CD133 Low Cells Research Articles

Role of CD133/NRF2 Axis in the Development of Colon Cancer Stem Cell-Like Properties

Cancer stem cells (CSCs) exhibit intrinsic therapy/stress resistance, which often cause cancer recurrence after therapy. In this study, we investigated the potential relationship between the cluster of differentiation (CD)-133, a CSC marker of colon cancer, and nuclear factor erythroid 2-like 2 (NFE2L2; NRF2), a master transcription factor for the regulation of multiple antioxidant genes. In the first model of CSC, a sphere culture of the colorectal cell line HCT116, showed increased levels of CD133 and NRF2. Silencing of CD133 reduced the levels of CSC markers, such as Kruppel-like factor 4 (KLF4) and ATP-binding cassette subfamily G member 2 (ABCG2), and further suppressed the expression levels of NRF2 and its target genes. As a potential molecular link, CD133-mediated activation of phosphoinositide 3-kinase/serine-threonine kinase (PI3K/AKT) signaling appears to increase the NRF2 protein levels via phosphorylation and the consequent inhibition of glycogen synthase kinase (GSK)-3β. Additionally, NRF2-silenced HCT116 cells showed attenuated sphere formation capacity and reduced CSC markers expression, indicating the critical role of the NRF2 pathway in the development of CSC-like properties. As a second model of CSC, the CD133high cell population was isolated from HCT116 cells. CSC-like properties, including sphere formation, motility, migration, colony formation, and anticancer resistance, were enhanced in the CD133high population compared to CD133low HCT116 cells. Levels of NRF2, which were elevated in CD133high HCT116, were suppressed by CD133-silencing. In line with these, the analysis of The Cancer Genome Atlas (TCGA) database showed that high levels of CD133 expression are correlated with increased NRF2 signaling, and alterations in CD133 gene or expression are associated with unfavorable clinical outcome in colorectal carcinoma patients. These results indicate that the CD133/NRF2 axis contributes to the development of CSC-like properties in colon cancer cells, and that PI3K/AKT signaling activation is involved in CD133-mediated NRF2 activation.

Design, Characterization, and Evaluation of scFvCD133/rGelonin: A CD133-Targeting Recombinant Immunotoxin for Use in Combination with Photochemical Internalization.

The objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133high and CD133low cell lines and a CD133neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133high cells compared to CD133low cells. No cytotoxic responses were detected in either CD133high or CD133low cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133high and CD133low cancer cells.

Abstract 2890: Trifluridine efficacy in cancer stem cell

Abstract Background: Cancer stem cells (CSC) have been involved in disease recurrence, metastasis, and therapeutic resistance, but effective anti-cancer agent for these cells is not still currently available. Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer who have been previously treated with fluoropyrimidine, oxaliplatin and irinotecan based chemotherapy etc. and improved overall survival. Here we showed the efficacy of FTD in CD44 and CD133 high expressing (CD44high/CD133high) cancer cells which has CSC like property. Method: CD44high /CD133high and CD44low /CD133low DLD-1 cells were separated by Fluorescence-Activated Cell Sorting (FACS) and subjected to sphere formation assay. Anti-proliferative and anti-sphere forming effects of FTD in CD44high /CD133high cells were measured. Then, 150 days FTD long exposed colorectal cell line's CSC markers CD44 and CD133 were measured by flow cytometry and its tumor initiating property in in vitro and in vivo were evaluated. Results: CD44 high /CD133 high DLD-1 cells formed much more sphere than CD44low /CD133low cells significantly. Compared to unsorted cells, CD44high /CD133high cells displayed 5-FU resistance (IC50 value of 5-FU for unsorted and CD44 high /CD133 high cells were 2.5 μM and 5.5 μM respectively) but not FTD resistance (IC50 value of FTD for unsorted and CD44 high /CD133 high cells were 8.9 μM and 10.7 μM, respectively) in in vitro proliferation assay. In addition, FTD treatment in a subtoxic concentration 1 μM for DLD-1 cells disrupted sphere formation. FTD-long exposed DLD-1 cell lines showed decreased CD44high /CD133high population compared to its parental cell line from 11.3% to 0.7%, and showed decreased sphere-forming efficacy. In addition, FTD-long exposed DLD-1 cell lines decreased tumor forming potential in in vivo. These results suggest that FTD is effective for CSC like cells and that long exposure to FTD might lead CSC depletion. Conclusion: FTD was proved to be effective for CSC like CD44high /CD133high cells. It suggests that FTD might be useful for CSC targeted chemotherapy in case of CD44 and CD133 are highly expressed subtype of tumors. Citation Format: Kenta Tsunekuni, Masamitsu Konno, Jun Koseki, Ayumu Asai, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Trifluridine efficacy in cancer stem cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2890.

