Stem-like Cell Lines Research Articles

GE-11 * THE FLORIDA CENTER FOR BRAIN TUMOR RESEARCH (FCBTR): BUILDING INFRASTRUCTURE TO FIND A CURE

INTRODUCTION: High quality tumor samples and DNA, coupled with annotated clinical data are the building blocks of basic, translational, and clinical research but are often difficult to obtain in a high-quality and standardized format and are expensive to maintain. In 2006, the legislature of the State of Florida appropriated funding for the Florida Center for Brain Tumor Research (FCBTR) to establish a coordinated biorepository and annotated clinical database among the state's public and private universities, hospitals, and biomedical industries. METHODS: Infrastructure for the collection, storage, and distribution of tumor and normal tissues and DNA is maintained by the FCBTR. Central coordination and annotated clinical database management is facilitated at the University of Florida. Samples are collected and distributed under institutional IRB approval. Participating member institutions (n = 8) are represented on the FCBTR Scientific Advisory Council through gubernatorial or state legislature appointed representatives that provide governing oversight of FCBTR operations. In collaboration with ABC2, peer-reviewed FCBTR pilot grants have been awarded to investigators throughout the state of Florida to support brain tumor research initiatives. RESULTS: To date, 8506 samples from 1100 patients have been collected under IRB approval by the FCBTR. Eight research awards to neuro-oncology investigators have been funded through the pilot award program. Distribution of FCBTR-maintained tissues has supported over thirty publications, submissions to The Cancer Genome Atlas, and several awarded and pending NIH grants. A NIH R24 (Resource-Related Research Project) grant to establish, characterize, and distribute primary glioma stem cell-like lines (GSCs) has been awarded to UF, with the FCBTR functioning as the coordinating center for deposition and distribution of GSC resources. CONCLUSIONS: State support for multi-institutional biorepositories is a model that should be considered by state legislatures for advancement of brain tumor research and other disease-related research initiatives.

CS-11 * AN RNAi KINOME SCREEN OF GLIOBLASTOMA STEM CELLS UNDER NORMOXIA AND HYPOXIA

Glioblastoma stem like cells are known to have phenotypes and gene expression patterns distinct from traditional glioma cell lines grown in serum. They also may have increased resistance to drugs and radiation and are likely to be responsible for tumor recurrence following therapy. As a result, it is important to identify therapeutic targets for these cells. To do this, we have conducted RNAi screens in glioblastoma stem cells against the human kinome since kinases are druggable targets and many kinase inhibitors are known. In addition, glioblastomas often have significant regions of hypoxia and glioblastoma stem cells tend to be resistant to hypoxia. We have therefore conducted shRNA screens in both normoxic and hypoxic conditions. We have screened 2 independent cell lines to date and are working on others. Defining a hit as a gene that has at least 2 independent shRNAs that give at least 50% growth inhibition as compared to controls, we found that 79 and 84 of (501 kinase genes) are hits under either normoxia or hypoxia for the GS6-22 and GS7-2 stem-like cell lines, respectively. We found that 56 and 63 kinases were hits under both conditions. 15 and 16 genes gave preferential inhibition under normoxia and 8 and 4 genes preferentially inhibited under hypoxia. 31 kinases were inhibitory in both cell lines. Thus, there is considerable variation between cell lines. Several of the identified hits are well known to have a role in glioblastoma such as EGFR and CDK4, and were inhibitory in both cell lines. About a third of the hits have previously been implicated in glioblastoma. One of the hits we have independently validated is the SMG1 kinase is preferentially inhibitory in hypoxia in only one of the cell lines. Together, these data give support to the idea of developing personalized therapies for glioblastoma.

Proscillaridin A is cytotoxic for glioblastoma cell lines and controls tumor xenograft growth in vivo.

