Human chloromas express VEGF and VEGF receptors.
Human leukemias are not only localized to the bone marrow and peripheral circulation but may also infiltrate tissues and form solid masses referred to as chloromas.
Immunohistochemical staining of human chloromas with antibodies specific for VEGFR-1 or VEGFR-2 revealed that, besides staining the endothelial lining of blood vessels, these receptors were also expressed by a subset of the leukemic cells (Figure , a and b). The staining localized to the cell membrane, and positive cells appeared scattered throughout the sections (Figure , c and d). Overall, there were more VEGFR-2–positive than VEGFR-1–positive areas in the sections analyzed. These receptors were primarily detected in different areas of the tumor, suggesting that the staining pattern of VEGFR-1 and VEGFR-2 may identify different cell populations. Furthermore, using a double immunostaining technique, it was shown that, on the sections analyzed, VEGFR-2–positive leukemic cells also stained for VEGF (Figure , e and f). Importantly, stromal cells did not stain for VEGFR-2 but expressed VEGF (Figure e), whereas endothelium shows a reverse staining pattern, staining for VEGFR-2 but not for VEGF (Figure f).
Figure 1 Human chloromas express FLT-1/VEGFR-1 and KDR/VEGFR-2. Sections from human chloromas were stained by immunohistochemistry, as described in Methods. (a) Control IgG, showing little nonspecific staining (×400); (b) factor VIII staining, showing (more ...) Primary leukemias and leukemic cell lines produce VEGF and express VEGF receptors.
Three leukemic cell lines and ten primary leukemias were analyzed for the production of VEGF and for the expression of VEGFR-2 by ELISA and RT-PCR, respectively.
All leukemic cells described in this study produced variable amounts of VEGF in vitro, as determined by ELISA (Table ). However, only 50% of primary leukemias and two of three cell lines expressed VEGFR-2 at the mRNA level (Figure and Table ). All VEGFR-2–positive leukemias were also positive for VEGFR-1 (Table ).
VEGF production and VEGF receptor expression of three leukemic cell lines (HL-60, HEL, and K562) and ten primary leukemia samples (samples 1–10)
Representative RT-PCR analysis for VEGFR-2 and VEGFR-1, on leukemic cell lines. The cell lines HL-60 and HEL express VEGFR-1 and VEGFR-2, as detected by RT-PCR. K562 cells were VEGFR-2 negative. β-actin was used as an internal control.
Leukemic cells express functional VEGF receptors.
VEGF induced receptor phosphorylation on leukemic cells, as demonstrated by immunoprecipitation and Western blotting, confirming that these are functional, signaling receptors. VEGF165 induced, in a dose-dependent manner (20–50 ng/mL), an increase in VEGFR-1 and VEGFR-2 phosphorylation on VEGF receptor–positive leukemic cell lines and primary leukemias, but not on fibroblasts or VEGFR-negative leukemic cells (Figure ). In the absence of exogenous VEGF, leukemic cells had baseline VEGFR-1 and VEGFR-2 phosphorylation (Figure ), which may be due to the production of VEGF and expression of its receptors by the same cells.
Figure 3 VEGF165 induced dose-dependent VEGFR-2 phosphorylation on HL-60 and HEL cells and primary leukemias 1, 2, and 3. Receptor tyrosine phosphorylation was done as described in Methods. Fibroblasts were used as negative controls and showed no sign of VEGFR-2 (more ...) VEGF mediates leukemic cell proliferation in vitro in an autocrine manner.
In the absence of exogenous growth factors (serum-free conditions), leukemic cells show a modest increase in proliferation over a period of 2–3 days (Figure a). IMC-1C11 (1 μg/mL) blocked proliferation of VEGFR-2–positive leukemias, such as HL-60, HEL, and primary samples 1 and 2, by 40–60% after 48–72 hours, but had no effect on VEGFR-2–negative cells such as K562 and primary samples 6 and 7 (P < 0.01; Figure a). mAb to VEGFR-1 (clone 6.12), used at the same concentration, had no effect on leukemic cell proliferation (data not shown). These data show that under serum-free conditions, on VEGF-producing, VEGFR-2–positive leukemias, blockade of VEGFR-2 signaling decreases leukemic cell proliferation.
Figure 4 (a) Leukemic cell growth in serum-free media. Treatment of VEGFR-2–positive cells (HL-60, HEL, and primary samples 1 and 2), but not of VEGFR-2–negative cells (K562 and samples 6 and 7), with IMC-1C11 decreases cell growth (AP < (more ...) VEGF induces leukemic cell proliferation, an effect mediated through VEGFR-2.
