Oxidative stress regulates expression of VEGFR1 in myeloid cells: link to tumor-induced immune suppression in renal cell carcinoma.
ABSTRACT Metastatic renal cell carcinoma (RCC) associates with overproduction of vascular endothelial growth factor (VEGF) due to the mutation/inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Herein we demonstrate that implantation of human RCC tumor cells into athymic nude mice promotes the appearance of VEGF receptor 1 (VEGFR1)/CD11b double-positive myeloid cells in peripheral blood. Avastin-mediated VEGF neutralization was capable of significantly reducing the numbers of circulating VEGFR1+ myeloid cells. Conversely, up-regulation of VEGFR1 by myeloid cells could also be achieved in vitro by coculturing bone marrow cells with RCC-conditioned medium or by short-term exposure of naive myeloid cells to oxidative stress. Treatment of myeloid cells with H2O2, lipid peroxidation product 4-hydroxy-2(E)-nonenal, or an inhibitor of thioredoxin reductase all resulted in increased expression of VEGFR1. Furthermore, after exposure to oxidative stress, myeloid cells acquire immunosuppressive features and become capable of inhibiting T cell proliferation. Data suggest that tumor-induced oxidative stress may promote both VEGFR1 up-regulation and immunosuppressive function in bone marrow-derived myeloid cells. Analysis of tumor tissue and peripheral blood from patients with metastatic RCC revealed that VEGFR1+ cells can be also found in cancer patients. Restoration of immunocompetence in metastatic RCC patients by pharmacological elimination of VEGFR1+ cells may have a significant impact on the therapeutic efficacy of cancer vaccines or other immune-based therapies.
- SourceAvailable from: Roberto Ruiu[Show abstract] [Hide abstract]
ABSTRACT: The tyrosine kinase human epidermal growth factor receptor 2 (HER2) gene is amplified in approximately 20% of human breast cancers and is associated with an aggressive clinical course and the early development of metastasis. Its crucial role in tumor growth and progression makes HER2 a prototypic oncoantigen, the targeting of which may be critical for the development of effective anticancer therapies. The setup of anti-HER2 targeting strategies has revolutionized the clinical outcome of HER2(+) breast cancer. However, their initial success has been overshadowed by the onset of pharmacological resistance that renders them ineffective. Since the tumor microenvironment (TME) plays a crucial role in drug resistance, the design of more effective anticancer therapies should depend on the targeting of both cancer cells and their TME as a whole. In this review, starting from the successful know-how obtained with a HER2(+) mouse model of mammary carcinogenesis, the BALB-neuT mice, we discuss the role of TME in mammary tumor development. Indeed, a deeper knowledge of antigens critical for cancer outbreak and progression and of the mechanisms that regulate the interplay between cancer and stromal cell populations could advise promising ways for the development of the best anticancer strategy.BioMed Research International 01/2014; 2014:534969. · 2.71 Impact Factor
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ABSTRACT: The progressive conversion of normal cells into cancer cells is characterized by the acquisition of eight hallmarks. Among these criteria, the capability of the cancer cell to avoid the immune destruction has been noted. Thus, tumors develop mechanisms to become invisible to the immune system, such as the induction of immunosuppressive cells, which are able to inhibit the development of an efficient immune response. Molecules produced in the tumor microenvironment are involved in the occurrence of an immunosuppressive microenvironment. Recently, it has been shown that vascular endothelial growth factor A (VEGF-A) exhibits immunosuppressive properties in addition to its pro-angiogenic activities. VEGF-A can induce the accumulation of immature dendritic cells, myeloid-derived suppressor cells, regulatory T cells, and inhibit the migration of T lymphocytes to the tumor. Other pro-angiogenic factors such as placental growth factor (PlGF) could also participate in tumor-induced immunosuppression, but only few works have been performed on this point. Here, we review the impact of pro-angiogenic factors (especially VEGF-A) on immune cells. Anti-angiogenic molecules, which target VEGF-A/VEGFR axis, have been developed in the last decades and are commonly used to treat cancer patients. These drugs have anti-angiogenic properties but can also counteract the tumor-induced immunosuppression. Based on these immunomodulatory properties, anti-angiogenic molecules could be efficiently associated with immunotherapeutic strategies in preclinical models. These combinations are currently under investigation in cancer patients.Frontiers in Oncology 01/2014; 4:70.
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ABSTRACT: Dendritic cells are an important target in cancer immunotherapy based on their critical role in antigen presentation and response to tumor development. The capacity of dendritic cells to stimulate anti-tumor immunity has led investigators to use these cells to mediate anti-tumor responses in a number of clinical trials. However, these trials have had mixed results. The typical method for generation of ex vivo dendritic cells starts with the purification of CD14(+) cells. Our studies identified a deficiency in the ability to generate mature dendritic cell using CD14(+) cells from cancer patients that corresponded with an increased population of monocytes with altered surface marker expression (CD14(+)HLA-DR(lo/neg)). Further studies identified systemic immune suppression and increased concentrations of CD14(+)HLA-DR(lo/neg) monocytes capable of inhibiting T-cell proliferation and DC maturation. Together, these findings strongly suggest that protocols aimed at immune stimulation via monocytes/dendritic cells, if optimized on normal monocytes or in systems without these suppressive monocytes, are unlikely to engender effective DC maturation in vitro or efficiently trigger DC maturation in vivo. This highlights the importance of developing optimal protocols for stimulating DCs in the context of significantly altered monocyte phenotypes often seen in cancer patients.Frontiers in Immunology 01/2014; 5:147.
