Efficient acquisition of dual metastasis organotropism
to bone and lung through stable spontaneous
fusion between MDA-MB-231 variants
Xin Luaand Yibin Kanga,b,1
aDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544; andbBreast Cancer Program, The Cancer Institute of New Jersey,
New Brunswick, NJ 08903
Edited by Joan Massagué, Memorial Sloan-Kettering Cancer Center, New York, New York, and approved April 16, 2009 (received for review January 6, 2009)
Cell fusion is involved in many critical developmental processes,
including zygote formation and organogenesis of placenta, bone,
and skeletal muscle. In adult tissues, cell fusion has been shown to
play an active role in tissue regeneration and repair, and its
frequency of occurrence is significantly increased during chronic
inflammation. Fusion between tumor cells and normal cells, or
among tumor cells themselves, has also been speculated to con-
tribute to tumor initiation, as well as phenotypic evolution during
cancer progression and metastasis. Here, we show that dual
metastasis organotropisms can be acquired in the same cell
through in vitro or in vivo spontaneous fusion between bone- and
lung-tropic sublines of the MDA-MB-231 human breast cancer cell
line. The synkaryonic hybrids assimilate organ-specific metastasis
gene signatures from both parental cells and are genetically and
phenotypically stable. Our study suggests cell fusion as an efficient
means of phenotypic evolution during tumor progression and
additionally demonstrates the compatibility of different metasta-
breast cancer ? cell fusion ? nuclear reprogramming ? genomic instability ?
highly malignant tumors (1, 2). Likewise, metastasis organotro-
pism—the capability of tumor cells to colonize specific target
organs—is thought to emerge via acquisition of distinct sets of
organ-specific metastasis genes in metastatic variants that are
most adapted to different target organ microenvironments
through Darwinian selection (3). Indeed, genomic profiling of
metastatic variants selected in vivo in mouse models of breast
cancer has unveiled 2 separate sets of genes that promote
metastasis to bone and lung, respectively (4, 5), although it is
unclear whether such distinct organ-specific metastasis gene
signatures can coexist in the same cell to give rise to tumor cells
capable of colonizing both organs. An alternative theory of
metastasis progression has also been proposed that argues for
rapid acquisition of metastatic phenotypes through fusion be-
tween tumor cells or between tumor cells and certain normal
cells, such as macrophages (6–9), rather than requiring the
progressive accumulation of independent genetic or epigenetic
alterations in a single cell lineage. Given that a 1-cm3tumor of
?109cells is estimated to harbor as many as 105proliferating
hybrid cells produced by spontaneous cell fusion (6, 10), the
contribution of cell fusion to the phenotypic evolution of tumors
cannot be overlooked.
In this study, we used a well-characterized model system of
cell fusion between bone-tropic and lung-tropic cancer cells,
both in vitro and in vivo, generates stable hybrids with dual
metastasis tropism to both organs. In addition to directly dem-
onstrating the role of cell fusion in the rapid acquisition of
complex metastasis properties, our study also discovered a
he development of cancer is believed to be driven by the
progressive accumulation of numerous genetic and epige-
surprisingly high level of chromosomal and phenotypic stability
in hybrids during long-term passage in vitro and in vivo, despite
the existence of amplified numbers of centrioles in these cells as
a consequence of cell fusion. These results suggest a potentially
important role of cell fusion in the progression and phenotypic
diversity of cancer.