Addition of exogenous VEGF165 to leukemic cell cultures increased the proliferation of VEGFR-2–positive leukemias and leukemic cell lines (Figure b). IMC-1C11, used at 1 μg/mL, blocked this increase in proliferation (Figure b; P < 0.05). mAb to VEGFR-1 (used at the same concentrations) had no effect on VEGF165-induced leukemic cell proliferation (data not shown), suggesting that on these cells the mitogenic effects of VEGF165 are mediated mainly through VEGFR-2. Addition of exogenous VEGF to VEGFR-2–negative cells, such as the K562 cell line, had no effect on cell proliferation (Figure b). Similar results were seen with the VEGFR-2–negative primary leukemic samples 6–10 (data not shown).
VEGF induces MMP secretion/production by leukemic cells.
Without stimulation, in serum-free conditions, leukemic cells release variable amounts of MMPs into the cell culture supernatant (Figure ). This suggests that MMP production by leukemic cells may identify a leukemic subtype or subpopulation with a more invasive phenotype. On primary leukemias and/or cell lines investigated, the myelo-monocytic subtypes (shown in Figure : HL-60 cells, samples 2 and 3) have consistently shown a higher level of basal MMP production and release. Incubation with VEGF165 increased MMP-9 release by leukemic cells (Figure a) over an 18-hour period. Quantification of VEGF165-induced increases in secreted MMP-9, by densitometry, indicated that this effect was significant (Figure b). On primary leukemias, MMP-9 was the main MMP released into the culture supernatants (Figure ). The levels of TIMP-1 produced by leukemic cells were also investigated, by Western blotting, but showed only a minor variation after stimulation with VEGF165 (data not shown).
Figure 5 VEGF165 induces MMP secretion by leukemic cells. (a) Zymographic analysis of leukemic cell supernatants, with or without VEGF165 stimulation for 18 hours. (b) Quantification of the gelatinolytic activity detected on the culture supernatants. Incubation (more ...) VEGF induces leukemic cell migration through Matrigel-coated transwells, an effect mediated through VEGFR-1 and VEGFR-2.
We investigated whether VEGF165 induced a more invasive phenotype on leukemic cells. This was demonstrated using a migration system in which transwell inserts were coated with a thin layer of Matrigel, a model of invasion through the basement membrane. VEGF165 induced trans-basement membrane migration of HL-60 cells and primary leukemias (Figure ). This process requires MMP production and activation, as the VEGF-induced cell migration was blocked by recombinant human TIMP-1 (Figure ). TIMP-1 had no effect on leukemic cell migration through bare (uncoated) transwells (data not shown).
Figure 6 VEGF165 induces leukemic cell migration through Matrigel-coated transwells, a process mediated through VEGFR-2 and VEGFR-1. Migration of HL-60 cells and four primary leukemias through Matrigel-coated transwells, in response to 200 ng/mL VEGF165, is shown. (more ...)
As shown here, the mitogenic effects of VEGF165 on leukemic cells were mediated through VEGFR-2. However, in the migration system used in our studies, incubation of HL-60 cells with IMC-1C11 (at 1 μg/mL) could only partially (40%) block VEGF165-induced migration through Matrigel (Figure ). On the other hand, an mAb to VEGFR-1, used at the same concentration, significantly blocked (60–70%) HL-60 cell migration (Figure ; P < 0.05), suggesting that VEGF165 may induce MMP activation and cell migration by interacting with both receptors. In fact, incubation of HL-60 cells with VEGFR-1 mAb and IMC-1C11 blocked VEGF165-induced migration more effectively (70–80%) than either antibody alone (Figure ). On primary leukemias, both VEGFR-1 mAb and IMC-1C11 showed, overall, comparable migration-blocking effects (Figure ). As shown for the HL-60 cells, incubation of primary leukemias with both antibodies was more effective than either antibody alone, blocking VEGF165-induced migration by 80% (Figure ).
Leukemic cells release VEGF in vivo.
Using NOD-SCID mice as an in vivo model, we investigated whether VEGFR-2–positive leukemias, HL-60, HEL, and a primary leukemia, released VEGF in vivo. As shown in Figure a, human VEGF plasma levels increased 7 and 14 days after HL-60 cell injection. Importantly, murine VEGF plasma levels remained very low (at or below the assay detection level; Figure a) throughout the experiment. Similar results were obtained using the HEL cell line (data not shown) and a primary leukemia sample (Figure b). If left untreated, mice injected with this primary leukemia had increased circulating human VEGF levels 14 days after inoculation (Figure b). However, similarly to the HL-60 model, murine VEGF plasma levels did not increase in these mice (data not shown).
