Stem Cell Surface Marker CD133 Research Articles

Role of mitochondria in endogenous renal repair.

Renal tubules have potential to regenerate and repair after mild-to-moderate injury. Proliferation of tubular epithelial cells represents the initial step of this reparative process. Although for many years, it was believed that proliferating cells originated from a pre-existing intra-tubular stem cell population, there is now consensus that surviving tubular epithelial cells acquire progenitor properties to regenerate the damaged kidney. Scattered tubular-like cells (STCs) are dedifferentiated adult renal tubular epithelial cells that arise upon injury and contribute to renal self-healing and recovery by replacing lost neighboring tubular epithelial cells. These cells are characterized by the co-expression of the stem cell surface markers CD133 and CD24, as well as mesenchymal and kidney injury markers. Previous studies have shown that exogenous delivery of STCs ameliorates renal injury and dysfunction in murine models of acute kidney injury, underscoring the regenerative potential of this endogenous repair system. Although STCs contain fewer mitochondria than their surrounding terminally differentiated tubular epithelial cells, these organelles modulate several important cellular functions, and their integrity and function are critical to preserve the reparative capacity of STCs. Recent data suggest that the microenviroment induced by cardiovascular risk factors, such as obesity, hypertension, and renal ischemia may compromise STC mitochondrial integrity and function, limiting the capacity of these cells to repair injured renal tubules. This review summarizes current knowledge of the contribution of STCs to kidney repair and discusses recent insight into the key role of mitochondria in modulating STC function and their vulnerability in the setting of cardiovascular disease.

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.

A novel approach for transforming breast cancer stem cells into endothelial cells.

Tumor vascular endothelial cells play a pivotal in the tumor microenvironment, influencing the proliferation, invasion, and metastasis of tumor progression. The present study investigated a novel method for inducing the transformation of breast cancer stem cells into endothelial cells, providing a cellular model investigating anti-angiogenic mechanisms in vitro. The breast cancer cell line MCF-7 was used, and the expression of CD133 was initially detected using flow cytometry. CD133+ breast cancer cells were purified using immunomagnetic bead sorting technology, yielding an MCF-7CD133+ subpopulation. The proliferation ability of these cells was assessed using an MTT assay, while their microsphere formation ability was evaluated using a microsphere formation assay. Post-transformation in an optimized endothelial cell culture medium, expression of endothelial cell markers CD31 and CD105 were detected using flow cytometry. Endothelial cell tube formation assays and DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) assays were employed to analyze the endothelial cell function of the MCF-7CD133+ cells. MDM2/CEN12 gene amplification was detected through fluorescence in situ hybridization (FISH). The MCF-7 breast cancer cell line exhibited 1.7±0.3% trace cells expressing the stem cell surface marker CD133. After anti-CD133 immunomagnetic bead sorting, MCF-7CD133+ and MCF-7CD133- subpopulation cells were obtained, with CD133 expression rates of 85.6±2.8 and 0.18±0.08%, respectively. MTT assay results demonstrated that, after 7 days, the proliferation rate of MCF-7CD133+ cells was significantly higher compared with MCF-7CD133- cells. MCF-7CD133+ subpopulation cells displayed strong stem cell characteristics, growing in suspension in serum-free media and forming tumor cell spheres. In contrast, MCF-7CD133- cells failed to form microspheres. After culturing cells in endothelial cell differentiation and maintenance media, the percentage of MCF-7CD133+ cells before and after endothelial cell culture was 0.3±0.16 and 81.4±8.37% for CD31+ cells and 0.2±0.08 and 83.8±7.24% for CD105+ cells, respectively. Vascular-like structure formation and Ac-LDL phagocytosis with red fluorescence in the tube formation assays confirmed endothelial cell function in the MCF-7CD133+ cells. FISH was used to verify MDM2/CEN12 gene amplification in the induced MCF-7CD133+ cells, indicating tumor cell characteristics. The modified endothelial cell transformation medium effectively induced differentiated tumor stem cells to express vascular endothelial cell markers and exhibit endothelial functions, ideal for in vitro anti-angiogenesis research.

Biological evaluation of [4-(4-aminophenyl)-1-(4-fluorophenyl)-1H-pyrrol-3-yl](3,4,5-trimethoxyphenyl)methanone as potential antineoplastic agent in 2D and 3D breast cancer models.

