Quiescent Cancer Stem Cells Research Articles

321. Oncolytic Adenovirus Expressing IFN as a Tool To Eliminate Pancreatic Cancer Stem Cells

Pancreatic cancer is the 4th leading cause of cancer related death in the US, and curative resection of tumors is the most effective treatment against the disease. Unfortunately, the majority of patients are diagnosed in the advanced stage of the disease, and palliative treatment with Gemcitabine is the therapy of choice. In both cases, curative resection of tumors or chemotherapy, effectiveness can be limited by the presence of pancreatic cancer stem cells. Cancer stem cells (CSC) are correlated with drug resistance, tumor recurrence, and metastasis. CSC are usually located in the hypoxic center of the tumors in a quiescent stage and are not affected by conventional chemotherapeutics that target highly replicating cells, such as Gemcitabine. CSC are also called tumor initiating cells (TIC), and as low as 100 cells are reported to form tumors in pre-clinical models.Inclusion of IFN alpha (IFN) in combination therapy protocols against pancreatic cancer can be highly beneficial to tackle this problem. IFN is reported to be cytotoxic to cancer cells, have antiangiogenic properties, stimulate anti-tumor immunity, and sensitize cancer cells to chemoradiation. In addition, IFN is reported to induce activation of quiescent CSC making them susceptible to chemotherapy drugs that target highly replicating cells. Phase II and III clinical trials combining IFN with 5-FU and radiation in an adjuvant therapy setting reported a 35% increase in the five year overall survival of pancreatic cancer patients. Stimulated by the promising results reported in IFN clinical trials we developed an oncolytic adenovirus expressing IFN (OAd-IFN). Our aim is to use the virus to improve IFN therapeutic effects in combination therapy by restricting high levels of IFN expression to the tumor. In vitro data testing triple combination of OAd-IFN + radiation+ 5-FU using MIA PACA-2 and S2103 pancreatic cancer cells in colony formation assay (CFA) showed that OAd-IFN combination was more efficacious in inhibiting colony formation than triple combination with OAd-LUC, a counterpart of OAd-IFN expressing luciferase. Triple combination with OAd-IFN was also more efficient than double therapies with 5-FU+radiation, OAd-LUC + radiation, and OAd-LUC+5-FU. As CFA is an assay frequently used to access the proliferating capacity of single cells in vitro, our data suggests that IFN expressed virus decreased the number of TIC/CSC. This is supported by our in vivo studies showing that inclusion of OAd-IFN in combination with radiation or with 5-FU+radiation results in superior tumor shrinkage than all therapies not involving IFN or including OAd-LUC. In addition, tumors treated with OAd-IFN combination have a longer recurrence interval compared with all other treatments suggesting reduced levels of TIC.Although further studies are necessary to understand the impact of our OAd-IFN virus in pancreatic cancer stem cells, we believe IFN expressed by our virus made quiescent CSC more susceptible to treatments. Because our virus can express high levels of IFN restricted to the tumor site, we believe that a higher percentage of these cells will be affected by IFN delaying clinical recurrence of tumors, and improving therapy effectiveness.

