Download High Phosphoantigen Levels in Bisphosphonate-Treated

Published OnlineFirst June 6, 2011; DOI:10.1158/0008-5472.CAN-10-3862
Cancer
Research
Therapeutics, Targets, and Chemical Biology
High Phosphoantigen Levels in Bisphosphonate-Treated
Human Breast Tumors Promote Vg9Vd2 T-Cell Chemotaxis
and Cytotoxicity In Vivo
ne Benzaïd1,2, Hannu Mo
€ nkko
€ nen1,2, Verena Stresing1,2, Edith Bonnelye1,2, Jonathan Green4,
Ismahe
4
zardin1,2
€ nkko
€ nen , Jean-Louis Touraine3, and Philippe Cle
Jukka Mo
Abstract
The nitrogen-containing bisphosphonate zoledronic acid (ZOL), a potent inhibitor of farnesyl pyrophosphate
synthase, blocks the mevalonate pathway, leading to intracellular accumulation of isopentenyl pyrophosphate/
triphosphoric acid I-adenosin-50 -yl ester 3-(3-methylbut-3-enyl) ester (IPP/ApppI) mevalonate metabolites. IPP/
ApppI accumulation in ZOL-treated cancer cells may be recognized by Vg9Vd2 T cells as tumor phosphoantigens in vitro. However, the significance of these findings in vivo remains largely unknown. In this study, we
investigated the correlation between the anticancer activities of Vg9Vd2 T cells and the intracellular IPP/ApppI
levels in ZOL-treated breast cancer cells in vitro and in vivo. We found marked differences in IPP/ApppI
production among different human breast cancer cell lines post-ZOL treatment. Coculture with purified human
Vg9Vd2 T cells led to IPP/ApppI-dependent near-complete killing of ZOL-treated breast cancer cells. In ZOLtreated mice bearing subcutaneous breast cancer xenografts, Vg9Vd2 T cells infiltrated and inhibited growth of
tumors that produced high IPP/ApppI levels, but not those expressing low IPP/ApppI levels. Moreover, IPP/
ApppI not only accumulated in cancer cells but it was also secreted, promoting Vg9Vd2 T-cell chemotaxis to the
tumor. Without Vg9Vd2 T-cell expansion, ZOL did not inhibit tumor growth. These findings suggest that
cancers-producing high IPP/ApppI levels after ZOL treatment are most likely to benefit from Vg9Vd2 T-cell–
mediated immunotherapy. Cancer Res; 71(13); 4562–72. 2011 AACR.
Introduction
Nitrogen-containing bisphosphonates (NBP) are potent
inhibitors of osteoclast-mediated bone resorption and can
treat bone-loss disorders such as postmenopausal osteoporosis (1), aromatase inhibitor–associated bone loss, and cancer
treatment–induced bone loss (2). In preclinical models, NBPs
have direct and indirect antitumor properties (3). NBPs inhibit
farnesyl pyrophosphate synthase (FPPS), thus blocking the
mevalonate pathway and preventing prenylation of G-proteins
(e.g., Ras and Rho). In addition to effects on bone, this can
Authors' Affiliations: 1INSERM, UMR 1033, F-69372 Lyon; 2University of
Lyon, F-69008 Lyon; 3Laboratory of Immunology and Transplantation,
Edouard Herriot Hospital, Lyon, France; 4Novartis Pharma AG, Basel,
Switzerland; and 5School of Pharmacy, University of Eastern Finland,
Kuopio, Finland
€ nkko
€ nen have contributed equally to this work.
Note: I. Benzaïd and H. Mo
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
zardin INSERM, UMR1033, UFR de
Corresponding Author: Philippe Cle
decine Lyon-Est (domaine Lae
€nnec) Rue G. Paradin, 69372 Lyon cedex
Me
08, France. Phone: 33-4-78-78-57-37; Fax: 33-4-78-77-87-72; E-mail:
[email protected]
doi: 10.1158/0008-5472.CAN-10-3862
2011 American Association for Cancer Research.
4562
inhibit tumor-cell invasion, proliferation, and adhesion (3).
Additional studies suggest that NBPs may also inhibit angiogenesis (3). Importantly, there is now clinical evidence that
adding the NBP zoledronic acid (ZOL) to endocrine therapy
improves disease-free survival in estrogen-responsive early
breast cancer (4,5) and that adding ZOL to neoadjuvant
chemotherapy reduces residual invasive breast cancer tumor
size (6). How ZOL mediates this activity is not understood.
Emerging data suggest that NBPs have immunomodulatory
properties (7,8), which may have therapeutic applications.
Some NBPs can induce expansion of human Vg9Vd2 T cells,
the dominant subset of gd T cells (8–10). NBP internalization
by peripheral blood mononuclear cells (PBMC) inhibits FPPS,
leading to intracellular accumulation of isopentenyl pyrophosphate (IPP) that, in turn, stimulates Vg9Vd2 T-cell proliferation in a Vg9Vd2 T-cell receptor (TCR)-dependent manner
(11,12). The ATP analogue ApppI [triphosphoric acid I-adenosin-50 -yl ester 3-(3-methylbut-3-enyl) ester], which results
from covalent binding of IPP and AMP (13), can also act as a
phosphoantigen, stimulating the expansion of Vg9Vd2 T cells
from PBMCs (14).
In preclinical models, Vg9Vd2 T cells activated by NBPs
have potent antitumor effects in vitro (7, 15–18), and Vg9Vd2
T cells expanded in vitro maintain antitumor activity upon
adoptive transfer into immunodeficient mice inoculated
with melanoma or pancreatic adenocarcinoma cells (15).
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IPP/ApppI Mediates Vg9Vd2 T-cell Migration and Cytotoxicity
ZOL also enhances Vg9Vd2 T-cell cytotoxicity in experimental models of chronic myelogenous leukemia and lung, colon,
or bladder cancer (18–21). In addition, clinical evidence
suggests anticancer effects of the Vg9Vd2 T cells in patients
with lymphoid malignancies treated with the NBP pamidronate þ interleukin-2 (IL-2; refs. 7, 22), and in patients with
castration-resistant prostate cancer or advanced metastatic
breast cancer treated with ZOL þ IL-2 (23,24). A single dose
of ZOL has also been shown to durably activate gd T cells in
patients with breast cancer (25).
