breast cancer cell lines. Of seven PIK3CA-mutant
breast cancer lines, six were responsive to BYL719
(fig. S15). In addition to their characteristic PIK3CA
mutation, two lines harbored mutations of unknown importance in FGFR4 (Y367C; MDA-MB-
453 cells) and in FGFR2 (K570E; EFM-19 cells).
The former showed cooperative cytotoxicity by
BYL719 and AZD4547, whereas the latter was insensitive to FGFR inhibition (fig. S15). One of five
PIK3CA-mutant breast cancer lines without an
FGFR gene mutation showed modest sensitivity
to AZD4547 (CAL51), whereas the other four were
resistant. Thus, the combination of genotyping
and functional testing for drug susceptibility is
essential to defining therapeutically relevant driver
mutations in both breast cancer cell lines and CTC
In vitro screening of additional drugs for co-operation with PIK3CA-targeted agents identified
inhibitors of the insulin-like growth factor receptor 1 (IGF1R, inhibitors OSI906 and BMS754807)
and HSP90 (inhibitor STA9090, Ganetespib)
(Fig. 3C). Although neither of these is mutated in
BRx-07 cells, IGF1R has been implicated in modulating signaling loops that mitigate sensitivity to
PI3K inhibitors (24), and HSP90 is involved in
stabilization of mutant kinases (20). To extend
drug sensitivity studies to mouse xenografts, we
generated BRx-07–derived mammary tumors and
treated these with BYL719, AZD4547, the two agents
in combination, or diluent control. In vivo tumor
suppression was observed after treatment with
either drug individually, whereas the combination completely abrogated tumor growth (Fig. 3D).
In this proof-of-concept study, we have shown
that the culture of tumor cells circulating in the
blood of patients with breast cancer provides an
opportunity to study patterns of drug susceptibility, linked to the genetic context that is unique
to an individual tumor. In patients with hormone-responsive breast cancer, most of whom have
bone metastases that are not readily biopsied,
the ability to noninvasively and repeatedly analyze live tumor cells shed into the blood from
multiple metastatic lesions may enable monitoring of emerging subclones with altered mutational
and drug sensitivity profiles. The successful culture of CTCs stems partly from the application of
a microfluidic device capable of effectively depleting leukocytes from a blood specimen while
preserving viable tumor cells for ex vivo expansion
(3). The proliferation of cultured CTCs as nonadherent spheres differs from that of characteristic epithelial cancer cell cultures and may reflect
intrinsic properties of tumor cells that remain
viable in the bloodstream after loss of attachment
to basement membrane. A recent report documented direct inoculation of the mouse femur
with blood-derived cancer cells from a patient
who had very high numbers of CTCs, but in vitro
culture was not successful (25). Our results differ
from the adherent in vitro CTC cultures described
by Zhang et al. (26), but these lines appear to share
the identical TP53, BRAF, and KRAS genotype of
the highly tumorigenic MDA-MB- 231 cell line.
Optimization of CTC culture conditions will be
needed before this strategy can be incorporated
into clinical practice. In addition, further char-
acterization of the nonadherent CTC-derived cell
lines described here will be required to define
how they differ from cells cultured from primary
tumor biopsies or directly implanted into mouse
models (4, 14). In the future, strategies such as that
described here may be an essential component
of “precision medicine” in oncology, where treat-
ment decisions are based on evolving tumor mu-
tational profiles and drug sensitivity patterns in
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We are grateful to all the patients who participated in this study.
We thank Dr. Lecia Sequist for coordinating the clinical studies,
A. McGovern, C. Hart, and the Massachusetts General Hospital (MGH)
clinical research coordinators P. Spuhler, A. Shah, J. Ciciliano, and V. Pai
for bioengineering technical support; R. Milano, K. Lynch, H. Robinson,
and M. Liebers for technical support; L. Collins (Beth Israel Deaconess
Medical Center) for providing pathological specimens; and L. Libby for
mouse studies. N. Aceto is a fellow of the Human Frontiers Science
Program, the Swiss National Science Foundation, and the Swiss
Foundation for Grants in Biology and Medicine. This work was supported
by grants from the Breast Cancer Research Foundation (D.A.H), Stand
Up to Cancer (D.A.H., M. T., S.M.), the Wellcome Trust (D.A.H., C. B.),
National Foundation for Cancer Research (D.A.H.), NIH CA129933
(D.A.H.), NIBIB EB008047 (M. T., D. A. H.), Susan G. Komen for the Cure
KG09042 (S.M.), National Cancer Institute–MGH Proton Federal Share
Program (S.M.), the MGH-Johnson and Johnson Center for Excellence
in C TCs (M. T., S.M.), and the Howard Hughes Medical Institute (M. Y.,
D.A.H.). A.J.I. holds equity in, and is a paid consultant for, Enzymatics, Inc.
M. T., D.A.H., and the Massachusetts General Hospital have filed for
patent protection for the CTC-iChip technology. RNA-Seq reads have been
deposited into Gene Expression Omnibus: uncultured CTCs (accession
no. GSE51827); the six cultured CTC lines (accession no. GSE55807).
Materials and Methods
Figs. S1 to S15
Tables S1 to S3
13 January 2014; accepted 10 June 2014
BACTERIAL CELL WALL
MurJ is the flippase of lipid-linked
precursors for peptidoglycan biogenesis
Lok-To Sham,1 Emily K. Butler,2 Matthew D. Lebar,3 Daniel Kahne,3,4
Thomas G. Bernhardt,1 Natividad Ruiz2*
Peptidoglycan (PG) is a polysaccharide matrix that protects bacteria from osmotic lysis.
Inhibition of its biogenesis is a proven strategy for killing bacteria with antibiotics. The assembly
of PG requires disaccharide-pentapeptide building blocks attached to a polyisoprene lipid
carrier called lipid II. Although the stages of lipid II synthesis are known, the identity of the
essential flippase that translocates it across the cytoplasmic membrane for PG polymerization
is unclear. We developed an assay for lipid II flippase activity and used a chemical genetic
strategy to rapidly and specifically block flippase function. We combined these approaches to
demonstrate that MurJ is the lipid II flippase in Escherichia coli.
Bacteria use polyisoprenoid-linked oligo- saccharides to assemble the essential pep- tidoglycan (PG) matrix that surrounds their cytoplasmic membrane and fortifies their cell envelope against high internal
osmotic pressure (1). The building block of PG
is a disaccharide-pentapeptide that is synthe-
sized at the cytoplasmic leaflet of the inner mem-
brane (IM) as a precursor known as lipid II
(Fig. 1A) (1, 2). This precursor must be flipped
across the membrane for cell wall synthesis. The
identity of the lipid II flippase has been contro-
versial, with the debate centered on two candi-
dates: MurJ-like and FtsW/RodA-like proteins
1Department of Microbiology and Immunobiology, Harvard
Medical School, Boston, MA 02115, USA. 2Department of
Microbiology, Ohio State University, Columbus, OH 43210,
USA. 3Department of Chemistry and Chemical Biology,
Harvard University, Cambridge, MA 02138, USA.
4Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
*Corresponding author. E-mail: thomas_bernhardt@hms.
harvard.edu ( T.G.B.); firstname.lastname@example.org (N.R.)