Glioblastoma is the most frequent primary brain tumor in adults. Because of molecular and cellular heterogeneity, high proliferation rate and significant invasive ability, prognosis of patients is poor. Recent therapeutic advances increased median overall survival but tumor recurrence remains inevitable. In this context, we used a high throughput screening approach to bring out novel compounds with anti-proliferative and anti-migratory properties for glioblastoma treatment. Screening of the Prestwick chemical library® of 1120 molecules identified proscillaridin A, a cardiac glycoside inhibitor of the Na(+)/K(+) ATPase pump, with most significant effects on glioblastoma cell lines. In vitro effects of proscillaridin A were evaluated on GBM6 and GBM9 stem-like cell lines and on U87-MG and U251-MG cell lines. We showed that proscillaridin A displayed cytotoxic properties, triggered cell death, induced G2/M phase blockade in all the glioblastoma cell lines and impaired GBM stem self-renewal capacity even at low concentrations. Heterotopic and orthotopic xenotransplantations were used to confirm in vivo anticancer effects of proscillaridin A that both controls xenograft growth and improves mice survival. Altogether, results suggest that proscillaridin A is a promising candidate as cancer therapies in glioblastoma. This sustains previous reports showing that cardiac glycosides act as anticancer drugs in other cancers.

Abstract 1920: Stem-like triple negative breast cancer cells exhibit a distinct response to selectin/selectin ligand interactions

Abstract Circulating tumor cells (CTCs) often over-express sialofucosylated glycans, which bind to E-selectin presented on vascular endothelial cells. This interaction facilitates the initial tethering and rolling adhesion events in CTC extravasation during cancer metastasis. Though selectins and their ligands are well-established cell adhesion molecules, relatively little is known about their roles as cell signaling molecules for regulating growth, migration, and apoptosis in breast cancer (BC). To investigate the effects of selectin/selectin ligand interactions on BC cell signaling and adhesion, Hs578T, and MDA-MB-231 stem-like triple negative cell lines and BT-20, MCF-7, and ZR-75-1 non-stem-like hormone-receptor-positive cell lines were grown on tissue culture plates pre-treated with recombinant E-selectin or perfused over E-selectin-presenting substrate at physiologically relevant laminar flow conditions. In culture, cell lines demonstrated a dose dependent response on substrates pre-treated with increasing concentrations (1-10 µg/ml) of recombinant E-selectin. More specifically, cell lines exhibited little change on substrate treated with low concentrations of recombinant E-selectin (1-5 µg/ml) relative to human Fc fragment and fibronectin controls, yet displayed varying degrees of membrane blebbing and, in some cases, aggregate formation and reduced proliferation on substrate treated with 10 µg/ml of recombinant E-selectin. Notably, the stem-like triple negative cell lines, specifically Hs578T cells, had the most pronounced response to culture on E-selectin substrate as defined by blebbing and formation of cell aggregates. Furthermore, when Hs578T cells were cultured on substrate with adjacent areas treated with either recombinant E-selectin (10 µg/ml) or fibronectin (molar equivalent), cells failed to attach and grow on E-selectin but successfully seeded and proliferated in culture on fibronectin. In the laminar flow assay, Hs578T and MDA-MB-231 stem-like triple negative cell lines had four fold fewer adhesions on E-selectin substrate relative to the adhesion of BT-20, MCF-7, and ZR-75-1 non-stem-like hormone-receptor-positive cell lines. Moreover, the average rolling velocity of the stem-like triple negative cell lines on E-selectin substrate was significantly greater than the average rolling velocities of the other cell lines, which illustrates that under dynamic flow conditions stem-like triple negative BC cell lines have relatively fewer and weaker interactions with E-selectin. These data show that BC cells, especially Hs578T stem-like triple negative BC cells, differ in response to selectin/selectin ligand interaction under static and dynamic conditions, and illustrate that threshold levels of selectin/selectin ligand interaction may regulate cell signaling in certain types of BC cell lines. Thus targeting these interactions may lead to novel BC therapeutics. Citation Format: Grady E. Carlson, Luis F. Delgadillo, Emily A. Blaha, Fabian Benencia, Monica M. Burdick. Stem-like triple negative breast cancer cells exhibit a distinct response to selectin/selectin ligand interactions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1920. doi:10.1158/1538-7445.AM2014-1920

