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Tumor Biology |
Divisions of Hematology-Oncology [L. F., C. R., F. P., G. M., F. B.], Experimental Oncology [A. G.], and Pathology [G. P.], IRCCS European Institute of Oncology, 20141 Milan, Italy, and Second Division of Pathology, IRCCS Maggiore Hospital, 20100 Milan, Italy [N. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clinical and laboratory studies have already generated robust evidence indicating that angiogenesis supports solid tumor viability and growth (5) . More recent studies have suggested that angiogenesis may play a crucial role also in ALL (6) , myelodysplastic syndromes (7) , lymphoma (8) , and myeloma (9) . Furthermore, albeit indirect, evidence of angiogenesis in human hematopoietic malignancies comes from the observation that VEGF is expressed by some AML cells and leukemic cell lines and possibly acts as a paracrine growth factor in AML development (10 , 11) . VEGF is currently considered the most relevant and the most endothelium specific of the already known angiogenic growth factors, and it appears to be involved in the vascular phase of many different neoplastic diseases (5 , 12) . Moreover, tumor-derived VEGF might inhibit dendritic cell maturation and facilitate tumor evasion from immune control (13 , 14) . Because Tokunaga et al. (15) reported recently that xenotransplantability of human colon cancers in SCID mice seems to be affected by VEGF expression, we were interested in evaluating the impact of VEGF in xenotransplantability of human hematological malignancies and related angiogenesis. In parallel, the role of the other key angiogenic regulatory factor bFGF (5 , 6) was similarly investigated.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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U-937, K-562, QD1-EIO, BC1-EIO, Jurkat, CEM, MOLT-4, Karpas 299, Namalwa, HS Sultan, L-363, and RAP1-EIO cells were seeded at 300 x 103/ml in RPMI-10% FBS. In vitro VEGF and bFGF production were evaluated after 3-day culture by means of commercial ELISA kits (R&D, Abingdon, United Kingdom) as we described previously (16) . Analyses and calibrations were carried out in duplicate; values of intra- and interassay variation were within the range given by the manufacturers, i.e., 3–6% and 4–9%, respectively.
Detection of bFGF, VEGF, and Related Receptors by RT-PCR.
Total RNA isolated from growing cells was treated with a reverse
transcriptase enzyme (SuperScript II; Life Technologies, Inc.,
Gaithersburg, MD). The cDNA generated following this approach was
amplified by PCR using Taq DNA polymerase (Life Technologies, Inc.).
Molecular expression of bFGF, VEGF, and related receptors KDR and Flt-1
was evaluated by already described primers (10
, 11)
. VEGF
primers designed by Fiedler et al. (10)
corresponded to sequences in the untranslated 5' and 3' region,
resulting in amplification of four different splice variants of a size
of 516, 648, 720, and 771 bp. VEGF primers designed by Bellamy et
al. (11)
were able to span intron-exon borders to
distinguish amplified cDNA from genomic DNA and depicted VEGF 121 (408
bp) and VEGF 165 (541 bp). We used primers specific for KDR (two
rounds), Flt-1 (two rounds), and actin (one round, positive control) as
described by Fiedler et al. (10)
. Related PCR
products sizes were, for the KDR outer primer pair, 591 bp, and for the
KDR inner primer pair, 213 bp; for the Flt-1 outer primer pair, 555 bp,
and for the Flt-1 inner primer pair, 196 bp; and for actin, 619 bp.
RT-PCR analysis of bFGF transcripts was performed by means of primers
described by Bellamy et al. (11)
, which
generated 237-bp transcripts. PCR-amplified products were stained with
ethidium bromide and evaluated by 2% agarose-gel electrophoresis.
Appropriate control reactions always remained negative, and RNA
isolated from HUVECs was used as a positive control for KDR and Flt-1.
Quantitative Expression of VEGF Receptors.
We used the Fluorokine kit (R&D) to determine quantitatively the
percentage of cells expressing VEGF receptors. Briefly, 500 x 103 cells were washed twice in PBS to
remove any residual growth factor from the culture medium and incubated
with biotinylated VEGF. As a control, some cells were stained with
biotinylated negative reagent provided by the manufacturer. Cells were
then directly incubated with avidin-FITC. Cells expressing VEGF
receptors were fluorescently stained, with the intensity of staining
proportional to the density of the receptors. Relative receptor density
was then determined by FC using a FACScalibur (Becton Dickinson,
Mountain View, CA). According to the manufacturer’s suggestions, we
confirmed VEGF specificity by observing a reduction of signal intensity
when the staining reaction was carried out in the presence of a
blocking anti-VEGF antibody (R&D).
In Vitro Inhibition of Cell Proliferation in the
Presence of a VEGF Antagonist.
