"Normal and neoplastic non-stem cells can spontaneously convert to a stem-like state".
Christine L. Chaffer a, b, Ines Brueckmann a, Christina Scheel a, b, Alicia J. Kaestli a, Paul A. Wiggins a, Leonardo O. Rodrigues a, b, Mary Brooks a, b, Ferenc Reinhardt a, b, Ying Su c, Korrnelia Polyak c, Lisa M. Arendt d, e, Charlotte Kuperwasser d, e, Brian Bierie a, b, and Robert A. Weinberg a, b, f, 1
a Whitehead Institute for Biomedical
Research, Cambridge, MA 02142;
b Ludwig MIT Center for Molecular
Oncology, Cambridge, MA 02139;
c Department of Medical Oncology,
Dana-Farber Cancer Institute, Boston, MA 02115;
d Department of Anatomy and Cellular
Biology, Sackler School, Tufts University School of Medicine, Boston,
MA 02111;
e Molecular Oncology Research Institute,
Tufts Medical Center, Boston, MA 02111;
f Department of Biology, Massachusetts
Institute of Technology, Cambridge, MA 02139
Contributed by Robert A. Weinberg, March 2, 2011 (sent for review December 8, 2010).
Current models of stem cell biology assume that normal and neoplastic stem cells reside at the apices of hierarchies and differentiate into non-stem progeny in a uni-directional manner. Here we identify a sub-population of basal-like human mammary epithelial cells that departs from that assumption, spontaneously de-differentiating into stem-like cells. Moreover, oncogenic transformation enhances the spontaneous conversion, so that non-stem cancer cells give rise to cancer stem cell (CSC)-like cells in vitro and in vivo. We further show that the differentiation state of normal cells-of-origin is a strong determinant of post-transformation behavior. These findings demonstrate that normal and CSC-like cells can arise de novo from more differentiated cell types and that hierarchical models of mammary stem cell biology should encompass bi-directional inter-conversions between stem and non-stem compartments. The observed plasticity may allow derivation of patient-specific adult stem cells without genetic manipulation and holds important implications for therapeutic strategies to eradicate cancer.
* breast cancer
* de-differentiation
1 To whom correspondence should be addressed. E-mail: weinberg@wi.mit.edu
Author contributions: C.L.C. and R.A.W. designed research; C.L.C., I.B., A.J.K., M.B., F.R., Y.S., L.M.A., and B.B. performed research; P.A.W. and L.O.R. contributed new analytic tools; C.L.C., C.S., P.A.W., L.O.R., K.P., and C.K. analyzed data; C.L.C. led the project; R.A.W. provided overall project guidance and scientific discussion; and C.L.C. and R.A.W. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at: http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1102454108/-/DCSupplemental
Introduction:
Tissue-specific stem cells exist in many adult tissues and can be
identified and isolated using specific antigen profiles.
Their
potential utility in regenerative medicine holds great promise.
However, our current ability to isolate and propagate adult stem
cells for such purposes is limited (1). This limitation
is due in
part to the paucity of stem cells in most epithelial tissues and
our
fragmentary understanding of the survival, proliferation, and
differentiation signals that these cells receive in highly specialized
stem-cell niches (2, 3).
The discovery of stem-like cells in a number of human solid
tumor types has suggested a central role for stem cells in tumorigenesis.
Thus, stem-like cells, often termed cancer stem cells
(CSCs), have been defined experimentally by their ability
to seed
new tumors and to spawn non-CSC populations lacking tumor-initiating
ability. CSC subpopulations have now been identified
in a variety of malignancies (4). Importantly,
CSC-rich tumors
are associated with aggressive disease and poor prognosis (5),
indicating that an understanding of their biology is pertinent to
developing effective therapies.
Both normal and neoplastic stem cells are thought to be self-renewing
and to reside at the apex of a cellular hierarchy. Through
asymmetric division, these stem cells generate more differentiated
progeny that lack self-renewal capacity (6, 7).
Intra-tumor heterogeneity
may thus derive from neoplastic cells at various differentiation
stages. In the case of human mammary epithelial cells
(HMECs), the structure of the associated stem-cell hierarchy
is yet
to be definitively described. It is becoming apparent, however,
that
the differentiation states of cells-of-origin can influence
the organization
of derived neoplastic cell populations (8, 9).
These hypotheses have been difficult to validate because of
the apparent critical role of the stem-cell niche and associated
micro-environment in the survival and differentiation of both
normal and neoplastic stem cells. However, evidence of normal
and neoplastic cells with stem-cell properties residing naturally
among populations of epithelial cells propagated in culture has
been reported (10–12).
