Human embryonic stem cell microenvironment
suppresses the tumorigenic phenotype
of aggressive cancer cells
Lynne-Marie Postovit*, Naira V. Margaryan, Elisabeth A. Seftor, Dawn A. Kirschmann, Alina Lipavsky,
William W. Wheaton, Daniel E. Abbott, Richard E. B. Seftor, and Mary J. C. Hendrix†
Program in Cancer Biology and Epigenomics, Children’s Memorial Research Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60614
Communicated by Richard D. Klausner, The Column Group, Seattle, WA, January 21, 2008 (received for review July 1, 2007)
Embryonic stem cells sustain a microenvironment that facilitates a
balance of self-renewal and differentiation. Aggressive cancer
cells, expressing a multipotent, embryonic cell-like phenotype,
engage in a dynamic reciprocity with a microenvironment that
promotes plasticity and tumorigenicity. However, the cancer-
associated milieu lacks the appropriate regulatory mechanisms to
maintain a normal cellular phenotype. Previous work from our
laboratory reported that aggressive melanoma and breast carci-
noma express the embryonic morphogen Nodal, which is essential
for human embryonic stem cell (hESC) pluripotency. Based on the
aberrant expression of this embryonic plasticity gene by tumor
cells, this current study tested whether these cells could respond to
regulatory cues controlling the Nodal signaling pathway, which
might be sequestered within the microenvironment of hESCs,
resulting in the suppression of the tumorigenic phenotype. Spe-
cifically, we discovered that metastatic tumor cells do not express
the inhibitor to Nodal, Lefty, allowing them to overexpress this
embryonic morphogen in an unregulated manner. However, ex-
posure of the tumor cells to a hESC microenvironment (containing
Lefty) leads to a dramatic down-regulation in their Nodal expres-
sion concomitant with a reduction in clonogenicity and tumori-
with the secretion of Lefty, exclusive to hESCs, because it is not
detected in other stem cell types, normal cell types, or tropho-
blasts. The tumor-suppressive effects of the hESC microenviron-
ment, by neutralizing the expression of Nodal in aggressive tumor
cells, provide previously unexplored therapeutic modalities for
Lefty ? Nodal ? melanoma ? breast carcinoma
phenotype of stem cells and cancer cells is profoundly influenced
by the microenvironment. During embryogenesis, precursor cells
are specified to particular fates through the delivery of signaling
molecules, and malignant cells similarly release and receive cues
that promote tumor growth and metastasis. There also is a con-
vergence between cancer cells and stem cells in the molecular
messengers they implement to regulate self-renewal and cell fate.
These stem-cell-associated factors include members of the Notch,
(1–7). Particularly noteworthy are recent findings involving Nodal,
an embryonic morphogen belonging to the TGF-? superfamily (4).
Overexpression of Nodal prevents hESC differentiation, and in-
hibiting Nodal signaling in metastatic melanoma cells results in
Furthermore, we have shown that the Nodal secreted by metastatic
melanoma cells is functional, as demonstrated by the induction of
Nodal-responsive genes and ectopic tissues in embryonic zebrafish
transplanted with these tumor cells (4).
etastatic cancer cells resemble stem cells in their ability to
self-renew and to derive a diverse progeny. Moreover, the
The multipotent phenotype of metastatic cancer cells permits
them to respond to cues normally restricted to developmental
processes (1). Hence, we hypothesized that embryonic microenvi-
ronments, which are inherently permissive to normal stem cell
differentiation, may be used to reprogram (i.e., redifferentiate)
cancer cells toward a benign phenotype. Indeed, embryonic mi-
croenvironments have been shown to inhibit the tumorigenicity of
a variety of cancer cell lines (1, 9–11). For example, the mouse
to a nontumorigenic phenotype capable of differentiating into
normal tissues (11), and extracts derived from zebrafish embryos
cell types. Moreover, we have demonstrated that exposure of
metastatic melanoma cells to an embryonic zebrafish microenvi-
ronment, before gastrulation, results in their reprogramming to-
ward a nontumorigenic phenotype (12, 13) and that metastatic
melanoma cells transplanted into developing chick embryos are
capable of following neural crest migration pathways, resulting in a
loss of tumorigenicity and the acquisition of a neural crest-like
To elucidate the mechanisms by which embryonic microenvi-
ronments reprogram cancer cells to a more differentiated, less
aggressive phenotype, so that humanized therapeutic modalities
the capacity of hESC-derived factors to epigenetically influence
metastatic cancer cells (1, 14). Using this approach, we previously
determined that exposure of melanoma cells to a hESC microen-
vironment results in the reexpression of melanocyte-specific mark-
ers (illustrative of redifferentiation) and a reduction in invasive
potential (1, 14). However, the molecular underpinnings of the
elusive. In the current study, we have discovered that hESC
microenvironments suppress the tumorigenic phenotype of human
is exclusive to hESCs and not other stem cell types derived from
amniotic fluid, cord blood, or adult bone marrow. Mechanistically,
the hESC microenvironment specifically neutralizes the aberrant
expression of Nodal in metastatic melanoma and breast carcinoma
Author contributions: N.V.M. and E.A.S. contributed equally to this work. L.-M.P., E.A.S.,
R.E.B.S., and M.J.C.H. designed research; L.-M.P., N.V.M., E.A.S., D.A.K., A.L., W.W.W.,
D.E.A., and R.E.B.S. performed research; L.-M.P., E.A.S., and R.E.B.S. contributed new
and L.-M.P., R.E.B.S., and M.J.C.H. wrote the paper.
