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.

Full text

Spherical Cancer Models in Tumor
Biology
1
Louis-Bastien Weiswald *
,†,‡,§
, Dominique Bellet
‡,¶
and Virginie Dangles-Marie
§, #
*Division of Gastroenterology, Department of Medicine,
University of British Columbia, Vancouver, British Columbia,
Canada;
†
Michael Smith Genome Sciences Center, British
Columbia Cancer Agency, Vancouver, British Columbia,
Canada;
‡
Laboratoire d’Oncobiologie, Hôpital René
Huguenin, Institut Curie, St Cloud, France;
§
Université Paris
Descartes, Faculté de Pharmacie de Paris, Sorbonne Paris
Cité, Paris, France;
¶
Université Paris Descartes, Faculté des
Sciences Pharmaceutiques et Biologiques, UMR 8151
CNRS—U1022 Inserm, Sorbonne Paris Cité, Paris, France;
#
Département de Recherche Translationnelle, Research
Center, Institut Curie, Paris, France
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.
Neoplasia (2015) 17, 1–15
www.neoplasia.com
Volume 17 Number 1 January 2015 pp. 1–15 1
Address all correspondence to: Louis-Bastien Weiswald, Michael Smith Genome
Sciences Center, British Columbia Cancer Agency, 675 West 10th Ave, Vancouver,
British Columbia, Canada V5Z 1L3.
E-mail: lweiswald@bcgsc.ca
Address all correspondence to: Virginie Dangles-Marie, Research Center, Institut
Curie, 12 rue Lhomond, F-75005 Paris, France.
1
Work of the laboratory on spheres is supported by a Genevieve and Jean-Paul Driot
Transformative Research Grant, a Philippe and Laurent Bloch Cancer Research Grant,
a Hassan Hachem Translational Medicine Grant, a Sally Paget-Brown Translational
Research Grant, the Institut National du Cancer and Cancéropôle Ile de France
(COLOMETASTEM grant), and GEFLUC (Grant 5/188).
Received 2 May 2014; Revised 29 November 2014; Accepted 4 December 2014
© 2014 Neoplasia Press, Inc. Published by Elsevier Inc. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by/3.0/).
1476-5586/15
http://dx.doi.org/10.1016/j.neo.2014.12.004

Introduction
Solid tumors grow in a three-dimensional (3D) spatial conformation,
resulting in a heterogeneous exposure to oxygen and nutrients as well
as to other physical and chemical stresses. Proliferation and hypoxia
are mutually exclusive in vivo, except in areas subjected to transient
changes in perfusion where nonproliferating but viable hypoxic tumor
cells have also been identified [1]. This diffusion-limited distribution
of oxygen, nutrients, metabolites, and signalling molecules is not
mimicked in two-dimensional (2D) monolayer cultures [2].In
addition to possible induction of chemical gradients in 3D structures,
it is now well admitted that the 3D cell–cell interaction per se
influences cell structure, adhesion, mechanotransduction, and
signaling in response to soluble factors which in turn regulate overall
cell function in ways that differ dramatically from traditional 2D
culture formats [3]. Thus, the study of cells in a 3D context can
provide insights not observed in traditional 2D monolayers. To
successfully investigate the pathobiology of human cancer, it is
necessary to maintain or recreate in culture the typical 3D
architecture of the tissue. To date, numerous 3D models have been
specifically developed in cancer research to take into account these
tumor architectural features in biological processes to as great an
extent possible. These models are based on different approaches as
illustrated by the multicellular tumor spheroid model (MCTS) [4],
organotypic slices of cancer tissue [5], multilayered cell cultures [6],
and scaffolds [7]. Continuous progress in tissue engineering,
including development of various 3D scaffolds and bioreactor
systems, has improved the diversity, fidelity, and capacity of culture
models for use in cancer research [8].
The 3D microenvironment enables mimicking the different types
of cell heterogeneity observed in vivo in different contexts. Thus, 3D
systems formed only by cancer cells and homotypic cell–cell adhesion
may display different phenotypes like those of quiescent versus
proliferating cells depending upon the chemically induced gradients
[2]. More sophisticated 3D systems combining cancer and stromal
cells could emphasize the importance of heterotypic cross talk [9,10].
Among the numerous 3D models, we focus here only on spherical
cancer models. All these spherelike structures are characterized by
their well-rounded morphology, the presence of cancer cells, and the
capacity to be maintained as free-floating cultures. Consequently,
multilayered tumor cell cultures, tumor slices, organoids, or 3D
cultures within reconstituted basement membrane do not fit in with
these features and will not be described here (for a review on 3D
models, [2,9]).
Spherical cancer models other than the MCTS model have been
described and used in cancer research. Initially, development of the
MCTS model was largely due to the work of Sutherland’s group in
the early 70s [11,12]. A decade later, the group of Rolf Bjerkvig
introduced a new model of sphere referred to as the organotypic
multicellular spheroid (OMS), easily achieved by the simple cutting
of cancer tissues [13]. Histologically, the OMSs closely resemble the
tumor in vivo, with the presence of capillaries maintained for several
weeks in culture [14]. The 2000s witnessed the emergence of a new
3D sphere model, the tumorospheres, for studying and expanding the
cancer stem cell (CSC) population. More recently, tissue-derived
tumor spheres (TDTSs) were obtained by partial dissociation of
tumor tissue, enabling maintaining cell–cell contact of cancer cells
[15,16]. Originally, such structures had been observed in a limited
number of studies performed for in vitro human colon cancer cell
lines establishing [17–19]. Thus, TDTSs have been largely
characterized for colorectal cancer, as demonstrated by the work of
Kondo’s group on cancer tissue–originated spheroids (CTOSs) [16]
and that of our group on colospheres [15,20]. However, TDTSs
could also be obtained from dissociation of various types of cancer
tissues including lung, bladder, prostate, or breast cancer tissue and
uveal melanoma (personal observation and [16,21]).
At present, given the rapid development of CSC as spheres and the
absence of a well-defined terminology for spherical cancer models,
clarification of the different spherical cancer models already used and
of their application to cancer research is warranted. Thus, we present
here the different terms used to designate these 3D systems, their
culture techniques, and their major characteristics. We will then
review their major fields of application to cancer research:
chemotherapy and radiotherapy cell responses, tumorigenicity,
migration, and invasion processes.
It is now possible to classify all spherical cancer models into four
groups: 1) multicellular tumor spheroids, generated in nonadherent
conditions from single-cell suspension; 2) tumorospheres, models of
CSC culture and expansion; 3) tissue-derived tumor spheres,
formed only by cancer cells after partial dissociation of cancer tissues;
and 4) organotypic multicellular spheroids, generated by cutting
cancer tissue under nonadherent conditions.
