Spherical cancer models in tumor biology

Abstract

Three-dimensional (3D) in vitro models have been used in cancer research as an intermediate model between in vitro cancer cell line cultures and in vivo tumor. Spherical cancer models represent major 3D in vitro models that have been described over the past 4 decades. These models have gained popularity in cancer stem cell research using tumorospheres. Thus, it is crucial to define and clarify the different spherical cancer models thus far described. Here, we focus on in vitro multicellular spheres used in cancer research. All these spherelike structures are characterized by their well-rounded shape, the presence of cancer cells, and their capacity to be maintained as free-floating cultures. We propose a rational classification of the four most commonly used spherical cancer models in cancer research based on culture methods for obtaining them and on subsequent differences in sphere biology: the multicellular tumor spheroid model, first described in the early 70s and obtained by culture of cancer cell lines under nonadherent conditions; tumorospheres, a model of cancer stem cell expansion established in a serum-free medium supplemented with growth factors; tissue-derived tumor spheres and organotypic multicellular spheroids, obtained by tumor tissue mechanical dissociation and cutting. In addition, we describe their applications to and interest in cancer research; in particular, we describe their contribution to chemoresistance, radioresistance, tumorigenicity, and invasion and migration studies. Although these models share a common 3D conformation, each displays its own intrinsic properties. Therefore, the most relevant spherical cancer model must be carefully selected, as a function of the study aim and cancer type.

Figure 1

Steps for formation of spherical cancer models. (A) Multicellular tumor spheroids are obtained after aggregation and compaction of cell suspension cultured in nonadherent conditions. (B) Tumorospheres are formed by clonal proliferation in low-adherent conditions and with stem cell medium. (C) Tissue-derived tumor spheres are generated through partial dissociation of tumor tissue and compaction/remodeling. (D) Organotypic multicellular spheroids are formed from cutting tumor tissue in nonadherent conditions that rounded up during the culture.

Figure 2

MCTSs, tumorospheres, TDTSs, and OMSs form very tightly packed spherical cancer structures. MCTSs could be obtained by different techniques. (A) Phase-contrast micrograph of MCTS formed by the hanging drop method with human breast cancer MCF7 cells cocultured with normal human dermal fibroblasts; (B) Hematoxylin staining and (C) anti-Ki67 immunostaining of MCTS formed by human colorectal cancer HCT116 cells. (D, F) MCTS formed by human colon cancer HT29 cells on agarose: phase-contrast micrograph (D) and immunostaining against carcinoembryonic antigen on paraffin-section (F). Confocal picture (E) of human colorectal MCTS stained with DAPI (blue) and phalloidin (magenta) according to confocal staining protocol described in . (G) Phase-contrast micrograph of encapsulated MCTS obtained with mouse colorectal cancer CT26 cells. (H) Confocal images of CT26 encapsulated MCTS after cryosection and immunolabeling for DAPI (blue), KI67 (magenta), and fibronectin (red). Phase contrast microscopy (I) and anti-CK20–stained section (J) of tumorosphere from patient colorectal tumors. Nuclei in blue (DAPI), no CK20 staining. TDTS derived from colorectal cancer tissue (K–N): phase-contrast micrograph (K), confocal (L) DAPI (blue), phalloidin (magenta), anti–E-cadherin (yellow). Hematoxylin–eosin staining (M) and anti-CK20 immunostaining (N). Hematoxylin–eosin staining (O), anti-CK20 and anti-CD68 immunostaining (P) in OMSs derived from patient colorectal tumors. CK20 is an intermediate filament protein whose presence is essentially restricted to differentiated cells from gastric and intestinal epithelium and urothelium. Source of pictures: (A-C) Courtesy of Jens M. Kelm, InSphero AG, Schlieren, Switzerland; (G–H) Alessandri K, Sarangi BR, Gurchenkov VV, Sinha B, Kießling TR, Fetler L, Rico F, Scheuring S, Lamaze C, Simon A, Geraldo S, Vignjevic D, Doméjean H, Rolland L, Funfak A, Bibette J, Bremond N, and Nassoy P (2013). Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro. Proc Natl Acad Sci U S A 110, 14843–14848 ; (I) ; (J) Vermeulen L, Todaro M, de Sousa Mello F, Sprick MR, Kemper K, Perez Alea M, Richel DJ, Stassi G, and Medema JP (2008). Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proc Natl Acad Sci U S A 105, 13427–13432. Copyright (2008) National Academy of Sciences, U.S.A. ; (O–Q) .