Prospective Identification of Glioblastoma Cells
Generating Dormant Tumors
Ronit Satchi-Fainaro1., Shiran Ferber1., Ehud Segal1, Lili Ma2, Niharika Dixit2, Ambreen Ijaz2,
Lynn Hlatky2, Amir Abdollahi2,3, Nava Almog2*
1Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel, 2Center of Cancer Systems Biology, Steward Research &
Specialty Projects Corp., St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Massachusetts, United States of America, 3Department of Radiation
Oncology, German Cancer Research Center and University of Heidelberg Medical School, Heidelberg, Germany
Although dormant tumors are highly prevalent within the human population, the underlying mechanisms are still mostly
unknown. We have previously identified the consensus gene expression pattern of dormant tumors. Here, we show that this
gene expression signature could be used for the isolation and identification of clones which generate dormant tumors. We
established single cell-derived clones from the aggressive tumor-generating U-87 MG human glioblastoma cell line. Based
only on the expression pattern of genes which were previously shown to be associated with tumor dormancy, we identified
clones which generate dormant tumors. We show that very high expression levels of thrombospondin and high expression
levels of angiomotin and insulin-like growth factor binding protein 5 (IGFBP5), together with low levels of endothelial
specific marker (ESM) 1 and epithelial growth factor receptor (EGFR) characterize the clone which generates dormant U-
87 MG derived glioblastomas. These tumors remained indolent both in subcutaneous and orthotopic intracranial sites, in
spite of a high prevalence of proliferating cells. We further show that tumor cells which form U-87 MG derived dormant
tumors have an impaired angiogenesis potential both in vitro and in vivo and have a slower invasion capacity. This work
demonstrates that fast-growing tumors contain tumor cells that when isolated will form dormant tumors and serves as a
proof-of-concept for the use of transcriptome profiles in the identification of such cells. Isolating the tumor cells that form
dormant tumors will facilitate understanding of the underlying mechanisms of dormant micro-metastases, late recurrence,
and changes in rate of tumor progression.
Citation: Satchi-Fainaro R, Ferber S, Segal E, Ma L, Dixit N, et al. (2012) Prospective Identification of Glioblastoma Cells Generating Dormant Tumors. PLoS
ONE 7(9): e44395. doi:10.1371/journal.pone.0044395
Editor: Javier S. Castresana, University of Navarra, Spain
Received February 16, 2012; Accepted August 3, 2012; Published September 6, 2012
Copyright: ? 2012 Satchi-Fainaro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Authors wish to thank Janusz Weremowicz and Clare Lamont for assisting with animal work. The project described was supported (in part) by Award
Number U54CA149233 from the National Cancer Institute (LH). The content is solely the responsibility of the authors and does not necessarily represent the
official views of the National Cancer Institute or the National Institutes of Health. This study was supported (in part) by grant no. 5145–300000 from the Chief
Scientist Office of the Ministry of Health, Israel, by the Israel Science Foundation (grant No. 1309/10), The Swiss Bridge Award and The Israel Cancer Research Fund
given to RSF. The authors disclose any commercial affiliations or financial interests that may be considered conflict of interest regarding this manuscript. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
A dormant phase during tumor progression is highly prevalent,
yet it is one of the most neglected areas in cancer research and the
associated biological mechanisms are still mostly unknown [1,2].
Cancer dormancy is a stage in which tumors are kept occult and
asymptomatic for a prolonged period of time [3,4]. It is present as
one of the earliest stages in tumor development, as micro-
metastasis in distant organs, and as minimal residual disease left
after surgical removal or treatment of primary tumors. Dormant
tumors are usually only a few millimeters diameter in size and are,
therefore, undetectable by most imaging technologies currently in
use [5,6]. They can, however, switch to become fast-growing,
clinically-apparent, and potentially lethal.