Targeting tubulin polymerization and depolymerization represents a promising approach to treat solid tumors. In this study, we investigated the molecular mechanisms underlying the anticancer effects of a structurally novel tubulin inhibitor, [4-(4-aminophenyl)-1-(4-fluorophenyl)-1H-pyrrol-3-yl](3,4,5-trimethoxyphenyl)methanone (ARDAP), in two- and three-dimensional MCF-7 breast cancer models. At sub-cytotoxic concentrations, ARDAP showed a marked decrease in cell proliferation, colony formation, and ATP intracellular content in MCF-7 cells, by acting through a cytostatic mechanism. Additionally, drug exposure caused blockage of the epithelial-to-mesenchymal transition (EMT). In 3D cell culture, ARDAP negatively affected tumor spheroid growth, with inhibition of spheroid formation and reduction of ATP concentration levels. Notably, ARDAP exposure promoted the differentiation of MCF-7 cells by inducing: (i) expression decrease of Oct4 and Sox2 stemness markers, both in 2D and 3D models, and (ii) downregulation of the stem cell surface marker CD133 in 2D cell cultures. Interestingly, treated MCF7 cells displayed a major sensitivity to cytotoxic effects of the conventional chemotherapeutic drug doxorubicin. In addition, although exhibiting growth inhibitory effects against breast cancer cells, ARDAP showed insignificant harm to MCF10A healthy cells. Collectively, our results highlight the potential of ARDAP to emerge as a new chemotherapeutic agent or adjuvant compound in chemotherapeutic treatments.

SPRED2: A Novel Regulator of Epithelial-Mesenchymal Transition and Stemness in Hepatocellular Carcinoma Cells.

The downregulation of SPRED2, a negative regulator of the ERK1/2 pathway, was previously detected in human cancers; however, the biological consequence remains unknown. Here, we investigated the effects of SPRED2 loss on hepatocellular carcinoma (HCC) cell function. Human HCC cell lines, expressing various levels of SPRED2 and SPRED2 knockdown, increased ERK1/2 activation. SPRED2-knockout (KO)-HepG2 cells displayed an elongated spindle shape with increased cell migration/invasion and cadherin switching, with features of epithelial-mesenchymal transition (EMT). SPRED2-KO cells demonstrated a higher ability to form spheres and colonies, expressed higher levels of stemness markers and were more resistant to cisplatin. Interestingly, SPRED2-KO cells also expressed higher levels of the stem cell surface markers CD44 and CD90. When CD44+CD90+ and CD44-CD90- populations from WT cells were analyzed, a lower level of SPRED2 and higher levels of stem cell markers were detected in CD44+CD90+ cells. Further, endogenous SPRED2 expression decreased when WT cells were cultured in 3D, but was restored in 2D culture. Finally, the levels of SPRED2 in clinical HCC tissues were significantly lower than those in adjacent non-HCC tissues and were negatively associated with progression-free survival. Thus, the downregulation of SPRED2 in HCC promotes EMT and stemness through the activation of the ERK1/2 pathway, and leads to more malignant phenotypes.

Temporomandibular Joint Fibrocartilage Contains CD105 Positive Mouse Mesenchymal Stem/Progenitor Cells with Increased Chondrogenic Potential.

A specific type of mesenchymal stem/progenitor cells (MSPCs), CD105+ is reported to aid in cartilage regeneration through TGF-β/Smad2-signalling. The purpose of this study was to identify and characterize CD105+ MSPCs in temporomandibular joint (TMJ) cartilage. MSPCs were isolated from mouse TMJ condyle explants and evaluated for their clonogenicity and pluripotential abilities. MSPC were examined for CD105 antigen using immunohistochemistry and flow cytometry. Immunohistochemistry revealed presence of CD105+ MSPCs in the proliferative zone of condyle's cartilage. Only 0.2% of isolated MSPCs exhibited CD105, along with the stem cell surface markers CD44 and Sca-1. In CD105+ MSPCs, intracellular immunostaining revealed significantly higher (p < 0.05) protein levels of collagen type 1, 2, proteoglycan 4. Ability for chondrogenic differentiation was found to be significantly higher (p < 0.05) after 4weeks compared to CD105- cells, using alcian blue staining. CD105+ cells were found to resemble an early MSPC subgroup with significantly higher gene expression of biglycan, proteoglycan 4, collagen type 2, Gli2, Sox5 (p < 0.001) and Sox9 (p < 0.05). In contrast, significantly lower levels of Runx2 (p < 0.05), Osterix, Trps1, Col10a1 (p < 0.01), Ihh (p < 0.001) related to chondrocyte senescence and commitment to osteogenic lineage, were observed compared to CD105- cells. The study showed the existence of a CD105+ MSPC subgroup within TMJ fibrocartilage that may be activated to aid in fibrocartilage repair.

Activation of Glucocorticoid Receptor Inhibits the Stem-Like Properties of Bladder Cancer via Inactivating the β-Catenin Pathway.

