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. 2021 May 27:11:601668.
doi: 10.3389/fonc.2021.601668. eCollection 2021.

Association of Preoperative NANOG-Positive Circulating Tumor Cell Levels With Recurrence of Hepatocellular Carcinoma

Yongrong Lei  1 , Xishu Wang  2 , Heng Sun  1 , Yuna Fu  1 , Yichen Tian  1 , Ludi Yang  2 , Jianhua Wang  1 , Feng Xia  2
Affiliations

Association of Preoperative NANOG-Positive Circulating Tumor Cell Levels With Recurrence of Hepatocellular Carcinoma

Yongrong Lei et al. Front Oncol. .

Erratum in

Abstract

Background: Cancer stem cells (CSCs) and Circulating tumor cells (CTCs) have been proposed as fundamental causes for the recurrence of hepatocellular carcinoma (HCC). CTCs isolated from patients with HCC illustrate a unique Nanog expression profile analysis. The aim of this study was to enhance the prediction of recurrence and prognosis of the CTC phenotype in patients with HCC by combining Nanog expression into a combined forecasting model.

Subjects materials and methods: We collected 320 blood samples from 160 patients with HCC cancer before surgery and used CanPatrol™ CTC enrichment technology and in situ hybridization (ISH) to enrich and detect CTCs and CSCs. Nanog expression in all CTCs was also determined. In addition, RT-PCR and immunohistochemistry were used to study the expression of Nanog, E-Cadherin, and N-Cadherin in liver cancer tissues and to conduct clinical correlation studies.

Results: The numbers of EpCAM mRNA+ CTCs and Nanog mRNA+ CTCs were strongly correlated with postoperative HCC recurrence (CTC number (P = 0.03), the total number of mixed CTCS (P = 0.02), and Nanog> 6.7 (P = 0.001), with Nanog > 6.7 (P = 0.0003, HR = 2.33) being the most crucial marker. There are significant differences in the expression of Nanog on different types of CTC: most Epithelial CTCs do not express Nanog, while most of Mixed CTC and Mesenchymal CTC express Nanog, and their positive rates are 38.7%, 66.7%, and 88.7%, respectively, (P=0.0001). Moreover, both CTC (≤/> 13.3) and Nanog (≤/>6.7) expression were significantly correlated with BCLC stage, vascular invasion, tumor size, and Hbv-DNA (all P < 0.05). In the young group and the old group, patients with higher Nanog expression had a higher recurrence rate. (P < 0.001).

Conclusions: The number of Nanog-positive cells showed positive correlation with the poor prognosis of HCC patients. The detection and analysis of CTC markers (EpCAM and CK8, 18, CD45 Vimentin,Twist and 19) and CSCs markers (NANOG) are of great value in the evaluation of tumor progression.

Keywords: cancer stem cells; circulating tumor cells; epithelial-mesenchymal; hepatocellular carcinoma; recurrence.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Use RNA-ISH technology to enrich and analyze CTCs in blood samples of HCC patients (A) The expression of epithelial marker mRNA (EpCAM and E-cadherin) and (B) mesenchymal marker mRNA (Twist and vimentin) and CSC marker mRNA (nanog) were validated by qPCR, p<0.05; (C) The protein expression levels of epithelial markers (EpCAM and E-cadherin), p<0.001. (D–G) The total number of CTCs, epithelial CTCs, mixed CTCs, and mesenchymal CTCs correlated with BCLC staging, p<0.05. **represents P<0.01, *represents P<0.05.
Figure 2
Figure 2
Association of CTC counts and subtypes with early clinical characteristics. (A, B) Tumor size was connected with the number of mixed CTCs and the number of mesenchymal CTCs, p<0.05; (C, D) alanine transaminase (ALT) and HBV DNA were correlated with the mixed CTC number, p<0.05; (E, F) PIVKA and aspartate amino transferase (AST) are related to mesenchymal CTC number, p<0.05.
Figure 3
Figure 3
The expression of Nanog and CTCs in liver cancer peripheral blood and clinical liver cancer tissues. (A) Expression differences of E-cadherin, N-cadherin, and Nanog in 7 pairs of cancer and adjacent tissues (*P < 0.05 , 2-△△Ct). (B) The correlation between the phenotype of CTC and the Nanog positive rate in peripheral blood. (C–E) Correlation between phenotype and number of Nanog in peripheral blood. (F, G) Immunochemistry : Expression of E-cadherin, N-cadherin and Nanog in liver cancer tissues and their paired adjacent tissues (magnification, ×100 and ×400). ***P < 0.001.
Figure 4
Figure 4
The clinicopathological features of patients are related to the expression of Nanog (A) BCLC staging, (B) tumor size, (C) AST, (D) HBV DNA, and (E) ALT are associated with Nanog.
Figure 5
Figure 5
Nanog+ CTC expression is positively correlated with recurrence (A) CTC number, (B) Nanog count, (C) The higher the expression of Mixed CTC number and Nanog, the higher the early recurrence rate of liver cancer; (D) Kaplan–Meier estimates of disease-free survival (E) (p<0.001) and overall (F) (p<0.001) survival of 160 HCC patients with high-level Nanog (≥500 μm2/field) and low-level Nanog (< 500 μm2/field).
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