Spontaneous Cell Fusion Generates Synkaryonic Hybrids That Inherit
Chromosomal Abnormalities of Parental Cells. To examine the
genetic and phenotypic consequences of cell fusion between
tumor cells with distinct metastasis characteristics, we used 2
previously reported sublines of the human breast cancer cell line
MDA-MB-231: the bone-metastatic SCP2 and the lung-
metastatic LM2 (4, 5). These 2 cell lines were renamed as Bm
1A). To isolate spontaneously fused cells from the coculture of
these 2 cell lines, we labeled Bm with a GFP-Fluc (firefly
luciferase) fusion protein expression construct with a puromycin
resistance marker and Lm with RFP-Rluc (Renilla luciferase)
and hygromycin as markers. These 2 cell lines were cocultured
for 1 day without any fusogenic reagents and hybrid cells were
selected by either dual drug selection or dual color fluorescence-
activated cell sorting (FACS) (Fig. 1A). After 4 rounds of cell
sorting, we obtained a relatively pure GFP?/RFP?population
hygromycin dual drug selection gave rise to a population with
95.4% GFP?/RFP?cells (named as BLmDrug) (Fig. 1A). We also
isolated BBm and LLm hybrids (resulting from self-fusion of Bm
and Lm cells) by using the same approach. The fused cells are
synkaryons with enlarged nuclear and cell sizes (Fig. S1 A–C),
but with growth doubling times similar to the parental cell lines
(Bm 34.6h, Lm 36.0h, BLmFACS36.2h, and BLmDrug34.2h). Flow
cytometric DNA content analysis and karyotyping showed
nearly doubled DNA content and chromosome numbers in
hybrids compared with the parental cell lines (Fig. 1B). Spectral
karyotyping (SKY) further revealed that BLm hybrids adopted
all major chromosomal abnormalities (translocations and dele-
tions) of both parental cell lines (Figs. 1 C and D and S1D).
To test whether spontaneous cell fusion also occurs in vivo, we
injected an equal mixture of Bm and Lm cells s.c. into nude mice.
When the tumor diameter reached 10 mm, a single-cell suspen-
sion was made from the tumor by mincing and digestion with
Author contributions: X.L. and Y.K. designed research; X.L. performed research; X.L. and
Y.K. analyzed data; and X.L. and Y.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the Gene
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
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collagenase A. The cells were plated directly into dual drug
selective media at a very low density (104cells/10-cm dish) to
select for surviving colonies for 14 days. By using this approach,
the frequency of in vivo spontaneous fusion resulting in viable
progenies was estimated to be 2.0 ? 0.8 ? 10?5in s.c. tumors,
very similar to the in vitro fusion frequency of 2.6 ? 0.7 ? 10?5
(see SI Materials and Methods, data reported as the mean ? SD),
and is consistent with previously reported frequencies in other
tumor models (11).
Fusion Between Bone- and Lung-Tropic Metastatic Cells Produced
Dual-Tropic Hybrids. Parental Bm, Lm, and hybrids BBm, LLm,
BLmDrug, and BLmFACSwere injected into nude mice through
either left cardiac ventricles or tail veins to test their bone or lung
metastasis abilities, respectively. Metastatic progression was
measured by weekly bioluminescence imaging (BLI) and ana-
lyzed with Kaplan-Meier curves (Fig. 2). Bm and BBm formed
aggressive bone metastases but no lung metastasis. Conversely,
Lm and LLm displayed high metastasis potential to lung and a
much weaker ability to form bone metastasis. In contrast,
BLmDrugand BLmFACSshowed strong metastasis ability to both
bone and lung, indicating that BLm cells have inherited metas-
tasis phenotypes from both parental cell lines. Formation of
massive metastases in both bone and lung by the hybrid cells
were confirmed by X-ray and histological analyses of the lesions
(Fig. 2 C and F). The acquisition of dual metastasis organotro-
pism was also confirmed when bone and lung metastasis assays
were performed with 2 hybrid clones (BLmDrug-i.v.#1 and
BLmDrug-i.v.#2) obtained from in vivo fusion by Bm and Lm
cells in s.c. tumors as described above (Fig. S2).