Abstract 4092: Long-lived Dclk1+ cells serve as colon cancer initiating cells

Abstract Introduction: In the rapidly proliferating gastrointestinal epithelium, long-lived tissue stem cells, characterized by multipotentiality and self-renewing ability, remain the most likely cellular origin for cancer. Previous studies have suggested actively cycling Lgr5+ stem cells are one cellular origin for intestinal adenomas. However, it has also recently been suggested that non-Lgr5+ cells may also contribute to the cellular origin of colorectal cancer. Doublecortin-like kinase 1 (Dclk1) protein is a gastrointestinal tuft cell marker that has been proposed to identify quiescent stem cells and cancer stem cells that sustain tumor growth. The role of Dclk1+ tuft cells within the gastrointestinal epithelium and their potential to function as cancer-initiating cells, however, remain poorly understood. Here, we used Dclk1(BAC)-CreERT;ROSA26rLacZ mice crossed to APCff mice to examine whether Dclk1+ cells contribute to colonic tumor formation. Methods: To recapitulate the endogenous expression pattern of Dclk1, we used a BAC strategy and generated a transgenic mouse with a Tamoxifen inducible Cre under the control of the Dclk1 promoter (Dclk1-BAC-Cre-ERT). Dclk1-CreERT mice were crossed to both ROSA26rLacZ and APCff mice and treated with tamoxifen (6 mg p.o.). Dclk1+ lineage tracing was assessed by X-gal staining. To examine the contribution of the Dclk1+ cells to colonic tumorigenesis, we treated Dclk1(BAC)-CreERT;ROSA26rLacZ; APCff mice with DSS (3% in drinking water) to induce colitis. Mice were sacrificed 3-4 months after DSS weeks to assess for tumor formation and X-gal staining performed to stain for the Dclk1+ cell lineage. Results: Dclk1-BAC-CreERT genetic lineage tracing demonstrated that a subpopulation of Dclk1+ cells is extremely long-lived and shows rare stem cell abilities. Moreover, genetic ablation reveals a pivotal role for Dclk1+ tuft cells in the response to intestinal and colonic injury. Surprisingly, conditional loss of APC in Dclk1+ cells is not sufficient to drive colonic carcinogenesis, whereas induction of DSS colitis in Dclk1-CreERT; APCflox/flox mice leads to the development of poorly differentiated colonic adenocarcinoma. Importantly, colonic tumor formation occurs even when the onset of colitis is delayed for up to 3 months after APC loss in Dclk1+ cells. Conclusions. Thus, our data define a novel intestinal Dclk1+ tuft cell population that is long-lived, quiescent and important for intestinal homeostasis and regeneration. Long-lived Dclk1+ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve as a potent cellular origin of colon cancer. Citation Format: Samuel Asfaha, Christoph Benedikt Westphalen, Yoku Hayakawa, Yoshihiro Takemoto, Dana J. Lukin, Wanda Setlik, Helen Remotti, Ashlesha Muley, Xiaowei Chen, Randal May, Courtney W. Houchen, James G. Fox, Michael D. Gershon, Michael Quante, Timothy Wang. Long-lived Dclk1+ cells serve as colon cancer initiating cells. [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 4092. doi:10.1158/1538-7445.AM2014-4092

Su1729 Targeting Both Active and Quiescent Cancer (stem) Cells Using Combined Therapy

BACKGROUND: Recurrence of colorectal cancers after surgery and chemotherapy may be partly explained by the presence of chemoresistant cancer stem cells (CSC). Given the highly heterogeneous nature of CSCs it is possible that further characterization might identify a subset of CSC that eventually develop micro-metastases. Galectin-3 (Gal3) has been implicated in tumor progression, metastasis, and drug-resistance. We have previously reported that cell surface Gal3 renders colon cancer cells resistant to TNF-related Apoptosis-Inducing Ligand (TRAIL). Here, our aim was to determine how Gal3 is related to expression of stem cell markers and chemoresistance in different colon cancer cell populations. METHODS: We used the highly metastatic colon cancer cell line LiM6 and its TRAIL-resistant derivative LIM6-TR as cellular model. CSC populations were identified and isolated based on the expression of 5 established CSC-Markers CD24, CD44, CD166, EpCAM, and ALDH. Surface Gal3 in these CSCs was monitored by flow cytometry. Self-renewal, a functional CSC hallmark of CSC, was measured in sphere-formation assays. Response to TRAIL wasmeasured by APO-BRDU TUNEL assays. RESULTS: Only 15% of LIM6 and LIM6-TR cells were CSC as defined by the 5 markers (CD24+, CD44+, CD166+, EpCAM+, ALDH+). Surface Gal3 analysis identified two subpopulations within the CSC pool of LiM6 (54% Gal3+, 46% Gal3 negative). In contrast, CSC from LIM6-TR were uniformly Gal3+. Of note, not all Gal3+ colorectal cells can be attributed with CSC properties, since only 25-30% of Gal3+ CRCs express the remaining CSC markers simultaneously. Sphere assays demonstrated that the parental LIM6-TR had a 2-fold higher sphere forming ability than LIM6. The isolated Gal3+ CSCs formed 3.3-fold more spheres than parental Lim6 cells, while a 90% decrease in sphere formation was observed in the GAL3 negative CSC subpopulation. Depletion of Gal3 in LiM6-TR cells resulted in the loss of the Gal3+ CSC subpopulation, diminished sphere formation, and resensitization to TRAIL. Sphere formation of GAL+ CSCs in the presence of the Gal3 inhibitor LacNAc [5mM] led to a 60% reduction in sphere-formation. CONCLUSIONS: Our findings indicate that surface Gal3 is responsible for high sphere formation ability and TRAIL-resistance in a subpopulation of CSC. Further experiments will determine whether surface Gal3 in combination with other CSC markers can help to identify metastatic and chemoresistant CSCs in the circulation of CRC patients.