Cancer cells may be especially vulnerable to Vg9Vd2 T cells
(10, 11), even though the antitumor activity of Vg9Vd2 T cells
against different tumor cells treated with NBPs varies (26).
Interestingly, IPP/ApppI production after NBP treatment in
different cell lines also varies (27), and these mevalonate
metabolites could be recognized by Vg9Vd2 T cells as tumor
phosphoantigens (10, 14), suggesting that the anticancer
potency of Vg9Vd2 T cells might depend on intracellular
IPP/ApppI levels in NBP-treated tumors. However, there is
no in vivo evidence that a NBP can induce IPP/ApppI accumulation in tumors. In addition, the reasons why IPP/ApppIproducing tumors may become vulnerable to Vg9Vd2 T cells
in vivo remain unexplored. In the present study, the correlation between the potency of Vg9Vd2 T cells to kill cancer cells
and intracellular IPP/ApppI levels in ZOL-treated breast
cancer cells was investigated in vitro and in vivo.
Materials and Methods
Drugs, cell culture, and animals
Unlabeled and carbon 14 (14C)–labeled ZOL was provided
by Novartis Pharma AG. Recombinant IL-2 was provided by
Novartis Pharmaceuticals Ltd. Sterile stock bisphosphonate
solutions were prepared in PBS (pH 7.4; Invitrogen).
Five-week-old female nonobese diabetic severe combined
immunodeficient (NOD/SCID) mice were purchased from
Charles River Laboratories. All procedures involving animals,
including their housing and care, the method by which they
were killed, and experimental protocols, were conducted in
accordance with a code of practice established by the local
ethical committee (CREEA: Comite Regional d’Etique pour
l’Experimentation Animale).
Blood was donated by healthy volunteers or obtained from
the Blood Transfusion Center (Etablissement Français du
Sang). Human PBMCs were isolated after Ficoll–Paque (Amersham Biosciences) density gradient centrifugation. The
Vg9Vd2 T cells were expanded by exposing PBMCs to 10mmol/L ZOL plus 100-U IL-2 for 14 days. Populations of
Vg9Vd2 T cells were purified by positive selection of TCR
gd cells using immunomagnetic cell sorting (AutoMACS Pro
Separator, Miltenyi Biotec).
Human breast cancer cell lines (T47D, MCF-7, BT-474, ZR75–1, MDA-MB-231, MDA-MB-435, MDA-MB-435s) were
obtained from the American Type Culture Collection
(ATCC)-LGC Promochem and used within 6 months. All cell
lines were authenticated using short tandem repeat analysis.
The human estrogen-receptor (ER)-negative B02 breast cancer
cell line, a subpopulation of MDA-MB-231, was prepared as
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previously described (28). PBMCs and ZR-75–1 breast cancer
cells were cultured in RPMI-1640 medium (ATCC) supplemented with 10% fetal bovine serum (Invitrogen) and penicillin/streptomycin (Invitrogen). BT-474 breast cancer cells
were cultured in Hybri-Care medium (ATCC) supplemented
with 1.5 g/L NaHCO3, 10% fetal bovine serum and penicillin/
streptomycin (Invitrogen). Other breast cancer cell lines were
cultured in Dulbecco's modified Eagle's medium (DMEM)
medium (Invitrogen) supplemented with 10% fetal bovine
serum and penicillin/streptomycin.
In vitro and in vivo Vg9Vd2 T-cell expansion and IFN-g
measurement
PBMCs were seeded in 24-well plates (1 106 cells/well) and
then treated with vehicle (PBS) or ZOL (1, 10, or 100 mmol/L)
IL-2 100 U/mL for 7, 14, 21, and 28 days. IL-2 was renewed at
day 4 and then every 3 days. In vivo expansion of Vg9Vd2 T cells
was carried out in NOD/SCID mice. PBMCs (3.5 107) were
inoculated intraperitoneally (i.p.), and mice were treated with
vehicle or ZOL (3, 10, or 30 mg/kg), i.p., and 10,000-U IL-2. Mice
then received IL-2 plus vehicle or ZOL every 2 days for 14 days.
On day 14, mice were sacrificed and peritoneal cells collected
for analysis of Vg9Vd2 T cells. Percentages of Vg9Vd2 T cells
in PBMCs and peritoneal cells were determined by flow cytometry (FACScan, Becton Dickinson) after immunostaining
with anti–CD3-PE (BD Pharmingen) and anti–Vd2-TCR-FITC
antibodies (Beckman Coulter). Human IFN-g in the serum
from mice was measured using a commercial ELISA kit
(Biosource).
IPP/ApppI analysis
ZOL-induced IPP/ApppI production was measured in
PBMCs and breast cancer cells in vitro and in s.c. tumors
in vivo. PBMCs (3 106) were exposed to PBS or ZOL (1, 10, or,
100 mmol/L) plus 100 U/mL IL-2, then cultured on 6-well
plates for 1, 2, 4, 7, 11, and 14 days. T47D, MCF-7, and B02
breast cancer cells were seeded in 6-well plates at 1 106
cells/well overnight. Cells were pulse-treated with 25-mmol/L
ZOL for 1 hour, then incubated without drug for 0 to 42 hours.
Cells were then scraped and washed in PBS and extracted
using ice-cold acetonitrile (300 mL) and water (200 mL) containing 0.25-mmol/L NaF and Na3VO4 to prevent degradation
of IPP and ApppI. Tumor xenografts were collected from mice
after sacrifice and snap-frozen in liquid nitrogen, pulverized,
and extracted using ice-cold acetonitrile. IPP and ApppI in cell
extracts were quantified by high-performance liquid chromatography negative ion electrospray ionization mass spectrometry (HPLC-ESI-MS; ref. 13).
Cellular uptake assay for ZOL
T47D, MCF-7, and B02 cells were seeded overnight to 10-cm
Petri dishes at 4 106 cells/dish and then treated with
14
C-labeled ZOL for 1 hour. Cells were then rinsed with
PBS, scraped, and extracted with acetonitrile and water.
