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Published OnlineFirst April 6, 2016; DOI: 10.1158/0008-5472.CAN-15-2998
Cancer
Research
Microenvironment and Immunology
Endothelial Side Population Cells Contribute
to Tumor Angiogenesis and Antiangiogenic
Drug Resistance
Hisamichi Naito, Taku Wakabayashi, Hiroyasu Kidoya, Fumitaka Muramatsu,
Kazuhiro Takara, Daisuke Eino, Keitaro Yamane, Tomohiro Iba, and
Nobuyuki Takakura
Abstract
Angiogenesis plays a crucial role in tumor growth, with an
undisputed contribution of resident endothelial cells (EC) to
new blood vessels in the tumor. Here, we report the definition
of a small population of vascular-resident stem/progenitor–like
EC that contributes predominantly to new blood vessel formation in the tumor. Although the surface markers of this population are similar to other ECs, those from the lung vasculature
possess colony-forming ability in vitro and contribute to angiogenesis in vivo. These specific ECs actively proliferate in lung
tumors, and the percentage of this population significantly
increases in the tumor vasculature relative to normal lung
tissue. Using genetic recombination and bone marrow trans-
plant models, we show that these cells are phenotypically true
ECs and do not originate from hematopoietic cells. After
treatment of tumors with antiangiogenic drugs, these specific
ECs selectively survived and remained in the tumor. Together,
our results established that ECs in the peripheral vasculature
are heterogeneous and that stem/progenitor–like ECs play an
indispensable role in tumor angiogenesis as EC-supplying
cells. The lack of susceptibility of these ECs to antiangiogenic
drugs may account for resistance of the tumor to this drug type.
Thus, inhibiting these ECs might provide a promising strategy
to overcome antiangiogenic drug resistance. Cancer Res; 76(11);
Introduction
respond only minimally (4). Moreover, inhibition of VEGF/
VEGFR induces pruning of abnormal tumor blood vessels while
maintaining less abnormal blood vessels, which in turn continue to provide a blood supply to the tumor (5). Several
mechanisms of resistance to antiangiogenic drugs have been
proposed, but little attention has been paid to the heterogeneity of resident vascular endothelial cells (EC). New blood
vessels in the tumor were originally considered to be formed by
angiogenesis. However, in 1997, the concept was proposed that
vasculogenesis, that is, the recruitment of bone marrow–
derived endothelial precursor cells (EPC) into new blood
vessels, also persists into adult life and contributes to the
formation of new blood vessels (6). Notably, it has been shown
that bone marrow–derived ECs could constitute >50% of all
ECs in the tumor vasculature (7). In contrast, recent reports
have failed to show a direct contribution of EPCs to newly
formed ECs (8–10), suggesting an exclusive role of resident ECs
for neovascular formation.
The adult lung has long been viewed as a very quiescent
tissue, with pulmonary circulation incapable of supporting the
growth of new vessels. However, accumulating evidence suggests that the lung harbors tissue-resident stem cells and has
remarkable reparative capacity (11, 12). Moreover, the existence of vascular-resident progenitor cells that are able to
differentiate into ECs has been proposed (13). However, their
characteristics and contribution to the tumor vasculature have
not been clearly defined. In earlier work, we identified stem/
progenitor–like ECs in peripheral blood vessels based on their
ability to efflux Hoechst 33342 dye (14). Cells that do this are
termed side population (SP) cells because of their characteristic
Lung cancer is the leading cause of cancer death worldwide.
Although there have been recent advances in diagnosis and
treatment, the prognosis is still very poor. Angiogenesis, the
development of new blood vessels from preexisting vessels
supplying oxygen and nutrients to the tumor, is regarded as
a hallmark of cancer development (1). Therapeutic strategies
aimed at destroying newly formed blood vessels with antiangiogenic drugs have now been adopted clinically for many
types of solid tumors, including lung cancer. Many antiangiogenic agents have been developed targeting VEGF itself or
VEGFRs and downstream signaling pathways (2). Although
therapy with these agents has resulted in retardation of tumor
progression in some patients, the results are more modest than
expected (3). This may be for several reasons, including the
possibility that many cancer patients are intrinsically refractory
to, or develop resistance to, antiangiogenic therapy and
Department of Signal Transduction, Research Institute for Microbial
Diseases, Osaka University, Suita, Osaka, Japan.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Authors: Nobuyuki Takakura, Research Institute for Microbial
Diseases, Osaka University, 3-1 Yamada-Oka, Suita-shi, Osaka 565-0871, Japan.
Phone: 816-6879-8316; Fax: 816-6879-8314; E-mail: [email protected];
and Hisamichi Naito, [email protected]
doi: 10.1158/0008-5472.CAN-15-2998
2016 American Association for Cancer Research.
3200–10. 2016 AACR.