280 Molecular and Functional Characterization of Quiescent and Proliferative Cancer Stem Cells in Human Squamous Cell Carcinoma in Vivo

280 Molecular and Functional Characterization of Quiescent and Proliferative Cancer Stem Cells in Human Squamous Cell Carcinoma in Vivo

The Society of Academic and Research Surgery

Abstract The Annual Meeting of the Society of Academic and Research Surgery was held at the University of Nottingham Medical School, Nottingham, on Wednesday 4th and Thursday 5th January 2012 under the Presidency of Professor J Andrew Bradley. The Patey Prize was awarded to Miss M Mallik from the University Department of Surgery, Addenbrooke's Hospital, Cambridge Biomedical Research Centre, Cambridge, for her presentation entitled REGULATORY B CELLS INDUCE LONG-TERM ALLOGRAFT SURVIVAL IN A MOUSE MODEL OF CHRONIC REJECTION. The Norman Williams Prize for the best clinical paper was awarded to Miss S A Hosgood from University of Leicester, Department of 31s, Transplant Group for her presentation entitled PLACEBO CONTROLLED DOUBLE BLIND RANDOMISED CLINICAL TRIAL OF TRANSVERSUS ABDOMINIS PLANE BLOCK IN LIVE DONOR NEPHRECTOMY. The Kevin Burnand Prize for the best experimental/basic science paper was awarded to Mr S Buczacki from Cancer Research UK, Cambridge Research Institute, Cambridge, for his presentation entitled CHARACTERISATION OF QUIESCENT INTESTINAL STEM AND CANCER STEM CELLS. The President's Poster Prize was awarded to Mr S Awad from Division of Gastrointestinal Surgery, Nottingham University Hospitals, Queen's Medical Centre, Nottingham for his poster presentation entitled PREOPERATIVE ORAL CARBOHYDRATE LOADING IN ELECTIVE SURGERY: A META-ANALYSIS.

Cancer Stem Cells

The cancer stem cell (CSC) hypothesis has had a major effect on the fields of cancer cell biology and clinical oncology. CSCs were originally described in hematologic malignancies, and subsequently in a variety of solid tumors. Their unique biological characteristics, including self-renewal capability, stem cell signaling pathways, relative quiescence and resistance to standard chemotherapy and radiotherapy are providing researchers and clinicians with new challenges. One important outcome of this new perspective on tumors is the recognition that effective treatment approaches will need to target both the rapidly proliferating bulk tumor cells, and the quiescent CSCs, which contain the ability to reestablish the malignancy when treatment is withdrawn. The clinical laboratory will undoubtedly see an influx of new molecular and histopathological tests to augment initial diagnosis, treatment decisions, and prognostic monitoring of cancer patients related to identifying and quantifying these as CSCs.

Abstract C131: Two-step strategy for efficient killing of multiple myeloma stem cells using Auger-electrons emitting nucleoside analogue 5-Iod-4'-thio-2'-desoxyuridin (I-125-ITdU).