Oxidative Stress Regulates Expression of VEGFR1 in Myeloid
Cells: Link to Tumor-Induced Immune Suppression in Renal
Sergei Kusmartsev,2* Evgeniy Eruslanov,* Hubert Ku ¨bler,* Timothy Tseng,‡Yoshihisa Sakai,*
Zhen Su,* Sergei Kaliberov,§Axel Heiser,* Charles Rosser,* Philip Dahm,* Dietmar Siemann,†
and Johannes Vieweg*
Metastatic renal cell carcinoma (RCC) associates with overproduction of vascular endothelial growth factor (VEGF) due to the
mutation/inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Herein we demonstrate that implantation of
human RCC tumor cells into athymic nude mice promotes the appearance of VEGF receptor 1 (VEGFR1)/CD11b double-positive
myeloid cells in peripheral blood. Avastin-mediated VEGF neutralization was capable of significantly reducing the numbers of
circulating VEGFR1?myeloid cells. Conversely, up-regulation of VEGFR1 by myeloid cells could also be achieved in vitro by
coculturing bone marrow cells with RCC-conditioned medium or by short-term exposure of naive myeloid cells to oxidative stress.
Treatment of myeloid cells with H2O2, lipid peroxidation product 4-hydroxy-2(E)-nonenal, or an inhibitor of thioredoxin reductase
all resulted in increased expression of VEGFR1. Furthermore, after exposure to oxidative stress, myeloid cells acquire immuno-
suppressive features and become capable of inhibiting T cell proliferation. Data suggest that tumor-induced oxidative stress may
promote both VEGFR1 up-regulation and immunosuppressive function in bone marrow-derived myeloid cells. Analysis of tumor
tissue and peripheral blood from patients with metastatic RCC revealed that VEGFR1?cells can be also found in cancer patients.
Restoration of immunocompetence in metastatic RCC patients by pharmacological elimination of VEGFR1?cells may have a
significant impact on the therapeutic efficacy of cancer vaccines or other immune-based therapies. The Journal of Immunology,
2008, 181: 346–353.
tokine-based approaches (1). Despite their tremendous promise,
current vaccine strategies have, unfortunately, shown only limited
success in clinical settings (2, 3). Particularly in RCC, multiple
immunosuppressive mechanisms considerably dampen antitumor
responses and weaken the activity of current immunotherapeutic
treatment regimens (4–7). Therefore, new strategies will be nec-
essary to reverse tumor-mediated immune suppression before im-
munotherapy is applied to cancer patients.
Metastatic RCC is associated with overproduction of vascular
endothelial growth factor (VEGF) due to mutation/inactivation of
etastatic renal cell carcinoma (RCC)3represents a pro-
totypical malignancy for the application of immuno-
therapy, including active, passive, and nonspecific cy-
the VHL tumor suppressor gene (8, 9). This proangiogenic growth
factor plays a crucial role in the development of tumor neovascu-
lature (10). Tumor-derived VEGF also affects functions of immune
system. Thus, it has been established that VEGF is involved in
tumor-induced abnormalities of dendritic cell (DC) differentiation,
and an inverse correlation between the density of DC and the ex-
pression of VEGF has been demonstrated within tumor tissue and
peripheral blood of cancer patients (11, 12). The elevated level of
circulating VEGF in cancer patients was also closely correlated
with an increased number of immature myeloid cells in peripheral
Recent studies suggest a direct role of bone marrow-derived
VEGF receptor 1 (VEGFR1)-positive myeloid cells in the induc-
tion of neovascularization (13, 14) and promotion of the metastatic
process (15). VEGFR1 can be found in the tumor tissue and lymph
nodes of cancer patients with lung cancer, breast cancer (15), and
lymphomas (16). However, to date there is little known regarding
the presence of VEGFR1/CD11b cells in cancer patients with met-
astatic RCC and their role in tumor-induced immune suppression.
Herein we demonstrate that inoculation of human RCC cells
into nude mice induces the appearance of VEGFR1?CXCR4?
CD11b?myeloid cells in peripheral blood. Expression of
VEGFR1 by myeloid cells could also be achieved in vitro by
coculturing bone marrow cells in the presence of a tumor-condi-
tioned medium, or by exposing naive myeloid cells to oxidative
stress. Furthermore, after exposure to oxidative stress, myeloid
cells have acquired immunosuppressive features and have become
capable of inhibiting T cell proliferation. These data suggest that
tumor-induced oxidative stress may promote both VEGFR1 up-
regulation and immunosuppressive features in bone marrow-de-
rived myeloid cells. Analysis of tumor tissue and peripheral blood
*Department of Urology, College of Medicine and†Department of Pharmacology and
Experimental Therapeutics, University of Florida, Gainesville, FL 32610;‡Depart-
ment of Surgery, Duke University, Durham, NC 27710; and§Department of Radiation
Oncology, University of Alabama, Birmingham, AL 35294
Received for publication October 25, 2007. Accepted for publication April 24, 2008.
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.
1This work was supported by grants from the Department of Health, Florida Bio-
medical Research Program (to S.K.) and the National Cancer Institute (to J.V.).