Extracts were separated by centrifugation (14,000g, 2 minutes). Precipitates were analyzed for total protein content by a
modified Bradford procedure. The soluble acetonitrile/water
extracts were evaporated and redissolved in 120-mL Milli-Q
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Benzaïd et al.
water, then mixed with OptiPhase HiSafe3 scintillation cocktail (PerkinElmer Wallac) and evaluated by liquid scintillation
(TriLux Microbeta, PerkinElmer Wallac).
TV ¼ (m12m2)/2. At the end of the protocols, mice were
sacrificed and tumors collected for immunohistochemistry
and real-time PCR.
3-Hydroxy-3-methylglutaryl-coenzyme A reductase
analysis by Western blotting
T47D, MCF-7, and B02 cells were treated with vehicle (0.5%
DMSO) or 0.3-mmol/L lovastatin for 24 hours and then lysed in
100-mL cell lysis buffer [1% (vol/vol) Nonidet P-40 in 0.05M
Tris-HCL containing 0.15-M NaCl, 1-mM EDTA, 0.05-M NaF
and a cocktail of protease inhibitors (1-mM PMSF, 1-mg/ml
aprotinin, 1-mg/mL leupeptin and 1-mmol/L NA3VO4)]. For
each sample, 50-mg protein was used for SDS-polyacrylamide
gel electrophoresis, and transferred onto a polyvinylidene
difluoride (PVDF) membrane (Invitrogen), which was then
washed with PBS/0.1% Tween and incubated overnight at 4 C
with a rabbit polyclonal anti–3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase Ab (1/500 in 5% nonfat
milk; Upstate). After washing, membranes were incubated
with HRP-conjugated antirabbit Ab (GE Healthcare) and
developed using Western Lightning Chemiluminescence (PerkinElmer LAS Inc.). As a control, washed membranes were
incubated with horseradish peroxidase (HRP)-conjugated
antimouse Ab (GE Healthcare) and then developed as before.
Densitometric analysis of blots was carried out using Quantity
One software (Bio-Rad Laboratories).
Immunohistochemistry
Tumors were embedded in Tissue-Tek (Sakura), snap-frozen in liquid nitrogen-cooled isopentane, and stored at–70 C.
Frozen 7-mm sections were cut in a cryostat, air-dried, and
fixed in cold acetone. After washing in PBS, tissue sections
were preincubated with 1% goat serum. For detection of
Vg9Vd2 T cells, tumor sections were incubated with a
fluorescein isothiocyanate (FITC)-conjugated antihuman
TCR d2 monoclonal Ab, diluted 1:20 in PBS/1% BSA/0.3%
Triton X-100. Cell nuclei were stained with 40 ,6-diamidino2-phenylindole (DAPI; Roche). Tissue sections were rewashed
and visualized by fluorescence microscopy (Axioskop 40;
Zeiss). To measure tumor-cell proliferation, tumor sections
were incubated with a rabbit polyclonal anti–Ki-67 Ab
(Abcam). Experiments were carried out as previously
described (28). The mitotic index was expressed as percentage
of Ki-67–positive nuclei.
Cytotoxicity assay
T47D, MCF-7, and B02 breast cancer cells had different
growth rates in culture. For cytotoxicity assessments, it was
therefore necessary to adjust the cell number at the beginning
of the experiment in order to have cell monolayers at a similar
confluence at the time of the coculture with Vg9Vd2 T cells. In
this respect, T47D (2 104 cells/well), MCF-7 (2 104 cells/
well), and B02 (7 103 cells/well) breast cancer cells were
incubated overnight, then treated for 1 hour with vehicle (PBS)
or ZOL (1–25 mmol/L). Cell monolayers were then washed; 18
hours later, breast cancer cells were cocultured with or without purified Vg9Vd2 T cells (cancer cell:Vg9Vd2 T-cell ratio
was 1:12.5 or 1:25) for 4 hours or 24 hours. Viability was
assessed by MTT assay.
Experimental tumorigenesis
Five-week-old female NOD/SCID mice were injected s.c. in
the flank with 5 106 B02 or T47D cells in 100-mL PBS. For the
ER-positive T47D cells, host mice were implanted with s.c. 60day–release pellets containing 1.7-mg 17b-estradiol (Innovative Research of America) 4 days before tumor-cell inoculation. Four weeks later, when B02 and T47D tumors had
reached a volume of 50 mL, mice were randomly assigned
to 4 treatment groups (n ¼ 6–7 mice/group): placebo (PBS);
ZOL at 30 mg/kg body weight; human PBMCs (3.5 107)
injected i.p. plus 10,000-U IL-2 administered alone or with 30mg/kg ZOL. In the relevant groups, IL-2 and ZOL in 0.5-mL PBS
were administered i.p. every 2 days for 14 days. Tumor size was
calculated by external measurement of the width (m1)
and length (m2) of s.c. tumor xenografts using a Vernier
caliper. Tumor volume (TV) was calculated using the equation
4564
Real-time PCR
Total RNA was extracted from T47D and B02 tumors and
infiltrating Vg9Vd2 T cells. Tumors were homogenized with a
Polytron device (Kinematica). RNA was extracted using the
nucleospin RNA II kit (Macherey–Nagel) followed by DNase
digestion (Macherey-Nagel). Samples of total RNA (1.5–5 mg)
were reverse-transcribed using Superscript II (Invitrogen).
real-time PCR was carried out (IQ SYBR Green, Bio-Rad) with
primers specific for the human housekeeping gene L32 (100
bp), a ribosomal protein used as an internal standard:
50 caaggagctggaagtgctgt; 30 cagctctttccacgatggc, and human
TCR Vd2 (162 bp): 50 caaaccatccgtttttgtc; 30 cttgacag cattgtacttcc. We confirmed the linear range of amplification for
all primers and products. Real-time reverse transcriptase
(RT)-PCR was carried out using the Mastercycler EP system
(SYBR Green; Realplex2). Amplimers were quantified in triplicate samples in the pool of 3 independent tumors for each
gene and normalized to corresponding L32 values.