Abstract Background: Despite advances in treatment, multiple myeloma (MM) remains an incurable disease. Tumor relapse is at least in part due to the existence of drug resistant cancer stem cells (CSCs). A new therapeutic paradigm that effectively eradicates bulk MM cells and CSC needs to be developed. Auger-electrons emitting nucleoside analogues are attractive for nano-irradiation therapy when incorporated into the DNA. For this proposal, the quiescent CSCs need to be awaked. Although dormant hematopoietic stem cells (HSC) are mostly highly resistant to therapeutic regiments, they can be activated by pro-drug 5-FU to enter active cell cycle and to generate proliferative, therapy sensitive progenitor and mature cells. In analogy to HSC, the activation of highly resistant dormant CSC may present the crucial step for achieving long-term cure of cancers. Therefore in this study we evaluated the therapeutic potential of I-125-ITdU for targeting of multiple myeloma CSCs after priming with a potent thymidylate synthase (TS) inhibitor FdUrd, an active metabolite of 5-FU. Methods: For CSCs isolation, KMS12BM cells were incubated with CD138 and CD27 MicroBeads. The CD Marker expression was investigated by flow cytometry. For FdUrd treatment, cells were incubated with 0.1 μM FdUrd for 2 d. The expression level of aldehyde dehydrogenase (ALDH) was evaluated using Aldefluor assay by flow cytometry. Cell cycle was investigated by Nicoletti. The uptake and DNA incorporation of I-125-ITdU (50kBq/2*10∘4 cells) were assessed after 4 d using gamma counter. Apoptotic cells were identified using Annexin-V by flow cytometry. Results: The purity of isolated CSC was more than 97% (CD27+CD138−). FdUrd treatment of CD27+CD138− cells yielded a decreased ALDH activity (65.2% vs. 29.8%). Furthermore, FdUrd induced CSC to enter the cell cycle (48.5% vs. 18.7% and 29.2% vs. 47.7% of cells in G0/G1 and S phase, respectively) and increased the CD138 expression. The treatment with a non-toxic dose of FdUrd was essential for effective incorporation of ITdU and inducing of irreparable DNA damage. The activation with FdUrd increased the cellular uptake by a factor of 25. KMS12BM CSC incorporated 46.3% ± 2.8% of internalized activity into DNA. Most important, I-125-ITdU showed a potent antimyeloma effect on isolated CSC. More than 95% of treated cells were detected as apoptotic. Conclusion: This is the first report to demonstrate that DNA is a promising target for endo-radio-therapy of CSC of multiple myeloma using two-step-strategy: FdUrd for activating and Auger-radiation emitting thymidine analogue ITdU for eliminating of malign cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C131.

Extinction models for cancer stem cell therapy

Cells with stem cell-like properties are now viewed as initiating and sustaining many cancers. This suggests that cancer can be cured by driving these cancer stem cells to extinction. The problem with this strategy is that ordinary stem cells are apt to be killed in the process. This paper sets bounds on the killing differential (difference between death rates of cancer stem cells and normal stem cells) that must exist for the survival of an adequate number of normal stem cells. Our main tools are birth–death Markov chains in continuous time. In this framework, we investigate the extinction times of cancer stem cells and normal stem cells. Application of extreme value theory from mathematical statistics yields an accurate asymptotic distribution and corresponding moments for both extinction times. We compare these distributions for the two cell populations as a function of the killing rates. Perhaps a more telling comparison involves the number of normal stem cells N H at the extinction time of the cancer stem cells. Conditioning on the asymptotic time to extinction of the cancer stem cells allows us to calculate the asymptotic mean and variance of N H . The full distribution of N H can be retrieved by the finite Fourier transform and, in some parameter regimes, by an eigenfunction expansion. Finally, we discuss the impact of quiescence (the resting state) on stem cell dynamics. Quiescence can act as a sanctuary for cancer stem cells and imperils the proposed therapy. We approach the complication of quiescence via multitype branching process models and stochastic simulation. Improvements to the τ-leaping method of stochastic simulation make it a versatile tool in this context. We conclude that the proposed therapy must target quiescent cancer stem cells as well as actively dividing cancer stem cells. The current cancer models demonstrate the virtue of attacking the same quantitative questions from a variety of modeling, mathematical, and computational perspectives.

Stem cells, quiescence and rectal carcinoma: an unexplored relationship and potential therapeutic target.

