Cancer Stem Cell Hierarchy Research Articles

Novel Strategy for Lineage Tracing of Cancer Stem Cells

The cancer stem cell (CSC) theory hypothesizes a subset of the tumorigenic cells able to self‐renew and wholly reconstitute original tumors by lineage restriction. We developed a lentiviral barcoding system as a means of genetic lineage tracing of cell subpopulations sorted by surface marker expression to investigate the proliferation kinetics, plasticity and clonality of tumor‐initiating cells (TICs). The abundance of each barcode in the reconstituted tumor reflects the frequency of TIC progeny in individual cell fractions. We determined that the CD29H/CD24H subfraction consistently out‐competes other subfractions while forming populations of different immune‐profiles, indicating that this subfraction retains the two cardinal features of self‐renewal and ability to give rise to progeny through lineage restriction ascribed to TICs, and suggests that not a lot of plasticity happens between non‐TICs and TICs. Also, iPCR was successfully applied into the lentiviral barcoding system, enabling the analysis of the TIC's clonality over serial transplant passages to understand the CSC's hierarchy. This novel lentiviral barcoding strategy provides future framework for defining specific biomarkers and evaluating therapeutics directed towards CSCs. This lineage tracing methodology should be applicable not only to genetically engineered mouse models but also to primary human breast cancer xenografts.

Abstract 3476: Hyaluronan-mediated motility receptor as a novel target for inhibiting glioblastoma stem cells

Abstract Introduction: Glioblastoma stem cells (GSCs) are a subpopulation of tumor cells that promote tumor invasion and angiogenesis, therapeutic resistance and tumor recurrence. Here, we study the function of hyaluronan-mediated motility receptor (HMMR) in human GSCs. HMMR is hyper-expressed in human glioblastoma samples relative to lower-grade glioma and non-neoplastic specimens. Hyper-expression of HMMR also correlates with poor survival in glioma patients. HMMR is a multifunctional oncogenic protein that plays essential roles in tumorgenesis. Extracellular HMMR forms a complex with CD44 that upon binding to hyaluronan activates intracellular signaling pathways that regulate tumor cell survival, proliferation and invasion. Intracellular HMMR associates with microtubules, interacts with the mitotic spindle and contributes to tumor progression by promoting genomic instability. HMMR and CD44 are two ubiquitous receptors for hyaluronan, which is a prominent component of the microenvironment in most malignant tumors. CD44 has been identified as a cancer stem cell marker and CD44 directly regulates cancer stem cells in a variety of cancers including glioblastoma. Critical evidence regarding the function of HMMR in actual glioblastoma tumors and in the context of tumor-initiating GSCs is still lacking. Results: We found that HMMR is ubiquitously expressed in a panel of human glioblastoma-derived neurosphere cultures that are enriched for GSCs. Targeting HMMR by lentivirus-mediated shRNA delivery potently disrupted in vitro self-renewal of GSCs and inhibited the expression of key GSC markers, such as CD133, SOX2 and Olig2. HMMR knockdown also blocked the in vivo tumorigenic potential of GSCs in mouse orthotopic xenograft model. Notably, disruption of HMMR in GSCs attenuated the activity of Notch signaling, which supports the cancer stem cell hierarchy in glioblastoma and various other cancer types. Significance: This research identifies HMMR as a novel therapeutic target in the treatment of glioblastoma by depleting the tumor-initiating GSCs. It also introduces a novel hypothesis regarding the mechanistic crosstalk between HMMR-mediated signaling and Notch signaling pathway. Understanding this crosstalk between HMMR and Notch signaling will have a significant impact on current efforts to target Notch driven mechanisms for GBM therapy. Overall, these studies will lay an essential foundation for the development of HMMR-targeting strategies, such as HMMR inhibitors or monoclonal antibodies, as new therapeutic approaches targeting glioblastoma. 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 3476. doi:1538-7445.AM2012-3476

Molecular pathology of tumor‐initiating cells: Lessons from Philadelphia chromosome‐positive leukemia

Recent improvements in cell purification and transplantation techniques have contributed to the identification of cell populations known as tumor-initiating cells (TIC). This discovery has led to the 'cancer stem cell hierarchy' concept, which holds that tumors are organized as a hierarchy of malignant tissues sustained by such TIC. However, this concept remains controversial. In this review, we examine recent advances in cancer stem cell research that have been generated from studies of Philadelphia (Ph) chromosome-positive leukemia. The abnormal Ph chromosome, which arises from a translocation creating the BCR-ABL1 fusion gene, is most commonly associated with chronic myelogenous leukemia (CML) and precursor B cell acute lymphoblastic leukemia (B-ALL). Examination of the pathophysiology of these diseases has provided interesting insights into not only the hierarchy of leukemia stem cells but also their clonal evolution. Both shared and unique regulatory mechanisms affecting normal and CML stem cell behavior have been identified. On the other hand, genetic diversity in specific clones of Ph(+) B-ALL that drive clonal evolution has recently come to light. Our expanding knowledge of the biology of TIC contributes to the progress of cancer research, and may open the door to new concepts in cancer therapy.