2Address correspondence and reprint requests to Dr. Sergei Kusmartsev, University
of Florida, Cancer and Genetics Research Center, 1376 Mowry Road, Room 459, P.O.
Box 103633, Gainesville, FL 32610. E-mail address: email@example.com
3Abbreviations used in this paper: RCC, renal cell carcinoma; BSO, L-buthionine-
(S,R)-sulfoximine; DC, dendritic cell; DCNB, 1,2-dichloro-4-nitrobenzene; 4-HNE,
4-hydroxy-2(E)-nonenal; GSH, glutathione; GSH-ME, glutathione methylester;
VEGF, vascular endothelial growth factor; VEGFR1, VEGF receptor 1; VHL, von
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
from patients with metastatic RCC revealed that VEGFR1?cells
can be also found in cancer patients.
Materials and Methods
Peripheral blood and tumor tissue were collected from 23 patients with
RCC at the Department of Surgery, Duke University Medical Center
(Durham, NC) and the Department of Urology, College of Medicine, Uni-
versity of Florida (Gainesville, FL). All specimens were obtained follow-
ing informed consent and approval by institutional review board. PBMC
from patients and healthy donors were separated by Lymphoprep gradient
Mice and tumor cell lines
We obtained female BALB/c wild-type mice and athymic nu/nu mice from
the National Cancer Institute (Frederick, MD) and used them at 6–10 wk
of age. Human RCC cell line Caki-1 was provided by D. Siemann (Uni-
versity of Florida, Gainesville, FL), and mouse renal tumor cell line Renca
was obtained from J. Finke (Lerner Research Institute, Cleveland, OH).
Caki-1 tumor cells (5 ? 106) were inoculated into nude mice orthotopically
or i.m., whereas Renca tumor cells (5 ? 105) were s.c. injected into
Reagents and Abs
Avastin (bevacizumab) was purchased from Genentech. 4-hydroxy-2(E)-
nonenal (4-HNE), 1,2-dichloro-4-nitrobenzene (DCNB), and L-buthi-
onine-(S,R)-sulfoximine (BSO) were obtained from Sigma-Aldrich. Glutathi-
one methylester (GSH-ME) was purchased from Calbiochem. GM-CSF was
obtained from Immunex, and IL-4 was purchased from R&D Systems.
Purified CD3e (clone 145–2C11), CD28 (37.51), and Gr-1 (RB6–8C5)
were purchased from BD Biosciences. The following fluorochrome-con-
jugated Abs were used for flow cytometry: anti-mouse and anti-human
VEGFR1 (R&D Systems) and CD11b, CXCR4, CD15, and CD33 (all from
CD11b?cells from were isolated from bone marrow by positive selection
using MACS microbeads against CD11b (Miltenyi Biotec). VEGFR1?
cells also were isolated from peripheral blood of RCC patients using
MACS microbeads. Freshly isolated PBMC were first analyzed by flow
cytometry for presence of VEGFR1?cells. PBMC from “positive” patients
were resuspended in cold MACS buffer and incubated with mixture
VEGFR1-PE Abs (R&D Systems) for 15 min on ice. After washing with
cold MACS, buffer cells were incubated with PE-microbeads (Miltenyi
Biotec) and subsequently subjected to positive selection of VEGFR1?cells
on a MACS column according to the manufacturer’s instructions (Miltenyi
DC generation and MLR assay
Dendritic cells were generated from peripheral blood-derived monocytes.
Briefly, isolated PBMC were plated into 6-well plates in a serum-free me-
dium and incubated at 37°C for 2 h. Then, nonadherent cells were removed
and the adherent cells were cultured in complete RPMI 1640 medium sup-
plemented with GM-CSF and IL-4 for 7 days. T lymphocytes were purified
from PBMC using T cell enrichment columns (R&D Systems). To set up
MLR, 2 ? 105T cells derived from a RCC patient and 2 ? 104allogeneic
DCs were plated in each well of 96-well round-flat-bottom plates. Inhibi-
tory activity of VEGFR1?cells was evaluated by adding increasing num-
bers of RCC patient-derived myeloid cells to the MLR reaction. After 96 h
of culture, 1 ?Ci of [3H]thymidine was added to each well and incubated
for an additional 18 h. Tritium thymidine uptake was analyzed by liquid
Oxidative stress in vitro
Experiments were performed in DMEM (Invitrogen) supplemented with
2% FCS. Freshly derived naive murine bone marrow cells or PBMC from
healthy volunteers were exposed to H2O2(1–50 ?M) for 45–60 min, to
DCNB (1–30 ?M) for 2–3 h, and to BSO (25–400 ?g/ml) overnight. Cells
were then collected, washed with cold PBS with 1% FCS, and immediately
analyzed by flow cytometry or used for functional studies. In some exper-
iments, before application of oxidative stress, bone marrow cells were pre-
incubated with GSH-ME (1–5 mM) for 30 min before being exposed to
A total of 1 ? 106cells were suspended in PBS buffer and incubated for
20 min at 4°C with the Ab and then washed twice with cold PBS. Fluo-
rochrome-conjugated Abs as well as isotypic control Abs were used for cell
staining. FACS data were acquired using a FACSCalibur flow cytometer
(BD Biosciences) and were analyzed using CellQuest software (BD Bio-
sciences). Results were expressed as the percentage of positive cells and
mean fluorescence intensity.
Proliferation and suppression assays
For T cell activation experiments, freshly isolated naive splenocytes (2 ?
