Cancer Stem Cell Surface Markers Research Articles

LncRNA NEAT1 Promotes the Cancer Stem Cell-Like Properties of HCC by miR-128-3p/GP73 Axis

In this study, we investigated the role of long non-coding RNA NEAT1 in hepatocellular carcinoma (HCC) and its impact on liver cancer cell behavior, particularly their cancer stem cell (CSC)-like properties. We observed elevated NEAT1 levels in HCC cell lines and tissues, and this upregulation was associated with adverse clinical characteristics and poor prognosis in HCC patients. Through in vitro experiments, we found that silencing NEAT1 in HCC cells led to decreased cell proliferation, migration, invasion, and inhibited epithelial-mesenchymal transition (EMT) in Huh7 cells. Additionally, the unique surface markers of CSCs were downregulated upon NEAT1 knockdown. Further investigation revealed that NEAT1 regulates the expression of GP73 by directly binding to miR-128-3p, functioning as a competitive ceRNA to suppress miR-128-3p expression. Rescue experiments showed that inhibiting miR-128-3p expression reversed the effects of NEAT1 knockdown on HCC cell proliferation,migration, and invasion. In summary, our findings demonstrate that lncRNA NEAT1 plays a pivotal role in promoting HCC progression and enhancing CSC properties by upregulating GP7 through competitive binding to miR-128-3p. This insight into the underlying mechanism may have promising implications for the treatment of HCC.

Anti-IL-8 monoclonal antibodies inhibits the autophagic activity and cancer stem cells maintenance within breast cancer tumor microenvironment.

Breast cancer tumor microenvironment (TME) is a promising target for immunotherapy. Autophagy, and cancer stem cells (CSCs) maintenance are essential processes involved in tumorigenesis, tumor survival, invasion, and treatment resistance. Overexpression of angiogenic chemokine interleukin-8 (IL-8) in breast cancer TME is associated with oncogenic signaling pathways, increased tumor growth, metastasis, and poor prognosis. Thus, we aimed to investigate the possible anti-tumor effect of neutralizing antibodies against IL-8 by evaluating its efficacy on autophagic activity and breast CSC maintenance. IL-8 monoclonal antibody supplemented tumor tissue culture systems from 15 females undergoing mastectomy were used to evaluate the expression of LC3B as a specific biomarker of autophagy and CD44, CD24 as cell surface markers of breast CSCs using immunofluorescence technique. Our results revealed that anti-IL-8 mAb significantly decreased the level of LC3B in the cultured tumor tissues compared to its non-significant decrease in the normal breast tissues.Anti-IL-8 mAb also significantly decreased the CD44 expression in either breast tumors or normal cultured tissues. While it caused a non-significant decrease in CD24 expression in cultured breast tumor tissue and a significant decrease in its expression in the corresponding normal ones. Anti-IL-8 monoclonal antibody exhibits promising immunotherapeutic properties through targeting both autophagy and CSCs maintenance within breast cancer TME.

Abstract 5440: TM4SF1 maintains cancer stemness in cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is an aggressive liver malignancy with increased incidence globally but poor prognosis. Cancer stemness plays critical roles on tumor initiation, recurrence, and resistance to therapy. However, the mechanism of stemness maintenance in CCA has been less explored. Previously, we have reported TM4SF1 is associated with poor survival in CCA and high expression of TM4SF1 tends to promote tumorigenesis. In this study, we found that the expression of TM4SF1 gene is upregulated in tumor tissues compared to adjacent normal tissues in CCA based on bulk RNA sequencing dataset from The Cancer Genome Atlas (TGCA) CCA cohort. There is a positive correlation between the expression of TM4SF1 and stemness genes, ITGB3, SOX9, ALDH1A3, and ITGB1. Interestingly, knockdown of TM4SF1 with siRNA in CCA cell line HuCCT-1 cells demonstrated a decrease in expression of cancer stem cell surface markers, CD24 and Prom1. Furthermore, with a CRISPR-based lentivirus construct to knockdown TM4SF1 gene in HuCCT-1 cells in vitro, we found that spheroid formation in TM4SF1 knockdown was attenuated in comparison to TM4SF1 wild type. In addition, the results from flow cytometry and RT-PCR showed a corresponding decrease in CD24 and CD133 expression with TM4SF1 knockdown when comparing to TM4SF1 wild type. In conclusion, TM4SF1 is associated with the stemness of CCA and could be a therapeutic target for CCA therapy to overcome therapy resistance in future. Citation Format: Xiao Bin Zhu, Jihye Golino, Xin Wang, Changqing Xie. TM4SF1 maintains cancer stemness in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5440.

