Putative Cancer Stem Cell Populations Research Articles

Abstract 5338: Pancreatic cancer tumor initiating cells are marked by the presence of cell surface associated acetylated alpha tubulin

Abstract The function of tubulin acetylation in the context of cancer initiation and progression remains unknown. In studying the frequency of primary cilia in early pancreatic cancer, we discovered an increase in acetylated alpha-tubulin labeling (AAT) labeling in murine PanIN, even in the absence of primary cilia. We therefore hypothesized that human pancreatic ductal adenocarcinoma (PDAC) cells may upregulate expression of AAT as they progress from non-invasive to invasive and metastatic disease. Performing confocal microscopy on a human PanIN (n=23) and metastatic PDAC (n=10) tissue arrays, we have determined that in 15% of PanIN lesions, 13-30% of the cells have detectable levels of AAT, with a primarily cytoplasmic/cell membrane pattern of labeling. In contrast, over 80% of metastatic PDAC cells are AAT-positive. Surprisingly, high-resolution 3D confocal imaging of both fixed and unfixed human PDAC cell lines demonstrated immunoreactive AAT to be present on the cell surface of unfixed cells, as previously reported in human leukemia cells (Quillen, JCB 1985). To evaluate the functional significance of this finding, we developed a FACS-based strategy to isolate viable PDAC cells based on levels of cell surface AAT. Applying this strategy to four different human pancreatic cancer cell lines and three early passage human xenografts, we have determined that 5-10% of human pancreatic cancer cells have detectable levels of cell-surface AAT. PDAC cells positive for cell surface AAT are markedly enriched in cells also positive for either CD133 or CD24/CD44, representing putative pancreatic cancer stem cell populations. We have functionally analyzed the tumor-initiating capacity of cell surface AAT+ cells using in vitro tumor sphere experiments. In this assay, cell surface AAT+ cells show a statistically significant 100-fold increase in tumor sphere-forming capacity relative to cell surface AAT-negative cells. Tumor sphere-forming capacity was significantly reduced by neutralizing anti-AAT antibodies, as well as by an siRNA targeting α-tubulin K40 acetyltransferase (αTAT). We further evaluated the functional significance of cell surface AAT by analyzing the in vivo tumor initiating capacities of cell surface AAT-positive and AAT-negative cells from a human PDAC xenograft. Cell surface AAT-positive cells exhibited significantly increased tumor initiating frequency relative to the AAT-negative population (**p<0.0000539). AAT-positive cells had a tumor-initiating frequency similar to an Aldefluor-bright population, suggesting that the presence of cell surface acetylated alpha tubulin may mark a pancreatic cancer tumor initiating population. These data provide important new information regarding cellular heterogeneity in human pancreatic cancer, and suggest that novel strategies targeting cell surface tubulin may have therapeutic utility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5338. doi:1538-7445.AM2012-5338

Abstract 1346: Induction of multiple myeloma in a novel humanized bone marrow xenograft model does not depend on CD138 expression

Abstract Multiple myeloma (MM) is an incurable B lineage malignancy characterized by an accumulation of monoclonal plasma cells in the bone marrow (BM) and is responsible for 20% of deaths from hematological cancers. MM patients present with an overproduction of monoclonal immunoglobulin, anemia, and hypercalcemia resulting in susceptibility to infection and end organ damage. Patients develop lytic bone lesions resulting in pathological fractures and significant pain. Plasmacytomas, solid tumors comprised of a single plasma cell clone, and extramedullary MM, presenting with lesions in the liver, lungs, and other organs, have also been reported in patients. Despite the development of potent new therapies, the median survival of MM patients remains 3-5 years, suggesting that a drug-resistant cancer stem cell (CSC) may be responsible for the inevitable relapse. We developed a clinically relevant humanized BM murine model of MM utilizing the NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mouse line (huBM/NSG model), sensitive enough to study the behavior of the putative CSC populations. To recapitulate the clinical symptoms of MM varying MM cell lines and primary cells from BM biopsies were co-transplanted with human BM stromal cells via an intratibial injection. Upon engraftment (5-15 weeks), mice developed systemic MM, plasmacytoma, and extramedullary disease. huBM/NSG mice developed symptoms of MM, including lytic bone lesions, elevated serum calcium, and low hemoglobin, mimicking the human presentation of MM with bone lesions, hypercalcemia, and anemia. In contrast to MM cell lines that induced disease without the support of human BM stroma, primary MM cells were able to engraft mouse bones and cause disease only when co-injected with human BM stromal cells. To determine whether CD138 can be used as a reliable marker for the MM-CSC, MM cells were sorted into CD138 positive and negative populations and injected into huBM/NSG mice to determine engraftment efficiency of each fraction. Surprisingly, both CD138+ and CD138- cell populations were able to engraft MM in huBM/NSG mice in primary and secondary transplants. Thus, it appears that CD138 expression is irrelevant for the engraftment of MM cells and subsequent initiation of the disease, suggesting that the MM-CSCs are present in both CD138+ and CD138- fractions. Taken together our study presents a new in vivo model of MM where the human BM microenvironment is reconstructed in the mouse faithfully recapitulating MM as it presents in human patients. We further establish that human stromal support is essential for the engraftment of primary MM cells, historically a challenging process. Furthermore, our data support the emerging findings that CD138 is not a reliable marker of the MM-CSCs, suggesting that the previously demonstrated differential engraftment of selected populations is likely an artifact of the less sensitive animal models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1346. doi:1538-7445.AM2012-1346

P1-02-02: Zoledronic Acid Reverses the Epithelial-Mesenchymal Transition While Inhibiting the Tumor Initiating Cell Population of Highly Tumorigenic Breast Cancer Cell Lines.

