Ex vivo culture of circulating breast
tumor cells for individualized testing
of drug susceptibility
Min Yu,1,2 Aditya Bardia,1,3 Nicola Aceto,1 Francesca Bersani,1 Marissa W. Madden,1
Maria C. Donaldson,1 Rushil Desai,1 Huili Zhu,1 Valentine Comaills,1 Zongli Zheng,1,4,5
Ben S. Wittner,1 Petar Stojanov,6 Elena Brachtel,4 Dennis Sgroi,1,4 Ravi Kapur,7
Toshihiro Shioda,1,3 David T. Ting,1,3 Sridhar Ramaswamy,1,3 Gad Getz,1,4,6 A. John Iafrate,1,4
Cyril Benes,1,3 Mehmet Toner,7,8 Shyamala Maheswaran,1,8† Daniel A. Haber1,2,3†
Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients
with solid tumors. It has been proposed that the isolation, ex vivo culture, and characterization
of CTCs may provide an opportunity to noninvasively monitor the changing patterns of drug
susceptibility in individual patients as their tumors acquire new mutations. In a proof-of-concept
study, we established CTC cultures from six patients with estrogen receptor–positive breast cancer.
Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines
revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen
receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among
others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new
therapeutic targets. With optimization of CTC culture conditions, this strategy may help identify
the best therapies for individual cancer patients over the course of their disease.
Circulating tumor cells (CTCs) are present in the blood of many patients with solid tu- mors. Most of these cells, which are thought o be involved in metastasis, die in the circulation, presumably due to the loss of
matrix-derived survival signals or circulatory shear
stress. Nonetheless, if CTCs can be isolated from
cancer patients as viable cells that can be geno-
typed and functionally characterized over the
course of therapy, they have the potential to iden-
tify treatments that most effectively target the
evolving mutational profile of the primary tumor
(1). The isolation of viable CTCs is technically
challenging: Most methods yield low numbers of
partially purified CTCs that are fixed before iso-
lation, damaged during the cell purification pro-
cess, or irreversibly immobilized on an adherent
matrix [see review (2)]. We recently reported a
microfluidic technology, the CTC-iChip, which
efficiently depletes normal blood cells, leaving
behind unmanipulated CTCs (3). The cytological
appearance, staining properties, and intact RNA
evident within a subset of CTCs isolated by means
of this tumor antigen-agnostic CTC isolation plat-
form suggested that the cells may be viable.
To investigate whether the CTCs were in fact
viable, we applied the CTC-iChip to blood samples from patients with metastatic estrogen receptor (ER)–positive breast cancer. After testing
a range of culture conditions (4–7) (see supplementary methods), we found that CTCs proliferated best as tumor spheres when cultured in
serum-free media supplemented with epidermal
growth factor (EGF) and basic fibroblast growth
factor (FGF) (8) under hypoxic conditions (4% O2)
(Fig. 1A). Nonadherent culture conditions were
critical, because CTCs senesced after a few cell
divisions in adherent monolayer culture (fig.
S1). We established long-term oligoclonal CTC
1Massachusetts General Hospital Cancer Center, Harvard
Medical School, Charlestown, MA 02129, USA. 2Howard
Hughes Medical Institute, Chevy Chase, MD 20815, USA.
3Department of Medicine, Harvard Medical School,
Charlestown, MA 02129, USA. 4Department of Pathology,
Harvard Medical School, Charlestown, MA 02129, USA.
5Department of Medical Epidemiology and Biostatistics,
Karolinska Insitutet, Stockholm, Sweden. 6Broad Institute of
Harvard and MIT, Cambridge, MA 02142, USA. 7Center for
Bioengineering in Medicine, Harvard Medical School,
Charlestown, MA 02129, USA. 8Department of Surgery,
Harvard Medical School, Charlestown, MA 02129, USA.
*Present address: Department of Stem Cell Biology and Regenerative
Medicine, University of Southern California, Los Angeles, CA 90033,
USA. †Corresponding author. E-mail: firstname.lastname@example.org.
harvard.edu (S.M.); email@example.com (D.A.H.)
Fig. 1. Ex vivo expansion
of breast cancer CTCs. (A)
Representative images of
nonadherent CTC culture
(BRx-07). Top: Phase contrast.
Scale bar, 100 mm. Middle:
for cytokeratin (CK, red), Ki67
(yellow), CD45 (green), nuclei
(DAPI), blue]. Scale bar, 20 mm.
Bottom: Light microscopic
imaging with Papanicolaou
staining. Comparable images
for uncultured primary CTCs
are shown in the insets.
Scale bar, 20 mm. (B) (Left)
showing growth of NSG mouse
xenografts, after implantation
of 20,000 cultured CTCs
(BRx-07) into the mammary
fat pad. (Right) Quantification
of bioluminescent signals
for BRx-07–derived mouse
xenografts (mean T SD, n = 6).
(C) Histology of matched
primary breast tumors, cultured CTCs, and CTC-derived mouse xenografts for two CTC lines. All panels show cellular staining with hematoxylin (blue) and
immunohistochemical staining for ER expression (brown). Scale bar, 20 mm.