Transwell migration assay
T47D, MCF-7, and B02 breast cancer cells were seeded
overnight to 6-well plates at 106 cells/well. The cells were
then treated for 1 hour with PBS or 25-mmol/L ZOL 10-mmol/L lovastatin. Cells were then washed twice with
PBS and cultured for 18 hours in DMEM þ 0.25% BSA. The
conditioned medium from treated or untreated breast cancer
cell lines was collected and centrifuged for use in migration
assays. Migration assays were carried out in 24-well Transwell
plates (Corning Costar) with 5-mm pore polycarbonate membrane. Before migration assays, Vg9Vd2 T cells were enriched
by PBMC culture with 10-mmol/L ZOL plus 100 U IL-2 for 10 to
14 days. Purified populations of Vg9Vd2 T cells obtained by
positive selection of TCR gd were resuspended at 3 106 cells/
100 mL in assay buffer. A cell suspension (100 mL) was placed in
the upper chamber of the Transwell inserts, and 600 mL of
conditioned medium from breast cancer cell lines, plus
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IPP/ApppI Mediates Vg9Vd2 T-cell Migration and Cytotoxicity
1 mmol/L ApppI or 0.5 mmol/L IPP, were placed in the lower
chamber. After incubation for 4 or 24 hours at 37 C, cells from
the lower chamber were counted. Basal cell-culture medium
was used as a control to measure random migration of Vg9Vd2
T cells in the absence of chemoattractant. The control was set
to 100%, and the results obtained in the presence of a chemoattractant were expressed as a percentage of the control.
Human cytokines released from vehicle- and ZOL-treated
T47D and B02 cells were detected using RayBio Human Cytokine Ab Array C series 1000, designed to detect 120 cytokines
(Ray Biotech). The array membranes were incubated overnight
with conditioned medium (1.2 mL/1.2 mg protein) from cultured T47D and B02 cells treated or untreated with ZOL.
Detection of immunoreactive spots was carried out using an
enhanced chemiluminescence detection system.
Human chemokines IL-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1; eBioscience) released from vehicleand ZOL-treated T47D, MCF-7, and B02 cells were measured
by ELISA.
A
Statistical analysis
All data were analyzed using StatView software (version 5.0;
SAS Institute Inc.). Pairwise comparisons were carried out
by conducting a nonparametric Mann–Whitney U test.
P values < 0.05 were considered statistically significant. All
statistical tests were two-sided.
Results and Discussion
ZOL induces human Vg9Vd2 T-cell expansion in vitro
and in vivo
ZOL stimulated, in a time- and dose-dependent manner,
expansion of Vg9Vd2 T cells from human PBMCs, peaking at
day 14 with a 10-mmol/L ZOL concentration (Fig. 1A). These
results are in accordance with previous reports showing that
NBPs stimulate proliferation of Vg9Vd2 T cells in vitro (29). In
addition, mass spectrometry revealed rapid IPP accumulation
in PBMCs after 1–2 days of ZOL treatment (Fig. 1B), whereas
ApppI was detected after 2–4 days (Fig. 1C).
B
C
90
80
10 μmol/L ZOL+IL-2
70
100 μmol/L ZOL+IL-2
40
20
10 μmol/L ZOL
100 μmol/L ZOL
60
50
40
30
20
10
14
21
28
2
nd
4
104
E
100 μmol/L ZOL
12
10
8
6
4
ndnd
7
11
ndnd nd
14
0
ndnd
1
nd
nd
2
4
7
ndnd
nd nd
11
14
Time, days
102
16% Vγ9Vδ2 T
101
102
103
2.7% Vγ9Vδ2 T
101
Tlymphocytes
10 μmol/L ZOL
14
Time, days
Time, days
D
16
104
7
1
nd
103
0
1 μmol/L ZOL
18
2
nd
0
0
20
1 μmol/L ZOL
6
60
1 μmol/L ZOL+IL-2
Apppl, pmol/106 cells
IL-2
IPP, pmol/10 cells
Proportion of Vγ9Vδ2T cells, %
80
101
102
103
104
101
102
103
104
Vγ9Vδ2 T cells
Figure 1. ZOL-induced Vg9Vd2 T-cell expansion in vitro and in vivo and IPP/ApppI production in human PBMCs. A, PBMCs were treated with 1, 10, or
100 µmol/L of ZOL plus 100 U/mL of IL-2, or with IL-2 alone. Vg9Vd2 T-cell proliferation was analyzed by flow cytometry after 0- to 28-day culture. Data
are expressed as mean SD of triplicate cultures from a representative donor. B and C, accumulation of IPP (B) and ApppI (C) in PBMCs after 1- to
14-day culture, detection limit 1 pmol/mg protein. Data are mean SD of duplicate cultures (representative donor). D and E, dot plot of flow cytometry
from peritoneal cells of NOD/SCID mice receiving PBMCs and 10,000 U of IL-2 alone (D) or with 30 µg/kg of ZOL (E), both every 2 days for 14 days. Plots
are representative of 5 mice. nd, not detected.
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Published OnlineFirst June 6, 2011; DOI:10.1158/0008-5472.CAN-10-3862
Benzaïd et al.
No reactivity of murine gd T cells to phosphoantigens such
as IPP or ApppI has yet been shown, and human gd T cells do
not recognize cells from animals other than humans (11, 30).
Thus, murine PBMCs treated with an amino-bisphosphonate
do not activate murine or human gd T cells. The effect of ZOL
on Vg9Vd2 T-cell expansion was therefore studied in vivo after
i.p. injection of human PBMCs into NOD/SCID mice and ZOL
þ IL-2 treatment. ZOL stimulated expansion of Vg9Vd2 T cells
such that they comprised up to 16% of T lymphocytes, versus
2.7% for IL-2 alone (Fig. 1D and E). This is explained by the fact
that ZOL is internalized by antigen-presenting cells from
human PBMCs where it induces the intracellular accumulation of IPP/ApppI and subsequent activation of Vg9Vd2 T-cell
expansion (12), making our animal model most suitable for
studying the role of ZOL in cancer immunotherapy in vivo.