Abstract Breast cancer remains the second leading cause of cancer related death amongst women in the United States. This is largely due to metastasis of cancer cells from the primary tumor to other parts of the body, and to the putative cancer stem cell population. Both are driven by the epithelial-mesenchymal transition (EMT), a cellular process whereby cancer cells of epithelial origin lose their epithelial characteristics and acquire a mesenchymal phenotype. Cells which undergo EMT tend to be motile and invasive, and therefore can metastasize to other parts of the body. EMT has also been implicated in the generation of cells expressing the cancer stem cell phenotype. As metastatic disease and the cancer stem cell are difficult to eliminate, more effective therapy is required. Zoledronic acid, originally indicated for use in the treatment of osteoporosis, has been reported to inhibit the growth of breast cancer cells. The mechanism of this effect however has yet to be determined. In preliminary data studies, treatment with zoledronic acid was found to reduce activation of nuclear factor kappa-b, an established regulator of EMT. These findings suggest that treatment with zoledronic acid may reverse EMT in breast cancer cells, driving them to express a more epithelial phenotype. To test this hypothesis, the highly metastatic, triple negative breast cancer cell lines MDA-MB-231 and Hs578t, which largely express mesenchymal proteins, were used to measure cell viability and changes in protein and mRNA expression following treatment with zoledronic acid. Dose response analysis for inhibition of cell viability showed an IC50 of approximately 2μM in the Hs578t and 6 μM in the MDA-MB-231 cell line. Zoledronic acid treated cells displayed a decreased mesenchymal phenotype, as evidenced by reduced expression of mesenchymal markers N-cadherin (75% reduction, p<0.076; 65% reduction, p<0.005) and TWIST (67% reduction, p<0.08; 64% reduction, p<0.009) in MDA-MB-231 and Hs578t cells, respectively. This was accompanied by a subsequent increase in epithelial phenotype as evidenced by increased expression of epithelial marker E-cadherin (223% increase, p<0.006) in MDA-MB-231 cells. To further elucidate the effects on the mesenchymal and epithelial phenotypes of these cells, surface expression of CD24 and CD44 was measured by flow cytometry. While vehicle treated samples of both cell lines stained positive for CD44 and negative for CD24, zoledronic acid treatment decreased CD44 expression. As both increased the ratio of surface expression of CD44high/CD24low and mammosphere formation are characteristics of the breast cancer stem cell, effects of zoledronic acid on this subpopulation in Hs578t cells were determined. Cells pretreated with zoledronic acid were seeded under mammosphere conditions and allowed to propagate for 7 days. Zoledronic acid treated cells formed significantly fewer mammospheres (86% reduction, p<0.041), while the ones that formed were smaller in size. These findings suggest that zoledronic acid is able to reverse the epithelial mesenchymal transition, which may reduce the tumor initiating capacity of highly metastatic cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-02.

An increase in cancer stem cell population after primary systemic therapy is a poor prognostic factor in breast cancer

Background:The cancer stem cell (CSC) hypothesis has important clinical implications for cancer therapeutics because of the proposed role of CSCs in chemoresistance. The aim of this study was to investigate changes in the CSC populations before and after primary systemic therapy (PST) and their prognostic role in human breast cancer.Methods:Paired samples (before and after PST) of breast cancer tissue were obtained from clinical stage II or III patients (n=92) undergoing PST with the regimen of doxorubicin plus docetaxel (AD) (n=50) or doxorubicin plus cyclophosphamide (AC) (n=42) and subsequent breast resection. The proportions of putative CSCs with CD44+/CD24− or aldehyde dehydrogenase 1+ (ALDH1+) phenotypes were determined by immunohistochemistry.Results:A higher proportion of CD44+/CD24− tumour cells and ALDH1 positivity in pre-chemotherapy tissue was correlated with higher histologic grade, oestrogen receptor (ER) negativity, high Ki-67 proliferation index and basal-like subtype of breast cancer. Aldehyde dehydrogenase 1 positivity in pre-chemotherapy biopsy was also associated with a higher rate of pathologic complete response following PST. In comparisons of putative CSC populations before and after PST, the proportions of CD44+/CD24− and ALDH1+ tumour cells were significantly increased after PST. The cases with increased CD44+/CD24− tumour cell populations after PST showed high Ki-67 proliferation index in post-chemotherapy specimens and those with increased ALDH1+ tumour cell population after PST were associated with ER negativity and p53 overexpression. Furthermore, cases showing such an increase had significantly shorter disease-free survival time than those with no change or a reduced number of CSCs, and the survival difference was most notable with regard to the changes of ALDH1+ tumour cell population in the patients who received AC regimen.Conclusion:The present study provides the clinical evidence that the putative CSCs in breast cancer are chemoresistant and are associated with tumour progression, emphasising the need for targeting of CSCs in the breast cancer therapeutics.

Abstract 3332: Obesity drives epithelial/mesenchymal transition and tumor progression in a novel Wnt-1 mammary cancer model