Cancer stem cell subsets and their relationships

Emerging evidence suggests that cancer stem cells account for the initiation and progression of cancer. While many types of cancer stem cells with specific markers have been isolated and identified, a variety of differences among them began to be appreciated. Cancer stem cells are hierarchical populations that consist of precancerous stem cells, primary cancer stem cells, migrating cancer stem cells and chemoradioresistant cancer stem cells, playing different roles in cancer initiation and progression. Here we propose a new concept "horizontal hierarchy of cancer stem cells" to distinguish them from vertical hierarchy cancer stem cells, cancer transient-amplifying cells and cancer differentiated cells, and summarize our current understanding of these subsets of cancer stem cells with the aim to open up novel therapeutic strategies for cancer based on this understanding.

Abstract 2694: DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked is a CD133+ tumor-specific protein and induces antitumor immunity

Abstract Cancer cells that exclusively maintain the ability of self-renewal and differentiation are termed cancer stem cells (CSCs). It is still controversial if the classical CSC hierarchy exists in all of solid tumors, however, accumulating evidence suggests that heterogeneity within cancer cells exists and that cancer survives as the cells with CSC features during potentially lethal stresses, including chemotherapy, radiation treatment, and molecular targeting therapy. Although it is necessary to eradicate CSCs to obtain cure of cancer, effective treatment has not been elucidated. Interestingly, most of the identified immunogenic tumor antigens are cancer/testis (CT) antigens. One of the reasons why CT antigens are immunogenic is that they are unlikely to maintain peripheral tolerance, owing to restricted expression in the testis and in immortal malignant cells, because Tregs are maintained with antigen stimulation by dendritic cells (DCs) that acquire dying cells in the steady state. Since CSCs are highly immortal, it is possible that they possess immunogenic antigens that are not expressed in differentiated cancer cells or normal epithelial cells, and that these antigens may be ideal therapeutic targets for cancer treatment. Recently it was reported that CT antigens are mainly expressed in CSCs. We isolated CD133+ tumor cells, which possessed CSC properties, from B16 melanoma cells. We found that CSC-specific LN T cells primed by the CD133+ tumor vaccine mediated potent antitumor therapeutic efficacy by eradicating CSCs in tumor, thereby curing parental melanomas that comprised <1% CSCs. Interestingly, CD133+ tumor antigens tended to prime type 17 helper T (Th17) cells and Th1 cells, but not Th2 cells. Our proteome analyses revealed that DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked (DDX3X) is one of CD133+ melanoma-specific proteins. B16 tumor cells didn't lose CD133 expression by DDX3X knock down but lost the ability to proliferate in an anchoring independent manner. We examined DDX3X expression in 6 human cancer cell lines. HCT116 (colon cancer), and 87.5 (small cell lung cancer), were CD133+ and S2 (small cell lung cancer), A549 (lung cancer), PC9 (lung cancer), and WM115 (melanoma) were CD133-. HCT116 and 87.5 expressed DDX3X but all of other CD133- cancer cells didn't. The LN T cells draining DDX3X vaccines exhibited specific IFNγ and IL-17 release upon CD133+ tumor stimulation. A DDX3X vaccination induced antitumor protective immunity against parental melanoma. These results indicate that anti-CSC, especially anti-DDX3X, immunotherapy is a promising treatment option in the clinical setting. 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 2694. doi:10.1158/1538-7445.AM2011-2694

Microvesicles as mediators of intercellular communication in cancer—the emerging science of cellular ‘debris’

