Abstract
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(pos)) primary tumors, HK2 enables resolving cytokeratin-negative (HK2(high)/CK(neg)) CTCs as a prevalent population in about half of the peripheral blood samples with positive CTC counts. However, HK2(high)/CK(neg) tumor cells are a minority population in pleural effusions and cerebrospinal fluids. Single-cell analysis shows that HK2(high)/CK(neg) CTCs exhibit smaller sizes but consistent copy number variation profiles compared with CK(pos) 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(high)/CK(neg) CTCs display metastasis and EGFR inhibitor resistance-related molecular signatures and are selectively enriched in patients with EGFR(L858R) driver oncogene mutation as opposed to EGFR(19Del) , which is more frequently found in patients with prevalent CK(pos) CTCs in the blood. Consistently, treatment-naïve patients with a larger number or proportion of HK2(high)/CK(neg) 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.