Abstract Epidemiological evidence suggests a potential role of obesity in regulating clinical subtype, differentiation status, and prognosis of breast cancer. Specifically, an elevated waist-hip ratio is associated with increased risk and progression of basal-like breast cancer, an aggressive form characterized by heterogeneous tumors typically enriched in a putative breast cancer stem cell (BCSC) population. Therefore, we hypothesized that obesity regulates a plastic population of multipotent malignant cells. To test this hypothesis, we generated and characterized two distinct murine mammary tumor cell lines derived from MMTV-Wnt-1 transgenic mice. M-Wnt cells displayed a mesenchymal morphology while E-Wnt cells had an epithelial morphology. M-Wnt cells harbored a large CD44+/CD24- putative cancer stem cell population (62% +7.8), had significant mammosphere forming capacity (>30% of cells form mammospheres, p<0.0001), and increased ALDH activity (7% are ALDH+, p=0.0004). M-Wnt cells have increased migration (scratch assay) and invasion in vitro (226-fold higher after 30h, p<0.0001). As few as 50 unsorted M-Wnt cells, injected into C57BL/6 mice were capable of forming a tumor. Microarray analysis revealed that M-Wnt cells display gene expression profiles virtually identical to human Claudin-low breast tumors, while E-Wnt cells clustered with basal-like tumors. EMT and stem cell gene expression patterns of M-Wnt cells were maintained in vivo, including decreased E-cadherin and increased N-cadherin, fibronectin, vimentin, SNAIL, TWIST, SLUG, FOXC2, and TGF-β (p<0.05 for all). Using these cell lines in vivo, we tested the hypothesis that energy balance modulation, through diet-induced obesity (DIO) and calorie restriction (CR), regulates the BCSC population. We found that M-Wnt tumors, transplanted into ovariecomized syngeneic female C57BL/6 mice, grew at significantly different growth rates depending on the diet treatment (DIO > Control, p=0.011; CR < Control, p=0.012), while E-Wnt tumors were only affected by CR (CR < Control p=0.001; no difference between DIO and Control tumors). DIO enhanced M-Wnt tumor progression, adipocyte infiltration, and central necrosis and drove EMT (decreased E-Cadherin and increased fibronectin and N-Cadherin expression) through modulation of key BCSC associated genes, including increased TGF-β, SNAIL, FOXC2, and Oct4 (p<0.05 for all). In conclusion, we found that the mesenchymal M-Wnt cell line is highly responsive to changes in dietary energy balance status relative to the E-Wnt differentiated epithelial cell line, suggesting that energy balance modulation (ie, CR and obesity) regulates tumor growth through direct regulation of a BCSC population, possibly in response to alterations in TGF-β signaling. 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 3332. doi:10.1158/1538-7445.AM2011-3332

Abstract 5199: Primary culture of human midgut carcinoid cell lines and isolation of a putative carcinoid stem cell population

Abstract Background: Research in carcinoid and neuroendocrine tumors (NETs) of midgut origin is limited by a lack of cell lines. The existing foregut NET lines, BON and H727, are phenotypically distinct from midgut NETs highlighting the importance of establishing midgut derived NET cell lines. Therapeutic options are limited for midgut NETs. NET cell lines are difficult to culture for long periods (thus few cell lines). We are developing methods to improve primary and prolonged NET cell culture in an attempt to identify novel therapeutic approaches. Along these lines, we are conducting studies to identify a putative cancer stem cell population in human NET specimens to provide new insights regarding potential targets in NETs. Methods: Sterile human midgut NET surgical specimens were digested to a single cell suspension and fibroblast depleted with magnetic beads. The NET cells were maintained on collagen I coated plates for primary culture. A portion of the primary cell culture was used for FACS by the Aldefluor assay to isolate potential cancer stem cells (CSCs). Sorted cells were grown in serum-free media on low attachment plates to assess sphere formation and preserved for RNA isolation and subsequent gene array. Subcutaneous xenografts in nude and NOD/SCID gamma mice using fresh tumor implants and NET cell suspensions were performed as potential means to further propagate NET cells. Results: The NET markers, somatostatin receptor 2 and chromogranin A, were detectable in cultures from a midgut carcinoid liver metastasis at passage 3 and 5, as well as in one mouse xenograft. While retention of NET markers from xenograft implants is promising, tumor growth remains limited and slow, with tumors in 5/59 mice over 4-10 months. We are now studying tumor xenografts in more immunodeficient NOD/SCID gamma mice with current patient samples to potentially enhance xenograft growth. FACS sorting identified an ALDH+ population of NET cells, which formed spheres (a characteristic of CSCs) more frequently than ALDH(−) negative cells. A FACS sorted midgut sample plated at 10k cells/well in sphere forming media produced spheres as early as 8 days in the ALDH+ population, while the ALDH- cells did not form spheres (sham sorted cells formed rare spheres), which was also observed in other samples where viable cells were obtained. Additionally, the ALDH+ cells from this population were cutlured on plastic and remained viable for 4 weeks, while the ALDH- population was not viable in culture. RNA isolation for gene array is underway for the ALDH+ population to characterize these cells in midgut carcinoids. Conclusions: We have developed a system allowing for NET primary culture for up to 4 weeks. We have also identified a putative CSC population in NETs. These techniques are the first step in obtaining cells for in vitro studies and in identifying novel therapeutic targets for NETs. Supported by the The Raymond and Beverly Sackler Foundation. 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 5199. doi:10.1158/1538-7445.AM2011-5199

