Imaging and Analysis of 3D Tumor Spheroids Enriched for a Cancer Stem Cell Phenotype

The Department of Experimental Therapeutics, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Journal of Biomolecular Screening (Impact Factor: 2.42). 08/2010; 15(7):820-9. DOI: 10.1177/1087057110376541
Source: PubMed


Tumors that display a highly metastatic phenotype contain subpopulations of cells that display characteristics similar to embryonic stem cells. These cells exhibit the ability to undergo self-renewal; slowly replicate to retain a nucleoside analog label, leading to their definition as "label-retaining cells"; express specific surface markers such as CD44(+)/CD24(-/low) and CD133; and can give rise to cells of different lineages (i.e., they exhibit multipotency). Based on these characteristics, as well as their demonstrated ability to give rise to tumors in vivo, these cells have been defined as tumor-initiating cells (TICs), tumor-propagating cells, or cancer stem cells (CSCs). These cells are highly resistant to chemotherapeutic agents and radiation and are believed to be responsible for the development of both primary tumors and metastatic lesions at sites distant from the primary tumor. Established cancer cell lines contain CSCs, which can be propagated in vitro using defined conditions, to form 3D tumor spheroids. Because the vast majority of studies to identify cancer-associated genes and therapeutic targets use adherent cells grown in 2 dimensions on a plastic substrate, the multicellular composition of these 3D tumor spheroids presents both challenges and opportunities for their imaging and characterization. The authors describe approaches to image and analyze the properties of CSCs within 3D tumor spheroids, which can serve as the basis for defining the gene and protein signatures of CSCs and to develop therapeutic strategies that will effectively target this critically important population of cells that may be responsible for tumor progression.

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    • "A challenge has been encountered with regard to the enrichment of CSCs from the established cell lines of a variety of solid tumors that develop as three-dimensional (3D) cell cultures. The 3D spheroid model is a new technique for the propagation of cells in vitro using serum-free medium and cultured under low-adherence conditions (25). An additional usage of spheroids constitutes the liquid overlay technique, namely multicellular tumor spheroids (26) "
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    ABSTRACT: Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133(high)/CD44(high) DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133(high)/CD44(high) population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 α1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.
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    • "Another group reported that CD44+ and aldehyde dehydrogenase positive (ALDH+) cells had enriched self-renewal and tumorigenic capacity,37 whereas two other reports agree that ALDH defines a population of ovarian cancer stem cells that acquire more tumorigenic capacity if concurrently expressing CD133 (ie, ALDH+/CD133+).38,39 According to the cancer-stem cell theory, these ovarian cancer progenitor cells also depict reduced sensitivity to platinum-taxane therapy.33,36–44 The cancer-initiating cell enrichment might be even higher after chemotherapy in the hypoxic conditions normally found within solid tumors.45 "
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    • "It is more likely that the quiescent inner cells express these " stemness " genes, considering that embryonic stem cells are low-proliferating. Some novel techniques, such as the use of a thermoreversible CyGEL™ reagent that stabilizes live 3D tumor spheroids (Robertson et al., 2010), may help to locate the region highly expressing iPS-related genes. "
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    ABSTRACT: We aimed to perform a preliminary study of the association between induced pluripotent stem cell (iPS)-related genes and biological behavior of human colorectal cancer (CRC) cells, and the potential for developing anti-cancer drugs targeting these genes. We used real-time reverse transcriptase polymerase chain reaction (RT-PCR) to evaluate the transcript levels of iPS-related genes NANOG, OCT4, SOX2, C-MYC and KLF4 in CRC cell lines and cancer stem cells (CSCs)-enriched tumor spheres. NANOG was knockdowned in CRC cell line SW620 by lentiviral transduction. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, plate colony formation, and a mouse xenograft model were used to evaluate alterations in biological behavior in NANOG-knockdown SW620 cells. Also, mock-knockdown and NANOG-knockdown cells were treated with 5-fluorouracil (5-FU) and survival rate was measured by MTT assay to evaluate drug sensitivity. A significant difference in the transcript levels of iPS-related genes between tumor spheres and their parental bulky cells was observed. NANOG knockdown suppressed proliferation, colony formation, and in vivo tumorigenicity but increased the sensitivity to 5-FU of SW620 cells. 5-FU treatment greatly inhibited the expression of the major stemness-associated genes NANOG, OCT4, and SOX2. These results collectively suggest an overlap between iPS-related genes and CSCs in CRC. Quenching a certain gene NANOG may truncate the aggressiveness of CRC cells.
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