Abstract
The cancer stem cell paradigm is the idea that subpopulations of cancer cells exist within a tumor that are capable of self-renewal, multipotency, proliferation, and tumor maintenance. This subpopulation of cells is believed to be responsible for tumor cell treatment resistance and tumor recurrence. In neuroblastoma, the phenotypic characteristics identifying cancer stem cells include expression of certain cell surface markers, expression of certain cytoplasmic and nuclear proteins, efflux of Hoechst 33,342 dye, and the ability to grow as spheres in culture. These cells reside in specialized microenvironments which are composed of non-tumor host cells, immune cells, tumor stromal cells, the extracellular matrix, and cell adhesion molecules. The identification of neuroblastoma cancer stem cell populations and the contribution of the various elements of the tumor microenvironment that support these cells is discussed in this chapter.
Keywords
Chemoresistance, Microenvironment, Stem cells
Cancer Stem Cells
The cancer stem cell (CSC) paradigm is the idea that subpopulations of cancer cells exist within a tumor that has capabilities similar to a normal stem cell: self-renewal, multipotency, proliferation, and tumor maintenance . This concept that tumors may develop from stem cells is not a new one. The embryonal rest theory from the early 19th century stated that embryonic tissues persisting in adults could be induced to proliferate, leading to an abnormal growth of cells . It was not until the 1960s that tritium-labeling studies allowed researchers to study leukemia and find a primitive-appearing subset of cells with unique cell cycle properties . Around the same time, researchers examining malignant teratoma found that its development was driven by primitive cells resembling stem cells . In the mid-1990s, more data in leukemia showed that the subpopulation of immature cells with expression of CD34 + CD38 − cell surface markers was tumorigenic when implanted into immunosuppressed mice, whereas the subpopulations lacking that cell surface marker combination were not tumorigenic, further supporting the idea that a fraction of cancer cells might have properties similar to stem cells that allowed tumor initiation . In the same year, the concept of CSCs was translated to solid tumors including teratoma, hepatocellular carcinoma, gastrointestinal adenocarcinomas, breast cancer, prostate cancer, and lung cancer . Since then, researchers have identified tumor-initiating cells and CSCs in many types of solid tumors including breast, prostate, colon, liver, lung, and pancreas . More recently, researchers have discovered the presence of stem cells in neuroblastoma . CSCs are believed to play a key role in tumor initiation, maintenance, recurrence, and therapy resistance . Therefore, therapeutics designed to target this cell population may lead to more effective treatments for multiple aspects of neuroblastoma.
Heterogeneity and CSCs in Neuroblastoma
Like many other cancers, neuroblastoma is a heterogeneous tumor. Given its origin from the neural crest, any cell phenotype derived from the neural crest may be within a given tumor including neuroblasts, glial cells, chondrocytes, and even melanocytes. Three distinct phenotypic groups of cells—N-, S-, and I-type cells—have been identified . N-type cells, or sympathoadrenal neuroblasts, are tumorigenic and can be induced to differentiate or de-differentiate. S-type cells are nontumorigenic, nonneuronal cells that are flattened in appearance and adhere readily to a substrate. The I-type cell has an intermediate morphology with short neurite-like processes and tumorigenesis resembling N-type cells but strong adhesion to a substrate similar to S-type cells. These I-type cells are capable of self-renewal and may become either N- or S-type cells, leading researchers to believe that the I-type cells are the neuroblastoma CSCs . These cells are readily tumorigenic; even I-type cells without MYCN amplification are more tumorigenic than MYCN-amplified N-type cells .
Identification of CSCs in Neuroblastoma
A hallmark of the CSC paradigm is that CSCs can be identified by phenotypic characteristics. In neuroblastoma, these include expression of certain cell surface markers associated with tissue-specific stem cells, expression of certain cytoplasmic and nuclear proteins, efflux of Hoechst 33,342 dye, and the ability to grow as spheres in culture.
Cell-Surface Markers
Multiple cell surface proteins have been proposed as markers for neuroblastoma CSCs ( Table 11.1 ). CD133 or prominin-1 is one such protein that has been found to be a marker for CSCs in a variety of cancers and is expressed in hematopoietic and central nervous system stem cells . The normal function and purpose of CD133 remain unclear, although it appears to play a role in early retinal development—a mutation in an Indian family yielding a truncated CD133 protein has caused retinal degeneration and mice lacking CD133 have photoreceptor degeneration and blindness . The malignant neuroblastoma cells with an I-type phenotype have been demonstrated to express significantly higher amounts of CD133 mRNA compared to N- and S-type cells, providing further evidence that the I-type cells are the CSCs in neuroblastoma . Isolation of neuroblastoma cells expressing CD133 exhibited CSC properties . Additionally, CD133 knockdown in neuroblastoma cells decreased sphere formation, one of the hallmarks of CSCs, and induced differentiation, indicating that CD133 plays a role in neuroblastoma stemness . Clinically, the CD133 expression is associated with worse outcomes in patients with neuroblastoma .
| Cellular Localization | CSC Marker |
|---|---|
| Cell surface |
|
| Intracellular |
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| Other |
|
CD114, or granulocyte colony stimulating factor receptor, is another surface marker for CSCs in neuroblastoma. Cells expressing CD114 are highly tumorigenic, self-renewing, and their progenies are more differentiated . Their gene expression is similar to that of early neural crest cells, whereas neuroblastoma cells lacking CD114 expression have gene expression patterns more similar to differentiated migratory crest cells. The population that expresses CD114 is distinct from that which expresses CD133.
