Downregulation of MUC15 by miR-183-5p.1 promotes liver tumor-initiating cells properties and tumorigenesis via regulating c-MET/PI3K/AKT/SOX2 axis

Mucin 15 (MUC15) is reportedly aberrant in human malignancies, including hepatocellular carcinoma (HCC). However, the role of MUC15 in the regulation of liver tumor-initiating cells (T-ICs) remains unknown. Here, we report that expression of MUC15 is downregulated in liver T-ICs, chemoresistance and recurrent HCC samples. Functional studies reveal that MUC15 inhibits hepatoma cells self-renewal, malignant proliferation, tumorigenicity, and chemoresistance. Mechanistically, MUC15 interacts with c-MET and subsequently inactivates the PI3K/AKT/SOX2 signaling pathway. Moreover, we find that miR-183-5p.1 directly targets MUC15 3′-UTR in liver T-ICs. Coincidentally, SOX2 feedback inhibits MUC15 expression by directly transactivating miR-183-5p.1, thus completing a feedforward regulatory circuit in liver T-ICs. Importantly, MUC15/c-MET/PI3K/AKT/SOX2 axis determines the responses of hepatoma cells to lenvatinib treatment, and MUC15 overexpression abrogated lenvatinib resistance. Analysis of patient cohort, patient-derived tumor organoids and patient-derived xenografts further suggests that the MUC15 may predict lenvatinib benefits in HCC patients. Collectively, our findings suggest the crucial role of the miR-183-5p.1/MUC15/c-MET/PI3K/AKT/SOX2 regulatory circuit in regulating liver T-ICs properties, suggesting potential therapeutic targets for HCC.Subject terms: Cancer stem cells, Tumour biomarkers, Liver cancer     

Selective small molecule stat3 inhibitor reduces breast cancer tumor-initiating cells and improves recurrence free survival in a human-xenograft model

Metastasis and disease relapse are hypothesized to result from tumor initiating cells (TICs). Previously, we have defined a CD44+/CD24-/low mammosphere-forming tumorigenic 493-gene signature in breast cancer. Stat3 was identified as a critical node in self-renewal based on an ongoing lentiviral shRNA screen being conducted in two breast cancer cell lines SUM159 and BT549. In corroborating work, targeting the SH2 domain of Stat3 with a novel small molecule decreased the percentage of cells expressing TIC markers (CD44+/CD24-/low and ALDH+) and mammosphere formation in p-Stat3 overexpressing human breast cancer xenografts in SCID-beige mice. Importantly, we observed a four-fold improvement in the 30-day recurrence-free survival relative to docetaxel alone with the addition of the Stat3 inhibitor in the chemoresistant tumor model. Thus, these findings provide a strong impetus for the development of selective Stat3 inhibitors in order to improve survival in patients with p-Stat3 overexpressing tumors.     

Down-regulated expression of the protein-tyrosine phosphatase 1B (PTP1B) is associated with aggressive clinicopathologic features and poor prognosis in hepatocellular carcinoma

The protein-tyrosine phosphatase 1B (PTP1B) is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and can exert both tumor suppressing and tumor promoting effects in different cancers depending on the substrate involved and the cellular context. However, the expression level and function of PTP1B in hepatocellular carcinoma (HCC) remain unclear. In this study, PTP1B expression was detected by immunohistochemistry in normal liver tissue (n=16) and hepatocellular carcinoma (n=169). The correlations between PTP1B expression level and clinicopathologic features and patient survival were also analyzed. One hundred and eleven of 169 HCC patients (65.7%) had negative or low PTP1B expression in tumorous tissues, whereas normal tissues always expressed strong PTP1B. Decreased PTP1B expression was significantly associated with aggressive clinicopathologic features and poor prognosis. Immunohistochemistry also showed that low PTP1B expression level was correlated with high percentage of OV6(+) tumor-initiating cells (T-ICs) and high frequency of nuclear β-Catenin expression in HCC specimens. Our findings demonstrate for the first time that the loss of inhibitory effect of PTP1B may contribute to progression and invasion of HCC through activation of Wnt/β-Catenin signaling and expansion of liver T-ICs. PTP1B may serve as a valuable prognostic biomarker and potential therapeutic target in HCC.     