Up-modulation of PLC-\u03b22 reduces the number and malignancy of triple-negative breast tumor cells with a CD133+/EpCAM+ phenotype: a promising target for preventing progression of TNBC

BackgroundThe malignant potential of triple negative breast cancer (TNBC) is also dependent on a sub-population of cells with a stem-like phenotype. Among the cancer stem cell markers, CD133 and EpCAM strongly correlate with breast tumor aggressiveness, suggesting that simultaneous targeting of the two surface antigens may be beneficial in treatment of TNBC. Since in TNBC-derived cells we demonstrated that PLC-β2 induces the conversion of CD133high to CD133low cells, here we explored its possible role in down-modulating the expression of both CD133 and EpCAM and, ultimately, in reducing the number of TNBC cells with a stem-like phenotype.MethodsA magnetic step-by-step cell isolation with antibodies directed against CD133 and/or EpCAM was performed on the TNBC-derived MDA-MB-231 cell line. In the same cell model, PLC-β2 was over-expressed or down-modulated and cell proliferation and invasion capability were evaluated by Real-time cell assays. The surface expression of CD133, EpCAM and CD44 in the different experimental conditions were measured by multi-color flow cytometry immunophenotyping.ResultsA CD133+/EpCAM+ sub-population with high proliferation rate and invasion capability is present in the MDA-MB-231 cell line. Over-expression of PLC-β2 in CD133+/EpCAM+ cells reduced the surface expression of both CD133 and EpCAM, as well as proliferation and invasion capability of this cellular subset. On the other hand, the up-modulation of PLC-β2 in the whole MDA-MB-231 cell population reduced the number of cells with a CD44+/CD133+/EpCAM+ stem-like phenotype.ConclusionsSince selective targeting of the cells with the highest aggressive potential may have a great clinical importance for TNBC, the up-modulation of PLC-β2, reducing the number of cells with a stem-like phenotype, may be a promising goal for novel therapies aimed to prevent the progression of aggressive breast tumors.

Abstract B30: Prrx1 isoforms regulate pancreatic cancer stem cell functions during pancreatic cancer progression

