Abstract
BackgroundCirculating tumor cells (CTCs) has been demonstrated as a promising liquid biopsy marker for breast cancer (BC). However, the intra-patient heterogeneity of CTCs remains a challenge to clinical application. We aim at profiling aggressive CTCs subpopulation in BC utilizing the distinctive metabolic reprogramming which is a hallmark of metastatic tumor cells.MethodsOncomine, TCGA and Kaplan–Meier plotter databases were utilized to analyze expression and survival relevance of the previously screened metastasis-promoting metabolic markers (PGK1/G6PD) in BC patients. CTCs detection and metabolic classification were performed through micro-filtration and multiple RNA in situ hybridization using CD45 and PGK1/G6PD probes. Blood samples were collected from 64 BC patients before treatment for CTCs analysis. Patient characteristics were recorded to evaluate clinical applications of CTCs metabolic subtypes, as well as morphological EMT subtypes classified by epithelial (EpCAM/CKs) and mesenchymal (Vimentin/Twist) markers.ResultsPGK1 and G6PD expressions were up-regulated in invasive BC tissues compared with normal mammary tissues. Increased tissue expressions of PGK1 or G6PD indicated shortened overall and relapse-free survival of BC patients (P < 0.001). Blood GM+CTCs (DAPI+CD45−PGK1/G6PD+) was detectable (range 0–54 cells/5 mL) in 61.8% of tCTCs > 0 patients. Increased GM+CTCs number and positive rate were correlated with tumor metastasis and progression (P < 0.05). The GM+CTCs ≥ 2/5 mL level presented superior AUC of ROC at 0.854 (95% CI 0.741–0.968) in the diagnosis of BC metastasis (sensitivity/specificity: 66.7%/91.3%), compared with that of tCTCs (0.779) and CTCs-EMT subtypes (E-CTCs 0.645, H-CTCs 0.727 and M-CTCs 0.697). Moreover, GM+CTCs+ group had inferior survival with decreased 2 years-PFS proportion (18.5%) than GM+CTCs− group (87.9%; P = 0.001).ConclusionsThis work establishes a PGK1/G6PD-based method for CTCs metabolic classification to identify the aggressive CTCs subpopulation. Metabolically active GM+CTCs subtype is suggested a favorable biomarker of distant metastasis and prognosis in BC patients.
Highlights
Circulating tumor cells (CTCs) has been demonstrated as a promising liquid biopsy marker for breast cancer (BC)
Through a multi-factor weighted model enrolling the array results, cell validation, expression rates in CTCs and metastasis-related functions reported by literature, Phosphoglycerate kinase 1 (PGK1) and glucose-6-phosphate dehydrogenase (G6PD) were determined the optimal metabolic markers related to tumor metastasis [20]
Before the markers were set for CTCs classification, we validated their relevance with BC using the Oncomine, The Cancer Genome Atlas (TCGA) and Kaplan–Meier plotter databases
Summary
Circulating tumor cells (CTCs) has been demonstrated as a promising liquid biopsy marker for breast cancer (BC). The intra-patient heterogeneity of CTCs remains a challenge to clinical application. We aim at profiling aggressive CTCs subpopulation in BC utilizing the distinctive metabolic reprogramming which is a hallmark of metastatic tumor cells. Circulating tumor cells (CTCs) attracts widespread concern as a biomarker for prognosis and monitoring in BC [2,3,4] and other tumors [5, 6], benefiting from the easy operation of sampling compared with tissue biopsy. Recent experimental studies presented that only a few CTCs population could succeed in metastases formation in mouse models, revealing intra-patient heterogeneity of CTCs [7, 8]. How to identify the aggressive CTCs subpopulation is an urgent yet challenging problem. Phenotypic analysis of CTCs could throw light on their outcome in metastasis to further provide information for disease assessment
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