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/
June 9, 2009 ?
vol. 106 ?
no. 23 ?
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.
1. Chin L, Gray JW (2008) Translating insights from the cancer genome into clinical
practice. Nature 452:553–563.
2. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70.
3. Fidler IJ (2003) The pathogenesis of cancer metastasis: The ‘seed and soil’ hypothesis
revisited. Nat Rev Cancer 3:453–458.
Cancer Cell 3:537–549.
5. Minn AJ, et al. (2005) Genes that mediate breast cancer metastasis to lung. Nature
6. Duelli D, Lazebnik Y (2003) Cell fusion: A hidden enemy? Cancer Cell 3:445–448.
7. Pawelek JM, Chakraborty AK (2008) Fusion of tumour cells with bone marrow-derived
cells: A unifying explanation for metastasis. Nat Rev Cancer 8:377–386.
stem cell: Current controversies and new insights. Nat Rev Cancer 5:899–904.
9. Larizza L, Schirrmacher V (1984) Somatic cell fusion as a source of genetic rearrange-
ment leading to metastatic variants. Cancer Metastasis Rev 3:193–222.
10. Fortuna MB, Dewey MJ, Furmanski P (1989) Cell fusion in tumor development and
progression: Occurrence of cell fusion in primary methylcholanthrene-induced tumor-
igenesis. Int J Cancer 44:731–737.
11. Miller FR, McInerney D, Rogers C, Miller BE (1988) Spontaneous fusion between
metastatic mammary tumor subpopulations. J Cell Biochem 36:129–136.
Nat Rev Cancer 2:815–825.
13. Ogle BM, Cascalho M, Platt JL (2005) Biological implications of cell fusion. Nat Rev Mol
Cell Biol 6:567–575.
14. Singec I, Snyder EY (2008) Inflammation as a matchmaker: Revisiting cell fusion. Nat
Cell Biol 10:503–505.
15. Bjornson CR, Rietze RL, Reynolds BA, Magli MC, Vescovi AL (1999) Turning brain into
blood: A hematopoietic fate adopted by adult neural stem cells in vivo. Science
16. Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR (2000) Turning blood into
brain: Cells bearing neuronal antigens generated in vivo from bone marrow. Science
17. Brazelton TR, Rossi FM, Keshet GI, Blau HM (2000) From marrow to brain: Expression
of neuronal phenotypes in adult mice. Science 290:1775–1779.
19. Terada N, et al. (2002) Bone marrow cells adopt the phenotype of other cells by
spontaneous cell fusion. Nature 416:542–545.
20. Wang X, et al. (2003) Cell fusion is the principal source of bone-marrow-derived
hepatocytes. Nature 422:897–901.
21. Alvarez-Dolado M, et al. (2003) Fusion of bone-marrow-derived cells with Purkinje
neurons, cardiomyocytes, and hepatocytes. Nature 425:968–973.
22. Cowan CA, Atienza J, Melton DA, Eggan K (2005) Nuclear reprogramming of somatic
cells after fusion with human embryonic stem cells. Science 309:1369–1373.
23. Silva J, Chambers I, Pollard S, Smith A (2006) Nanog promotes transfer of pluripotency
after cell fusion. Nature 441:997–1001.
24. Johansson CB, et al. (2008) Extensive fusion of haematopoietic cells with Purkinje
neurons in response to chronic inflammation. Nat Cell Biol 10:575–583.
25. Nygren JM, et al. (2008) Myeloid and lymphoid contribution to non-haematopoietic
26. Coussens LM, Werb Z (2002) Inflammation and cancer. Nature 420:860–867.
27. Duelli DM, et al. (2007) A virus causes cancer by inducing massive chromosomal
instability through cell fusion. Curr Biol 17:431–437.
28. Duelli D, Lazebnik Y (2007) Cell-to-cell fusion as a link between viruses and cancer. Nat
Rev Cancer 7(12):968–976.
29. Overholtzer M, et al. (2007) A nonapoptotic cell death process, entosis, that occurs by
cell-in-cell invasion. Cell 131(5):966–979.
marrow transplant recipient. Bone Marrow Transplant 34:183–186.
31. Yilmaz Y, Lazova R, Qumsiyeh M, Cooper D, Pawelek J (2005) Donor Y chromosome in
renal carcinoma cells of a female BMT recipient: Visualization of putative BMT-tumor
hybrids by FISH. Bone Marrow Transplant 35:1021–1024.
32. Johnson RT, Rao PN (1970) Mammalian cell fusion: Induction of premature chromo-
some condensation in interphase nuclei. Nature 226:717–722.
33. Atkin NB (1979) Premature chromosome condensation in carcinoma of the bladder:
Presumptive evidence for fusion of normal and malignant cells. Cytogenet Cell Genet
34. Kovacs G (1985) Premature chromosome condensation: Evidence for in vivo cell fusion
in human malignant tumours. Int J Cancer 36:637–641.
35. Williams DM, Scott CD, Beck TM (1976) Premature chromosome condensation in
human leukemia. Blood 47:687–693.
36. Kang HS, Youn YK, Oh SK, Choe KJ, Noh DY (2000) Flow cytometric analysis of primary
tumors and their corresponding metastatic nodes in breast cancer. Breast Cancer Res
37. Isharwal S, et al. (2008) Prognostic value of Her-2/neu and DNA index for progression,
metastasis and prostate cancer-specific death in men with long-term follow-up after
radical prostatectomy. Int J Cancer 123:2636–2643.
38. Horn LC, Raptis G, Nenning H (2002) DNA cytometric analysis of surgically treated
squamous cell cancer of the uterine cervix, stage pT1b1-pT2b. Anal Quant Cytol Histol
39. Gupta GP, Massague ´ J (2006) Cancer metastasis: Building a framework. Cell 127:679–
40. Wei Y, Au JL-S (2005) Role of tumor microenvironment in mediating chemoresistance.
Cancer growth and progression. In Integration/Interaction of Oncologic Growth, ed
Meadows GG (Springer, New York), Vol 15, pp 285–321.
41. Stanbridge EJ, et al. (1982) Human cell hybrids: Analysis of transformation and tumor-
igenicity. Science 215:252–259.
42. Miller FR, Mohamed AN, McEachern D (1989) Production of a more aggressive tumor
cell variant by spontaneous fusion of two mouse tumor subpopulations. Cancer Res
43. Brinkley BR (2001) Managing the centrosome numbers game: From chaos to stability
in cancer cell division. Trends Cell Biol 11:18–21.
45. Ray P, De A, Min J-J, Tsien RY, Gambhir SS (2004) Imaging tri-fusion multimodality
reporter gene expression in living subjects. Cancer Res 64:1323–1330.
46. Ponomarev V, et al. (2004) A novel triple-modality reporter gene for whole-body
fluorescent, bioluminescent, and nuclear noninvasive imaging. Eur J Nucl Med Mol
47. Bartos JD, et al. (2004) Genomic heterogeneity and instability in colorectal cancer:
Spectral karyotyping, glutathione transferase-Ml and ras. Mutat Res 568:283–292.
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