Hexokinase 2 discerns a novel circulating tumor cell population associated with poor prognosis in lung cancer patients.

Unlike other epithelial cancer types, circulating tumor cells (CTCs) are less frequently detected in the peripheral blood of non-small cell lung cancer (NSCLC) patients using epithelial marker-based detection approaches despite the aggressive nature of NSCLC. Here, we demonstrate hexokinase-2 (HK2) as a metabolic function-associated marker for the detection of CTCs. In 59 NSCLC patients bearing cytokeratin-positive (CK) primary tumors, HK2 enables resolving cytokeratin-negative (HK2/CK) CTCs as a prevalent population in about half of the peripheral blood samples with positive CTC counts. However, HK2/CK tumor cells are a minority population in pleural effusions and cerebrospinal fluids. Single-cell analysis shows that HK2/CK CTCs exhibit smaller sizes but consistent copy number variation profiles compared with CK counterparts. Single-cell transcriptome profiling reveals that CK expression levels of CTCs are independent of their epithelial-to-mesenchymal transition (EMT) status, challenging the long-standing association between CK expression and EMT. HK2/CK CTCs display metastasis and EGFR inhibitor resistance-related molecular signatures and are selectively enriched in patients with driver oncogene mutation as opposed to , which is more frequently found in patients with prevalent CK CTCs in the blood. Consistently, treatment-naïve patients with a larger number or proportion of HK2/CK CTCs in the blood exhibit poor therapy response and shorter progression-free survival. Collectively, our approach resolves a more complete spectrum of CTCs in NSCLC that can potentially be exploited to identify patient prognosis before therapy.