Abstract SY24-01: Pluripotency genes as targets for cancer prevention

Abstract Recent studies have suggested an overlap between the core pathways that maintain pluripotency in embryonal stem (ES) or induced pluripotency stem (iPS) cells and those implicated in oncogenesis. The presence of ES/iPS signature genes can be demonstrated in diverse cancers and correlates with a poorer outcome. The ES pathways have been shown to be particularly expressed in the putative cancer stem cell (CSC) population within the tumor bulk. Therefore pathways that regulate stemness are being actively targeted in human cancer. Here, I will discuss evidence for immune-mediated targeting of the core ES genes, such as OCT4 and SOX2 in humans. Initial studies suggested that humoral and cellular responses against SOX2 can be detected in patients with preneoplastic monoclonal gammopathy of undetermined significance (MGUS). Presence of anti-SOX2 T cell responses correlated with reduced risk of progression to clinical myeloma. Surprisingly, T cell responses against OCT4, a common marker of ES cells, can be readily detected in healthy human donors. OCT4-specific T cell response resides predominantly in the CD4+ T cell compartment and comprises of CD45RO+ memory T cells. These T cells can be readily expanded in culture using autologous dendritic cells loaded with OCT4-expressing germ cell tumors. In contrast to the finding with healthy donors, T cell immunity to OCT4 is deficient in freshly isolated circulating T cells from patients with germ cell tumors (GCTs). However, these T cells can be observed after chemotherapy-induced cell death of tumor cells in vivo. The capacity of the human immune system to target both of the core pluripotency genes (SOX2 and OCT4) may facilitate harnessing the immune system against GCTs and putative CSCs in diverse tumor types. ES-associated genes such as OCT4 seem to be dispensable for the function of adult stem cells in mice, suggesting that targeting these genes in the setting of cancer might not carry adverse effects against normal adult stem cells. As these immune responses seem to be engaged during early stages of carcinogenesis, including in preneoplastic states, we suggest that they may also be particularly attractive targets for immune-mediated cancer prevention in diverse high-risk settings. 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 SY24-01. doi:10.1158/1538-7445.AM2011-SY24-01

Abstract 2449: Pancreatic cancer tumor initiating cells are marked by the presence of a primary cilium

Abstract Primary cilia are microtubule rich projections eminating from the apical surface of epithelial cells. Primary cilia are responsible for the transduction of growth factor and morphogenetic signaling pathways, including the hedgehog, wnt and platelet-derived growth factor pathways. Initial observations in the field of pancreatic cancer have suggested the absence of primary cilia in human pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma. While absent in the majority of pancreatic cancer cells, primary cilia have been identified in differentiated acinar cells undergoing an acinar to ductal metaplasia. This metaplasia is well characterized in pancreatic injury and regeneration models and accelerates tumor formation in the presence of acinar specific KrasG12D mutations. Thus, we are addressing the hypothesis that primary cilia, while not expressed in bulk tumor populations, are maintained in pancreatic tumor-initiating cells. To address the hypothesis, we have used confocal microscopy to quantify the percentage of cells within low grade human PanIN lesions that express a primary cilium. Performing confocal microscopy on a human PanIN tissue array (n=23), we have determined that in 15% of PanIN lesions, 13-30% of the cells are expressing a primary cilium, as is evidenced by staining for acetylated alpha tubulin. We have also developed a sorting strategy using an acetylated alpha tubulin antibody to isolate ciliated cells by Fluorescence Activated Cell Sorting (FACS) analysis. Using this protocol, we have discovered that human pancreatic cancer cells bearing a primary cilium are markedly enriched in cells also positive for either CD133 or CD24/CD44, representing putative pancreatic cancer stem cell populations. We have functionally analyzed the tumor-initiating capacity of ciliated vs non-ciliated cells using tumor sphere experiments. In this assay, ciliated cells show a significant increase (100 fold) in tumor sphere-forming capacity, suggesting that the presence of a primary cilium may mark a pancreatic cancer tumor initiating population. These data are informative regarding the earliest initiating events in pancreatic cancer. 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 2449. doi:10.1158/1538-7445.AM2011-2449

Abstract 3331: An increase in cancer stem cell population after primary systemic therapy is a poor prognostic factor in breast cancer

Abstract Introduction: The cancer stem cell (CSC) hypothesis has important clinical implications for cancer therapeutics because of the proposed role of CSCs in chemoresistance. The aim of this study was to investigate changes in the CSC populations before and after primary systemic therapy (PST) and their prognostic role in human breast cancer. Methods: Paired samples (before and after PST) of breast cancer tissue were obtained from clinical stage II or III patients (n=92) undergoing PST and subsequent breast resection. The proportions of putative CSCs with CD44+/CD24- or ALDH1+ phenotypes were determined by immunohistochemistry. Results: A higher proportion of CD44+/CD24- tumor cells and ALDH1 positivity in pre-chemotherapy biopsy was correlated with higher histologic grade, estrogen receptor negativity, high Ki-67 proliferation index and basal-like subtype of breast cancer. ALDH1 positivity in pre-chemotherapy biopsy was also associated with a higher rate of pathologic complete response (pCR) following PST. In comparisons of putative CSC populations before and after PST, the proportions of CD44+/CD24- and ALDH1+ tumor cells were significantly increased after PST. The cases with increased CD44+/CD24- tumor cell populations after PST showed high Ki-67 proliferation index in post-chemotherapy specimens and those with increased ALDH1+ tumor cell population after PST were associated with estrogen receptor negativity and p53 overexpression. Furthermore, cases showing such an increase had significantly shorter disease-free survival time than those with no change or a reduced number of CSCs. Conclusions: The present study provides the clinical evidence that the putative CSCs in breast cancer are chemoresistant and are associated with tumor progression, emphasizing the need for targeting of CSCs in the breast cancer therapeutics. 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 3331. doi:10.1158/1538-7445.AM2011-3331

The Cancer Stem Cell Hypothesis: Failures and Pitfalls

Based on the clonal evolution model and the assumption that the vast majority of tumor cells are able to propagate and drive tumor growth, the goal of cancer treatment has traditionally been to kill all cancerous cells. This theory has been challenged recently by the cancer stem cell (CSC) hypothesis, that a rare population of tumor cells, with stem cell characteristics, is responsible for tumor growth, resistance, and recurrence. Evidence for putative CSCs has been described in blood, breast, lung, prostate, colon, liver, pancreas, and brain. This new hypothesis would propose that indiscriminate killing of cancer cells would not be as effective as selective targeting of the cells that are driving long-term growth (ie, the CSCs) and that treatment failure is often the result of CSCs escaping traditional therapies.The CSC hypothesis has gained a great deal of attention because of the identification of a new target that may be responsible for poor outcomes of many aggressive cancers, including malignant glioma. As attractive as this hypothesis sounds, especially when applied to tumors that respond poorly to current treatments, we will argue in this article that the proposal of a stemlike cell that initiates and drives solid tissue cancer growth and is responsible for therapeutic failure is far from proven. We will present the point of view that for most advanced solid tissue cancers such as glioblastoma multiforme, targeting a putative rare CSC population will have little effect on patient outcomes. This review will cover problems with the CSC hypothesis, including applicability of the hierarchical model, inconsistencies with xenotransplantation data, and nonspecificity of CSC markers.

