A Novel In Vitro Model of Human Mesothelioma for
Studying Tumor Biology and Apoptotic Resistance
Ki-Up Kim*, Shannon M. Wilson*, Keith S. Abayasiriwardana, Rodney Collins, Lars Fjellbirkeland,
Zhidong Xu, David M. Jablons, Stephen L. Nishimura, and V. Courtney Broaddus
Lung Biology Center or Department of Pathology, San Francisco General Hospital, University of California San Francisco; and Cancer Research
Institute, UCSF Comprehensive Cancer Center, University of California San Francisco, San Francisco, California
Like many tumors, malignant mesothelioma exhibits significant
chemoresistance and resistance to apoptosis in vivo that is not seen
in current in vitro models. To study the mechanisms of this multicel-
lular resistance, biologically relevant in vitro models are necessary.
grown in vitro as tumor fragment spheroids. After 5–10 d in culture,
fragments from each of 15 human mesothelioma tumors rounded
into spheroids. The tumor fragment spheroids maintained multiple
characteristics of the original tumors for up to 3 mo including the
presence of viable mesothelioma cells, macrophages, and a collagen-
rich stroma. In 14-d-old spheroids, mesothelioma cells showed the
same proliferation rate and expression of a death receptor, DR5,
as in the original tumor. To determine responses to treatment,
we treated tumor fragment spheroids grown from three separate
tumors with agents, TNF-related apoptosis-inducing ligand (TRAIL)
plus cycloheximide, that induced near total apoptosis in three hu-
man mesothelioma cell lines (M28, REN, MS-1) grown as mono-
layers (94 ?6% apoptosis; mean ? SEM). Compared with mesothe-
lioma cells in monolayers, mesothelioma cells in the spheroids were
resistant to TRAIL plus cycloheximide (32 ? 4% apoptosis; mean ?
SEM). Apoptotic resistance of mesothelioma cells was significantly
reduced by inhibiting either the PI3K/Akt pathway with LY294002
(47 ? 6% apoptosis) or the mTOR pathway with rapamycin (50 ?
17% apoptosis). We conclude that human mesothelioma can be
maintained in vitro in a biologically relevant model that exhibits
apoptotic resistance, thereby permitting study of its tumor biology
and of novel approaches to therapy.
resistance; PI3K/Akt survival pathway; TNF-related apoptosis-inducing
ligand (TRAIL); tumor-associated macrophage; tumor fragment spheroid
collagen; death receptor DR5; mTOR; multicellular
Resistance to apoptosis, or programmed cell death, is now consid-
ered to be a critical step in the generation and maintenance of
cancer (1). Resistance to apoptosis may underlie the resistance
of tumors to chemotherapy and radiotherapy (2). Mechanisms
of resistance have been identified on a cellular level, for example
via P-glycoprotein efflux pumps, DNA repair mechanisms, or
from expression of anti-apoptotic proteins such as Bcl-2 (3).
Additional mechanisms of resistance are now recognized to
involve stimuli from the cell’s external environment, termed
multicellular resistance (3). These multicellular resistance mech-
anisms have been attributed to cell–cell contacts, cell–matrix
contacts, and the three-dimensional shape found in tissues but
(Received in original form November 14, 2004 and in final form July 30, 2005)
*These authors contributed equally to this work.
The research was supported by an NIH RO1 NCI-095671 grant (V.C.B.) and
a grant from the Norwegian Cancer Society (L.F.).
Correspondence and requests for reprints should be addressed to V. Courtney
Broaddus, M.D., Lung Biology Center, Box 0854 UCSF, San Francisco, CA 94143-
0854. E-mail: firstname.lastname@example.org
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2004-0355OC on August 25, 2005
Internet address: www.atsjournals.org
Vol 33. pp 541–548, 2005
not in monolayer cultures. Other mechanisms of multicellular
resistance may derive from resident nonmalignant cells, such as
tumor-associated macrophages (4). To study multicellular resis-
tance, systems other than monolayer cultures of highly selected
homogeneous immortal cell lines are needed.