105/well) were cultured in 96-well round-bottom plates in complete culture
medium containing soluble or plate-bound anti-CD3 (1 ?g/ml) and soluble
anti-CD28 (5 ?g/ml). CD11b?cells derived from naive murine bone mar-
row cells were exposed to H2O2(20 ?M) for 45 min or DCNB (30 ?M)
for 2 h. Then, bone marrow-derived cells were collected, washed with PBS,
suspended in complete culture media, and mixed with graded numbers of
naive splenocytes in 96-well plates. Proliferation of CD3 T cells and CD4
and CD8 T cells was assessed by incorporation of BrdU in DNA by flow
RCC tissues were fixed and embedded in paraffin. Endogenous peroxidase
activity was blocked by treating tissue sections with H2O2in methanol for
20 min. The following procedure was performed using a HRP-3,3-diami-
nobenzidine (DAB) cell and tissue staining Kit (R&D Systems). The sec-
tions were incubated with serum-blocking reagent for 30 min to block
nonspecific binding of Ig. Avidin/biotin blocking was performed using
avidin and biotin-blocking reagents. Then, sections were incubated over-
night with the first primary mouse anti-human VEGFR1 (Flt-1) (R&D
Systems). The following day, sections were incubated for 60 min with
biotinylated secondary Ab. Then, high-sensitivity streptavidin conjugated
to HRP was added. Thirty minutes later, the sections were incubated with
DAB chromogen solution for 5–20 min and counterstained with hematox-
ylin. Dehydration was performed through an ascending alcohol series. Tis-
sue sections were cleared in xylene and mounted in xylene-based mounting
media. Examination of the tissue sections was performed with an Axioplan
2 upright microscope at magnification ?200.
Matrigel plug assay
Athymic nude mice were injected s.c. at the midpoint of their right back
sides with 0.3 ml Matrigel (BD Biosciences) together or without VEGF.
After 6 days, the animals were euthanized and dissected, and the implants
were photographed. Implants, together with associated skin, were fixed for
60 min in 10% formalin and embedded in paraffin. Sections were cut, the
paraffin was removed, and sections were then treated with 0.1% trypsin for
30 min at 37°C to enhance Ag availability to anti-VEGFR1 mAb. Bound
rabbit anti-rat secondary Ab, coupled with HRP (Vector Laboratories), was
visualized with True Blue peroxidase substrate (Kirkegaard & Perry Lab-
oratories). Sections were counterstained with eosin Y (Richard-Allan
Concentrations of VEGF in blood serum obtained from patients with RCC
and from healthy donors were measured using commercial ELISA kits
(R&D Systems) following the manufacturer’s instructions.
The statistical significance between values was determined by the Student
t test. All data were expressed as the means ? SD. Probability values
?0.05 were considered nonsignificant. Significant values were expressed
as ?, p ? 0.05. The immunohistochemistry images and flow cytometry data
shown are representative of at least three separate determinations.
Growth of renal cell carcinoma in mice associates with
appearance of VEGFR1?cells in peripheral blood
Caki-1 renal carcinoma cells were implanted into athymic nude
mice, and 3 wk after tumor inoculation peripheral blood cells were
analyzed for the presence of double-positive VEGFR1/CD11b my-
eloid cells. Results of representative experiments are shown in Fig.
1A. Data obtained demonstrate that orthotopic (intrarenal) inocu-
lation of RCC tumor cells in mice promotes a marked increase of
347The Journal of Immunology
VEGFR1 expression in the circulating CD11b myeloid cell pop-
ulation. Importantly, the numbers of VEEGFR1/CD11b double-
positive cells in circulation were dependent on tumor size, indi-
cating the possible role of tumor-secreted factor(s) in the
mobilization of VEGFR1?myeloid cells. We also observed that
most of VEGFR1?cells in peripheral blood also coexpressed
TGF-?RII and CXCR4 (Fig. 1B), a receptor for the chemokine
CXCL12, also known as SDF-1. However these cells did not ex-
press IL-4R?, which is known to be expressed on myeloid-derived
To test whether renal carcinoma-derived factors could stimulate
VEGFR1 expression in myeloid cells in vitro, we cocultured naive
murine bone marrow cells with a 20% tumor-conditioned medium
obtained from RCC Caki-1, colon carcinoma CT-26, or prostate
tumor RM-9. Results clearly demonstrate that renal tumor cell su-
pernatant promotes significant increase (?3-fold) of VEGFR1 ex-
pression in CD11b cells (Fig. 1C). We also observed up-regulation
of VEGFR1 in human CD11b cells when human PBMC from
healthy volunteer blood were cultured in the presence of Caki-1
renal tumor cell supernatant (Fig. 1D). Collectively, these data
demonstrate that RCC up-regulates VEGFR1 expression in CD11b
myeloid cells and that these double-positive cells can be found in
the peripheral blood after inoculation of renal tumor into mice.
Oxidative stress up-regulates expression of VEGFR1 by CD11b
It has been established that expression of VEGFR1 is regulated by
hypoxia in a hypoxia-induced factor (HIF)-dependent manner (17).