Interpretation of the finding that hybrid cells can form ag-
gressive metastases in both organs may be complicated by the
fact that the hybrids may contain a very small fraction of unfused
lung. This possibility was ruled out by 2 pieces of evidence. First,
firefly luciferase and Renilla luciferase BLI performed on con-
secutive days showed exactly matched metastasis locations in the
mice injected with BLmDrug(Fig. S3). This result suggested that
most, if not all, of metastases were derived from hybrids with
dual markers instead of rare singly labeled parental cells that
survived the drug selection. A more rigorous test was performed
by analyzing single cell isolates from BLm. By using single-cell
sorting, we isolated 21 single cell clones from BLmDrug. We
randomly picked 5 clones (#6, #7, #8, #12, and #18) to test
their tissue-specific metastasis abilities (Fig. S4). All of the
clones were bone metastatic, although clones #6 and #7 were
not as strong as the pooled BLmDrugpopulation. All of the clones
except clone #7 were also lung metastatic, with clone #18
showing an even more aggressive phenotype than the pooled
population. Taken together, these results indicated that both
bone and lung metastasis phenotypes can be efficiently acquired
in a single tumor cell through cell fusion.
Bone and Lung Metastasis Gene Signatures Are Coexpressed by
Hybrids. As cell fusion did not confer growth advantages, we
reasoned that the acquired dual metastasis tropism may be
because of the coexpression of tissue-specific metastasis genes
that have been previously defined for both parental cell lines (4,
5). We performed microarray analyses on various cell lines
bone and lung metastasis gene signatures to perform unsuper-
vised clustering of these cell lines as well as several previously
characterized MDA-MB-231 sublines with different metastasis
organotropisms (Fig. 3 A and B). Both signatures were able to
segregate various cell lines into a lowly metastatic group and a
highly metastatic group. BLm and its single cell-derived clones
were consistently clustered into the strongly metastatic groups
based on either signature, although not all of the genes in each
signature retained their expression level in the BLm cells. As
expected, the self-fusion hybrids, BBm and LLm, always clus-
or drug selection to isolate spontaneous fusion hybrids (BLmFACSor BLmDrug) from the coculture of Lm and Bm cells that were differentially labeled with various
markers. The fluorescence dot plots for BLmDrugand BLmFACSrepresent the results after 1 round of drug selection and 4 rounds of FACS, respectively. (B) DNA
content and chromosome numbers of the parental and hybrids evaluated by flow cytometric (propidium iodide staining) and karyotyping (Giemsa staining)
methods (n ? haploid chromosome number of MDA-MB-231). (C) Representative SKY image showing the chromosomal composition of BLmDrug. (D) Summary
of chromosomal translocations and deletions in Bm, Lm, and BLmDrug.
www.pnas.org?cgi?doi?10.1073?pnas.0900108106 Lu and Kang
When the expression profiles of MDA-MB-231 variants and
hybrids were scored for their similarity to the lung and bone
metastasis signatures, the hybrids were positive for both signa-
tures, albeit with scores slightly lower than those from the
sublines with only one metastasis organ tropism (Fig. 3C).
to bone (A) or the lung (D).*, P ? 0.05 with log-rank test when compared with Lm (A) and Bm (D) (n ? 8 in A or n ? 5 in D). (B and E) BLI images of representative
mice from each experimental group showing development of metastases in the hindlimbs (B) or the lungs (E). (C) X-ray radiography of the hindlimbs and H&E
staining of the femurs from mice injected with parental and BLm cells. Osteolytic bone lesions are marked with arrows, and metastases are marked with dotted
lines. (F) Photographs and H&E staining of the lung from mice injected with parental and BLm cells. Metastasis nodules are indicated with dotted lines in the
H&E images. (Scale bar, 500 ?m in C and F.)
sublines is shown with different metastatic abilities by using the bone-metastasis gene signature (A) or the lung-metastasis gene signature (B). Cell lines under
the red line are strongly metastatic, whereas those under the green line are weakly metastatic (4, 5). ‘‘Parental’’ refers to the original MDA-MB-231 cell line
3481, 4173, 4175, 4180, and 4142 were obtained by in vivo selection (4, 5). In both heatmaps, the BLm lines clustered together with the strongly metastatic
sublines. (C) The metastasis signature correlation score analysis shows the grouping of the weakly metastatic cell lines (negative for both signatures), cell lines
are the same as in A and B. (D) Northern blot showing BLm cells coexpress representative lung- and bone-metastatic signature genes. MMP1 is a shared gene
for both signatures.