The authors declare no conflict of interest.
*Present address: Department of Anatomy and Cell Biology, Schulich School of Medicine
and Dentistry, University of Western Ontario, Medical Sciences Building, Room 438,
London, ON, Canada N6A 5C1.
†To whom correspondence should be addressed at: Children’s Memorial Research Center,
Feinberg School of Medicine, Northwestern University, 2300 Children’s Plaza, Box 222,
Chicago, IL 60614-3394. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
March 18, 2008 ?
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no. 11 ?
cells, reprogramming them to a less aggressive phenotype exem-
plified by diminished clonogenicity and tumorigenicity (with apop-
tosis). Moreover, we uncovered hESC-secreted Lefty (an inhibitor
of Nodal signaling) as an important mediator of these phenomena.
The microenvironment of hESCs provides a previously unexplored
therapeutic entity for the regulation of aberrantly expressed em-
bryonic factor(s) in aggressive tumor cells.
Characterization of Nodal Signaling Pathway Members in Human
Normal Cells, Metastatic Cancer Cells, Human Embryonic Stem Cells
(hESC), and Other Stem Cell Types. To better characterize the role of
Nodal in the maintenance of pluripotency and tumorigenicity, we
examined the expression of key components of the Nodal signaling
pathway in human normal, neoplastic and stem cell types. Western
blot analyses revealed that in a manner similar to hESCs (MEL-2,
H1, and H9), metastatic melanoma (C8161) and breast carcinoma
(MDA-MB-231) cells express Nodal protein at ?48 kDa (Fig. 1A).
myoepithelial cells (Hs 578 Bst), and primary human mammary
epithelial cells (HMEpC)], in which Nodal was not detected. Using
RT-PCR, we determined that Nodal is expressed in additional
melanoma (WM278), and breast carcinoma (T47D, MDA-MB-
468, MDA-MB-330, and ZR75–30) cell lines [supporting informa-
tion (SI) Fig. 5A]. Nodal propagates its signal by binding to a
receptor complex comprised of Cripto and a heterodimer of type
I (ALK 4/7) and type II (ActRIIB) activin-like kinase receptors.
Genetic studies in zebrafish and mice have determined that Cripto
directly associates with ALK 4 and Nodal and that these associa-
tions facilitate the ability of Nodal to propagate its signal (15, 16).
Using Western blot analysis and immunofluorescence microscopy,
we determined that hESCs uniformly express high levels of Cripto
at ?35 kDa; however, only a low level of Cripto was heteroge-
neously expressed in C8161 and MDA-MB-231 cells (Fig. 1 A and
B). Moreover, only one additional melanoma line (WM278) and
two additional breast carcinoma lines (T47D and ZR75–30) exam-
the TGF-? superfamily, spatially and temporally antagonize Nodal
in embryological systems (17). Moreover, the Lefty genes are
downstream targets of Nodal signaling, thereby providing a pow-
erful negative-feedback loop for this pathway (17). By using West-
ern blot analysis we determined that, in accordance with previously
published accounts of Lefty protein processing patterns, hESCs
express Lefty protein at ?42, 34 and 28 kDa (17, 18). In contrast,
Lefty is not expressed by metastatic breast carcinoma and mela-
noma cells or by corresponding normal somatic cell types (Fig. 1A
and SI Fig. 5A). To visualize the localization of Lefty and Nodal in
hESC colonies and their microenvironment, we performed immu-
nofluorescence localization with confocal microscopy. Using this
method, we determined that Lefty protein localizes to the areas
where hESCs contact the underlying Matrigel matrix, and that
hESC-derived Lefty permeates into this microenvironment (Fig.