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a propensity for metastasis and drug resistance. To overcome this and improve the outcome of patients with PDAC, there is an urgent need to elucidate the molecular mechanisms of metastasis in which cancer stem cells (CSCs) may play a role. Prrx1, a homeobox transcription factor has been identified by us, as a key molecule involved in pancreatic development, regeneration and early carcinogenesis (Reichert et al. Genes and Development 2013). Furthermore, we have reported recently that Prrx1 isoform switching governs EMT-MET plasticity to regulate PDAC invasion and metastasis (Takano et al. Genes and Development 2016). Herein, we examined that the correlation between the two Prrx1 isoforms and putative CSC markers to investigate the role of Prrx1 on pancreatic CSC functions. Materials and Methods: Mouse pancreatic ductal cell lines were isolated and established from Pdx1Cre mice (normal pancreatic ductal cells), Pdx1Cre;KrasG12D/+ mice (PanIN cells), Pdx1Cre;KrasG12D/+;p53R175H/+ mice (primary and metastatic PDAC cells). Either Prrx1a and Prrx1b was overexpressed by lentiviral transduction in each cell line with controls. Prrx1 knockdown was done in primary PDAC cells and their matched liver metastatic cells by two isoform specific siRNAs. We immunophenotyped putative pancreatic CSCs using antibodies against CSC markers (CD133, CD44, CD24 and Epcam) by flow cytometry. These subpopulations of cells were evaluated in 3D cultures, invasion assays and single-pancreatosphere formation assays. Additionally, we evaluated the expression of pancreatic CSC markers in Prrx1a and Prrx1b overexpressing and knockdown cells by qRT-PCR and flow cytometry. Results: CD133High cells and CD44HighCD24High cells are expressed highly in metastatic PDAC cells compared to primary PDAC cells. CD133High cells form spheroid cysts and CD133Low cells form spindle-shaped cells in 3D culture. CD133High cells demonstrate more invasiveness and higher self-renewal capacity compared to CD133 Low cells. The percentage of CD133High cells, CD44HighCD24High cells and EpCAMHigh cells are increased significantly in Prrx1a overexpressing primary PDAC cells. However, these cells are decreased in Prrx1a knockdown metastatic cells. Contrary to this, these subpopulation of cells are significantly decreased in Prrx1b overexpressing primary PDAC cells, and increased in Prrx1b knockdown metastatic cells. These data suggest that Prrx1a might regulate pancreatic CSC functions in metastatic PDAC cells. Conclusion: Prrx1a may be associated with a subpopulation of pancreatic cancer cells that have features of cancer stem cells. This may influence metastatic properties of pancreatic cancer cells to the liver. Citation Format: Shigetsugu Takano, Maximilian Reichert, Hideyuki Yoshitomi, Basil Bakir, Koushik K. Das, Steffen Heeg, Shingo Kagawa, Hiroaki Shimizu, Masayuki Ohtsuka, Katsunori Furukawa, Masaru Miyazaki, Anil K. Rustgi.{Authors}. Prrx1 isoforms regulate pancreatic cancer stem cell functions during pancreatic cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B30.

Potential Role of CD133 Expression in the Susceptibility of Human Liver Cancer Stem-Like Cells to TRAIL.

Hepatocellular carcinoma (HCC) is one of the most common malignancies, with a poor prognosis and high recurrence rate. In the present study, we identified CD133, one of the markers of cancer stem cells, as a novel molecular target of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In four human HCC cell lines established from primary HCC tumors, we found that CD133-high human liver cancer stem-like cells (CD133hi) derived from the SNU-475 cell line were highly susceptible to TRAIL compared to other HCC cell lines with a small population of CD133. CD133hi SNU-475 cells showed upregulation of TRAIL receptor DR5 and stemness-related genes such as c-Myc and ABC transporters compared to their CD133-low (CD133lo) cells. Hypersensitivity of CD133hi cells to TRAIL was associated with c-Myc-mediated upregulation of DR5 and downregulation of c-FLIPL in the cells. Knockdown of CD133 expression in CD133hi cells resulted in the downregulation of c-Myc, and depletion of c-Myc caused a decrease in the cell surface expression of DR5 and an increase in the expression of c-FLIPL and, consequently, attenuated TRAIL-induced cytotoxicity and apoptosis of CD133hi cells. These results suggest that TRAIL may provide a new strategy for CD133hi CSCs of HCC-targeted therapies and, potentially, for therapies of other CD133-expressing types of cancer.

Effect of MUC1/β-catenin interaction on the tumorigenic capacity of pancreatic CD133+ cells.

Despite the fact that the biological function of cluster of differentiation (CD)133 remains unclear, this glycoprotein is currently used in the identification and isolation of tumor-initiating cells from certain malignant tumors, including pancreatic cancer. In the present study, the involvement of mucin 1 (MUC1) in the signaling pathways of a highly tumorigenic CD133+ cellular subpopulation sorted from the pancreatic cancer cell line HPAF-II was evaluated. The expression of MUC1-cytoplasmic domain (MUC1-CD) and oncogenic signaling transducers (epidermal growth factor receptor, protein kinase C delta, glycogen synthase kinase 3 beta and growth factor receptor-bound protein 2), as well as the association between MUC1 and β-catenin, were characterized in HPAF-II CD133+ and CD133low cell subpopulations and in tumor xenografts generated from these cells. Compared with HPAF CD133low cells, HPAF-II CD133+ cancer cells exhibited increased tumorigenic potential in immunocompromised mice, which was associated with overexpression of MUC1 and with the accordingly altered expression profile of MUC1-associated signaling partners. Additionally, MUC1-CD/β-catenin interactions were increased both in the HPAF-II CD133+ cell subpopulation and derived tumor xenografts compared with HPAF CD133low cells. These results suggest that, in comparison with HPAF CD133low cells, CD133+ cells exhibit higher expression of MUC1, which contributes to their tumorigenic phenotype through increased interaction between MUC1-CD and β-catenin, which in turn modulates oncogenic signaling cascades.