Helicobacter pylori CagA protein induces gastric cancer stem cell-like properties through the Akt/FOXO3a axis.

The presence of Helicobacter pylori (H. pylori) infection poses a substantial risk for the development of gastric adenocarcinoma. The primary mechanism through which H. pylori exerts its bacterial virulence is the cytotoxin CagA. This cytotoxin has the potential to induce inter-epithelial mesenchymal transition, proliferation, metastasis, and the acquisition of stem cell-like properties in gastric cancer (GC) cells infected with CagA-positive H. pylori. Cancer stem cells (CSCs) represent a distinct population of cells capable of self-renewal and generating heterogeneous tumor cells. Despite evidence showing that CagA can induce CSCs-like characteristics in GC cells, the precise mechanism through which CagA triggers the development of GC stem cells (GCSCs) remains uncertain. This study reveals that CagA-positive GC cells infected with H. pylori exhibit CSCs-like properties, such as heightened expression of CD44, a specific surface marker for CSCs, and increased ability to form tumor spheroids. Furthermore, we have observed that H. pylori activates the PI3K/Akt signaling pathway in a CagA-dependent manner, and our findings suggest that this activation is associated with the CSCs-like characteristics induced by H. pylori. The cytotoxin CagA, which is released during H. pylori infection, triggers the activation of the PI3K/Akt signaling pathway in a CagA-dependent manner. Additionally, CagA inhibits the transcription of FOXO3a and relocates it from the nucleus to the cytoplasm by activating the PI3K/Akt pathway. Furthermore, the regulatory function of the Akt/FOXO3a axis in the transformation of GC cells into a stemness state was successfully demonstrated.

Astragali Radix-Curcumae Rhizoma inhibits colon cancer progression and enhances 5-FU efficacy by regulating hypoxia-inducible factors and tumor stem cells

The animal and cell models were used in this study to investigate the mechanism of Astragali Radix-Curcumae Rhizoma(HQEZ) in inhibiting colon cancer progression and enhancing the efficacy of 5-fluorouracil(5-FU) by regulating hypoxia-inducible factors and tumor stem cells. The animal model was established by subcutaneous transplantation of colon cancer HCT116 cells in nude mice, and 24 successfully modeled mice were randomized into model, 5-FU, HQEZ, and 5-FU+HQEZ groups. The tumor volume was measured every two days. Western blot was employed to measure the protein levels of epidermal growth factor receptor(EGFR), dihydropyrimidine dehydrogenase(DPYD), and thymidylate synthase(TYMS), the key targets of the hypoxic core region, as well as the hypoxia-inducible factors HIF-1α and HIF-2α and the cancer stem cell surface marker CD133 and SRY-box transcription factor 2(SOX2). The results of animal experiments showed that HQEZ slowed down the tumor growth and significantly increased the tumor inhibition rate of 5-FU. Compared with the model group, HQEZ significantly down-regulated the protein levels of EGFR and DPYD, and 5-FU+HQEZ significantly down-regulated the protein levels of EGFR and TYMS in tumors. Compared with the model group, HQEZ significantly down-regulated the protein levels of HIF-1α, HIF-2α, SOX2, and CD133 in the hypoxic core region. Compared with the 5-FU group, 5-FU+HQEZ lowered the protein levels of HIF-1α, HIF-2α, and SOX2. The cell experiments showed that the protein le-vels of HIF-1α and HIF-2α in HCT116 cells elevated significantly after low oxygen treatment. Compared with 5-FU(1.38 μmol·L~(-1)) alone, HQEZ(40 mg·mL~(-1)) and 5-FU+HQEZ significantly down-regulated the protein levels of HIF-1α, HIF-2α, and TYMS. In conclusion, HQEZ can inhibit the expression of hypoxia-responsive molecules in colon cancer cells and reduce the properties of cancer stem cells, thereby enhancing the therapeutic effect of 5-FU on colon cancer.

