Subpopulation Of Stem-like Cells Research Articles

Abstract 1958: Benztropine enhances temozolomide sensitivity by restricting the growth of drug-resistant glioma stem cells

Abstract Glioblastomas (GBMs) are the most aggressive type of primary brain tumor in adults, the second leading cause of cancer-related death in children, and are incurable using currently available treatment modalities. GBMs are characterized by the presence of primitive glioma stem-like cell subpopulations that resist chemotherapy and sustain disease progression. In this study, we found that Benztropine, a muscarinic acetylcholine receptor (mAChR) inhibitor, suppresses glioma cell proliferation and sensitizes these cells to treatment with Temozolomide. Modeling the development of Temozolomide resistance in vitro, we identified a subpopulation of glioma cells, defined by the co-expression of CD44, CD15, and PDGFRa that was resistant to Temozolomide. This chemo-resistant subpopulation was phenotypically similar to oligodendroglial progenitor cells (OPC-like); a stem-like cell population important in the initiation and progression of glioblastoma known to be modulated by mAChR signaling. The addition of the mAChR inhibitor, benztropine, dramatically reduced the emergence of this Temozolomide-resistant sub-population. To begin understanding the mechanism of action of benztropine on GBM cells, we analyzed the effect of this drug on a panel of mitotic drivers that have been implicated in glioma initiation and progression. Sub-lethal doses of Benztropine alone or in combination with temozolomide repressed the PDGF-signaling axis as characterized by a significant reduction of PDGFA secretion and PDGFRA activation. We also developed an in vivo model of Temozolomide resistance to determine whether Benztropine could improve the survival of animals harboring glioma xenografts that had recurred following treatment with Temozolomide. These in vivo studies demonstrated that Benztropine alone improves animal survival as compared with vehicle controls and that Benztropine combined with Temozolomide reduces glioma growth following initial Temozolomide treatment and improves survival in mice xenografted with human GBM cells. Taken together, our results suggest that Benztropine might be targeting glioma stem cells by altering the glioma stem cell microenvironment. These findings support a potential role for the use of adjuvant Benztropine for the clinical management of GBM. Citation Format: Damian A. Almiron Bonnin, Myung Chang Lee, Joseph M. Howard, Alison L. Young, Matthew C. Havrda. Benztropine enhances temozolomide sensitivity by restricting the growth of drug-resistant glioma stem cells [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 1958.

Zeb1 potentiates genome-wide gene transcription with Lef1 to promote glioblastoma cell invasion.

Glioblastoma is the most common and aggressive brain tumor, with a subpopulation of stem-like cells thought to mediate its recurring behavior and therapeutic resistance. The epithelial-mesenchymal transition (EMT) inducing factor Zeb1 was linked to tumor initiation, invasion, and resistance to therapy in glioblastoma, but how Zeb1 functions at molecular level and what genes it regulates remain poorly understood. Contrary to the common view that EMT factors act as transcriptional repressors, here we show that genome-wide binding of Zeb1 associates with both activation and repression of gene expression in glioblastoma stem-like cells. Transcriptional repression requires direct DNA binding of Zeb1, while indirect recruitment to regulatory regions by the Wnt pathway effector Lef1 results in gene activation, independently of Wnt signaling. Amongst glioblastoma genes activated by Zeb1 are predicted mediators of tumor cell migration and invasion, including the guanine nucleotide exchange factor Prex1, whose elevated expression is predictive of shorter glioblastoma patient survival. Prex1 promotes invasiveness of glioblastoma cells invivo highlighting the importance of Zeb1/Lef1 gene regulatory mechanisms in gliomagenesis.