Terminology
Historically, the multicellular tumor spheroid model was introduced
by radiobiologists in the early 70s and was mainly developed via a
wide diversity of cancer cell lines [22]. However, other types of
emerging “cancer spheres”have been recently reported and, apart
from their 3D morphology, share little with the first MCTS
established by Sutherland. Unfortunately, the absence of strict
nomenclature by the authors does not enable clear identification of
their spheres and leads to some confusions and misunderstandings.
The terms “spheroid”and “sphere,”respectively, are not consistent
throughout the literature, and this is critical to the rational use of
spherical cancer models. In general, several terms are used to refer to
the culture of well-rounded 3D cancer structures: spheroids,
organoids, and spheres (Table 1). For example, “organoid”(meaning
miniorganlike) should be dedicated to 3D culture of normal cells and
tissue [23–25], but this term has also been used for the 3D structure
spontaneously formed by colon carcinoma cell line LIM1863 [26],
for 3D cultures of tumor cells embedded in basement membrane–like
gel [27], and even for the classical model of MCTS [28]. Likewise, the
term “aggregate”was primarily used to describe loose packages of cells
and to distinguish them from compact spherical cultures [29].
Unfortunately, some so-called “spheres”and “spheroids”in the
literature are no more than loose aggregates that easily detach, cannot
be manipulated or transferred, and lack not only true spherical
geometry but possibly also cell–cell and cell–matrix interactions,
impacting biological properties.
The term “sphere”has been recently applied to normal and CSC
culture and expansion. Indeed, the terms “tumorspheres”[30,31],
“tumorospheres”[32,33], and “oncospheres”[34] were used to
describe CSC spheres issued from different types of cancer having a
large panel of derived names. Spheres from brain and breast (normal
or cancer) stem cell culture were termed “neurospheres”[35,36] and
“mammospheres”[37,38], respectively, related to their tissue of
origin. Thus, CSC spheres from colon cancer were referred to as
“colon cancer spheres”by the authors who first described them [39].
2Cancer Spheres in Tumor Biology Weiswald et al. Neoplasia Vol. 17, No. 1, 2015

However, the latter spheres have also been termed “spheroids”[40],
“colonospheres”[41,42], and more recently “colospheres”[43,44].
OMSs are clearly distinct from the classical model of MCTS. OMSs,
also designated as “biopsy spheroids”[45],“organotypic spheroids”[13],
and “tumor fragment spheroids”[46], are generated directly from cut
tissues, whereas MCTSs are established from single-cell suspensions of
cancer cells. Ovarian carcinoma ascites fluids can lead to “spherules”[47]
or “ovarian carcinoma ascites spheroids”[48], a specific term that prevents
confusion with MCTSs from ovarian carcinoma cell lines.
Preparation of Spherical Cancer Models
The four spherical cancer models are derived from various cancer cell
sources with different preparation protocols. Medium composition,
culture surface, cell density, time required for formation, origin and
handling of tumor material are the major parameters (Table 2,Figure 1).
Multicellular Tumor Spheroids
MCTS are generated from single-cell suspension culture in
conventional fetal bovine serum (FBS)–supplemented medium
without a supply of an exogenous extracellular matrix (ECM). Such
cell cultures often originate from cancer cell lines but rarely from
tumor cell suspensions derived from tumor tissue. Nevertheless, not
all cell lines were able to generate compact MCTSs [49].
Various methods can be used for MCTS cultures (reviewed in
[50]), but the core principle remains the same based on anchorage-
independent methodology. By providing conditions in which
adhesive forces between cells are greater than for the substrate they
are plated on, tumor cells were prevented from adhering to underlying
tissue culture plastic, with the aim of promoting cell–cell adhesion,
leading to well-rounded spherical structure (Figures 1Aand 2,A-H).
In this case, traditional culture medium supplemented with FBS is
used. Depending on the cell line and system used, MCTSs can be
obtained after 1 to 7 days of culture.
Nonadherent conditions could be induced in rotating systems,
including gyratory shakers and spinner flasks. These protocols
enabled production of large pools of MCTSs of various sizes but
necessitated determination of optimal cell concentration, and the cells
required vast quantity of media for growth.
MCTS are easily generated by the liquid overlay technique that
prevents matrix deposition. Tumor cells are placed on tissue culture
plastic (well plates, flasks, or dishes) covered with a thin layer of inert
substrate, agar [51], agarose [52], or polyHEMA [53]. This thin film
is allowed to dry before addition of medium, in which cancer cells
grow without adhering, thus promoting cell aggregation and
compaction (Figure 2,D–F). Spheroid formation can also be
achieved by seeding cells in ultra–low attachment plates without any
coating, as the polystyrene surface offers low-adhesion properties [54].
Use of the liquid overlay technique in 96-well plates leads to
reproducible formation of one single MCTS per well, homogeneous
in size. A small working volume and possibly automation make these
cells ideal for high-throughput screening [49].
Another method, the “hanging drop method,”has been described
for generating MCTSs (Figure 2,A–C)[55]. Drops of cell suspension
up to 30 μl in size are deposited on a dish lid. Upon inversion of the
tray, cells accumulate at the free liquid–air interface to form a single
MCTS. This method avoids coating of plates and effects on cells due
to substratum contact but requires MCTS transfer for further
investigation. The InSphero AG (Schlieren, Switzerland) plate system
permits simple transfer of the MCTS from the drop in the well for
drug incubation [9,55]. A large amount of regular spheres can be
obtained and manipulated in this way.
Table 2. Methods Used to Generate the Different Cancer Sphere Models.
Tumor Sphere Model Tumor Material Culture Conditions Time Required for Tightly
Packed Sphere Formation
References
Multicellular tumor spheroids Single-cell suspension from permanent cancer cell
lines (rarely from dissociated cancer tissue)
▪Medium with FBS w.o. any additional growth factor 1-7 d [49,50,55]
▪Nonadherent conditions (inert matrix agarose like polyH,
agarose; hanging drop; spinner)
Tumorospheres Single-cell suspension from permanent cell lines,
tumor tissue, or blood
▪Serum-free medium with EGF and FGF-2 5-7 d until 1-2 mo [38,40,67,71,112]
▪Low-attachment plastic
▪Clonal density
▪Potential preliminary cell sorting
Organotypic multicellular
spheroids
Cut and minced tumor tissue ▪Medium with FCS and nonessential amino acids w.o.
any additional growth factor
2-5 d until 12-18 d [83,84]
▪Nonadherent conditions
Tissue-derived tumor spheres Partially mechanically or enzymatically
dissociated tumor tissue
▪Medium with FCS w.o. any additional growth factor
(colospheres) or serum-free medium with EGF and FGF-2 (CTOSs)
1-3 d [15,16,20,21]
▪Culture-treated plastic then non- adherent conditions
FGF-2, fibroblast growth factor 2; w.o., without.