CD117, or c-kit or mast/stem-cell growth factor receptor, is another putative cell surface marker for neuroblastoma CSCs. CD117 mRNA is abundant in I-type neuroblastoma cells, which are believed to represent CSCs . Additionally, CD117 has been demonstrated to be present in neuroblastoma tumor specimens .
Neuroblastoma tumor specimens have also been shown to express ATP-binding cassette (ABC) subfamily G member 2, or ABCG2, which is a protein that transports various molecules across membranes . Given its function and location on the cell surface, ABCG2 plays a role in drug efflux and chemoresistance. Sphere-forming neuroblastoma cells believed to be CSCs, express ABCG2 . Additionally, side-population neuroblastoma cells also believed to be CSCs, express ABCG2 .
Frizzled receptor 6 (FZD6) is a transmembrane Wnt receptor that predicts poor survival in neuroblastoma. FZD6-positive neuroblastoma cells form spheres in culture, have increased invasive ability, and are resistant to chemotherapeutics, which are all indicators that FZD6 is a marker of CSCs .
Intracellular Proteins
Some intracellular proteins have also been deemed putative markers of CSCs in neuroblastoma. Nestin is an intermediate filament protein that is present in neural stem cells during normal development. Nestin has been demonstrated to be expressed in multiple types of malignancies and coexpressed with other CSC markers including CD133 and ABCG2 .
The aldehyde dehydrogenase (ALDH) enzyme has been implicated as a stem cell marker in multiple cancers, including neuroblastoma. ALDH activity is associated with poor survival, high-risk prognostic factors, and drug resistance in neuroblastoma . Additionally, ALDH expression was found to be associated with growth and dedifferentiation of neuroblastoma in vivo .
Hoechst/Side Population
A technique initially optimized for hematopoietic cells, the Hoechst side population analysis has been utilized to identify CSCs in neuroblastoma . Hoechst 33,342 DNA-binding dye is passively taken up by neuroblastoma cells, and the CSCs are capable of dye efflux via the ATP-Binding Cassette (ABC) transporters. Those cells lacking Hoechst dye may be isolated using fluorescence-activated cell sorting to yield a side population fraction, which is believed to be CSCs. The size, proliferation, and colony-forming efficiency of the side population fraction in relapsed neuroblastoma cell lines is increased compared to that prior to treatment, indicating the role of side population cells in tumor recurrence .
Microenvironment
CSCs reside in niches, specialized microenvironments that support CSC stemness through cell-to-cell and paracrine interactions. The CSC niche is part of the overall tumor microenvironment and is characterized by infiltrating immune cells, extracellular matrix (ECM) remodeling, and hypoxia . While the mechanisms by which these characteristics support and initiate CSCs have yet to be fully elucidated, some of the known interactions of CSCs with the neuroblastoma tumor microenvironment will be discussed in the following sections. Such interactions may provide future targets for neuroblastoma therapy .
Tumor-Associated Macrophages (TAMs) and Cancer-Associated Fibroblasts (CAFs)
It is well known that the tumor microenvironment includes nontumor host cells known as tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) . Monocytes differentiate into two classes of macrophages: M1 and M2. M1 macrophages stimulate the immune system providing defense against microorganisms and neoplastic cells. M2 macrophages promote angiogenesis, debris removal, and wound repair. M2 macrophages also down-regulate M1-mediated inflammatory responses . In the tumor microenvironment, tumor cells recruit monocytes via CCL2, M-CSF, and VEGF. Further signaling by factors such as IL-3 then induce monocyte differentiation into TAMs, which are believed to resemble M2 macrophages that promote tumor cell growth and progression . TAM cell markers include CD68, CD163, and CD204 while alpha smooth muscle actin is a marker of CAFs .
Patient-derived xenograft models of metastatic neuroblastoma have recapitulated the tumor microenvironment with rich vascularization, pericyte coverage, TAMs, CAFs, and ECM components along with CD45 + lymphoid cells and lymphatic vessels . Immunohistochemical analysis of neuroblastoma tumor samples demonstrated TAMs and CAFs occurrence in the same vicinity. Coculturing of tumor cells with TAMs led to an increase in tumor cell invasiveness, while co-culturing of tumor cells with CAFs increased tumor cell proliferation. The number of TAMs and CAFs have been shown to correlate with the clinical stage, MYCN amplification, bone marrow metastasis, histological classification, and risk classification . Tissue microarray analysis of neuroblastoma samples also demonstrated a higher degree of TAMs in metastatic disease and an association with clinical stage. Non-MYCN amplified neuroblastoma samples from children greater than 18 months of age demonstrated higher expression of inflammation-related genes such as CD14, CD16, CD33, IL-6R, and IL-10 , when compared to those children diagnosed at less than 18 months of age, suggesting that TAMs might contribute to the diversity in outcomes . CAFs were found to be significantly upregulated in Schwannian stroma-poor neuroblastoma samples versus Schwannian stroma-rich samples and were also associated with microvascular proliferation .
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