Combination of metformin and 9-cis retinoic acid increases apoptosis in C6 glioma stem-like cells

Glioblastoma (GBM) is the most commonly diagnosed type of brain cancer and the leading cause of brain cancer-related death. GBM contains a subpopulation of tumor-propagating glioblastoma stem-like cells that are thought to drive cancer progression and recurrence. Although several clinical trials are ongoing to explore new chemotherapeutic agents to treat GBM, the use of metformin (Met), a first-line drug for type 2 diabetes mellitus, in cancer remains controversial. Here, we show that combining Met with 9-cis retinoic acid (9-cis RA) reduced the proliferation rate of C6-GSCs (glioblastoma stem-like cells) in vitro. The results of flow cytometric analysis showed that treatment with 9-cis RA for 24 h induced 4.5% early and 38.0% late apoptosis in C6-GSCs. Twenty-four hours of Met treatment induced 23.6% early and 33.5% late apoptosis in C6-GSCs. Combination of Met and 9-cis RA treatment significantly increased both early and late apoptosis to 30.4% and 55.4%, respectively. The present findings suggest that not only 9-cis RA but also Met has the potential to induce early and late apoptotic GSCs death by affecting the functional cytoplasmic and nuclear organelles. At the protein level, there was increased cleaved caspase-3 but decreased procaspase-3 expression in Met-, 9-cis RA- and Met+9-cis RA-treated C6 GSCs, as detected by western blotting. The ratio of cleaved caspase-3/procaspase-3 was 1.6 times higher in Met+9-cis RA-treated groups compared to control. Ultimately, a combination of Met and 9-cis RA might be a possible therapeutic target for the treatment of GBM.

MP70-11 REVERSE OF DOCETAXEL RESISTANCE IN PROSTATE CANCER VIA NOTCH SIGNALING INHIBITION

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2018MP70-11 REVERSE OF DOCETAXEL RESISTANCE IN PROSTATE CANCER VIA NOTCH SIGNALING INHIBITION Qiu Shi, Xiang Tu, Qiang Wei, and Lu Yang Qiu ShiQiu Shi More articles by this author , Xiang TuXiang Tu More articles by this author , Qiang WeiQiang Wei More articles by this author , and Lu YangLu Yang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2255AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Chemotherapeutic Docetaxel (Doc) is the standard of care for the hormone resistant prostate cancer (HRPC). However, many HRPC patients do not respond to Doc chemotherapy in a long-term treatment because the cancerous tissue acquires resistance to Doc treatment. The molecular mechanism underlying the development of Doc resistance in HRPC remains unclear. METHODS Human prostate cancer cell line DU145 were purchased and Doc resistant human prostate cancer cell line DU145/adr were generated by culturing cells with Docetaxel in a dose-escalation manner. Cell viability was determined by MTT assay kit. After enumeration of tumor stem-like cells in 3D fibrin gels, CD133 positive cells were selected by fluorescence-activated cell sorting. A Notch signaling pathway inhibitor was applied to combine with Doc to overcome the Doc resistance in HRPC. The ability to release doxorubicin from DU145 and DU145/adr cells was detected by HPLC. Quantitative real-time PCR and western blotting were performed for the detection of the target genes and proteins. RESULTS A subpopulation of prostate cancer cells, owing enhanced stem-like properties, was found in both prostate cancer cell line and HRPC patient tumor tissues, showed high resistance to Doc. The subpopulation of Doc resistance cells has up-regulation of Notch2 and activation of Notch signaling pathway, leading to the high tumor initiating ability and colony formation capability. Most importantly, the Notch signaling pathway up-regulates the expression of drug resistant associated gene ABCB1, which encodes P-GP, resulting in the Doc resistance. CONCLUSIONS Our results illustrated the role of a stem cell-like subpopulation of prostate cancer cells in Doc-resistance that is dependent on Notch signaling pathway activation. Importantly, regulation of Notch signaling was evident in samples derived from the tumors of HRPC patients indicating the clinical relevance of Notch as a potential Doc resistance biomarker. Furthermore, inhibition of Notch signaling pathway in Doc treatment regimens can re-sensitize cancer cells, revealing a potential therapeutic strategy in cases of HRPC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e938 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Qiu Shi More articles by this author Xiang Tu More articles by this author Qiang Wei More articles by this author Lu Yang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Distinctive epigenomes characterize glioma stem cells and their response to differentiation cues