1C). This is in contrast to Nodal protein, which localizes to the
surface of hESC colonies, and is secreted into the media. These
results were confirmed with Western blot analyses, which demon-
strated that Lefty protein can be detected in Matrigel conditioned
by H9 hESCs (H9 CMTX; Fig. 1C). Furthermore, neither Nodal
nor Lefty is found in unconditioned control Matrigel alone (data
We next sought to elucidate the expression of Nodal, Lefty, and
Cripto in other human stem cell types and in first trimester human
cytotrophoblast cells (HTR-8/SVneo). Western blot analyses re-
vealed that umbilical-cord-derived mesenchymal stem cells (MSC;
SC00125) and adult MSCs do not express Nodal and Cripto, and
that although amniotic-fluid-derived stem cells (GM00473,
GM00957A) and cytotrophoblast cells express Cripto, only the
latter expresses appreciable levels of Nodal (SI Fig. 5B). Of note, in
contrast to hESCs, none of the other stem cell lines examined
expressed an appreciable level of Lefty protein (SI Fig. 5B). To
analyses of Nodal, Lefty, and Cripto in: MEL-2, H1, and H9 hESCs; C8161, human
metastatic melanoma cells; normal human melanocytes; MDA-MB-231, human
metastatic breast carcinoma cells; Hs 578 Bst normal human myoepithelial cells;
and HMEpC normal human mammary epithelial cells. Actin is used as a loading
control. (B) Immunofluorescence localization of Cripto (red) in ?100% of H9
counterstained blue with DAPI. (Scale bar: 10 ?m.) (C) Immunofluorescence
with its underlying matrix. Dashed line designates the cell-matrix interface, and
on the right illustrate Lefty and Nodal at the cell surface (arrow) and the cell-
colonies stained with Lefty (distributed in the matrix microenvironment) and
model outlining how hESCs may reprogram metastatic cancer cells by inhibiting
Nodal signaling. Nodal initiates a signaling cascade by binding to a receptor
complex consisting of Cripto, type I (ALK 4/7), and type II (ActRIIB) activin-like
antagonizes the Nodal signaling pathway by interacting with Nodal and/or
Cripto. Like hESCs, cancer cells also express Nodal, whereas unlike hESCs they do
not express Lefty. HESC-derived Lefty, found in hESC conditioned matrices
ming toward a less malignant phenotype. Other tumor suppressive factors also
may be deposited by the hESCs.
www.pnas.org?cgi?doi?10.1073?pnas.0800467105Postovit et al.
confirm the apparent exclusivity of Lefty expression to hESCs, we
performed real-time RT-PCR. Using this method, we determined
and trophoblast cells do not express appreciable levels of Lefty
study expressed a comparably insignificant level of Lefty (SI Fig.
5C). We previously demonstrated that Nodal expression is posi-
tively correlated with melanoma progression, such that Nodal
protein is not expressed in normal melanocytes or radial growth
phase melanomas, but is present in vertical growth phase and
metastatic lesions (4). The current study characterized Nodal
localization (previously unexplored) in breast tissues: Using immu-
nohistochemical analysis of a human breast tissue microarray
(TMA) we observed that Nodal protein is absent in normal breast
tissue, and that its expression is positively correlated with breast
cancer progression (P ? 0.05; SI Fig. 5D and SI Table 1). Given the
significant observation that like hESCs, cancer cells express Nodal,
although unlike hESCs, they do not express Lefty, we hypothesized
that hESC-derived Lefty and possibly other tumor-suppressive
factors found in hESC-conditioned matrices (CMTX), may inhibit
Nodal signaling in cancer cells (Fig. 1D). We further proposed that
reprogram these tumor cell types toward a less tumorigenic
Exposure of Cancer Cells to hESC-Derived Factors, Particularly Lefty,
Results in Decreased Nodal Expression Concomitant with Reduced
Clonogenicity. To examine the effect of hESC-derived Lefty on
tumor cell phenotype, we used Dynabeads covalently coupled to
anti-Lefty antibody to isolate Lefty from hESCs cultured on a
feeder-free Matrigel matrix. The specificity and utility of the
Western blot analysis and corresponding Coomassie stained SDS/
PAGE of known rLefty-A and -B standards whose sequences were
subsequently confirmed by liquid chromatography/mass spectrom-
etry (LC/MS; SI Fig. 6A). Purified hESC-derived Lefty was seeded
into Matrigel and the effects of the ‘‘Lefty-containing’’ matrix on
cancer cell phenotype were determined. Western blot analysis
revealed that hESC-derived Lefty significantly diminished Nodal
protein expression in C8161 and MDA-MB-231 cells (Fig. 2A).
To further substantiate the specificity of these results, the cancer
cells were also exposed to Matrigel conditioned by hESCs in which
Lefty protein expression was knocked down with FITC-tagged
Morpholinos specific for Lefty-A and Lefty-B (MOLEFTY). [The
fluorescently tagged Morpholinos could be detected microscopi-
cally in ?75% of the hESC colonies treated (SI Fig. 6B Upper), and
Western blot analysis confirmed the efficient knock down of Lefty
protein in hESCs for up to 3 days (SI Fig. 6B Lower)]. The
expression of Oct-3/4 and Nanog, representative of pluripotency,
was not affected during this time, and morphology of the hESC
colonies was not altered (SI Fig. 6C). Thus, although MOLEFTY
knocked down Lefty protein in the hESCs, it did not induce stem
analysis revealed that C8161 and MDA-MB-231 cells up-regulate
Nodal protein expression in response to this ‘‘H9 Lefty-deficient’’
mRNA expression (SI Fig. 6D), and hESCs treated with MOLEFTY
in accordance with the positive feed-back loop that characterizes
the Nodal signaling pathway. In contrast to rNodal, which has
Lefty protein (rLefty) is unable to inhibit Nodal signaling (17). We
confirmed these results and extended them to discover that
rLefty-B can inhibit Nodal protein in C8161 cells, but at a non-
physiological dose (1,000 ng/ml) (SI Fig. 6E). In an effort to
understand the disparate results between hESC-derived Lefty and
rLefty on Nodal signaling, we analyzed glycoprotein content in
rLefty-B, rLefty-A and a lysate from the H9 hESCs plus their
CMTX. We determined that in an apparent contrast to the rLefty
proteins, H9-derived Lefty is glycosylated (SI Fig. 6F).