Table 1. Confusing Terminology to Depict the Different Models of Cancer Spheres.
Cancer Sphere Models Alternative Names
Multicellular tumor spheroids Spheroids [171]
Tumoroids [172]
Mixed spheroids [173]
Nodules [174]
Heterospheroids [175]
Organoids [28]
Tumorospheres Spheroids [40]
Colospheres [43,44]
Spheres [30,34,36,38,39]
Tumorspheres [30]
Oncospheres [34]
Xenospheres (from patient tumor-derived xenografts) [176]
Neurospheres (normal and malignant brain) [35,36]
Mammospheres (normal and malignant breast) [37,38]
Colon cancer spheres (colon cancer) [39]
Tissue-derived tumor spheres Colospheres [15,20]
Cancer tissue–originated spheroids [16]
Spheroids [21]
Organotypic multicellular spheroids Biopsy spheroids [85]
Organotypic spheroids [177]
Organotypic tumor spheroids [84]
Fragment spheroids [50]
Primary spheroids [178]
Ovarian carcinoma ascites spheroids [48]
Spherule [47]
Neoplasia Vol. 17, No. 1, 2015 Cancer Spheres in Tumor Biology Weiswald et al. 3

More recently, several studies reported production of MCTSs using
microcapsules with alginate-based membranes (Figure 2,G–H)
[56,57]. Tumor cells are encapsulated in cellulose-based microparticles,
with a narrow size distribution, via a peroxidase-catalyzed reaction in a
water-immiscible fluid under laminar flow. Next, the microparticles are
coated with an alginate-based gel several dozen micrometers thick via
the same enzymatic reaction in water-immiscible fluid. Finally, the
cellulose-based microparticles are degraded using cellulase to prepare
MCTS formation [58]. The alginate encapsulation approach enables
preparation of MCTSs in large quantities of a well-defined size,
compatible with high-throughput screening [59]. This method also
enables studying cancer cell lines unable to form MCTS by the
techniques described above. However, the alginate membrane would
reduce oxygen, nutrient supply, and contact between cells, and this
membrane might introduce a bias. To address the latter issues,
Alessandri et al. developed an aqueous core enclosed by a hydrogel shell
in gentle, oil-free conditions. The permeability of the gel allowed
free flow of nutrients into the capsule and cell proliferation in a scaffold
free-environment [59]. This promising approach, inspired by flavor
pearls from molecular gastronomy, must nevertheless prove its
biological relevance.
Likewise, some artificial MCTSs have been obtained using
methylcellulose, a temperature-sensitive polymer used in several
neural tissue engineering applications [60] that gathers the
tumor cells.
Whereas the first MCTSs formed by monoculture of cancer cells
mimicked micrometastasis or tumor emboli, heterotypic MCTS
rapidly appeared to take into account the presence of noncancerous
cells in tumor tissue. Diverse spheroid coculturing strategies of tumor
and stromal cell types lead to the study of heterologous interactions in
tumor tissue. The most frequent cell types used for tumor cell
coculturing are immune cells, fibroblasts, and endothelial cells.
Stromal cell suspensions can be cocultured with compact MCTS,
leading to invasion of MCTS (immune cells [61–63], endothelial cells
[64]), or they can be seeded together in starting cell suspensions with
tumor cells [9]. Mixed MCTS testing with tumor and endothelial
cells for the study of tumor angiogenesis had had little success [65]
until Timmins et al. obtained in vitro microvascularized tumors [64].
They introduced endothelial cells into preformed HCT116 MCTS in
hanging drops; the endothelial cells formed secondary aggregates and
then migrated into the MCTS, establishing tubular networks and
luminal structures.
The MCTS model has now been well characterized, and new
protocols for obtaining them have been reported [55,59]. The latter
are obtained from a wide range of cancer cell lines and are currently
used in numerous cell biology studies. Micropatterning and
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.
4Cancer Spheres in Tumor Biology Weiswald et al. Neoplasia Vol. 17, No. 1, 2015

microfluidics technologies offer the exciting prospect of standardized
MCTS mass production to tackle high-throughput screening
applications using more sophisticated MCTS coculture models,
more closely reflecting tumor tissues composed of tumor and various
stromal cell types.
Tumorospheres
CSC culture as a free-floating sphere (tumorosphere) was first
described in brain tumors by Singh et al. [36]. Initially, the
quantification and characterization of such floating spherical
aggregates had been developed for normal neural stem cells grown
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 [171]. (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 [59]; (I) [40]; (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. [110];(O–Q) [84].
Neoplasia Vol. 17, No. 1, 2015 Cancer Spheres in Tumor Biology Weiswald et al. 5

as neurospheres, in which a single cell is able to give rise to a sphere by
clonal expansion [35,66]. In the ensuing years, tumorospheres were
developed from a wide range of solid tumors, including breast [38],
lung [67], colon [39], prostate [68], pancreas [69], and ovarian [70]
cancers, under that same assumption that “sphere assays”enable
measuring self-renewal capacity.
Methods for CSC isolation and expansion as spheres do not greatly
differ from one cancer tissue origin to another. The first step requires
mechanical and enzymatic dissociation of the tumor sample in single-
cell suspensions. CSC culture can also be performed on cancer cell
lines, facilitating the dissociation step. Recently, circulating breast
tumor cells from patients have been reported to proliferate as
tumorospheres. Consequently, the first step aims here to depleting
other blood cells using a microfluidic technology [71]. Next, the cell
suspension is cultivated at low density in specific medium called
“stem cell medium”in low-adherent conditions to promote extensive
proliferation as clonal nonadherent spherical clusters (Figure 1B).
Stem cell medium is devoid of FBS and supplemented with several
factors that favor stem cell growth, including basic fibroblast growth
factor and epidermal growth factor (EGF). Hydrocortisone, insulin,
and progesterone, known to induce stem cell proliferation, can be
added to the medium, as is with the case for heparin, which stabilizes
the association between basic fibroblast growth factor and its receptor
[72]. Likewise, optimal growth as spheroids has been obtained with
leukemia inhibitory factor [73]. Depending on the cancer type, other
growth factors can be preferentially added, like Wnt3A for colorectal
cancer via the inhibitory effect of Wnt on enterocyte differentiation
[74]. Moreover, inhibition of adhesion induces death through anoikis
in nonmalignant and differentiated cells. Under these culture
conditions, undifferentiated tumor cells proliferate and grow as
floating clusters termed tumorospheres (Figure 2,I–J).