Specifically, unlike the KDR/Flk-1 (VEGFR2) gene, the Flt-1
(VEGFR1) receptor gene is directly up-regulated by hypoxia via a
hypoxia-inducible enhancer element located at positions ?976 to
?937 of the Flt-1 promoter. More recently, HIF-? stabilization
expression was shown to be dependent on reactive oxygen species
production (reviewed in Ref. 18). To test whether exposure of
myeloid cells to oxidants may regulate expression of VEGFR1, we
incubated murine bone marrow cells and human PBMC in the
presence of H2O2. Fig. 2 illustrates that a 45-min treatment with
in nude mice promotes an appearance of VEGFR1?
CD11b?cells in peripheral blood. A and B, One million
Caki-1 RCC cells were orthotopically inoculated into
nude mice. Twenty-one days after inoculation, tumor-
bearing and control tumor-free mice were sacrificed. Tu-
mor size was measured using calipers. Peripheral blood
was collected, depleted for RBC, and immediately
stained with mAbs. Gated CD11b?cells were analyzed
for coexpression of VEGFR1 (A) or for CXR4, TGF-
?RII, and IL-4R? (B). C, Culturing of bone marrow
cells in the presence of Caki-1 tumor-conditioned me-
dium induces up-regulation of VEGFR1. Murine bone
marrow cells were depleted for erythrocytes and cul-
tured in complete medium (control group) or in the pres-
ence of 20% tumor-conditioned medium (renal carci-
noma Caki-1, colon carcinoma CT-26, or prostate tumor
RM-9) for 48 h. Cells were collected, washed, stained
with Abs, and analyzed by flow cytometry for expres-
sion of VEGFR1 by CD11b myeloid cells. D, RCC cells
up-regulate expression of VEGFR1 in human CD11b
cells. PBMC from healthy volunteers were cultured in
the presence of Caki-1 RCC cell supernatant (20%).
Twenty-four hours later, cells were collected and
washed with PBS. Cells were stained, CD11b?cells
were gated, and the expression of VEGFR1 in these
cells was assessed by flow cytometry.
Inoculation of RCC tumor cells Caki-1
duces expression of VEGFR1 in my-
eloid cells. PBMC from healthy volun-
teers and bone marrow cells from naive
mice were suspended in DMEM sup-
plemented with 2% FCS. Cells were
exposed to H2O2(0.7–17 ?M) for 45
min at 37°C. Cells were then washed
with cold PBS, stained with VEGFR1
and CD11b Abs, and immediately an-
alyzed by flow cytometry. Experiments
were repeated three times.
348 OXIDATIVE STRESS AND IMMUNE SUPPRESSION IN RENAL CANCER
H2O2leads to up-regulation of VEGFR1 expression in both mu-
rine and human CD11b cells in a dose-dependent manner. Con-
centrations of H2O2up to 30 ?M did not induce cell death as
determined by trypan blue exclusion, whereas 50 ?M caused
?50% cell death (data not shown). Collectively, these data indi-
cate a role for oxidative stress in the increased cell surface expres-
sion of VEGFR1.
Thioredoxin reductase is involved in regulation of VEGFR1
Both cell susceptibility to oxidative stress and intracellular levels
of H2O2are controlled by two major antioxidant systems: gluta-
thione peroxidase/glutaredoxin/glutathione and thioredoxin reduc-
tase/thiredoxin (19). Because these proteins are involved in intra-
cellular catabolism of H2O2, which in turn regulates VEGFR1
expression, we asked whether one of the major antioxidant systems
could be involved in regulation of VEGFR1 receptor in myeloid
cells. First, we tested whether depletion of intracellular glutathione
(GSH) could mimic the effect of H2O2and affect VEGR1 expres-
sion. As shown in Fig. 3 (upper left panel), overnight treatment of
bone marrow cells with BSO did not affect VEGFR1 expression.
We also examined whether addition of reduced form of GSH (i.e.,
GSH-ME) could affect oxidative stress-induced VEGFR1 expres-
sion. Pretreatment of bone marrow cells with GSH-ME just before
its exposure to H2O2could not prevent VEGFR1 expression by
oxidative stress (Fig. 3, upper right panel). Taken together, these
results suggest that a glutathione/glutathione reductase system is
not involved in mechanisms of regulating VEGFR1 expression in
We next asked whether exposure of cells to the thioredoxin
reductase inhibitor DCNB could mimic the effect of H2O2on
VEGFR1 expression. Fig. 3 (lower left panel) shows that the in-
cubation of bone marrow cells with DCNB for 2 h leads to up-
regulation of VEGFR1 in CD11b myeloid cells. We also tested the
effect of alternative agents known to inhibit thioredoxin reductase
acivity. 4-HNE is the end-product of lipid peroxidation and effi-
ciently inhibits thioredoxin reductase activity (20). The exposure
of murine bone marrow cells to 4-HNE resulted in dose-dependent
up-regulation of VEGFR1 (Fig. 3, lower right panel). Collectively,
these data support the idea that thioredoxin reductase in involved
in mechanisms of VEGFR1 expression regulation in bone marrow-
derived myeloid cells.