In light of the latter observations, we undertook to study the
biology of sub-populations of HMECs that exist in culture
and
share certain properties with either stem-like cells or their more
differentiated derivatives. We identified an unexpected degree
of plasticity between stem-like and non-stem cell compartments,
leading to the demonstration that differentiated cell types can
convert to stem-like cells. Moreover, these observations
hold
true for the neoplastic counterparts of such cells.
Results:
Enrichment of a Rare Floating Population of Cells from Cultured Human Mammary Epithelial Cells.
In the work described below we
used primary HMECs as well as HMECs immortalized with
human telomerase (hTERT) that are termed here HME cells
(13, 14). We observed that populations of HME
cells cultured
in their normal mammary epithelial growth medium contained
a small proportion of cells that grew as floating cells above
the
majority population of adherent cells. These populations, termed
here HME–floating population of cells (HME-flopcs), were
collected from the conditioned media of HME cells through
centrifugation and introduced into new culture dishes, yielding
fully viable, adherent cell populations.
The epithelial nature of HME and HME-flopc cells was confirmed
by the expression of cytokeratins and low vimentin expression
(Fig. 1C) and by formation of acinar structures
when
plated at clonal density on a layer of Matrigel (15).
These polarized
epithelial structures contained cells expressing vimentin,
cytokeratins, and MUC1, the latter marking luminal epithelial
cells in the mammary gland (Fig. S1C). However,
under two-dimensional
(2D) culture conditions, HME cells formed a typical
epithelial monolayer with junctional E-cadherin, b-catenin,
and
ZO-1, whereas HME-flopc cells did not, despite similarly high
levels of those proteins (Fig. 1 B and C). Thus,
HME-flopc cells
were stably morphologically distinct from the bulk HME cells.
Fig. 1. Enrichment of a rare population of floating cells from
cultured mammary epithelial cells.
Fig. 1. Enrichment of a rare population of floating cells from cultured mammary epithelial cells.
(A) Schematic illustrating the derivation of HME- flopcs and single-cell clones (SCCs), as well as the antigen profiles of the major cell subpopulations described in this paper.
(B) Phase contrast micrographs (20×) of HME and HME-flopc populations in 2D culture. Immuno-fluorescence of HME and HME-flopc cells is shown, for E-cadherin (green), b-catenin (red), and ZO-1 [nuclei stained blue with 4,6-diamidino-2-phenylindole (DAPI)].
(C) Immunoblot with antibodies to cytokeratins 14, 18 and
19, vimentin, E-cadherin and b-catenin
(total and active forms).
(D) Flow cytometry plots for CD44, CD24, and ESA on bulk HME and HME-flopc cells.
Gating is set to unstained control cells.
Fig. S1. (A) Bright phase micrographs (20×) showing morphology of two HME-flopc-CD44 lo single-cell clones (SCCs, FL1 and FL2) and two HME-flopc-CD44 hi SCCs (FH1 and FH2). Immunofluorescence (20×) demonstrates that E-cadherin, b-catenin, and ZO-1 rarely localize to cell–cell junctions in HME-flopc SCCs.
(B) Flow cytometry analysis to determine ESA expression in the various cell populations, demonstrating that HME bulk cells contain ESA-positive and -negative cells, whereas bulk HME-flopc cells and SCCs are largely ESA negative.
(C) 3D culture of mammary epithelial cells (HME and HME-flopc) on Matrigel illustrating their epithelial origins. Phase contrast micrographs (20×), hematoxylin staining (40×), and immunofluorescence (40×) are shown for cytokeratin, vimentin, and MUC1.
(D) Gene set enrichment analysis comparing gene expression signatures
of pure HME-flopc-CD44 hi cells (SCC FH1) and bulk HME-flopc
cells to published gene expression profiles of primary luminal colony-forming
cells (CFCs), bipotent CFCs, differentiated luminal cells, and differentiated
myoepithelial cells (19).
HME-flopc-CD44 hi cells are enriched for bipotent and
luminal CFC gene signatures.
(E) Flow cytometry plots showing CD44/CD24 profile of HME-flopc SCCs.
The most un-anticipated discovery that has emerged from this
study is the plasticity that we can now ascribe to human
mammary
epithelial cells. We have shown that differentiated mammary
epithelial cells can convert to a stem-like state, doing
so in
an apparent stochastic manner in vitro. This conversion occurs
in transformed and non-transformed HMECs isolated from cell
lines and primary tissue. In each case, the conversion proceeded
without genetic manipulation.
These findings represent a profound divergence from the
currently accepted uni-directional hierarchical model of mammary
epithelial cells and have widespread implications for the
use of cultured cells. In mammalian cells, the idea that non-stem
cells de-differentiate to form functional stem cells has
been restricted
to the notion that progenitor cells can re-acquire stem
cell activity in mouse differentiating spermatogonia (26).