Since delayed disease recurrence, common in breast cancer,
colon cancer and other tumor types, can be explained by the
concept of tumor dormancy [7,8], eradicating dormant tumors is
currently a major challenge in cancer treatment [9–12]. Tumors
can remain occult and asymptomatic for years, or even decades,
while certain molecular and cellular mechanisms either halt, or are
insufficient to enable, tumor progression and mass expansion.
Clinical data and experimental models have led to the develop-
ment of the concepts of cellular dormancy [13–16] and tumor
dormancy [17–20]. Tumor cell dormancy is observed when
solitary disseminated cancer cells either circulate in the blood
system or settle at secondary sites, and is often associated with
quiescence. Whereas, tumor dormancy is observed when tumors,
as clusters of cells, do not expand in size over a long period of time.
Clearly, dormancy of cancerous lesions depends on crucial signals
from the microenvironment and the tumor stroma [4,16,18,21–
28]. Such signals can induce tumor cell quiescence. Alternatively,
systemic influences – such as the immune system of the host,
hormonal control, or the blockage or insufficiency of tumor
angiogenesis potential – can result in dormant tumors in which cell
proliferation is balanced by cell death.
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A lack of suitable experimental models and limited clinical
access to dormant tumors are two of the major obstacles in the
advancement of research on tumor dormancy . We have
previously established in vivo models of human breast cancer,
glioblastoma, osteosarcoma, and liposarcoma dormancy in severe
combined immunodeficient (SCID) mice [30,31]. These models
were all derived from human tumor cell lines isolated from cancer
patients and no artificial genetic modifications were made to
generate the cell lines that form dormant or fast-growing tumors
when injected into SCID mice. Tumor dormancy in these models
was associated with an impaired angiogenic potential resulting in a
delayed expansion of tumor mass. A high proliferation rate of
tumor cells in dormant tumors is balanced by apoptosis and cell
death. Using these models, we have shown that viable and
metabolically-active, non-angiogenic, microscopic dormant tu-
mors can reside in mice for very long periods of time until they
spontaneously switch to become fast-growing, angiogenic tumors
Next, we sought to identify the molecular determinants of
human tumor dormancy. Using genome-wide expression profiling
assays to compare gene expression profiles in dormant and fast-
growing tumors from our human breast cancer, glioblastoma,
osteosarcoma, and liposarcoma models, we looked for genes with
similar patterns of expression across all tumor types. The
consensus signature of human tumor dormancy was then
determined based on genes that were differentially expressed
between dormant and fast-growing tumors in the same pattern in
all tumor types analyzed . For example: in all dormant tumors,
high expression of thrombospondin and angiomotin with con-
comitant low expression of CD73 and epidermal growth factor
receptor (EGFR) were observed.
Tumor cells are well known to be heterogeneous with respect to
a wide variety of characteristics such as metastatic activity,
angiogenic potential, proliferation rate, and enzymatic activity
. Here, we set out to test whether the tumor dormancy gene
signature that we previously identified can be used for isolation of
tumor cells that will form non-angiogenic dormant tumors. Hence,
this approach can lead to further and deeper understanding of the
molecular mechanisms underlying human tumor dormancy.
Single cell derived clones were generated using a limiting
dilution method from the parental U-87 MG human glioblastoma
cell line. Thirteen clones were chosen according to similar rapid
kinetics of colony formation in tissue culture wells. RNA was
extracted from each clone and the relative expression level of
Thrombospondin (TSP-1), a well-known endogenous inhibitor of
angiogenesis that has been shown to be elevated in all dormant
tumors , was determined using real time PCR (Fig. 1A). When
compared with the expression level of the parental U-87 MG cell
line, most (10 out of 13) of the clones had lower TSP-1 expression,
while only 3 clones (#1, #2 and #6) had elevated TSP levels.
Clone #1 had a significant increase in TSP level (over 25-fold
higher expression than in parental U-87 MG cell line).