Background: Glucocorticoid receptor (GR) signaling pathway has been shown to involve epithelial -to- mesenchymal transition which was implicated in the regulation of bladder cancer stem cells (CSCs) in our previous study. Herein, we aim to figure out how GR affects the stem-like properties of bladder cancer cells.Methods: We used dexamethasone (DEX) treatment or gene-knockdown/-knockout techniques to activate or silence the GR pathway, respectively. Then we applied immunohistochemical staining and flow cytometry to assess the associations between the expression levels of GR and a stem cell surface marker CD44. Stem-like properties were assessed by reactive oxygen species (ROS), sphere-formation and side population assays. The expression levels of cancer stem cell-associated molecules were assessed by quantitative PCR and Western blotting. Tumor growth was compared using mouse xenograft models.Results: In GR-positive bladder cancer cells, DEX significantly reduced the expression of CD44 as well as pluripotency transcription factors including β-catenin and its downstream target (C-MYC, Snail, and OCT-4), the rate of sphere formation, and the proportion of side populations, and induced the intracellular levels of ROS. By contrast, GR silencing in bladder cancer cells showed the opposite effects. In xenograft-bearing mice, GR silencing resulted in the enhancement of tumor growth.Conclusions: These data suggested that GR activity was inversely associated with the stem-like properties of bladder cancer cells, potentially via inactivating the β-catenin pathway.

Metformin treatment decreases the expression of cancer stem cell marker CD44 and stemness related gene expression in primary oral cancer cells

ObjectiveMetformin, a common drug for diabetes treatment has shown promising characteristics against wide types of cancer cells in vitro as well as in vivo in the context of halted growth of cancer. But, it was unclear whether cancer stem cells are affected by the metformin treatment. Here, we attempt to find out the effect of metformin on cancer stem cell marker CD44 and stemness related transcription factors including OCT4, SOX2, NANOG, c-Myc and KLF4. Materials and MethodsWe prepared single-cell suspension from primary oral tumors and subjected the cells to grow in vitro. Gene expression of transcription factors was assessed by real-time PCR. Further, the expression of CD44 was checked by flow Cytometry. ResultsMetformin showed downregulation in the gene expressions of stemness related transcription factors OCT4, SOX2, NANOG, c-Myc, and KLF4 in a dose-dependent as well as time-dependent manner. Also, the most effective concentration of metformin at 25 μM was found to decrease the expression of CD44 in the primary tumor cells in a time-dependent manner. ConclusionContinuous treatment of lower concentrations of metformin decreases the expression of cancer stem cell markers at the transcription level and cancer stem cell-surface marker CD44 in primary oral cancer cells.

Inducing differentiation of human urine-derived stem cells into hepatocyte-like cells by coculturing with human hepatocyte L02 cells.

To investigate the possibility of inducing differentiation of human urine-derived stem cells (hUSCs) into hepatocyte-like cells by coculturing with human hepatocyte L02 cells in vitro. HUSCs were isolated from fresh urine samples collected from healthy adult volunteers by centrifugation. Cells were observed under an inverted phase contrast microscope, and proliferative activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Stem cell surface markers were detected by flow cytometry. HUSCs were induced to differentiate into hepatocyte-like cells by coculturing with human hepatocyte L02 cells, which were confirmed by cellular morphology, messenger RNA expression of albumin (ALB), α-fetoprotein (AFP) and hepatocyte cytochrome P450 (CYP450) analyzed with quantitative reverse transcription polymerase chain reaction and the expression of glycogen detected by glycogen staining kits at 5, 10, and 15 days after coculturing. HUSCs from urine were successfully isolated and cultured in vitro. At passages 3, the growth curve of hUSCs was S-shaped with good proliferation activity. Mesenchymal stem cell surface markers CD44 and CD90 were detected positive by flow cytometry. CD31 for endothelial cells and CD34 for hematopoietic stem cell markers were not detected. HUSCs gained the cellular morphology and function of hepatocyte cells including higher expression of several hepatocyte-specific genes such as ALB and some CYP450, lower expression of AFP and positive glycogen expression (P < .05) in coculturing with human hepatocyte L02 cells for 10-15d. HUSCs can be induced to differentiate into hepatocyte-like cells by coculturing with human hepatocyte L02 cells for a certain number of days.

In vitro enhancement and functional characterization of neurite outgrowth by undifferentiated adipose-derived stem cells.

