Inhibition of both paracrine and autocrine VEGF?
VEGFR-2 signaling pathways is essential to
induce long-term remission of
xenotransplanted human leukemias
Sergio Dias*†, Koichi Hattori*†‡, Beate Heissig†, Zhenping Zhu§, Yan Wu§, Larry Witte§, Daniel J. Hicklin§,
Masatoshi Tateno¶, Peter Bohlen§, Malcolm A. S. Moore‡, and Shahin Rafii†?
†Division of Hematology-Oncology, Weill Medical College of Cornell University, New York, NY 10021;§ImClone Systems Incorporated, New York, NY 10014;
‡Sloan-Kettering Institute for Cancer Research, New York, NY 10021; and¶Sapporo City General Hospital, 060-8604 Sapporo, Japan
Edited by M. Judah Folkman, Harvard Medical School, Boston, MA, and approved July 12, 2001 (received for review March 9, 2001)
Antiangiogenic agents block the effects of tumor-derived angio-
genic factors (paracrine factors), such as vascular endothelial
growth factor (VEGF), on endothelial cells (EC), inhibiting the
growth of solid tumors. However, whether inhibition of angio-
We recently have shown that certain leukemias not only produce
VEGF but also selectively express functional VEGF receptors (VEG-
FRs), such as VEGFR-2 (Flk-1, KDR) and VEGFR1 (Flt1), resulting in
the generation of an autocrine loop. Here, we examined the
relative contribution of paracrine (EC-dependent) and autocrine
(EC-independent) VEGF?VEGFR signaling pathways, by using a
human leukemia model, where autocrine and paracrine VEGF?
VEGFR loops could be selectively inhibited by neutralizing mAbs
specific for murine EC (paracrine pathway) or human tumor (au-
tocrine) VEGFRs. Blocking either the paracrine or the autocrine
VEGF?VEGFR-2 pathway delayed leukemic growth and engraft-
ment in vivo, but failed to cure inoculated mice. Long-term remis-
sion with no evidence of disease was achieved only if mice were
treated with mAbs against both murine and human VEGFR-2,
whereas mAbs against human or murine VEGFR-1 had no effect on
mice survival. Therefore, effective antiangiogenic therapies to
treat VEGF-producing, VEGFR-expressing leukemias may require
blocking both paracrine and autocrine VEGF?VEGFR-2 angiogenic
loops to achieve remission and long-term cure.
neutralizing vascular endothelial growth factor receptor
(VEGFR)-specific antibodies, results in delayed growth of solid
tumor cell lines implanted into mice (1). However, the exact
mechanism whereby inhibition of the VEGF-VEGFR axis re-
sults in the regression of tumor tissue is not well studied.
There is accumulating evidence that angiogenesis supports the
growth of solid tumors in vitro and in vivo. It has been suggested
that as the tumor endothelial mass expands, in response to
tumor-derived angiogenic factors such as VEGF, it supports
tumor growth in a paracrine fashion. However, demonstration
that angiogenesis also plays a role in liquid tumors such as
leukemia has not been rigorously examined. Nevertheless, there
are numerous reports suggesting there is increased bone marrow
angiogenesis in patients with different hematological malignan-
cies such as acute lymphoblastic leukemias (2–6). Moreover,
leukemic cell release of angiogenic growth factors such as VEGF
portends poor clinical outcome and progression of the disease
(5). Similar to solid tumor growth, in response to leukemia-
derived angiogenic factors such as VEGF, it has been suggested
that the proliferating bone marrow endothelial mass may release
growth factors that support leukemic cell growth in a paracrine
fashion (7). Therefore, blocking VEGF signaling on endothelial
cells (EC) may reduce growth factor production, thereby retard-
agents that block endothelial proliferation, such as with
ing leukemic growth. However, thus far, studies in a clinical
setting have failed to demonstrate whether blocking angiogen-
esis results in delayed human leukemic growth in vivo.