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Endothelial SP Cells in Tumor Angiogenesis
appearance in flow cytometry. SP cells appear as a discrete
subpopulation at the side of main population (MP) cells. The
Hoechst method was first developed as a purification method
for hematopoietic stem cells (15) and is now applied to a wide
variety of tissue-resident stem cells and cancer cells (16, 17). We
showed that these endothelial-SP (EC-SP) cells possess EC
colony–forming potential in vitro and contribute to angiogenesis by generating functional mature blood vessels when transplanted into an ischemic limb. In this study, we characterize
EC-SP cells in the lung vasculature and examine the origin of
these cells using a Cre/loxP–based lineage-tracing system
and bone marrow transplantation model. Furthermore, we
show a contribution of EC-SP cells to the tumor vasculature
and their potential role in resistance to small-molecule antiangiogenic drugs. Our data suggest that this heterogeneity of
ECs may be a mechanism contributing to resistance to antiangiogenic therapy.
Materials and Methods
Mice
C57BL/6, DBA2, and C57BL/6-Tg (CAG-EGFP) mice
[enhanced GFP (EGFP) mice that express GFP in all tissues]
were purchased from Japan SLC. Mice 8 to 12 weeks of age were
used for all experiments. VE-Cadherin (BAC) CreERT2 (18) and
Flox-CAT-EGFP mice (19, 20) were provided by Drs. Yoshiaki
Kubota (Keio University, Tokyo, Japan) and Toshio Suda
(Kumamoto University, Kumamoto, Japan), respectively.
Vav1-Cre mice were purchased from The Jackson Laboratory.
VE-Cadherin (BAC) CreERT2 mice were crossed with FloxCAT-EGFP mice, and recombination was induced by intraperitoneal injection of tamoxifen (Sigma) at adult ages (older than
2 months). All experimental procedures in this study were
approved by the institutional Animal Care and Use Committee
of Osaka University (Osaka, Japan).
Cell preparation
Cells from lung and tumor were isolated as described previously, with slight modification (21). Briefly, mice were euthanized and organs were excised, minced, and digested with Dispase
II (Roche Applied Science), collagenase (Wako), and type II
collagenase (Worthington Biochemical Corp.) with continuous
shaking at 37 C. The digested tissue was passed through 40-mm
filters to yield single-cell suspensions. Red blood cells (RBC) were
lysed with ACK buffer (0.15 mol/L NH4Cl, 10 mmol/L KHCO3,
and 0.1 mmol/L Na2-EDTA).
Flow cytometry
Hoechst staining was performed as described previously (14).
Briefly, cell-surface antigen staining was performed, and cell
suspensions were incubated with Hoechst 33342 (5 mg/mL;
Sigma) at 37 C for 90 minutes in DMEM [2% FCS (Sigma), 1
mmol/L HEPES (Sigma)] at a concentration of 106 nucleated
cells/mL in the presence or absence of verapamil (50 mmol/L;
Sigma). Cell-surface antigen staining was performed as described
previously (22). The mAbs used in immunofluorescence staining
were anti-CD31, -CD34, -CD44, -CD45, -VE-cadherin, -Flk1,
-Sca1, and -c-Kit mAbs (BD Biosciences). Respective isotype
controls (BD Biosciences) were used as negative controls. Propidium iodide (PI, 2 mg/mL; Sigma) was added before FACS analysis
to exclude dead cells. Flow cytometry of stained cells was per-
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formed on a SORP FACSAria (BD Biosciences), and data were
analyzed using FlowJo Software (Tree Star Software). A UV laser
(emitting at 355 nm) was used to excite the Hoechst dye. To
analyze cell-cycle status using Pyronin Y (Sigma), cells were first
stained with Hoechst 33342 at 37 C for 45 minutes, and then
0.5 mg/mL of Pyronin Y was added and incubation continued
at 37 C for another 45 minutes.
Cell culture
Lewis lung carcinoma (LLC; RIKEN Cell Bank) was maintained in DMEM (Sigma) supplemented with 10% FCS and 1%
penicillin/streptomycin (Life Technologies). The KLN205
(mouse squamous cell carcinoma; RIKEN Cell Bank) cell line
was cultured in aMEM (Sigma) supplemented with 10% FCS,
1% penicillin/streptomycin, 2 mmol/L L-glutamine (Life technologies), and 1% nonessential amino acids (Life technologies). The OP9 stromal cells (RIKEN Cell Bank) were maintained in DMEM with 20% FCS, 2 mmol/L L-glutamine, and 1%
penicillin/streptomycin. Cell lines utilized are mycoplasma
free, authenticated by supplier based on morphology, growth
curve analysis, and isoenzyme analysis, and were passaged for
fewer than 6 months after resuscitation.
EC colony–forming assay and in vitro VEGFR inhibition assay
The sorted EC-SP or -MP cells were seeded into plates and
cocultured on OP9 stromal cells in RPMI (Sigma), supplemented
with 10% FCS and 105 mol/L 2-mercaptoethanol (Life Technologies). VEGF (10 ng/mL; PeproTech) was added every three
days. For the in vitro VEGFR inhibition assay, vandetanib (LC
Laboratories) was added to the EC-SP or -MP coculture dishes at
different concentrations. For the drug accumulation study, nonspecific inhibitors of the ATP-binding cassette (ABC) transporter,
cyclosporine A (10 nmol/L, Sigma) or verapamil (5 nmol/L) was
added to the VEGFR inhibition assay. Cells were fixed for immunostaining after 10 days.
