Background
Breast cancer (BC) is the most common cancer in women and accounts for 24.2% of female new cases worldwide (Global Cancer Statistics 2018) [
1]. Although the development of modern medical technology has improved the therapeutic effect of BC, tumor-related death caused by metastasis and recurrence remains major trouble in clinical management. Recently, circulating tumor cells (CTCs) attracts widespread concern as a biomarker for prognosis and monitoring in BC [
2‐
4] and other tumors [
5,
6], benefiting from the easy operation of sampling compared with tissue biopsy. However, 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.
Research on molecular phenotypes of CTCs has emerged over the last decade. The occurrence of epithelial-mesenchymal transition (EMT) in CTCs [
9,
10] results in epithelial-, mesenchymal- and hybrid-subtypes of CTCs. The clinical analysis showed that mesenchymal- and hybrid-CTCs predicted higher metastasis risk and shorter relapse-free survival (RFS) than epithelial-CTCs [
11,
12], demonstrating that CTCs undergoing EMT are behaviorally more invasive. Variations in the expression of hormone receptors (ER/PR), HER-2 and CA15-3 in tumor samples are also investigated as classification markers of CTCs [
13‐
15]. Aktas et al. [
13] profiled ER/PR/HER-2 expression in metastatic BC patients and observed 43–67% of the concordant expression of these markers between CTCs and metastases. Nonetheless, controversy exists about the disease relevance of these CTCs subtypes such as EMT classification, due to the dynamic EMT balance involved in complex metastasis cascade [
16]. The mesenchymal features of tumor cells might disappear once new colonization occurs. Hence, it is desirable to develop new markers to profile the activity and function of CTCs.
Metabolic reprogramming is a vital feature of cancer cells, characterized by enhanced glycolysis, the pentose phosphate pathway, and glutaminolysis [
17]. The reprogrammed metabolism equipped tumor cells with the rapid generation of energy and precursor molecules, which are essential for cell proliferation and metastasis. Our early research and other’s report demonstrated that the metabolic transition from oxidative phosphorylation to aerobic glycolysis plays a vital role in cell invasion [
18,
19]. Accordingly, we hypothesize that the metabolic features could be functional markers for CTCs classification, considering the decisive roles of the metabolic switch in cell behavior. Previously we screened the differentially expressed metabolic genes in high and low metastatic cancer cells. PGK1 and G6PD were found closely correlated with the metastatic potential of tumor cells, and CTCs from prostate cancer patients presented heterogeneous expression of these genes [
20]. Increased expression of PGK1 and G6PD, as critical regulators involved in glycolysis and the pentose phosphate pathway, respectively, reveals active glucose metabolism of tumor cells. Studies demonstrated that up-regulated PGK1 and G6PD in BC tissues is associated with a high risk of recurrent metastasis and poor survival of patients. The mechanisms include the enhancement of energy supply and activation of the HIF-1α pathway in cell migration and invasion [
21,
22]. Therefore, PGK1 and G6PD are potential markers to profile the metabolic activity of CTCs and further indicate the functional CTCs subtypes for BC patients. The clinical relevance of these subtypes remains to be explored.
In this work, we aim to establish a metabolic typing method for CTCs based on the combined PGK1/G6PD markers and investigate the clinical significance of CTCs metabolic classification in BC. Characterization of CTCs metabolism is expected to bring to light the metabolic and functional heterogeneities of CTCs, and further promote the better application of CTCs phenotypic analysis.
Discussion
The predictive and prognostic roles of CTCs have been recently investigated in the BC setting. However, the heterogeneity of CTCs remains a challenge for the clinical application of CTCs test. Here, we used PGK1/G6PD markers to design a metabolic typing method for CTCs by integrating the membrane filtration, fluorescent staining, and multi-mRNA-ISH techniques. We determined the positive/negative criteria for the CTCs metabolic subtypes and evaluated their clinical relevance in BC patients. Comparisons between EMT and metabolic subtypes of CTCs were performed to assess their significance in predicting BC metastasis and prognosis.