As a functional correlate, we determined that exposure of C8161
and MDA-MB-231 cells to H9 Lefty-derived matrix significantly
reduces anchorage-independent growth, and that this inhibition of
Nodal (rNodal; 100 ng/ml) (Fig. 2C). Furthermore, exposure of
C8161 and MDA-MB-231 cells to H9 CMTX reduced their ability
to undergo anchorage-independent growth but this inhibition of in
vitro clonogenicity was only partially rescued by the inclusion of
rNodal (100 ng/ml) (Fig. 2D). In addition, hESC supernatant
inhibited anchorage-independent growth, even when Lefty was
decreased via the aforementioned Dynabead isolation, although
not to the same level as purified Lefty. Collectively, these findings
suggest that hESC-derived Lefty has important anti-tumorigenic
potential, but that it is not the only tumor suppressive factor within
this unique embryonic microenvironment.
We next determined the effects of H9 CMTX on Nodal expres-
that exposure to H9 CMTX down-regulates Nodal mRNA expres-
sion in both C8161 and MDA-MB-231 cells, and that this effect is
reversible over time (Fig. 3A). Exposure to H9 CMTX similarly
sion concomitant with reduced anchorage-independent growth. (A) Western
purified from hESCs (H9-derived Lefty). MDA-MB-231 cells were allowed to
recover on fresh Matrigel for 2 days before analysis. Actin is used as a loading
MOControlor MOLeftyand Nodal protein levels in C8161 and MDA-MB-231 cells
cultured for 3 days on Matrigel conditioned by hESCs treated with either
MOControlor MOLefty. Numbers represent percentage change between MOControl
and MOLeftygroup determined by using densitometric analysis. (C) Relative
colony formation of C8161 and MDA-MB-231 cells cultured on soft agar for 14
days after 3 days of exposure to control Matrigel, Matrigel seeded with Lefty
purified from hESCs (H9-derived Lefty), or Matrigel seeded with Lefty-reduced
ng/ml). Bars represent mean normalized colony formation ? SD, and values
indicated by an asterisk (*) are significantly different from the colony-forming
MDA-MB-231 cells cultured on soft agar for 14 days after 3 days of exposure to
control Matrigel or to H9 CMTX. Assays were conducted in the presence or
absence of rNodal (100 ng/ml). Bars represent mean normalized colony forma-
tion ? SD, and values indicated by an asterisk (*) are significantly different from
the colony-forming ability of control cells whereas values indicted by a double
cells and H9 CMTX treated cells (n ? 12, P ? 0.05).
Exposure of cancer cells to hESC-derived Lefty decreases Nodal expres-
Postovit et al.PNAS ?
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decreases Nodal protein expression in these cells (SI Fig. 7A).
Exposure of C8161 cells to matrices conditioned by adult bone-
marrow-derived stem cells (MSC), amniotic-fluid-derived stem
cells (GM00473, GM00957A), or cytotrophoblast cells (HTR-8/
and SI Fig. 7B), thus illuminating the exclusivity of the epigenetic
influence of the hESC microenvironment.
The Microenvironment of hESCs Inhibits Melanoma and Breast Carci-
examine the effects of the hESC microenvironment on the in vivo
tumorigenicity of melanoma and breast carcinoma cells. Exposure
of C8161 and MDA-MB-231 cells to H9 CMTX resulted in a
to unconditioned Matrigel (Fig. 4 A and B). To establish a mech-
anism for the reduction in tumorigenicity, we examined the effects
of this treatment on in vivo tumor cell proliferation and apoptosis.
Using immunohistochemical staining for Ki67 as a measure of
proliferation, and terminal deoxynucleotidyl transferase biotin-
dUTP nick-end labeling (TUNEL) as a measure of apoptosis, we
found that exposure to hESC CMTX decreased proliferation and
increased apoptosis in C8161 and MDA-MB-231 cells (Fig. 4C and
SI Fig. 8). These in vivo data confirm the tumor suppressive effects
cancer cells and implicate the potential involvement of apoptotic
documented the ability of embryonic microenvironments to repro-
gram cancer cells toward a benign phenotype (1, 19); however, the
mechanisms underlying this phenomenon have remained largely
illusive. This study applied an in vitro hESC model to specifically
demonstrate the anti-tumorigenic properties of the human embry-
onic microenvironment, and the possible underlying mechanisms.
Of significance, our results indicate that: (i) The tumor suppressive
effects of the microenvironment are exclusive to hESCs, but not
other stem cell types; (ii) the hESC microenvironment neutralizes
the expression of the embryonic morphogen Nodal in metastatic
melanoma and breast carcinoma cells, reprogramming them to a
less aggressive phenotype exemplified by diminished clonogenicity
and tumorigenicity (with increased apoptosis); and (iii) Lefty, an
inhibitor of the Nodal signaling pathway, is secreted into the hESC
microenvironment and is an important mediator of these tumor-
suppressive effects. These findings illuminate an embryological
signaling pathway that is aberrantly reexpressed in metastatic
melanoma (neural crest derived) and breast carcinoma cells (epi-
thelial origin) that could be targeted for the reprogramming of
aggressive tumor cells with unique embryonic regulator(s).