Murine bone marrow transplantation model
Eight- to 12-week-old C57BL/6 mice underwent bone marrow
transplantation from age-matched donor EGFP mice as described
previously (23). Briefly, bone marrow cells were obtained by
flushing the tibias and femurs. RBCs were depleted using ACK
buffer. Transplantation was performed into C57BL/6 mice lethally irradiated with 10.0 Gy by intravenous infusion of approximately 1 106 donor RBC-lysed bone marrow cells. At 24 weeks
after transplantation, by which time recipient bone marrow was
reconstituted, the mice were used for the experiments. The percent
reconstitution of the bone marrow was confirmed in all mice at
the time of the experiment.
Quantitative reverse-transcription PCR
RNA was extracted using RNeasy Mini Kits (Qiagen), and cDNA
was generated using reverse transcriptase from the ExScript RT
Reagent Kit (TaKaRa). Real-time PCR was performed using a
Stratagene Mx3000P (Stratagene). PCR was performed on cDNA
using specific primers. The sequences of the gene-specific primers
were as follows: 50 -TGG CAA AGT GGA GAT TGT TGC C-30 and 50 AAG ATG GTG ATG GGC TTC CCG-30 for GAPDH; 50 -CCA GCA
GTC AGT GTG CTT ACA-30 and 50 -GCC ACT CCA TGG ATA ATA
GCA-30 for ABCG2; 50 -CCA GCA GTC AGT GTG CTT ACA-30 and
50 -GCC ACT CCA TGG ATA ATA GCA-30 for ABCB1a; and 50 -TGA
TCA TCA GCA ACA GCA GTC-30 and 50 -TGA AAC CTG GAT GTA
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Naito et al.
GGC AAC-30 for ABCB1b. Expression level of the target gene was
normalized to the GAPDH level in each sample.
Tumor model
LLC or KLN205 cells (2 105 ) were mixed with 30-mL
Matrigel (BD Biosciences) and injected into the lung intercostally. Tumors were studied 2 weeks after implantation when
they were 5 to 10 mm in diameter. The lung metastasis model
was established by injecting 1 105 LLC cells intravenously. For
the tumor cell and EC-SP or -MP cell coinoculation assays, 2 105 LLC cells and 1 104 EC-SP or -MP cells sorted from EGFP
mice were mixed together and injected into the right lungs of
wild-type mice. After 10 days, those tumors >3 mm in diameter
were fixed and prepared for immunostaining. The number of
GFPþ vascular colonies was counted in three different sections
of each tumor. The sum of these counts was taken as the number
of colonies in that tumor. For drug treatment, vandetanib and
axitinib (Selleck Chemicals) were used as angiogenesis inhibitors as described previously (24). Vandetanib was dissolved in
DMSO (Sigma)–PEG-400 (Sigma; 1:1) and injected at a dose of
30 mg/kg i.p. for 3 days. Axitinib was dissolved in a solution of
PEG-400–acidified (pH 2–3) water (3:7) and injected at a dose
of 25 mg/kg i.p. for 3 days.
Immunohistochemical and immunofluorescence staining
For IHC, anti-CD31 antibody (BD Biosciences) was used for
staining, and biotin-conjugated polyclonal anti-rat IgG (Dako)
was used as the secondary antibody. Biotinylated secondary
antibodies were developed using ABC Kits (Vector Laboratories). DAB/NiCl2 (Sigma) was used for the color reaction. For
immunofluorescence studies, anti-CD31 antibody, Cy3-conjugated anti-SMA antibody (Sigma), and anti-GFP antibody
(Invitrogen) were used for staining and anti-rat IgG Alexa
Fluor-546, -647 and anti-rabbit IgG Alexa Fluor-488 (Invitrogen) as the secondary antibodies. Cell nuclei were visualized
with TO-PRO-3 (Invitrogen) or Hoechst dye (Sigma). Samples
were imaged using an Olympus IX-70 and Leica TCS/SP5
confocal microscope. Images were processed with the Leica
Application Suite (Leica) and Adobe Photoshop CS6 software
(Adobe Systems).
Statistical analysis
All data are presented as the mean SEM. Statistical
analyses were performed using Statcel 3 (OMS). Data were
analyzed by ANOVA, followed by Tukey–Kramer multiple
comparison tests. When only two groups were compared, the
two-sided Student t test was used. P < 0.01 was considered
significant.
Results
Identification and characterization of lung EC-SP cells
We first performed flow cytometric analysis of cells isolated
from adult mouse lung to identify EC-SP cells in the lung
vasculature using a UV laser emitting at 355 nm. Among cells
positive for the EC marker CD31 and negative for the hematopoietic cell marker CD45 (CD31þCD45 ECs; Fig. 1A), 0.67 0.24% were found in the Hoechst low-fluorescent SP fraction of the dot plot relative to Hoechst bright-fluorescent
MP cells (Fig. 1B), in line with our previous work (14). Low
intracellular accumulation of Hoechst is characteristic of SP
3202 Cancer Res; 76(11) June 1, 2016
cells, and this cell population disappears when cells are treated
with ABC transporter inhibitors, such as verapamil (Fig. 1C).