Moreover, although ZOL was administered more frequently in
our model than the current clinical dosing regimen in humans,
the dose given (30 mg/kg) was lower than the dosing in
humans (100 mg/kg), supporting the clinical relevance of
our findings.
luminal breast cancer cell lines studied (T47D, MCF-7, BT-474,
ZR-75–1) produced IPP/ApppI, whereas none (0/4) of the
basal breast cancer cell lines tested (MDA-MB-231, B02,
MDA-MB-435, and MDA-MB-435s) produced these phosphoantigens. In addition, we found that IPP/ApppI accumulation in ZOL-treated T47D cells was time-dependent, with IPP
reaching a maximum concentration at 12 hours (1,052 pmol/
mg protein) and ApppI peaking at 24 hours (1,302 pmol/mg
protein) (Fig. 2B). These results were in line with our previous
findings showing that the basal MCF-10A and MDA-MB-436
breast cell lines produced low levels of IPP/ApppI after ZOL
treatment (27). The higher IPP/ApppI production in luminal
versus basal breast cancer cell lines is, to our knowledge, a
novel observation.
Three breast cancer cell lines having a high (T47D), medium
(MCF-7), or low (B02) production of phosphoantigens were
then selected for subsequent experiments. Fluid-phase endocytosis is the major route by which NBPs are internalized in
macrophages and osteoclasts (31). Variations in endocytosis
between the cancer cell lines might explain differences in IPP/
ApppI production induced by ZOL. Uptake of 14C-labeled ZOL
was therefore measured in T47D, MCF-7, and B02 cells. Uptake
was similar in T47D and MCF-7 cells, but 3-fold lower in B02
cells (Fig. 2C). In addition, we found a strong correlation
between HMG-CoA reductase expression, a mevalonate pathway enzyme upstream of FPP synthase, and ZOL-induced IPP/
ApppI accumulation (Fig. 2D). As shown by Western blotting,
lovastatin (an HMG-CoA reductase inhibitor) substantially
increased HMG-CoA reductase protein levels in ER-positive
T47D and MCF-7 cells (Fig. 2D). By contrast, HMG-CoA
reductase was undetectable, and only a faint protein band
was observed in lovastatin-treated ER-negative B02 cells
ZOL induces IPP/ApppI production in human breast
cancer cells in vitro
Prior studies showed the intracellular accumulation of IPP
and ApppI in NBP-treated cancer cell lines in vitro (10, 27). In
our study, ZOL-induced IPP/ApppI production was quantified
in a series of human breast cancer cell lines having a luminal
[ER-positive and/or progesterone receptor (PR)-positive and
Ki-67-positive] or basal (ER-, PR-, and HER2-negative) molecular subtype. Mass spectrometry analysis showed that IPP/
ApppI levels varied substantially among breast cancer cell
lines after ZOL treatment (Fig. 2A). Specifically, 3 of the 4
B
IPP
Apppl
1,400
1,200
1,000
800
600
400
200
C
<1 <1
0
nd
nd
1,500
T47D, pmol/mg protein
1,600
IPP
Apppl
1,200
900
600
300
0
T4
7D
M
C
FBT 7
-4
7
ZR 4
M
DA -75
M -MB 1
DA
-4
-M 35
M B-4
DA
35
s
M MB
DA
-2
3
-M
1
BBO
2
Accumulation, pmol/mg protein
A
0
4
8 12 16 20 24 28 32 36 40 44
Time, h
D
Cellular uptake,
pmol/mg protein
40
30
18
15
0
97
100
8
HMG-CoAR
20
Tubulin
10
0
4566
Band density
T47D MCF-7
T47D
MCF-7
BO2
Basal
BO2 T47D MCF-7
Lovastatin
BO2
Figure 2. IPP/ApppI accumulation
induced by ZOL depends on
activity of the mevalonate
pathway and cellular uptake of
ZOL. A, breast cancer cells were
treated with 25-mmol/L ZOL for 1
hour, then cultured without drug
for 18 hours (mean SD, n ¼ 12–
21); detection limit was 1 pmol/mg
protein. B, T47D cells were treated
with 25-mmol/L ZOL for 1 hour and
samples collected 0 to 42 hours
after drug removal (mean SD,
n ¼ 3). C, cellular uptake of
14
C-labeled ZOL 1 hour after
25-mmol/L ZOL treatment (mean
SD, n ¼ 3). D, bands of HMGCoA reductase (HMG-CoAR) and
tubulin by Western blotting in
cancer cell lines after treatment
with 0.5% DMSO (basal) or 0.3mmol/L lovastatin for 24 hours.
Band density of HMG-CoAR is
normalized by tubulin.
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IPP/ApppI Mediates Vg9Vd2 T-cell Migration and Cytotoxicity
addition, Vg9Vd2 T cells caused ZOL-treated MCF-7 cell
death; concentrations of ZOL 10 mmol/L were required to
prime MCF-7 cells for Vg9Vd2 T-cell–mediated cytotoxicity
(Fig. 3D). In sharp contrast, Vg9Vd2 T cells were not cytotoxic against ZOL-treated B02 cells (Fig. 3F). Although
human cancer cell lines exhibit susceptibility to Vg9Vd2
T-cell–mediated cytotoxicity upon NBP treatment, the in
vitro antitumor activity of Vg9Vd2 T cells against tumor cells
treated with NBPs varies greatly (10, 11, 15). For example,
Vg9Vd2 T cells fail to efficiently kill a variety of human renal
(ACHN, Caki-2, A-704) and gastric (MKN45, MKN74) cancer
cell lines after NBP pretreatment (11). Here, our results
strongly suggest that ZOL-induced IPP/ApppI accumulation
in breast cancer cells was responsible for Vg9Vd2 T-cell–
mediated cytotoxicity. In this respect, cotreatment of T47D
cells with ZOL þ lovastatin almost completely eliminated
IPP/ApppI phosphoantigen production by these cells and
substantially reduced Vg9Vd2 T-cell cytotoxicity (Supplementary Fig. S1). It is therefore most conceivable that the
lack of cytotoxicity of Vg9Vd2 T cells against some cancer
cell lines is attributable to a low intracellular accumulation
of phosphoantigens upon NBP treatment of cancer cells,
likely because of the low activity level of the mevalonate
pathway.