Role of CD44(high)/CD133(high) HCT-116 cells in the tumorigenesis of colon cancer.

This study aimed to explore cell surface biomarkers related to cancer stem cells (CSCs) and their role in the tumorigenesis of colon cancer. Various colon cancer cell lines were screened for CD133 and CD44 expression. CD44high/CD133high and CD44low/CD133low cells were separately isolated by Fluorescence-Activated Cell Sorting (FACS). The cell proliferation, colony formation, cell cycle characteristics, and tumorigenic properties in CD44high/CD133high and CD44low/CD133low cells were investigated through in vitro experiments and in vivo tumor xenograft models. The expression profiles of stem cell-related genes were examined by RT-PCR. With HCT-116 cells, flow cytometry analysis revealed that CD44high/CD133high cells had higher proliferation potency than CD44low/CD133low cells. Compared to CD44low/CD133low cells, CD44high/CD133high cells had more stem cell-related genes, and displayed increased tumorigenic ability. In summary, CD44high/CD133high cells isolated from HCT-116 cells harbor CSC properties that may be related to the tumor growth of colon cancer. These results suggest that CD44 and CD133 could be strong markers of colorectal cancer stem cells.

FoxO1-negative cells are cancer stem-like cells in pancreatic ductal adenocarcinoma.

Flow cytometry assays using aldehyde dehydrogenase (ALDH) activity or CD133 positivity to isolate cancer stem cells (CSCs) are widely applied but have limitations. Thus, characterization of CSC makers for a specific cancer is potentially important. We have previously shown that miR-21 regulates cancer cell growth via FoxO1 in pancreatic ductal adenocarcinoma (PDAC). Here, we areported evidence of FoxO1-negative PDAC cells as CSCs in PDAC. Both ALDH-high and CD133-high cell fractions isolated from PDAC of the patients expressed high levels of miR-21 and null FoxO1. Cultured PDAC cells were virally transduced with GFP under FoxO1 promoter. GFP (FoxO1)-null PDAC cells expressed high levels of miR-21, and grew more quickly than FoxO1-positive PDAC cells. Moreover, the fold increases in growth of FoxO1-negative vs FoxO1-positive cells were greater than CD133-high vs CD133-low cells, or ALDH-high vs ALDH-low cells. Further, FoxO1-negative cells formed tumor spheres in culture and developed tumors after serial adoptive transplantation into NOD/SCID mice, while the FoxO1-positive cells did not. Finally, selective elimination of FoxO1-negative cells completely inhibited the growth of PDAC cells. Together, these data suggest that FoxO1-negative cells as CSCs in PDAC, and targeting FoxO1-negative cells in PDAC may provide better therapeutic outcome.

In triple negative breast tumor cells, PLC-β2 promotes the conversion of CD133high to CD133low phenotype and reduces the CD133-related invasiveness

