Self-Renewal and CSCs In Vitro Enrichment: Growth as Floating Spheres

Abstract

Cancer stem cells (CSC) are a vital component to the progression and reoccurrence of cancers, making them a primary target of study for both fundamental understanding of cancer biology and the development of effective and targeted treatments. CSCs reside in a complex 3D microenvironment, and the 3D spheroids are an indispensable tool in tumor biology due to their 3D structure and replication of the tumor microenvironment. Within this chapter the methodology for CSC isolation, suspension culture in hanging drop model, and characterization assays for CSC are described. First, the methodology for identifying and isolating CSCs from patient tumors, ascites, or cancer cell lines is described through the use of FACS analysis. Next, a detailed description of 3D hanging drop model for generating CSC spheroids is provided, followed by maintenance and monitoring techniques for extended 3D culture. Analysis methods are described for the quantification of CSC spheroid proliferation and viability tracking, throughout culture by on-plate alamarBlue fluorescence. Additional viability assays are described utilizing confocal microscopy with Live/Dead Viability/Cytotoxicity Kit. The characterization of CSCs populations within spheroids is described through FACS analysis. Further, an immunohistochemistry procedure is described for cell-cell and cell-matrix interaction assessment. Finally, several notes and tips for successful experiments with 3D CSC spheroids on the hanging drop model are provided. These methods are not only applicable to CSCs within a variety of tumor cell types, for not only understanding the fundamental tumor biology, but also for drug screening and development of preclinical chemotherapeutic strategies.

A novel model of liver cancer stem cells developed from induced pluripotent stem cells

“…4c), while the GFPnegative fibroblast-like cells could not survive in non-adhesive condition of hanging drop, which would provide a small number of cells such as CSCs with a three-dimensional isolated suspension environment where they were maintained as spheroids. 21 miPS-Huh7cmP1 cells showed significantly high spheroid-forming potential when compared to miPS-Huh7cm cells or Huh7 cells. Also, the sphere size was larger in miPS-Huh7cm P1 when compared to miPS-Huh7cm as well as Huh7 cells (Fig.…”

Review: Mechanotransduction in ovarian cancer: Shearing into the unknown

“…With this combinatory approach, it will be possible to gain a more complete picture of the cancer microenvironment and ascertain potential avenues of treatment. Additionally, non-cell factors such as chemotaxis, 130 3D culture 94,[131][132][133][134][135][136] , and hypoxia 137 should be considered for future investigations, in conjunction with mechanical cues to create a microenvironment that can more fully recapitulate in vivo conditions. The study of ovarian cancer mechanotransduction promises to improve patient treatment through future investigations that utilize designs pertinent to the specific microenvironment.…”

PI3K in stemness regulation: from development to cancer

“…Such studies are inherently difficult to perform because CSCs are thought to represent rare cell populations in most tumours [25]. They may be enriched by using in vitro cancer spheroid models [40], but will still fall short of capturing the evolution of the stemness phenotype upon induction of oncogenic PI3K signalling in otherwise normal cells. While adult stem cells may represent an alternative option, hyperactivation of PI3K signalling in these cells is often associated with stem cell exhaustion and terminal differentiation (see section 'A note on context').…”