Two types of in vitro models used to study the complex
resistancefound intumors aremulticellular spheroidsandtumor
fragment spheroids (5, 6). In the first, cells are allowed to grow
into three-dimensional structures called multicellular spheroids
(5). In these structures, certain features contributing to multicel-
lular resistance can be studied including cell–cell contact and a
three-dimensional architecture (7). These multicellular spher-
oids, though still artificial, can recreate a resistance not found
in monolayers and have been used particularly in studies of
resistance to radiotherapy (8). However, these structures still
lack much of the complexity of the original tumor because they
are generated from cell lines, themselves highly selected clonal
subpopulations derived from the original tumor cells, and they
contain none of the original stroma or other cell types found in
tumors. In the second major in vitro model, small fragments
of the original tumor tissue are allowed to grow into three-
dimensional structures called tumor fragment spheroids. In the
tumor fragment spheroid model, there is the potential for pre-
serving the original heterogeneity of the tumor, including the
actual,not selected,tumorcells, residentnonmalignant cells,and
the tumor extracellular matrix. In recent years, the interactions
have been recognized as important in cell growth, migration,
and differentiation, as well as in survival and resistance to apo-
ptosis (9, 10).
Tumor fragment spheroids have been generated successfully
mainly from glioblastoma (11, 12), meningioma (13, 14), head
and neck squamous tumors (15), and lung cancer (16). In several
markers and viability better than either monolayers or multicel-
lular spheroids (12–14, 17). In a few cases, the effect of different
treatments has been studied—for example, on the proliferation
or histology of the tumor (13, 18–20). We are not aware of
any study in which tumor fragment spheroids were specifically
examined for apoptosis, although this in vitro model has much
promise for such studies. Studies of in vitro tumor responses
to treatments may be particularly relevant in infrequent and
recalcitrant tumors in which novel treatments can be tested.
One such tumor, malignant mesothelioma, is an aggressive
cancer that has not responded to standard therapies (21) and is
considered highly resistant to apoptosis (22). Despite its increas-
ingfrequency, mesothelioma remains an unusual tumor and thus
difficult to study in large clinical trials. Cell lines and animal
models have greatly advanced the understanding of this tumor;
however, an in vitro human tumor model could be particularly
useful for screening and testing treatment strategies. An in vitro
mesothelioma modelderived from a patient’s tumor could possi-
bly be used to test targeted therapy designed for the individual
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Instudies usingmesothelioma cell linesgrownasmonolayers,
we havesuccessfullyovercomeapoptotic resistanceby activating
both the death receptor and DNA damage apoptotic pathways
(24, 25). Whereas activating either pathway alone failed to in-
duce apoptosis, activating the two pathways together produced
a synergistic apoptosis (24). The damaging agent sensitized the
mesothelioma cell lines to the death receptor ligand, TNF-
related apoptosis-inducing ligand (TRAIL), and induced syner-
gistic apoptosis via interactions at the level of the mitochondria
(25, 26). Because TRAIL, unlike other death receptor ligands
such as fas ligand and TNF, has been largely safe and effective
in in vivo studies (27), TRAIL is considered a potentially valu-
able agent for therapy (28). We thus wished to test this combina-
torial treatment approach using TRAIL in a more biologically
relevant model of mesothelioma.
In this study, we asked whether tumor fragment spheroids
could be generated from human mesothelioma tissue and
whether these spheroids could be used to study responses of
mesothelioma to treatment. Using tumors from 15 patients, we
developed approaches to allow spheroid formation in vitro and
examined the spheroids serially over a period of up to three
each tumor and maintained multiple characteristics of the origi-
nal tumor, including mesothelioma cell viability, proliferation,
and expression of the TRAIL receptor, DR5, as well as the
presence of tumor-associated macrophages and a collagen-rich
stroma. When compared with mesothelioma cell lines grown in
monolayer culture, the mesothelioma cells in tumor fragment
spheroids were resistant to apoptosis induced by the potent
combinatorial stimulus, TRAIL plus cycloheximide. Efforts to
block various anti-apoptotic pathways showed a contribution of
the PI3K and mTOR pathways to the resistance. We conclude
that human mesothelioma can be maintained as a complex, via-
ble, biologically relevant system that will expand opportunities
to study tumor biology and responses to therapy.
MATERIALS AND METHODS
Antibodies and Reagents
Antibodies were used at the indicated dilutions to detect cytokeratin
(1:200, AE1/AE3; DakoCytomation, Carpinteria, CA), calretinin
(1:200, M7245; DakoCytomation), Ki67 (1:200, MIB1; DakoCytoma-
tion), TRAIL death receptor-2 or DR5 (1:200; Calbiochem, San Diego,
CA),CD68, amacrophagemarker, (1:200,PG-M1 clone; DakoCytoma-
tion), cleaved caspase 3 (1:100, AB3623; Chemicon, Temecula, CA),
phospho-Akt (1:50, Ser473, 736E11; Cell Signaling, Beverly, MA), and
phospho-S6 kinase (1:500, Thr389 [1A5]; Cell Signaling). TRAIL (375-
TEC) was purchased from R&D Systems (Rosen, MN). Inhibitors,
LY294002 and rapamycin, were purchased fromSigma (St.Louis, MO).