Oxidative stress promotes immune-suppressive features of
Reactive oxygen species and oxidative stress are involved in
mechanisms of tumor-induced immune suppression mediated by
myeloid-derived suppressor cells (21, 22). To examine the direct
role of oxidative stress in the induction of immune suppression, we
exposed bone marrow-derived CD11b cells to H2O2or thioredoxin
reductase inhibitor DCNB and then tested exposed cells for the
ability to inhibit T cell proliferation induced by CD3/CD28 stim-
ulation. T cell proliferation was measured by flow cytometry using
BrdU assay. As shown in Fig. 4, A and B, control untreated bone
marrow myeloid cells did not affect CD3/CD28-induced T cell
response, whereas both H2O2- and DNCB-treated myeloid cells
efficiently inhibited proliferation of CD3 (Fig. 4A) and CD4 and
CD8 T cells (Fig. 4B) in a dose-dependent fashion. VEGFR1?
cells isolated from bone marrow, which has been preexposed to
low dose of H2O2,also suppressed T cell response (Fig. 4C). Col-
lectively, the obtained data indicate that exposure of myeloid cells
to oxidative stress leads to up-regulation of VEGFR1 and pro-
motes its immunosuppressive features.
Presence of VEGFR1?cells in cancer patients with metastatic
Recent publications demonstrate that VEGFR1?cells could be
recruited in tumor sites including lung cancer, breast cancer, and
lymphomas. To examine whether VEGFR1?cells can be found in
human RCC tumor tissue, we evaluated the expression of
VEGFR1 in surgically dissected cancer tissue by immunohisto-
chemistry. As shown in Fig. 5, A and B, VEGFR1?cells are
present only in tumor tissue but not in “normal” benign tissue
cells were cultured overnight in DMEM supplemented with 2% FCS in presence of BSO (0.025–0.4 mg). Cells were then collected, washed with PBS,
and the expression of VEGFR1 by myeloid cells was measured (left upper panel). To provide bone marrow cells with additional reduced GSH before
application of oxidative stress, cells first were preincubated with GSH-ME (1–5 mM) for 30 min before being exposed to H2O2(15 ?M, 1 h). Cells were
then collected, washed with PBS, and the expression of VEGFR1 by myeloid cells was measured (right upper panel). For treatment with DCNB, bone
marrow cells were incubated in DMEM supplemented with 2% FCS in the presence of DCNB (1–30 ?M) for 2 h. Cells were then collected, washed, and
analyzed by flow cytometry for VEGFR1 expression (left lower panel). For exposure to 4-HNE, bone marrow cells were incubated in DMEM supplemented
with 2% FCS in the presence of 4-HNE (1–30 ?M) for 2 h. Cells were then collected, washed with PBS, and the expression of VEGFR1 by myeloid cells
was measured (right lower panel). All experiments were repeated at least twice.
Thioredoxin reductase/thioredoxin system is involved in regulation of VEGFR1 expression. To deplete GSH, freshly derived bone marrow
349 The Journal of Immunology
(Fig. 5D). Tumor cells themselves do not express VEGFR1, sug-
gesting that these VEGFR1?cells are recruited.
Recruitment of VEGFR1?cells into a tumor can be mediated by
tumor-produced VEGF (13, 23). Measurement of VEGF levels in
the blood serum of RCC patients demonstrated that they have an
increased serum level of VEGF compared with normal healthy
controls (Fig. 5E). To see whether increased levels of VEGF may
have association with numbers of VEGFR1?myeloid cells in the
peripheral blood, we have analyzed blood of 23 cancer patients
with metastatic RCC. We found that about half of observed RCC
patients (11 out 23) with metastatic RCC showed increased ex-
pression of VEGFR1 in peripheral blood as compared with healthy
donors. The level of expression of VEGFR1 in “positive patients”
was significantly varied. The representative data are shown in Fig.
5F. Importantly, most VEGFR1?cells also coexpressed myeloid
marker CD11b (?2-integrin), suggesting that the expression of
VEGFR1 in peripheral blood of RCC patients is limited to the
myeloid cell lineage. To examine whether VEGFR1?cells derived
suppressive. CD11b cells derived from naive murine bone marrow cells
were exposed to H2O2(20 ?M) for 45 min or DCNB (30 ?M) for 2 h. Cells
were then collected, washed with PBS, suspended in complete culture me-
dia, and mixed with naive syngeneic splenocytes in a graded manner in
96-well plates. Cultures were stimulated with anti-CD3 (1 ?g/ml) and anti-
CD28 (5 ?g/ml) Abs and cultured for 48 h at 37°C. Proliferation of CD3
T cells (A) and CD4 and CD8 T cells (B) was assessed by incorporation of
BrdU in DNA by flow cytometry. In separate experiments, naive bone
marrow cells were exposed to 6 ?M of H2O2for 45 min, and then
VEGFR1?cells were isolated with magnetic beads and added to the CD3/
CD28-stimulated lymphocyte as described above (C). Proliferation of CD3
T lymphocytes was evaluated by flow cytometry using BrdU assay. All
experiments were repeated twice.
Oxidative stress enables myeloid cells to become immuno-
astatic RCC. A–D, Presence of VEGFR1?cells in human tumor tissue.
Surgically dissected renal carcinoma tumor tissues were fixed and embed-
ded in paraffin. For detection of VEGFR1?cells by immunohistochemis-
try, primary mouse anti-human VEGFR1 (Flt-1) was used (A, B, and D).