Activation by intravesicular amino acids of system L-mediated serine uptake into human placental microvillous plasma membrane vesicles

s / Placenta 35 (2014) A1eA112 A99 Results: IF dams consumed 29±1% less food and gained less weight (P Ala>BCH>Pro>Gly>MeAIB>Met. Conclusion: The identity of amino acids present on the internal face of MVM dictates serine uptake capacity across MVM. The broad internal amino acid selectivity by system L in MVM indicates that both the composition and concentration of freely-exchangeable amino acids within syncytiotrophoblast is likely to regulate system L exchange capacity in MVM. Collectively these observations provide further experimental support for previous model predictions regarding serine uptake by system L exchange transporters. P2.123-N. STATUS ANALYSIS OF TUMOUR ASSOCIATED FACTORS IN HUMAN PLACENTAE AND STEM-LIKE CELLS DERIVED FROM PLACENTAL CELL LINES Rham Balahmar, Shiva Sivasubramaniam Nottingham Trent university,

Proteomic analysis underlines the usefulness of both primary adherent and stem-like cell lines for studying proteins involved in human glioblastoma.

Primary cell lines enriched in cancer stem cells (CSC) have become popular models for testing new drugs for glioblastoma. In this proteomic study on an important number of cell lines obtained either as adherent cells in the presence of serum (a classic way to derive cell lines) or as neurospheres (enriched in CSC), we show that each type of cell line displays different GBM-specific features, highlighting that these two culture types are complementary tools for drug screening.

DIFFERENT MECHANISMS OF TUMOR GROWTH INHIBITION BY BLOCKADE OF IGF-IR IN U87-INDUCED AND STEM-LIKE CELL-INDUCED GLIOBLASTOMAS

BACKGROUND: Insulin-like growth factor receptor I (IGF-IR) signaling can contribute to the formation, maintenance, and/or progression of many diverse tumor types, including glioblastomas. We investigated the effect of an IGF-IR blocking antibody (IMC-A12) on tumors derived either from U87 cells or from glioblastoma stem-like (GS) cells in vitro and in vivo. METHODS: Two different xenograft models were established in nude mice. U87 cells were used to generate solid tumors, and the glioblastoma stem-like cell line GS-12 was used to generate diffusely invasive tumors. In both models, IMC-A12 was administered intratumorally via osmotic mini-pumps with catheters for 3-4 weeks. Tumor size, proliferation, apoptosis and angiogenesis were evaluated histologically and immunohistochemically. Receptor expression and activation were analyzed by flow cytometry and immunoprecipitation. Proliferation, apoptosis and migration were studied in vitro. RESULTS: Growth of U87 and of GS-12 tumors was inhibited by 75% and by 50%, respectively. In the solid, non-invasively growing U87 tumors, the intratumoral blood vessel density was reduced significantly, whereas the tumor cell proliferation and apoptosis rates did not differ from controls. In contrast, in the highly invasive GS-12 model, IMC-A12 treatment had no effect on tumor neovascularization, while the proportion of proliferating cells was decreased by 30% compared to controls. We therefore hypothesized that IMC-A12 interfered directly with the stem-like GS-12 glioblastoma cells but not with U87 cells. FACS analyses revealed that only 6% of U87 cells expressed IGF-IR, whereas 87% of GS-12 cells expressed the receptor. IGF-I and IGF-II-induced receptor activation was blocked by IMC-A12 in vitro, and the antibody reduced proliferation of GS-12 cells by 40% and migration by 27%. In contrast, IMC-A12 had no effect on the proliferation or migration of U87 cells. CONCLUSIONS: IGF-IR blockade can inhibit glioblastoma growth in vivo by different mechanisms. In the highly invasive GS-12 model it inhibits proliferation and migration directly. In contrast, in highly angiogenic tumors derived from U87 cells, which barely express IGF-IR, it acts by blocking neovascularization. These findings suggest that the IGF-IR signaling pathway might be useful to target both the highly proliferative angiogenesis-dependent solid tumor component as well as the non-angiogenic infiltrative tumors cells at the invasive edge in glioblastomas. SECONDARY CATEGORY: Tumor Biology.

Derivation and long-term culture of an embryonic stem cell-like line from zebrafish blastomeres under feeder-free condition.

Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.

Simvastatin radiosensitizes differentiated and stem-like breast cancer cell lines and is associated with improved local control in inflammatory breast cancer patients treated with postmastectomy radiation.