To evaluate the proliferative capacity of lung EC-SP cells in vitro,
we cultured them on OP9 stromal cells, which can support
EC growth (25). After 10 days, EC-SP cells generated a higher
number of CD31þ EC colonies than did EC-MP cells
(Fig. 1D–F). These data document the presence of EC-SP cells
in the lung vasculature and their potential to generate EC
colonies in vitro.
Next, we characterized the phenotype of lung EC-SP cells
and found that they express the EC markers VE-cadherin, Flk1,
Sca1, and CD34, as do EC-MP cells (Fig. 1G). The CD44
antigen, which is expressed by EC-MP cells but not by EC-SP
cells isolated from limb muscle (14), was not expressed
by either EC-SP or EC-MP cells of the lung. The c-Kit antigen,
a well-known marker for hematopoietic progenitor cells, was
widely expressed by EC-MP cells but not EC-SP cells (Fig. 1G).
We next analyzed the expression of a set of genes known to be
arterial or venous markers. The expression of the arterial markers Ephrin B2, Hey1, and Hey2 was lower in EC-SP than
EC-MP cells. In contrast, venous markers EphB4 and COUPTF2 did not differ in the two groups. Glycam1, which is
upregulated in lower limb EC-SP cells (14), was not detectable
in either lung EC-SP or -MP cells (Fig. 1H). These results
indicate that EC-SP cells in the lung vasculature are phenotypically indistinguishable from MP cells by well-known EC
markers. Furthermore, the EC-SP markers found in lower limb
EC-SP cells are not applicable to lung EC-SP cells.
EC-SP cells are vascular-resident ECs and are not derived from
bone marrow
We next examined whether lung EC-SP cells are committed
to the EC lineage by analyzing the lung vasculature of VEcadherin CreERT2 (BAC)/Flox-CAT-EGFP mice. As expected,
GFP was detected only in the ECs at the innermost layer of
peripheral blood vessels by immunostaining (Fig. 2A and B)
and not in peripheral blood (Fig. 2C) in these mice. Among
CD31þCD45 cells, >90% were GFPþ (Fig. 2C and D), suggesting that most ECs successfully underwent Cre-lox recombination. EC-SP cells are present in the GFPþCD31þCD45 EC
fraction (Fig. 2E). When cultured on OP9 stromal cells, these
GFPþ SP cells generated EC colonies (Fig. 2F). Previous work
by other investigators has demonstrated that lung SP cells from
adult mice are composed of CD45þ and CD45 SP cells (26).
They also showed that the small percentage of CD45 SP cells
is derived from the bone marrow (27). Therefore, we examined
whether lung EC-SP cells originate from bone marrow by
analyzing wild-type mice that had undergone bone marrow
transplantation from EGFP donor mice. Unlike in CD45þ lung
hematopoietic cells, there were no highly GFPþ cells in the
CD31þCD45EC fraction nor in the EC-SP fraction. Most of
the EC-SP cells were negative for GFP, although 3.12 0.28%
were weakly positive (GFPdim; Fig. 2G). To evaluate colonyforming capacity of GFPdim and GFP EC-SP cells, we cultured
each fraction on OP9 stromal cells. After 10 days, only GFP
EC-SP cells generated CD31þ colonies in numbers comparable
with wild-type mice (Fig. 2H and I). These results indicate that
EC-SP cells are phenotypically identical to terminally differentiated ECs in the endothelial-specific gene recombination
model and that colony-forming EC-SP cells do not originate
from bone marrow.
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Endothelial SP Cells in Tumor Angiogenesis
Figure 1.
þ
Identification and characterization of EC-SP cells in the lung vasculature. A, FACS analysis of lung ECs from DBA2 mice. CD31 CD45 ECs are gated in the
box. B, Hoechst analysis of ECs gated in A. Among ECs, 0.67 0.24% were in the SP gate. C, disappearance of EC-SP cells on verapamil treatment.
D and E, endothelial colony formation of EC-SP (D) and -MP (E) cells on OP9 feeder cells. Cells were stained with anti-CD31 antibody. Arrows, EC
þ
colonies. Inset, representative pattern of EC colony in higher magnification. F, quantitative evaluation of the number of CD31 EC colonies from
1,000 EC-SP or -MP cells (n > 7). G, histogram showing expression levels of surface markers in lung EC-SP (red line), -MP (black line) cells, and the
negative control (dotted gray line). H, quantitative RT-PCR analysis of mRNA in lung EC-SP and -MP cells, corrected for expression of the control gene
GAPDH. The arterial markers, EphrinB2, Hey1, and Hey2, were lower in EC-SP cells (n ¼ 6). Error bars, SEM. , P < 0.01; , P < 0.05. Scale bars, 5 mm
(D and E). N.D., not determined; N.S., not significant.