We recently have shown that certain leukemic cells not only
produce VEGF, but have also acquired the capacity to express
functional VEGFRs (such as VEGFR-2), which results in the
generation of an endothelial-independent autocrine loop that
supports leukemic survival and migration in vivo (8). Therefore,
in VEGF-producing, VEGFR-expressing leukemias, generation
of VEGF?VEGFR autocrine (endothelial-independent) and
paracrine (endothelial-dependent) loops, may contribute toward
leukemic growth. In the present report we assessed the relative
contribution of paracrine and autocrine VEGF?VEGFR signal-
ing pathways to the growth of human leukemias in vivo. This was
achieved by using neutralizing mAbs specific for human or
mouse VEGFRs. We demonstrate that targeting the paracrine
(endothelial-dependent) or the autocrine (endothelial-
independent) VEGF?VEGFR-2 pathway delays leukemic
growth, but it is not sufficient to cure inoculated mice. Long-
term remission with no evidence of leukemia was achieved only
if mice were treated with mAbs against both murine and human
VEGFR-2, whereas mAbs against murine or human VEGFR-1
had no effect. Therefore, effective antiangiogenic therapies
should target both endothelial-dependent and endothelial-
independent signaling pathways to achieve remission.
Materials and Methods
All materials were obtained from Sigma, unless indicated
from American Type Tissue Culture Collection) were cultured
in RPMI (Life Technologies, Grand Island, NY) medium,
supplemented with 10% FBS, penicillin, and streptomycin.
Primary leukemic samples were isolated from the peripheral
blood of leukemic patients, and cultured overnight in serum-free
RPMI. For each in vivo experiment, viable leukemic cells were
counted and ressuspended in serum-free RPMI.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; EC, en-
granulocyte–macrophage colony-stimulating factor.
*S.D. and K.H. contributed equally to this work.
?To whom reprint requests should be addressed at: Cornell University Medical College,
Division of Hematology-Oncology, 1300 York Avenue, Room D601, New York, NY 10021.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
September 11, 2001 ?
vol. 98 ?
no. 19 ?
Human umbilical vein EC (HUVEC) and bone marrow-derived
EC (BMEC) were cultured in complete endothelial medium as
described (21). For leukemia proliferation experiments, EC were
cultured in 6-well plates (Costar) in complete EC medium until the
start of the experiment. Before starting the proliferation experi-
ments, EC were placed in serum-free medium.
Endothelium?Leukemia Cocultures. Leukemic cells were cultured
alone, or in the presence of a confluent layer of HUVEC or
BMEC in serum-free medium. To avoid direct cell?cell contact,
1 ? 105leukemic cells were placed on transwell (Costar) inserts
with a pore size of 0.4 ?m. Viable cells were counted by trypan
blue exclusion, using a hemacytometer.
Cytokine ELISA. Conditioned medium was collected from BMEC
or HUVEC monolayers, cultured in serum-free conditions over
a 48-h period. Granulocyte–macrophage colony-stimulating fac-
tor (GM-CSF) and IL-6 levels were measured by ELISA,
following conventional protocols (Cytokine Core Laboratory,
Antibodies. Neutralizing mAbs against human (clone IMC-1C11)
or mouse (clone DC101) VEGFR-2 (KDR?Flk-1) and against
human (clone 6.12) or mouse (clone mF-1) VEGFR-1 were
provided by ImClone Systems. These antibodies are specific for
one or the other VEGFR and have strict species specificity. A
human Ig preparation (Bayer, Elkhart, IN) was used at the same
dose as the neutralizing antibodies, as a negative control.