MG132, a proteasome inhibitor, was purchased from EMD Biosciences
(San Diego, CA).
Single immunohistochemical staining was performed with the EnVi-
sion plus kit with peroxidase detection (DakoCytomation), and double
immunohistochemical staining with the EnVision Doublestain System
with peroxidase/alkaline phosphatase detection (DakoCytomation).
Control staining was performed with omission of the primary antibody
to confirm specificity for each antibody.
Formation of Tumor Fragment Spheroids
Fifteen freshly resected tumor samples were obtained under approval
from theCommittee on Human Research at the University of California,
San Francisco. Tumor pathology was later determined to be epithelial
(n ? 9), sarcomatous (n ? 4), or biphasic (n ? 2) by examination of
stained sections by one of our authors (S.L.N.). The tissue was asep-
tically transferred to a container containing Dulbecco’s modification of
Eagle’s medium (DMEM; Difco Laboratories, Sparks, MD) supple-
mented with 10% heat inactivated FCS, penicillin (100 IU/ml), and
(Fisher Scientific, Fair Lawn, NJ) and embedded in paraffin for histo-
For spheroid culture, tumor tissue was diced finely with scalpels to
pieces smaller than 1 mm in diameter that were suspended in medium
in 10-cm plates coated with 0.8% agar (Agar Noble; Sigma). Some
tumor fragments were maintained in DMEM and some in a mesothelial
cell–specific media, LHC-MM (Lechner and LaVeck medium; Bio-
source International, Camarillo, CA). The volume of overlay media
was 15 ml, and half the volume of the overlay media was changed twice
a week. Individual cells or clumps of cells obtained at the time of tumor
harvest were also plated onto uncoated 10-cm plates in an attempt to
grow cells in monolayer culture. The cultures were maintained at 37?C
in 5% CO2with 100% relative humidity. The agar-coated plates were
regularly observed using an inverted phase microscope during the incu-
bation period, up to a maximum of 3 mo. Spheroids were collected at
different time points, fixed in 10% formalin, and embedded in paraffin.
Tumor fragments grown in LHC-MM failed to form spheroids con-
sistently; therefore, we have limited our report to the spheroids grown
Identification and Characterization of
Mesothelioma Cells in Spheroids
To confirm the presence of mesothelioma cells, the tumor fragment
spheroids were stained for the mesothelioma markers, cytokeratin or
calretinin. In brief, after deparaffinization and rehydration, antigens
were retrieved by boiling in sodium citrate solution (pH 6.0) with 0.1%
Tween in a pressure cooker for 15 min. The slides were then cooled
at room temperature for 20 min. After antigen retrieval, sections were
blocked in hydrogen peroxide for 20min toremove endogenousperoxi-
dase. The primary antibody, anti-cytokeratin or anti-calretinin, was
applied for 1 h and the secondary antibody conjugated to a horseradish
peroxidase–labeled polymer was applied for 30 min and detected by
the 3?, 3?-diaminobenzidine tetrahydrochloride (DAB) method.
To assess expression of Ki67 or DR5 specifically in mesothelioma
cells, we used a double immunohistochemical staining approach (Dako-
Cytomation; Envision Doublestain System). After initial immunohisto-
chemical staining for Ki67 or DR5 with the peroxidase method as
described above, blocking was performed by protocol and the next
primary antibody, anti-cytokeratin, was applied, followed by a second-
ary antibody conjugated to an alkaline phosphatase–labeled polymer
and substrate chromagen (fast red).
Determination of Collagen and Macrophages in Spheroids
Tumor fragment spheroids of various ages werestained with hematoxy-
lin and eosin (H&E). Some were also stained using Gomori’s one-step
trichrome stain for collagen, by standard techniques.
Macrophages were detected using an antibody to CD68, a macro-
phage marker (29). To confirm that macrophages and mesothelioma
cellswereseparate populations,doublestainingforCD68and cytokera-
tin was also performed using the protocol described above.