Nodal is required to preserve the pluripotency of hESCs and our
recent work revealed that Nodal signaling maintains the dediffer-
entiated multipotent phenotype of highly metastatic C8161 mela-
noma cells (4, 20, 21). The present study showed that this protein
is restricted to aggressive cancer cells, embryonic stem cells, and
cell types or with more specified MSCs. Hence, Nodal expression
was restricted to embryonic and cancer cell types. Of note, the
Nodal gene has been sequenced in the hESCs and melanoma cells
used in this study, and no differences or point mutations have been
detected (M. Bento Soares, personal communication). This study
further demonstrated that Cripto, a prominent mediator of the
to multipotent cancer and stem cell fates. Previous studies, from
other laboratories, determined that like Nodal, Cripto is expressed
in cancer, specifically testicular, colon and breast cancer cells, but
is rarely detected in normal adult tissues (22–24). Furthermore,
Cripto is a stem cell marker for pluripotent hESCs and has been
associated with dedifferentiation, vascularization and invasion in
breast carcinoma cells (23, 25, 26). These functions mirror those of
Nodal in melanoma, inferring a convergence of their tumor-
promoting functions. Relative to hESCs, Cripto was very weakly
and heterogeneously expressed in the metastatic melanoma and
breast carcinoma cells used in this study. This finding is intriguing,
and raises the possibility that the Cripto-positive cells represent a
Nodal expression in melanoma and breast carcinoma cells. (A) Real-time RT-PCR
analysis of Nodal mRNA in C8161 and MDA-MB-231 cells exposed for 3 days to
CMTX). Some cancer cells exposed to H9 CMTX subsequently were recovered on
control Matrigel for 2 or 7 days before analysis. Gene levels were normalized by
using 18S, and bars represent mean gene expression normalized to Matrigel. (B)
Real-time RT-PCR analysis of Nodal mRNA in C8161 cells exposed for 3 days to
derived stem cells (GM00473/GM00957A CMTX) or trophoblast cells (HTR-8/
SVneo CMTX). Gene levels were normalized by using 18S, and bars represent
mean gene expression normalized to H9 Matrigel values.
The microenvironment of hESCs, containing Lefty, down-regulates
formation in a mouse injected with C8161 cells preexposed for 3 days to either a
or MDA-MB-231 cells preexposed for 3 days to either a control matrix (Matrigel)
or a matrix conditioned by hESCs (H9 CMTX) (n ? 10) (B). Values represent the
median tumor volume (mm3) ? interquartile range (A) or the mean tumor
volume (mm3) ? SE (B). Tumor volumes were significantly different at the time
MDA-MB-231-derived tumors determined by TUNEL staining. Before injection
into a mouse, C8161 and MDA-MB-231 cells were cultured for 3 days on control
or hESC-conditioned (H9 CMTX) matrices. Bars represent mean normalized val-
ues ? SD, and values indicated by an asterisk (*) are significantly different from
control values (P ? 0.05).
The microenvironment of hESCs inhibits tumorigenicity. In vivo tumor
www.pnas.org?cgi?doi?10.1073?pnas.0800467105Postovit et al.
unique side population. However, because of the disproportion-
ately low levels of Cripto observed in these cancer cells, we suggest
that it is unlikely that modulations in Cripto contributed signifi-
cantly to the results obtained in our study. Furthermore, we
activating the ALK 4/7 receptor complex via a Cripto-independent
mechanism (please refer to Fig. 1D).