Putative Cancer Stem Cell Markers are Frequently Expressed by Melanoma Cells in Vitro and in Situ but are also Present in Benign Differentiated Cells.

Currently, there remains an incomplete view of cancer stem cells (CSCs) in solid tumours. We studied a panel of putative CSC surface markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) in 40 established melanoma cell lines and four early-passage melanoma strains by flow cytometry. We additionally examined 40 formalin-fixed paraffin-embedded melanoma tissues using immunofluorescence microscopy. This was compared with their expression in healthy skin, normal differentiated melanocytes and fibroblasts. Most of the putative CSC markers were expressed by both melanoma cell lines and tissues. When present, these proteins were expressed by the majority of cells in the population. However, the expression of these markers by cells in healthy skin sections, normal differentiated melanocytes, and fibroblasts revealed that differentiated non-malignant cells also expressed CSC markers indicating that they lack of specificity for CSCs. Culturing cell lines under conditions more characteristic of the tumour microenvironment upregulated CSC marker expressions in a proportion of cell lines, which correlated with improved cell growth and viability. The testing of melanoma cell lines (n = 40), early-passage cell strains (n = 4), and melanoma tissues (n = 40) showed that several putative CSC markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) are commonly present in a large proportion of melanoma cells in vitro and in situ. Further, we showed that these putative markers lack specificity for CSCs because they are also expressed in differentiated non-malignant cell types (melanocytes, fibroblasts, and skin), which could limit their use as therapeutic targets. These data are consistent with the emerging notion of CSC plasticity and phenotype switching within cancer cell populations.

Effect of microserum environment stimulation on extraction and biological function of colorectal cancer stem cells

Background3D cancer stem cell (CSC) cultures are widely used as in vitro tumor models. In this study, we determined the effects of enriching HCT116 tumor spheres initially cultured in serum-free medium with different concentrations of serum, focusing on the effect of microserum environment stimulation on extraction and biological function of colorectal cancer stem cells (CCSCs).MethodsCCSCs were enriched in standard serum-free medium and serum-free medium with different concentrations of serum for 1 week. The expression of CSC-associated markers in CCSCs, and the presence and relative proportion of CSCs (CD133/CD44 cell sorting) were then determined to elucidate the effect of the microserum environment on the preservation of CSC-related features. Further, the tumorigenic capacity of CCSCs was evaluated in an immunodeficiency mouse model.ResultsOur data indicated that a significantly greater number of spheres with a greater size range and high viability without drastic alteration in biological and structural features, which maintained self‐renewal potential after sequential passages were formed after serum supplementation. Real-time analysis showed that both serum spheres and serum-free spheres displayed similar expression patterns for key stemness genes. Serum spheres showed higher expression of the CSC surface markers CD133 and CD44 than did CSCs spheres cultured in serum-free medium. Adherent cultures in complete medium could adapt to the serum-containing microenvironment faster and showed higher proliferation ability. The addition of serum induced EMT and promoted the migration and invasion of serum globular cells. Compared with serum-free cells and adherent cells, serum spheres showed higher tumor initiation ability.ConclusionsMicroserum environment stimulation could be an effective strategy for reliable enrichment of intact CCSCs, and a more efficient CSC enrichment method.

SOX9 knockout decreases stemness properties in colorectal cancer cells.

Colorectal cancer (CRC) is a leading cause of death worldwide. SRY-box transcription factor 9 (SOX9) participates in organogenesis and cell differentiation in normal tissues but has been involved in carcinogenesis development. Cancer stem cells (CSCs) are a small population of cells present in solid tumors that contribute to increased tumor heterogeneity, metastasis, chemoresistance, and relapse. CSCs have properties such as self-renewal and differentiation, which can be modulated by many factors. Currently, the role of SOX9 in the maintenance of the stem phenotype has not been well elucidated, thus, in this work we evaluated the effect of the absence of SOX9 in the stem phenotype of CRC cells. We knockout (KO) SOX9 in the undifferentiated CRC cell line HCT116 and evaluated their stemness properties using sphere formation assay, differentiation assay, and immunophenotyping. SOX9-KO affected the epithelial morphology of HCT116 cells and stemness characteristics such as its pluripotency signature with the increase of SOX2 as a compensatory mechanism to induce SOX9 expression, the increase of KLF4 as a differentiation feature, as well as the inhibition of the stem cell markers CD44 and CD73. In addition, SOX9-KO cells gain the epithelial-mesenchymal transition (EMT) phenotype with a significant upregulation of CDH2. Furthermore, our results showed a remarkable effect on first- and second-sphere formation, being SOX9-KO cells less capable of forming high-size-resistant spheres. Nevertheless, CSCs surface markers were not affected during the differentiation assay. Collectively, our findings supply evidence that SOX9 promotes the maintenance of stemness properties in CRC-CSCs.