Stem cells are responsible for maintaining differentiated cell numbers during normal physiology and at times of tissue stress. They have the unique capabilities of proliferation, self-renewal, clonogenicity and multi-potentiality. It is a widely held belief that stem-like cells, known as cancer stem cells (CSCs), maintain tumours. The majority of currently identified intestinal stem cell populations appear to be rapidly cycling. However, quiescent stem cell populations have been suggested to exist in both normal intestinal crypts and tumours. Quiescent CSCs may have particular significance in the modern management of colorectal cancer making their identification and characterisation a priority. In this review, we discuss the current evidence surrounding the identification and microenvironmental control of stem cell populations in intestinal crypts and tumours as well as exploring the evidence supporting the existence of a quiescent stem and CSC population in the gut and other tissues.

Stem Cell Quiescence

AbstractAdult stem cells are maintained in a quiescent state but are able to exit quiescence and rapidly expand and differentiate in response to stress. The quiescent state appears to be necessary for preserving the self-renewal of stem cells and is a critical factor in the resistance of cancer stem cells (CSCs) to chemotherapy and targeted therapies. Limited knowledge about quiescence mechanisms has prevented significant advances in targeting of drug-resistant quiescent CSCs populations in the clinic. Thus, an improved understanding of the molecular mechanisms of quiescence in adult stem cells is critical for the development of molecularly targeted therapies against quiescent CSCs in different cancers. Recent studies have provided a better understanding of the intrinsic and extrinsic regulatory mechanisms that control stem cell quiescence. It is now appreciated that the p53 gene plays a critical role in regulating stem cell quiescence. Other intrinsic regulatory mechanisms include the FoxO, HIF-1α, and NFATc1 transcription factors and signaling through ATM and mTOR. Extrinsic microenvironmental regulatory mechanisms include angiopoietin-1, TGF-β, bone morphogenic protein, thrombopoietin, N-cadherin, and integrin adhesion receptors; Wnt/β-catenin signaling; and osteopontin. In this article, we review current advances in understanding normal stem cell quiescence, their significance for CSC quiescence and drug resistance, and the potential clinical applications of these findings. Clin Cancer Res; 17(15); 4936–41. ©2011 AACR.

Quiescent stem cells in intestinal homeostasis and cancer

Adult stem cell niches are characterized by a dichotomy of cycling and quiescent stem cells: while the former are responsible for tissue turnover, their quiescent counterparts are thought to become active upon tissue injury thus underlying the regenerative response. Moreover, quiescence prevents adult stem cells from accumulating mutations thus ensuring a reservoir of unaltered stem cells. In the intestine, while cycling stem cells were shown to give rise to the main differentiated lineages, the identity of their quiescent equivalents remains to date elusive. This is of relevance for conditions such as Crohn's disease and ulcerative colitis where quiescent stem cells may underlie metaplasia and the increased cancer risk associated with chronic inflammation. Tumours are thought to share a comparable hierarchical structure of adult tissues with pluripotent and self-renewing cancer stem cells (CSCs) giving rise to more differentiated cellular types. As such, neoplastic lesions may encompass both cycling and quiescent CSCs. Because of their infrequent cycling, quiescent CSCs are refractory to chemo- and radiotherapy and are likely to play a role in tumour dissemination, dormancy and recurrence.

Abstract 2464: Targeting liver cancer stem cells for molecular imaging and therapeutic application