EC-SP cells are activated in the tumor and contribute to the
tumor vasculature
To study the potential of EC-SP cells in the lung to contribute
to the formation of new blood vessels in the tumor, we first
investigated their proliferative capacity in the tumor vasculature. We implanted LLC tumor cells orthotopically into the
lung, and after 14 days, tumors were dissected for Hoechst
analysis (Fig. 3A). In the tumor, the percentage of EC-SP cells
was higher (7.02 2.81%) than the control lung obtained from
the other side of the animal (Fig. 3B–E). We confirmed that
SP-ECs in the tumor were indeed SP cells by their response
to verapamil treatment (Fig. 3C and E). We next analyzed
the percentages of tumor EC-SP cells in the lung tumor metastasis model using LLC cells. The metastatic small nodules were
gathered, minced, and stained with Hoechst dye. In the CD31þ
CD45 EC fraction, the percentage of SP cells was significantly
higher (2.81 0.38%) than normal lung (Fig. 3F). To determine the mitotic state of these EC-SP and -MP cells, we next
performed cell-cycle analysis with Hoechst dye and the RNAbinding dye Pyronin Y to identify cells in G0 and G1 (28). In the
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normal lung, 96.6 1.4% of EC-SP cells and 96.7 1.0% of
EC-MP cells were in the Pyronin Y–negative G0 phase (Fig. 3G
and Supplementary Fig. S1A). In the tumor microenvironment,
the number of Pyronin Y–positive cells, reflecting the number of
cells in the active phase of the cell cycle, was significantly
increased compared with the normal steady state (Fig. 3H
and I and Supplementary Fig. S1B and S1C). Moreover, we
examined the expression of several key factors for the cell cycle
and for maintenance of quiescence using quantitative RT-PCR.
It has been reported that progression of the cell cycle is dependent on members of the Cip/Kip family of cyclin-dependent
kinase (CDK) inhibitors (p21, p27, and p57; ref. 29). Accordingly, the expression of all these factors was found to be
significantly lower in tumor EC-SP and -MP cells compared
with normal lung cells (Supplementary Fig. S1D–S1F). Furthermore, the percentage of Ki67-positive cells, indicating cell
proliferation (30), was higher in tumor EC-SP and -MP cells
compared with normal controls (Supplementary Fig. S1G).
These results indicate that both EC-SP and -MP cells in the
tumor are Ki67-positive actively proliferating cells in which
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Figure 2.
EC-SP cells are committed to the
endothelial lineage and do not
originate from bone marrow. A–F, data
using VE-cadherin promoter EGFP
mice. A, confocal microscopic image of
the lung of VE-cadherin promoter
EGFP mice stained with anti-CD31
antibody (red), TO-PRO-3 (blue), and
anti-GFP antibody (green). B, higher
power views of areas indicated by the
þ
box in A. Note that all the GFP cells are
þ
þ
CD31 , and most of the CD31 cells are
þ
GFP . However, there are a few GFP
þ
CD31 ECs (arrows), possibly occurring
by Cre leakage. C, FACS analysis of
þ
GFP cells from peripheral blood (red
þ
line) or lung CD31 CD45 ECs (black
line). D, representative dot plot of
þ
CD45 lung cells. GFP ECs and GFP
ECs are indicated by the box. E,
þ
þ
Hoechst analysis of GFP CD31 CD45
þ
ECs. F, GFP EC-SP cells indicated by
the red-gated region in E were cultured
on OP9 stromal cells and stained with
anti-CD31 antibody and anti-GFP
antibody. G–I, analysis using GFP-BMT
mice. G, FACS analysis of GFP-BMT
mice. Histogram showing GFP intensity
of lung hematopoietic cells (dot line),
þ
CD31 CD45 ECs (black line), and
EC-SP cells (red line). H and I, GFP
dim
EC-SP cells and GFP EC-SP cells (red
arrow region in G) were cultured on
OP9 stromal cells and stained with
CD31 antibody. Scale bars, 100 mm (A),
20 mm (B), 500 mm (F), and 5 mm
(H and I). BMT, bone marrow
transplantation.
p21, p27, and p57 were downregulated. Next, to study whether
the origin of tumor ECs is EC-SP cells, we first cultured sorted
tumor EC-SP and EC-MP cells on OP9 feeder cells to evaluate
their colony-forming capacity. After 10 days, tumor EC-SP cells
gave rise to EC colonies comparable with normal lung EC-SP
cells (Fig. 3J). On the other hand, although cell-cycle analysis
revealed that EC-MP cells were actively cycling, they generated
significantly lower numbers of EC colonies, and the colony size
was smaller (Fig. 3K and L). Furthermore, we inoculated tumor
cells with normal lung EC-SP or -MP cells to confirm their
contribution to the tumor vasculature in vivo. Lung EC-SP cells
were isolated from EGFP mice and inoculated together with LLC
tumor cells into the right lungs of wild-type mice. As expected,
many large GFPþ colony-like vascular structures were formed in
the EC-SP–coinoculated tumor. On the other hand, EC-MP cells
only formed a few GFPþ small dots (Fig. 4A and B). These
results indicate that although both EC-SP and -MP cells are
activated in the tumor, the former generate more ECs than the
latter and effectively contribute to the new blood vessels being
formed in the tumor microenvironment.