BackgroundGlioma stem cells (GSCs) are a subpopulation of stem-like cells that contribute to glioblastoma (GBM) aggressiveness, recurrence, and resistance to radiation and chemotherapy. Therapeutically targeting the GSC population may improve patient survival, but unique vulnerabilities need to be identified.ResultsWe isolate GSCs from well-characterized GBM patient-derived xenografts (PDX), characterize their stemness properties using immunofluorescence staining, profile their epigenome including 5mC, 5hmC, 5fC/5caC, and two enhancer marks, and define their transcriptome. Fetal brain-derived neural stem/progenitor cells are used as a comparison to define potential unique and common molecular features between these different brain-derived cells with stem properties. Our integrative study reveals that abnormal expression of ten-eleven-translocation (TET) family members correlates with global levels of 5mC and 5fC/5caC and may be responsible for the distinct levels of these marks between glioma and neural stem cells. Heterogenous transcriptome and epigenome signatures among GSCs converge on several genes and pathways, including DNA damage response and cell proliferation, which are highly correlated with TET expression. Distinct enhancer landscapes are also strongly associated with differential gene regulation between glioma and neural stem cells; they exhibit unique co-localization patterns with DNA epigenetic mark switching events. Upon differentiation, glioma and neural stem cells exhibit distinct responses with regard to TET expression and DNA mark changes in the genome and GSCs fail to properly remodel their epigenome.ConclusionsOur integrative epigenomic and transcriptomic characterization reveals fundamentally distinct yet potentially targetable biologic features of GSCs that result from their distinct epigenomic landscapes.

In silico selection approach to develop DNA aptamers for a stem-like cell subpopulation of non-small lung cancer adenocarcinoma cell line A549

BackgroundDetection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells.Materials and methodsThe human adenocarcinoma cell line A549 was used for selection in seven cell SELEX cycles. We used human blood leukocytes for negative selection, and lung stem cell protein marker CD90 antibody binding A549 cells for positive selection.ResultsThe obtained oligonucleotide sequences after the seventh SELEX cycle were subjected to in silico selection analysis based on three independent types of bioinformatics approaches, selecting two closely related aptamer candidates in terms of consensus sequences, structural motifs, binding affinity (Kd) and stability (ΔG). We selected and identified the aptamer A155_18 with very good binding characteristics to A459 cells, selected for CD90 antibody binding. The calculated phylogenetic tree showed that aptamers A155_18 and the known A549 cell aptamer S6 have a close structural relationship. MEME sequence analysis showed that they share two unique motifs, not present in other sequences.ConclusionsThe novel aptamer A155_18 has strong binding affinity for A549 lung carcinoma cell line subpopulation that is expressing stem cell marker CD90, indicating a possible stemness, characteristic for the A459 line, or a subpopulation present within this cell line. This aptamer can be applied as diagnostic tool, identifying NSLC circulating cells.

Crizotinib and erlotinib inhibits growth of c-Met+/EGFRvIII+ primary human glioblastoma xenografts

ObjectivesReceptor tyrosine kinases (RTK), such as c-Met and epidermal growth factor receptor (EGFR), are implicated in the malignant progression of glioblastoma. Studies show that RTK systems can co-modulate distinct and overlapping oncogenic downstream signaling pathways. EGFRvIII, a constitutively activated EGFR deletion mutant variant, leads to increased tumor growth and diminishes the tumor growth response to HGF: c-Met pathway inhibitor therapy. Conversely, activation of the c-Met pathway diminishes the tumor growth response to EGFR pathway inhibitors. Previously we reported that EGFRvIII and c-Met pathway inhibitors synergize to inhibit tumor growth in isogenic GBM cell lines engineered to express EGFRvIII. More recently, studies suggest that despite targeting RTK signaling in glioblastoma multiforme, a subpopulation of stem-like tumor-propagating cells can persist to replenish the tumor cell population leading to tumor recurrence. Patients and MethodsMayo 39 and Mayo 59 xenograft lines were cultured and xenografts were maintained. Subcutaneous xenograft lines were serially passaged in nude mice to generate subcutaneous xenografts. Xenografts were implanted in 6–8 week old nude mice. Once tumors reached a substantial size (150 mm3), mice were randomly divided into 4 groups: 1) control vehicle, 2) Crizotinib (crizo), 3) Erlotinib (erlot), or 4) Crizotinib + Erlotinib, (n = 5 per group). ResultsCrizotinib (c-Met pathway inhibitor) and Erlotinib (EGFR pathway inhibitor) in combination significantly inhibited tumor growth, phospho-EGFRvIII, phospho-Met, phospho-AKT, phospho-MAPK, and neurosphere growth in Mayo 39 and Mayo 59 primary GBM subcutaneous xenografts. The expression of the stem cell markers Nestin, Musashi, Olig 2 and Sox2 were also significantly down-regulated by c-Met inhibition, but no additive down-regulation was seen by co-treatment with Erlotinib. ConclusionsThese results are consistent with and corroborate our previous findings demonstrating that targeting these two parallel pathways with c-Met and EGFR inhibitor therapy provides substantial anti-tumor activity in glioblastoma models.

