Study On CDCP1 Distribution And Regulatory Mechanisms On The Cell Membrane Of Non-small Cell Lung Cancer By Super-resolution Fluorescent Microscope

Background:Lung carcinoma is characterized by high morbidity and mortality.Targeted therapy,as a precise treatment method,is one of the most important methods for clinical treatment of lung carcinoma.Because of the drug resistance of targeted therapy,it is of great significance to find new therapeutic targets and solve the problem of drug resistance to improve the prognosis of lung cancer patients.CDCP1 is a type I single-channel transmembrane protein that is involved in a variety of important biological processes including proliferation,anchorage-free survival,and metastasis of tumor cells.Previous studies have shown that plasma level of CDCP1 is abnormal increased before the diagnosis of lung cancer;moreover,CDCP1 is up-regulated in lung cancer,and its expression level is obviously associated with disease-free survival,overall survival and other prognostic indicators;and CDCP1-targeted therapy can inhibit tumor progression and restore sensitivity to agents such as EGFR TKI.In conclusion,CDCP1 is expected to be a biomarker and target for lung cancer screening,diagnosis,treatment,and has an indicator role in risk stratification and prognosis evaluation.Therefore,it is important to further investigate the function and related mechanisms of CDCP1 in lung cancer.The plasma membrane is an essential component of the cell,and it is also a vital place for many cancer-related proteins to function,interact and trigger a variety of biochemical reactions.Membrane proteins are the main carriers of biofilm functions,and the information transmission between cells or with the external environment is often carried out through membrane proteins.However,the membrane proteins distributed unevenly and they often arranged into complexes,clusters or other highly ordered forms to perform their corresponding functions.Hence,the study of the proteins’ distribution and its regulatory mechanism on the cell membrane surface will help us to understand its function and the mechanism as cancer-promoting molecules more comprehensively and deeply.Due to the light diffraction limit,biological structures smaller than 200nm,such as protein molecules,cannot be observed by ordinary microscopy.However,super-resolution microscopy can break the diffraction limit and increase the resolution to 20nm.Therefore,it is suitable for observing the localization,distribution differences and regulatory mechanisms of proteins at the single-molecule level.Methods and content:In this study,we used super-resolution fluorescent microscope as the main research method,and the ImageJ and SR-Tesseler software for image reconstruction and data analysis before quantified and evaluated;in addition,we also used bioinformatics tools,western blot and other traditional experimental methods as auxiliary research tools to verify the expression and functional phenotype of CDCP1.In the first part of this paper,we analyzed the level of CDCP1 mRNA in non-small cell lung cancer(NSCLC)and its relationship with prognosis using TCGA database.Then,we used super-resolution microscopy to observe the spatial distribution of CDCP1 in non-small cell lung cancer and normal lung cells at different levels.By analyzing the differences of CDCP1 expression and distribution,the distribution characteristics of CDCP1 on lung cancer cell membrane were obtained.In the second part of the study,we regulated the activity and functional state(positive and negative)of CDCP1,and further observed its spatial distribution by using super-resolution microscopy.By combining biochemical and molecular biological methods,we revealed the regularities between functional activities and distribution patterns of CDCP1.In the third part of the study,we investigated the distribution and changes of CDCP1 in gefitinib(EGFR TKI)-resistant and non-resistant cell lines by using super-resolution microscopy.Based on the relationship between protein activity and distribution,we further analyzed how CDCP1 plays a role in gefitinib resistance by regulating its distribution pattern.Results:(1)The expression level of CDCP1 is dramatically increased and is related with poor prognosis in lung cancer;knockdown of CDCP1 reduced the migration ability of lung cancer cells.We used dSTORM to observe the spatial distribution pattern of CDCP1 at different levels from cultured cell lines to extracted primary cells,and then pathological tissue sections,and found that cancer cells could generate more and larger protein clusters containing more CDCP1 molecules than normal cells.This feature is more evident in primary cells and tissues,and the distribution of proteins in pathological tissue shows obvious heterogeneity.(2)When the activity of CDCP1 is inhibited,its clustering ability is also inhibited,resulting in the formation of smaller,fewer and more sparse protein clusters.This distribution pattern prevents it from exerting its cancer-promoting function.On the contrary,when the expression level of CDCP1 is increased or its function is promoted,more and denser clusters can be formed as functional domains to exert their functions.(3)The CDCP1 expression is related to the resistance of EGFR TKI(gefitinib).In non-drug resistant cells,gefitinib significantly decreased the expression level of CDCP1 and its clustering ability.In resistant cells,however,the use of gefitinib induced increased CDCP1 expression levels,even at very low concentrations.CDCP1 could exert its function of promoting drug resistance by up-regulating its expression and forming larger and denser protein clusters.Knocking out CDCP1 in drug-resistant cells can improve its sensitivity to gefitinib.Conclusions:(1)CDCP1 is distributed in an almost dispersed form in normal lung tissue and therefore does not perform its cancer-promoting function;while in the patient’s tumor tissue,it is often distributed as protein clusters that can exert their cancer-promoting effects.(2)The cells containing protein clusters of CDCP1 with more,larger and and higher degree of aggregation in tumor tissue may be more prone to invasion and metastasis.(3)CDCP1 is involved in the resistance of gefitinib,and the effect of gefitinib on the expression and distribution of CDCP1 is related to the sensitivity of cells to gefitinib.Therefore,CDCP1 may be involved in drug resistance by regulating the distribution of its protein clusters.