Phenotypic and functional characterization of subpopulation of Imatinib resistant chronic myeloid leukemia cell line

PurposeChronic myeloid leukemia (CML) is a hematological malignancy characterized by the presence of BCR-ABL protein. Imatinib (IMA) is considered as the first line therapy in management of CML which particularly targets the BCR-ABL tyrosine kinase protein. However, emergence of resistance to IMA hinders its clinical efficiency. Hence, identifying novel targets for therapeutic approaches in CML treatment is of great importance. Here, we characterize a new subpopulation of highly adherent IMA-resistant CML cells that express stemness and adhesion markers compared to naive counterparts. Materials and methodsWe performed several experimental assays including FISH, flow cytometry, and gene expression assays. Additionally, bioinformatics analysis was performed by normalized web-available microarray data (GSE120932) to revalidate and introduce probable biomarkers. Protein-protein interactions (PPI) network was analyzed by the STRING database employing Cytoscape v3.8.2. ResultsOur findings demonstrated that constant exposure to 5 ​μM IMA led to development of the adherent phenotype (K562R-adh). FISH and BCR-ABL expression analysis indicated that K562R-adh cells were derived from the original cells (K562R). In order to determine the role of various genes involved in epithelial–mesenchymal transition (EMT) and stem cell characterization, up/down-regulation of various genes including cancer stem cell (CSC), adhesion and cell surface markers and integrins were observed which was similar to the findings of the GSE120932 dataset. ConclusionTreating CML patients with tyrosine kinase inhibitors (TKIs) as well as targeting adhesion molecules deemed to be effective approaches in prevention of IMA resistance emergence which in turn may provide promising effects in the clinical management of CML patients.

Expression and Prognostic Value of Lgr5 in Patients with Recurrent Nasopharyngeal Carcinoma.

As a cancer stem cells (CSCs) surface marker, Lgr5 plays an important role in the signal transduction of cancer cells and is a potential biomarker for cancer diagnosis and prognosis. However, the expression and prognostic value of Lgr5 in recurrent nasopharyngeal carcinoma (rNPC) remains ambiguous. We used RNA sequencing to screen differentially expressed mRNAs in eleven specimens of rNPC tissues and five fresh adjacent normal tissue samples and the CSC marker, Lgr5, was identified. The expression level of Lgr5 in rNPC samples was also detected by immunohistochemistry and Western blot assay. The chi-square test was used to analyze the relationship between the clinicopathological variables and the immunostaining of Lgr5. The Log-rank method was used for prognosis analysis. The Cox regression model was used for univariate and multivariate analysis. Significantly elevated expression of Lgr5 in the rNPC tissues was observed compared to the normal tissues using RNA sequencing, Western blot and immunohistochemistry. The expression of Lgr5 was significantly correlated with the T stage (P=0.014). High Lgr5 expression (P=0.007), tumor necrosis (P=0.013) and WHO type II (P=0.043) in rNPC patients exhibited worse overall survival (OS). Lgr5 expression was proved to be an independent risk factor for OS (P=0.035) in multivariate analyses, and had promising predictive value for survival and recurrence in rNPC patients (area under the ROC curve: 0.711 and 0.665, P=0.017 and 0.028, respectively). Lgr5 as a CSC marker is a promising therapeutic target and could be employed to predict the survival prognosis of rNPC patients.