Abstract Conventional therapies target rapidly proliferating non-tumorigenic cells and spare the relatively quiescent cancer stem cells (CSCs) and the CSCs will remain viable after therapy and re-establish the tumor. Thus developing therapeutic strategies to target CSCs to prevent tumor recurrence will be vital for cancer therapy. To date, it has been shown that CSCs in hepatocellular carcinoma (HCC) can be identified by several cell surface antigens CD133, CD90, CD44, OV6 and the epithelial cell adhesion molecule (EpCAM). Here, we developed a novel strategy which targeted CSCs by anti-CD44 antibody mediated nanoparticles delivery system loaded with a triple fusion gene containing the suicide gene-herpes simplex virus truncated thymidine kinase (TTK), renilla luciferase (RL) and RFP (TF, RL-RFP-TTK). The in situ liver cancer model was established by injection of 1.0×105 HepG2 cells, which carry a reporter system encoding the genes of firefly luciferase (FL) and GFP into the liver of NOD/SCID mice. The mice were subsequently treated with ganciclovir (GCV). Then the growth status of tumor was monitored by the optical bioluminescence imaging of firefly luciferase(FL) and the specific targeting of the nanoparticles was tracked by imaging of renilla luciferase(RL). Our results demonstrated that the anti-CD44 antibody mediated nanoparticles loaded with TTK could specifically target the CSCs of HCC, and thereafter were endocytosed by the plasma membrane to transport the triple fusion (TF, RL-RFP-TTK) into the cells, resulted in the apoptosis of the targeted cells. Taken together, our study demonstrated a novel therapeutic strategy by targeted CSCs, we also developed a useful multimodality imaging techniques to monitor HepG2 cells’ fate in vivo and assessed the targeting efficacy of the nanoparticles. This may open a new application for the treatment of the HCC by targeting CSCs. 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 2464. doi:10.1158/1538-7445.AM2011-2464

Abstract 3317: Single-cell invasion and chemoresistance of slowly proliferating brain-tumor stem cells

Abstract Despite intense efforts in basic research and clinical medicine, glioblastoma (GBM) remains one of the most lethal types of cancer. In particular, tumor recurrence after surgical resection, aggressive chemotherapy, and targeted radiation remains an insurmountable obstacle. Recurrence may be attributed to residual cancer stem cells that re-initiate tumor growth after primary clinical intervention. Here, we provide new evidence for a subpopulation of relatively quiescent and chemoresistant cancer stem cells that invade deeply into the parenchyma. We have identified a critical transcription factor that is upregulated in these glioma stem cells, which regulates epithelial-mesenchymal transition (EMT) in other tumors. Knockdown of this transcription factor results in an increased sensitivity to chemotherapeutic agents and reduced tumor cell invasion. We hypothesize that single cell invasion of malignant gliomas is regulated by similar molecular pathways as EMT and metastasis in solid tissue tumors. These pathways allow slowly proliferating glioma stem cells to leave the site of the primary tumor, invade deeply into the surrounding parenchyma and, by virtue of their resistance to chemotherapeutic agents, evade all types of classical brain tumor therapy. This offers a potential novel model for GBM recurrence, and a potential new therapeutic target. 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 3317. doi:10.1158/1538-7445.AM2011-3317

Quiescent, Slow-Cycling Stem Cell Populations in Cancer: A Review of the Evidence and Discussion of Significance

Long-lived cancer stem cells (CSCs) with indefinite proliferative potential have been identified in multiple epithelial cancer types. These cells are likely derived from transformed adult stem cells and are thought to share many characteristics with their parental population, including a quiescent slow-cycling phenotype. Various label-retaining techniques have been used to identify normal slow cycling adult stem cell populations and offer a unique methodology to functionally identify and isolate cancer stem cells. The quiescent nature of CSCs represents an inherent mechanism that at least partially explains chemotherapy resistance and recurrence in posttherapy cancer patients. Isolating and understanding the cell cycle regulatory mechanisms of quiescent cancer cells will be a key component to creation of future therapies that better target CSCs and totally eradicate tumors. Here we review the evidence for quiescent CSC populations and explore potential cell cycle regulators that may serve as future targets for elimination of these cells.