Therapy-Senescent Lymphoma Cells Bear Potentially Detrimental Stem-Cell Characteristics.

AbstractAbstract 2836Premature senescence is a stress-inducible terminal cell-cycle arrest, which, like apoptosis, prevents propagation of aberrantly proliferating cells. DNA-damaging chemotherapy induces cellular senescence, particularly in cells where apoptosis is no longer available (e.g. due to Bcl2 over-expression). Since senescent cells remain vital, it is critical to understand their long-term fate and ultimate impact on outcome. Given the potential reversibility of senescence by additional genetic events, assessment of a senescence-associated tumorigenic capability appears to be crucial for the development of safe pro-senescent treatment regimens. Since “stemness” is a property that – either confined to a putative cancer stem cell population or endowed in all neoplastic cells – enables malignant transformation and propagation, we decided to investigate features of stemness in senescent Eμ-myc-transgenic B-cell lymphomas.Here, we compared expression changes of stem-cell related gene sets in proliferating vs. treatment-induced senescent (TIS) Eμ-myc lymphoma cells. The clonogenicity and proliferation capacity were assessed in cells released from TIS by acute inactivation of downstream senescence gatekeeper proteins, such as Suv39h1, Klf4 or p53.Our data show that embryonic stem cell-like (ESCL) gene expression signatures, associated with aggressive tumor behavior decrease with TIS induction in Eμ-myc lymphoma cells. However, the adult tissue stem cell (ATSC) gene signature is highly up-regulated, providing the senescent lymphoma cells with an alternative stemness-maintaining prospective. Similar conversion from an ESCL- to an ATSC-governed program is observed in hematopoietic stem cells shifting from an actively proliferating to a quiescent state. TIS cells presented with high expression levels of reprogramming factors (Klf4 and Sox2), which have the potential to initiate ESCL program, and exhibited up-regulation of several transcripts associated with invasive neoplastic features, like Twist1/2, Snai1/2, Cdh2 or Mmp2These findings suggest that senescent cells retain or even increase their initial malignant capacity by promoting a latent stemness program that is kept in check by cellular senescence. Indeed, upon inactivation of the senescence barrier, cells resumed cycling, and showed an increased clonogenic potential compared to matching proliferating lymphoma cells that had not encountered senescence. The relevance of these genetic changes for tumor progression in vivo is currently under investigation.These results suggest a more complex relation between senescence and stemness than intuitively expected. The intended execution of the senescence response evokes critical changes in other fundamental, cancer-related cellular programs, opening the possibility of arresting neoplastic cells on one side, but simultaneously promoting aggressive cancer features, like self-renewal, invasion and motility that may become harmful upon disruption of the “senescence shield”. This calls for a thorough functional reassessment of the senescent phenotype, before employing senescence as a therapeutic option. Disclosures:No relevant conflicts of interest to declare.

Preferential antitumor effect of the Src inhibitor dasatinib associated with a decreased proportion of aldehyde dehydrogenase 1-positive cells in breast cancer cells of the basal B subtype.

BackgroundRecent studies have suggested that the Src inhibitor dasatinib preferentially inhibits the growth of breast cancer cells of the basal-like subtype. To clarify this finding and further investigate combined antitumor effects of dasatinib with cytotoxic agents, a panel of breast cancer cell lines of various subtypes was treated with dasatinib and/or chemotherapeutic agents.MethodsSeven human breast cancer cell lines were treated with dasatinib and/or seven chemotherapeutic agents. Effects of the treatments on c-Src activation, cell growth, cell cycle, apoptosis and the proportion of aldehyde dehydrogenase (ALDH) 1-positive cells were examined.ResultsThe 50%-growth inhibitory concentrations (IC50s) of dasatinib were much lower in two basal B cell lines than those in the other cell lines. The IC50s of chemotherapeutic agents were not substantially different among the cell lines. Dasatinib enhanced antitumor activity of etoposide in the basal B cell lines. Dasatinib induced a G1-S blockade with a slight apoptosis, and a combined treatment of dasatinib with etoposide also induced a G1-S blockade in the basal B cell lines. Dasatinib decreased the expression levels of phosphorylated Src in all cell lines. Interestingly, dasatinib significantly decreased the proportion of ALDH1-positive cells in the basal B cell lines but not in the other cell lines.ConclusionsThe present study indicates that dasatinib preferentially inhibits the growth of breast cancer cells of the basal B subtype associated with a significant loss of putative cancer stem cell population. A combined use of dasatinib with etoposide additively inhibits their growth. Further studies targeting breast cancers of the basal B subtype using dasatinib with cytotoxic agents are warranted.

