Studying The Distribution And Mechanism Of Trop2 On Non-small Cell Lung Cancer Cell Membranes By Super-resolution Imaging

In worldwide,non-small cell lung cancer(NSCLC)has been the leading cause of cancer deaths.Before the introduction of tyrosine kinase inhibitors(TKIs),most advanced NSCLC patients who received platinum-based chemotherapy had an overall survival of less than one year.In the 1970 s,patients’ baseline performance,disease severity and weight loss,etc.were the main predictors of survival.With continuous research of abnormal molecules that lead to tumor growth and survival,the treatment of NSCLC has been greatly changed.This means the treatment of NSCLC is moving into an age based on the molecular characteristics of tumors,and personalized molecular targeted therapy and clinical applications are coming true.NSCLC is a multi-step and complex biological process,and may last from several years to decades.The specific proteins and genes always participate together and eventually lead to the occurrence and development of lung cancers.In addition,the normal or abnormal expression of proteins at the molecular level will also affect the growth and survival of the cells,and then every transition from somatic cells to cancer cells would affect the development of cancers.Cell membrane is an important part of cell life and it is also the place where many biochemical processes occur.Cell membrane proteins play important roles in a large number of physiological processes.Therefore,researches which analyzing the different expression of proteins on membrane will provide clues to understand the molecular regulation mechanism of tumors,and further realize potential of diagnostic and therapeutic in the treatment of solid tumors.In recent decades,studies on cell signaling pathways had made great progress.Although many proteins in important signaling pathways have been identified,improving the understanding of biological processes at the molecular level will help us describe a more detailed scene on cell membrane and understand the mechanism of lung cancer more completely.The membrane protein Trop2 was first discovered as a biomarker of invasive trophoblast cells,and then many studies have found that it plays driving roles in a variety of tumors,which can endow cancer cells with the ability to proliferate and invade,and also participate in cancer metastasis.Therefore,studying the expression and mechanism of Trop2 in NSCLC will help to broaden the potential therapeutic targets of NSCLC.Since the molecular size of membrane protein is generally tens of nanometers,which is much smaller than the diffraction limit of ordinary optical microscopes,it cannot be directly analyzed.However,in recent years,the direct stochastic optical reconstruction microscopy(dSTORM)has broken the limitation of diffraction,and help us convenient complete nanoscale high-resolution imaging.Therefore,we use super-resolution imaging to study the distribution pattern of Trop2 on NSCLC cell membranes and mechanisms,which will make an important contribution to elucidating the mechanism of specific proteins in lung cancer.In experimental preparation stage,we first confirmed the basic conditions such as the number of original frames of the image collected by dSTORM,the reconstruction of super-resolution data,and the calculation method of localization density.Then we used Nano J-SQUIRREL and SR-Tesseler to evaluate and quantify the image.The experiment one,we used dSTORM to explore the distribution pattern of Trop2 on cell membranes in three main NSCLC cell lines and HBE cell line.We reconstructed the subcellular structure model of Trop2 on membranes.It was found that compared with the HBE cells,Trop2 distributed more molecules on the membranes of NSCLC cells.Meanwhile,the distribution pattern of Trop2 was also different between different cell lines,cancer cells generated more and larger clusters consisting of more molecules than normal cells.The experiment two,we confirmed the distribution pattern in primary lung cancer cells of human was consistent with the cell lines,which reminded us the overexpress of Trop2 and clustering distribution on cell membranes have potential diagnostic value for lung cancer.The experiment three,we used dSTORM to study the distribution mechanism of Trop2 on cell membranes.First,we studied the influence of cell polarity on the distribution characteristics of Trop2.We found that Trop2 had higher density and tended to form more and larger clusters on the apical membranes than on the basal membranes in both A549 cells and HBE cells.We also found that the apical clusters contained more molecules than the basal clusters.This means that cell polarity affects the heterogeneous distribution of membrane proteins.Then,we used dual-color imaging and depletion of lipid rafts by dSTORM to investigate the relationship of Trop2 and lipid rafts.The results indicated that there was a high degree of colocalization between Trop2 clusters and lipid rafts,and depletion of lipid rafts will destroy the distribution pattern of Trop2,and further reduce the amount of surface Trop2 and degree of Trop2 clustering.In addition,Trop2 was also related to actin cytoskeleton on the membranes of NSCLC cells.The Trop2 clustering was limited when actin was disrupted,such as the decrease of the clusters number and area.However,the effect of actin cytoskeleton on Trop2 clustering was less than the lipid rafts.At last,in order to further explore whether there is abnormal activation of Trop2 in NSCLC,we chose IGF-1 to interact with Trop2,and explored the relationship between its activation state and Trop2 distribution.We found that IGF-1 stimulation promoted the expression level of Trop2,induced Trop2 to form larger clusters,and increased the number of molecules in clusters in HBE cells.However,there was only the increase of Trop2 expression level in cancer cells.Thus,we conclude that Trop2 is in an active state in cancer cells and exists in the form of clusters.The experiment four,we studied the expression of downstream related signal proteins and cell migration and invasion after the clustering pattern was disrupted.We found that the downstream signal proteins such as ERK1/2,p ERK,Cyclin D1,and Cyclin E1 were all decreased after Trop2 cluster distribution was disrupted.Moreover,the corresponding cancer cell migration and invasion ability were also restricted.However,the downstream signal proteins and cell invasion ability were increased after IGF-1 treatment,suggesting that Trop2 clustering on membranes has an important effect on downstream related signal proteins and cell phenotype.Overall,our research studied the Trop2 distribution and regulation mechanism on NSCLC cell membranes by dSTORM,and connected the location,distribution characteristics and molecular composition of Trop2 to its functions.The works will help us understand the structural characteristics,localization changes and interaction relationship of Trop2 in lung cancers on a nanoscale scale,and lay a foundation for further revealing the mechanism of complex signal transduction pathways.