Investigation Of CD47 Distribution And Regulatory Mechanisms On The Membrane Of Non-small Cell Lung Cancer Cells By Super-resolution Fluorescence Microscopy

As an inhibitory node in the clearance of target cells by immune cells,the immune checkpoint plays an important role in regulating the strength and persistence of the immune response.Tumor cells gain immune resistance by hijacking this inhibitory pathway and achieve tumor immune escape.Immune checkpoint inhibitors restore the aggressiveness of immune cells against tumors by blocking the interaction of immune checkpoints with ligands on tumor cells.The survival benefits shown by acquired immune checkpoint inhibitors in the treatment of non-small cell lung cancer,an important tool in the immunotherapy of malignant tumors,have driven researchers to explore multiple immune checkpoint inhibitors.As a representative of the innate immune checkpoint,previous studies on CD47 have focused on its expression and antitumor effects of inhibitors,while less is known about its spatial structure,distribution,aggregation and other structural characteristics.At the same time,the abnormalized assembly of proteins on cell membranes is closely linked to the development of diseases.In order to understand the link between structural variations and functional abnormalities of the CD47 transmembrane protein in tumor immune escape,we used an efficient and convenient direct observation method.Therefore,in order to understand the relationship between structural changes and functional abnormalities of CD47 transmembrane protein in tumor immune escape,we used the most convenient and effective direct observation method to investigate.However,the size of the protein is below the diffraction limit of ordinary light microscopy resolution and its detailed structural features cannot be clearly revealed.With the introduction of super-resolution fluorescence microscopy that breaks the limits of optical diffraction,great progress has been made in the fields of spatial structure,distribution patterns,and intermolecular interactions of cell membrane proteins.The direct Stochastic Optical Reconstruction Microscopy（d STORM）is the most widely used microscope,with its high resolution,ease of operation and relatively easy construction conditions.In this paper,we have studied CD47 on the membrane of non-small cell lung cancer cells using d STORM imaging technique,and the results are as follows:1.CD47 is heterogeneously distributed on the membrane surface of both normal and cancer cells,either sporadically in dots or in clusters.Compared to normal cells,cancer cells formed denser clusters of CD47 protein,with a larger average cluster size and higher aggregation,but there were no significant differences in CD47 protein expression and distribution between cancer cells.The results from primary cells also confirm that CD47 is expressed in higher amounts on cancer cells than on normal cells,while forming larger and more abundant protein clusters.This work demonstrates that CD47 normally maintains immune homeostasis as small and sparse protein clusters in normal cells,while in cancer cells it provides a structural basis for immune escape by increasing the degree of protein aggregation to respond more rapidly to external signals.2.The functional units on the cell membrane are tightly connected structurally and functionally related to each other.To determine the mechanism of CD47 aggregation and distribution,we intervened on CD47-associated proteins and functional regions and observed changes in CD47 with d STORM super-resolution microscopy.The co-localization of CD47 with lipid rafts was shown by two-colour d STORM imaging,and the stabilization of lipid raft structure contributed to the aggregation of CD47 into larger protein clusters as determined by cholesterol depletion and recovery experiments.Meanwhile,the degree of CD47 aggregation was significantly reduced after treatment with CB and the N-acetyl-β-D-gulcosaminides,NAG.The experiments demonstrated that cytoskeleton and N-glycosylation are indispensable for CD47 clustering.A degree of co-localization of CD47 with integrinα_vβ₃ was determined by two-colour d STORM imaging.Gene silencing of integrinα_vβ₃ was able to significantly attenuate CD47 aggregation.These experimental results suggest that the structural integrity of the CD47-associated complex is a key factor in supporting CD47 aggregation into clusters.3.The expression of CD47 on tumor cells is regulated by multiple factors.The TNF-NFκB signaling pathway,a key bridge mediating the relationship between inflammation and tumor,also has an important influence on the degree of aggregation and distribution patterns of CD47.The changes in CD47 expression and distribution were observed by adding stimulators and inhibitors of the TNF-NFκB inflammatory pathway to the A549 cell line.When the signaling pathway is activated,it promotes the expression of CD47 and facilitates the aggregation of CD47 protein into larger clusters.When the signaling pathway was inhibited,CD47 expression and aggregation tended to decrease,and this inhibition was also effective in chemo-resistant cancer cells.We also observed a similar regulation of integrin α_vβ₃ by this signaling pathway and influenced the degree of co-localization of CD47 and integrin α_vβ₃.These results suggest that the TNF-NFκB inflammatory pathway is important for CD47 protein clustering itself and produces significant regulation of the CD47 complex by affecting the expression and distribution of integrin α_vβ₃.