Abstract 3347: Enrichment of a CD44+/CD24- putative cancer stem cell population drives tumor progression and growth factor independence in a mouse model of postmenopausal breast cancer

Abstract MMTV-Wnt-1 transgenic mice develop heterogeneous mammary tumors that accurately recapitulate human basal-like breast cancer. Additionally, targeted alterations in the Wnt signaling pathway in mammary epithelial cells preferentially induce mammary cancers from progenitor cells. Therefore, we hypothesized that MMTV-Wnt-1 mammary tumors harbor cells with cancer stem cell characteristics. To test this hypothesis, we characterized (in vitro and in vivo) two newly generated, clonal mammary epithelial cell lines (WA4 and WG4) from MMTV-Wnt-1 transgenic mouse tumors. The cell lines were analyzed using flow cytometry for CD44 and CD24 cell surface markers (a CD44+/CD24- profile is indicative of putative human and murine mammary cancer stem cells), mammosphere formation (low density seeding on non-adherent plates in stem-cell media), in vitro scratch assay (to measure wound healing), and by an in vitro Matrigel invasion chamber assay (to quantitate cell invasion). Finally, unsorted WA4 and WG4 cell lines were transplanted into syngeneic mammary fat pads of female ovariectomized (to model the postmenopausal state) C57BL/6 mice (1×10^7 cells/mouse; n=5/cell line), and tumor burden was assessed after 3 weeks. Tumors were analyzed by qRT-PCR and immunohistochemistry (IHC) for pathological and genetic markers of progression. Flow cytometric analysis demonstrated that the percentage of CD44+/CD24- cells was 62.2% in the WA4 line and 2.4% in the WG4 line. Serum starvation enriched the WA4 line for CD44+/CD24- cells (from 62.2% to 82.0%), without altering the composition of the WG4 line. Approximately 30% of unsorted WA4 cells, but <1% of unsorted WG4 cells, formed mammospheres. Additionally, the WA4 line showed increases in wound healing by scratch assay and significant increases in invasion (226-fold increase versus WG4 cells after 30 hours). In vivo, the WA4 cell line was associated with larger, more aggressive tumors on average (+/− SD) than the WG4 line (2.09 +/− 0.68 g and 0.03 +/− 0.02 g, respectively, p<0.0001). WA4 tumors were characterized by increased cellular proliferation, microvascular proliferation, and a basal appearance lacking ductal structures. WA4 tumors also demonstrated a loss of ERα/β expression (qRT-PCR), lacked IGF1-R activation (by IHC), and showed an increase in Wnt-1 progenitor signaling (qRT-PCR). In conclusion, MMTV-Wnt-1 transgenic mouse mammary tumors harbor a CD44+/CD24- putative cancer stem cell population, and an enrichment of this population corresponds with a more aggressive tumor phenotype in a mouse model of postmenopausal breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3347.

Abstract 5044: Dual GSK-3β/CDK inhibitors induce multiple myeloma cell death and eliminate putative cancer stem cell populations

Abstract Multiple myeloma (MM) is a plasma cell malignancy that recurs in 100% of patients despite high treatment response rates. The incurable nature of the disease and the low proliferative capacity of terminally differentiated MM plasma cells supports the existence of a cancer stem cell (CSC) component - a cell population with self-renewal and drug resistance properties capable of repopulating the disease after seemingly effective treatment and periods of undetectable disease. It is imperative to develop new therapies that target both terminally differentiated MM plasma cells and MM CSCs to extend disease-free survival and control the disease. We tested the ability of c-Myc-inducing compounds, such as GSK-3β and dual GSK-3β/CDK inhibitors, to enhance the activity of bortezomib against advanced MM cell lines (RPM-I8226 and NCI-H929). We have shown that this class of inhibitors has anti-tumor effects and sensitizes numerous solid malignancies to apoptosis-inducing agents (Mayes P.A., Dolloff N.G. et al., in revision). Moreover, c-Myc expression increases protein translation, and in doing so, enhances the cytotoxicity of proteasome inhibitors such as bortezomib (Nawrocki et al., 2008). We found that GSK-3β inhibitors (lithium choloride and sc-24020) failed to induce apoptosis or reduce viability of MM cell lines or to enhance the activity of bortezomib. By contrast, the dual GSK-3β/CDK inhibitors, alsterpaullone and the nucleoside analogue SLM3 (Mayes P.A., Dolloff, N.G. et al., in revision), were highly toxic to MM cells both alone and in combination with bortezomib. In addition to inducing massive apoptosis and reducing MM cell viability, alsterpaullone and SLM3 eliminated the presence of dye-effluxing side-populations, that have been shown to exhibit CSC behavior in vitro and in animal models of MM. By comparison, bortezomib, adriamycin and melphalan increased the percentage of MM side-population cells, suggesting that MM CSC cells are more resistant to these agents compared to their non-CSC counterparts. Interestingly, alsterpaullone and SLM3 eliminate a distinctly large sub-population of MM cells that are enriched in side-population cells, providing a further characterization of the MM CSC phenotype. We propose that using GSK-3β/CDK inhibitors may be effective therapies, as they target tumorigenic CSC populations, which are responsible for disease relapse. We are currently testing this hypothesis in mouse models of MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5044.