Leukemic Growth in Vivo. Nonobese diabetic immunocompromised
(NOD-SCID) mice, age-matched (6–8 weeks) and sex-matched,
were used in all experiments. Briefly, mice were irradiated with 3.5
Gy from a137Cs g-ray source at a dose rate of ?0.90 Gy?min and
intravenously inoculated with 1 ? 107HL-60 cells in 0.2 ml
divided into four groups of eight mice. One group was treated with
400 ?g human IgG thrice weekly (control), one group received
IMC-1C11 alone (400 ?g?mouse thrice weekly), one was treated
was treated with IMC-1C11 and DC101 twice weekly, at the same
dosage. Each experiment was done twice. Mice were evaluated for
any signs of toxicity throughout the experiment, and survival was
recorded. At days 7, 14, 21, 42, 60, and 120 after the start of the
and peripheral blood were collected for histology and quantifica-
tion of plasma VEGF levels and circulating leukemic cells, respec-
Bone Marrow Leukemic Cell Content. The presence of human
leukemia cells in the bone marrow of inoculated mice was
quantified by flow cytometry. Briefly, surgically removed tibias
day 21 after the start of the experiment were flushed from their
bone marrow content with a 19-g syringe, and the single cell
suspension was stained for the presence of leukemic cells.
Antibodies used for the FACS analysis were: mAb against
human CD15 [phycoerythrin (PE)-labeled, clone DU-HL60–3,
Sigma]; mAb against human VEGFR-2 (clone 6.64, ImClone
Systems), with a secondary goat anti-mouse IgG PE-labeled
antibody (Kirkegaard & Perry Laboratories); mAb against mu-
rine VEGFR-2 (FITC-labeled DC101) and an isotypic control,
MSIgG1 (FITC?PE-labeled clone 2T8–2F5, Coulter). The per-
VEGFR-2?cells in mouse bone marrow samples was deter-
mined by using a Coulter Elite flow cytometer. These results are
representative of three mice?treatment group. Each experiment
was done twice.
VEGF Plasma Levels. Human and murine-VEGF plasma levels
according to the manufacturer’s instructions. These assays had
a sensitivity of 7.5 pg?ml. Each sample was analyzed in duplicate,
and two samples (a total of six mice) were assayed per time point.
The results shown are representative of three mice?time point
and two separate experiments.
Statistical Analysis. The FACS results are expressed as mean ?
SEM. Statistical analyses were performed by using the unpaired
two-tailed Student’s t test. For survival analysis, the nonpara-
metric one-tailed Mann–Whitney u test was used.
EC Support Leukemic Cell Growth in a Paracrine Fashion. Freshly
isolated leukemias, or their cell line counterpart, if cultured in
serum-free condition survive and even show a modest increase
in proliferation over a 48-h period (ref. 8 and Fig. 1). Earlier, we
by the generation of an autocrine VEGF?VEGFR-2 loop (8).
However, if cultured in the presence of bone marrow-derived or
umbilical vein EC, leukemic cells proliferate significantly more
(Fig. 1). This finding suggested that EC support leukemia growth
and survival in vitro, perhaps by secreting specific growth factors
into the cell culture supernatants. Therefore, we analyzed EC
supernatants for the presence of growth factors that promote
leukemic growth, such as GM-CSF, IL-6, or IL-8 (Table 1). In
the present study, both endothelial sources released significant
amounts of GM-CSF, IL-6, or IL-8 into the cell culture super-
natants, as measured by ELISA (Table 1). This finding indicates
that EC may support leukemia survival and proliferation in a
lines (HL-60) in the presence of BMEC or HUVEC resulted in a remarkable
increase in leukemic proliferation over a 48-h period (P ? 0.05). As control,
leukemic cells were incubated with IL-6 (10 ng?ml) and GM-CSF (10 ng?ml),
complete medium or in serum-free conditions. Results shown represent the
average leukemia proliferation with three primary leukemias. Each experi-
ment was done in triplicate.
Incubation of freshly isolated monocytic leukemic cells (M5) or cell
Table 1. GM-CSF, IL-6, and IL-8 production
373 ? 78.3
292 ? 21.