BackgroundBeyond its possible correlation with stemness of tumor cells, CD133/prominin1 is considered an important marker in breast cancer, since it correlates with tumor size, metastasis and clinical stage of triple-negative breast cancers (TNBC), to date the highest risk breast neoplasia.MethodsTo study the correlation between the levels of CD133 expression and the biology of breast-derived cells, CD133low and CD133high cell subpopulations isolated from triple negative MDA-MB-231 cells were compared in terms of malignant properties and protein expression.ResultsHigh expression of CD133 characterizes cells with larger adhesion area, lower proliferation rate and reduced migration speed, indicative of a less undifferentiated phenotype. Conversely, when compared with CD133low cells, CD133high cells show higher invasive capability and increased expression of proteins involved in metastasis and drug-resistance of breast tumors. Among the signalling proteins examined, PLC-β2 expression inversely correlates with the levels of CD133 and has a role in inducing the CD133high cells to CD133low cells conversion, suggesting that, in TNBC cells, the de-regulation of this PLC isoform is responsible of the switch from an early to a mature tumoral phenotype also by reducing the expression of CD133.ConclusionsSince CD133 plays a role in determining the invasiveness of CD133high cells, it may constitute an attractive target to reduce the metastatic potential of TNBC. In addition, our data showing that the forced up-regulation of PLC-β2 counteracts the invasiveness of CD133-positive MDA-MB-231 cells might contribute to identify unexplored key steps responsible for the TNBC high malignancy, to be considered for potential therapeutic strategies.

Abstract A49: Anti-DLL4 antibodies inhibit cancer stem cells in small cell lung cancer.

Abstract Small cell lung cancer (SCLC) is a very aggressive lung cancer with features suggesting enrichment in cancer stem cells (CSCs). Delta-like ligand 4 (DLL4) is a membrane bound ligand for Notch receptors critical for functional angiogenesis. Blocking DLL4 signaling increases the density of nonfunctional blood vessels and hypoxia of tumors, and thereby inhibits growth of tumor xenografts in mice. In addition, growing evidence implicates DLL4 Notch signaling pathway in the maintenance of CSCs. Here we investigated the anti-CSC activity of anti-DLL4 mAbs using in vitro and in vivo models of SCLC. Biochemical and flow cytometry analyses revealed that multiple SCLC cell lines express DLL4, and notably, SCLC spheres cultured under CSC-enriching conditions express higher levels of DLL4. Prior to determining the ability of anti-DLL4 mAb in inhibiting CSC in vivo, we first characterized the phenotype of CSCs in the SCLC cell line NCI-H69. NCI-H69 cells enriched for high CD133 expression were more tumorigenic and expressed higher levels of Nanog, Oct 3/4, and EZH2, which are genes crucial for maintenance of CSCs, than cells with low or negative levels of CD133. Moreover, RNA level of DLL4 was found to be three fold higher in CD133 high cells compared to CD133 low cells, suggesting that expression of DLL4 correlates with the CSC phenotype. In vivo, an anti-DLL4 antibody leads to inhibition of NCI-H69 tumor growth when the antibody is administered as a single agent, or in combination with cisplatin+etoposide or topotecan, chemotherapy agents commonly used in treatment of SCLC. To determine if blockade of DLL4 inhibits CSCs in vivo, NCI-H69 tumor xenografts treated with anti-DLL4 mAb were analyzed by flow cytometry. A subset of dissociated tumor cells expressed CD133 and DLL4, and a reduction of these populations of cells was observed in the anti-DLL4 antibody treated groups. In summary, these studies highlight that, in addition to vascular expression, (1) DLL4 is frequently expressed in SCLC cells, (2) DLL4 expression correlates with a CSC phenotype, and (3) that DLL4 blockade using an anti-DLL4 mAb results in inhibition of CSCs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A49. Citation Format: Patrick Strout, Martin Korade, Ching Ching Leow, Ivan Inigo, Suneetha Thomas, Elaine Hurt, Jon Chesebrough, Adeela Kamal, Song Cho. Anti-DLL4 antibodies inhibit cancer stem cells in small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A49.