Intermediate sorting steps on the basis of putative CSC markers by
fluorescent-activated cell sorting or magnetic-activated cell sorting can
be performed after dissociation so as to enrich the CSC population
and eliminate stromal cells. To detect these CSCs, several markers
have been proposed depending on the tumor type, including CD44,
CD133, aldehyde dehydrogenase, CD90, ABCG2, and ABCB5
(ABC for ATP-binding cassette), with some of them being more
specific than others, but no single marker yet exists that has
established itself for identifying CSCs with adequate sensitivity and
specificity [75].
As pertinently discussed by Pastrana et al. [76], the tumorosphere
forming assay must be carefully interpreted. It is widely admitted that
each tumorosphere is derived from a single cell and is therefore clonal.
Nevertheless, various cell densities are used if cells need to be seeded
at extremely low densities to avoid cell fusion and aggregation [77].
Indeed, in-depth analyses in normal neurospheres clearly demon-
strated that neurospheres were not clonal aggregates because of
fusion-induced “growth”[77]. Semisolid methylcellulose used to
grow putative clonal tumorospheres [78] also failed to prevent cell
aggregation [77]. In addition to cell density, critical parameters such
as medium composition, volume, surface area of the culture dish, and
duration are also to be taken into account.
Tissue-Derived Tumor Spheres
In contrast to MCTS obtained from single-cell suspensions,
TDTSs are obtained from partially dissociated cancer tissue. This
model includes CTOSs [16], MARY-X spheroids [79], and our
colospheres [15,20]. Colospheres were generated by finely cutting the
tissue sample with a scalpel blade and then crushing it with a striated
plunger from a disposable syringe. The resulting pieces are cultivated
in a cell culture–treated flask with classical medium supplemented in
FBS. Colospheres are formed in 1 day after cell remodeling and
compaction (Figures 1Cand 2,K–N) and are isolated by passing the
bulk in cell strainers. This TDTS model was obtained in around 50%
of colorectal tumor specimens and was related to tumor aggressiveness
[15]. Nevertheless, the frequency of colospheres attained 95% (19/
20) in colorectal cancer patient–derived xenografts (personal data).
Colospheres can be kept for at least 2 weeks in culture in growth
medium and nonadherent conditions to prevent plastic attachment
[20]; moreover, they can be cryopreserved as whole sphere structures
in freezing medium (personal data). Only tumor tissue dissociation
produces colospheres; none were obtained from nontumoral tissue
counterpart, showing that strong interaction between epithelial cells is
not sufficient to give rise to TDTS.
As performed for generation of CTOSs, an intermediate step of
incubation with liberase after mechanical dissociation is required. Next,
aggregates are filtered by a cell strainer before culture in suspension with
stem cell medium (without FBS and supplemented in growth factor) or in
Matrigel with a medium supplemented with FBS. CTOSs were observed
in 98% of samples, including endoscopic biopsy samples [16].These
structures can be passaged by dividing them mechanically, allowing
expansion in vitro. Similarly to colosphere protocol, nontumoral colonic
mucosa never gave rise to CTOS.
MARY-X spheroids were obtained from a patient inflammatory breast
cancer xenograft. Upon mincing, the tumor cells are released into the
medium supplemented with FBS as sheets of cells and single cells to form
tight, compact clumps or aggregates of cells. These structures can be
maintained in culture for periods of up to 3 months [80].
Organotypic Multicellular Spheroids
The OMS model is very similar to the explant model, which
consists of culturing ex vivo fragments of tumors [81,82] without
dissociation, in contrast to TDTSs. The method for establishing
OMS in culture from tumor tissue is simple. The tumor fragment is
cut with a scalpel into pieces (0.3 to 0.8 mm in diameter) and
incubated in tissue culture flasks previously coated with 0.75% agar
in medium. Growth medium is supplemented with 10% FBS and
with an excess of nonessential amino acids. Tissue fragments are
cultured for 12 to 18 days [83] or 2–5 days [84] until they round up
to form OMS (Figures 1Dand 2,O–Q). At that time, they can be
isolated from tissue debris with a pipette by visual control using a
microscope. The OMS can be further cultured in new agar-coated
flask with medium. The growth medium of the overlay suspension is
usually changed once a week, and the OMSs can be kept in culture for
several weeks [13,83]. OMSs can be stored as frozen stocks. As a
typical example, cryopreserved glioma OMSs remain viable, retain
their histological characteristics, and display only minor phenotypic
and genotypic changes after thawing [85].
OMSs have been successfully generated from glioblastoma [86,87],
meningioma [88], mesothelioma [46], head and neck squamous
tumor [89], lung cancer [90], bladder cancer [91], and colorectal
cancer [84]. Spheroids isolated from ovarian carcinoma ascites fluid
are a special case in the OMS family. Unlike the other OMSs, they are
not generated after tissue processing but are isolated directly from
patient effusions [92].
6Cancer Spheres in Tumor Biology Weiswald et al. Neoplasia Vol. 17, No. 1, 2015

Biology of Spherical Cancer Models
Multicellular Tumor Spheroids
MCTSs can attain 1 to 3 mm in diameter [93,94] and are
somewhat compact depending on the cancer cell line [29]. In large
MCTSs (N500 μm diameter), a necrotic core is surrounded by a
viable rim, with an inner layer of quiescent cells and an outer layer of
proliferating cells, as described in microregion of tumor in vivo. Such
tumor cell heterogeneity is due to growth factor deprivation, nutrients
and oxygen gradients, and catabolites accumulation.
Moreover, the dynamic of the MCTS growth rate reproduces that
of solid tumor in vivo characterized by an early exponential phase
followed by a period of delayed growth. Whereas monolayer cultures
grow exponentially, MCTS and in vivo tumor are characterized by
exponential cell proliferation followed by a phase of declining growth
rate associated with an increase in nonproliferating and necrotic cells
[4]. Ewing tumor MCTSs have been demonstrated to be more closely
related to patient tumors in their proliferative index but also to cell
morphology, cell–cell junctions, and ERK1/2 MAPK and PI3K ±
AKT pathway activation [95].
Spatial organization of MCTSs is based on cell interactions
differing from those existing in flat monolayer culture. Indeed,
differential expression of proteins implicated in cell–cell and cell–
matrix interactions has been revealed through monolayer–MCTS
transition. Thus, in several studies, E-cadherin was found to be
overexpressed in MCTSs compared to monolayer cells [96–100],in
the manner of CD44 and EpCAM in gastric cancer MCTSs [29].In
contrast, integrin β1, β4, and α6 subunits were downregulated in
MCTSs from epidermoid carcinoma cell line A541 [101]. Dynamic
analysis of hepatoma MCTS formation has shown the fundamental
role of E-cadherin and β1-integrin in cell aggregation and MCTS
compaction [102].