Metabolic reprogramming is a hallmark of cancer cells involved in the promotion of tumorigenesis and progression [
30]. PGK1 and G6PD, as crucial metabolic markers of glycolysis and the pentose phosphate pathway, have been demonstrated to favor metastasis of various cancers [
31‐
35]. In vitro and in vivo studies showed that knockdown of PGK1 reduced proliferation and metastasis of SNU449 and HCCLM3 cells, indicating a driving role of PGK1 in hepatocellular carcinoma progression [
31]. Similar studies from Yu et al. [
32] and Ahmad et al. [
33] presented that PGK1 enhanced metastasis of lung and colon cancer through the activated AKT/mTOR pathway and JUN/FOS pathway. Overexpression of G6PD could regulate the Notch1/HES-1 pathway in MCF-7 and MDA-MB-231 cells to promote BC metastasis [
34]. Mele et al. [
35] used Polydatin (100 mg/kg) to block G6PD and the pentose phosphate pathway and observed about 80% inhibition of lymph node metastases in a metastatic mouse model. In the present work, we found PGK1 and G6PD were markedly up-regulated in invasive BC tissues compared with normal mammary tissues and indicated shorter RFS and OS of BC patients. The data were parallel with previous clinical researches, which demonstrated PGK1 and G6PD could predict a high risk of recurrent metastasis and poor prognosis in BC [
22,
36]. These results confirmed the significance of PGK1 and G6PD in BC metastasis and indicated their feasibility for CTCs metabolic classification.
The optimized operation flow for CTCs analysis used in this work facilitates synchronous identification and classification of CTCs. This system presented favorable sensitivity for CTCs capture representing a prevalence of 85.9% in BC patients, which was better than reports on the FDA-approved CellSearch method in BC (38.0–56.7%) [
37,
38]. Moreover, leukocytes were eliminated by firstly size-based filtration and secondly CD45 marker recognition to guarantee the purity of CTCs, equipping the system with higher specificity than the size-based or negative-enriched methods alone. We found the increased tCTCs number was markedly associated with distant metastasis and unfavorable pathological grading, suggesting the importance of CTCs in promoting metastasis and disease progression. In the updated TNM staging for BC, the NCCN guidelines have added a new M0 (i +) category which is defined as “no clinical or radiographic evidence of distant metastases, but deposits of detected tumor cells in the circulation fluids” [
39]. Remarkably, in spite of the low proportion of GM
+CTCs in tCTCs (median 9.4%), GM
+CTCs presented close relevance with metastasis as well as lymph node status and clinical stage. These results provided support for Yu’s [
7] research which demonstrated only a small part of CTCs could finally form metastases and suggested the vital role of GM
+CTCs in promoting metastasis and disease progression.
Although various methods have been developed for CTCs detection, there is a lack of uniform cut-off value for clinical utility. Most CellSearch studies set the standard at 5 CTCs/7.5 mL for BC ever since Cristofanilli’s group demonstrated that the baseline level of ≥ 5 CTCs/7.5 mL was associated with shorter PFS and OS [
40,
41]. Reported standards for other methods include ≥ 1, ≥ 2, and ≥ 3 CTCs in 5–7.5 mL of blood sample [
20,
40,
42]. It is reasonable to define different positive criteria of CTCs for diverse methods since the compositions of captured CTCs are not the same depending on the enrichment principle. Here, we determined the positive threshold of CTCs parameters using the Youden Index curve, which could suggest the best cut-off value by fitting optimal sensitivity and specificity [
28]. By the threshold of tCTCs ≥ 3/5 mL and GM
+CTCs ≥ 2/5 mL, we observed favorable performance of GM
+CTCs and tCTCs in the diagnosis of BC metastasis. Though tCTCs presented higher sensitivity than GM
+CTCs (77.8% versus 66.7%), GM
+CTCs presented higher specificity than tCTCs (91.3% versus 71.8%). The data verified our hypothesis that metabolically active CTCs might represent the aggressive CTCs subpopulations in cancer metastasis. Thus GM
+CTCs turned out to be a more specific marker than tCTCs. This phenomenon could also account for the inconsistent results that advanced clinical stage and lymph node status were relevant to increased GM
+CTCs but not tCTCs. Since only a small part of the CTCs are functionally active [
7,
8], the non-aggressive CTCs such GM
−CTCs may be confounding factors to affect the correlation analysis. Overall, these results indicated GM
+CTCs could be a powerful marker of BC metastasis as a supplement for the tCTCs test.