A human Flt-1/Fc chimeric protein binding VEGF with high affinity and
known to inhibit VEGF-dependent HUVEC proliferation (17)
was added to cell cultures at concentrations ranging from 0.5 to 2
µg/ml. After 5-h culture at 37°C, the extent of cell proliferation
was evaluated by means of a standard
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
(Sigma Chemical Co., St. Louis, MO).
Animal Studies.
NOD/SCID mice, 6–8 weeks of age, received injections i.p. with
10 x 106 cells and were
evaluated daily for a maximum of 120 days for tumor growth.
Tumor-bearing mice were sacrificed by CO2
inhalation, and solid tumor, ascites fluid, PB, and BM studied by FC
and IHC. All procedures involving animals were done in accordance with
national and international laws and policies. Human tumor engraftment
was confirmed by FC and IHC evaluation of the cell phenotype
(e.g., human CD13, CD15, and CD45 for the U-937 cell line).
Angiogenesis in solid tumors and BM was evaluated by means of
monoclonal antibodies against human and murine vWF (clone 4F9; Coulter,
Miami, FL), murine CD31 (clone MEC 13.3; PharMingen, San Diego, CA),
and murine CD34 (clone RAM34; PharMingen). MVD was evaluated by light
microscopy as described previously (7)
. Briefly, for each
case, one to three H&E-stained slides were evaluated at low
magnification (x40 and x100) to detect the area with the highest MVD
(hot spot). Three microscopic fields were then examined in this area at
x250 magnification (each field representing an area of 0.72
mm2), and the mean MVD value was recorded. Any
endothelial cell or endothelial cell cluster that was clearly separated
from adjacent microvessels was considered a single, countable
microvessel. In FC studies, 100–500 x 103 cells were incubated at 22°C for 30 min in
PBS-1% BSA with monoclonal antibodies. By means of FACScalibur (Becton
Dickinson), the percentage of stained cells was determined as compared
with phycoerythrin- or FITC-conjugated isotypic control. A portion of
each sample was incubated with the appropriate isotype control
antibodies to establish the background level of nonspecific staining,
and positivity was defined as being greater than nonspecific background
staining. According to Philpott et al. (18)
,
7AAD was used in FC evaluations to depict apoptotic and dead cells.
Endothelial cells were enumerated as vWF+/7AAD- and as
CD31+/CD34+/7AAD- cells. Mice without visible signs of engraftment
were sacrificed within day 120. In these negative mice, necropsy was
performed to further investigate, by means of FC and IHC, the presence
of solid tumors and/or human cells in the PB and BM.
Statistical Analysis.
Statistical comparisons were performed using the t test and
ANOVA, when data were normally distributed, and the nonparametric
analyses of Spearman and Mann-Whitney when data were not normally
distributed. Values of P < 0.05 were
considered as statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Table 1
, VEGF mRNA expression was found in 11 of 12 lines, Flt-1 was found in
12 of 12 lines, and KDR was found in 2 of 12 lines. VEGF primers
designed by Fiedler et al. (10)
resulted in
amplification of the four different splice variants in K-562 cells.
Other lines expressed one to three different variants. VEGF primers
designed by Bellamy et al. (11)
depicted both
VEGF 121 and 165 in all VEGF-expressing lines. In contrast to VEGF,
bFGF mRNA was found in 1 of 12 lines. Similarly, ELISA quantification
of in vitro VEGF and bFGF production during cell culture
indicated that 10 of 12 and 2 of 12 lines produced an amount of VEGF
and bFGF above the detection threshold of our methods (15 and 10 pg/ml,
respectively). As well as in previous studies (10
, 11)
,
VEGF and related receptors were found to be expressed in most
hematopoietic malignancies. Accordingly, this growth factor may play a
significant role in leukemia and lymphoma pathogenesis.
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indicated that in all of the 12 tested lines, >90% cells were able
to bind VEGF. Quantitative differences in the expression of VEGF
receptors were not significant. Cell culture studies in the presence of
2 µg/ml of the Flt-1/Fc chimera (known to inhibit VEGF-dependent
HUVEC proliferation) indicated that VEGF deprivation from the culture
medium was associated with 5–44% inhibition of cell proliferation
(Fig. 3, A and B)
. Interestingly, a strong inverse
correlation was found between VEGF production and the extent of
inhibition of cell proliferation (r = -0.772; P = 0.008; Fig. 3C
).
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, 10 of 12 evaluated cell lines were able to engraft NOD/SCID mice,
whereas engraftment was not observed in recipient of BC1-EIO and Jurkat
cells. Engraftment failure was defined as the absence of solid tumors,
ascites, or human cells in the PB or BM. The efficiency of engraftment
ranged from 100% (mice transplanted with Karpas 299, Namalwa, HS
Sultan, and L-363 cells) to 11% (1 of 9 mice transplanted with K-562
cells). K-562, CEM, MOLT-4, Karpas 299, Namalwa, HS sultan, L-363, and
RAP1-EIO cells engrafted as i.p. solid tumors in the abdomen in a
median of 24, 42, 60, 24, 18, 29, 32, and 20 days, respectively. U-937
cells engrafted in a median of 18 days as solid tumors in 9 of 18
animals and as ascites in the remaining cases. A mean of 2 ± 1% U-937 cells were found in the PB and BM of engrafted
animals. QD1-EIO cells were found in the PB, BM, spleen, pancreas, and
liver of 5 of 10 transplanted animals in a median of 63 days, with
human QD1-EIO cells representing 30–65% of all BM cells in engrafted
animals.