In embryological systems Nodal is regulated via a positive
cell fate decisions, endogenous inhibitors such as Lefty-A, Lefty-B,
and Cerberus, are used (1, 15). In conjunction with Nodal and Oct
3/4, Lefty-A and -B are among the most enriched genes expressed
in hESCs, exemplifying the putative importance of these inhibitors
somatic cells used in this study express Lefty. In fact Lefty expres-
sion was unique to hESCs: Other human stem cell types (including
those derived from amniotic fluid, umbilical cord blood, and adult
bone marrow) and embryo-associated human cytotrophoblast cells
did not express appreciable levels of this protein. Furthermore,
unlike H9 CMTX, matrices conditioned by these more committed
stem cell lineages were unable to inhibit Nodal expression in
aggressive melanoma cells. These results highlight the inherent
differences between hESCs and other stem cell types, and suggest
that certain therapeutic modalities may be derived exclusively from
The results of our study demonstrate that exposure to a hESC
microenvironment down-regulates Nodal signaling in melanoma
and breast cancer cells, associated with a reduction in clonoge-
nicity and tumorigenicity. However, this suppressive effect is
transient and reversible. To decipher the molecular underpinnings
of the suppressive effects of the hESC microenvironment on tumor
cells, we focused on the deposition of Lefty (Nodal’s inhibitor
cells that do not express Lefty could in fact respond to Lefty
produced by hESCs. In a manner similar to H9 CMTX, exposure
to H9-derived Lefty down-regulated Nodal expression in mela-
noma and breast carcinoma cells, and decreased clonogenicity in
both tumor cell types as well. We were able to rescue the effect of
H9-derived Lefty on clonogenicity with rNodal, suggesting that the
results obtained with this treatment were due specifically to a
reduction in Nodal. Further confirmation of these findings showed
H9 hESCs treated with MOLEFTYwere unable to inhibit Nodal
expression in the C8161 and MDA-MB-231 cells. In fact, exposure
of these tumor cells to matrix conditioned by the Lefty-deficient
hESCs resulted in a significant up-regulation of Nodal expression,
which we postulate is because of a feed-forward response of
hESCs. Finally, exposure to H9 CMTX reduced anchorage-
independent growth, but in contrast to purified hESC-derived
Lefty, this effect could not be completely rescued by the inclusion
of rNodal. Collectively, these results suggest that Lefty plays an
important role in the ability of the hESC microenvironment to
suppress the tumorigenic phenotype, but that it is not the only
Lefty protein to hESC cultures, even at a high, nonphysiological
dose of 200 ng/ml, failed to inhibit Nodal and induce hESC
differentiation (17). Our results showed that although Nodal could
be inhibited in C8161 melanoma cells, it required ?1,000 ng/ml of
CMTX) inhibited Nodal in C8161 and MDA-MB-231 cells at a
significantly lower concentration (estimated in the range of 20–50
ng/ml). These observations led us to examine whether rLefty is
significantly different from hESC-derived Lefty in terms of glyco-
sylation because glycosylation is typical for proteins made by
eukaryote cells, but not characteristic of proteins made recombi-
nantly (e.g., through bacterial transduction). We found that com-
mercially available rLefty A and rLefty B are not glycosylated by
using the Pro-Q Emerald 300 staining kit, whereas Lefty recovered
from hESCs (plus CMTX) is glyosylated, which could ultimately
affect how this protein functions physiologically, and may provide
a possible explanation for these disparate observations.
Although exposure to a hESC microenvironment inhibits Nodal
expression and tumorigenicity in both melanoma and breast car-
cinoma cells, the breast cancer cells undergo a more complex
reprogramming event. Although melanoma cells respond to the
hESC-derived factors within 3 days, the breast carcinoma cells
require 2 additional days to achieve the most significant down-
regulation in Nodal. This discrepancy is likely attributable to
differences in signaling mechanisms between these two cell types.
For example, although C8161 cells do not express TGF-?1, which
actively employ this system, enabling the continued phosphoryla-
tion of Smad-2, which may sustain Nodal expression for a longer
duration (4, 28). Despite the inherent differences between neural
crest-derived melanoma cells and epithelial-derived breast carci-
noma cells, these divergent tumor types both underwent apoptosis
after exposure to hESC CMTX. This remarkable similarity is likely
attributable to the commonality of plasticity (e.g., the aberrant and
unregulated expression of Nodal) that indiscriminately unifies
highly aggressive cancer cells, regardless of their origin.
This study used an innovative approach to address whether
aggressive cancer cells, expressing a plastic, embryonic-like pheno-
type, could respond to regulatory cues that control cell fate
determination within the microenvironment of hESCs and other
stem cell types. We revealed that aggressive cancer cells, but not
normal cell types, express the embryonic morphogen Nodal, which
is essential to the maintenance of hESC pluripotency, but unlike
hESCs, the tumor cells lack Lefty, a negative regulator of Nodal.
Down-regulation of Nodal expression via exposure to the hESC
microenvironment (containing Lefty) resulted in a reduction in
clonogenicity and tumorigenesis accompanied by an increase in
exclusivity of the hESC microenvironment to down-regulate Nodal
expression in aggressive tumor cells, because it is not observed with
other stem cell types (isolated form amniotic fluid, cord blood, or
adult bone marrow) or trophoblasts, which do not express Lefty.
Understanding the tumor-suppressive effect(s) of the hESC micro-
environment, together with the importance of its influence on the
Nodal signaling pathway, may offer new therapeutic strategies to
inhibit tumor progression.
Materials and Methods
Cell Culture. Human cutaneous melanoma (C8161, WM-278) and breast carci-
noma (MDA-MB-231, MCF-7, T47D, MDA-MB-468, MDA-MB-330 and ZR75–30)
lines were maintained as described (29–31). Normal human neonatal epidermal
ican Type Culture Collection (ATCC)] and primary mammary epithelial cells
(HMEpC; Cell Applications) were maintained as per distributor instructions. Live
Institute for Medical Research), amniotic-fluid-derived stem cells (GM00473,
GM00957A) and adult bone-marrow-derived MSCs (Stem Cell Technologies)
were maintained under the recommended conditions. HTR-8/SVneo is a well
characterized human extravillous cytotrophoblast cell line, and was maintained
as described in ref. 32. H1 and H9 hESCs (WiCell, Madison WI) and MEL-2 hESCs
(Millipore) were maintained as described in ref. 14. For conditioned matrix
experiments, hESCs were maintained in stem cell medium preconditioned on
irradiated mouse embryonic fibroblasts (CF-1, ATCC) as described in ref. 14.