EC-SP cells are resistant to antiangiogenic therapy
Next, we compared the effectiveness of antiangiogenic drugs on
tumor EC-SP and -MP cells. After 3-day treatment of tumorbearing mice with the small-molecule tyrosine kinase inhibitors
3204 Cancer Res; 76(11) June 1, 2016
(TKI) axitinib or vandetanib targeting primarily the VEGFR, the
tumor vascularity decreased by 59% and 61%, respectively
(Fig. 5A–D). Although tumor vascular density decreased on
treatment with these two drugs, FACS analysis revealed that the
percentage of EC-SP cells in the tumor ECs increased to 17.6 6.3% and 19.9 6.5%, respectively (Fig. 5E–H). To determine the
sensitivity of EC-SP and -MP cells to these antiangiogenic drugs,
we next cultured them on OP9 stromal cells together with different doses of vandetanib. Addition of a high concentration
(100–500 nmol/L) of vandetanib inhibited colony formation
by both EC-SP and -MP cells (Fig. 6A). However, although colony
formation by EC-MP cells was significantly inhibited, EC-SP
cells maintained their colony formation potential at a low concentration (20 nmol/L) of vandetanib (Fig. 6B). It has been
reported that several drug transporters are highly expressed
in tissue-resident stem cells and cancer stem-like cells that have
an SP phenotype (31, 32). The ABC transporter family of proteins is also well characterized for its ability to efflux a wide
range of small molecules and drugs. It was also reported that
tumor ECs are resistant to low-dose chemotherapy through
expression of ABCB1 (33). Therefore, to clarify the mechanism
responsible for this differential sensitivity to antiangiogenic
drugs, we investigated the expression of mRNA for the ABC
drug transporters. The level of expression of mRNA for several
ABC drug transporters was indeed significantly higher in tumor
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Endothelial SP Cells in Tumor Angiogenesis
Figure 3.
þ
EC-SP cells proliferate in the tumor. A, gross appearance of the orthotopic lung tumor inoculation model using LLC cells. B–E, Hoechst analysis of CD31 CD45
ECs derived from lung tumor observed in A. B, tumor EC-SP cells. C, disappearance of EC-SP cells treated with verapamil. D, EC-SP cells in the other
side of the lung described as control in A. Note that the percentage of the EC-SP cells is comparable with wild-type mice. E, quantification of the
þ
percentage of EC-SP cells shown in B–D (n ¼ 5). F, Hoechst analysis of C31 CD45 ECs of tumor metastasis model using LLC cells. G and H, representative
dot plot of EC-SP cells with Pyronin Y and Hoechst staining of ECs in normal lung and lung tumor. I, quantification of the number of Pyronin Y–positive
þ
(PY ) cells relative to normal lung (n ¼ 5). J–L, endothelial colony formation by tumor-derived EC-SP and -MP cells cultured on OP9 feeder cells.
J and K, ECs were stained with anti-CD31 antibody. Note that tumor EC-SP cells generated larger EC colonies. L, quantification of the number of colonies
(n > 5). Scale bars, 5 mm (A), 500 mm (J and K). Data, mean SEM. , P < 0.01.
EC-SP cells (Fig. 6D). Next, to confirm that antiangiogenic drugs
were actually exported through these ABC transporters, tumor ECSP and -MP cells were cultured on OP9 stromal cells, together with
a low concentration (20 nmol/L) of vandetanib with or without
ABC transporter inhibitors. Although addition of verapamil or
cyclosporine A, which are known to inhibit the multiple drug
transporters, including ABCB1, itself partially blocked the appearance of EC-SP–derived colonies, a combination of these drugs
with vandetanib blocked colony formation more markedly (Fig.
6E and F). These results indicate that EC-SP cells are VEGF
dependent, as is the case for regular ECs. However, in the presence
of a low concentration of an antiangiogenic inhibitor, EC-SP cells
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are more resistant to the drugs, at least in part via ABC drug
transporter activity.
Furthermore, we examined the origin of tumor EC-SP cells
before and after antiangiogenic therapy to exclude the possibility that the increasing EC-SP cell population after antiangiogenic therapy derived from hematopoietic lineage cells. We
analyzed tumor-bearing Vav1 Cre/Flox-CAT-EGFP (Vav1-GFP)
mice and VE-cadherin CreERT2 (BAC)/Flox-CAT-EGFP (VEcadherin-GFP) mice. In these transgenic mice, hematopoietic
lineage cells are GFPþ in Vav1-GFP mice, and endothelial
lineage cells are GFPþ in VE-cadherin-GFP mice. FACS analysis
of cells from tumors generated in VE-cadherin-GFP mice
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Naito et al.
Figure 4.
EC-SP cells contribute to tumor angiogenesis. A, IHC on sections from LLC tumors generated by orthotopic transplantation of LLC cells, together with
lung EC-SP or -MP cells sorted from EGFP mice. Expression of CD31 (blue), GFP (green), and SMA (red) is shown. The higher magnifications of the
areas indicated by the box in the left panel are shown in the middle panel. Dotted lines show the border between normal lung tissue and tumor. Note that
þ
þ
þ
GFP EC-SP cells clearly contribute to the blood vessels as ECs. White arrow (right), GFP CD31 cells in the EC-MP cells of the transplanted tumor.