Reported rates of local failure after adjuvant radiation for women with inflammatory breast cancer (IBC) and triple-negative non-IBC are higher than those of women with receptor-expressing non-IBC. These high rates of locoregional recurrence are potentially influenced by the contribution of radioresistant cancer stem cells to these cancers. Statins have been shown to target stem cells and improve disease-free survival among IBC patients. We examined simvastatin radiosensitization of multiple subtypes of breast cancer cell lines in vitro in monolayer and mammosphere-based clonogenic assays and examined the therapeutic benefit of statin use on local control after postmastectomy radiation (PMRT) among IBC patients. We found that simvastatin radiosensitizes mammosphere-initiating cells (MICs) of IBC cell lines (MDA-IBC3, SUM149, SUM190) and of the metaplastic, non-IBC triple-negative receptor cell line (SUM159). However, simvastatin radioprotects MICs of non-IBC cell lines MCF-7 and SKBR3. In a retrospective clinical study of 519 IBC patients treated with PMRT, 53 patients used a statin. On univariate analysis, actuarial 3-year local recurrence-free survival (LRFS) was higher among statin users, and on multivariate analysis, triple negative breast cancer, absence of lymphatic invasion, neoadjuvant pathological tumor response to preoperative chemotherapy, and statin use were independently associated with higher LRFS. In conclusion, patients with IBC and triple-negative non-IBC breast cancer have the highest rates of local failure, and there are no available known radiosensitizers. We report significant improvement in local control after PMRT among statin users with IBC and significant radiosensitization across triple-negative and IBC cell lines of multiple subtypes using simvastatin. These data suggest that simvastatin should be justified as a radiosensitizing agent by a prospective clinical trial.

Engineering neuronal networks on photolithographically defined, biologically activated silicon substrates

BackgroundTo interface neurons with silicon semiconductors is a bioengineering challenge. Such constructs provide a platform for understanding neuronal coding and learning, an arena for modelling disease, and ultimately a step towards creating intelligent neuroprostheses. A prerequisite is the ability to dictate the spatial organisation of neuronal cells, in a context in which electrical and synaptic behaviour can be assessed. Our aim was to pattern cells on silicon, leading the way to the eventual incorporation of a wide range of biomicroelectromechanical systems for cellular interrogation. MethodsA predetermined design of the chlorine-containing polmer parylene-C was photolithographically printed onto oxidised silicon wafers (in a microelectronics cleanroom facility). Parylene-patterned chips were activated by acid etching and then incubated in fetal calf serum. The resulting substrate became differentially cell-adhesive (parylene) or repulsive (SiO2) for certain cell types. A human glioma-derived stem-like cell line that had been isolated during debulking surgery was cultured on-chip, growing selectively on parylene. Neurons (Lund Human Mesencephalic [LUHMES] cell line) were then plated, adhering to the glial template and extending neurites to connect with adjacent cells to form a network. The effect of parylene design on neuronal network configuration was assessed by use of a pattern of circular nodes with four orthogonal spokes printed in arrays of three different configurations (node diameter 50 μm, spoke length 125 μm; node 100 μm, spoke 100 μm; node 250 μm, spoke 100 μm). FindingsTen different cell lines were assessed on the parylene–silicon cell-patterning platform. Cell adhesion behaviour was heterogeneous, with only select cell lines patterning at high resolution. LUHMES neurons in isolation failed to pattern. However, in co-culture with glioma-derived stem-like cells (which pattern accurately), the neurons adhered and differentiated to form networks. Neurite directionality differed significantly according to parylene design (Kolmogorov-Smirnov 250 μm vs 100 μm nodes D=0·204, p=0·001; 100 μm vs 50 μm nodes D=0·147, p=0·028), allowing creation of reticular neuronal networks. InterpretationThe cell adhesion molecule profile of different cell lines affects their behaviour on the parylene–silicon platform. By consolidating DNA microarray data from cell types with contrasting patterning behaviour, we hope to correlate gene expression profiles with a cell's tendency to adhere to, or be repulsed by, the two contrasting substrates. The co-culture approach we describe has allowed creation of defined neuronal networks on silicon. The next steps include assessing long-term viability and functionality of patterned networks, while also improving control over their spatial organisation. FundingWellcome Trust.