Synthesis of ECM components and their receptors can be largely
influenced by culture conditions [103]. Thus, a thick ECM-like
filament network has been reported to be associated with the surface
itself. Moreover, the presence of ECM components (fibronectin,
laminin, collagen, and glycosaminoglycans) has been demonstrated in
MCTSs from human thyroid cancer and glioma cell lines [104].
MCTSs at least partially recapitulate differentiation of the parent
tumor (Table 3), in contrast to monolayer cultures. Generally, cells are
more strongly differentiated in MCTS cultures than in monolayer
cultures. Outer layers of MCTS from hepatoma cell lines are
differentiated into smooth tightly packed polarized cells [102],and
microvilli were observed on the surface of MCTSs from colon cancer
cell lines [15,105]. Furthermore, MCTSs contain pseudoglandular
structures with lumen, very similar to the characteristics of the original
adenocarcinoma specimens [106]. Similarly, features of histological
differentiation characteristic of primary ovarian carcinomas are not
present in monolayer cultures but are restored in MCTSs [107].
Tumorospheres
Little is known about the biology of tumorospheres; indeed, contrary
to the use of other sphere models, that of the tumorosphere does not seek
to mimic cancer tissues but rather to study CSC properties (Table 3): it is
admitted that tumorospheres do not fully replicate the 3D structure and
environment of an in vivo tumor [108].
The CSC model postulates that tumors are organized hierarchically
with a subset of rare tumor cells, which possess self-renewal and
multilineage differentiation potential. Because of the general lack of
cell-surface markers and the absence of a distinct and discernible
morphological phenotype related to the instability of the CSC
phenotype, CSCs have typically been defined and studied on the basis
of functional assays in relation to these properties. The gold standard
for evaluating the presence of CSCs is transplantation of a few cells
into an immunocompromised mouse: CSCs have the unique capacity
to form tumors in serial xenotransplantation assays after injection of
low number of cells and to reestablish, at each in vivo passage, the
hierarchical cell organization and heterogeneity of the parental tumor
[75].In vitro methods have been developed as attractive surrogate
assays to measure their ability to form in vitro tumorospheres
(clonogenic) when plated at low density in nonadherent cultures in
“sphere”-forming assays. Histological examination of tumorospheres
demonstrated the absence of nonneoplastic cells in patient-derived
tumor spheres obtained without sorting step [109].
The CSC capacity for multilineage differentiation was assessed in vivo
by testing the ability to reproduce tumor heterogeneity in a xenograft
assay and/or in vitro by testing the capacity of tumorospheres to
differentiate under differentiating conditions. In stem cell medium,
patient-derived tumorosphere cells maintain their sphere morphology and
remain poorly differentiated, with little or no expression of differentiation
markers that include cytokeratin (CK) 20 in colon cancers [110];GFAP
or β-tubulin 3 in brain tumors [36];CK18,CK14,andα-SMA in breast
cancers [38]; and CK7 and CA-125 in ovarian cancers [70].After
withdrawal of growth factors and addition of 10% FBS on tissue-culture–
treated plastics or in Matrigel, floating cells were able to adhere and to
strongly differentiate with expression of differentiation markers. In
contrast, the addition of myofibroblast-derived factors prevented
differentiation of colon CSCs [111].
It is noteworthy that single neurospheres from normal brain
contain stem cells, progenitors, and differentiated cells, as observed in
the tumorospheres. Tumorospheres are not homogeneous structures
enriched with undifferentiated cells but rather comprise a range of
morphologically distinct entities displaying inter- and intrasphere
molecular heterogeneity, including variable expression of markers of
differentiation [112]. Several studies have demonstrated clonal
Table 3. Comparison of Various Tumor-Related Parameters in the Four Cancer Sphere Models.
Multicellular
Tumor Spheroids
Tumorospheres OMSs TDTSs
Cancer sphere culture
Success rate of initiation + + +/−+
Ease of maintenance ++ + +/−+/−
Genetic manipulation ++ + ND ND
Sphere composition
Tumor heterogeneity +/−−++ ++
Tumor–stroma interaction + −++ −
Immune system +/−−+−
Characteristics of original tumor + −++ ++
Application fields
Tumor growth ++ + + +/−
Survival ++ −+/−+
Hypoxia ++ −++
Cancer stemness −++ +/−−
Migration/invasion + −++
In vivo tumorigenicity +/−++ + +
Personalized medicine + + ++ ++
High-throughput drug screening ++ + −+/−
Low-throughput drug screening ++ ++ + ++
Radiosensitivity ++ +/−+ND
Parameters are appreciated as best (++), suitable (+), possible (+/−), and unsuitable (−). ND,
Not determined.
Neoplasia Vol. 17, No. 1, 2015 Cancer Spheres in Tumor Biology Weiswald et al. 7

heterogeneity among tumorospheres. Thus, in the case of patients
with colorectal cancer, three different types of CSC, all undifferen-
tiated, were resolved on the basis of clonal tumorosphere cultures
from individual patient tumors (one cell per well was seeded in a 96-
well plate) [109]: 1) a rare subset of CSCs that maintained tumor
growth on serial transplantations; 2) a subset of tumor-initiating cells
with limited self-renewal capacity; the latter contribute to tumor
formation only in primary mice and are therefore not consistently
defined as CSCs; and 3) a more latent subset of CSCs apparently
activated in second or tertiary transplantation assays. Likewise, in
PTEN-deficient glioblastoma, a series of phenotypically distinct self-
renewing cells was observed in both CD133 + and CD133 −fractions
[113]. Clearly, the tumorosphere assay selectively enriches for the
growth of CSC, although it is noteworthy that these spheres also
contain more differentiated tumor cells [114].
A recent work [115] reports that the tumorosphere assay enriches CSC
population in a cell line–dependent manner and that the conventional
monolayer culture might maintain a CSC phenotype more effectively
than the tumorosphere depending on the cancer cell line.
To date, no study has precisely determined how many cells within
tumorospheres are actually CSCs: the gold standard for evaluating the
presence of CSC remains a comparison between a putative CSC
population and unselected cancer cells in in vivo assays. Readout of the
tumorosphere assay involves the number and size of the spheres; thus, the
evaluation of the CSC presence is subject to discussion. Indeed, quiescent
stem cells may not divide to form tumorospheres because the assays used
do not provide as-yet-unknown key components of the in vivo niche
required for activation of dormant stem cells. Likewise, the size may
simply reflect growth factor responsiveness.
Tissue-Derived Tumor Spheres
Initially, the observation of such structures was reported in a limited
number of studies performed for the in vitro human colon cancer cell lines
establishing [17–19]. Thus, TDTSs have been largely characterized for
colorectal cancer, as demonstrated by the work of Kondo’sgroupon
CTOSs [16] and that of our group on colospheres [15,20]. However,
TDTSs were also obtained from dissociation of various types of cancer
tissues including lung, bladder, prostate, and breast cancer tissue and uveal
melanoma (personal observation and [16,21].