Adipose‑derived stem cells(ASCs) can easily be obtained and expanded invitro for use in autologous cell therapy. Via their production of cytokines and neurotrophic factors, transplanted ASCs provide neuroprotection, neovascularization and induction of axonal sprouting. However, the influencing mechanism of undifferentiated ASCs on nerve regeneration is currently only partially understood. In the present study, undifferentiated ASCs and cutaneous primary afferent dorsal root ganglion(DRG) neurons were co‑cultured in order to investigate their interaction. ASCs were isolated from adult rat fat tissue. The presence of characteristic stem cell markers was determined by flow cytometry in three subsequent passages. Adipogenic, osteogenic, chondrogenic and glial differentiation was performed in order to evaluate their differentiation capacity. A direct co‑culture system with DRG cells was established to determine the effect of undifferentiated pluripotent ASCs on neurite elongation. Neurite outgrowth, length and number was examined in the co‑culture and compared with single‑culture cells and cells stimulated with nerve growth factor (NGF). In ASC cultures, NGF expression was assessed by ELISA. The present results demonstrated that the specific mesenchymal stem cell surface markers CD44, CD73 and CD90 were detected in all three subsequent passages of the isolated ASCs. In accordance, ASC differentiation into adipogenic, osteogenic, chondrogenic and Schwann cell phenotype was conducted successfully. Neurite outgrowth of DRG neurons was enhanced following co‑culture with ASCs, resulting in increased neurite length after 24h of cultivation. Furthermore, neurite outgrowth of DRG neurons was directed towards the undifferentiated ASC and direct cell‑to‑cell contact was observed. In summary, the results of the present study revealed an interaction between the two cell types with guidance of neurite growth towards the undifferentiated ASC. These findings suggest that the use of undifferentiated ASC optimizing tissue‑engineered constructs may be promising for peripheral nerve repair.

Abstract A040: Targeting the prostate cancer stem cell niche with the STAT3 inhibitor galiellalactone

Abstract Introduction and Objectives: Cancer stem-like cells (CSCs) represent a small subpopulation of largely quiescent cells that reside within tumors. Several studies have demonstrated that this population is more resistant to current therapies and is therefore directly responsible for tumor recurrence. Recent evidence suggests that the transcription factor STAT3 is crucial to the survival of stem cells in prostate cancer (PCa). Thus, inhibition of activated STAT3 (pSTAT3) is a valid strategy to selectively target PCa CSCs. We have previously shown that pSTAT3 blockade, by galielallactone (GL), not only reduces proliferation and induces apoptosis of prostate cancer cells in vitro, but it also inhibits the growth of prostate tumors and the metastatic spread to regional and distal lymph nodes, in vivo. Here we performed experiments aimed at studying the effect of the STAT3 inhibitor, GL, on PCa CSCs, as a promising therapeutic approach for prostate cancer patients. Materials and Methods: The expression of stem, basal, and luminal cell surface markers (CD133/CD44/CD24) was analyzed by FACS on DU145 and PC3 cells following treatment with GL. DU145 cells were sorted, based on the expression of the stem cell surface marker CD133, and then plated for further analysis. Expression of pSTAT3 was studied by IHC, on sorted cells, and the effect of GL on clonogenic recovery was determined. Furthermore, the effect of GL on sphere formation efficiency was studied on the CD133 population. The compound galiellalactone was obtained from Glactone Pharma (Sweden). Results: Our results show that the CSCs population (CD133+/CD44+) expresses high levels of activated pSTAT3 compared to the CD44+/CD24+ cell population. Treatment with GL decreased the number of CSCs in DU145 cells after 48h, while the number of CD44+/CD24+ cells was not significantly affected. Importantly, treatment with GL had no effect on CD133+/CD44+ or CD44+/CD24+ populations in PC3 cells, which do not express pSTAT3. Treatment with GL significantly reduced the ability of CSCs to form colonies and to generate prostate-derived spheres. Interestingly, the CSCs population (CD133+) was more sensitive to GL in terms of colony- and sphere-forming efficiency compared to the CD133- population. Conclusion: This study demonstrates that the STAT3 inhibitor galiellalactone can specifically target the prostate cancer stem-like cell population in vitro, implying that pSTAT3 inhibition by GL could represent a valid therapeutic strategy to antagonize CSCs in human prostate cancer. Future experiments will be aimed at validating these data and relating the clonogenic inhibition to the effects of pSTAT3 blockade by GL on tumor initiation by prostate cancer stem-like cells in vivo. Citation Format: Giacomo Canesin, Anne T. Collins, Rebecka Hellsten, Norman J. Maitland, Anders Bjartell. Targeting the prostate cancer stem cell niche with the STAT3 inhibitor galiellalactone [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A040.