CD117/c-kit in Cancer Stem Cell-Mediated Progression and Therapeutic Resistance.

Metastasis is the primary cause of cancer patient morbidity and mortality, but due to persisting gaps in our knowledge, it remains untreatable. Metastases often occur as patient tumors progress or recur after initial therapy. Tumor recurrence at the primary site may be driven by a cancer stem-like cell or tumor progenitor cell, while recurrence at a secondary site is driven by metastatic cancer stem cells or metastasis-initiating cells. Ongoing efforts are aimed at identifying and characterizing these stem-like cells driving recurrence and metastasis. One potential marker for the cancer stem-like cell subpopulation is CD117/c-kit, a tyrosine kinase receptor associated with cancer progression and normal stem cell maintenance. Further, activation of CD117 by its ligand stem cell factor (SCF; kit ligand) in the progenitor cell niche stimulates several signaling pathways driving proliferation, survival, and migration. This review examines evidence that the SCF/CD117 signaling axis may contribute to the control of cancer progression through the regulation of stemness and resistance to tyrosine kinase inhibitors.

Current perspectives on the crosstalk between lung cancer stem cells and cancer-associated fibroblasts.

Lung cancer, in particular non-small cell lung carcinoma (NSCLC), is the second most common cancer in both men and women and the leading cause of cancer-related deaths worldwide. Its prognosis and diagnosis are determined by several driver mutations and diverse risk factors (e.g. smoking). While immunotherapy has proven effective in some patients, treatment of NSCLC using conventional chemotherapy is largely ineffective. The latter is believed to be due to the existence of a subpopulation of stem-like, highly tumorigenic and chemoresistant cells within the tumor population known as cancer stem cells (CSC). To complicate the situation, CSCs interact with the tumor microenvironment, which include cancer-associated fibroblasts (CAFs), immune cells, endothelial cells, growth factors, cytokines and connective tissue components, which via a dynamic crosstalk, composed of proteins and exosomes, activates the CSC compartment. In this review, we analyze the crosstalk between CSCs and CAFs, the primary component of the NSCLC microenvironment, at the molecular and extracellular level and contemplate therapies to disrupt this communication.

Elimination of stem-like cancer cell side-population by auranofin through modulation of ROS and glycolysis

Cancer side-population (SP) represents a sub-population of stem-like cancer cells that have an important role in drug resistance due to their high expression of the ATP-binding cassette transporter ABCG2 involved in drug export. Auranofin (AF), a clinical drug of gold complex that is used in treatment of rheumatoid arthritis, has been reported inducing tumor antiproliferation. However, whether AF can impact SP cells remains unclear. Our study showed that AF caused a depletion of SP cells and a downregulation of stem cell markers, and impaired their ability to form tumor colonies in vitro and incidence to develop tumors in vivo of lung cancer cells. Reactive oxygen species (ROS) had an important role in mediating AF-induced depletion of SP cells, which could be reversed by antioxidant NAC. Further study revealed that AF could also cause ATP depletion by inhibition of glycolysis. The depletion of cellular ATP might impair the function of ABCG2 pump, leading to increased drug accumulation within the cells and thus enhancing anticancer activity of chemotherapeutic agents such as adriamycin. Synergistic effect of AF and adriamycin was demonstrated both in vitro and in vivo. Simultaneous increase of ROS and inhibition of glycolysis is a novel strategy to eliminate stem-like cancer cells. Combination of AF with adriamycin seems to be promising to enhance therapeutic effectiveness.