For detection of myeloid cells, tissues were stained with anti-CD11b Ab
(C). Examination of the tissue sections was performed with an Axioplan 2
upright microscope with a magnification of ?200. Brown-colored cell
clusters represent the VEGFR1?cells. E, Increased concentration of VEGF
in the blood of cancer patients with RCC. Blood serum was obtained from
10 cancer patients and 8 healthy volunteers. Concentration of VEGF was
measured by ELISA. F, VEGFR1/CD11b double-positive cells in periph-
eral blood of RCC patients. Freshly isolated PBMC from donors and pa-
tients were stained with VEGFR1-PE and CD11b-allophycocyanin Abs
and analyzed for presence of VEGFR1?CD11b?cells. G, Inhibition of
allogeneic T cell proliferative response in presence of VEGFR1?myeloid
cells is shown. Graded numbers of purified VEGFR1?cells from a patient
with metastatic RCC were added to a mixture of 2 ? 105T cells from the
same patient and 2 ? 104allogeneic DCs. Cells were cocultured for 4 days,
and T cell proliferation was measured by [3H]thymidine incorporation.
Experiments were repeated twice.
VEGFR1?myeloid cells are present in patients with met-
350OXIDATIVE STRESS AND IMMUNE SUPPRESSION IN RENAL CANCER
from RCC cancer patients can directly inhibit T cell immune re-
sponse, we purified these cells and tested their ability to inhibit
allogeneic T cell response in MLR. Purified VEGFR1?and T cells
from RCC patients were cocultured with allogeneic DCs, and the
proliferative T cell response was measured 96 h later. Fig. 5G
shows that VEGFR1?cells readily inhibited in vitro T cell im-
mune response in a dose-dependent fashion. Collectively, our re-
sults suggest that metastatic RCC recruits immunosuppressive my-
eloid cells through overproduction of VEGF and complementary
up-regulation of VEGFR1 by hematopoietic myeloid cells.
Administration of anti-VEGF Ab (Avastin) prevents an
appearance of VEGFR1?myeloid cells in peripheral blood of
Excessive production of VEGF has shown to be responsible for
recruitment of myeloid VEGFR1?cells (13, 15, 24). It has also
been previously demonstrated that the humanized anti-VEGF Ab,
Avastin (bevacuzimab), can effectively neutralize tumor-secreted
VEGF. To test whether administration of Avastin could reduce the
number of VEGFR1?myeloid cells, we inoculated human RCC
tumor cells Caki-1 into athymic nude mice following injection
with Avastin. Flow cytometry analysis of peripheral blood (Fig.
6A) revealed a significant reduction of VEGFR1/CD11b double-
positive cells in tumor-bearing mice treated with Avastin as com-
pared with untreated group. Most of these VEGFR1?cells also
coexpressed ligand for SDF-1 chemokine CXCR4. Importantly,
that injection of matrigel into nude mice also induced recruitment
of VEGFR1?cells into matrigel plugs (Fig. 6B, left panel). When
matrigel was additionally mixed before implantation into mice
with VEGF (Fig. 6B, right panel), we observed a substantial in-
crease of recruited VEGFR1?cells. Taken together, these data
support the role of VEGF in mobilization and recruitment of
The incidence of RCC has been increasing during the past two
decades, with 38,890 new diagnoses expected in the United States
in 2006 (25). About one-third of these cases will present the met-
astatic disease uniformly resistant to radiation and chemotherapy.
At the same time, RCC represents one of the most receptive can-
cers to immunotherapy. However, the clinical response is limited,
mostly due to tumor-induced immune suppression. Tumor-induced
immune suppression represents a major obstacle for successful
RCC is characterized by the overproduction of VEGF due to
inactivation/mutation of VHL tumor suppressor gene. Overproduc-
tion of VEGF by malignant cells accounts for promotion of tumor
growth via angiogenic and mitogenic effects (10). Most proangio-
genic activity of VEGF is transduced via VEGFR2 (KDR, Flk-1),
which is predominantly expressed on endothelial cells and its pre-
cursors. The role of another VEGF receptor, VEGFR1, is less well
understood. However, there is growing evidence that VEGFR1 has
a significant role in the recruitment of bone marrow-derived cells
that may home in on the tumor vasculature and promote angio-
genesis. Furthermore, recent findings suggest a direct role of
VEGFR1?bone marrow-derived myeloid cells in the initiation
and regulation of the cancer metastasis process (15). Targeting of
VEGFR1 with a specific Ab found in mice with transplanted lung
carcinoma prevented the metastasis development. Preclinical data
also suggest that anti-VEGFR1 Ab administration has a significant
antiinflammatory effect (26).
In this study we demonstrate that orthotopic inoculation of hu-
CD11b?myeloid cells in peripheral blood. Most of the VEGFR1?
cells in peripheral blood also coexpressed TGF-?RII and CXCR4,
a receptor for the chemokine CXCL12 (SDF-1). Because SDF-1
can be induced in malignant tissues in a VEGF-dependent manner
(13), this finding supports a contribution of both VEGF and SDF-1
signaling pathways in the mobilization of myeloid cells in tumor
host bearing RCC. Coexpression of VEGFR1 and CXCR4 by
CD11b cells in RCC could potentially shed a light on a mechanism
of recruitment of VEGFR1?cells in tumor site; however, a sep-
arate study has to be designed to clarify this point.
Our clinical data demonstrate that VEGFR1?cells can also be
found in peripheral blood and tumor tissue obtained from cancer
patients with metastatic RCC. About half of analyzed cancer pa-
tients showed increased numbers of VEGFR1/CD11b double-pos-
itive cells in peripheral blood, whereas all tested tumor tissues
were positive for VEGFR1. RCC tumor cells do not express
VEGFR1, suggesting that these VEGFR1?cells are recruited.