(Fig. 2D). These results agree with data reported by Borgquist
and colleagues (32) who showed that high HMG-CoA reductase expression correlated positively with ER expression in
tumor tissue from patients with breast cancer. Altogether,
these results show that IPP/ApppI production in breast cancer
cells depends on cellular uptake of NBP and on the activity of
the mevalonate pathway. Moreover, we suggest that luminal
neoplastic mammary epithelial cells might be more sensitive
to bisphosphonate therapy than basal neoplastic mammary
epithelial cells.
Vg9Vd2 T-cell cytotoxicity correlates with ZOL–induced
IPP/ApppI accumulation in breast cancer cells in vitro
and in vivo
The antitumor potency of human Vg9Vd2 T cells against
ZOL-treated breast cancer cell lines was first examined in vitro
(Figs. 3A–3F). Incubation for 1h with ZOL (1–25 mmol/L) did
not affect survival of T47D, MCF-7, and B02 cells (Figs. 3A, 3C,
and 3E, respectively), whereas these bisphosphonate concentrations did induce intracellular IPP/ApppI accumulation in
T47D and MCF-7 cells (Figs. 2A-B). Coculture of ZOL-treated
T47D cells with purified Vg9Vd2 T cells led to dose-dependent
cancer-cell death, which was statistically significant with ZOL
at concentrations as low as 1 mmol/L for 1 hour (Fig. 3B). In
– γδ T cells
+ γδ T cells
B
A
140
120
80
60
40
0
CTR
1
5
10
15
120
100
80
60
40
CTR
1
3
5
10
15
25
*
80
**
**
60
40
20
CTR
1
3
5
10
15
ZOL, μmol/L
E
0
25
CTR
1
120
100
100
Viability, %
125
75
50
3
5
10
15
25
15
25
ZOL, μmol/L
F
25
80
60
40
20
CTR
1
3
5
10
ZOL, μmol/L
www.aacrjournals.org
**
ZOL, μmol/L
100
0
**
**
D
20
BO2
**
40
120
0
**
60
0
25
Viability, %
Viability, %
3
ZOL, μmol/L
C
MCF-7
**
80
20
20
Viability, %
Figure 3. ZOL-induced IPP/ApppI
accumulation in breast cancer
cells correlates with Vg9Vd2 Tcell–mediated cancer-cell death.
T47D, MCF-7, and B02 cells were
pretreated with control (CTR; PBS)
or ZOL 1 to 25 mmol/L for 1 hour,
then cultured without drug for 18
hours. After incubation, cells were
cocultured without (A, C, E) or with
(B, D,F) purified Vg9Vd2 T cells for
24 hours. The ratio of cancer cells
to Vg9Vd2 T cells was 1:12.5.
A—F are representative of 3
independent experiments
(mean SD; *, P < 0.05;
**, P < 0.01).
100
100
Viability, %
T47D
Viability, %
120
15
25
0
CTR
1
3
5
10
ZOL, μmol/L
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Benzaïd et al.
We next conducted in vivo experiments to examine the
contribution of IPP/ApppI accumulation in breast tumors to
Vg9Vd2 T-cell cytotoxicity. Earlier experimental in vivo studies
used human Vg9Vd2 T cells expanded in vitro and then
purified before inoculation (15, 18, 19, 20). Here, we showed
that ZOL þ IL-2 treatment induced Vg9Vd2 T-cell expansion
from human PBMCs injected i.p. into NOD/SCID mice
(Fig. 1E). In the clinic, it has been recently shown that the
treatment of patients with advanced breast cancer with ZOL
and low-dose IL-2 triggers Vg9Vd2 T-cell amplification in vivo
(24). We therefore chose to use this therapeutic strategy to
treat tumor-bearing animals. NOD/SCID mice bearing sub-
cutaneous breast cancer xenografts were inoculated with
human PBMCs, with expansion of Vg9Vd2 T cells in vivo
(Fig. 4). Treatment with ZOL alone or PBMCþIL-2 did not
inhibit s.c. tumor growth (Fig. 4A and B). In contrast, PBMC þ
IL-2 þ ZOL completely blocked growth progression of T47D
tumors, compared with that observed in placebo-treated
animals (P < 0.01; Fig. 4A). In addition, in situ immunodetection of the proliferation marker Ki-67 nuclear antigen in T47D
tumors from mice treated with PBMC þ IL-2 þ ZOL showed
drastic reduction in proliferative index versus tumors from
placebo-treated mice (Fig. 4C). However, this treatment did
not inhibit growth or proliferative index of B02 s.c. tumors
RT-PCR
Human PBMCs
Tumor cells
IHC
2 Weeks
4 Weeks
ZOL
±
IL-2
A
B
T47D tumors
ns
100
**
50
W4 W6
Placebo
W4 W6
ZOL
W4 W6
PBMC
+IL-2
Ki67 index, (%)
C
21
18
15
12
9
6
3
0
W4 W6
PBMC
+IL-2
+ZOL
Tumor volume (mm3)
ns
150
0
BO2 Tumors
250
ns
**
Placebo
ZOL
E
PBMC
+IL-2
PBMC
+IL-2
+ZOL
150
100
50
40
35
30
25
20
15
10
5
0
4
3
2
ns
1
Placebo
ZOL
ns
PBMC
+IL-2
PBMC
+IL-2
+ZOL
x40
Relative Vγ9Vδ2
expression
Relative Vγ9Vδ2
expression
W4 W6
PBMC
+IL-2
W4 W6
PBMC
+IL-2
+ZOL
*
Placebo
ns
ns
ZOL
PBMC
+IL-2
PBMC
+IL-2
+ZOL
ns
ns
ns
ZOL
PBMC
+IL-2
PBMC
+IL-2
+ZOL
6
**
5
G
4568
W4 W6
ZOL
F
6
0
W4 W6
Placebo
D
ns
ns
ns
ns
200
0
Ki67 index, (%)
Tumor volume (mm3)
200
5
4
3
2
1
0
Placebo
Figure 4. Cytotoxic effect of
Vg9Vd2 T cells against T47D and
B02 breast cancer s.c. tumor
xenografts. Mice were inoculated
s.c. with T47D or B02 cells. Four
weeks after tumor cell inoculation,
when tumors became palpable,
mice were engrafted with PBMCs
from healthy human adult blood
donors. In addition, mice were
treated with IL-2 ZOL (30 mg/kg)
every 2 days for 2 weeks. T47D (A)
and B02 (B) tumor growth was
determined at weeks 4 and 6 after
tumor cell inoculation, by
measurement of the tumor volume
with a Vernier caliper. T47D (C)
and B02 (D) Ki-67 indices
were assessed by
immunohistochemistry. The
relative expression of Vg9Vd2 T
cells in T47D (E) and B02 (F)
tumors was assessed by RT-PCR.