Since a high TSP level could suggest slow kinetics of tumor
growth, we chose to focus our analysis on three clones with varying
TSP levels: Clone #1, with the highest TSP level, Clone #6 with
an intermediate TSP level, and Clone #7 with a very low TSP
level (marked by arrows in Fig. 1A). We postulated that Clone #1
might generate dormant tumors, whereas clones #6 and #7
would generate intermediate or fast-growing tumors, respectively.
We then continued to analyze the expression levels of several
tumor dormancy-associated genes we had previously identified
. First, we tested the expression levels in the three clones of the
additional genes that were previously shown to be upregulated in
dormant tumors (Fig. 1B). Clearly, angiomotin (Amot) and
IGFBP5 levels were upregulated only in Clone #1. Notably,
IGFBP5 expression was around 1000-fold higher than in the
parental U-87 MG cell line. Expression of TGF-b2 was upregu-
lated in all clones tested.
Genes previously shown to be elevated in fast-growing tumors
were expected to be observed as downregulated in tumor cells that
form dormant or slow-growing tumors. Such downregulation was
indeed observed in Clone #1 for CD73, EGFR, and most
significantly for ESM-1 (Fig. 1B), strengthening our prediction that
this clone could generate dormant tumors.
Tumor growth patterns were then analyzed in SCID mice.
Equal numbers of cells were injected subcutaneously (s.c.) from
each clone and from the parental U-87 MG cell line, and tumor
growth was monitored (Fig. 2A). As expected, the parental U-
87 MG cells generated very small tumors (volume below
100 mm3), which after 3–4 weeks initiated rapid growth. Similar
‘bi-phasic’ growth kinetics were observed for tumors generated
from clones #6 and #7. Although Clone #6, which had an
intermediate level of TSP, initially formed tumors larger than the
parental cell line, its tumors grew slower in the rapid growth phase.
Clone #7, which had a very low level of TSP, formed tumors
smaller than those generated by the parental cell line or by Clone
#6. Importantly, Clone #1 formed dormant tumors which
remained indolent and were barely detectable by gross examina-
tion throughout the experiment (Fig. 2B). This confirmed our
hypothesis that the parental U-87 MG cell line contains cells
which when isolated will form dormant tumors, and that Clone
#1 was generated from such cells.
At the end point of the experiment, tumors generated by clones
#6 and #7 were clearly smaller than those generated by the
parental U-87 MG cells. Although smaller in mass, Clone #6 and
Clone #7 tumors were highly vascularized and tightly capsulated,
similar to tumors generated from parental U-87 MG cells (Fig. 2C).
In contrast, tumors generated from Clone #1 could be detected
only after flipping the skin and seemed avascular. These tumors
were occasionally found attached to the muscle tissue instead of
the skin, like most tumors from U-87 MG, Clone #6, and Clone
#7 (Fig. 2C).
The fate of indolent tumors generated by Clone #1 was
analyzed by following their tumor growth over a prolonged period
of time lasting more than 200 days (Fig. S1). As expected, while U-
87 MG tumors grew rapidly in the first 3–4 weeks after
inoculation, tumors from Clone #1 remained undetectable for
over 70 days. Three of the four tumors from Clone #1 eventually
emerged from dormancy and initiated growth at 81, 122, and 127
days post inoculation (Fig. S1). One mouse injected with Clone #1
cells never developed any detectable tumors during the 270 days of
the experiment (data not shown). Tumors that originated from
Clone #1 cells remained at the site of injection in a constant small
size without expanding in mass for a long period of time (i.e.,
dormant). Importantly, once these tumors emerged from dorman-
cy and started growing, the growth rate could be as rapid as in the
parental U-87 MG cell line derived tumors.
To evaluate tumor properties in their orthotopic microenviron-
ment in a non-invasive manner, both Clone #1 and U-87 MG
parental cells were infected with mCherry as previously described
. Then, in order to assure that the infection did not alter tumor
characteristics, SCID mice were inoculated s.c. with either cell
line, and dormancy periods were monitored and compared.