440 ? 15
100.2 ? 2.8
96.3 ? 3.9
410 ? 22.3
and is shown as pg?ml per 106cells.
www.pnas.org?cgi?doi?10.1073?pnas.191117498Dias et al.
paracrine fashion, by secreting specific growth factors that will
subsequently act on the leukemic cells. Importantly, a constant
source of growth factors appears to be critical for the survival
and proliferation of the leukemic cells, because addition of
exogenous GM-CSF or IL-6 to leukemic cultures was less
effective at promoting leukemia proliferation (Fig. 1).
As they expand, the leukemic cells produce angiogenic growth
factors such as VEGF and fibroblast growth factor, which
stimulate both proliferation as well as growth factor production
by the endothelial layer. Notably, activation of HUVEC or
BMEC by angiogenic growth factors such as VEGF in vitro
significantly increased the production of GM-CSF, IL-6, and
IL-8, mimicking the coculture system described above (data not
shown). Taken together, these results suggest that generation of
such paracrine loops in vivo may be essential for leukemia
engraftment and proliferation of a malignant clone.
Next, we examined the relative contribution of the paracrine
(endothelial-derived) as well as autocrine (endothelial-
independent) loops toward leukemic growth and engraftment
Autocrine and Paracrine VEGF?VEGFR2 Signaling Pathways Are Essen-
tial for Leukemic Cell Growth. Sublethally irradiated NOD?SCID
mice inoculated with HL-60 leukemic cells, if left untreated, or
(KDR?Flk-1) and human (clone 6.12) and mouse (mF1) VEGFR-1 (FLT-1). Mice treated with DC101 survived two times more than control (P ? 0.05), whereas those
treated with IMC-1C11 survived three times more (P ? 0.005). Mice treated with mF1 or 6.12 showed no increase in survival as compared with control mice. All
mice treated with IMC-1C11 ? DC101 survived up to day 100, and 40% survived beyond day 200. (B) HL-60 injection into NOD-SCID mice induces high levels of
IMC-1C11, DC101, or IMC-1C11 ? DC101 had reduced circulating human VEGF levels up to day 35 (P ? 0.01).
(A) Mouse survival (%) after i.v. injection of HL-60 leukemia cells. Neutralizing mAbs against human (clone IMC-1C11) or mouse (clone DC101) VEGFR-2
Dias et al. PNAS ?
September 11, 2001 ?
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treated with an irrelevant human IgG, die within 2–3 weeks (Fig.
2A, mean survival time ? 18.2 ? 1.3 days. In these mice, clinical
signs and symptoms of acute leukemia, such as severe anemia,
lymphadenopathy, and cachexia became apparent within 2
weeks. As with leukemic patients (3), clinical progression in
inoculated mice is characterized by increased circulating human
VEGF levels. As shown in Fig. 2B, human, but not murine,
VEGF plasma levels increased in inoculated mice with disease
To investigate whether the growth of leukemia in vivo is angio-
tumor infiltrate (arrow) in the spleen of these mice. The bone marrow (ii) has evidence of increased angiogenesis and is largely replaced by tumor cells. Finally,
the lungs of these mice were largely replaced by tumor cells (iii), and the liver showed extensive areas of invading tumor cells (iv). (Magnifications: i–iii, ?; iv,
?400.) (B) Histology of livers (i, ii, and iv–ix) and spleen (iii) of HL-60-inoculated, antibody-treated mice. DC101-treated mice, day 21 postinoculation, had some
sign of leukemic infiltrates in the liver (arrow, i and ii) and the spleen was largely replaced by tumor cells (iii). On the other hand, in IMC-1C11-treated mice tumor
infiltration was localized to the perivascular area of the liver on days 21 and 42 (iv–vi). Finally, IMC-1C11 ? DC101-treated mice had no evidence of metastatic disease
on days 21 and 42 (vii and viii) but one small micrometastasis could be detected on day 60 (ix). (Magnifications: i, iii, iv, v, vii, viii, and ix, ?200; ii and vi, ?400.)