Coexpression of both bone and lung metastasis signatures in the BLm hybrid cells. (A and B) Hierarchical clustering of the BLm cells and MDA-MB-231
Lu and KangPNAS ?
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Northern blot analysis confirmed the coexpression of typical
the dual metastasis organ tropism displayed by BLm reflects the
coexpression of distinct sets of tissue-specific metastasis genes
and that fusion as a cellular process itself did not significantly
alter gene expression or metastatic behavior.
Chromosomal and Phenotypic Stability of Hybrids. Cell fusion may
result in dramatic chromosomal instability and hybrid cells may
gradually lose extra chromosomes through reductive mitosis
(12). We tested the long-term genomic and phenotypic stability
of BLm by culturing the cells continuously for nearly a year (up
to the time of manuscript preparation). Remarkably, BLmFACS,
BLmDrugand its clones maintained the nearly combined genome
size (Fig. 4A) and the expression of the characteristic metastasis
genes (Fig. 4B) throughout the duration of the experiment. In
addition, the tissue-specific metastasis ability to both bone and
lung was maintained in BLmDrugcells that were cultured for 1,
3, and 6 months (Fig. 4 C and D). To test whether hybrids also
maintained the chromosomal and phenotypic stability in vivo
during metastasis assays in mice, we isolated tumor cells (1241R
and 1241L) from bone metastases formed by BLmDrug and
confirmed their sustained strong metastatic ability to both
organs (Fig. 4 C and D). DNA content analysis of 1241R, 1241L
and their secondary in vivo derivatives (1241R-d and 1241L-d)
consistently revealed a combined genome size (Fig. 4E).
Although cell fusion is generally considered to be a highly
specialized event that only occurs in limited scenarios during
development (13, 14), a broader role for cell fusion in repair and
regeneration of adult tissues has been increasingly recognized
since the discovery of cell fusion as one of the underlying
mechanisms of the so-called ‘‘transdifferentiation’’ phenome-
non—the ability of one lineage of differentiated cells, such as
bone marrow cells, to give rise to a different lineage of cells, such
as liver or neural cells (15–21). In recent years, cell fusion has
also become an important research platform to study epigenetic
reprogramming, cell fate manipulation and pluripotency (14, 22,
23). In cancer research, cell fusion has been speculated to play
important roles in several aspects of tumor progression, includ-
ing generation of cancer stem cells (8), acquisition of metastasis
ability (7), and multidrug resistance (11). Cancer cells can have
considerably high rate (up to 1%) of fusion in vivo in experi-
mental tumor models (10). Recent findings that chronic inflam-
mation dramatically increases the frequency of cell fusion be-
tween hematopoietic cells with various cell lineages (24, 25) may
help explain the high fusogenicity of tumor cells, as inflamma-
tion is often associated with the tumor microenvironment (26).
Fusion between cancer cells may be further facilitated by fuso-
genic viruses (27, 28) and entosis (29). Despite the technical
difficulty in directly detecting cell fusion in cancer patients,
several clinical observations support the existence and potential
importance of cell fusion in cancer development. First, in kidney
cancer patients who were also recipients of previous bone
marrow transplantation, the renal cell carcinoma cells were
found to contain genetic material from both the recipients and
the donor (30, 31). Secondly, premature chromosome conden-
sation, a consequence of fusion of cells at different stages of cell
cycle, has been observed in a large number of tumor types
(32–35). Finally, binucleated and multinucleated cells are fre-
quently observed in many types of tumors (9), and increased
DNA content and aneuploidy in tumor cells, which may result
(36–38). Indeed, we estimated that cell fusion accounts for
65–88% of the hyperploid cells in our current experimental
setting (see calculation in SI Materials and Methods and Fig. S6
and Table S1).