Novel anti-glioblastoma agents and therapeutic combinations identified from a collection of FDA approved drugs

BackgroundGlioblastoma (GBM) is a therapeutic challenge, associated with high mortality. More effective GBM therapeutic options are urgently needed. Hence, we screened a large multi-class drug panel comprising the NIH clinical collection (NCC) that includes 446 FDA-approved drugs, with the goal of identifying new GBM therapeutics for rapid entry into clinical trials for GBM.MethodsScreens using human GBM cell lines revealed 22 drugs with potent anti-GBM activity, including serotonergic blockers, cholesterol-lowering agents (statins), antineoplastics, anti-infective, anti-inflammatories, and hormonal modulators. We tested the 8 most potent drugs using patient-derived GBM cancer stem cell-like lines. Notably, the statins were active in vitro; they inhibited GBM cell proliferation and induced cellular autophagy. Moreover, the statins enhanced, by 40-70 fold, the pro-apoptotic activity of irinotecan, a topoisomerase 1 inhibitor currently used to treat a variety of cancers including GBM. Our data suggest that the mechanism of action of statins was prevention of multi-drug resistance protein MDR-1 glycosylation. This drug combination was synergistic in inhibiting tumor growth in vivo. Compared to animals treated with high dose irinotecan, the drug combination showed significantly less toxicity.ResultsOur data identifies a novel combination from among FDA-approved drugs. In addition, this combination is safer and well tolerated compared to single agent irinotecan.ConclusionsOur study newly identifies several FDA-approved compounds that may potentially be useful in GBM treatment. Our findings provide the basis for the rational combination of statins and topoisomerase inhibitors in GBM.

Low-dose triptolide in combination with idarubicin induces apoptosis in AML leukemic stem-like KG1a cell line by modulation of the intrinsic and extrinsic factors

Leukemia stem cells (LSCs) are considered to be the main reason for relapse and are also regarded as a major hurdle for the success of acute myeloid leukemia chemotherapy. Thus, new drugs targeting LSCs are urgently needed. Triptolide (TPL) is cytotoxic to LSCs. Low dose of TPL enhances the cytotoxicity of idarubicin (IDA) in LSCs. In this study, the ability of TPL to induce apoptosis in leukemic stem cell (LSC)-like cells derived from acute myeloid leukemia cell line KG1a was investigated. LSC-like cells sorted from KG1a were subjected to cell cycle analysis and different treatments, and then followed by in vitro methyl thiazole tetrazolium bromide cytotoxicity assay. The effects of different drug combinations on cell viability, intracellular reactive-oxygen species (ROS) activity, colony-forming ability and apoptotic status were also examined. Combination index-isobologram analysis indicates a synergistic effect between TPL and IDA, which inhibits the colony-forming ability of LSC-like cells and induces their apoptosis. We further investigated the expression of Nrf2, HIF-1α and their downstream target genes. LSC-like cells treated with both TPL and IDA have increased levels of ROS, decreased expression of Nrf2 and HIF-1α pathways. Our findings indicate that the synergistic cytotoxicity of TPL and IDA in LSCs-like cells may attribute to both induction of ROS and inhibition of the Nrf2 and HIF-1α pathways.

Podocalyxin-Like Protein Is Expressed in Glioblastoma Multiforme Stem-Like Cells and Is Associated with Poor Outcome

Glioblastoma multiforme (GBM) is the most common primary malignant adult brain tumor and is associated with poor survival. Recently, stem-like cell populations have been identified in numerous malignancies including GBM. To identify genes whose expression is changed with differentiation, we compared transcript profiles from a GBM oncosphere line before and after differentiation. Bioinformatic analysis of the gene expression profiles identified podocalyxin-like protein (PODXL), a protein highly expressed in human embryonic stem cells, as a potential marker of undifferentiated GBM stem-like cells. The loss of PODXL expression upon differentiation of GBM stem-like cell lines was confirmed by quantitative real-time PCR and flow cytometry. Analytical flow cytometry of numerous GBM oncosphere lines demonstrated PODXL expression in all lines examined. Knockdown studies and flow cytometric cell sorting experiments demonstrated that PODXL is involved in GBM stem-like cell proliferation and oncosphere formation. Compared to PODXL-negative cells, PODXL-positive cells had increased expression of the progenitor/stem cell markers Musashi1, SOX2, and BMI1. Finally, PODXL expression directly correlated with increasing glioma grade and was a marker for poor outcome in patients with GBM. In summary, we have demonstrated that PODXL is expressed in GBM stem-like cells and is involved in cell proliferation and oncosphere formation. Moreover, high PODXL expression correlates with increasing glioma grade and decreased overall survival in patients with GBM.