(A) Histology of spleen (i), bone marrow (ii), lung (iii), and liver (iv) from control HL-60-injected mice day 21 postleukemia inoculation. (i) Note a massive
www.pnas.org?cgi?doi?10.1073?pnas.191117498 Dias et al.
genesis-dependent, or is promoted solely via an autocrine VEGF?
human VEGFR stimulation, we used neutralizing mAbs to murine
(clone DC101) or human (clone IMC-1C11) VEGFR-2 and mAb
to murine (clone mF1) or human (clone 6.12) VEGFR-1. These
VEGF-induced receptor phosphorylation and endothelial prolif-
eration (9–11). In addition, we have previously shown that the
HL-60 leukemic cells used in our studies express functional
solid tumor models (10).
Treatment of leukemic mice with DC101, three times a week,
at 800 ?g?injection (a treatment schedule determined for other
tumor models), prolonged survival by more than 2-fold (Fig. 2A,
mean survival time ? 36.8 ? 6.3 days, P ? 0.05 compared with
the control group), but the mice eventually died within 42 days.
Treatment with IMC-1C11 (three times a week, at 400 ?g?
injection) prolonged survival by more than 3-fold (Fig. 2A, mean
survival time ? 70.3 ? 3.3 days, P ? 0.01) compared with the
human IgG-treated group but, again, all of the mice still died
within 70–80 days. The results obtained by treating leukemic
mice with DC101 show that blocking VEGF-induced angiogen-
esis in vivo, via interaction with murine VEGFR-2, delays
leukemic growth, but is not sufficient to eradicate the disease.
Targeting the autocrine VEGF?VEGFR-2 loop on leukemic
cells with IMC-1C11 significantly blocked leukemic growth and
invasiveness, but also failed to induce long-term remission of
inoculated mice. Notably, administration of either mF1 or 6.12
to leukemia-inoculated mice, to target the paracrine or the
mouse survival. Mice treated with either mF1 or 6.12 died
because of leukemia proliferation 23 and 25 days after the start
of the experiment, similarly to those in the control group (Fig.
2A). These results demonstrate that the VEGF?VEGFR-2 loops
are critical for leukemia proliferation in vivo, whereas VEGFR-1
appears to play a marginal role.
Coadministration of IMC-1C11 and DC101 had a synergistic
effect on survival of leukemic mice. Eighty percent (10?12, and
11?12 in a total of two experiments, P ? 0.001) of the mice
treated with both antibodies survived beyond 100 days (Fig. 2A).
Moreover, 40% of these mice achieved prolonged remission (up
to 200 days, Fig. 2A) with no clinical symptoms or histological
evidence of disease. Given the overall short life expectancy of
NOD-SCID mice (?10–12 months), this result is highly signif-
icant. As predicted, plasma levels of human VEGF remained
lower in the DC101 ? IMC-1C11-treated group, presumably
reflecting slower tumor progression (Fig. 2B).
Targeting Autocrine and Paracrine VEGF?VEGFR-2 Angiogenic Path-
ways Blocks Leukemia Invasiveness. We determined whether treat-
ment of leukemic mice with the anti-VEGFR-2 antibodies also
blocked the dissemination of the disease. Gross inspection of
control mice on days 7 and 14 revealed marked lymphadenop-
athy and infiltration of HL-60 cells into the bone marrow, spleen,
liver, and lung. Infiltrating HL-60 cells also were found in the
liver, spleen, bone marrow, and lungs of control mice on day 21
(Fig. 3A). In addition, bone marrow sections showed abundant
vascularization, suggesting increased angiogenesis (Fig. 3A).
Similarly, leukemia mice treated with mF1 or 6.12 also had
evidence of leukemia proliferation in peripheral organs and
bone marrow (data not shown), which correlated with the
inability of these antibodies to delay leukemic growth. In con-
trast, all organs from IMC-1C11- or DC101-treated mice were
free of HL-60 cells microscopically on days 7 and 14, and, even
after 21 days, tumor infiltration was limited to the perivascular
areas of the liver (Fig. 3B). DC101-treated mice did eventually
develop lymphadenopathy and massive tumor infiltration of the
spleen (Fig. 3B), at which point they succumbed to the prolif-
erating leukemia. In mice treated with IMC-1C11, tumor infil-
tration was reduced and still limited to the perivascular area of
the liver on day 42 (Fig. 3B). However, in mice treated with both
antibodies, there was no evidence of disseminated leukemia until
day 60 (Fig. 3B), at which time selected liver sections had
evidence of micrometastases (Fig. 3B). All mice in this group
survived beyond 100 days and, upon achieving remission, had no
evidence of metastatic disease up to day 120 (last time point
analyzed, data not shown).