Impact of Cystic Fibrosis Transmembrane Conductance Regulator on Malignant Properties of KRAS Mutant Lung Adenocarcinoma A549 Cells

背景与目的肺癌发病率逐年上升，有必要寻找新型的治疗靶点，而最新研究发现囊状纤维化跨膜转导调节子（cystic fibrosis transmembrane conductance regulator, CFTR）与多种肿瘤的发生和恶性转化有关。本研究探讨CFTR对肺癌A549细胞恶性特性的影响。方法应用CCK8细胞增殖实验、细胞划痕实验、Transwell细胞侵袭实验以及克隆形成实验等方法分别检测CFTR的表达对非小细胞肺癌A549细胞的增殖、迁移、侵袭等细胞恶性特性的影响。同时通过免疫印迹（Western blot）分析CFTR基因表达对肿瘤干细胞相关转录因子表达的影响。结果过表达CFTR基因显著抑制A549细胞的增殖、迁移、侵袭和克隆形成等肿瘤恶性特征，而RNA干扰A549细胞CFTR的表达导致细胞上述特征的明显增强。免疫印迹实验进一步发现CFTR基因过表达抑制A549细胞中干细胞相关转录因子SOX2和OCT3/4，以及细胞表面CD133蛋白的表达；相反，RNA干扰A549细胞中CFTR基因的表达增加SOX2、OCT4和CD133的表达。然而，免疫印迹和流式细胞术发现CFTR基因表达对A549细胞肺癌干细胞标志乙醛脱氢酶1的表达和阳性细胞数量无显著影响。结论CFTR基因在肺癌A549细胞中具有抑制细胞恶性特征的作用，提示其可能是肺腺癌治疗的一个新的靶点，但其对其他肺腺癌细胞的作用与分子机制还有待进一步研究。

Protein arginine methyltransferase 8 gene enhances the colon cancer stem cell (CSC) function by upregulating the pluripotency transcription factor.

Objective: Cancer stem cells play a crucial role in tumor multidrug resistance and metastasis, which can produce heterogeneous tumor cells and have self-renewal ability. The related literature reported that PRMT8 was overexpressed in tumor stem cells and pluripotent stem cells. However, it's unclear how PRMT8 acts on the stemness of colon tumor cells. This study is designed to detect functions by transfecting with PRMT8 plasmid to colon cancer cells.Methods: In this study we investigated colon cancer cell sphere and its differential expression of PRMT8 compared with colon cancer cells grown by static adherence. RKO Sphere formation assay was used to identify CSCs and verified PRMT8 and pluripotent transcription factors SOX2, OCT4, Nanog expression level in colon cell sphere. Colon cancer cell HCT-8 and RKO up-regulated PRMT8 expression by being transfected with PRMT8 plasmid to evaluate its effect on the stemness of colon tumor cell.Results: In RKO cell sphere, stem cell surface marker CD133 and CD44 were highly expressed. And PRMT8, SOX2, OCT4 and Nanog were also highly expressed in RKO cell sphere. After PRMT8 was up-regulated in HCT-8 and RKO cells, flow cytometry proved that PRMT8 group cells have a significant increase of the side population (SP) cells with cancer stem cell surface markers CD133 and CD44. And overexpression of PRMT8 in HCT-8 and RKO cells facilitated their aggressive traits, which contained proliferation, invasion and migration, as well as leading to their drug resistance. PRMT8 may play a role in colon cancer stem cells (CSC) through its regulation of pluripotent transcription factors, such as Nanog Homeobox (Nanog), octamer-binding transcription factor-4 (Oct4) and SRY-related high-mobility-group(HMG)-box protein-2 (Sox2).Conclusion: PRMT8 may promote the formation of colon cancer stem cells and, thus, be considered a potential therapeutic target for the treatment of malignant colon tumor.

Abstract 1904: Novel 5-fluorouracil-thymoquinone hybrid kills colon cancer stem cells