Abstract 5048: Combatting pancreatic cancer stem cells by novel titanium peroxides nanoparticles combined with X-ray radiation

Abstract Background: Radiotherapy is widely used as the main treatment for multiple malignancies. However, several types of cancers, including pancreatic cancer, show resistance to radiation therapy. The presence of cancer stem cells (CSCs) are the major cause of radiation resistance in pancreatic cancer. Whereas, titanium peroxides nanoparticles (TiOxNPs) which are a modified form of titanium oxide nanoparticles promote enhanced remarkable efficacy of radiation therapy. Therefore, we examined the efficacy of TiOxNPs as radiosensitizers to eradicate pancreatic cancer stem cells. Methods and materials: In vitro, Sphere-forming assay, survival assay, migration, and invasion assay were evaluated after using TiOxNPs prior to radiation exposure to pancreatic cancer cell line. In vivo, tumor-bearing nude mice were injected by TiOxNPs either intratumoral or intravenous one hour prior to radiation treatment, and the tumor volume, body weight, and mice survival was calculated. In addition, proteins-related stemness were measured in vitro and in vivo to evaluate the usage of TiOxNPs as radiosensitizers to pancreatic CSCs. Moreover, we planned to evaluate the mechanism beyond the efficacy of TiOxNPs as radiosensitizers to CSCs by detecting reactive oxygen species (ROS) production, mitochondrial function, and the phosphorylation of some signaling proteins. We found that TiOxNPs combined with ionizing radiation showed anti-cancer effects on radioresistant CSCs both in vitro and in vivo. Results: In vitro, a marked reduction in growth was detected after exposing the TiOxNPs-treated cells to radiation therapy, specifically with a 5Gy dose compared with 2Gy dose. TiOxNPs exhibited a synergistic effect with radiation on pancreatic CSC-enriched spheres by downregulating self-renewal regulatory factors and CSC surface markers. In vivo, we first established an aggressive xenograft by injecting MIA PaCa-2 sphere cells into the flank region of BALB/c nude mice, and found that animals treated with combined TiOxNPs and irradiation showed a dramatic reduction in tumor volume and weight compared to the untreated group. Moreover, combined treatment suppressed epithelial-mesenchymal transition, migration, and invasion properties in primary and aggressive pancreatic cancer cells by reducing the expression of proteins relevant to these processes. Radiosensitizing TiOxNPs suppressed pancreatic xenograft outgrowth after primary or dissociating sphere MIA PaCa-2 cell implantation. It is assumed that synergy is created by inactivating the AKT signaling pathway and producing unbearable amounts of ROS. Conclusion: Our findings showed that using TiOxNPs in combination with radiation might be a favorable therapeutic approach to eradicate pancreatic CSCs. Citation Format: Mohammed Salah, Hiroki Kawaguchi, Hiroaki Akasaka, Yasuyuki Shimizu, Kenta Morita, Yuya Nishimura, Hikaru Kubota, Tomomi Sogawa, Naritoshi Mukumoto, Chiaki Ogino, Ryohei Sasaki. Combatting pancreatic cancer stem cells by novel titanium peroxides nanoparticles combined with X-ray radiation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5048.