Surface proteomic analysis of differentiated versus stem‐like osteosarcoma human cells

Cancer stem cell characterization represents a breakthrough in cancer research. Despite evidence showing the existence and the role of cancer stem cells in osteosarcoma (OS) onset and progression, little is known about their specific surface phenotype. To address this issue, we carried out a cytometric analysis with an antibody-array comprising 245 membrane proteins comparing the stem and differentiated OS cells. As experimental model, we chose the stem-like cell line 3aminobenzamide-OS and its parental, differentiated, cell line MG63. We identified 50 differentially expressed, 23 homogeneously expressed, and 172 not expressed proteins in the two cell line models, thus defining a surface protein signature specific for each of them. Furthermore, we selected ERK1/2 (p44/42 mitogen-activated protein kinases) as a potential pathway correlated with processes that characterize tumorigenic potential and stemness of 3aminobenzamide-OS cells.

Expansive growth of two glioblastoma stem-like cell lines is mediated by bFGF and not by EGF

BackgroundPatient-derived glioblastoma (GBM) stem-like cells (GSCs) represent a valuable model for basic and therapeutic research. GSCs are usually propagated in serum-free Neural Basal medium supplemented with bFGF and EGF. Yet, the exact influence of these growth factors on GSCs is still unclear. Recently it was suggested that GBM stem-like cells with amplified EGFR should be cultured in stem cell medium without EGF, as the presence of EGF induced rapid loss of EGFR amplification. However, patient biopsies are usually taken into culture before their genomic profiles are defined. Thus, an important question remains whether GBM cells without EGFR amplification also can be cultured in stem cell medium without EGF.Meterials and methodsTo address this question, we used two heterogeneous glioblastoma GSC lines (NCH421k and NCH644) that lack EGFR amplification.ResultsAlthough both cell lines showed very low EGFR expression under standard growth conditions, bFGF stimulation induced higher expression of EGFR in NCH644. In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF. Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect. We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).ConclusionsWe demonstrate that GSC cultures without EGFR amplification can be maintained and expanded with bFGF, while the addition of EGF has no significant effect and therefore can be omitted.

Alteration of cancer stem cell‐like phenotype by histone deacetylase inhibitors in squamous cell carcinoma of the head and neck

Recent progression in the understanding of stem cell biology has greatly facilitated the identification and characterization of cancer stem cells (CSCs). Moreover, evidence has accumulated indicating that conventional cancer treatments are potentially ineffective against CSCs. Histone deacetylase inhibitors (HDACi) have multiple biologic effects consequent to alterations in the patterns of acetylation of histones and are a promising new group of anticancer agents. In this study, we investigated the effects of two HDACi, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), on two CD44+ cancer stem-like cell lines from squamous cell carcinoma of the head and neck (SCCHN) cultured in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. Histone deacetylase inhibitors inhibited the growth of SCCHN cell lines in a dose-dependent manner as measured by MTS assays. Moreover, HDACi induced cell cycle arrest and apoptosis in these SCCHN cell lines. Interestingly, the expression of cancer stem cell markers, CD44 and ABCG2, on SCCHN cell lines was decreased by HDACi treatment. In addition, HDACi decreased mRNA expression levels of stemness-related genes and suppressed the epithelial-mesencymal transition phenotype of CSCs. As expected, the combination of HDACi and chemotherapeutic agents, including cisplatin and docetaxel, had a synergistic effect on SCCHN cell lines. Taken together, our data indicate that HDACi not only inhibit the growth of SCCHN cell lines by inducing apoptosis and cell cycle arrest, but also alter the cancer stem cell phenotype in SCCHN, raising the possibility that HDACi may have therapeutic potential for cancer stem cells of SCCHN.