Insights into the cell of origin in breast cancer and breast cancer stem cells

The precise cell types that give rise to tumors and mechanisms that underpin tumor heterogeneity are poorly understood. There is increasing evidence to suggest that diverse solid tumors are hierarchically organized and may be sustained by a distinct subpopulation of cancer stem cells (CSCs). The CSC hypothesis provides an attractive cellular mechanism that can account for the therapeutic refractoriness and dormant behavior exhibited by many tumor types. Breast cancer was the first solid malignancy from which CSCs were identified and isolated. Direct evidence for the CSC hypothesis has also recently emerged from mouse models of mammary tumorigenesis, although alternative models to explain heterogeneity also seem to apply. Our group has found that the luminal epithelial progenitor marker CD61/beta3 integrin identified a CSC population in mammary tumors from MMTV-wnt-1 mice. However, no CSCs could be identified in the more homogeneous MMTV-neu/erbB2 model, suggesting an alternate (clonal evolution or stochastic) model of tumorigenesis. It seems likely that both paradigms of tumor propagation exist in human cancer. From a clinical perspective, the CSC concept has significant implications. Quiescent CSCs are thought to be more resistant to chemotherapy and targeted therapy. Enrichment of putative CSCs has been noted in studies of chemotherapy-treated patients, lending support to the CSC hypothesis and their potential role in chemoresistance. Although many unresolved questions on CSCs remain, ongoing efforts to identify and characterize CSCs continue to be an important area of investigation, with the potential to identify novel tumor targeting strategies.

Cancer Stem Cells and Aneuploid Populations within Developing Tumors Are the Major Determinants of Tumor Dormancy

Tumor formation involves substantial cell division and genetic instability, but the relationship between quiescent cancer stem cells (CSC) and dividing progenitors in these events is poorly understood. Likewise, the implication of aneuploid cells in solid tumors is uncertain. CSCs are postulated to contribute to tumor dormancy and present a formidable obstacle in limiting treatment outcomes for a majority of cancers, whereas the genetic heterogeneity conjured by aneuploid cells may influence tumor drug resistance. However, direct confirmation of these events remains forthcoming. In the present study, we addressed the identification of tumor dormancy in terms of isolation of therapy-refractory residual tumor cells from tumors that persist in a state of quiescence as label-retaining cells. The choices of label were PKH67/PKH26 dyes that irreversibly bind to the lipid bilayer on cell membranes and get equally partitioned among daughter cells subsequent to each cell division. Consequent characterization revealed that label-retaining cells encompass two different populations capable of remaining in a state of quiescence, i.e., stem-like cells and aneuploid cells. The former express a reversibility of quiescence through retention of functionality and also exhibit therapeutic refractoriness; the latter seem to be either quiescent or proliferation-arrested at steady-state. Subsequent to exposure to selective pressure of chemotherapy, a fraction of these cells may acquire the potential to proliferate in a drug-refractory manner and acquire stem-like characteristics. Collectively, the findings of the present study reveal that tumor-derived CSCs and aneuploid populations contribute to drug resistance and tumor dormancy in cancer progression.

Preclinical development of cancer stem cell drugs

Background: Despite recent progress in cancer treatment, the current cancer chemotherapy can mainly produce remission but often fails to cure cancer due to the existence of cancer stem cells. The emerging cancer stem cell hypothesis offers new insight into the failure of current cancer drugs and suggests new approaches for improved understanding of cancer biology and cancer drug development. Objective: In this review, we discuss the concept of cancer stem cells, origin of cancer stem cells and different approaches for isolating or enriching cancer stem cells. We also review the resistance of cancer stem cells to standard chemotherapy and radiation therapy and potential mechanisms for the resistance. Finally, based on the current knowledge on cancer stem cells, we discuss potential approaches for developing new drugs that target cancer stem cells and propose new methods for evaluation of cancer stem cell drugs. Conclusion: Improved cancer treatment is likely to be achieved by a combination of drugs that kill both replicating cancer cells and more quiescent cancer stem cells.

P53 Regulates Hematopoietic Stem Cell Quiescence

The importance of the p53 protein in the cellular response to DNA damage is well known, but its function during steady-state hematopoiesis has not been established. We have defined a critical role of p53 in regulating hematopoietic stem cell quiescence, especially in promoting the enhanced quiescence seen in HSCs that lack the MEF/ELF4 transcription factor. Transcription profiling of HSCs isolated from wild-type and p53 null mice identified Gfi-1 and Necdin as p53 target genes, and using lentiviral vectors to upregulate or knockdown the expression of these genes, we show their importance in regulating HSC quiescence. Establishing the role of p53 (and its target genes) in controlling the cell-cycle entry of HSCs may lead to therapeutic strategies capable of eliminating quiescent cancer (stem) cells.