Treatment of Spheroids with TRAIL plus Cycloheximide
At 14 d of culture, we selected rounded spheroids from three separate
tumor samples, and plated 15–20 in each well of a 6-well agar-coated
plate in new media for 24 h. Three human mesothelioma cell lines,
M28 (obtained from Dr. Brenda Gerwin, NCI), REN (obtained from
Dr. Roy Smythe, Texas A&M University), and MS-1 (obtained from
Dr. Steven Idell, University of Texas at Tyler) grown as monolayers,
were also studied in parallel. Mesothelioma cell lines were plated in
6-well plates the day before study to achieve either 60–70% confluence
or, in an attempt to maximize apoptotic resistance, to achieve complete
confluence. Tumor fragment spheroids and the mesothelioma cell
monolayers were then treated either with nothing (control) or with
agents that are known to induce synergistic and potent apoptosis,
TRAIL (5 ng/ml) plus cycloheximide (10 ?g/ml). Doses of TRAIL
are in the high range of clinically used concentrations (27, 30), while
cycloheximide, a protein synthesis inhibitor, is known to enhance
TRAIL-induced apoptosis (24). Because each agent alone failed to
induce significant apoptosis in mesothelioma cell monolayers in our
prior studies, TRAIL and cycloheximide were not used individually.
Initially, both the tumor fragment spheroids and the cells were treated
for 24 h. However, because the spheroids had no obvious apoptotic
Kim, Wilson, Abayasiriwardana, et al.: In Vitro Model of Human Mesothelioma 543
response, tumor fragment spheroids and cell monolayers were treated
for 48 h with two treatment doses, one given at 0 h and another at 24 h.
Data are reported for both monolayers and tumor fragment spheroids
treated identically for 48 h. In separate studies, additional spheroids
were treated with inhibitors of putative resistance pathways including
LY294002 (a PI3K inhibitor, 100 ?M), rapamycin (an mTOR inhibitor,
5 nM), or MG132 (a proteasome inhibitor, 100 ?M). These doses were
selected from separate experiments in cell lines or spheroids for the
ability to enhance the apoptosis of TRAIL or TRAIL plus cyclohexi-
mide without toxicity (data not shown). These agents were added to
spheroids alone or together with TRAIL and cycloheximide at the start
of the experiment and again at 24 h, for a total exposure of 48 h.
Tumor fragment spheroids were collected immediately after treat-
ment, fixed in 10% formalin, and embedded in paraffin. After rehydra-
tion and antigen retrieval as above, specimens were doubly stained for
immunofluorescent detectionofbothcleavedcaspase3and cytokeratin.
The slides were first incubated with a rabbit polyclonal antibody to
cleaved caspase 3 (Chemicon) followed by the primary murine mono-
clonal antibodies to cytokeratin. After washing, the cytokeratin was
detected with a secondary biotinylated sheep anti-mouse antibody
(Amersham Biosciences Corp., Piscataway, NJ), followed by washing,
and then a streptavidin-conjugated Oregon Green 488 (Molecular
Probes, Eugene, OR). The cleaved caspase 3 was separately detected
using a secondary goat anti-rabbit IgG conjugated with Alex Fluor 594
(Molecular Probes). Positive immunofluorescent staining for cytokera-
tin wasgreen andcleaved caspase3wasred. Cellstreatedasmonolayers
were stained with a nuclear stain, 4?,6-diamidino-2-phenylindole (DAPI;
Vector Laboratories, Burlingame, CA), and examined by microscopy for
the dense, shrunken nuclear morphology characteristic of apoptosis, as
previously described (24).
To confirm the activity of the survival pathways and their inhibition
by the kinase inhibitors, mesothelioma spheroids exposed to TRAIL
plus cycloheximide with and without inhibitors were stained for expres-
sion of p-Akt or p-S6 kinase, downstream targets of PI3K and mTOR,
respectively. Intensity of staining of individual cells was assessed by an
observer blinded to the experimental condition on a scale of 0–3 for
cytoplasmic staining. An average staining intensity for each spheroid
was calculated for each of five different spheroids from two tumors.
Fluorescent images were captured using a fluorescent microscope
(Zeiss, Gottingen, Germany) and image acquisition software (Spot Ad-
vanced, Chantilly, VA), with standardized acquisition times for the
different colors. The separate images were then merged. The gain and
contrast of each image was adjusted until the background was black.
Methods for Quantifying Proliferation,
DR5 Expression, and Apoptosis
For quantification purposes, images of stained spheroids were captured
as above, overlaid with a grid, and examined by independent observers
blinded to the experimental conditions.
To determine the proliferation rate of mesothelioma cells, three
observers analyzed spheroids from three different tumors. An average
of 300 cytokeratin-positive cells from between 5 and 10 spheroids were
counted from each tumor for each time point. Positivity for cytokeratin
was identified as pink (fast red; DakoCytomation) cytoplasmic staining
above background. Cytokeratin-positive cells with visible nuclei were
counted as Ki67-positive (brown) or Ki67-negative (not brown). The
mesothelioma cell proliferation rate was determined as the percentage
of all cytokeratin-positive cells with Ki67-positive nuclei.