Self-renewal and circulating capacities of metastatic hepatocarcinoma cells required for collaboration between TM4SF5 and CD44

Tumor metastasis involves circulating and tumor-initiating capacities of metastatic cancer cells. Hepatic TM4SF5 promotes EMT for malignant growth and migration. Hepatocellular carcinoma (HCC) biomarkers remain unexplored for metastatic potential throughout metastasis. Here, novel TM4SF5/CD44 interaction-mediated self-renewal and circulating tumor cell (CTC) capacities were mechanistically explored. TM4SF5-dependent sphere growth was correlated with CD133⁺, CD24^-, ALDH activity, and a physical association between CD44 and TM4SF5. The TM4SF5/CD44 interaction activated c-Src/STAT3/ Twist1/ B mi1 signaling for spheroid formation, while disturbing the interaction, expression, or activity of any component in this signaling pathway inhibited spheroid formation. In serial xenografts of less than 5,000 cells/injection, TM4SF5-positive tumors exhibited locally-increased CD44 expression, suggesting tumor cell differentiation. TM4SF5-positive cells were identified circulating in blood 4 to 6 weeks after orthotopic liver-injection. Anti-TM4SF reagents blocked their metastasis to distal intestinal organs. Altogether, our results provide evidence that TM4SF5 promotes self-renewal and CTC properties supported by CD133⁺/TM4SF5⁺/CD44^{+(TM4SF5-bound)}/ALDH⁺/ CD24^- markers during HCC metastasis. [BMB Reports 2015; 48(3): 127-128]     

miR-130b Promotes CD133(+) liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear protein 1

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%-13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133? counterparts, CD133(+) cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133(+) cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133(+) tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133(+) and CD133? cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133(+) TICs. Functional studies on miR-130b lentiviral-transduced CD133? cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133(+) TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133? cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133(+) liver TICs, in part, via silencing TP53INP1.     

Inhibition of mitochondrial respiration overcomes hepatocellular carcinoma chemoresistance

The clinical management of advanced hepatocellular carcinoma (HCC) is challenging due to its resistance to chemotherapy. In our work, we demonstrate that an antiparasitic drug atovaquone at clinically relevant concentrations is active against chemoresistant HCC. We show that atovaquone inhibits proliferation and induces apoptosis in not only HCC parental cells but also cells exposed to long time culture of chemotherapeutic agents. Consistently, the combination of atovaquone with cisplatin or doxorubicin achieved remarkably greater efficacy than single drug alone. Mechanistically, atovaquone overcomes HCC chemoresistance via supressing mitochondrial respiration and inducing oxidative stress. Atovaquone but not cisplatin or doxorubicin is ineffective in mitochondrial respiration-deficient ρ0, confirming mitochondria as a specific upstream target of atovaquone. Interestingly, we show that prolonged exposure of HCC cells to chemotherapeutic agents induces higher level of mitochondrial respiration, suggesting that tumors which develop chemoresistance after chemotherapy might be more dependent on mitochondrial respiration than primary tumors and explaining the sensitivity of chemoresistant HCC cells to atovaquone. We further show that atovaquone at tolerable does significantly inhibits chemoresistant HCC growth in mice throughout the duration of treatment. In line with in vitro data, we observe the increased oxidative stress in atovaquone-treated tumors. Our findings highlight the dependency of chemoresistant HCC on mitochondrial respiration and demonstrate that atovaquone is a potential drug to overcome HCC chemoresistance.     

Human ESC self-renewal promoting microRNAs induce epithelial-mesenchymal transition in hepatocytes by controlling the PTEN and TGFβ tumor suppressor signaling pathways

The self-renewal capacity ascribed to embryonic stem cells (ESC) is reminiscent of cancer cell proliferation, raising speculation that a common network of genes may regulate these traits. A search for general regulators of these traits yielded a set of microRNAs for which expression is highly enriched in human ESCs and liver cancer cells (HCC) but attenuated in differentiated quiescent hepatocytes. Here, we show that these microRNAs promote hESC self-renewal, as well as HCC proliferation, and when overexpressed in normally quiescent hepatocytes, induce proliferation and activate cancer signaling pathways. Proliferation in hepatocytes is mediated through translational repression of Pten, Tgfbr2, Klf11, and Cdkn1a, which collectively dysregulates the PI3K/AKT/mTOR and TGFβ tumor suppressor signaling pathways. Furthermore, aberrant expression of these miRNAs is observed in human liver tumor tissues and induces epithelial-mesenchymal transition in hepatocytes. These findings suggest that microRNAs that are essential in normal development as promoters of ESC self-renewal are frequently upregulated in human liver tumors and harbor neoplastic transformation potential when they escape silencing in quiescent human hepatocytes.     