To determine the effects of anti-VEGFR-2 antibody therapy
on the capacity of human leukemic cells to remain engrafted, we
examined the bone marrow cells of leukemic mice by flow
cytometric analysis, using human-specific antibodies to leukemic
(CD15) and endothelial (VEGFR-2) markers. Twenty one days
after the start of the experiment, there were large numbers of
human-CD15?, VEGFR-2?cells in the control group treated
with human-IgG (Fig. 3, human CD15?, 7% of total bone
marrow cells; VEGFR-2?, 5% of bone marrow cells). On the
other hand, in IMC-1C11, DC101- or IMC-1C11 ? DC101-
treated mice, at the same time point, the number of leukemic
cells in the bone marrow was significantly reduced, with human
CD15?VEGFR-2?cells comprising less than 1% of the total
bone marrow cells. Similar results were obtained in peripheral
blood samples from the same mice (data not shown). In addition,
to confirm that leukemia engraftment increases bone marrow
of VEGFR-2?(Flk-1?) murine EC. Twenty one days after the
start of the experiment, the percentage of murine VEGFR-2?
mice (baseline levels), to over 1.4% in human IgG-treated,
leukemia-inoculated mice (Fig. 4). This result correlated with
the bone marrow histology (Fig. 2A) and suggests that bone
marrow angiogenesis increases after inoculation with leukemic
cells. The percentage of murine VEGFR-2?cells in bone
marrow aspirates was reduced to baseline levels in IMC-1C11-,
DC101-, and IMC-1C11 ? DC101-treated mice (Fig. 4). Similar
results were obtained in mice treated with IMC-1C11 alone (up
to day 60) or the combination of IMC-1C11 ? DC101 up to day
120 (data not shown).
(noninoculated) and HL-60 inoculated NOD-SCID mice. HL-60-injected mice
had a 2-fold increase in the number of Flk-1-positive cells, compared with
normal mice, reflecting an increase in bone marrow angiogenesis after leu-
kemia inoculation. DC101-, IMC-1C11-, and DC101 ? IMC-1C11-treated mice
had a significantly lower percentage of human cells in the bone marrow than
untreated mice (P ? 0.001).
Leukemic cell engraftment (%) in the bone marrow of normal
Dias et al.PNAS ?
September 11, 2001 ?
vol. 98 ?
no. 19 ?
Discussion Download full-text
It has been recently shown in murine models of human solid
tumors that combining antiangiogenesis agents, such as the mAb
to VEGFR-2 (DC101), with standard chemotherapy or radio-
therapy increases therapeutic efficacy (12, 13). In a separate
study, metronomic administration of chemotherapeutic drugs to
mice bearing different tumors was shown to be more effective
than bulk chemotherapy, because it produced antitumor as well
as antiangiogenesis effects (14). These studies highlighted the
importance of the autocrine (targeting tumor cells) and para-
crine (targeting tumor angiogenesis) components of tumor
growth, but have not identified any particular molecule(s) or
factor(s) involved in this processes. In addition, thus far a clear
demonstration that angiogenesis also plays an important role in
the progression of liquid tumors such as acute leukemias has not
In the present report, we have shown, using an in vivo model
of human leukemia, that blocking angiogenesis induced by the
interaction of leukemia-derived VEGF with murine VEGFR-2
delays leukemic growth, but is not sufficient for its eradication
from inoculated mice. These results demonstrate that targeting
VEGF-induced angiogenesis may be at least partially effective at
delaying leukemia progression. In addition, these in vivo results
correlate with the ability of endothelial layers to support leu-
kemia proliferation in the absence of any exogenous growth
factors in vitro. The generation of such endothelial-leukemia
paracrine loops may be of critical importance for the establish-
ment of the disease. On the other hand, earlier we showed that
subsets of leukemias survive and proliferate in serum-free
conditions, in an endothelial-independent manner, because of
the existence of a VEGF?VEGFR-2 autocrine loop (8).