Photochemical internalization (PCI) of immunotoxins targeting CD133 is specific and highly potent at femtomolar levels in cells with cancer stem cell properties

CD133 is a putative cancer stem cell (CSC) marker for a number of different cancers and is suggested to be a therapeutic target. Since also normal stem cells express CD133 it is of paramount importance that targeting strategies provide a specific and efficient delivery of cytotoxic drugs in only CD133-positive CSCs. In this study, we have employed photochemical internalization (PCI), a minimally invasive method for light-controlled, specific delivery of membrane-impermeable macromolecules from endocytic vesicles to the cytosol, to specifically target CD133-positive cancer cells. We demonstrate that PCI increases the cytotoxic effect of an immunotoxin (IT) targeting CD133-expressing cancer cells of colon (WiDr and HCT116) and pancreas (BxPC-3) origin. The IT consisted of the mAb CD133/1 (AC133) bound to the ribosome inactivating plant toxin saporin (anti-CD133/1-sap). We show that TPCS2a-PCI of anti-CD133/1-sap is specific, and highly cytotoxic at femto-molar concentrations. Specific binding and uptake of CD133/1, was shown by fluorescence microscopy and co-localization with TPCS2a in endosomes/lysosomes was determined by confocal microscopy. CD133high WiDr cells, isolated by fluorescence activated cell sorting, had a 7-fold higher capacity to initiate spheroids than CD133low cells (P<0.001) and were resistant to photodynamic therapy (PDT). However, PDT-resistance was bypassed by the PCI strategy. Tumor initiation and aggressive growth in athymic nude mice was obtained with only 10 CD133high cells in contrast to CD133low cells where substantially higher cell numbers were needed.The excellent high efficacy and selectivity of eliminating CD133-expressing cells by PCI warrant further pre-clinical evaluations of this novel therapeutic approach.

In triple negative breast tumor cells, PLC-β2 promotes the conversion of CD133high to CD133low phenotype and reduces the CD133-related invasiveness

Beyond its possible relationship with stemness of tumor cells, CD133/prominin is a highly glycosylated trans-membrane protein that correlates with tumor size, metastasis and clinical stage of triple negative breast cancers (TNBC), that represent 20% of all breast tumors and have a particularly worse clinical outcome than other tumor subtypes [1]. The correlation between the levels of CD133 expression and the biology of breast tumor cells was studied in CD133low and CD133high cell subpopulations isolated from MDA-MB-231 cells (ER-, PR-, HER2-). High expression of CD133 characterizes a small percentage of cells with larger adhesion area, lower proliferation rate, higher invasion capability and increased expression of proteins involved in metastasis and drug resistance of breast cancers. PLC-b2 expression, that plays a crucial role in malignancy of breast tumor cells [2, 3], inversely correlates with the levels of CD133 and has a role in inducing the CD133high cells to CD133low cells conversion. The forced up-regulation of PLC-b2 counteracts the invasiveness of CD133high MDAMB- 231, suggesting that, in TNBC, the de-regulation of this PLC isozyme is responsible of the switch from an early to a mature tumoral phenotype also by reducing the expression of CD133. These data might contribute to identify unexplored key steps in TNBC malignancy, to be considered for potential therapeutic strategies.

CD133 induces tumour-initiating properties in HEK293 cells

The pentaspan protein CD133 (Prominin-1) is part of the signature of tumour-initiating cells for various cancer entities. The aim of the present study was to investigate the impact of ectopic CD133 expression on tumourigenic properties of otherwise CD133-negative, non-tumourigenic cells in vitro and in vivo. CD133 was stably transfected into human embryonic kidney 293 (HEK293) which was then sorted for the expression of CD133. The effects of CD133 on cell proliferation were assessed upon standard cell counting of sorted cells at various time points. Severe combined immunodeficient (SCID) mice (n = 30) were injected with HEK293 CD133high and CD133low transfectants (5 × 103, 1 × 105, or 5 × 106 cells per injection). The expression of CD133, Ki67, CD44s, CD44v6, and EpCAM was analysed upon immunohistochemical staining of cryosections with specific antibodies. In vitro, ectopic expression of CD133 did influence neither cell proliferation nor cell cycle distribution of otherwise CD133-negative HEK293 cells. However, CD133high cells generated tumours in vivo in SCID mice with at least 1,000-fold increased frequency compared to CD133low cells. Tumour load was also significantly increased in CD133high cells as compared to those tumours formed by high numbers of CD133low cells. Immunohistochemistry stainings disclosed no changes in Ki67, CD44s, CD44v6, or EpCAM once tumours were formed by either cell type. CD133 induces tumour-initiating properties in HEK293 cells in vivo and is potentially involved in the regulation of tumourigenicity. Future research will aim at the elucidation of molecular mechanisms of CD133-induced tumourigenicity.