Tumors generated by cells from Clone #1 remained dormant and
avascular for more than 70 days, while tumors generated from the
Gene Expression Pattern of Dormant Glioblastomas
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Figure 1. Gene expression analysis of single cell-derived clones from U-87 MG glioblastoma cell line. All RT-PCR measurements were
normalized according to expression in the parental U-87 MG cell line. A. Thrombospondin-1 (TSP-1) relative level in U-87 MG derived clones. B.
Gene Expression Pattern of Dormant Glioblastomas
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parental U-87 MG cell line were highly vascularized and palpable
20 days following inoculation (Fig. 3A–3B). Following the escape
from dormancy, mCherry-labeled Clone #1 tumors showed a
similar tumor growth rate pattern to mCherry-labeled U-87 MG
tumors (Fig. 3A–3B).
CellvizioH imaging of the vasculature of U-87 MG tumors
revealed enlarged, highly tangled, and non-continuous vessels with
wider lumen and blunt ends, leakage, and sluggish blood flow.
These are typical signs of the enhanced permeability and retention
(EPR) effect phenomenon for macromolecules such as Dextran-
FITC at 70 kDa size used here (Fig. 3C). In contrast, blood vessels
in Clone #1 tumors at a size of 2 mm3are almost non-appearing.
For comparison, normal vessels in healthy tissues adjacent to these
tumors are shown to have continuous blood flow and a regular
shape with anastomosis.
To compare the angiogenic potential, in vivo analysis of size-
matched tumors (,2 mm3) from U-87 MG and Clone #1 was
performed (Fig. 4). The presence of the tumors at the site of
injection was validated by the mCherry fluorescent signal and later
by H&E staining (Fig. 4A–4B). U-87 MG tumors were signifi-
Expression level of genes previously shown to be upregulated in dormant tumors is shown on the left panel. Expression level of genes previously
shown to be upregulated in fast-growing tumors is shown on the right panel.
Figure 2. Comparison of tumor growth patterns and characteristics. A. Tumor growth kinetics of U-87 MG derived clones in SCID mice. B.
Tumor size of U-87 MG clones 40 days following tumor cell inoculation. While tumors generated from U-87 MG parental cell line, Clone #6, or Clone
#7 were clearly detected, the tumors generated from Clone #1 were undetectable by gross examination. Tumor growth analysis comparing U-
87 MG and Clone #1 derived tumors was repeated in independent settings three times. C. Tumors generated from the U-87 MG parental cell line
and derived clones 47 days after injection. Scale bars represent 1 cm.
Gene Expression Pattern of Dormant Glioblastomas
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cantly more vascularized compared with size-matched tumors
generated from Clone #1 cells, as observed by gross examination
of the tumors after flipping the skin, by analysis of CD34
positively-stained cells and by presence of blood vessels in H&E
staining (marked with arrows) in the U-87 tumor sections (Fig. 4A–
4B and Fig. S2). Microbubbles contrast-enhanced ultrasound (US)
Figure 3. Tumor growth patterns of mCherry-labeled tumor cells. A. Size of U-87 MG (red, n=3) and Clone #1 (black, n=3) tumors
measured by caliper (left panel) and by non-invasive CRI MaestroTMimaging system (right panel). B. Forty days post subcutaneous inoculation of
mCherry-labeled U-87 MG and Clone #1 cells into SCID mice, U-87 MG cells established vascularized and palpable tumors (,1200 mm3), whereas
Clone #1 tumors remained avascular and non-palpable (left panel), but detectable by non-invasive CRI MaestroTMimaging system (right panel). C.
Fiber confocal microscopy imaging of U-87 MG and Clone #1 tumor vasculature, as well as abdominal blood vessels, on day 40.
Gene Expression Pattern of Dormant Glioblastomas
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