Compared with mutation-based phenotypic evolution during
cancer progression, the main advantage of cell fusion is its
efficiency in generating drastic genomic changes and phenotypic
heterogeneity (11). Although fusion of 2 genetically identical
tumor cells may not benefit tumor cell progression, fusion
between tumor cells with distinct phenotypes can generate
hybrids with diversified phenotypes to allow better adaptation to
various secondary environments during metastasis or to survive
different therapeutic challenges. In this study, we derived spon-
taneous fusion hybrids from the mixed population of 2 breast
cancer sublines with distinct organ-specific metastasis propen-
sities (Bm and Lm) and found that the hybrids (BLm) efficiently
acquired the ability to metastasize to both organs (Fig. 2).
BLmDrugand its single cell derived clones measured after 1, 3, and 6 months of culture (n ? haploid number of chromosomes in MDA-MB-231). (B) Expression
of selected genes from the lung and bone metastasis signatures in the pooled BLmDrugat 3 time points during the long-term in vitro culture. (C) Kaplan-Meier
curves showing bone metastasis (Left) and lung metastasis (Right) developed after injection of BLmDrugcultured in vitro for 1, 3, or 6 months, or isolated from
bone metastases formed by BLmDrug(1241L, 1241R).*, P ? 0.05 with log-rank test when compared with Lm or Bm (n ? 8 for bone metastasis assays and n ? 5
(n ? haploid chromosome number of MDA-MB-231).
Genomic and phenotypic stability of hybrid BLm cells during long-term in vitro culture and in vivo passage. (A) DNA content histograms of the pooled
www.pnas.org?cgi?doi?10.1073?pnas.0900108106Lu and Kang
Similar results were observed when hybrid cells were obtained
from the fusion of Lm and SCP28, another bone-tropic meta-
static subline of MDA-MB-231 (Fig. S7).
The gain of dual tropism in the BLm cells is not simply due to
larger cell size and increased mechanical arrests in capillary beds
of distant organs, because hybrids of identical parental cells
(LLm and BBm) did not show significant change in metastasis
propensity. Instead, dual metastasis organotropism of hybrids is
likely because of the expression of both sets of genes important
for metastasis to each organ. It should be noted that the modest
but statistically insignificant (P ? 0.0849, log-rank test) increase
of bone metastatic potential by LLm as compared with Lm (Fig.
3D) may be because of elevated expression of 2 bone metastasis
genes, MMP1 and IL11. The ability of hybrid cancer cells to
retain the genomic, transcriptomic, and phenotypic characteris-
tics of both parents without a significant bias (Figs. 3 and S7C)
is distinctively different from the dramatic nuclear reprogram-
ming of differentiated somatic cells to the embryonic state after
their fusion with embryonic stem (ES) cells (22, 23). The
difference in transcriptional reprogramming in hybrids may be in
part because of the difference in epigenetic regulation between
the somatic/ES cell hybrids and the BLm hybrids. Although ES
cells may reprogram the epigenetic regulation landscape (e.g.,
state after fusion with fibroblasts (22), coexistence of epigenetic
regulatory mechanisms from both fusion partners in the BLm
hybrids may explain the moderately elevated expression of both
sets of organ-specific metastasis genes in the hybrids (Fig. 3 A
and D). Although the level of expression of these genes in the
hybrid are modest compared with the generally higher level of
organotropic gene expression in Bm or Lm cells, it is sufficient
to promote metastasis of hybrids to both bone and lung.
The fact that cell fusion can generate tumor cells with dual
metastasis tropism to bone and lung indicates that different
metastasis organotropism and the related metastasis signatures
can coexist in the same tumor cell to facilitate simultaneous
metastasis to multiple organs (4, 5, 39). Hybrid cells with
multiple metastasis organotropism may confer several advan-
tages for disseminated tumor cells to proliferate and survive
therapeutic treatments. First, tumor cells with multiple metas-
tasis organotropism will be able to colonize several different
organs when they are disseminated systemically through blood
circulation, whereas tumor cells with only a single metastasis
organotropism can only colonize one organ and will be lost when
they are distributed to an inhospitable microenvironment.
Therefore, fusion between tumor cells with different metastatic
ability can increase the efficiency of metastatic dissemination.