Abstract 4105: Improvement of CD133-specific chimeric antigen receptor T cells by secreting anti-PD-L1 single-chain variable fragment against cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is rare but hard-to-treat solid cancer affecting people worldwide. Cancer stem cells (CSCs) in CCA contribute to tumor heterogeneity and resistance to conventional therapies. Thus, CSCs are notable targets for cancer immunotherapy. CD133 is a potential CSC surface marker overexpressed in various cancers. T cell-based immunotherapy, namely chimeric antigen receptor (CAR) T-cell targeting CD133 has shown potential in eradication of some solid tumors including CCA. Nevertheless, anti-tumor function of CAR T cell could be decreased under immunosuppressive tumor microenvironment (TME). Thus, to improve its function under TME, the next-generation of CD133-specific CAR T-cell is required. We therefore constructed two lentiviral vectors containing: (i) anti-CD133 single-chain variable fragment (scFv), 4-1BB costimulatory molecule, and CD3ζ (133CAR), and (ii) anti-CD133 scFv, 4-1BB, and CD3ζ in tandem with anti-PD-L1 scFv (133CARsL). The lentiviruses were transduced to generate CD133-specific CAR T cells expressing the second-generation (133CART) or anti-CD133 CAR with self-secreting anti-PD-L1 scFv (133CARTsL). The latter was able to simultaneously target CD133 and PD-L1 molecules on CCA cells. CCA cells endogenously expressing CD133 and PD-L1 were cocultured with either 133CART or 133CARTsL. In short-term coculture, the antitumor effects of 133CARTsL were not different from that of 133CART. However, in long-term coculture and after tumor re-challenge, 133CARTsL showed lower expression of programmed cell death protein (PD-1) and better cytotoxic function against high CD133 and PD-L1 CCA cells. The higher potential of 133CARTsL with reduced exhaustion profile would offer great promise for application of CD133-targeting CAR T cells in solid tumors. Citation Format: Thanich Sangsuwannukul, Kamonlapat Supimon, Thaweesak Chieochansin, Kornkan Choomee, Jatuporn Sujjitjoon, Mutita Junking, Pa-thai Yenchitsomanus. Improvement of CD133-specific chimeric antigen receptor T cells by secreting anti-PD-L1 single-chain variable fragment against cholangiocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4105.

Investigating the resistance mechanism of 5-fluorouracil in colorectal cancer based on surface markers of cancer stemness and cytokine level: A pre-clinical study.

Colorectal cancer (CRC) is the deadliest malignancy in the world. The first-line chemotherapy used for CRC is 5-fluorouracil (5-FU). 5-FU completely eradicates rapidly proliferating and terminally differentiated tumor cells but fails to target cancer stem cells (CSCs). As a result, the tumor may shrink temporarily, but remnant CSC multiplies and forms a tumor again more aggressively. The recurrence and resistance lead to metastasis. CRC was induced in 12 Sprague-Dawley (RPCP/IAEC/2019-20/R2) rats by 1,2 dimethyl hydrazine. Later, animals were treated with 5-FU for 7 weeks at a 10 mg/kg dose by the subcutaneous route. At the end of treatment, half population was sacrificed (6), whereas the remaining half (6) was left without treatment of 5-FU for 5 weeks and then sacrificed. Parameters such as body weight, complete blood count (CBC), immune cell subset (CD4, CD8, and NK cells), colon length to weight index, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) level, occult blood in stool, tumor multiplicity, and liver metastasis were estimated. In addition, the dissected colon was fixed in formalin and sent to the histology lab for hematoxylin-eosin staining and immunohistochemistry at both intervals. All blood and tissue-based markers have shown significant differences (p < 0.05) between the animals sacrificed at the end of the 27th week and the end of the 32nd week for 5-FU treatment. It can be concluded that 5-FU up-regulates inflammatory cytokines and cell surface markers of CSC that promote CRC stemness via the Wnt/β-catenin pathway. Also, involvement of Nf-κB, fibronectin, MMP-9, and RANKL leads to tumorigenesis, disease aggressiveness, metastasis, and resistance.

Promoter Methylation of Cancer Stem Cell Surface Markers as an Epigenetic Biomarker for Prognosis of Oral Squamous Cell Carcinoma.

Growing evidence suggests that genetic and epigenetic factors, including environmental factors, contribute to the development of oral squamous cell carcinoma (OSCC). Here, we investigated the transcriptional silencing of the CD24, CD44, CD133, and CD147 genes, which are well-known cancer stem cell surface markers in various cancer types, including OSCC. We first examined the correlation between the transcriptional expression level and reactivation by 5-aza-2′-deoxycytidine (5-aza-dC) and the promoter methylation levels of the four genes in several OSCC cell lines. We observed promoter hypermethylation for the CD24, CD133, and CD147 genes at 70%, 75%, and 70%, respectively, in OSCC cell lines compared to normal oral mucosa tissues (<53%), indicating that this methylation pattern is cancer-specific, which was confirmed by bisulfite sequencing analysis. More specifically, the expression and methylation profiles of CD133 and CD147 extracted from The Cancer Genome Atlas (TCGA) database were negatively correlated, supporting their epigenetic regulation in primary OSCC tumors. The methylation status of CD133 and CD147 was associated with poor survival in patients with OSCC using the TCGA database. Our findings provide additional insight into the abnormal DNA methylation of CD133 and that CD147 could be used for the diagnosis and therapeutic treatment of patients with OSCC.