These data are consistent with observations made by Kaplan et al.
(15) showing that VEGFR1?cells in tumor tissue localize in close
proximity to each other and form cell clusters.
We show herein that up-regulation of VEGFR1 expression in
myeloid cells in vitro can be achieved through culturing naive
bone marrow cells in the presence of tumor cell supernatant or,
alternatively, through the exposure of myeloid cells to oxidative
in mice with renal carcinoma. A, Anti-VEGF therapy reduces the number
of circulating VEGFR1?myeloid cells in mice with transplanted human
RCC. One million Caki-1 RCC cells were inoculated i.m. into nude mice
(n ? 8). On day 6, after tumor cell inoculation, four mice were i.p. injected
with Avastin (100 mg/kg) and four mice were injected with PBS. On day
10, after inoculation of tumors, all mice were sacrificed. Collected periph-
eral blood cells were depleted of erythrocytes, stained with allophycocya-
nin-VEGFR1, PerCp-CD11b, and PE-CXCR4 mAbs, and analyzed by flow
cytometry. B, Recruitment of VEGFR1?cells in implanted matrigel plugs.
Matrigel was premixed with recombinant human VEGF (100 ng) or an
equal volume of PBS and then inoculated subcutaneously into six athymic
nude mice. After 6 days, the animals were euthanized and dissected. Im-
plants were fixed for 60 min in 10% formalin and embedded in paraffin.
Presence of VEGFR1?cells in matrigel plugs was analyzed by immuno-
histochemistry. Brown-colored cells represent VEGFR1?cells.
VEGF regulates mobilization of VEGFR1?myeloid cells
351 The Journal of Immunology
stress, particularly H2O2. Importantly, that exposure of bone mar-
row-derived myeloid cells to the H2O2or inhibitor of thioredoxin
reductase DCNB promoted immunosuppressive function of my-
eloid cells, enabling them to inhibit T cell immune response.
Several tumor-derived growth factors such platelet-derived
growth factor, epidermal growth factor, and VEGF are known to
stimulate the intracellular production of reactive oxygen species
upon binding with specific receptor (reviewed in Ref. 27). Because
oxidative stress promotes up-regulation of VEGFR1 expression in
myeloid cells,we suggest that constant production of growth fac-
tors by malignant cells may affect expression of VEGFR1 in the
tumor microenvironment. Tumor cells are known to induce local
oxidative stress via enhanced production of H2O2as well as a
variety of lipid peroxide products, including 4-HNE (19). Taken
together, these factors may promote in the tumor microenviron-
ment specific local conditions that favor up-regulation of VEGFR1
in recruited myeloid cells and, more importantly, may induce their
immunosuppressive function, thus promoting tumor evasion from
This suggestion could also be supported by the following facts:
1) intratumoral VEGFR1?cells were observed in virtually all
tested tumors; 2) purified intratumoral myeloid cells, including
tumor-associated macrophages, represent a very potent immuno-
suppressive cell subpopulation (28–30). Recently, an important
role of VEGFR1?myeloid cells in initiating a premetastatic niche
has been demonstrated (15). To extend our hypothesis, we suggest
that the presence of immunosuppressive VEGFR1?cells in a
premetastatic niche could help incoming tumor cells survive by
inducing local immune suppression via inhibition of effector im-
mune cells and by helping to evade immune system control, thus
promoting metastasis growth. The bone marrow-derived myeloid
cells play an important role in tumor-induced immune suppression
and immune tolerance (31–34). Therefore, the immunosuppressive
features of VEGFR1?myeloid cells indicate the involvement of
these cells in tumor-induced immune suppression in cancer pa-
tients with RCC. However, it is at present unclear whether tumor-
derived VEGF uses a reactive oxygen species-dependent mecha-
nism to induce up-regulation of its own receptor, or whether
VEGFR1 expression is required for the recruitment of VEGFR1?
cells into tumor.
One of the emerging therapeutic approaches for RCC is antian-
giogenic anti-VEGF therapy. Recently, several new drugs that tar-
get VEGF, its receptors, and VEGF signaling pathways have been
introduced for cancer therapy, including metastatic RCC (35, 36).
However, anti-VEGF therapy was viewed solely as antiangio-
genic, with the most effects occurring at the local, intratumoral
level. In this study we demonstrate that anti-VEGF therapy with
Avastin reduces the numbers of VEGFR1?myeloid cells in pe-
ripheral blood and may have a significant impact on the efficacy of
Overall, our findings may have several implications for meta-
static RCC. Thus, although our study does not rule out every con-
ceivable pathway, they provide a clear relationship between oxi-
dative stress, increased surface VEGFR1 expression, and
immunosuppressive function of bone marrow-derived myeloid
cells. Our data also suggest the targeting of the VEGF-VEGFR
axis as not only an antiangiogenic therapy but also as an instru-
ment to enhance the effect of cancer immunotherapy for metastatic
RCC. Collectively, an increased level of VEGFR1?expression by
CD11b?myeloid cells in RCC patients may have a significant
impact on the progress and outcome of cancer via regulation of
angiogenesis, immunity, and metastatic processes.
We thank Suzanne Fesperman and Dawn Alayon for excellent assistance
with manuscript preparation.
The authors have no financial conflicts of interest.
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353The Journal of Immunology