Effect of the STAT3 inhibitor STX-0119 on the proliferation of cancer stem-like cells derived from recurrent glioblastoma

Signal transducer and activator of transcription (STAT) 3, a member of a family of DNA-binding molecules, is a potential target in the treatment of cancer. The highly phosphorylated STAT3 in cancer cells contributes to numerous physiological and oncogenic signaling pathways. Furthermore, a significant association between STAT3 signaling and glioblastoma multiforme stem-like cell (GBM-SC) development and maintenance has been demonstrated in recent studies. Previously, we reported a novel small molecule inhibitor of STAT3 dimerization, STX-0119, as a cancer therapeutic. In the present study, we focused on cancer stem-like cells derived from recurrent GBM patients and investigated the efficacy of STX-0119. Three GBM stem cell lines showed many stem cell markers such as CD133, EGFR, Nanog, Olig2, nestin and Yamanaka factors (c-myc, KLF4, Oct3/4 and SOX2) compared with parental cell lines. These cell lines also formed tumors in vivo and had similar histological to surgically resected tumors. STAT3 phosphorylation was activated more in the GBM-SC lines than serum-derived GB cell lines. The growth inhibitory effect of STX-0119 on GBM-SCs was moderate (IC50 15-44 µM) and stronger compared to that of WP1066 in two cell lines. On the other hand, the effect of temozolomide was weak in all the cell lines (IC50 53-226 µM). Notably, STX-0119 demonstrated strong inhibition of the expression of STAT3 target genes (c-myc, survivin, cyclin D1, HIF-1α and VEGF) and stem cell-associated genes (CD44, Nanog, nestin and CD133) as well as the induction of apoptosis in one stem-like cell line. Interestingly, VEGFR2 mRNA was also remarkably inhibited by STX-0119. In a model using transplantable stem-like cell lines in vivo GB-SCC010 and 026, STX-0119 inhibited the growth of GBM-SCs at 80 mg/kg. STX-0119, an inhibitor of STAT3, may serve as a novel therapeutic compound against GBM-SCs even in temozolomide-resistant GBM patients and has the potential for GBM-SC-specific therapeutics in combination with temozolomide plus radiation therapy.

Abstract 3763: Identification of gpA33 as a marker expressed on colon cancer stem cell lines.

Abstract Introduction: Recent studies have implied that solid tumors including colon cancer, can arise from tissue-specific stem cells. Defined serum-free culture conditions, employed to select for fetal colon epithelial progenitor cells, were utilized to isolate and expand a sub-population of epithelial cells from human tumors resected from a repertoire of colon cancer patients (Roberts 2011, AACR #5211). These cancer stem-like cell (CSLC) lines are stable homogenous populations that, when cultured under conditions that promote colon crypt differentiation, differentiate into organoids containing the 3 principal cell types seen in the colon and in differentiated colorectal tumors. In vivo they form tumors that fully recapitulate the morphologic and phenotypic characteristics of the patients’ original tumors. While any consistent definition of cancer stem cell properties remains elusive, these CSLC providing unique tools to interrogate the biology of CSC or tumor initiating cells, and identify CSC-expressed surface antigens. Methods: Cell immunizations were performed with colon CSLC. mAbs were analyzed by IHC to determine their reactivity toward normal and cancer tissues as well as a panel of cancer lines including colon CSLC. Antitumor bioactivity screens were followed by antigen identification of promising candidates. Affymetrix U133 micro-array analysis was performed to profile gene expression and Taqman analyses performed to identify differentially expressed genes compared to non-CSLC control populations. Results: mAbs from CSLC immunizations included a subset that displayed uniform binding across the complete panel of CSLC lines while displaying high penetrance of expression on both primary and metastatic colon cancer tissues. Expression cloning identified the antigen as glycoprotein A33 (gpA33), which was confirmed by IP and SPR analyses. Transcriptional profiling confirmed elevated gpA33 expression across the panel of colon CSLC in addition to revealing enhanced LGR5, ASCL2 and SOX9 expression, consistent with the CSLC having a colon stem cell origin. The uniform expression of gpA33 on putative cancer stem cell populations is distinguished from other cell surface molecules generally regarded as “canonical” CSC markers, including CD133 and CD44, which are variably expressed. Conclusion: gpA33 is an antigen with excellent expression penetrance in colon cancer and colon cancer-derived CSLC. As shown here, immunizations with CSLC lines can yield antibodies specific for colon cancer antigens that may also represent CSC expressed markers. This provides an example how new therapeutic antibody candidates can be generated to recognize and target both differentiated cancer cells as well as their associated CSC population. Citation Format: Jonathan Li, Claudia B. Fieger, Penny Roberts, Doug Smith, Monica Licea, Jeff Hooley, Francine Chen, Kathy King, Jennie Mather, Ezio Bonvini, Deryk Loo, Paul A. Moore. Identification of gpA33 as a marker expressed on colon cancer stem cell lines. [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 3763. doi:10.1158/1538-7445.AM2013-3763