Cancer Stem Cells: A Step Toward the Cure

This special issue of Journal of Clinical Oncology is devoted to the emerging field of cancer stem cells (CSCs). The goal of this article is to introduce general concepts of CSC biology to clinicians, and to provide a framework for their understanding the CSC-based approach to the development of novel diagnostics, therapeutics, and prevention strategies in oncology. This new approach differs from the traditional one because the latter is based on the long-held concept that tumor formation and growth are due to increased proliferation of cells in cancers compared with normal tissues. This traditional view is supported by molecular oncology research during the last 50 years, which showed that tumors undergo a series of genetic events (mutations) that result in the activation or overexpression of genes promoting proliferation (eg, oncogenes), the silencing of genes involved in inhibition of proliferation (eg, tumor suppressor genes), and the development of the ability of cancer cells to elude apoptosis. The consequence is the development and unchecked growth of tumors and their progression to metastases. Based on this traditional concept of tumor growth, it has been disappointing that therapies designed to kill proliferating cells often fail to cure cancer patients. Furthermore, during tumor development, tissues accumulate a series of mutations over years or even decades, but, because of tissue renewal, most cells in tissues are lost or eliminated in just a brief time, typically days or weeks, and with their loss, any mutations they have acquired would also be lost. Thus, new mechanisms need to be considered to explain human tumorigenesis. One explanation is that tumorigenic mutations occur in cells that are few in number but reside long term in tissues. But that is not sufficient to explain tumorigenesis. To be truly tumorigenic, this population of rare, mutated cells would have to self-renew, clonally expand, and acquire additional mutations. It is now widely believed that these long-lived, uncommon cells are tissue stem cells (SCs) or cells derived from them that acquire the ability to self-renew. When mutated, they can become CSCs. Both normal SCs and CSCs may account for only a small fraction of cells ( 1%) in any given tissue or tumor. By definition, SCs are self-renewing. Mounting evidence presented in the articles in this special issue suggests that CSCs initiate and drive tumor growth. This is nothing less than a paradigm shift. It is a shift to the view that, first, cancers originate in tissue stem or progenitor cells through dysregulation of the self-renewal process. Second, throughout tumorigenesis, CSCs drive tumor growth. Third, current chemotherapeutic agents and radiation therapy largely target proliferating and differentiated cells that form the bulk of the tumor but not the relatively quiescent CSCs. Also, SCs are drug resistant and CSCs appear to be so too. Thus, this quiescence and resistance may account for many treatment failures. Fourth, if this is the case, then the only effective way to treat cancer is to target the CSC population. Although this view may seem novel, the basic idea that SCs are the cells of clonal origin of malignancies has existed for at least several decades, if not longer, particularly for leukemias and teratomas. Early attempts—beginning in the 1970s—to directly measure SCs in tumors used several approaches. One was mousespleen colony assays (eg, injection of lymphoma cells into mice with the appearance of lymphoma colonies in the spleen). A second was the counting of metastatic lung colonies after intravenous inoculation of tumor cells into mice. A third was tumor colony formation in vitro. However, this latter method produced mixed results for human tumors. SCs have also been known for decades to radiation biologists. More recent evidence, as presented in the articles in this special issue, supports an SC origin for solid tumors as well. In the following paragraphs, we discuss the properties of normal SCs and CSCs, and their possible roles in carcinogenesis and in mediating tumor behavior.

Targeting the Untargetable: Oncolytic Virotherapy for the Cancer Stem Cell

The biological and therapeutic importance of cancer stem cells has become increasingly clear over the past several years.1 The success or failure of cancer treatment approaches may be influenced greatly by the presence and treatment sensitivity of these cells. Cancer cures may therefore require effective targeting and destruction of the cancer stem cell population. For therapeutics to be effective against such cells, they must be effective against quiescent, apoptosis-resistant, and drug transporter–overexpressing cells. Further complicating this challenge, normal stem cells share many of these same features, to the extent that selectively targeting the cancer stem cell while leaving the normal stem cells unharmed is a difficult proposition. In this issue of Molecular Therapy, Eriksson and colleagues report proof-of-concept studies for the efficacy of oncolytic virotherapy against cancer stem cells.2