G, presence of Vg9Vd2 T cells in
T47D tumors from the placebo
and PBMCþIL-2þZOL groups.
Tumor sections were stained with
TCR Vd2 (green) and DAPI (nuclei,
blue). Results are mean SD of 6
to 7 mice per group. ns, not
significant; *, P < 0.05; **, P < 0.01
compared with placebo.
x40
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IPP/ApppI Mediates Vg9Vd2 T-cell Migration and Cytotoxicity
Human PBMCs
MS
2 Weeks
ZOL
±
IL-2
T47D Tumors
Tumor volume (mm3) after
2-week treatment
200
ns
150
ns
**
50
0
Placebo
ZOL
PBMC
+IL-2
100
Standards
Apppl
IPP
PBMC
+IL-2
+ZOL
NH2
22
80
O
40
20
O
OH
O
OH
O
O
OH
OH
O
O
O
O
O
60
OH
O CH2
O
OH
H
H
4x
159
A
H
H
OH OH
Placebo
100
80
60
40
20
B
100
Relative abundance (%)
4 Weeks
Relative abundance (%)
Tumor cells
Relative abundance (%) Relative abundance (%)
48 hours after the last ZOL dose in PBMC þ IL-2 þ ZOLtreated mice (0.7 pmol; 16 nmol/L), whereas ApppI levels were
measured (0.11 pmol; 2.5 nmol/L; Fig. 5C). Of note, IPP and
ApppI were also detected in T47D tumor extracts from mice
treated with ZOL alone. In contrast, ZOL-induced IPP/ApppI
was not detected in B02 tumor xenografts (not shown).
These data, to the best of our knowledge, show for the first
time that s.c. tumors can produce IPP/ApppI after ZOL
administration, showing the uptake of the NBP into tumor
cells in vivo. If IPP/ApppI had been produced by tumorinfiltrating cells (e.g., macrophages), we would have expected
IPP/ApppI also to be produced in B02 tumors, contrary to
what we observed in vivo. It is interesting that Guenther and
colleagues (33) recently reported, based on their use of a
mouse xenograft model for plasmacytoma, that the treatment
of animals with ZOL leads to accumulation of unprenylated
Rap1A (a surrogate marker of the inhibitory effect of NBP on
the mevalonate pathway) in plasma cell tumors ex vivo. These
findings (33), along with our present data, provide evidence for
(Fig. 4B and D). Of note, Vg9Vd2 T-cell infiltrates were
detected in T47D but not B02 tumors from mice treated with
PBMC þ IL-2 þ ZOL (Fig. 4E–G), as judged by both RT–PCR
and immunohistochemistry. Moreover, serum levels of human
IFN-g were high in T47D-tumor-bearing mice treated with
PBMCs þ IL-2 þ ZOL (942 240 pg/mL, n ¼ 5) and moderate
in animals treated with PBMCs þ IL-2 (563 370 pg/mL, n ¼
5), whereas the cytokine was not detected in animals treated
with placebo or ZOL alone, showing that Vg9Vd2 T cells were
activated in vivo.
Because Vg9Vd2 T cells infiltrated s.c. T47D tumor xenografts in mice treated with PBMC þ IL-2 þ ZOL (Fig. 4E and
4G), IPP/ApppI was measured in tumor extracts by mass
spectrometry. As expected, IPP and ApppI were barely detectable in T47D tumors from placebo-treated mice (Fig. 5A),
whereas IPP levels were substantial 24 hours after the last ZOL
dose in mice treated with PBMC þ IL-2þ ZOL (3.7 pmol;
82 nmol/L) (Fig. 5B). By contrast, ApppI remained undetectable at 24 hours. Conversely, lower IPP levels were detected at
ZOL, 24 h
100
80
60
40
20
C
ZOL, 48 h
100
80
60
40
20
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
Time (min)
Time (min)
IPP, m/z 245 → 159
Apppl, m/z 574 → 408
Figure 5. Mass spectrometry analysis of IPP and ApppI in T47D breast cancer s.c. tumor xenografts. Identification of IPP and ApppI in T47D tumors
of placebo- and ZOL-treated mice was carried out by high-performance liquid chromatography negative ion electrospray-ionization mass spectrometry.
Selected reaction monitoring chromatograms are shown. They correspond to tumor extracts obtained from placebo-treated mice (A) and animals that received
PBMC þ IL-2 þ ZOL whose tumors were collected 24 hours (B) and 48 hours (C) after the last ZOL injection. Similar chromatograms were obtained with tumor
extracts from mice treated with ZOL alone. Tumor extracts were spiked with 1.35-pmol IPP and 0.11-pmol ApppI. IPP and ApppI chromatograms are drawn
on the same scale (standards).