Genotype analysis of tumor‐initiating cells expressing CD133 in neuroblastoma

Neuroblastoma (NB) is the most common and lethal extracranial solid tumor of childhood. Despite aggressive therapy, more than half of the children with advanced NB will die of uncontrolled metastatic disease. After chemotherapy, tumor-initiating cells (TICs) could persist, cause relapses and metastasis. The aim of this study is to demonstrate the tumor-initiating properties of CD133high NB cells and to identify new specific genetic abnormalities. Isolation of the CD133high cell population from NB cell lines was followed by neurosphere formation, soft agar assays, and orthotopic injections in NOD/SCID/IL2Rγc-null mice. A differential genotyping analysis was performed with Affymetrix SNP 6.0 arrays on CD133low and CD133high populations and the frequency of the abnormalities of 36 NB tumors was determined. Our results show that CD133high NB cells possess tumor-initiating properties, as CD133high cells formed significantly more neurospheres and produced significantly more colonies in soft agar than CD133low. Injection of 500 CD133high cells was sufficient to generate primary tumors and frequent metastases in mice. Differential genotyping analysis demonstrated two common regions with gains (16p13.3 and 19p13.3) including the gene EFNA2 in the CD133high population, and two with loss of heterozygosity (16q12.1 and 21q21.3) in the CD133low population. The gain of EFNA2 correlated with increased expression of the corresponding protein. These abnormalities were found in NB samples and some were significantly correlated with CD133 expression. Our results show that CD133high NB cells have TICs properties and present different genotyping characteristics compared to CD133low cells. Our findings reveal insights into new therapeutic targets in NB TICs.

CD133 expression is associated with poor outcome in neuroblastoma via chemoresistance mediated by the AKT pathway

Neuroblastoma is a frequent childhood cancer with a heterogeneous prognosis. CD133 expression is an independent prognostic marker for a low survival rate in several cancers. The aim of this study was to determine the prognostic value of CD133 expression in a large cohort of neuroblastoma cases, to define the chemoresistance of neuroblastoma cells expressing CD133, and to determine whether this chemoresistance is regulated by activation of the AKT pathway. Two hundred and eighty samples of neuroblastoma were screened for CD133 expression. The sensitivity of purified CD133+ neuroblastoma cells isolated from two human cell lines to doxorubicin, vincristine and cisplatin, as single agents or in combination with LY294002, an AKT inhibitor, was evaluated in vitro. CD133 expression was found in 100 of 280 tumours. There was a significant association between CD133 expression and the following poor prognosis covariates: age, International Neuroblastoma Staging System stage, MYCN amplification, and phospho-AKT (pAKT) expression. Patients with CD133- tumours had significantly better 3-year event-free and overall survival than patients with CD133+ tumours. In a multivariate model, CD133 expression was independently associated with decreased overall survival. CD133(high) neuroblastoma cells were significantly resistant to chemotherapy as compared with CD133(low) cells. Treatment of unsorted neuroblastoma cells with the three anticancer drugs significantly enriched the CD133+ subpopulation. CD133(high) cells expressed significantly higher levels of pAKT than CD133(low) cells. LY294002 treatment abolished the preferential survival of CD133(high) cells. CD133 is associated with in-vitro resistance to chemotherapy involving activation of the AKT pathway.