microRNA-448 inhibits stemness maintenance and self-renewal of hepatocellular carcinoma stem cells through the MAGEA6-mediated AMPK signaling pathway

Hepatocellular carcinoma (HCC) occurs mainly in patients with chronic liver disease and cirrhosis. Increasing evidence has identified the involvement of microRNAs (miRNAs) acting as essential regulators in the progression of HCC. As predicted by microarray analysis, miR-448 might potentially affect HCC progression by regulating the melanoma-associated antigen (MAGEA). Therefore, the present investigation focused on exploring whether or not miR-448 and MAGEA6 were involved in the self-renewal and stemness maintenance of HCC stem cells. The interaction among miR-448, MAGEA6, and the AMPK signaling pathway was evaluated. It was noted that miR-448 targeted and downregulated MAGEA6, thus activating the AMP-activated protein kinase (AMPK) signaling pathway in HCC. Furthermore, for the purpose of exploring the functional relevance of MAGEA6 and miR-448 on the sphere formation, colony formation, and invasion and migration of HCC stem cells, the CD133⁺CD44 ⁺ HCC stem cells were sorted and treated with the mimic or inhibitor of miR-448, small interfering RNA (siRNA) against MAGEA6 or an AMPK activator AICAR. MAGEA6 silencing or miR-448 overexpression was demonstrated to inhibit the abilities of sphere formation, colony formation, cell migration, and invasion of HCC cells. Afterwards, a rescue experiment was conducted and revealed that MAGEA6 silencing reversed the effects of miR-448 inhibitor on stemness maintenance and self-renewal of HCC stem cells. Finally, after the in vivo experiment was carried out, miR-448 was observed to restrain the tumor formation and stemness in vivo. Altogether, miR-448 activates the AMPK signaling pathway by downregulating MAGEA6, thus inhibiting the stemness maintenance and self-renewal of HCC stem cells, which identifies miR-448 as a new therapeutic strategy for HCC.     

TSSC3 overexpression reduces stemness and induces apoptosis of osteosarcoma tumor-initiating cells

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents, typically presenting with poor prognosis. Recent studies suggested that tumor initiating cells (T-ICs) drive tumor formation and relapse or metastasis and are relatively resistant to cell death induced by conventional chemo- and radiotherapies. Therefore, the poor prognosis of OS appears to be associated with T-ICs. Here, we enriched T-ICs in OS cell lines and evaluated whether the imprinted gene TSSC3 (tumor-suppressing STF cDNA 3) associated with apoptosis could affect T-ICs in OS. Sarcosphere selection and serial clone-forming unit assays were successfully used to enrich T-ICs from OS cell lines. Enrichment of T-ICs from a malignantly transformed hFOB1.19 osteoblast cell line (MThFOB1.19) indicated that OS T-ICs could originate from differentiated cells, and most of these MThFOB1.19 cells showed stem-like features. TSSC3 was expressed at a low level in T-ICs, while overexpression of TSSC3 could efficiently downregulate the expression of stem cell markers Nanog, Oct4 and Sox2 in T-ICs and decrease the clone formation rate, as well as downregulate tumorigenesis in MThFOB1.19 cells, supporting a suppressive role for TSSC3 in OS T-ICs. Furthermore, overexpression of TSSC3 was found to induce apoptosis of OS T-ICs through increasing cleaved caspase-3 (active form), increasing the release of Cyt c and decreasing pro-caspase-9 (pro-enzyme form), as well as disruption of the mitochondrial membrane potential (ΔΨ). Taken together, our findings provide preliminary evidence that TSSC3 inhibits OS tumorigenicity through reducing stemness and promoting apoptosis of T-ICs. Thus, targeting TSSC3 may be a promising approach to suppressing tumorigenicity in OS.     