Cancer cells emit a heterogeneous mixture of vesicular, organelle-like structures (microvesicles, MVs) into their surroundings including blood and body fluids. MVs are generated via diverse biological mechanisms triggered by pathways involved in oncogenic transformation, microenvironmental stimulation, cellular activation, stress, or death. Vesiculation events occur either at the plasma membrane (ectosomes, shed vesicles) or within endosomal structures (exosomes). MVs are increasingly recognized as mediators of intercellular communication due to their capacity to merge with and transfer a repertoire of bioactive molecular content (cargo) to recipient cells. Such processes may occur both locally and systemically, contributing to the formation of microenvironmental fields and niches. The bioactive cargo of MVs may include growth factors and their receptors, proteases, adhesion molecules, signalling molecules, as well as DNA, mRNA, and microRNA (miRs) sequences. Tumour cells emit large quantities of MVs containing procoagulant, growth regulatory and oncogenic cargo (oncosomes), which can be transferred throughout the cancer cell population and to non-transformed stromal cells, endothelial cells and possibly to the inflammatory infiltrates (oncogenic field effect). These events likely impact tumour invasion, angiogenesis, metastasis, drug resistance, and cancer stem cell hierarchy. Ongoing studies explore the molecular mechanisms and mediators of MV-based intercellular communication (cancer vesiculome) with the hope of using this information as a possible source of therapeutic targets and disease biomarkers in cancer.

Cancer Stem Cell Tumor Model Reveals Invasive Morphology and Increased Phenotypical Heterogeneity

The recently developed concept of cancer stem cells (CSC) sheds new light on various aspects of tumor growth and progression. Here, we present a mathematical model of malignancies to investigate how a hierarchical organized cancer cell population affects the fundamental properties of solid malignancies. We establish that tumors modeled in a CSC context more faithfully resemble human malignancies and show invasive behavior, whereas tumors without a CSC hierarchy do not. These findings are corroborated by in vitro studies. In addition, we provide evidence that the CSC model is accompanied by highly altered evolutionary dynamics compared with the ones predicted to exist in a stochastic, nonhierarchical tumor model. Our main findings indicate that the CSC model allows for significantly higher tumor heterogeneity, which may affect therapy resistance. Moreover, we show that therapy which fails to target the CSC population is not only unsuccessful in curing the patient, but also promotes malignant features in the recurring tumor. These include rapid expansion, increased invasion, and enhanced heterogeneity.

Maintenance of HCT116 colon cancer cell line conforms to a stochastic model but not a cancer stem cell model

The cancer stem cell (CSC) model, in which a small population of cells within a tumor possesses the ability to self-renew and reconstitute the phenotype of primary tumor, has gained wide acceptance based on evidence over the past decade. It has also been reported that cancer cell lines contain a CSC subpopulation. However, phenotypic differences between CSCs and non-CSCs in cancer cell lines are not better defined than in primary tumors. Furthermore, some cell lines do not have a CSC population, revealed as a side population and expression of CD133. Thus, the identification of CSCs in cancer cell lines remains elusive. Here, we investigated the CSC hierarchy within HCT116 colon cancer cells, which do not have a CD133-positive subpopulation. We examined the expression of alternative CSC markers epithelial specific antigen (ESA) and CD44 in floating-sphere-derived cells, which are known to be the cells of enriching CSCs. Sphere-derived HCT116 cells exhibited heterogeneous expression of ESA and CD44. The two major subpopulations of HCT116 sphere cells (ESA(low)CD44(-/low) and ESA(high)CD44(high)) exhibited a biological/proliferative hierarchy of sphere-forming and soft agar colony-forming activity. However, there was no difference between the two subpopulations in the incidence of xenograft tumors. When ESA(low)CD44(-/low) cells were allowed to aggregate and re-form floating-spheres, the biological/proliferative hierarchy of parental HCT116 spheres was reconstituted, in terms of ESA and CD44 expression. Thus, HCT116 cells have plasticity when they are set in floating-spheres, suggesting that maintenance of the HCT116 cell line conforms to a stochastic model, not a CSC model.

Multipotent CD15+ Cancer Stem Cells in Patched-1–Deficient Mouse Medulloblastoma

Subpopulations of tumorigenic cells have been identified in many human tumors, although these cells may not be very rare in some types of cancer. Here, we report that medulloblastomas arising from Patched-1-deficient mice contain a subpopulation of cells that show a neural precursor phenotype, clonogenic and multilineage differentiation capacity, activated Hedgehog signaling, wild-type Patched-1 expression, and the ability to initiate tumors following allogeneic orthotopic transplantation. The normal neural stem cell surface antigen CD15 enriches for the in vitro proliferative and in vivo tumorigenic potential from uncultured medulloblastomas, supporting the existence of a cancer stem cell hierarchy in this clinically relevant mouse model of cancer.