Recombinant Nodal and Lefty (R&D Systems) were diluted as per manufacturer
3D Conditioned Matrix Experiments. Conditioned matrices were prepared by
using hESCs, melanocytes, myoepithelial cells, amniotic-fluid-derived stem cells,
or trophoblast cells on growth factor-reduced Matrigel (14 mg/ml; BD Bio-
sciences) as described in ref. 14. Alternatively, hESC-derived Lefty protein was
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to specific matrices preconditioned by these cells for 3 to 4 days.
Immunoblotting. Protein lysates were prepared and quantified as described in
ref. 4. Equal amounts of protein were separated by SDS/polyacrylamide gel
electrophoresis (PAGE) under reducing conditions, and resolved proteins were
transferred onto Immobilon-P membranes (Millipore). Membranes were
blocked, incubated with primary antibody (SI Table 2), washed, and incubated
with the appropriate horseradish peroxidase-labeled secondary antibody. Sec-
Pierce) with exposure to autoradiography film (Midwest Scientific).
RNA Extraction and RT-PCR. RNA was isolated by using TRIzol (Invitrogen,
PCR was performed as described in ref. 4 by using TaqMan gene expression
human primer/probe sets for Lefty1/B (Hs00764128?s1), Nodal (Hs00250630?s1),
Nanog (Hs02387400?g1), and OCT 3/4 (Hs00999632?g1). Gene expression was
normalized to 18S rRNA (Hs99999901?s1). Data were analyzed by using the
Applied Biosystems Sequence Detection Software (Version 1.2.3). Semiquantita-
tive RT-PCR was performed for Lefty, Nodal, Cripto, and HPRT1 as outlined in SI
Immunofluorescence. Cells were fixed with 4% paraformaldehyde, made per-
meable with 20 mM Hepes, 0.5% Triton X-100 and blocked with serum-free
protein block (DAKO). Primary antibodies were diluted in antibody dilutent
conjugated secondary antibodies were used according to manufacturer recom-
mendations. Nuclei were stained with DAPI (0.1 mg/ml; Invitrogen/Molecular
Probes), and images were obtained by using confocal microscopy (Zeiss 510
META; Carl Zeiss).
Anchorage-Independent Growth Assays. Assays were conducted as described in
Glycoprotein Determination. Protein lysates underwent SDS/PAGE and transfer
and were stained for glycoproteins by using the Pro-Q Emerald 300 staining kit
an UV transilluminator and an image of the green fluorescing proteins were
protein was detected with immunoblotting. See SI Methods for more in depth
Experimental Orthotopic Tumor Models. Five-week-old mice were injected s.c.
in 50 ?l of complete RPMI were injected into the mammary fat pad of 6- to
8-week-old mice. When tumors became palpable measurements were taken
twice per week. All experiments involving animals were approved by the Insti-
tutional Animal Care and Use Committee at the Children’s Memorial Research
Immunohistochemistry. Immunohistochemical staining for Nodal in a breast
carcinoma progression tissue microarray (CBL-TMA-029; Creative Biolabs) was
performed as described in ref. 4. In addition, tissues from the orthotopic tumor
models were formalin-fixed and paraffin-embedded and immunohistochemical
staining on this tissue was conducted by using a Ki67-specific antibody (SI Table
2) or ChromPure Goat IgG (Jackson Laboratories) as described in ref. 4. TUNEL
assays to measure apoptosis were conducted as per manufacturer’s instructions
Lefty Knockdown. Fluorescein (FITC)-conjugated control (5?-CCTCTTACCTCAGT-
TACAATTTATA-3?), Lefty-A (5?-GCCACATGGTGCTGCCCTGGG-3?) and Lefty-B
(5?CTGCATGGTGCTGCCCTGGAGGA-3?) Morpholinos (20 ?M) (Gene Tools) were
delivered by using the scrape method (4). Cancer cells were sorted for FITC and
were allowed to recover for 1 day before experimentation.
Statistical Analyses. For tumor formation studies, we determined statistical
significance by using the Kruskal–Wallis one-way ANOVA on ranks, followed by
Dunn’s method or a one-way ANOVA followed by the Student–Newman–Keuls
method for pairwise multiple comparisons. For the clonogenic assays we deter-
mined statistical significance by using ANOVA followed by the Student–
used. In all cases, differences were statistically significant at P ? 0.05.
ACKNOWLEDGMENTS. We thank Drs. Zhila Khalkhali-Ellis, M. Bento Soares,
Luigi Strizzi, and David Salomon for helpful scientific discussions, and Paula
Pittock at the London Regional Proteomics Core at the University of Western
the Illinois Regenerative Medicine Institute, U.S. National Institutes of Health
(CA59702 and CA121205), and Charlotte Geyer Foundation (to M.J.C.H.), and a
Canadian Institutes of Health Research Postdoctoral Fellowship (to L.-M.P.).