As
such, our work demonstrates in mammalian cells that differentiated
epithelial cells can revert to a stem-like state.
Our findings also hold implications for the development of
anti-cancer therapeutics. As we previously reported, cells that
have been forced experimentally into a mesenchymal / stem-like
state can be used to screen for candidate therapeutic agents that
specifically target CSCs (27); the intent here
was to eliminate
these cells and thereby deprive tumors of their ability to regenerate
and thrive following initial therapy. However, if non-CSCs
can spontaneously de-differentiate into CSCs, then targeting
CSC populations will, on its own, be unlikely to yield durable
clinical responses, because the therapeutic eradication of existing
CSC populations might be followed by their regeneration from
non-CSCs within the tumor under treatment.
Given the present findings, the known ability of micro-environmental
signals to provoke epithelial–mesenchymal transitions
(EMTs) and the close connection between passage through
an
EMT and entrance into a stem-cell state, we suspect that
the
presently observed spontaneous conversion in vitro may be
augmented in vivo by contextual signals in the tumor micro-environment,
such as those that drive the EMT (28, 29). Relevant
here are studies demonstrating that hypoxia-inducible factors
(HIFs) can induce the EMT phenotype and promote metastasis
and the CSC phenotype (30, 31). Spontaneous
de-differentiation
in vivo may involve the reactivation of one or more of
the described
pluripotency factors (Oct4, Klf4, c-myc, and Sox-2) (32).
Hence, the representation of CSCs within tumor cell populations
is likely to be influenced both by contextual signals and by the
intrinsic phenotypic plasticity of these cells, as observed here.
The ability of non-CSCs to convert into CSCs in vivo might
resolve many of the current inconsistencies of the CSC model.
In
particular, the observed plasticity that was once reserved for CSCs
alone can now be associated with non-stem cells. As such,
CSC
populations may differ profoundly between various tumor types
according to the inherent plasticity of cells in their respective
nonstem fractions and their ability to spawn CSCs de novo.
The present observations lend further support to the emerging
view that the biological state of cells-of-origin is an important
determinant of the phenotype of their transformed derivatives
(8, 9), where experimental transformation of cells
that have a
phenotype related to that of mammary stem cells generates
cell
populations with a high frequency of tumor-initiating cells
(~1:1,420–1:1,804 cells) and metastasis, which contrasts with the
low tumor-initiating ability and non-metastatic nature of tumors
derived via transformation of more differentiated cell types.
The present findings hold the implication that patient- and
tissue-specific stem-like cells may one day be created in vitro
via
spontaneous conversion of a patient’s own terminally differentiated
epithelial cells, a process that would not require any genetic
alteration of these cells. Such stem-like cells could be
important
for regenerative therapies. Our results further emphasize the
pathological implications of cellular plasticity in cancer
development,
progression, and recurrence. Further research needs to be
undertaken to determine the mechanism underlying the de novo
generation of CSCs from non-CSCs in vivo, with the
promise
of potential novel targets for future cancer therapies aimed at
eradicating CSCs.
Materials and Methods
Detailed materials and methods are provided in SI Materials and Methods.
Cell Culture.
HME cells and all derivatives were cultured in MEGM media as
previously described (13). HMECs were isolated
from primary tissue as previously
described (33) and cultured in M87A+X (34).
A list of antibodies is
provided in Table S1.
Mammosphere Culture.
Mammosphere culture was performed as previously described (17).
Flow Cytometry.
Cells were prepared according to standard protocols.
Animal Studies.
Athymic female nude mice were 2–4 mo of age at time of
injections. Tumor cells were resuspended in 10% Matrigel/MEGM (20
mL)
for
mammary fat pad injections. GFP-positive lung metastases were counted
from individual lobes by fluorescent microscopy.
Statistical Analysis.
Data are presented as mean ± SEM. Student’s t test
(two-tailed)
was used to compare two groups (P < 0.05 was considered
significant)
unless otherwise indicated.
ACKNOWLEDGMENTS:
We thank the Core Facilities at Whitehead Institute for Biomedical Research at Massachusetts Institute of Technology, Koch Institute for Cancer Research, and Annie Gifford for technical assistance at Whitehead Institute for Biomedical Research.
This work was supported by the National Health and Medical Research
Council of Australia (C.L.C.),
National Institutes of Health Grant U54 CA12515, Massachusetts Institute
of Technology’s Ludwig Center for Molecular Oncology, the Breast Cancer
Research Foundation, the Advanced Medical Research Foundation, and a Department
of Defense Breast Cancer Research Program Idea Award. R.A.W. is an American
Cancer Society Research Professor and a Daniel K. Ludwig Foundation Cancer
Research Professor.
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