In the present study, we evaluated the relative contribution of
paracrine (endothelial-dependent) and autocrine (endothelial-
independent) loops in supporting leukemic growth and engraft-
ment in vivo. Notably, long-term remission with no evidence of
the disease could be achieved only if mice were treated with
neutralizing mAb against murine and human VEGFR-2, block-
ing the paracrine and autocrine VEGF?VEGFR-2 signaling
pathways. On the other hand, mAbs against murine or human
VEGFR-1 had no effect toward improving the survival of
leukemic mice, suggesting that the VEGF?VEGFR-2 pathway is
more important for the proliferation and engraftment of acute
leukemias in vivo. However, it is also possible that certain
leukemias may depend on VEGFR-1 signalling.
As suggested for solid tumors, our data suggest that thera-
chemotherapy, may be insufficient to completely eradicate a
proliferating leukemic mass. On the other hand, antiangiogen-
esis therapies target only tumor-induced neo-vascularization,
and a number of studies already have shown that this strategy
results in delayed growth of solid tumor cell lines implanted into
mice (15, 16). However, based on evidence shown here, antian-
giogenesis therapy targeting the paracrine loop alone may be
insufficient to completely eradicate proliferating, VEGF-
producing and VEGFR-expressing leukemias. Interestingly,
most solid tumors produce VEGF, and subsets of solid tumor
cells express at least one of its receptors (17–19), suggesting that
the observations described above may apply to other tumors as
Emerging data suggest that VEGF production by leukemia
cells may reflect their ability to engraft into mice in vivo (20).
Based on the results shown in the present report, we hypothesize
that VEGF production and VEGFR coexpression by leukemic
cells may identify a leukemic clone with the capacity to engraft
and proliferate in vivo. VEGF production has been correlated
with disease progression in patients with a variety of hemato-
logical malignancies (3–6), but the expression of VEGFRs on
it would be of interest to identify other proangiogenic or
hematopoietic cytokines that regulate VEGFR expression on
the leukemic cells. Also, VEGFR expression by leukemias may
correlate with leukemia progression or resistance to conven-
tional therapies. Effective therapeutic strategies against rapidly
proliferating acute leukemias may therefore be required to
regulate VEGF production and VEGFR expression by the
expanding leukemic clone.
Taken together, the data presented here, underscoring the
significance of autocrine and paracrine?angiogenic pathways,
may lay the foundation for a different therapeutic approach for
the treatment of VEGF-producing, VEGFR-expressing tumors,
in particular acute leukemias. In addition, it also introduces
additional paradigms in tumor angiogenesis that will facilitate
tailoring future clinical anti-angiogenesis trials based on expres-
sion pattern of VEGFR-1 or VEGFR-2.
S.R. is supported by a Translational Research Award from The Leuke-
mia and Lymphoma Society, National Heart, Lung, and Blood Institute
Grants R01 HL-58707 and R01 HL-61849, Research Scholar Grant from
American Cancer Society (RSG-01-091-01), the Dorothy Rodbell Foun-
dation for Sarcoma Research, and the Lupin Foundation. M.A.S.M. is
supported by National Heart, Lung, and Blood Institute Grant R01 HL-
61401 and the Gar Reichman Fund of Cancer Research Institute. B.H.
is the recipient of a fellowship from the Mildred Scheel Stiftung fur
Krebsforschung (Bonn, Germany). K.H. is the recipient of a fellowship
from the Uehara Memorial Foundation (Tokyo).
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