To determine the frequency of DR5 staining, two observers exam-
ined 14-d-old spheroids grown from four different tumors compared
with the original tumor. An average of 300 cells from between 5 and
10 spheroids were counted for each tumor at each time point. Positivity
for DR5 was determined by brown cytoplasmic or membrane staining
above background. The total cell number was counted as the number
of DAPI-positive nuclei. The frequency of DR5 expression was deter-
mined as the percentage of all DAPI-stained cells with DR5 expression.
To determine the percentage of mesothelioma cell apoptosis, two
observers analyzed spheroids from three different tumors treated with
apoptotic agents in separate experiments. An average of 200 and at
least 100 cytokeratin-positive cells from between 10 and 20 spheroids
were countedfor each experimentalcondition in eachtumor.Cytokeratin-
positive cells were identified as having cell-specific green staining above
background. All cytokeratin-positive cells were counted as either non-
apoptotic (green only) or as apoptotic if the red staining for cleaved
caspase 3 exceeded background levels. Mesothelioma cell apoptosis
was calculated as the percentage of all cytokeratin-positive cells with
staining for cleaved caspase 3.
Statistical analysis was performed using ANOVA with post hoc analysis
using Tukey’s test for parametric data (e.g., apoptotic responses to
treatment) or using the Mann-Whitney rank sum testfor nonparametric
data (e.g., intensity of staining) using GraphPad Prism version 4.0 for
Windows (GraphPad Software, San Diego, CA). Data are represented
as mean ? SEM or SD as appropriate for at least three separate
are representative of at least three experiments.
Mesothelioma Can Be Cultured as Tumor
Spheroids could be grown from all mesothelioma cases. Most
tumor fragments became rounded into spheroids by 5–10 d.
of individual cells and clumps of cells obtained from minced
tumor tissue werealso plated on plasticdishes. However,despite
to proliferate. No tumor cells were successfully propagated in
By hematoxylin and eosin staining, tumor fragments were
shape as the cells appeared at the periphery (Figures 1A–1G). By
inverted phase microscopy, cells could be seen as a bright border
at the periphery of the spheroids (Figure 1H). At least for 3 mo,
spheroids maintained their shape and size. Fragments that failed
to form spheroids were found to be acellular (data not shown).
Mesothelioma Cells Are Identified within
Tumor Fragment Spheroids
The majority of cells in spheroids were found to be cytokeratin-
and calretinin-positive, strongly supporting their identity as me-
sothelioma cells (Figures 2A–2D). Staining for cytokeratin and
calretinin was sustained up to at least 3 mo in culture (data not
By Gomori trichrome staining, there was strong staining for
collagen in the original tumor and in spheroids (Figures 2E and
2F). In spheroids, collagen was most apparent in the central,
less cellular region (Figure 2F).
Mesothelioma Cell Proliferation Is Maintained in
Tumor Fragment Spheroids
Cells in both the original tumor samples and in the tumor frag-
ment spheroids were shown to be proliferating by virtue of
positive staining for Ki67, a nuclear proliferation marker. The
proliferation was determined specifically for the mesothelioma
cells by double staining for Ki67 and cytokeratin (Figures 3A
and 3B). Mesothelioma cell proliferation was demonstrated for
at least 4 wk, in an experiment using mesothelioma tumor frag-
ment spheroids from three different tumors (Figure 3C). The
rate of proliferation of mesothelioma cells in the spheroids was
not significantly different from that in the original tumor at any
time (Figure 3C). Mesothelioma cell proliferation could still be
demonstrated at 3 mo (data not shown).
Macrophages Are Detected in Tumor Fragment Spheroids
Both the original tumor and spheroids grown from those tumors
contained numerous CD68-positive cells (Figure 4). In double
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Figure 1. Tumor fragment spheroids form from human mesothelioma
tissue. By hematoxylin and eosin staining, the parental tumor is shown
(A), followed by tumor fragment spheroids at Days 1 (B), 3 (C), 5 (D),
7 (E), 10 (F), and 14 (G). Tumor fragment spheroids had consistently
formed by Day 10 and showed little change in size during growth in
culture. At 2 wk of age, a tumor fragment spheroid is shown with cells
around the margin seen as a bright halo (H). Bar, 50 ?m.
staining experiments, these CD68-positive cells and cytokeratin-
positive cells were shown to be two separate populations,
oma cells, respectively. Both cell populations could be found in
the original tumor (Figures 4A and 4C) and in the tumor frag-
ment spheroids (Figures 4B and 4D).