Intrahepatic Tissue Implantation Represents a Favorable Approach for Establishing Orthotopic Transplantation Hepatocellular Carcinoma Mouse Models

Mouse models are commonly used for studying hepatocellular carcinoma (HCC) biology and exploring new therapeutic interventions. Currently three main modalities of HCC mouse models have been extensively employed in pre-clinical studies including chemically induced, transgenic and transplantation models. Among them, transplantation models are preferred for evaluating in vivo drug efficacy in pre-clinical settings given the short latency, uniformity in size and close resemblance to tumors in patients. However methods used for establishing orthotopic HCC transplantation mouse models are diverse and fragmentized without a comprehensive comparison. Here, we systemically evaluate four different approaches commonly used to establish HCC mice in preclinical studies, including intravenous, intrasplenic, intrahepatic inoculation of tumor cells and intrahepatic tissue implantation. Four parameters—the latency period, take rates, pathological features and metastatic rates—were evaluated side-by-side. 100% take rates were achieved in liver with intrahepatic, intrasplenic inoculation of tumor cells and intrahepatic tissue implantation. In contrast, no tumor in liver was observed with intravenous injection of tumor cells. Intrahepatic tissue implantation resulted in the shortest latency with 0.5cm (longitudinal diameter) tumors found in liver two weeks after implantation, compared to 0.1cm for intrahepatic inoculation of tumor cells. Approximately 0.1cm tumors were only visible at 4 weeks after intrasplenic inoculation. Uniform, focal and solitary tumors were formed with intrahepatic tissue implantation whereas multinodular, dispersed and non-uniform tumors produced with intrahepatic and intrasplenic inoculation of tumor cells. Notably, metastasis became visible in liver, peritoneum and mesenterium at 3 weeks post-implantation, and lung metastasis was visible after 7 weeks. T cell infiltration was evident in tumors, resembling the situation in HCC patients. Our study demonstrated that orthotopic HCC mouse models established via intrahepatic tissue implantation authentically reflect clinical manifestations in HCC patients pathologically and immunologically, suggesting intrahepatic tissue implantation is a preferable approach for establishing orthotopic HCC mouse models.     

The Clinical Significance of the CD163+ and CD68+ Macrophages in Patients with Hepatocellular Carcinoma

Our previous study has found that the abundance of peritumoral CD68⁺ macrophages was associated with poor prognosis in hepatocellular carcinoma (HCC) after resection. However, CD68 staining could not discriminate the protumoral or tumoricidal subpopulations from pan-macrophages. CD163 is a marker of alternatively activated macrophages. In this study, the clinical significance of CD163⁺ cells in tumors and peritumoral liver tissues was evaluated in a cohort of 295 patients with HCC after curative resection. We found that the density of CD163⁺ cells was well correlated with that of CD68⁺ cells in both tumors and peritumoral liver tissues but was much more. Immunostaining on consecutive sections and flow cytometry assay on surgical resected specimens further supported the findings that the CD163⁺ cells was more abundant than CD68⁺ cells. The density of peritumoral CD68⁺ cells was associated with poor recurrence-free survival (RFS) and poor overall survival (OS) (P = 0.004 and P = 0.001, respectively), whereas the CD163⁺ cells have no prognostic values either in tumors or in peritumoral liver tissues. In another cohort of 107 HCC patients, preoperative plasma concentration of soluble form of CD163 (sCD163) was associated with active hepatitis-related factors but not associated with the markers of tumor invasion. In conclusion, both the CD163⁺ cells local infiltration and plasma sCD163 were of limited significance in HCC, and they were more likely markers related to active hepatitis rather than tumor progression.     

High-throughput flow cytometry screening of human hepatocellular carcinoma reveals CD146 to be a novel marker of tumor-initiating cells

Hepatocellular carcinoma (HCC) remains a common and lethal cancer. Cancer stem cells, or tumor-initiating cells (TICs), are thought to contribute to the pathogenesis of HCC, but remain to be fully characterized. Unbiased screens of primary human HCC cells for the identification of novel HCC TIC markers have not been reported. We conducted high-throughput flow cytometry (HT-FC) profiling to characterize the expression of 375 CD antigens on tumor cells from 10 different human HCC samples. We selected 91 of these for further analysis based on HT-FC data that showed consistent expression in discrete, rare, sortable populations of HCC cells. Nine of these CD antigens demonstrated significantly increased expression in the EpCAM⁺ stem/progenitor fraction of a human HCC cell line and were further evaluated in primary human HCC tissues from 30 different patients. Of the nine tested, only CD146 demonstrated significantly increased expression in HCC tumor tissue as compared with matched adjacent non-tumor liver tissue. CD146⁺CD31^?CD45^? cells purified from HCC tumors and cell lines demonstrated a unique phenotype distinct from mesenchymal stem cells. As compared with other tumor cell fractions, CD146⁺CD31^?CD45^? cells showed significantly increased colony-forming capacity in vitro, consistent with TICs. This study demonstrates that HT-FC screening can be successfully applied to primary human HCC and reveals CD146 to be a novel TIC marker in this disease.     