Abstract 4344: Membrane-type 1 matrix metalloproteinase-mediated pro-invasive properties of neuroblastoma initiating cells

Abstract Introduction. Neuroblastoma is the most common and deadly extracranial solid tumour of childhood. Unlike localized neuroblastomas which have generally good prognosis, metastatic stages are associated with poor outcome. In this context, increasing evidences suggest that neuroblastoma may contain tumor-initiating cells (TICs) that cause relapse and metastasis. Our recent work indicates that CD133, a putative marker of TICs in various cancers, is associated with chemoresistance and poor outcome in neuroblastoma. In addition, we and others have demonstrated that membrane-type 1 matrix metalloproteinase (MT1-MMP) is associated with neuroblastoma progression and metastasis. Purpose. The purpose of this study was to characterize the tumor-initiating properties of CD133high neuroblastoma cells and determinate the role of MT1-MMP in pro-invasive properties of these cells. Methods/Results. Following CD133high cells selection from neuroblastoma cell lines (SK-N-DZ, SK-N-FI, SK-N-SH and SJ-NB10) with FACSAria cell sorter, we have examined their tumor-initiating features with neurosphere-forming assay in serum-free medium, colony-forming assay in soft agar and orthotopic transplantation into the adrenal gland (major site of primary neuroblastoma) of severe immunocompromised NOD/SCID/IL2Rγ- mice (NSG). Contrary to their counterpart CD133low, CD133high cells formed more colonies in soft agar and develop more neurospheres. Interestingly, orthotopic transplantation of as few as 500 CD133high cells into NSG mice resulted in tumor formation into the adrenal gland and dissemination to liver, lungs, brain and bone marrow, while matched control CD133low presented no tumor, suggesting that CD133high cells are TICs in neuroblastoma. Further western blot analyses of neuroblastoma cell lines revealed that CD133 expression correlated with that of MT1-MMP. In addition, CD133high cells growth within three-dimensional type I collagen matrices was markedly higher than CD133low cells and was significantly reduced by anti-MT1-MMP neutralizing antibodies. In the same vein, we performed cell migration assay in Boyden chamber and observed that CD133high cells exhibit more migratory abilities that CD133low cells, this migration being inhibited by anti-MT1-MMP neutralizing antibodies. Conclusions. Altogether, these findings strongly suggest that CD133-expressing TICs of neuroblastoma exhibit high pro-invasive capacities, requiring the involvement of MT1-MMP. Targeting pro-invasive capacities of neuroblastoma TICs with MMPs-activaed pro-drugs in combination with conventional therapy, represents an attractive therapeutic strategy aimed at eliminating both neuroblastoma TICs and the bulk of the tumour. Acknowledgment. Fondation Centre de cancérologie Charles-Bruneau. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4344. doi:10.1158/1538-7445.AM2011-4344

Recognition and Killing of Brain Tumor Stem-Like Initiating Cells by CD8+ Cytolytic T Cells

Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8(+) CTLs. Compared with serum-differentiated CD133(low) tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of antigens as evidenced by the killing of these cells by CTL when antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population.

Achaete-Scute Complex Homologue 1 Regulates Tumor-Initiating Capacity in Human Small Cell Lung Cancer

The basic helix-loop-helix transcription factor achaete-scute complex homologue 1 (ASCL1) is essential for the development of normal lung neuroendocrine cells as well as other endocrine and neural tissues. Small cell lung cancer (SCLC) and non-SCLC with neuroendocrine features express ASCL1, where the factor may play a role in the virulence and primitive neuroendocrine phenotype of these tumors. In this study, RNA interference knockdown of ASCL1 in cultured SCLC resulted in inhibition of soft agar clonogenic capacity and induction of apoptosis. cDNA microarray analyses bolstered by expression studies, flow cytometry, and chromatin immunoprecipitation identified two candidate stem cell marker genes, CD133 and aldehyde dehydrogenase 1A1 (ALDH1A1), to be directly regulated by ASCL1 in SCLC. In SCLC direct xenograft tumors, we detected a relatively abundant CD133(high)-ASCL1(high)-ALDH1(high) subpopulation with markedly enhanced tumorigenicity compared with cells with weak CD133 expression. Tumorigenicity in the CD133(high) subpopulation depended on continued ASCL1 expression. Whereas CD133(high) cells readily reconstituted the range of CD133 expression seen in the original xenograft tumor, CD133(low) cells could not. Our findings suggest that a broad range of SCLC cells has tumorigenic capacity rather than a small discrete population. Intrinsic tumor cell heterogeneity, including variation in key regulatory factors such as ASCL1, can modulate tumorigenicity in SCLC.