Expression of DR5 Is Maintained over 14 d
A major death receptor for TRAIL, DR5, was present in both
the original tumor and in the tumor fragment spheroids, in a
tochemical staining for DR5 and cytokeratin, DR5 co-localized
with mesothelioma cells (Figures 5C and 5D). Compared with
the original tumors, DR5 expression was not altered in the
spheroids. In four of the original tumors, DR5 was expressed
grown from these four different mesotheliomas, DR5 was ex-
pressed by 49 ? 13% of cells (P ? 0.05, n ? 4).
Mesothelioma Cell Monolayers Undergo Complete Apoptosis
with TRAIL plus Cycloheximide
Three mesothelioma cell lines (M28, REN, MS-1), grown to
60–70% confluence or to 100% confluence, were exposed to
nothing or to two doses of TRAIL plus cycloheximide over 48 h.
All cell lines showed complete apoptosis (94 ? 6%) as measured
Figure 2. Mesothelioma cells and collagen are identified in tumor frag-
ment spheroids. Mesothelioma cells were identified by immunohisto-
chemical stains for cytokeratin or calretinin. Shown is expression of cyto-
keratin (arrows) in a 2-wk-old spheroid (A) and a 6-wk-old spheroid (B)
from the same tumor, there is expression of cytokeratin (C) and of
calretinin (D) (arrows). By Gomori Trichrome staining, blue-green stain-
ing identifiesthecollagen-rich stroma present in boththe originalmeso-
thelioma tumor (E) and in the spheroids grown from this tumor at Day
14 (F). Bar, 50 ?m.
by characteristic nuclear morphology, compared with the un-
treated cells (6 ? 4%, mean ? SEM; n ? 3).
Mesothelioma Cells within Tumor Fragment Spheroids
Demonstrate Apoptotic Resistance
Tumor fragment spheroids were exposed to nothing or to two
doses of TRAIL plus cycloheximide over 48 h. By the use of
double staining for cytokeratin and cleaved caspase 3, apoptosis
could be identified in some, but not all, of the mesothelioma
cells in the treated spheroids (Figures 6A–6F). Apoptotic cells
were evenly distributed throughout the spheroid (data not
shown). Rapamycin appeared toincrease the apoptotic response
to TRAIL plus cycloheximide (Figures 6G–6I).
PI3K/Akt and mTOR Contribute to Apoptotic Resistance
Quantificationof apoptosisshowed that,incontrastto thenearly
complete apoptosis of the mesothelioma cell monolayers, TRAIL
plus cycloheximide induced apoptosis in 32 ? 4% (mean ?
SEM) of the mesothelioma cells within the spheroids versus
7 ? 1% apoptosis in untreated spheroids. Addition of a protea-
some inhibitor (MG132) had no effect on apoptosis (Figure 7A).
Rapamycin, an inhibitor of themTORpathway, hadno effect
on baseline apoptosis, but significantly increased apoptosis due
to TRAIL plus cycloheximide to 50 ? 17% (mean ? SEM)
(Figure 7A). LY294002, an inhibitor of the PI3K/Akt pathway,
increased the apoptosis in spheroids by itself and also enhanced
Kim, Wilson, Abayasiriwardana, et al.: In Vitro Model of Human Mesothelioma545
Figure 3. Mesothelioma cells proliferate within tumor fragment spher-
oids. By double immunochemical staining, cytokeratin-positive cells
(pink) could be examined for Ki67 staining (brown) so that proliferation
could be determined specifically for mesothelioma cells. Both the origi-
nal tumor (A) and a 10-d-old tumor fragment spheroid grown from it
from three tumors, proliferation of mesothelioma cells in the tumor
fragment spheroids over 4 wk was compared with that of the original
tumor. The proliferation rate was not significantly different from that
of the original tumor during the 4 wk. (Mean ? SD; n ? 3). Mesotheli-
oma cells continued to proliferate for at least 3 mo (data not shown).
the response to TRAIL plus cycloheximide to 47 ? 6% (mean ?
SEM) (Figure 7A).
For confirmation of the activity and the inhibition of these
survival pathways, spheroids exposed for 48 h to TRAIL plus
Figure 4. Macrophages are identified in tumor fragment spheroids.
Macrophages, staining with anti-CD68 (brown), were found in the
original tumors (A and C) and in tumor fragment spheroids (B and D)
grown from those tumors, shown at 2 wk (B) or at 3 mo of age (D)
(see arrows). The macrophages could be identified in close proximity
to the cytokeratin-positive mesothelioma cells (pink). Bar, 50 ?m.
Figure 5. MesotheliomacellsintumorfragmentspheroidsexpressDR5.