Ever since the existence of EMT in CTCs was highlighted [
9], several studies have profiled the EMT phenotypes of CTCs in primary and metastatic BC [
43‐
45]. For instance, Mego’s group evaluated the expression of EMT transcription factors (Twist, Snail1, Slug, and Zeb1) in CTCs by RT-PCR in 427 primary BC patients and found EMT-CTCs were associated with inferior prognosis [
43]. Results of the present work were in concordance with the above reports and our early studies on hepatocellular carcinoma and prostate cancer [
11,
20]. We found that BC metastasis was significantly correlated with increased H-CTCs and M-CTCs but not E-CTCs, and H-CTCs presented the best diagnosis performance among the three subtypes. Although the AUCs of EMT subtypes (0.645–0.727) was lower than that of GM
+CTCs (0.854), H-CTCs showed a favorable specificity of 84.8% (tCTCs 71.8% and GM
+CTCs 91.3%) in the metastasis diagnosis. This result also gives evidence for recent research by Liu’s group [
45]. They found epithelial-type CTCs with a restricted mesenchymal transition had the most potent ability of lung metastases formation in a BC mouse model. The hybrid CTCs (E
+M
+) might represent the most plastic cells in cancer metastasis [
46].
Concomitant metabolic reprogramming is induced during the activation of EMT and cancer metastasis. Compared with epithelial cells, mesenchymal tumor cells have different metabolic requirements to meet the increased energy demands for migration and invasion [
47]. Shaul’s group designed a mesenchymal-like metabolic gene expression signature by the database analysis on 978 human cancer cell lines. This signature includes 44 metabolic genes that are essential for EMT but not cell proliferation, involving metabolism regulation of glucose, lipid, and nucleotide [
48]. Noticeably, functional studies demonstrated that knockdown of PGK1 could reverse the EMT process to inhibit invasion of BC cells [
21]. An investigation on hepatocellular carcinoma indicated that G6PD could activate the STAT3 pathway to promote EMT and further favor cancer metastasis [
49]. Consistent with the above studies, we observed an association of GM
+CTCs number with H-CTCs (R
2 = 0.852,
P < 0.001) and M-CTCs (R
2 = 0.591,
P < 0.001) numbers, whereas no obvious correlation was observed between GM
+CTCs and E-CTCs (
P = 0.10; Additional file
8: Figure S2). Moreover, the proportions of GM
+CTCs in H-CTCs (51.8%) and M-CTCs (47.5%) were remarkably higher than those in E-CTCs (27.6%; Additional file
8: Figure S2). These results demonstrated an intensive correlation between CTCs metabolic subtypes and EMT subtypes. The two classifications of CTCs, representing respectively the functional and morphological features of heterogeneous CTCs, could synergistically enrich the significance of CTCs test.
Metastasis is the leading cause of recurrence and tumor-related death. Given the driving role of CTCs in cancer metastasis, we investigated the prognostic role of CTCs parameters. Positive tCTCs, M-CTCs, and GM
+CTCs presented a predictive function of increased progression ratio and decreased 2 years PFS ratio. Previous studies on the prognosis value of EMT CTCs showed controversial conclusions [
43,
50,
51]. Apart from the dynamic changes of EMT status occurred in CTCs dissemination, reasons for the conflicts include different detection methods involved in these researches and the lack of a uniform cut-off standard. Our study indicated GM
+CTCs to be a potential predictor for inferior prognosis in BC patients. In addition, the analysis of TCGA data revealed the up-regulation of PGK1 and G6PD in common cancers such as colon, lung and gastric cancers (Additional file
9: Figure S3). These results suggest the possibility of applying the PGK1/G6PD-based CTCs metabolic classification method in other cancer diseases besides BC, though further validation remains in need before practical applications.
One shortcoming of this study is the limited follow-up time (maximum 24 months), which results in the immaturity of overall survival investigation. Long term observational studies on this cohort as well as an expanded sample size are underway to explore the prognostic value of CTCs subtypes. Furthermore, direct detection of the metabolic phenotypic features of the CTCs subpopulation is needed in future research to validate the active metabolic level of GM+CTCs. The emerging techniques such as microfluidic chip, nanomaterials and AIEgens (luminogens with aggregation-induced emission) might provide vital ideas for convenient CTCs analysis methods which target on glucose uptake, oxygen consumption, lactate production, ATP synthesis or other key regulators of metabolism. With the technical development of single-cell sequencing, multi-omics analysis and three-dimensional cell culture, the in vivo and in vitro mechanism investigations on GM+CTCs are desirable to illustrate the CTCs-related metabolic reprogramming, which could further enrich the metastasis theory and promote the application of CTCs classification.
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