Secondly, because metastases in different organs often have
different responses to therapeutic treatments (40), tumor cells
with multiple metastasis organotropisms can survive in a differ-
ent organ and then quickly re-colonize the organ that has
positively responded to therapies. Finally, cell fusion could also
lead to combinatorial advantage in other phenotypes, like che-
moresistance, and may therefore, confer additional survival
advantage for hybrid cells.
We also found that hybrid cells resulted from spontaneous
fusion are remarkably robust in maintaining their genetic and
phenotypic stability, at least for several months in our current
experiment (Figs. 4 and S7 G and H). The chromosome stability
after cell fusion was also observed in various other experimental
systems, including fusion beween HeLa and fibroblasts (41),
between mouse mammary tumor cells (11, 42), or between ES
cells and fibroblasts (22). Cell fusion automatically leads to
increased number of centrosomes in the hybrids. Supernumerary
centrosomes can cause multipolar spindles in mitosis which will
result in either aborted mitosis or daughter cells with imbalanced
chromosome numbers (12, 43). However, the near complete
retention of all chromosomes from parent cell lines in our
hybrids and their remarkable chromosomal stability during
long-term passage suggested that they may have intrinsic mech-
the existence of additional centrosomes resulting from cell
fusion. Indeed, when we quantified the number of centrosomes
in postprophase mitosis in Bm, Lm, and various BLm cell lines
by staining the cells with the centrosome marker pericentrin, all
cells tested displayed a similar distribution of centrosome num-
bers: ?90% of cells in meta/ana/telophase harbor 2 centrosomes
as microtubule-organizing centers; ?10% of the cells have more
than 2 centrosomes, which may be accounted for by the basal
level of centrosome amplification present in MDA-MB-231 cells
(Fig. S8). This result suggests that BLm hybrids are able to
maintain bipolar cell division despite initially gaining additional
centrosomes after cell fusion. When we stained cells for both the
centrosome marker (pericentrin) and a centriole marker (cen-
trin) and examined the centriole distribution in the cells with 2
centrosomes during mitosis (Fig. S9), we observed 3 major
configurations: (i) 2 standard centrosomes each containing 2
centrioles; (ii) 1 or both centrosomes containing ?2 centrioles
(coalesced centrioles); and (iii) centrioles dispersed in the cy-
toplasm without pericentrolar materials (orphan centrioles) in
addition to the centrioles associated with the 2 centrosomes.
Therefore, we envision 3 corresponding scenarios that may have
allowed hybrids to divide successfully despite supernumerary
centrioles introduced by cell fusion: (i) hybrids retain only 2
structurally and functionally intact centrosomes by discarding
extra centrioles; (ii) multiple centrioles coalesce into 2 spindle,
similar to what was observed in neuroblastoma (43, 44); and (iii)
multipolar mitosis is suppressed by keeping extra centrioles in a
pericentriolar material-free, inactive status (orphan centrioles).
Interestingly, when these 3 configurations were quantified, a
significantly higher percentage (50.0%) of cells with orphan
centrioles was seen in BLmDrugcompared with Bm (22.1%) and
Lm (19.4%), perhaps representing a vestige of the fusion events
responsible for the formation of BLmDrug. Orphan centrioles
might therefore be an important approach for hybrids to main-
tain relative chromosomal stability following cell fusion.
Overall, our study demonstrates a role for cell fusion in the
rapid acquisition of complex malignant traits such as metastasis
organotropism in tumor cells. Genomic and phenotypic charac-
teristics of both parental cells were assimilated in the resultant
hybrids without an overall nuclear reprogramming toward a
predominant fusion partner and were stably maintained in
hybrids after long-term passage in vitro and in vivo. These
observations, together with recent findings of the involvement of
cell fusion in many physiological and pathological conditions,
suggest a potentially important role of cell fusion in cancer
progression that warrants further investigation.
Materials and Methods
Cell Culture. SCP2 (Bm) and LM2 (Lm) sublines were derived from the parental
cell line MDA-MB-231 (ATCC) (4, 5) and were generously provided by Joan
Massauge ´ (Sloan-Kettering Institute, New York). These sublines and their
fusion variants were maintained in DMEM with10% FBS and antibiotics
supplemented with appropriate selective drugs. H29 cells, a packaging cell
with 10% FBS, 1% glutamine, and antibiotics.