By staining for DR5 alone followed by a hematoxylin counterstain, DR5
was shown to be present in clusters of cells (arrow) in both the original
tumor (A) and in tumor fragment spheroids at Day 14 (B). By double
immunohistochemical staining for DR5 (brown) and cytokeratin (red)
without hematoxylin counterstaining, DR5 staining could be localized
(C) and the tumor fragment spheroid grown from it at Day 14 (D).
Bar, 50 ?m.
cycloheximide with or without inhibitors were stained for
p-Akt or p-S6 kinase, targets of the PI3K and mTOR pathways,
respectively. On a scale of 0–3 for intensity of cytoplasmic stain-
ing, the intensity of staining for p-Akt decreased significantly
with use of LY294002 from 2.5 ? 0.1 to 0.4 ? 0.2 (mean ?
SD, P ? 0.05) and the intensity of cell staining for p-S6 kinase
decreased with use of rapamycin from 2.9 ? 0.1 to 1.9 ? 0.1
(mean ? SD, P ? 0.05) (Figures 7B–7E).
Mesothelioma, a tumor with a high degree of resistance to ther-
apy, remains incurable despite aggressive therapy. Much of this
resistance to available chemotherapy or radiotherapy is thought
to be due to resistance to apoptosis (22). The mechanisms of
apoptosis are difficult to study in immortal cell lines, where
apoptotic mechanisms can be expectedto be altered significantly
compared with the original tissue. The complexity of the original
tumor is also lost in the selection of tumor cells for growth as
monolayers. Here, we have grown mesothelioma as fragments
in which the three-dimensional form of the tumor and its com-
plexity of cell types and extracellular matrix are maintained.
We have further shown that this in vitro model demonstrates
apoptotic resistance when compared with mesothelioma cell
lines in monolayer culture and thereby represents a more realis-
tic model of the tumor in vivo.
Tumor fragment spheroids retained many characteristics of
the original tumor, despite weeks to months in in vitro culture.
Inparticular, themesothelioma cells showedretention ofseveral
key features. First of all, mesothelioma cells in the spheroids
remained viable. Individual tumor cells or clumps of cells that
were plated on plastic on the first day of harvest never propa-
gated successfully, whereas tumor cells in spheroids remained
erate, at the same rate as in the original tumor, for at least
4 wk. Finally, the mesothelioma cells continued to express a
major TRAIL receptor, DR5, thus showing that the resistance
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Figure 6. Mesothelioma cell apoptosis can be identified
within spheroids. Spheroids were either not treated (A–C),
with TRAIL plus cycloheximide with rapamycin for 48 h
(G–I) and then stained for the presence of cytokeratin (green)
or cleaved caspase 3 (red). In the top panels (A, D, G), the
green represents cytokeratin, identifying the mesothelioma
cells; in the middle panels (B, E, H), red represents cleaved
caspase 3, identifying ongoing apoptosis; in the lower panels
tion of red with the green. Apoptotic cells can be identified
within the treated spheroids (E and H) and can be demonstrated
to be cytokeratin-positive in the merged images (F and I). Bar,
to exogenous TRAIL was not due to loss of TRAIL receptors.
In addition, the tumor fragment spheroids appeared to retain
key cell types and constituents. For example, it was interesting
that both the tumor fragment spheroids and the original meso-
of mesothelioma. In other tumors, tumor-associated macro-
phages havebeen recognized asimportant contributorsto tumor
Figure 7. Mesothelioma cell apoptosis within spheroids is enhanced by blocking PI3K/
Akt and mTOR pathways. (A) Apoptosis in treated spheroids quantified as the percent-
age of cytokeratin-positive cells with co-expression of cleaved, active caspase 3. Spher-
oids from three separate tumors were either not treated or treated with the strong
apoptotic stimulus, TRAIL plus cycloheximide, for 48 h. To determine a possible contri-
bution of known survival pathways, spheroids were also exposed to inhibitors of the
proteasome (MG132), the mTOR pathway (rapamycin), or the PI3K/Akt pathway
(LY294002). Apoptosis induced by TRAIL plus cycloheximide was enhanced by co-
treatment with rapamycin or with LY294002, while LY294002 also had an effect on
baseline apoptosis. (mean ? SEM; *different from untreated or inhibitor alone,†differ-
ent from TRAIL plus cycloheximide without inhibitor; P ? 0.05, n ? 3). (B–E) Immuno-
ness of inhibitors. In spheroids stained for p-Akt (B and C), spheroids treated with
TRAIL plus cycloheximide alone (B) demonstrated staining for p-Akt that was reduced
in spheroids co-treated with LY294002 (C). Similarly, in spheroids stained for p-
S6 kinase (D and E), spheroids treated with TRAIL plus cycloheximide alone (D)
demonstrated staining for p-S6 kinase, a target of mTOR, which was reduced in
spheroids co-treated with rapamycin (E).