B, quantification of the number of EC colonies in the tumor evaluated by microscopy (n > 3). Scale bars, 200 mm (A, right and left), 50 mm (A, middle).
Error bars, SEM. , P < 0.01.
revealed that the percentage of SP cells was increased in the
GFPþ cell fraction to the same extent as in wild-type mice. This
indicates that tumor EC-SP cells are true ECs (Fig. 7A). FACS
analysis of Vav1-GFP mice revealed that all the ECs, including
EC-SP cells, were GFP, showing that they are not of hematopoietic origin (Fig. 7B). Moreover, tumor GFPþ ECs isolated
Figure 5.
EC-SP cells are resistant to antiangiogenic therapy. A–C, confocal microscopic images of the blood vessels of KLN205 tumor treated with axitinib (A),
þ
vandetanib (B), or vehicle (C). Sections were stained with anti-CD31 antibody. D, bar graphs illustrate changes in vascular density evaluated by CD31
vessels in KLN205 tumors (n ¼ 4). E–G, Hoechst analysis of KLN205 tumor ECs treated with axitinib (E), vandetanib (F), or vehicle (G). Percentages of
EC-SP cells are significantly higher than control (H; n > 4). Scale bars, 200 mm (A–C). Error bars, SEM. , P < 0.01.
3206 Cancer Res; 76(11) June 1, 2016
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Endothelial SP Cells in Tumor Angiogenesis
Figure 6.
3
EC-SP cells are resistant to vandetanib treatment partly through ABC transporter activity. A, representative microscopic images of 10 EC-SP cells or
þ
-MP cells cultured for 10 days on OP9 stromal cells with vandetanib, stained with anti-CD31 antibody. Arrows (bottom), CD31 cells. Inset, higher
magnification of the area indicated by the box. B and C, quantification of the number of EC colonies in each well (n > 4). D, quantitative RT-PCR
analysis of ABC transporter mRNA in lung EC-SP and -MP cells, corrected for expression of the control gene GAPDH (n ¼ 4). E, representative
3
microscopic image of 10 EC-SP cells cultured for 10 days on OP9 stromal cells, together with 20 nm of vandetanib and verapamil or cyclosporine
þ
A. F, quantitative evaluation of the number of CD31 EC colonies of E (n > 4). Scale bars, 1 mm. Error bars, SEM. , P < 0.01. N.S., not significant.
from the VE-cadherin-GFP mice treated with antiangiogenic
drugs harbored high percentages of EC-SP cells (Fig. 7C). On
the other hand, none of the ECs, including EC-SP cells, were
GFPþ in Vav1-GFP mice treated with antiangiogenesis drugs
(Fig. 7D). These results suggest that tumor EC-SP cells originate
from resident ECs, not from hematopoietic cells, and selectively
survive after VEGFR inhibition via expression of drug
transporters.
Discussion
The presence of SP cells in the lung has been reported by several
groups (34–36). Although such studies indicated that some of
www.aacrjournals.org
these SP cells are positive for endothelial markers (34, 37), their
identity has not been clearly established. We previously reported
the existence of stem/progenitor–like endothelial SP cells by the
Hoechst method in the peripheral vasculature (14, 23). Therefore,
to determine the characteristics of lung SP cells within the ECs, we
gated the EC fraction to include cells expressing the endothelial
marker CD31 but to exclude CD45þ hematopoietic cells and then
applied the Hoechst assay to that fraction. We showed that ECs
indeed harbor SP cells. Among the cells positive for CD31 and
negative for CD45, about 0.7% were in the SP region; these cells
possessed colony-forming potential in the OP9 coculture model.
Since the concept of adult vasculogenesis by bone marrow derived
EPCs was proposed (6), the importance of their contribution to
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Naito et al.
Figure 7.
Tumor EC-SP cells do not originate from hematopoietic
cells. FACS analysis of tumor ECs from VE-cadherin
CreERT2 (BAC)/Flox-CAT-EGFP (VE-cadherin-GFP)
mice (A and C) and Vav1 Cre/Flox-CAT-EGFP (Vav1-GFP)
mice (B and D). Tumor EC-SP cells are present in the
þ
GFP EC fraction in VE-cadherin-GFP mice (A). Tumor
þ
CD31 CD45 ECs and EC-SP cells are GFP in Vav1-GFP
mice (B). Administration of vandetanib induces increase
þ
of the percentage of GFP tumor EC-SP cells in VEþ
cadherin-GFP mice (C). Correspondingly, tumor CD31
CD45 ECs and EC-SP cells after vandetanib treatment
are all GFP in Vav1-GFP mice (D).
new blood vessel formation has been intensively studied. The
formation of EC colonies is a feature shared by both EPCs and ECSP cells; however, we found that EC-SP cells are EC lineagecommitted cells, which do not originate from the bone marrow.
