USF2-Mediated APOE-Cholesterol Signaling Promotes CTC Clustering and Metastasis in Non-Small Cell Lung Cancer

Circulating tumor cell (CTC) clustering is a key driver of metastatic progression in non-small cell lung cancer (NSCLC), but the underlying molecular mechanisms remain largely unknown. Here, we performed single-cell proteomic sequencing on CTC clusters and individual CTCs isolated from lung cancer patients, identifying 912 proteins in total, of which 246 were significantly upregulated in clusters versus single CTCs. Enrichment analyses revealed a prominent involvement of lipid and cholesterol metabolism pathways. Apolipoprotein E (APOE) emerged as a central hub, exhibiting marked upregulation in CTC clusters. Functional experiments showed that cholesterol supplementation promoted cluster formation in NSCLC cell lines and accelerated metastatic dissemination in vivo, whereas APOE depletion impaired clustering capacity. Mechanistically, we identified USF2 as a potential transcriptional regulator of APOE through direct binding to the promoter, as supported by Chromatin Immunoprecipitation (ChIP) and reporter assays. Clinical validation in a cohort of 75 lung cancer patients demonstrated that elevated APOE expression was strongly associated with advanced pathological grade, distant metastasis, and poor survival. Taken together, these findings establish a novel USF2–APOE–cholesterol axis that governs CTC clustering and metastatic progression, positioning APOE as both a prognostic biomarker and a potential therapeutic target for NSCLC metastasis.