Generation of Hybrids by Spontaneous Cell Fusion. Bm and Lm cells were
labeled with different fluorescence, bioluminescence, and drug selection
makers before fusion. The retroviral construct pMSCV-hyg-hrl-mrfp-ttk was
reporter from pcDNA3.1-CMV-hrl-mrfp-ttk (45) to the BglII and HpaI sites of
pMSCV-hyg (Clontech). Retroviruses were generated from the H29 packing
cell lines and used to transduce Lm cells. Bm cells were first labeled with the
resistant to puromycin. To obtain fusion hybrids, 106RFP-Rluc/hyg labeled Lm
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cells were mixed with 106GFP-Fluc/pur labeled Bm cells in a 10-cm dish. After
24 h, the coculture was either selected with 1.5 mg/mL hygromycin B and 0.3
?g/mL puromycin to obtain dual resistant cells, or subjected to FACS to purify
with 70% ethanol, treated with 0.12 mg/mL RNase A (Sigma), stained with 8
?g/mL propidium iodide (Sigma), and analyzed by flow cytometry.
Karyotype Analysis. To obtain metaphase spreads to quantify chromosome
The cells were harvested, treated with 0.075 M KCl for 10 min, fixed with a
the SKY/FISH facility in the Roswell Park Cancer Institute as described (47).
Tumor Xenografts and Analysis. All procedures involving mice, such as housing
and care, and all experimental protocols were approved by Institutional
Animal Care and Use Committee (IACUC) of Princeton University. For intra-
cardiac injections, 105cells in PBS were injected into the left cardiac ventricle
of 4-week-old, female nude mice (NCI) as described (4). For i.v. injection, 2 ?
105cells in PBS were injected into the tail vein of nude mice as described (5).
Development of metastases in bone and lung was monitored by BLI with the
IVIS Imaging System (Xenogen) as described (4, 5). BLI analysis was performed
of interest. Metastasis status was recorded as positive when BLI signal was
for constructing Kaplan-Meier curves. X-ray radiography analysis of bone
xenograft model, mammary fat pad injections and tumor size measurements
were performed following the procedure described previously (5).
Accession Number. Microarray data reported herein have been deposited at
(http://www.ncbi.nlm.nih.gov/geo/) with the accession no. GSE14244.
Statistical Analysis. Results were reported as mean ? SD. Kaplan-Meier curves
were created by using Stata 7.0 software (Stata Corporation). Log-rank test
was used to calculate the statistic significance of difference between metas-
tasis curves. Other comparisons were performed by using unpaired 2-sided
Student’s t test without equal variance assumption or nonparametric Mann-
Additional experimental procedures and discussion, including measure-
the contribution of cell fusion to hyperploidy are listed in SI Materials and
ACKNOWLEDGMENTS. We thank C. DeCoste for assistance on FACS; Roswell
Park Cancer Institute for SKY analysis; G. Hu and M. Yuan for technical
assistance in statistical analysis and animal experiments; G. Hu, Y. Wei, M.A.
Blanco, and members of our laboratories for helpful discussions; T.A. Guise
and K.S. Mohammad for training and technical advice in bone histology; J.
Massague ´ (Sloan-Kettering Institute, New York) for Lm and Bm cell lines; R.
Blasberg (Sloan-Kettering Institute, New York) for SFG-NESTGL plasmid; S.S.
Gambhir (University of California, Los Angeles) for triple-reporter plasmids;
and J. L. Salisbury (Mayo Clinic, Rochester, MN) for centrin antibody. Y.K. is a
Champalimaud Investigator and was funded by grants from the Department
of Defense, The American Cancer Society, The National Institutes of Health,
and the New Jersey Commission on Cancer Research. X.L. is a recipient of a
Harold W. Dodds Fellowship from Princeton University.
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www.pnas.org?cgi?doi?10.1073?pnas.0900108106Lu and Kang