Apoptosis signaling in cancer stem cells

Since the discovery of specific populations of cells with stem-like characteristics in human leukemias, phenotypically and/or functionally similar tumor-promoting cells have been identified in a variety of human cancers. By dint of the similarities to normal human stem cells in terms of self-renewal, differentiation, long life span, and proliferative capacity, these defined populations of cells within the bulk tumor are referred to as “cancer stem cells (CSCs)”. The presence of CSCs has challenged the age-old dogma of carcinogenesis, which posits that all cells within a tissue retain the capacity to generate tumors. With respect to the frequency of CSCs, there is still a lack of consensus as in some recent models the notion that these cells constitute a very small proportion within the tumor has been challenged. Another issue that remains unresolved is the existence of a “global” marker, although reference has been made to the CD133⁺, CD34⁺CD38^?, and CD44⁺CD24^? populations as the functional stem-like cells in different cancers. Nevertheless, the identification of this sub-set within the bulk tumor and its contribution to chemotherapy resistance suggest that the CSCs could be the Achilles heel in terms of chemosensitization. Therefore, a paradigm is emerging that an effective therapeutic approach against cancers is to target this critical pool of cells that have the capacity to self-renew and proliferate as well as evade death signals. Here we provide a brief review of the literature vis a vis the various mechanisms of defective apoptotic signaling in CSCs with potential for therapeutic intervention.     

Inhibition of EZH2 Attenuates Sorafenib Resistance by Targeting NOTCH1 Activation-Dependent Liver Cancer Stem Cells via NOTCH1-Related MicroRNAs in Hepatocellular Carcinoma

Acquired resistance and intrinsic to sorafenib therapy represents a major hurdle in improving the management of advanced hepatocellular carcinoma (HCC), which has been recently shown to be associated with the emergence of liver cancer stem cells (CSCs). However, it remains largely unknown whether and how histone posttranslational modifications, especially H3K27me3, are causally linked to the maintenance of self-renewal ability in sorafenib-resistant HCC. Here, we found that NOTCH1 signaling was activated in sorafenib-resistant HCC cells and NOTCH1 activation conferred hepatoma cells sorafenib resistance through enhanced self-renewal and tumorigenecity. Besides, the overexpression of EZH2 was required for the emergence of cancer stem cells following prolonged sorafenib treatment. As such, modulating EZH2 expression or activity suppressed activation of NOTCH1 pathway by elevating the expression of NOTCH1-related microRNAs, hsa-miR-21-5p and has-miR-26a-1-5p, via H3K27me3, and consequently weakened self-renewal ability and tumorigenecity and restored the anti-tumor effects of sorafenib. Overall, our results highlight the role of EZH2/NICD1 axis, and also suggest that EZH2 and NOTCH1 pathway are rational targets for therapeutic intervention in sorafenib-resistant HCC.     

CD133和maspin在肝癌中的表达及其临床病理意义

目的 探讨肿瘤干细胞标志物CD133和maspin在肝细胞性肝癌（hepatocellular carcinoma HCC）中的表达情况及其临床病理意义.方法 采用免疫组织化学ElivisionTM plus法检测100例HCC组织和20例癌旁肝组织中CD133和maspin蛋白的表达情况.结果 在癌旁肝组织中CD133和maspin蛋白的阳性表达率分别为5.0%和100%,而在HCC组中其阳性表达率分别为51.0%和48.0%,两组之间差异有显著性（P〈0.01）.CD133蛋白的表达水平与肿瘤的Edmondson分级、淋巴结转移、肿瘤的数目和有无血管侵犯有关（全部P〈0.05）;maspin蛋白的表达水平与肿瘤的Edmondson分级、肿瘤的数目和有无血管侵犯有关（全部P〈0.05）.且CD133蛋白的表达与maspin蛋白的表达呈负相关（P〈0.01）.结论 CD133和maspin蛋白的表达与HCC组织的Edmondson分级、肿瘤数目和血管侵犯有关;CD133和maspin的联合检测对HCC的进展及预后判断有重要意义.     