Abstract 4889: Alteration of cancer stem cell-like phenotype by histone deacetylase inhibitors in squamous cell carcinoma of the head and neck.

Abstract Recent progression in the understanding of stem cell biology has greatly facilitated the identification and characterization of cancer stem cells (CSCs) from various tumors. In squamous cell carcinoma of the head and neck (SCCHN), various cell surface markers, including CD44, CD133 and ALDH1, have been identified as the potential markers to isolate CSCs. Evidence has accumulated indicating that conventional cancer treatments are potentially ineffective against CSCs. Histone deacetylase inhibitors (HDACi) have multiple biologic effects consequent to alteration in patterns of acetylation of histones and are promising new group of anticancer agents. In this study, we investigate the effects of two HDACi, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) on two CD44+ cancer stem-like cell lines from SCCHN cultured in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. SAHA and TSA inhibited the growth of SCCHN cell lines in a dose dependent manner as measured by MTS assays. Moreover, SAHA and TSA induced G2/M cell cycle arrest and apoptosis in these SCCHN cell lines. Interestingly, the expression of cancer stem cell markers, CD44 and ABCG2 on SCCHN cell lines were decreased by SAHA or TSA treatment. HDACi also decreased the mRNA expression levels of stemness-related genes (Notch and Nanog) and TGF-β. As expected, HDACi markedly increased the expression of E-cadherin. In addition, the combination of HDACi and chemotherapeutic agents, including cisplatin and docetaxel had a synergistic effect on SCCHN cell lines. Taken together, our data indicate that HDACi not only inhibit the growth of SCCHN cell lines by inducing apoptosis and cell cycle arrest, but also alter stem-like properties of the SCCHN cells, raising the possibility that HDACi may have therapeutic potentials for cancer stem cells of SCCHN. Citation Format: Koichi Sakakura, Takaaki Murata, Kazuaki Chikamatsu. Alteration of cancer stem cell-like phenotype by histone deacetylase inhibitors in squamous cell carcinoma of the head and neck. [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 4889. doi:10.1158/1538-7445.AM2013-4889

Differentiation of human osteosarcoma 3AB-OS stem-like cells in derivatives of the three primary germ layers as a useful <i>in vitro</i> model to develop several purposes

A number of solid tumors contain a distinct subpopulation of cells, termed cancer stem cells (CSCs) which represent the source for tissue renewal and hold malignant potential and which would be responsible for therapy resistance. Today, the winning goal in cancer research would be to find drugs to kill both cancer cells and cancer stem cells, while sparing normal cells. Osteosarcoma is an aggressive pediatric tumor of growing bones that, despite surgery and chemotherapy, is prone to relapse. We have recently selected from human osteosarcoma MG63 cells a cancer stem-like cell line (3AB-OS), which has unlimited proliferative potential, high levels of stemness-related markers, and in vivo tumorforming capacity in xenograft assays. Here, we have shown that 3AB-OS cells can differentiate in vitro into endoderm-, mesoderm-and ectoderm-derived lineages. Cell differentiation is morphological, molecular and functional. We propose that this model system of 3AB-OS differentiation in vitro might have a number of useful purposes, among which the study of molecular mechanisms of osteosarcoma origin, and the analysis of factors involved in specification of the various cell lineages. We still do not know either what are the shared and distinguishing characters between CSCs and normal stem cells, or what is the reason why the cancer stem cells, like the normal stem cells, have the ability to differentiate toward the derivatives of the primary germ layers. It is possible that each of the differentiation capability may be exploited by CSCs to supply their needs of growing and surviving in hostile microenvironment.