Super-Resolution Fluorescence Imaging Reveals the Role of CD24 Clusters in NSCLC Proliferation

CD24, a glycosylphosphatidylinositol-anchored cell surface protein, is frequently dysregulated in non-small-cell lung cancer (NSCLC) and contributes to tumor proliferation, metastasis, and immune evasion. However, the nanoscale spatial organization of CD24 and its functional relevance to NSCLC proliferation remain incompletely understood. Here, we employed direct stochastic optical reconstruction microscopy (dSTORM) to visualize CD24 clusters on the membranes of NSCLC cell lines, freshly isolated primary NSCLC cells, and clinical NSCLC tissues. Our results revealed that the increased clustering of CD24 promotes proliferation. Using an Asn36 single-point mutant that disrupts CD24 clustering without altering its expression level, we confirmed that CD24 clustering, rather than CD24 expression alone, drives NSCLC proliferation. More importantly, we found that the clustering disruption suppressed the NSCLC cell proliferation by reducing the phosphorylation of PI3K, AKT, and mTOR, which was consistent with the result of treating with PI3K inhibitor. Our findings highlight the role of CD24 nanoscale clustering in promoting NSCLC proliferation via the PI3K/AKT/mTOR axis, providing mechanistic insights into CD24-mediated tumorigenesis and identifying CD24 clustering as a potential prognostic biomarker and therapeutic target for NSCLC.