The critical role of CD133(+)CD44(+/high) tumor cells in hematogenous metastasis of liver cancers

Metastatic hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. However, the cell population responsible for its metastasis remains largely unknown. Here, we reported that CD133(+)CD44(+/high) defined a subgroup of tumor cells that was responsible for hematogenous metastasis of liver cancers. Immunohistochemical investigation of human HCC specimens revealed that the number of CD133(+) and CD44(+) HCC cells was increased and was associated with portal vein invasion. Purified CD133(+) or CD44(high) HCC cells were superior in clonogenic growth and vascular invasion, respectively. Thus, the combination of CD133 and CD44 was used to define a novel HCC sub-population. CD133(+)CD44(high), but not CD133(+)CD44(low/-), CD133(-)CD44(high) or CD133(-)CD44(low/-) xenografts, produced intrahepatic or lung metastasis in nude mice. Further analysis of human HCC samples by flow cytometry showed that the number of CD133(+)CD44(+) tumor cells was associated with portal vein metastasis. The cDNA microarray analysis of CD133(+)CD44(+) and CD133(+)CD44(-) tumor cells isolated from metastatic HCC patients revealed that these cells comprised of two different populations possessing distinct gene expression profiles. Our results suggest that CD133(+)CD44(+) tumor cells are a particular population responsible for hematogenous metastasis in liver cancers and that these cells might be targets for treatment of HCC metastasis.     

Doxorubicin and 5-fluorouracil resistant hepatic cancer cells demonstrate stem-like properties

The efficacy of hepatocellular carcinoma (HCC) treatment is very low because of the high percentage of recurrence and resistance to anticancer agents. Hepatic cancer stem cells (HCSCs) are considered the origin of such recurrence and resistance. Our aim was to evaluate the stemness of doxorubicin and 5-fluorouracil resistant hepatic cancer cells and establish the new method to isolate the HCSCs from primary cultured HCC tumors. HCC biopsies were used to establish primary cultures. Then, primary cells were selected for HCSCs by culture in medium supplemented with doxorubicin (0, 0.1, 0.25, 0.5 or 1 μg/mL), 5-fluorouracil (0, 0.1, 0.25, 0.5 or 1 μg/mL) or their combination. Selection was confirmed by detection of HCSC markers such as CD133, CD13, CD90, and the side population was identified by rhodamine 123 efflux. The cell population with the strongest expression of these markers was used to evaluate the cell cycle, gene expression profile, tumor sphere formation, marker protein expression, and in vivo tumorigenesis. Selective culture of primary cells in medium supplemented with 0.5 μg/mL doxorubicin and 1 μg/mL 5-fluorouracil selected cancer cells with the highest stemness properties. Selected cells strongly expressed CD13, CD133, CD90, and CD326, efflux rhodamine 123 and formed tumor spheres in suspension. Moreover, selected cells were induced to differentiate into cells with high expression of CD19 and AFP (alpha-fetoprotein), and importantly, could form tumors in NOD/SCID mice upon injection of 1 × 10⁵ cells/mouse. Selective culture with doxorubicin and 5-fluorouracil will enrich HCSCs, is an easy method to obtain HCSCs that can be used to develop better therapeutic strategies for patients with HCC, and particularly HCSC-targeting therapy.     

Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory, CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133(+) HCC CSC population is still heterogeneous, consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach, we compared protein profiles between CD133(+) and CD133(-) subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133(+) subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH(+) to be CD133(+), yet not all CD133(+) HCC cells were ALDH(+). Subsequent studies on purified subpopulations found CD133(+)ALDH(+) cells to be significantly more tumorigenic than their CD133(-)ALDH(+) or CD133(-)ALDH(-) counterparts, both in vitro and in vivo. These data, combined with those from our previous work, reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133(+)ALDH(+) > CD133(+)ALDH(-) > CD133(-)ALDH(-). ALDH, expressed along CD133, can more specifically characterize the tumorigenic liver CSC population.