CD44v6+ Hepatocellular Carcinoma Cells Maintain Stemness Properties through Met/cJun/Nanog Signaling.

Cancer stem cells (CSCs) are characterized by their self-renewal and differentiation abilities. CD44v6 is a novel CSC marker that can activate various signaling pathways. Here, we hypothesized that the HGF/Met signaling pathway promotes stemness properties in CD44v6+ hepatocellular carcinoma (HCC) cells via overexpression of the transcription factor, cJun, thus representing a valuable target for HCC therapy. Magnetic activated cell sorting was used to separate the CD44v6+ from CD44v6- cells, and Met levels were regulated using lentiviral particles and the selective Met inhibitor, PHA665752. An orthotopic liver xenograft tumor model was used to assess the self-renewal ability of CD44v6+ cells in immunodeficient NOD/SCID mice. Luciferase reporter and chromatin immunoprecipitation assays were also conducted using cJun-overexpressing 293 T cells to identify the exact binding site of cJun in the Nanog promoter. Our data demonstrate that CD44v6 is an ideal surface marker of liver CSCs. CD44v6+ HCC cells express higher levels of Met and possess self-renewal and tumor growth abilities. Xenograft liver tumors were smaller in nude mice injected with shMet HCC cells. Immunohistochemical analysis of liver tissue specimens revealed that high Met levels in HCC cells were associated with poor patient prognosis. Further, a cJun binding site was identified 1700 bp upstream of the Nanog transcription start site and mutation of the cJun binding site reduced Nanog expression. In conclusion, the HGF/Met signaling pathway is important for maintenance of stemness in CD44v6+ HCC cells by enhancing expression of cJun, which binds 1700 bp upstream of the Nanog transcription start site.

Cancer Stem Cells in Hepatocellular Carcinoma: Intrinsic and Extrinsic Molecular Mechanisms in Stemness Regulation.

Hepatocellular carcinoma (HCC) remains the most predominant type of liver cancer with an extremely poor prognosis due to its late diagnosis and high recurrence rate. One of the culprits for HCC recurrence and metastasis is the existence of cancer stem cells (CSCs), which are a small subset of cancer cells possessing robust stem cell properties within tumors. CSCs play crucial roles in tumor heterogeneity constitution, tumorigenesis, tumor relapse, metastasis, and resistance to anti-cancer therapies. Elucidation of how these CSCs maintain their stemness features is essential for the development of CSCs-based therapy. In this review, we summarize the present knowledge of intrinsic molecules and signaling pathways involved in hepatic CSCs, especially the CSC surface markers and associated signaling in regulating the stemness characteristics and the heterogeneous subpopulations within the CSC pool. In addition, we recapitulate the effects of crucial extrinsic cellular components in the tumor microenvironment, including stromal cells and immune cells, on the modulation of hepatic CSCs. Finally, we synopsize the currently valuable CSCs-targeted therapy strategies based on intervention in these intrinsic and extrinsic molecular mechanisms, in the hope of shedding light on better clinical management of HCC patients.

Properties of cancer stem cells

Adult stem cells (ASCs) are pluripotent and make up a small percentage of healthy tissue. ASCs are essential for tissue repair, regeneration and growth. ASCs divide into identical stem cells or transiently proliferating cells. The latter cells mature after a number of divisions and become adult differentiating cells. There are different types of cells in cancerous tissue, as in normal, healthy tissue. Most cancer tissues contain three types of cells: (I) cancer stem cells (CSC); (II) cancer initiating cells; and (III) “differentiated”, non-proliferating cancer cells. In the review, the authors present experimental evidences for the presence of CSC, describe cell surface and intracellular markers of CSC. These markers allow for the appropriate identification and isolation of CSC. In addition, the possible location of these cells and theories of their formation are considered. It should be noted, that the theories of CSC originating are in fact theories of neoplasm formation. Many factors and processes may be involved in the formation of CSC. These include: (I) mutations of oncogenes or tumor suppressor genes in spoczynadult differentiated cells; (II) expression of different regulatory RNA types (microRNAs or long non-coding RNAs); processes of epithelial-mesenchymal transition and mesenchymalepithelial transition; (IV) CSC formation from differentiated cancer cells in a consequence of cancer therapy; (V) formation of CSC in conditions of hypoxy and during cell aging. The presented work synthetically characterizes the features, localization and formation of cancer stem cells. Furthermore, the research on these cells is still ongoing and many properties of CSC have not been fully understood.