NKG2D ligands in glioma stem-like cells: expression in situ and in vitro.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor. Tumor stem cells have a major influence on tumor malignancy, and immunological escape mechanisms, involving the Natural Killer Group 2, member D (NKG2D) receptor-ligand-system, are key elements in tumor immuno-surveillance. We analyzed the expression profile and localization of NKG2D ligands (NKG2DL) and embryonic and neural stem cell markers in solid human GBM and stem-like cells isolated from glioma cell lines by qRT-PCR and immunohistochemistry, including quantitative analysis. We also evaluated the effect of Temozolomide (TMZ), the standard chemotherapeutic agent used in GBM therapy, on NKG2DL expression. NKG2DL-positive cells were mostly found scattered and isolated, were detectable in glial fibrillary acidic protein (GFAP)-positive tumor regions and partly in the penumbra of tumor vessels. NKG2DL were found in a distinct tumor stem-like cell subpopulation and were broadly costained with each other. Quantitative analysis revealed, that dependent on the individual NKG2DL investigated, cell portions costained with different stem cell markers varied between small (Musashi-1) and high (KLf-4) amounts. However, a costaining of NKG2DL with CD3γ, typically found in T cells, was also observable, whereas CD11b as a marker for tumor micoglia cells was only rarely costained with NKG2DL. Stem-like cells derived from the glioma cell lines T98G and U251MG showed a distinct expression pattern of NKG2DL and stem cell markers, which seemed to be balanced in a cell line-specific way. With differentiation, T98G displayed less NKG2DL, whereas in U251MG, only expression of most stem cell markers decreased. In addition, stimulation with TMZ led to a significant upregulation of NKG2DL in stem-like cells of both lines. As stem-like glioma cells tend to show a higher expression of NKG2DL than more differentiated tumor cells and TMZ treatment supports upregulation of NKG2DL, the NKG2D system might play an important role in tumor stem cell survival and in GBM therapy.

Molecular and functional analysis of anchorage independent, treatment-evasive neuroblastoma tumorspheres with enhanced malignant properties: A possible explanation for radio-therapy resistance.

Despite significant advances in cancer treatment and management, more than 60% of patients with neuroblastoma present with very poor prognosis in the form of metastatic and aggressive disease. Solid tumors including neuroblastoma are thought to be heterogeneous with a sub-population of stem-like cells that are treatment-evasive with highly malignant characteristics. We previously identified a phenomenon of reversible adaptive plasticity (RAP) between anchorage dependent (AD) cells and anchorage independent (AI) tumorspheres in neuroblastoma cell cultures. To expand our molecular characterization of the AI tumorspheres, we sought to define the comprehensive proteomic profile of murine AD and AI neuroblastoma cells. The proteomic profiles of the two phenotypic cell populations were compared to each other to determine the differential protein expression and molecular pathways of interest. We report exclusive or significant up-regulation of tumorigenic pathways expressed by the AI tumorspheres compared to the AD cancer cells. These pathways govern metastatic potential, enhanced malignancy and epithelial to mesenchymal transition. Furthermore, radio-therapy induced significant up-regulation of specific tumorigenic and proliferative proteins, namely survivin, CDC2 and the enzyme Poly [ADP-ribose] polymerase 1. Bio-functional characteristics of the AI tumorspheres were resistant to sutent inhibition of receptor tyrosine kinases (RTKs) as well as to 2.5 Gy radio-therapy as assessed by cell survival, proliferation, apoptosis and migration. Interestingly, PDGF-BB stimulation of the PDGFRβ led to transactivation of EGFR and VEGFR in AI tumorspheres more potently than in AD cells. Sutent inhibition of PDGFRβ abrogated this transactivation in both cell types. In addition, 48 h sutent treatment significantly down-regulated the protein expression of PDGFRβ, MYCN, SOX2 and Survivin in the AI tumorspheres and inhibited tumorsphere self-renewal. Radio-sensitivity in AI tumorspheres was enhanced when sutent treatment was combined with survivin knock-down. We conclude that AI tumorspheres have a differential protein expression compared to AD cancer cells that contribute to their malignant phenotype and radio-resistance. Specific targeting of both cellular phenotypes is needed to improve outcomes in neuroblastoma patients.