Abstract Objectives: Recently, it has been shown that a subset population of cancer cells in a tumor, the so-called cancer stem cells (CSCs), majorly contributes to chemoresistance and the ability of a tumor to form recurrences and metastases. Since the response to the standard chemotherapeutic drug 5-fluorouracil (5-FU) in patients with colorectal cancer (CRC) leads only to an inadequate response, we aimed to synthesize new hybrid molecules by combining 5-FU with the plant-derived drug thymoquinone (TQ). Methods: The cytotoxic effect of hybrids, the single drugs and the combination was investigated by crystal violet assays. HCT116, HT29 and 5-FU-resistant colorectal tumor cells were used to assess the spheroid forming capability after treatment with different concentrations of the hybrid and the single drugs by staining with the fluorescent stem cell marker CDy1 and Hoechst 3324 after 21 days of incubation. Western Blot analysis for stem cell surface marker CD133 were performed to verify these results. We have analyzed the anticancer effects of the single drugs TQ and 5-FU and the most effective hybrid on deregulation of cancer signaling pathways utilizing the NANOSTRING cancer pathway panel. In addition, we performed a single cell cloning from spheroids to generate monoclonal cell lines with enriched CSC populations. In vivo effects were studied in the chick chorioallantoic membrane (CAM) assay and in mouse xenografts. Results: The 5FU-TQ hybrid has an increased cytotoxic effect towards CRC cells when compared to single drug treatments. The spheroid formation assay revealed the capability of the hybrid to reduce sphere number and size at fairly low concentrations (10, 20 µM), however at 50 µM there was a complete suppression of spheroid growth. NANOSTRING analysis showed that gene expression of main components of the PI3K/AKT signaling pathway was significantly down-regulated after treatment with all drugs but most prominently with the new hybrid. After data processing we found that AKT3, IGF1, COL11A1, FGF10, GNG7, EPO genes were downregulated in hybrid-treated cells, while CDKN1A, BAX, JUN, MDM2, FAS were upregulated. This was verified via Western Blotting for a panel of markers. Next, we investigated the effectiveness of the hybrid to kill clones having an enriched CSC population. Indeed, CD133 protein levels were significantly decreased in hybrid-treated clones. Histological analysis of in vivo CAM grafts showed that HCT116 control cells developed a well vascularized tumor mass whereas HCT116 cells that were treated with the hybrid showed no more vital tumor cells, but extended areas of necrosis, calcification, and desmoplasia. In vivo mouse xenografts verified the potential of this hybrid as an antitumor agent reducing tumor volume significantly. Conclusion: Our findings strongly suggest that the newly synthesized 5-FU-TQ hybrid might become a promising anticancer drug by directly targeting CSCs. Citation Format: Benardina Ndreshkjana, Aysun Çapcı Karagöz, Volker Klein, Pithi Chanvorachote, Julienne K. Muenzner, Kerstin Huebner, Chuanpit Ninsontia, Sara Steinmann, Abbas Agaimy, Maamoun Fatfat, Hala Gali-Muhtasib, Svetlana B. Tsogoeva, Regine Schneider-Stock. Novel 5-fluorouracil-thymoquinone hybrid kills colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1904. doi:10.1158/1538-7445.AM2017-1904

Analysis of dysregulated long non-coding RNA expressions in glioblastoma cells

Long non coding RNAs (lncRNAs) are associated with various biological roles such as embryogenesis, stem cell biology, cellular development and present specific tissue expression profiles. Aberrant expression of lncRNAs are thought to play a critical role in the progression and development of various cancer types, including gliomas. Glioblastomas (GBM) are common and malignant primary brain tumours. Brain cancer stem cells (BCSC) are isolated from both low and high-grade tumours in adults and children, by cell fraction which express neuronal stem cell surface marker CD133. The purpose of this study was to investigate the expression profiles of lncRNAs in brain tumour cells and determine its potential biological function. For this purpose, U118MG-U87MG; GBM stem cell series were used. Human parental brain cancer cells were included as the control group; the expressions of disease related human lncRNA profiles were studied by LightCycler 480 real-time PCR. Expression profiles of 83 lncRNA genes were analyzed for a significant dysregulation, compared to the control cells. Among lncRNAs, 51 lncRNA genes down-regulated, while 8 lncRNA genes were up-regulated. PCAT-1 (−2.36), MEG3 (−5.34), HOTAIR (−2.48) lncRNAs showed low expression in glioblastoma compared to the human (parental) brain cancer stem cells, indicating their role as tumour suppressor genes on gliomas. As a result, significant changes for anti-cancer gene expressions were detected with disease-related human lncRNA array plates. Identification of novel target genes may lead to promising developments in human brain cancer treatment.