TDTSs are formed by tissue remodeling and compaction after
partial tissue dissociation and are exclusively composed of tumor cells
(Figures 1Cand 2L) without nonneoplastic cells [15,16,21,80]. This
might be explained by strong cell–cell interactions between carcinoma
cells. However, the interaction between epithelial cells could not per
se explain the formation of TDTS because none are obtained from
nontumoral mucosa. Thus, interactions between tumor cells and
nonneoplastic cells might be lost after partial dissociation, resulting in
the absence of stromal cells in TDTSs. Indeed, E-cadherin has been
shown to be involved in cell–cell interactions within TDTSs
[16,20,80,116], whereas E-cadherin/β-catenin complexes were
shown to be tethered to the cytoskeleton. Because this organization
has been demonstrated to strengthen cell–cell adhesion in other
systems [117], it can be postulated that the same is true for TDTSs.
Through their strong cell–cell interactions, colospheres, MARY-X
spheroids, and CTOSs escape anoikis and may remain viable for
several weeks in culture.
A major trait of TDTSs is their capacity to recapitulate avascular
tumor microregions. Colospheres and CTOSs have been shown to
mimic the parent tumor (Table 2) in terms of histological
characteristics, gene expression profiles, mutations in key genes, and
tumorigenic and metastatic properties [15,16,20,21,80].Thus,
MARY-X spheroids display polar architecture and internal lumenlike
structures (canalis) coated with microvilli as observed in lymphovas-
cular emboli in vivo. This type of architecture is not found in MCF-7
MCTSs [80]. In the same manner, colospheres retain the
differentiation level of the parent tumor, including glandularlike
structures and mucus production, in contrast to MCTSs formed by
cancer cell line from the same parent tumor [15]. Moreover, after 1
week of culture, fractions of proliferating cells in colospheres are
approximately the same as observed in vivo, whereas MCTSs from
cancer cell line possess extensive proliferative capacity, probably
because of selective pressure induced by culture (personal data).
However, colospheres show little volume growth even after 15 days in
culture. A potential explanation would be that active cell proliferation
is compensated by substantial cell death and/or cell shedding. In most
CTOSs, the increase in size slowed down after about 14 days of
culture in stem cell medium through AKT pathway activation. As in
the in vivo situation, proliferating cells predominantly localize to the
outer rim of the growing CTOSs [16].
Organotypic Multicellular Spheroids
From the various in vitro sphere models described here, OMSs seem to
be the 3D model which is closest to in vivo tumors, in light of the absence
of any dissociation process for obtaining these spheroids (Table 2).
This model is very close to both organ culture of tumors, wherein a
small piece of tissue is cultivated on the surface of the medium in a moist
gas phase [118], and the explant model, which consists of culturing tumor
fragments completely immersed in medium [119]. Precise characteristics
of OMS have not yet been reported, and most information on this model
was obtained by Rolf Bjerkvig’sgroup[83].
The morphology of OMSs is usually similar to that of the original
tumor tissue. OMSs recapitulate the original heterogeneity of the
tumor; they maintain the presence of macrophages and preserve
vessels with striated fibers of collagen in association with fibroblasts
(Figure 1D) that surround vascular elements [13]. Maintenance of the
stromal component within the OMS clearly makes OMSs distinct
from the TDTSs. It is noteworthy that OMSs obtained from glioma
biopsies show wide variation in central necrosis formation. In these
spheroids, cell cycle analyses reveal that the fractions of proliferating
cells (S and G
2
M) in the glioma OMSs after 3 weeks of culture are
approximately the same as those observed in the tumors in vivo. Even
after prolonged culture (~ 70 days), the cell cycle was unchanged.
Similarly, OMSs from bladder cancer display cell cycle distribution which
is similar to that observed in original tumors [83].Furthermore,mitotic
figures are frequently observed in OMS, demonstrating their growth
capacity. Nevertheless, as described for colospheres, no significant volume
growth was observed after several weeks in culture, suggesting cell loss
and/or cell shedding in these structures. This was demonstrated for one
glioblastoma in which cell shedding from each OMS was found to be
approximately 31 cells per hour [13].
Tumor cells that propagated as OMSs from biopsies show a
remarkable stability in ploidy even after long culture periods [83].
Moreover, genomic profiles of OMSs from glioblastomas are
genetically stable and more representative of the parent glioblastoma
than short-term primary cultures [120]. Nonetheless, this type of
spheroid has been reported in only a few types of cancer.
8Cancer Spheres in Tumor Biology Weiswald et al. Neoplasia Vol. 17, No. 1, 2015

Applications
Radioresistance
Radioresistance was the first application scope to be studied using
the MCTS model (Table 3). This powerful model is particularly well
adapted to ionizing radiation studies because tumor sensitivity to
ionizing radiation is controlled by parameters that include intercel-
lular contact and communication, oxygen, damage repair, and
apoptosis induction [121]. The first evidence of radioresistance in
MCTSs dates back to the early seventies and has been made on rodent
cell lines [122]. In general, survival of MCTS cells is better than that
of the same cells cultured in monolayers, and growth of MCTSs and
tumors in vivo after irradiation is very similar. MCTSs from the
WiDr colorectal cancer cell line thus appear to accurately model the
radiation sensitivity of WiDr tumors compared to cells in a monolayer
[123]. Using the MCTS model, resistance to radiotherapy has been
explained by hypoxia [124] and cell–cell contacts [122]. Santini and
colleagues thus demonstrated that an increase in compaction of
HT29 MCTSs was responsible for enhanced resistance to ionizing
radiation [117]. Moreover, proposed mechanisms for the “contact
effect”include gap junctional “reciprocity,”cell shape–mediated
changes in (repair-related) gene expression, and alterations in
chromatin packaging that influence DNA repair [125]. To our
knowledge, no radiosensitivity studies had previously been performed
on whole tumorospheres. CSC spheres have been consistently
dissociated before radiosensitivity assays [126]. Few radioresponse
studies have been performed on OMSs and then only from human
glioblastoma. Radiation induces minor effects on glioblastoma
OMSs, as observed in in vivo situation. In glioblastoma, radio-
resistance is related to the presence of blood vessels and hypoxia. In
contrast, most glioblastoma cell lines cultured as monolayers are
radiosensitive [127].
