is presented.Among 98 lung cancer patients,the detection rate of CTCs per 5ml blood was 86.7%,the number of detections was between 1-36,and the median was 2.CTCs have high levels of CNVs mutations and gene mutations sites analyzed by lung cancer-related 72 genes.CTCs showed gene variation characteristic of tumor cells.Part II:Gene level heterogeneity of lung cancer cells in single circulation CTCsFive CTCs of lung adenocarcinoma patients with EGFR mutation had been detected in lung tissue were selected.A total of 12 CTCs samples were sequenced for the 72 genes,and 9 CTCs of 4 patients finally obtained valid data.Based on the single-cell gene variation,the heterogeneity of gene mutations is analyzed in depth.All CTCs samples are detected by NGS sequencing method,and all tissues are detected by ARMS method.The results show that mutation heterogeneity is widespread,including:(1)Heterogeneity of mutation number,type and mutation site among CTCs of the same pathological type.Although the pathological type is consistent,the number of mutations in CTCs samples is quite different,and there is heterogeneity;(2)Heterogeneity of EGFR mutations between CTCs and tumor tissues in the same patient;(3)Heterogeneity of mutation number,type and mutation site between CTCs in the same patient Genes are ubiquitous.Part III:Heterogeneity of lung cancer tissues and CTCs at PD-L1 protein(1)With PD-L1-PE-CF594(M1H1)antibody for detection of PD-L1 protein expression,we established the detection method of CTC-PD-L1.The identification criteria of PD-L1~+CTCs are:Ep CAM~+/PD-L1~+/Hoechst~+/CD45~-.were set up.(2)The expression of PD-L1 in 13 human lung cancer cell lines was analyzed.The expression of PD-L1 in CTCs is obviously heterogeneous.PD-L1 is expressed differently in different lung cancer cell lines,with a total positive rate of 69.2%,mainly in adenocarcinoma.The expression of CTCs-PD-L1 in 57 patients was also heterogeneous,and the positive rate of PD-L1 expression was 0-100%.(3)IFN-?is the main PD-L1 expression regulating cytokine.Lung cancer cell expressing CD137L can promote IFN-?secretion in antigen-induced T lymphocyte,which is one of the definite mechanisms of up-regulation of PD-L1 expression in tumor cells.Conclusion:(i)A new CTCs staining technique has been established,which significantly improves the detection rate,and can further for analyzing the characteristics of CTCs tumor cells at the genetic level,that also providing a powerful means for study of gene mutation heterogeneity at individual CTCs.(ii)The single-cell gene sequencing shows that the tumor is highly heterogeneous,showing widespread differences in the number and type of lung cancer-related gene mutations,which may be related to drug resistance.Single-cell level studies have shown that individual CTCs are rich in genetic information,which is more valuable for in-depth analysis of tumor heterogeneity,and is potentially instructive for the design of targeted treatment and monitoring responses.The preliminary research data of this study still need to be refined to analyze the obtained biological information of CTCs.(iii)There are a clear differences in the expression of PD-L1 between CTCs in same patient,and there is also a difference in the expression of PD-L1 between CTCs and the matched tissues,all these indicating that clinical PD-L1 detection is difficult to accurately predict the tumor response to anti-PD-1 treatment.Lung cancer cell expressing CD137L,as a co-stimulatory molecule,could induce T cells to release IFN-?and then promotes PD-L1 expression in tumor cells,which is a potential tumor escape mechanism,indicating the combined therapeutic value of CD137 and PD-1/PD-L1 antibody.