In the lung vasculature, the existence of highly proliferative resident progenitor cells that possess an endothelial colony–forming
cell (ECFC)–like phenotype has been reported (38, 39). ECFCs
were originally described in human peripheral and umbilical
cord blood based on the timing of their emergence in culture
(40). Considering that ECFCs are believed to originate from
resident vascular endothelium (41), EC-SP cells may overlap
with this population. However, as with ECFC, we could not
find any specific or restricted molecules that permit discrimination of EC-SP cells from mature endothelium; identification of
specific markers for EC-SP cells within resident vascular endothelium will be required.
In the tumor vasculature, we found that the percentage of
EC-SP cells, which can generate EC colonies is increased and
that these cells are actively proliferating. Furthermore, we found
that tumor blood vessels are predominantly formed by EC-SP
cells in a transplantation model. There is a report demonstrating a major contribution of EPC to ECs in tumors (42), but
in another study, it was reported that bone marrow–derived
cells do not integrate, but associate with ECs and promote
angiogenesis only indirectly (9). Although there are controversies in the field of EC differentiation from EPCs, the formation
of new blood vessels as a result of the mitotic division of ECs
of existing blood vessels is beyond doubt (43). Our results
suggest that resident vascular EC-SP cells may play an important role in tumor angiogenesis as EC-supplying cells. However,
3208 Cancer Res; 76(11) June 1, 2016
it is possible that our in vitro culture system and cotransplantation model does not accurately reflect the tumor microenvironment; therefore, lineage tracing of EC-SP cells with specific
markers would be needed to provide definitive results.
Interestingly, the percentage of tumor EC-SP cells was increased after treatment with small-molecule TKIs of VEGF
receptors. We found that total blood vessels decreased to
about one third, and the percentage of EC-SP cells increased
2.5-fold after antiangiogenic therapy. This strongly suggests
that the number of EC-SP cells did not increase but remained
the same after therapy. Although the EC-SP phenotype cannot
be maintained in the OP9 coculture assay, EC colonies generated from EC-SP cells were more resistant to vandetanib, at least
partly via ABC transporter activity. Recently, development of
resistance to antiangiogenic agents has become apparent as a
clinical problem that must be overcome (4, 44). Several mechanisms for acquired resistance to antiangiogenic therapy, such
as mutation of tumor cells imbuing greater resistance to hypoxia (45, 46), the compensatory increased secretion of
multiple angiogenic factors by tumor cells and stromal cells
(47), or increased pericyte coverage that protects from vessel
regression (48) have all been suggested. Because EC-SP cells
selectively remain after antiangiogenic therapy, we propose that
this heterogeneity of ECs may be another mechanism for
acquired resistance. However, because axitinib is known to
inhibit the kinase activity of c-Kit in addition to the VEGF
receptors and PDGFR-b, it is possible that EC-MP cells, which
express c-Kit, are more sensitive to treatment with this drug.
In summary, we characterized vascular EC-SP cells in the lung
vasculature and documented their contribution to tumor
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Endothelial SP Cells in Tumor Angiogenesis
angiogenesis. We used the genetic recombination model to
demonstrate that these EC-SP cells are indeed ECs and do not
originate from hematopoietic cells. Furthermore, we showed
that tumor EC-SP cells selectively remained after treatment with
small-molecule antiangiogenic agents. Our data suggest that
targeting this specialized EC population may offer a new
strategy to overcome antiangiogenic drug resistance.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: H. Naito, N. Takakura
Development of methodology: H. Naito, N. Takakura
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): H. Naito, T. Wakabayashi, H. Kidoya, F. Muramatsu,
K. Takara, D. Eino, K. Yamane, T. Iba
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): H. Naito, T. Wakabayashi
Writing, review, and/or revision of the manuscript: H. Naito, N. Takakura
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): H. Kidoya, N. Takakura
Study supervision: H. Kidoya, N. Takakura
Acknowledgments
The authors thank Drs. Toshio Suda (Kumamoto University, Kumamoto,
Japan) and Yoshiaki Kubota (Keio University, Tokyo, Japan) for providing them
with Flox-CAT-EGFP mice and VE-cadherin (BAC) CreERT2 mice, respectively.
The authors also thank M. Ishida, N. Fujimoto, C. Takeshita, and K. Fukuhara for
technical assistance.
Grant Support
This work was supported by the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) Grant-in-Aid for Scientific Research on Innovative Areas grant number 22112005, the Japan Science and Technology Agency
(JST) Projects for Technological Development, Research Center Network for
Realization of Regenerative Medicine, Japan Society for the Promotion of
Science (JSPS) Grants-in-Aid for Young Scientists B grant number 25830080,
15K18409, Takeda Science Foundation, Foundation for Promotion of Cancer
Research in Japan, and Tokyo Biochemical Research Foundation.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received November 1, 2015; revised February 26, 2016; accepted March 19,
2016; published OnlineFirst April 6, 2016.
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Published OnlineFirst April 6, 2016; DOI: 10.1158/0008-5472.CAN-15-2998
Endothelial Side Population Cells Contribute to Tumor
Angiogenesis and Antiangiogenic Drug Resistance
Hisamichi Naito, Taku Wakabayashi, Hiroyasu Kidoya, et al.
Cancer Res 2016;76:3200-3210. Published OnlineFirst April 6, 2016.
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