1. Hendrix MJ, et al. (2007) Reprogramming metastatic tumour cells with embryonic
microenvironments. Nat Rev Cancer 7:246–255.
plasticity: Lessons from melanoma. Nat Rev Cancer 3:411–421.
of melanocytes to melanomas. Cancer Res 64:5270–5282.
signaling: Role in melanoma aggressiveness. Nat Med 12:925–932.
5. Bittner M, et al. (2000) Molecular classification of cutaneous malignant melanoma by
gene expression profiling. Nature 406:536–540.
6. Weeraratna AT, et al. (2002) Wnt5a signaling directly affects cell motility and invasion
of metastatic melanoma. Cancer Cell 1:279–288.
7. Balint K et al. (2005) Activation of Notch1 signaling is required for beta-catenin-
mediated human primary melanoma progression. J Clin Invest 115:3166–3176.
8. Vallier L, Alexander M, Pedersen R (2007) Conditional gene expression in human
embryonic stem cells. Stem Cells 25:1490–1497.
9. Pierce GB, Pantazis CG, Caldwell JE, Wells RS (1982) Specificity of the control of tumor
formation by the blastocyst. Cancer Res 42:1082–1087.
crest-like phenotype in an embryonic microenvironment. Proc Natl Acad Sci USA
11. Illmensee K, Mintz B (1976) Totipotency and normal differentiation of single terato-
12. Cucina A, et al. (2006) Zebrafish embryo proteins induce apoptosis in human colon
cancer cells (Caco2). Apoptosis 11:1615–1628.
13. Lee LM, Seftor EA, Bonde G, Cornell RA, Hendrix MJ (2005) The fate of human
malignant melanoma cells transplanted into zebrafish embryos: Assessment of migra-
tion and cell division in the absence of tumor formation Dev Dyn 233:1560–1570.
14. Postovit LM, Seftor EA, Seftor RE, Hendrix MJ (2006) A 3-D model to study the
epigenetic effects induced by the microenvironment of human embryonic stem cells
Stem Cells 24:501–505.
15. Schier AF (2003) Nodal signaling in vertebrate development. Annu Rev Cell Dev Biol
16. Yeo C, Whitman M (2001) Nodal signals to Smads through Cripto-dependent and
Cripto-independent mechanisms. Mol Cell 7:949–957.
17. Tabibzadeh S, Hemmati-Brivanlou A (2006) Lefty at the crossroads of ‘‘stemness’’ and
differentiative events. Stem Cells 24:1998–2006.
18. Ulloa L, et al. (2001) Lefty proteins exhibit unique processing and activate the MAPK
pathway. J Biol Chem 276:21387–21396.
19. Hochedlinger K, et al. (2004) Reprogramming of a melanoma genome by nuclear
transplantation. Genes Dev 16:1675–1685.
20. James D, Levine AJ, Besser D, Hemmati-Brivanlou A (2005) TGFbeta/activin/nodal
signaling is necessary for the maintenance of pluripotency in human embryonic stem
cells. Development 132:1273–1282.
21. Vallier L, Reynolds D, Pedersen RA (2004) Nodal inhibits differentiation of human embry-
onic stem cells along the neuroectodermal default pathway. Dev Biol 275:403–421.
cancer: A study with long-term survival. Eur J Surg Oncol 33:438–443.
23. Minchiotti G (2005) Nodal-dependant Cripto signaling in ES cells: From stem cells to
tumor biology. Oncogene 24:5668–5675.
24. Bianco C, et al. (2006) Identification of cripto-1 as a novel serologic marker for breast
and colon cancer. Clin Cancer Res 12:5158–5164.
25. Normanno N, et al. (2004) Cripto-1 overexpression leads to enhanced invasiveness and
resistance to anoikis in human MCF-7 breast cancer cells. J Cell Physiol 198:31–39.
26. Strizzi L, et al. (2004) Epithelial mesenchymal transition is a characteristic of hyperpla-
27. Sato N, et al. (2003) Molecular signature of human embryonic stem cells and its
comparison with the mouse. Dev Biol 260:404–413.
28. Arrick BA, Korc M, Derynck R (1990) Differential regulation of expression of three
transforming growth factor beta species in human breast cancer cell lines by estradiol.
Cancer Res 50:299–303.
29. Welch DR, et al. (1991) Characterization of a highly invasive and spontaneously
metastatic human malignant melanoma cell line. Int J Cancer 47:227–237.
30. Seftor EA, et al. (2002) Expression of multiple molecular phenotypes by aggressive
melanomatumorcells:roleinvasculogenicmimicry. CritRevOncolHematol 44:15–27.
31. Cailleau R, Young R, Olive M, Reeves WJ, Jr (1974) Breast tumor cell lines from pleural
effusions. J Natl Cancer Inst 53:661–674.
32. Graham CH, et al. (1993) Establishment and characterization of first trimester human
trophoblast cells with extended lifespan. Exp Cell Res 206:204–211.
www.pnas.org?cgi?doi?10.1073?pnas.0800467105Postovit et al.