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Benzaïd et al.
medium from T47D cells treated with ZOL þ lovastatin
resulted in less migration of Vg9Vd2 T cells (Fig. 6C). To
clarify whether IPP/ApppI acted as migration factors for
Vg9Vd2 T cells, we used mass spectrometry to measure IPP
levels in conditioned medium from ZOL-treated breast cancer
cell lines (T47D, MCF-7, B02). We found substantial IPP levels
in conditioned medium from ZOL-treated T47D and MCF-7
cells, but not B02 cells (data not shown). Moreover, 0.5-mmol/L
IPP or 1-mmol/L ApppI significantly stimulated gd T-cell
migration (Fig. 6C). Because ApppI can be converted into
IPP on hydrolysis by nucleotide pyrophosphatases (14), it also
may be processed into IPP for subsequent recognition by the
Vg9Vd2 TCR. The cytokines IL-6, IL-8, and MCP-1 are also gd
T-cell chemoattractants (30). However, we found no differences in these or any other cytokines in a human cytokine
antibody array of conditioned medium from untreated or
ZOL-treated T47D, MCF-7, and B02 cells (Fig. 6D). Although
B02 cells produced a greater range of cytokines than T47D
cells, cytokine profiles were similar for ZOL-treated or
a direct uptake of ZOL in tumor cells in vivo. However, in our
breast cancer model, ZOL treatment without PBMC þ IL-2
(i.e., without Vg9Vd2 T-cell expansion) did not inhibit tumor
growth, showing the important role of human Vg9Vd2 T cells
in the antitumor activity of NBPs.
IPP and ApppI released by breast cancer cells are
chemotactic factors for Vg9Vd2 T cells
In our model, ZOL was able to induce recruitment of
Vg9Vd2 T cells to T47D tumors. We therefore investigated
the ability of breast cancer cells treated in vitro with ZOL to
promote Vg9Vd2 T-cell migration. Using conditioned medium
from untreated breast cancer cell lines (T47D, MCF-7, B02)
in Transwell migration assays, we observed significantly
enhanced migration of Vg9Vd2 T cells after 4 hours (not
shown) and 24 hours (Fig. 6A; P < 0.05 and P < 0.01) versus
assay buffer. Migration of Vg9Vd2 T cells was much higher
with conditioned medium from ZOL-treated T47D and MCF-7
breast cancer cells (Fig. 6B and C). In addition, conditioned
B
180
**
200
*
Cell migration (%)
250
*
150
100
50
***
ns
140
120
100
80
60
40
Control
D
T47D, MCF-7,
CM
CM
–
+
–
T47D
CM
T47D
CTR
*
ZOL
+
–
MCF-7
CM
100
Angiogenin
+
Control –ZOL +ZOL +ZOL
+LOV
BO2
CM
–
1
1
Angiogenin
GDNF,
GM-CSF
IGFBP-1
–
+
2
2
MCP-1
–
2
+
2
GRO, GRO-α
IL-8
sTNF-R1
GRO, GRO-α
+
+
–
IL-6
+
MCP-1
TIMP-1, TIMP-2
–
Apppl
T47D, CM
IGFBP-4
+
IPP
E
+
–
ns
0
+
ZOL
CTR
1
*
150
B02
1
**
200
50
0
ZOL
BO2
CM
**
250
20
0
–
300
***
160
10,000
Secreted cytokine (pg/mL)
300
Cell migration (% of
control)
C
Cell migration (% of
control)
A
IL-6
IL-8
1,000
MCP-1
100
10
1
nd
T47D, CM
nd
MCF-7, CM
BO2, CM
uPAR, VEGF
TIMP-1, TIMP-2
Figure 6. IPP and ApppI induce chemotactic migration of Vg9Vd2 T cells. Purified Vg9Vd2 T cells isolated from ZOL-stimulated PBMCs by positive selection
of TCR-gd cells were tested in a Transwell assay for their capacity to migrate (A) toward conditioned medium (CM) from T47D, MCF-7, and B02
cell lines compared with assay medium alone; (B) toward CM from ZOL-treated T47D, MCF-7, and B02 cells versus CM from untreated cells; or
(C) toward CM from ZOL-treated T47D cells lovastatin and 0.5-mmol/L IPP or 1-mmol/L ApppI added in the medium, or compared with assay medium
alone. Data represent mean percentage migration from 2 independent experiments SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 using pairwise comparisons
with the assay medium or CM from untreated cells. ns, not significant. D, detection of cytokines produced in the supernatant by cultured, untreated
(control; CTR), and ZOL-treated T47D and B02 cells using 2 human cytokine antibody array membranes (1 and 2) designed to detect altogether
120 human cytokines. E, IL-6, IL-8, and MCP-1 secretion in CM of T47D, MCF-7, and B02 cells, using ELISA.
4570
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IPP/ApppI Mediates Vg9Vd2 T-cell Migration and Cytotoxicity
untreated cells (Fig. 6D). By ELISA, untreated B02 cells (control) produced more IL-8 and MCP-1 compared with T47D or
MCF-7 control cells (Fig. 6E); IL-6 was detected only in B02
cells.
In conclusion, our study shows for the first time that ZOL
induces the production of IPP/ApppI phosphoantigens by
breast tumors in vivo, which, in turn, promote chemotaxis
and cytotoxicity of Vg9Vd2 T cells. These results provide some
support for an adjuvant role of NBPs in breast cancer. There is
now clinical evidence that adding ZOL to endocrine therapy
improves disease-free survival in ER-responsive early breast
cancer (4,5). ZOL has also been shown to durably activate gd
T cells in patients with breast cancer (25). The immunomodulating role of NBPs on human gd T cells might explain, at
least in part, the anticancer effects of ZOL observed in these
adjuvant clinical trials. Overall, our findings suggest that
cancers producing high IPP/ApppI levels after ZOL treatment
are most likely to benefit from Vg9Vd2 T-cell–mediated
immunotherapy.
Disclosure of Potential Conflicts of Interest
P. Clezardin has served on advisory boards for Novartis and Amgen. J. Green
is a consultant for Novartis and holds stock in the company.
Acknowledgments
The authors thank Catherine Browning, PhD, ProEd Communications, Inc.,
Beachwood, Ohio, for her editorial assistance with this manuscript and Novartis
Pharmaceuticals Corporation for financial support. We also thank the nurses
from Edouard Herriot Hospital for their valuable help.
Grant Support
P. Clezardin has received lecture fees and a research grant from Novartis; and
is supported by INSERM, the University of Lyon, and the Ligue Regionale contre
le Cancer. H. M€
onkk€
onen is supported by Inserm (Poste Vert). J. M€
onkk€
onen is
supported by the Academy of Finland.
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 October 26, 2010; revised April 27, 2011; accepted May 4, 2011;
published OnlineFirst June 6, 2011.
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