Fluorescence-Activated Cell Sorting of Murine Mammary Cancer Stem-Like Cell Subpopulations with HIF Activity.

Fluorescence-activated cell sorting (FACS) is a common method to identify and to isolate subpopulations within a complex mixture of cells based on their light scatter and fluorescent staining profiles. FACS is widely used to enrich for normal tissue and tumor cells that have stem cell potential. Whereas FACS protocols using conventional breast cancer cell lines are relatively routine, additional technical challenges are encountered when sorting for cell populations from freshly digested solid tumors, particularly for use in downstream cancer stem cell (CSC) assays. First, it is more difficult to isolate live, single cells from whole tumors, and second, single tumor cells prepared from enzymatically digested tumors are typically more sensitive to cell death following the physical stresses of digestion, pipetting, and sorting. Herein methods are described that have been optimized to harvest and to FACS profile viable tumor epithelial cells digested from late-stage mammary tumors originating in the mouse mammary tumor virus (MMTV)-polyomavirus middle T antigen (PyMT) transgenic mouse. Protocols were designed to enrich for single, viable, MMTV-PyMT tumor cell populations sorted by FACS and to facilitate the collection of sorted cell subpopulations suitable for head-to-head comparison of CSC activity by tumorsphere assays in vitro or limiting dilution transplantation in vivo.

Tracking Functional Tumor Cell Subpopulations of Malignant Glioma by Phasor Fluorescence Lifetime Imaging Microscopy of NADH.

Intra-tumoral heterogeneity is associated with therapeutic resistance of cancer and there exists a need to non-invasively identify functional tumor subpopulations responsible for tumor recurrence. Reduced nicotinamide adenine dinucleotide (NADH) is a metabolic coenzyme essential in cellular respiration. Fluorescence lifetime imaging microscopy (FLIM) of NADH has been demonstrated to be a powerful label-free indicator for inferring metabolic states of living cells. Using FLIM, we identified a significant shift towards longer NADH fluorescence lifetimes, suggesting an increase in the fraction of protein-bound NADH, in the invasive stem-like tumor-initiating cell (STIC) subpopulation relative to the tumor mass-forming cell (TMC) subpopulation of malignant gliomas. By applying our previously studied model to transition glioma from a majority of STIC to a majority of TMC in serum-adherent culture conditions following serial passages, we compared changes in NADH states, cellular respirations (oxidative phosphorylation and glycolysis), EGFR expression, and cell-growth speed over passages. We identified a significant positive correlation between free-NADH fraction and cell growth, which was related to an increase of TMC fraction. In comparison, the increase of EGFR and cellular respirations preceded all these changes. In conclusion, FLIM of NADH provides a non-invasive method to monitor the dynamics of tumor heterogeneity before and after treatment.

Phenotypic Plasticity in Uveal Melanoma Is Not Restricted to a Tumor Subpopulation and Is Unrelated to Cancer Stem Cell Characteristics.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults and approximately half of those diagnosed will die of metastasis. This study investigates whether UM progression is driven by a subpopulation of stem-like cells, termed "cancer stem cells" (CSCs). Expression of postulated stem cell markers aldehyde dehydrogenase (ALDH), CD44, and CD133 was analyzed in UM cell lines and primary UM short-term cultures (STCs) established from tumor samples. Additionally, the notion of a "cellular hierarchy" within UM was investigated. Finally, the phenomenon of phenotypic plasticity in response to environmental factors was explored. We demonstrate that expression of ALDH, CD44, and CD133 does not select for a subpopulation of stem-like cells in either UM cell lines or UM STCs. Furthermore, there is an absence of a cellular hierarchy in cell lines and all cells in culture are able to drive tumor progression. Last, we show that established UM cell lines and UM STCs are plastic in nature and switch their phenotype in response to environmental stimuli. We hypothesize that this capacity to undergo phenotypic plasticity may be a consequence of neural crest lineage and renders the exploration of the CSC hypothesis extremely challenging in UM.

