Visualization of stem cell features in human hepatocellular carcinoma revealsin vivosignificance of tumor-host interaction and clinical course

Abstract

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies because of recurrence and/or metastasis even after curative resection. Emerging evidence suggests that tumor metastasis and recurrence might be driven by a small subpopulation of stemness cells, so-called cancer stem cells (CSCs). Previous investigations have revealed that glioma and breast CSCs exhibit intrinsically low proteasome activity and that breast CSCs also reportedly contain a lower reactive oxygen species (ROS) level than corresponding nontumorigenic cells. Here we visualized two stem cell features, low proteasome activity and low intracellular ROS, in HCC cells using two-color fluorescence activated cell sorting to isolate cells with stem cell features. These cells were then analyzed for their division behavior in normoxia and hypoxia, expression of stem cell markers, tumorigenicity, metastatic potential, specific gene expression signatures, and their clinical implications. A visualized small subpopulation of HCC cells demonstrated asymmetric divisions. Their remarkable tumorigenicity in nonobese diabetic/severe combined immunodeficient mice suggested the cancer initiation potential of these HCC CSCs. Comprehensive gene expression analysis revealed that chemokinerelated genes were up-regulated in the CSCs subpopulation. Our identified HCC CSCs facilitated the migration of macrophages in vitro and demonstrated metastatic potential by way of recruitment of macrophages in vivo. In patients who undergo curative operation for HCC, the CSC-specific gene signature in the liver microenvironment significantly correlates with recurrence. Conclusion: Based on these findings, the stem cell feature monitoring system proposed here is a promising tool to analyze the in vivo significance of CSC microenvironments in human HCCs. (HEPATOLOGY 2013;58:218-228) H epatocellular carcinoma (HCC) is one of the most common malignancies and the third leading cause of cancer death worldwide. 1 The primary curative treatment for HCC is surgical resection; however, even after curative resection patient prognosis remains poor because of frequent recurrence and/or metastasis. 2,3 Because cancer stem cells (CSCs) possess self-renewal capacity, multilineage potency, and increased tumorigenicity, it has been hypothesized that CSCs exist as a small population within the bulk tumors and play a critical role in cancer progression, metastasis, and recurrence. 4 Various tools have been reported for identification of the CSC population, including the cell surface markers CD44, CD133, CD90, and ESA/EpCAM. [5][6][7][8] In addition, specific stemness properties based on stem cell biology of their Abbreviations: CSC, cancer stem cells; FDR, false discovery rate; GSEA, gene set enrichment analysis; HCC, hepatocellular carcinoma; NOD/SCID, nonobese diabetic / severe combined immunodeficient; ODC, ornithine decarboxylase; ROS, reactive oxygen species.From the

Basic Science of Liver Cancer Stem Cells and Hepatocarcinogenesis

“…Gdeg high cells divided asymmetrically into a Gdeg high cell and a Gdeg low cell, but Gdeg low cells never divided into Gdeg high , only into more Gdeg low . As already discussed, the Gdeg high ROS low cells were significantly more tumourigenic in NOD/SCID xenoengraftment than unsorted cells (Muramatsu et al, 2013).…”

“…Finally, there is one report of xenoengraftment of LCSCLCs isolated from primary human HCC tumour (Muramatsu et al, 2013). These cells were enriched with a nice system to mark asymmetry of division (see Section 14.2.7).…”

“…Both studies capture live asymmetric division of putative LCSCs: from HCC cell lines in the first case (Hari et al, 2011;Xin et al, 2012) and from cultures established from four primary human resected HCCs, as well as from cell lines, in the second (Muramatsu et al, 2013). In the first case, capture was with confocal microscopy cinematography; in the second, with 21-hour timelapse video microscopy.…”

Cancer stem cells, cancer cell plasticity and radiation therapy

“…This unique reporter system for CSCs allows for identification, tracking and isolation of CSC populations over time without further manipulation of cells, in vitro and in vivo [46]. Other laboratories have employed our reporter system to identify CSC in lung cancer [50], pancreatic cancer [51] and hepatocellular carcinoma [52]. In breast cancer, cells with low proteasome activity partially overlap with ALDH1 + and CD24 low/− /CD44 high cells [53].…”

Developmental Stage-Specific Hepatocytes Induce Maturation of HepG2 Cells by Rebuilding the Regulatory Circuit

“…HCC cells own common characteristics that are often found to be hepatic stem/progenitor cell biomarkers (2,(6)(7)(8)(9)(10), although multiple environmental and genetic risk factors exist. HCC progression and embryonic liver development share many same properties, and a tumor may originate from progenitor/stem cells that receive abnormal proliferation and maturation signals (11)(12)(13).…”