Chemosensitivity
Decades of research have firmly established that, in a preclinical or
clinical setting, cancer cells grown in vitro as 3D MCTSs more
accurately mimic the drug sensitivity/resistance behavior of cancer cells
found in solid tumors in vivo than cancer cells cultured under
conventional 2D monolayer conditions. Experts in the field recom-
mend their use in major programs for drug screening and development
and frequently point out the rationale for using MCTSs in antitumor
drug testing [49] (Table 3). MCTSs have been and are still being used
for modeling and for studying multicellular resistance (for review,
[128]) due to different mechanisms: hypoxia [129], alteration of
chromatin structure or chromatin packaging [125], apoptosis inhibition
[130],cellcycle[131], and permeability [132]. Numerous anticancer
therapies have been evaluated in different cancer cell types in MCTS
and directly compared to the same cells grown in a 2D monolayer
format. Studies showed that tumor cells were less sensitive to anticancer
agents when evaluated in MCTSs compared to 2D culture
conditions [133]. However, a number of studies indicated that the
observed effects of anticancer agents against tumor cells in MCTS
culture were equal to, or more sensitive than, the same tumor cell type
cultured in a 2D monolayer format [134]. Because of formation of
HER2 homodimers, trastuzumab inhibits cell proliferation in SKBR3
and SKOV-3 cells when they are maintained as MCTSs as compared to
conventional 2D culture through formation of HER2 homodimers
[135]. Activation of HER2 in MCTS led to higher sensitivity to
trastuzumab while maintaining MCTS compaction.
Whether cell sensitivity to drugs/compounds is increased or decreased,
information obtained from use of these 3D cell cultures in cancer research
can potentially provide a more accurate representation of drug/compound
activity in vivo. In addition, the regular size and well-rounded shape, as
well as easy rapid handling, make MCTSs powerful tools for drug testing,
facilitating high-throughput screening [9,49,59].
Although recent evidence indicates that CSCs respond to
antitumor agents differently in vitro and in vivo [108], tumorospheres
are being increasingly used for studying the response to chemotherapy
because the remaining CSCs presumably trigger relapse after
treatment termination. Interestingly, Todaro and colleagues demon-
strated that tumorospheres from patient colon tumors are resistant to
5-FU and oxaliplatin through autocrine production of interleukin-4
[40]. An original study reports the proof of concept of culturing
circulating tumor cells from patients as tumorospheres to perform
personalized anticancer drug testing [71] (Table 3). Many studies use
tumorospheres derived from cancer cell lines to highlight the
resistance of CSC compared to the bulk tumor. Those studies
compared the anticancer drug response of tumorospheres to that of
adherent cells, referred to here as bulk tumors. However, as in the case
for the radioresponse, it is crucial to take into account the 3D aspect
of the tumorospheres because the resistance observed in CSCs could
be due to multicellular resistance and not to the intrinsic properties of
CSCs. Few studies have analyzed the effect of anticancer drugs on
OMSs [46,91,136], which could be explained by heterogeneity
between OMSs of the same tumor, thus complicating standardiza-
tion. Glioblastoma OMSs were used to demonstrate that NG2/
MPG-expressing tumors were more resistant to doxorubicin,
carboplatin, and etoposide [136], and OMSs from human
mesothelioma showed resistance to apoptosis after TRAIL-plus-
cycloheximide treatment, partly mediated by Akt/PI3k and mTOR
pathways [46]. From a personalized treatment perspective, study of
the response of TDTSs could predict a patient’s tumor response to
chemotherapy, and initial studies are promising (Table 3). Growth of
CTOSs from colorectal cancer specimens was dose-dependently
inhibited by 5-FU, whereas the response of CTOSs differed in
individual cases [16]. Using colospheres from colorectal cancer
patient–derived xenografts, our group showed a correlation between
the ex vivo colosphere response to 5-FU or irinotecan and the in vivo
xenograft response [20].
Migration and Invasion
The multistep process of metastasis can only be successful if the 3D
microenvironment is permissive for tumor cell invasion, metastatic
dissemination, and metastatic growth. Noncellular components of the
tumor microenvironment, such as ECM and hypoxia, critically drive
tumor progression via increased ECM deposition, cross-linking, and
remodeling [137]. Moreover, cadherins and integrins have been
linked to the metastatic process, and adhesion and the ECM molecule
expression pattern in the 3D environment resemble those of the
tumor in vivo. In addition, 3D conformation can induce expression
of proteins associated with metastasis, as suggested by enhancement of
carcinoembryonic antigen expression in the MIP-101 colorectal
cancer cell line grown as MCTSs [138]. Depending on the cell type
and tissue environment, cells can migrate in two major ways:
individually, when cell–cell junctions are absent, or collectively as
multicellular groups, when cell–cell adhesion is retained. This
multicellular migration mode, or “collective migration,”is commonly
used by carcinomas, which retain high or intermediate levels of
Neoplasia Vol. 17, No. 1, 2015 Cancer Spheres in Tumor Biology Weiswald et al. 9

differentiation [139]. Collectively, these data present spherical cancer
models as relevant in vitro models for studying invasion and
migration processes (Table 3). Numerous studies used MCTS for
invasion and migration assays, and recent publications have
demonstrated their great advantage in the evaluation of therapeutic
agents/drugs with antimigratory properties using rapid highly
reproducible 96-well plate–based technique [54,140]. MCTSs can
thus be embedded in different matrices [141,142] or seeded on top of
ECM [143]. Invasion has also been studied using MCTSs in different
coculture systems. MCTSs obtained from the MCF-7 cell line have
been confronted in vitro with chick heart fragments to study the effect
of retinoic acid on the invasion process [144]. Expression of antisense
uPAR and antisense uPA from a bicistronic adenoviral construct
inhibited invasion of fetal rat brain aggregates by cells from SNB19
MCTSs [145]. Invasion was also assessed using cultures of MCTSs
with mouse embryoid bodies [54], precultured reepithelialized
endometrial fragments [146], and organotypic brain slice cultures
[147]. Interestingly, two different sphere models obtained from the
same type of cancer displayed differing invasion and migration
properties. In a recent study, MCTSs obtained from the glioblastoma
U87MG cells were unable to invade the corticostriatal slice, in
contrast to glioblastoma OMSs. The U87MG cell line may have lost
the invasive features characteristic of glioblastomas as a result of
selective pressures and genetic drift [147]. Likewise, a given 3D model
of different tumor grade shows different properties, illustrating a poor
artifact effect due to the model used. Thus, OMSs have been shown
to mimic properties of their origin tumor. Using coculture of OMSs
from brain tumors with fetal rat brain aggregates, low-grade glioma
OMSs were less invasive than those obtained from the highly
malignant glioblastomas. Indeed, the invasiveness of the glioblastoma
OMSs was characterized by a gradual destruction of normal brain
tissue by tumor cells, followed by replacement of normal tissue by
these cells [148]. OMSs can also be embedded in matrix to study
migration and invasion of cells from the OMS. Not all the OMSs
from a given glioblastoma sample displayed the same capacity to
migrate in or invade ECM [86], reflecting inherent intratumoral
heterogeneity. This was confirmed by two studies in which the
metastatic potential of ovarian carcinoma ascites OMSs was assessed
using adhesion [48] and migration [149] assays in different matrices
and in normal human mesothelial cell monolayers. Taken together,
these observations strongly suggest that the metastatic potential of
OMSs is driven by both their microenvironment and their intrinsic
invasive properties.