biology, generally because of cytokine production that promotes
angiogenesis and supports the growth and survival of tumor cells
(4). The specificity of the CD68 marker for macrophages in our
study is supported by the use of an antibody to CD68 (clone
PG-M1) thought to be highly specific for macrophages (29); our
use of tissue, not cell culture (29); and the lack of staining for
cytokeratin in these cells. There still remained cell types not
staining for either marker that may represent fibroblasts or
Kim, Wilson, Abayasiriwardana, et al.: In Vitro Model of Human Mesothelioma 547
endothelial cells, cell types that have been identified in spheroids
from other tumors (31). Tumor fragment spheroids also contained
a large amount of collagen as in the original tumor. A collagen-
rich stroma has been shown to contribute to cell survival and
resistance to apoptosis in other models (32). The mesothelioma
tumor fragment spheroids retain a complexity representative of
theoriginal tumor,acomplexitythat constitutesbothachallenge
and an opportunity for study.
Mesothelioma may form similar structures in vivo, as has
been described in cytologic studies (33). Fragments of tumor
found in cytologic preparations of malignant pleural fluid are
strongly associated with malignancy, and are much more fre-
quently found in mesothelioma than in adenocarcinoma. These
fragments often have an internal structure composed of collagen
called a collagen core that, in one study, was found in 64% of
mesothelioma cases versus 4% of adenocarcinoma cases (34).
The source of these fragmentary tumor structures is unknown,
of papillary structures. This natural tendency of mesothelioma
to form tumor fragments may favor the formation of similar
structures in our in vitro culture system and explain the high
success rate of growing spheroids from this tumor.
not found in cell culture. First, because tissue presents a complex
mixture of cell types, identifying the cell of interest required use
of a specific biomarker. For apoptosis studies, this biomarker
should persist despite apoptosis. For our purposes, cytokeratin
was used as an identifying biomarker of mesothelioma cells, as
we have used successfully in an in vivo model of apoptosis in
the pleural space (35). Second, assays for apoptosis in tissue are
currently being reappraised. For example, TUNEL and the
in situ ligation assay may be nonspecific due to detection of
DNA strand breaks not solely due to apoptosis (36). Thus, we
used the detection of the caspase 3 cleavage fragment as a mea-
sure of activated “executioner” caspases and presumably an
irreversible and specific step in apoptosis (37).
Using this approach, we found that treatment induced apo-
ptosis in the mesothelioma cells within spheroids but that the
degree of apoptosis was significantly less than in cell lines grown
as monolayers. This resistance to apoptosis did not appear to
be due to reduced diffusion of the reagents into the spheroids
because resistant and apoptotic cells were equally distributed in
all regions of the spheroid and because the effect of the kinase
inhibitors on their targets was also homogeneous. Instead, we
found that resistance could be attributed in part to known sur-
Various survival or anti-apoptotic mechanisms have been
identified in tumors and shown to contribute to their survival
and resistance to therapy. We found that inhibitors of the PI3K
apoptosis in the human mesothelioma spheroids. Inhibition of
the proteasome did not enhance apoptosis in this system. The
PI3K/Akt pathway is a major survival pathway in several tumors
and has been shown to be active in mesothelioma cell lines
(38–40). mTOR, a kinase downstream of Akt, is best known as
a sensor of nutrients and a key initiator of cell cycle progression
and protein translation (41). A role in survival signaling has
been identified in overexpression studies in mice, where the
mTOR pathway was found to account for the major survival
effect of Akt (42). We found that inhibition of PI3K/Akt with
LY294002 increased apoptosis both as a single agent and to-
gether with TRAIL and cycloheximide. Inhibition of mTOR
did not increase apoptosis by itself but significantly increased
apoptosis due to TRAIL and cycloheximide in the human tumor
spheroids. This study is the first to our knowledge to show a
role for mTOR in mesothelioma. Because several inhibitors of
mTOR now exist and are entering clinical trials in oncology,
mTOR represents a potentially interesting target.
Despite efforts to maximize apoptosis in this system, a signifi-
Resistance may derive from interaction of mesothelioma cells with
the three-dimensional structure, the extracellular matrix, or the
products of nonmalignant cells within the tumor. This in vitro
model opens new avenues for the study of tumor biology and
resistance mechanisms active in human mesothelioma.
Conflict of Interest Statement: None of the authors have a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
Acknowledgments: The authors thank Dr. Kirk Jones for his assistance.
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