Abstract B05: Mesenchymal-driven hedgehog signaling drives tumor cell growth and is a potential new target for triple-negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a clinical therapeutic challenge due to the lack of receptors for estrogen, progesterone, and human epidermal growth factor receptor 2 which limits treatment options to chemotherapy and radiation. With current standard therapy less than 30% will survive the 5-year remission rate, especially Hispanic and African-American women where TNBC is more frequent and has the lowest (&amp;lt;14%) 5-year survival rates. Recent studies indicate that Hedgehog (Hh) signaling is active and correlates with reduced survival rates in TNBC patients. However, the role of the Hh signaling in the breast tumor microenvironment is not well understood. A main cellular mediator of Hh signaling is the adjacent mesenchyme which promotes tumor growth via a paracrine interaction, but which is also highly heterogeneous. As mesenchymal sub-types have been associated to specific pharmacological therapies, understanding of the mechanisms in mesenchymal-driven tumors is necessary for combinatorial pharmacological treatments that can increase survival rates and eliminate tumor relapse in patients. We developed a tumor-mesenchymal in vitro model to evaluate the role of mesenchymal cell sub-types from different sources in the proliferative potential and stem cell markers of breast cancer cells using a custom designed multiwell array. Cells were culture in adjacent compartments and active Hh signaling was confirmed by up-regulation of canonical Hh target genes Gli1, Patch1 and SMO. As a source of mesenchymal cell sub-types we evaluated TGF-β treated fibroblasts, cancer- associated and normal breast primary fibroblasts, and tumor epithelial cells that undergo epithelial-mesenchymal transition (EMT). Paracrine Hh signaling significantly increased proliferation (5-10%) of tumor cells after 72-96hrs in co-culture. Cells that undergo EMT promoted cell growth at higher rates than myofibroblasts. SHH treatment alone (without TGF-β pre-treatment) had an inhibitory effect in the proliferation rates of the adjacent epithelium compartment. Observed increase in tumor cell proliferation was abolished when treated with the Hh signaling inhibitor cyclopamine. Stem cell surface markers CD44+CD24- remained unchanged but a significant increase was observed in the proliferating CD44+/CD24+ sub-population. Similar results were observed in normal human breast cells suggesting that activation of Hh signaling in the mesenchyme is sufficient to promote cell growth in the epithelium. Computational analysis of a panel of genes associated to Hh signaling in breast cancer patients (&amp;gt;500 samples) show a strong correlation among TNBC patients and mesenchymal-driven Hh signaling in &amp;gt;80% of samples. We evaluated the distribution of mesenchymal cell-sub-types and stem cells among the sub-set of basal TNBC samples (82 samples). Gene markers associated to myofibroblasts, EMT, mesenchymal stem cell and cancer stem cell were found in samples with active Hh signaling and co-expressed in groups of two or more which represented the 45.6% of the total samples. The most abundant cell sub-type were cancer stem cells with 43% of the total samples and alone by itself in 13.9% of samples. Together, these results indicate that the Hh-pathway and adjacent tumor microenvironment are key players in the progression of TNBC. Mesenchymal cell component can modulate tumor growth behavior induced by Hh signaling and is a potential new clinical target for TNBC. Citation Format: Maribella Domenech, Karla P. Ramos, Wandaliz Torres, Fernando Boria. Mesenchymal-driven hedgehog signaling drives tumor cell growth and is a potential new target for triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B05.

Stem cell surface markers CD133 expression in hepatocellular carcinoma and as single prognostic factor for liver transplantation.

e15166 Background: Recent evidences indicate that CD133, a primitive haematopoietic and neuronal stem cell marker, is known to be present in cancer stem cells (CSCs) in hepatocellular carcinoma(HCC...

Characterization of glioma stem-like cells from human glioblastomas.

Glioma stem-like cells (GSCs) could have potential for tumorigenesis, treatment resistance, and tumor recurrence (GSC hypothesis). However, the mechanisms underlying such potential has remained elusive and few ultrastructural features of the cells have been reported in detail. We therefore undertook observations of the antigenic characteristics and ultrastructural features of GSCs isolated from human glioblastomas. Tumor spheres formed by variable numbers of cells, exhibiting a variable appearance in both their size and shape, were frequently seen in GSCs expressing the stem cell surface markers CD133 and CD15. Increased cell nucleus atypia, mitochondria, rough endoplasmic reticulum, coated vesicles, and microvilli, were noted in the GSCs. Furthermore, cells at division phases and different phases of the apoptotic process were occasionally observed. These findings could imply that GSCs have certain relations with human neural stem cells (NSCs) but are primitively different from undifferentiated NSCs. The data may provide support for the GSC hypothesis, and also facilitate the establishment of future glioblastoma treatments targeting GSCs.

Abstract 268: Circadian rhythms of glioma stem cells and progenitor cells.