Studies on the metastatic properties of TDTS have shown that
colospheres retain aggressiveness of the parent xenograft tumor in
contrast to MCTSs [15,20]. Furthermore, MARY-X spheroids have
been shown to mimic lymphovascular emboli in vivo, a structure
efficient at metastatic dissemination. Similarly to the tumor emboli,
MARY-X spheroids display a high level of E-cadherin [150] because
of altered trafficking [151].
Despite the emergence of the concept of “migrating cancer stem
cells”[152] and the association between EMT and stemness
properties [153], tumorospheres are not used in Matrigel assays for
dissemination monitoring but rather for differentiation induction, so
as to assess their capacity of multilineage differentiation [110].
CSCs/Tumorigenicity
Tumorosphere cultures have gained popularity as in vitro assays for
propagating and analyzing CSCs (Table 3). As already discussed
above, the “CSC sphere assay,”derived from the normal neural stem
cell assay, should be more carefully interpreted before conclusions can
be drawn concerning their stemlike properties. Moreover, the concept
of CSC itself remains subject to debate [154], and the difference
between the CSC, cancer-initiating cells, and highly proliferating cells
is ambiguous.
In breast cancer, use of serum media was demonstrated to promote
cell differentiation rather than an undifferentiated state. Clonal
dilutions used in tumorosphere-forming assays revealed the intrinsic
property of cancer stem/progenitor cells at surviving and growing in
serum-free suspension, whereas more differentiated cells undergo
anoikis and die under these conditions [32].
Recent data suggest that CSCs depend on a similar, permissive
environment, the CSC niche, to retain their exclusive ability to self-renew
and to give rise to more differentiated progenitor cells, while themselves
remaining in an undifferentiated state [155]. This CSC niche, by analogy
with the normal stem cell niche, is defined as a particular location or
microenvironment that maintains the properties of stem cell self-renewal
and multipotency. The CSC niche is composed of blood vessels, stromal
cells, and ECM components but also defined by its 3D organization.
Although tumorospheres are characterized by the absence of stromal cells
and the lack of exogenous ECM, it has been reported that cancer cells
themselves within tumorospheres are able to produce ECM components
like tenascin C to partially create their CSC niche [34]. Likewise, culture
medium can be supplemented with cytokines like SCF and G-CSF,
present in the bone marrow microenvironment, forming a metastatic
niche [156].
In general, the 3D local microenvironment in all spherical cancer
models could enhance cell survival through strong cell–cell contacts
and might, at least in the biggest ones, offer a hypoxic microenvi-
ronment favorable to cancer stemness [157]. Nevertheless, we
demonstrated using several human carcinoma cell lines that
MCTSs are not more tumorigenic than cell suspension when injected
into mice ([15] and personal data). However, the size of the injected
MCTSs (~ 150 μm) did not lead to a hypoxia microenvironment,
known to be associated with tumor development [158].
TDTSs have been reported to initiate tumors and to reproduce
characteristics of the original tumor [16,20,21,159]. Study of the
expression of potential CSC markers by colospheres and CTOSs has
shown that expression of CSC markers is maintained at a low level as
observed in the parent tumors. The CSC character is not lost during
culture, although colospheres remain largely differentiated. The
presence of several differentiated cell states is important because
cooperation between CSC and differentiated cells in drug resistance
mechanisms has been recently reported [160]. In contrast, MARY-X
spheroids contain a high percentage of cells expressing putative cancer
or normal stem cell markers related to the aggressive nature of
inflammatory breast cancer [159]. Similarly, OMSs can be cultured
without loss of tumorigenicity, even after cryopreservation [85].
Other Applications
MCTS have been used in many other applications, including
studies on hypoxia [161], tumor metabolism [162], penetration of
adenoviruses into tumors [163], and the effects of mechanical stress
upon tumor cells [164]. More complex sphere models have been
developed involving coculture with other cell types to study
interactions between the tumor and stroma in vitro. MCTSs can be
cultured with endothelial cells to study angiogenesis (for review, [65])
or with immune system cells (for review, [165]), such as dendritic
10 Cancer Spheres in Tumor Biology Weiswald et al. Neoplasia Vol. 17, No. 1, 2015

cells [166], macrophages [167], monocytes [63], and T lymphocytes
[61,62], for studying interactions between tumor cells and immune
cells. Moreover, MCTSs containing tumor cells and fibroblasts have
been generated to study influence of fibroblasts on the tumor
[168,169].The“minitumor spheroid model,”which is more
complex, includes endothelial cells, fibroblast and tumor cells [170].
Conclusion
We provide here a rational classification of the four most commonly
used spherical cancer models in cancer research: 1) the MCTS model,
described since the early 70s and obtained by culture of cancer cell
lines in nonadherent conditions; 2) tumorospheres, a model of CSC
expansion established in serum-free medium supplemented with
growth factors; and 3) TDTSs and 4) OMSs, obtained by tumor
tissue mechanical dissociation and cutting, respectively. These models
appear to closely resemble each other. Nevertheless, although they
share a common 3D conformation, these four models display their
own intrinsic properties. MCTSs arise from cell lines from all cancer
types and offer a very high level of reproducibility, but considerable
debate still continues to surround the usefulness and pertinence as
culture-adapted cell line models. Tumorospheres have been proven to
be an excellent model for enriching the CSC fraction but not for
studying intrinsic properties of CSCs related to their 3D architecture.
TDTSs and OMSs are relevant models for mimicking tumors, but
they are not susceptible to transfection and standardization. Finally,
depending on the study aim and the cancer type, the most relevant
sphere models must be carefully selected.
Acknowledgements
Work of the laboratory on spheres is supported by a Genevieve and
Jean-Paul Driot transformative research grant, a Philippe, Stéphanie
and Laurent Bloch cancer research grant, a Hassan Hachem
translational medicine grant, a Sally Paget-Brown translational
research grant, the Institut National du Cancer and Cancéropôle
Ile de France (COLOMETASTEM grant), and Groupement des
Entreprises Françaises dans la Lutte contre le Cancer (grant 5/188).
L.B.W. was supported by postdoctoral fellowships from the
Association pour la Recherche en cancérologie de Saint-Cloud
(ARCS) and the Canadian Institutes of Health Research (CIHR). We
thank Jerri Bram for her valuable help. We thank Jens M. Kelm,
Pierre Nassoy, Danijela Vignjevic, Connie R. Jiménez, and Uros
Rajcevic for sharing pictures of spherical cancer spheres.
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