Embryonic Stem Cell-Like Subpopulations in Venous Malformation.

Venous malformation (VM) consists of a network of ectatic anomalous thin-walled venous channels. A role for an activating TIE2 mutation in the development of the dilated luminal vessels in VM, and its proposed involvement of embryonic stem cells (ESCs), led us to investigate the expression of ESC markers in subcutaneous VM (SCVM) and intramuscular VM (IMVM). Formalin-fixed paraffin-embedded sections of SCVM from seven patients and IMVM samples from seven patients were analyzed for the expression of Nanog, pSTAT3, OCT4, SOX2, SALL4, and CD44, using 3,3'-diaminobenzidine (DAB) immunohistochemical (IHC) staining. All these samples did not express lymphatic marker D2-40. NanoString mRNA analysis and RT-PCR were performed on snap-frozen samples of SCVM (n = 3) and IMVM (n = 3) from the respective original cohorts of patients included in DAB IHC staining. To confirm co-expression of two proteins, immunofluorescent (IF) IHC staining on two representative samples of IMVM and SCVM samples from the original cohorts of patients included for DAB IHC staining was performed. DAB IHC staining demonstrated expression of all of the above ESC markers in both SCVM and IMVM samples. IF IHC staining showed that these markers were localized to the endothelium within these lesions and that Nanog, pSTAT3, SOX2, and CD44 were also expressed by cells outside of the endothelium. NanoString mRNA analysis confirmed transcription activation of pSTAT3, OCT4, and CD44. RT-qPCR confirmed transcription activation of Nanog, SOX2, and SALL4. Our findings support the presence of two ESC-like subpopulations, one within and one outside of the endothelium, of both SCVM and IMVM. Given that the endothelial ESC-like subpopulation expresses the more primitive marker, OCT4, it is exciting to speculate that they give rise to the non-endothelial subpopulation.

Effective treatment of drug resistant recurrent breast tumors harboring cancer stem-like cells by staurosporine/epirubicin co-loaded polymeric micelles

Breast cancer recurrence and resistance are associated with cancer stem-like cell (CSC) sub-populations. As conventional therapies fail to treat CSCs, institution of novel therapeutic strategies capable of eradicating both cancer cells and CSCs is central for achieving effective treatments with long-term survival. Here, we studied the ability of polymeric micelles cooperatively loading the cytotoxic drug epirubicin (Epi) and the CSC inhibitor staurosporine (STS) to treat breast tumors, particularly when tumors relapsed after chemotherapy. The STS/Epi-loaded micelles (STS/Epi/m) demonstrated potent therapeutic efficacy against both naïve orthotopic 4T1-luc breast tumors and their recurrent Epi-resistant counterparts, significantly prolonging survival. This efficacy enhancement of STS/Epi/m was correlated with the ability of the micelles to suppress the CSC-associated sub-populations of breast cancer, i.e. the aldehyde dehydrogenase-positive (ALDH+) population and the CD44+/CD24− fraction, in Epi-resistant cells and tumors. These results demonstrated STS/Epi/m as a promising strategy for effective management of breast cancer.

Targeting SRC Coactivators Blocks the Tumor-Initiating Capacity of Cancer Stem-like Cells.

Tumor-initiating cells (TIC) represent cancer stem-like cell (CSC) subpopulations within tumors that are thought to give rise to recurrent cancer after therapy. Identifying key regulators of TIC/CSC maintenance is essential for the development of therapeutics designed to limit recurrence. The steroid receptor coactivator 3 (SRC-3) is overexpressed in a wide range of cancers, driving tumor initiation, cell proliferation, and metastasis. Here we report that SRC-3 supports the TIC/CSC state and induces an epithelial-to-mesenchymal transition (EMT) by driving expression of the master EMT regulators and stem cell markers. We also show that inhibition of SRC-3 and SRC-1 with SI-2, a second-generation SRC-3/SRC-1 small-molecule inhibitor, targets the CSC/TIC population both in vitro and in vivo Collectively, these results identify SRC coactivators as regulators of stem-like capacity in cancer cells and that these coactivators can serve as potential therapeutic targets to prevent the recurrence of cancer. Cancer Res; 77(16); 4293-304. ©2017 AACR.