Abstract The circadian timing system generates and maintains daily rhythms in physiological processes throughout the body. Disruption of circadian timing is associated with higher cancer incidence and more malignant cancers, although the interactions between cancer and circadian rhythms remain unclear. Embryonic stem cells appear to lack endogenous circadian rhythms until they differentiate. We asked whether cancer stem cells (CSCs) also lack persistent circadian oscillations at the molecular level. An inability to generate circadian rhythms may be linked to the undifferentiated state of CSCs, and treatments that can induce clock gene expression might help to initiate rhythms and promote differentiation. Because CSCs appear to evade standard cancer therapies that are effective against non-CSCs new methods to force their differentiation would be useful. To examine the circadian properties of CSCs, we selected the C6 rat glioma cell line because it expresses a strong circadian rhythm in gene expression. C6 CSCs have been characterized through flow cytometry by the side-population cell sorting method that relies on the ability of CSCs to efflux the fluorescent dye Hoechst 33342. We developed a Hoechst dye-exclusion method to identify putative CSCs in C6 monolayer cultures. This cell subpopulation failed to show a circadian rhythm in nuclear transport of a fusion protein of GFP and the circadian clock component mPER2, while the remaining cells were rhythmic. Evidence that these non-rhythmic cells are CSCs was shown by their proliferation in a medium that promotes stem cell growth and their prevalence in neurospheres grown from the C6 line that are masses of CSCs and partially differentiated cells including progenitor cells. The spheres showed immunostaining for the stem cell surface marker CD-133 and they could be induced to form astrocyte and neuron-like cells according to the cell-specific markers GFAP and beta III-tubulin, respectively. To determine whether the neurospheres are capable of generating circadian rhythms, C6 cells expressing a firefly luciferase and mPER2 fusion protein under control by the mPer2 gene promoter were imaged after they formed neurospheres. Circadian rhythms in bioluminescence persisted when the cells were synchronized with a 2-hour pulse of forskolin that induces clock genes but not when the spheres were imaged without forskolin treatment. We interpret these results as indicating a lack of circadian clocks in CSCs but circadian clocks functioning in progenitor cells of neurospheres that require synchronization for coordinated circadian activity. Because CSCs in the microenvironment of tumors may also be arrhythmic, efforts to manipulate circadian clock genes should be examined when treating gliomas and possibly other cancers. Citation Format: Vishal P. Sharma, Michael E. Geusz. Circadian rhythms of glioma stem cells and progenitor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 268. doi:10.1158/1538-7445.AM2013-268

Abstract 3307: Transcriptional silencing of c-Myc by promoter-directed siRNA efficiently target prostate cancer stem cells.

Abstract Prostate cancer is the most frequent epithelial cancer in elderly men in Western countries. Depending on the stage, prostate cancer treatment requires surgery, hormone therapy, radio- and chemo-therapy. c-MYC is a transcription factor activated by mitogenic signaling pathways and playing a central function in stem cell biology. Over-activity of c-Myc plays a central role in tumorigenesis by affecting cell proliferation, metabolic adaptation and survival. Amplification of c-MYC is one of the most common genetic alterations occurring in cancer genomes. c-Myc expression is significantly elevated in invasive prostate adenocarcinomas compared to benign prostatic hyperplasia and normal prostate tissue. Particularly, c-Myc is highly expressed in prostate cancer cells having the CD44+/CD24- phenotype, which is described as a hallmark of cancer progenitor/stem cells (CSCs). Importantly, several studies link therapy resistance and disease progression to the CSC subpopulation within the highly heterogeneous cellular composition of the tumor. CSCs are likely the tumor initiating population that produces metastasis and disease recurrence. These lines of evidence suggest a central role of c-Myc in the maintenance of CSC compartment in human tumors and justify c-Myc as a therapeutic target. Previously, we showed that the c-Myc gene could be silenced with small interfering RNAs (siRNA) targeting its promoter. Interestingly, single transfection with myc targeting siRNA (myc-siRNA) induced long lasting effects on cell proliferation and clonogenicity, indicative of a persistent loss of proliferative and clonogenic potential. In this study, we investigated the effects of transcriptional silencing of c-Myc on the CSC subpopulation in human prostate cancer cell lines in vitro and in vivo. We found that treatment with myc-siRNA significantly reduced the fraction of CSCs defined by expression of stem cell surface markers CD44 and CD24, in vitro sphere forming ability and self-renewal. Furthermore, combined analysis of senescence and cell surface markers showed that senescence occurred prevalently in the CD44+/CD24- cell subpopulation leading to its depletion, and reduced self renewal capability, in vivo tumorigenicity and metastatic potential. Moreover, repeated intraperitoneal administration of myc-siRNA over a 4-week period reduced tumor growth in a xenogeneic prostate cancer model. These results are consistent with the role of c-Myc in the maintenance of the CSC subpopulation in human prostate tumors and suggest that c-Myc downregulation induces an irreversible loss of clonogenic and tumor-initiating capability linked to the induction of cell senescence in CSCs. Our findings show also that an RNAi-based transcriptional therapy directed to genes directly involved in the maintenance of the cancer stem cells could be an efficient approach to block progression and relapse of prostate cancer. Citation Format: Gianluca Civenni, Anastasia Malek, Domenico Albino, Manuela Sarti, Sandra Pinton, Stefano Di Marco, Giuseppina M. Carbone, Carlo V. Catapano. Transcriptional silencing of c-Myc by promoter-directed siRNA efficiently target prostate cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3307. doi:10.1158/1538-7445.AM2013-3307