Targeting Cancer Stem Cells: Therapeutic and diagnostic strategies by the virtue of nanoparticles.

Cancer stem cells (CSCs) are the subpopulation of cells present within a tumor with the properties of self-renewing, differentiating, and proliferating. Owing to the presence of ATP-binding cassette drug pumps and increased expression of anti-apoptotic proteins, the conventional chemotherapeutic agents have failed to eliminate CSCs resulting in relapse and resistance of cancer. Therefore, to obtain long-lasting clinical responses and avoid the recurrence of cancer, it is crucial to develop an efficient strategy targeting CSCs by either employing a differentiation therapy or specifically delivering drugs to CSCs. Several intracellular and extracellular cancer specific biomarkers are overexpressed by CSCs and are utilized as targets for the development of new approaches in the diagnosis and treatment of CSCs. Moreover, several nanostructured particles, alone or in combination with current treatment approaches, have been used to improve the detection, imaging, and targeting of CSCs, thus addressing the limitations of cancer therapies. Targeting CSC surface markers, stemness-related signaling pathways, and tumor microenvironmental signals has improved the detection and eradication of CSCs and, therefore, tumor diagnosis and treatment. This review summarizes a variety of promising nanoparticles targeting the surface biomarkers of CSCs for the detection and eradication of tumor-initiating stem cells, used in combination with other treatment regimens.

Reactive oxygen species-inducing titanium peroxide nanoparticles as promising radiosensitizers for eliminating pancreatic cancer stem cells

BackgroundDespite recent advances in radiotherapy, radioresistance in patients with pancreatic cancer remains a crucial dilemma for clinical treatment. Cancer stem cells (CSCs) represent a major factor in radioresistance. Developing a potent radiosensitizer may be a novel candidate for the eradication of pancreatic CSCs.MethodsCSCs were isolated from MIA PaCa-2 and PANC1 human pancreatic cancer cell lines. Titanium peroxide nanoparticles (TiOxNPs) were synthesized from titanium dioxide nanoparticles (TiO2NPs) and utilized as radiosensitizers when added one hour prior to radiation exposure. The antitumor activity of this novel therapeutic strategy was evaluated against well-established pancreatic CSCs model both in vitro and in vivo.ResultsIt is shown that TiOxNPs combined with ionizing radiation exhibit anti-cancer effects on radioresistant CSCs both in vitro and in vivo. TiOxNPs exhibited a synergistic effect with radiation on pancreatic CSC-enriched spheres by downregulating self-renewal regulatory factors and CSC surface markers. Moreover, combined treatment suppressed epithelial-mesenchymal transition, migration, and invasion properties in primary and aggressive pancreatic cancer cells by reducing the expression of proteins relevant to these processes. Notably, radiosensitizing TiOxNPs suppressed the growth of pancreatic xenografts following primary or dissociating sphere MIA PaCa-2 cell implantation. It is inferred that synergy is formed by generating intolerable levels of reactive oxygen species (ROS) and inactivating the AKT signaling pathway.ConclusionsOur data suggested the use of TiOxNPs in combination with radiation may be considered an attractive therapeutic strategy to eliminate pancreatic CSCs.

Cancer Stem Cell Markers for Urinary Carcinoma.

Cancer stem cell (CSC) refers to cancer cells with stem cell properties, that is, they have the ability of “self-renewal” and “differentiation.” Cancer stem cells exist in cancer cells and are the “culprit” of cancer recurrence and metastasis. It is difficult to be found because of its small amount, and it is difficult for anticancer drugs to produce effects on it. At present, the isolation and identification of cancer stem cells from many solid tumors are still quite difficult, mainly due to the lack of specific molecular markers of cancer stem cells. In this review, cancer stem cell surface markers and functional markers in urinary system were summarized. These markers can provide molecular targets for cancer therapy.