The effects of type I interferon on glioblastoma cancer stem cells

Glioblastomas (GBMs) are highly invasive brain tumors that are extremely deadly. The highly aggressive nature of GBM as well as its heterogeneity at the molecular and cellular levels has been attributed to a rare subpopulation of GBM stem-like cells (GSCs). Interferons (IFNs) are a family of endogenous antiviral proteins that have anticancer activity in vitro, and have been used clinically to treat GBM. IFN inhibits the proliferation of various established GBM cell lines, but the effects of IFNs on GSCs remain relatively unknown. The present study explored the effects of IFN on the proliferation and the differentiation capacity of GSCs isolated from GBM patient-derived xenolines (PDXs) grown as xenografts in immunocompromised mice. We show that IFN inhibits the proliferation of GSCs, inhibits the sphere forming capacity of GSCs that is a hallmark of cancer stem cells, and inhibits the ability of GSCs to differentiate into astrocytic cells. In addition, we show that IFN induces transient STAT3 activation in GSCs, while induction of astrocytic differentiation in GSCs results in sustained STAT3 activation.

Abstract 3899: Synthetic small molecule targeting CD24-/CD44+/ALDH+ cell population inhibits cancer stem cell activities in breast cancer

Abstract Purpose of the study: Cancer stem cells (CSCs) have been described as the “seed of tumor”, and have been suggested as the driving force of tumor relapses. In this study we aimed to isolate small molecules via screening of the combinatorial chemical library. The isolated molecules bind CD24-/CD44+/ALDH+ breast cancer cell subpopulation with high selectivity and specificity. Experimental design and plan: Through the unbiased cell based chemical library screening, we identified compound candidates that specifically bind the stem-like cancer cell subpopulations in breast cancer cells and tissues. By performing in vitro, tissue and in vivo-based assessments on ligand-binding specificity and functional activities, we ensured that the isolated ligands target tumorigenic stem-like breast cell or tissue preferentially. Summary of discoveries and findings: The in vitro compound binding validations proved the specificity of the isolated compound candidates toward breast cancer cells and tissues displaying cancer stem cell properties. The compound-targeting cell populations display significantly-higher expressions of the well-known stem cell markers than the unbound cell populations. On the other hand, a series of biological and biochemical functional assays also confirmed the compound candidates exert tumor-suppression activities on breast cancer cell lines, such as slow down proliferation and reduce cell viability. More importantly, our study demonstrated that both isolated compounds have preferential selectivity and higher binding affinity to the TNBC cells and tissues, which have been reported for higher percentage of stem-like cancer cell population detected than in non-TNBC cells or tissues. Through mouse xenograft study followed by histological analysis, we further confirmed the binding specificity of the compound towards cell line models could be retained in vivo in mouse xenograft model. Conclusion: We demonstrated our ligand screening technique is able to identify synthetic compounds targeting cell subgroups or sub-populations of defined interest within one particular type of tumor. High specificity of the isolated molecules could be utilized to develop chemical probes for quantification of percentage of CSC in the tumor, which facilitates clinical evaluation of tumor relapse risk. The screening strategy also provides a new methodology in discovering novel drug targeting CSC subpopulations, not only in breast tumor, but also other types of human cancers. Funding: This project is supported by the Cancer Prevention and Research Institute of Texas (CPRIT) grant, award ID: RP150713. Citation Format: Luxi Chen, Chao Long, Kha A. Tran, Jiyong Lee. Synthetic small molecule targeting CD24-/CD44+/ALDH+ cell population inhibits cancer stem cell activities in breast cancer [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 3899. doi:10.1158/1538-7445.AM2017-3899