Abstract SY33-03: Role of telomerase in normal and neoplastic stem cells

Abstract We have used hTERT (the catalytic component of telomerase) to immortalize a variety of human cell types (sometimes in combination with Cdk4 to bypass cell culture stress). Cell types immortalized include skin keratinocytes and fibroblasts, muscle satellite cells, breast epithelial and stromal cells, corneal epithelial cells and fibroblasts (keratocytes), and human colonic epithelial cells. In addition, we have immortalized human bronchial epithelial cells (HBEC) and have determined that these cells can terminally differentiate into both central and peripheral lung cell types. These immortalized HBECs have been used to study the molecular pathogenesis of lung cancer by stable “knock down” of TP53 and also by over-expression of C-myc and mutant K-rasV12. When these cells containing multiple genetic alterations are introduced into immunosuppressed mice, the experimentally transformed cells make tumors that represent several distinct histological types. This suggests that these immortalized and transformed HBECs have bronchiolar-alveolar stem-like characteristics that can differentiate into multiple lineages. Stem cells are defined by both their ability to make more stem cells (self renewal) and their ability to produce cells that can differentiate. Experimentally immortalized human bronchial epithelial cells fulfill this definition of normal stem cells by continuous self renewal and by retaining the capability of differentiating into several cell types. Since experimentally transformed cells make lung tumors representing several major lineages, this is also an indication that the HBECs are derived from a multi-potent lung stem cell. Similar to normal stem cells, cancer (initiating) stem cells also have the ability to self-renew as well as undergo differentiation to give rise to phenotypically diverse types of cancer cells. There is mounting evidence that these rare cancer stem cells may be multidrug resistant and responsible for tumor relapse and metastasis. Targeted cancer therapeutic approaches seek to identify pathways that are more tumor specific, resulting in fewer side-effects and that may produce long-term durable responses. Telomerase is a novel cancer therapeutic target since it is activated in the vast majority of human cancers and telomeres of almost all human tumor cells are maintained at short but stable lengths. In addition, telomerase is not expressed or is expressed at levels that do not fully maintain telomeres in normal tissues, and telomeres are generally longer in normal stem cells compared to cancer cells. This potentially provides a therapeutic advantage for targeting telomerase over approaches that affect both normal and cancer cells equally. We have previously reported that telomerase positive cancer cells that are experimentally induced to undergo quiescence, down regulate telomerase. As part of our anti-telomerase therapeutics program, we have addressed the following questions: Are putative cancer stem cell populations quiescent and do they have short or long telomeres? While the molecular characteristics of cancer stem cells are not completely defined and subject to some controversies, we have isolated and examined cells expressing these cancer stem cell putative markers reported for breast, brain, prostate, pancreas, and lung cancer. In each case we have observed that purified cancer stem cell populations are positive for telomerase activity, indicating they are not quiescent. In addition, a telomerase inhibitor currently being tested in clinical trials robustly inhibits the activity of telomerase in these sorted sub-populations of putative cancer stem cells as well as the mass population of cancer cells. Finally, we have observed that cancer stem cells have short telomeres in comparison to normal stem cells. These findings support the idea that there may be a therapeutic window of opportunity to target cancer stem cells by inhibiting telomerase, thus driving telomeres progressively shorter leading to cancer stem cell death, potentially without irreversible damage to normal stem cells. Cancer remains a major cause of death in spite of substantial progress towards understanding the molecular basis of many types of cancers. The discovery of new drugs is a high priority, and telomerase inhibitors have the potential to act by a novel mechanism that will provide new options for cancer therapy. A review of ongoing anti-telomerase clinical trials will be presented. In summary, telomerase inhibitors might not only directly limit or stop the growth of human tumors including cancer stem cells, but might also act in an additive or synergistic fashion with existing therapies to amplify their effectiveness. Citation Format: Jerry W. Shay. Role of telomerase in normal and neoplastic stem cells [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY33-03

Abstract 709: The fully human IGF-1 receptor antibody, CP-751,871, targets putative colon cancer stem cell populations

Abstract The identification of cancer stem cells (CSCs) has the potential to provide answers to questions of tumor pathogenesis, metastasis, and therapeutic resistance. We have previously shown that insulin-like growth factor 1 (IGF-1) enriches putative colon CSC populations, an important finding with the potential to explain the association of colon cancer risk and chemoresistance with the levels of circulating IGF-1 and obesity. Here, we have employed three established methods (side population (SP) analysis, aldehyde dehydrogenase activity (ALDH+), and CD133 expression) to analyze a panel of 8 human colon cancer cell lines for putative cancer stem cell properties. We have found enrichment of SP and/or ALDH+ populations across the panel of 8 colon cancer cell lines, as well as many lines originating from other tissues. The enrichment of SP cells by IGF-1 is independent of altered ABC transporter activity as measured by mitoxantrone efflux. Enrichment of CSCs by IGF-1 does not appear to depend on the activity of MAP kinases (based on inhibition with PD098059 and U0126); therefore, we are currently exploring dependence on alternative IGF signaling pathways downstream of Akt, including NF-κB and mTOR. While we continue our efforts to unravel the mechanism by which IGF-1 enriches CSCs we are also interested in the more immediate clinical implications of our findings. CP-751,871 is a fully human antibody with specificity to the IGF-1 receptor and is currently being tested in clinical trials for a variety of solid malignancies. We have found that CP-751,871 reduces both SP and ALDH+ CSC populations in multiple colon cancer cell lines in vitro, and has promising activity against these populations in pilot experiments in vivo. We are currently repeating these studies to yield conclusions supported by statistics. Interestingly, CP-751,871 blocks IGF-1 enrichment of the side population in DLD1 cells, but not in SW480 cells suggesting a potential role of alternative receptors, such as the insulin receptor in some cell lines, a possibility we are currently analyzing. These studies have the potential to affect clinical oncology treatment strategies as the role of CSCs in tumor growth is delineated. Our results suggest that CP-751,871 has preferential activity against putative CSC populations, and therefore, may benefit current standard chemotherapeutic regimens that target cycling cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 709.