Figure 7 (a) HL-60 injection into NOD-SCID mice induces high levels of human but not murine VEGF in mouse plasma. Mice were injected with 3 × 106 HL-60 cells (intravenously), and the human and murine VEGF plasma levels were determined at different time (more ...) IMC-1C11 blocks leukemic growth in vivo.
In the absence of any treatment, or treated with unrelated human IgG, mice injected with 3 × 106 HL-60 cells (intravenously) survived only 14–25 days (n = 8 for each treatment), highlighting the aggressive nature of these leukemic cells. However, mice treated with IMC-1C11 survived five times longer than untreated mice (Figure a; P < 0.005) and, as shown in Figure a, had reduced human VEGF plasma levels throughout the experiment. Similarly, untreated HEL-inoculated mice died within 45 days, whereas those treated with IMC-1C11 survived significantly longer (P < 0.05; Figure b).
Figure 8 (a) Mouse survival (%) after intravenous HL-60 injection. Mice were injected with 3 × 106 cells intravenously, and 4 days after leukemic cell injection they were left untreated (control, n = 8), or treated (n = 8) intraperitoneally (more ...)
These observations were extended to include a primary leukemia. In the absence of any treatment, mice inoculated with 5 × 106 primary leukemic cells died within 3 weeks. However, as shown for the two leukemic cell lines, IMC-1C11 prolonged the survival of primary leukemia-bearing mice (Figure c). This increase in survival was accompanied by a reduction in human VEGF plasma levels (Figure b). Given that IMC-1C11 is specific for human VEGFR-2 (KDR) and does not cross react with murine flk-1, the data suggest that treatment of inoculated mice with IMC-1C11 blocks leukemic growth by impeding leukemia-derived VEGF from interacting with VEGFR-2 on leukemic cells.
Decreased metastasis in mice treated with IMC-1C11.
As shown in vitro, VEGF can promote an invasive phenotype on VEGFR-2–positive leukemic cells, which may be one mechanism by which extramedullary leukemic masses are established. Using the in vivo models described here, it was next investigated whether VEGFR-2 blockade affected metastatic behavior.
In untreated HL-60 inoculated mice, histological analysis of the liver and spleen revealed the presence of metastatic lesions in both organs (Figure , b and d). Liver sections had several areas occupied by leukemic cells, with signs of active cellular proliferation (Figure b). Furthermore, the white pulp area of the spleen of these mice was largely replaced by leukemic cells (Figure d). However, 14 days after start of the experiment, IMC-1C11–treated mice had normal liver and spleen histology (Figure , a and c). In these mice, there was no evidence of metastatic lesions in the liver (Figure a), and the spleen appeared normal, with evidence of apoptosis (Figure c). Mice inoculated with the HEL cell line showed similar results. In control mice, 15 days after the start of the experiment, there were numerous metastatic foci in the spleen and lungs (more than three metastatic foci seen at 10× magnification). However, mice treated with IMC-1C11 had no evidence of metastasis (data not shown).
Figure 9 Histology of liver (a and b) and spleen (c and d) from HL-60–injected mice. Untreated (control) mice had evidence of metastatic disease in the liver (white and red arrows, b) and spleen (d). In contrast, mice treated with IMC-1C11 showed no evidence (more ...) Decreased circulating leukemic cells and reduced bone marrow leukemic cell engraftment in mice treated with IMC-1C11.
In HL-60–injected mice, untreated or treated with unrelated human IgG, 15 days after the start of the experiment, there were 6% human CD15-positive cells in the bone marrow, of which approximately 4% were VEGFR-2 positive (Figure a). However, mice treated with IMC-1C11 had tenfold less human cells in the bone marrow (Figure a). Similar results were obtained with the HEL leukemia cell line (Figure a). These cells, contrarily to HL-60, do not express CD15, but express VEGFR-2/KDR. As shown in Figure a, 15 days after the start of the experiment, there were tenfold more VEGFR-2–positive HEL cells in the bone marrow of untreated mice compared with those treated with IMC-1C11 (P < 0.005).
Figure 10 (a) Leukemic cell engraftment (%) in the bone marrow of HL-60– and HEL-inoculated mice. Day 15 after the start of the experiment, bone marrow cells from inoculated mice were stained for human CD15 and human VEGFR-2/KDR and quantified by (more ...)
We also quantified the number of circulating leukemic cells in mice inoculated with a primary leukemia. Fourteen days after the start of the experiment, untreated mice had 12% circulating CD45+VEGFR-2+ leukemic cells (Figure b). However, those treated with IMC-1C11, at the same time point, had less than 1% circulating leukemic cells (Figure b). Similar results were obtained with the two cell lines HL-60 and HEL (data not shown).