Abstract B66: Aggressiveness of MMTV-Wnt-1 mammary tumors corresponds with enrichment of a CD44+/CD24− putative cancer stem cell population

Abstract MMTV-Wnt-1 transgenic mice develop heterogeneous mammary tumors that accurately recapitulate human basal-like breast cancer. Additionally, targeted alterations in the Wnt signaling pathway in mammary epithelial cells preferentially induce mammary cancers from progenitor cells. Therefore, we hypothesized that MMTV-Wnt-1 mammary tumors harbor cells with cancer stem cell characteristics. To test this hypothesis, we characterized (in vitro and in vivo) two newly generated, clonal mammary epithelial cell lines (WA4 and WG4) from a MMTV-Wnt-1 transgenic mouse tumor. The cell lines were analyzed using flow cytometry for CD44 and CD24 cell surface markers (a CD44+/CD24− profile is indicative of putative human and murine mammary cancer stem cells), mammosphere formation (low density seeding on non-adherent plates in stem-cell media), in vitro scratch assay (to measure wound healing), and by an in vitro Matrigel invasion chamber assay (to quantitate cell invasion). Finally, unsorted WA4 and WG4 cell lines were transplanted into syngeneic mammary fat pads of C57BL/6 mice (1×10^7 cells/mouse; n=5/cell line), and tumor burden was assessed after 3 weeks. Flow cytometric analysis demonstrated that the percentage of CD44+/CD24− cells was 62.2% in the WA4 line and 2.4% in the WG4 line. Serum starvation enriched the WA4 line for CD44+/CD24− cells (from 62.2% to 82.0%), without altering the composition of the WG4 line. Approximately 30% of unsorted WA4 cells, but <1% of unsorted WG4 cells, formed mammospheres. Additionally, the WA4 line showed increases in wound healing by scratch assay and significant increases in invasion (226-fold increase versus WG4 cells after 30 hours). In vivo, the WA4 cell line was associated with larger, more aggressive tumors on average (+SD) than the WG4 line (2.09 +/−0.68 g and 0.03 +/−0.02 g, respectively, p<0.0001). WA4 tumors were characterized by increased cellular proliferation, microvascular proliferation, and a basal appearance lacking ductal structures. In conclusion, MMTV-Wnt-1 transgenic mouse mammary tumors harbor a CD44+/CD24− putative cancer stem cell population, and an increased percentage of this population corresponds with a more aggressive tumor phenotype. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B66.

Cancer Stem Cells and Epithelial Ovarian Cancer

The cancer stem cell hypothesis is becoming more widely accepted as a model for carcinogenesis. Tumours are heterogeneous both at the molecular and cellular level, containing a small population of cells that possess highly tumourigenic “stem-cell” properties. Cancer stem cells (CSCs), or tumour-initiating cells, have the ability to self-renew, generate xenografts reminiscent of the primary tumour that they were derived from, and are chemoresistant. The characterisation of the CSC population within a tumour that drives its growth could provide novel target therapeutics against these cells specifically, eradicating the cancer completely. There have been several reports describing the isolation of putative cancer stem cell populations in several cancers; however, no defined set of markers has been identified that conclusively characterises “stem-like” cancer cells. This paper highlights the current experimental approaches that have been used in the field and discusses their limitations, with specific emphasis on the identification and characterisation of the CSC population in epithelial ovarian cancer.

Zebrafish Tumor Assays: The State of Transplantation

Tumor transplant studies are important tools for studying cancer biology in a model organism. Transplantation is especially important for assaying tumor cell malignancy and migration capabilities, and is critical for identifying putative cancer stem cell populations. In this review, we discuss the current state of tumor transplantation studies performed in the zebrafish. We address several zebrafish-specific considerations for development of the transplant assay, including choosing recipient animals, transplant methods, and post-transplant observation. We also examine how the zebrafish is an advantageous model for transplantation, particularly with development of the translucent fish. Transplantation has already been critical for characterizing zebrafish models of leukemia, rhabdomyosarcoma, and melanoma. With further development of imaging techniques and other tools, zebrafish tumor transplantation will continue to contribute to our understanding of tumor cell biology.

Visualization and enrichment of live putative cancer stem cell populations following p53 inactivation or Bax deletion using non-toxic fluorescent dyes

Putative cancer stem cell (CSC) populations efflux dyes such as Hoecsht 33342 giving rise to side-populations (SP) that can be analyzed or isolated by flow cytometry. However, Hoecsht 33342 is highly toxic, more so to non-SP cells, and thus presents difficulties in interpreting in vivo studies where non-SP cells appear less tumorigenic than SP cells in immunodeficient mice. We searched for non- toxic dyes to circumvent this problem as well as to image these putative CSCs. We found that the fluorescent dye calcein, a product of intracellular Calcein AM cleavage, is effluxed by a small subpopulation, calcein low population (CloP). This population overlaps with SP and demonstrated long term cell viability, lack of cell stress, and proliferation in several cancer cell lines when stained whereas Hoecsht 33342 staining caused substantial apoptosis and ablated proliferation. We also found that the effluxed dye D-luciferin exhibits strong UV-fluorescence that can be imaged at cellular resolution and spatially overlaps with Calcein AM. In order to evaluate the hypothesis that p53 loss promotes enrichment of putative CSC populations we used Calcein AM, D-luciferin, and Mitotracker Red FM as a counterstain to visualize dye-effluxing cells. Using fluorescence microscopy and flow cytometry we observed increased dye-effluxing populations in DLD-1 colon tumor cells with mutant p53 versus wild-type (WT) p53-expressing HCT116 cells. Deletion of the wild-type p53 or pro-apoptotic Bax genes induced the putative CSC populations in the HCT116 background to significant levels. Restoration of WT p53 in HCT116 p53-/- cells by an adenovirus vector eliminated the putative CSC populations whereas a control adenovirus vector, Ad-LacZ, maintained the putative CSC population. Our results suggest it is possible to image and quantitatively analyze putative CSC populations within the tumor microenvironment and that loss of pro-apoptotic and tumor suppressing genes such as Bax or p53 enrich such tumor-prone populations.