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indicated that vWF+/7AAD- and
CD31+/CD34+/7AAD- cells ranged from 0.4 to 6% of all cells. The
correlation between the frequency of vWF+/7AAD- and of
CD31+/CD34+/7AAD- cells was significant (r = 0.697; P = 0.03). A slightly weaker
correlation was found between MVD and vWF+/7AAD- cells
(r = 0.596) and between MVD and
CD31+/CD34+/7AAD- cells (r = 0.487).
According to FC and MVD data, the extent of solid tumor angiogenesis
was significantly reduced in mice engrafted with MOLT-4 cells (Table 2
and Fig. 4
; P < 0.05). Similarly, both 7AAD staining
(Fig. 5)
and microscopy evaluation of apoptotic cells (Fig. 6, a and b)
indicated that the frequency of
apoptotic/dead cells in solid tumors was higher in mice engrafted with
CEM and MOLT-4 cells (P < 0.01).
Interestingly, these cell lines produced in vitro
significantly less VEGF when compared with other lines able to generate
solid tumors in NOD/SCID mice. A significant increase of both vWF+ and
CD31+/CD34+ cells was found in the BM of mice transplanted with U-937
cells (Fig. 4
, P < 0.05), i.e.,
the cell line producing the higher amount of VEGF, whereas both vWF+
and CD31+/CD34+ BM cells were not significantly increased in mice
engrafted with other cell lines. In fact, in 10 untreated NOD/SCID mice
evaluated as controls, vWF+ and CD31+/CD34+ BM cells were found to be
4 ± 2% and 4 ± 1%, respectively.
Interestingly, vessels in either U-937 and control mice had a
sinusoid-like morphology (Fig. 6c)
, whereas BM vessels in
mice transplanted with QD1-EIO cells infiltrating the BM environment
showed a sprouts-like morphology (Fig. 6d)
. It should be
noted that the latter vessel morphology was similar to the predominant
one we observed in BM biopsies from AML patients (7)
. FC
and IHC evaluation indicated that in engrafted as well as in
non-engrafted mice all of the endothelial cells were of murine origin.
Interestingly, the efficiency of engraftment, frequency of vWF+ and
CD31+/CD34+ cells, and MVD were similar in animals transplanted with
RAP1-EIO and QD1-EIO cells lines and in animals transplanted with
primary lymphoma and leukemia cells from the same patients (Table 2)
.
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), and a similarly significant, albeit inverse, correlation
was found between VEGF in vitro production and the median
time of tumor engraftment (r = -0.792;
P = 0.006; Fig. 7B
) and between
VEGF production and the frequency of apoptotic/dead cells in solid
tumors (r = -0.892; P = 0.007; Fig. 7C
). Along this line, VEGF production
correlated with the frequency of endothelial (CD31+/CD34+) cells in
solid tumors (r = 0.897;
P = 0.001; Fig. 7d
). As reported
in Table 3
, a significant correlation was also found between the
frequency of CD31+/CD34+ cells in solid tumors and the efficiency of
engraftment (r = 0.780;
P = 0.013). Furthermore, a significant
inverse correlation was found between the frequency of CD31+/CD34+
cells in solid tumors, the time of engraftment (r = -0.728; P = 0.025) and the frequency
of apoptotic/dead cells (r = -0.800;
P = 0.030), and between MVD and the frequency
of apoptotic/dead cells (r = -0.811;
P = 0.049). A correlation of borderline
significance (r = -0.512;
P = 0.088) was observed between the
expression of CD34 and CD117 antigens (found previously in normal
hematopoietic cells and AML progenitors with NOD/SCID engraftment
potential; Ref. 1
) and the efficiency of engraftment.
Conversely, a number of other variables including bFGF production, cell
line doubling time, c-myc, KDR, and EBV genome expression
did not correlate with the efficiency of engraftment, the speed of
engraftment, and the frequency of apoptotic/dead cells in solid tumors ( Table 3
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Data collected in the present study indicate that the NOD/SCID mouse model is useful to evaluate the xenotransplantability of a wide spectrum of hematological malignancies and possibly to ascertain crucial steps in leukemia and lymphoma growth as solid tumors or disseminated diseases. Along this line, our novel finding that in vitro VEGF production correlates with tumor engraftment efficiency, speed of engraftment, frequency of apoptotic/dead cells, and endothelial cells in solid tumors supports a possible role for tumor-derived VEGF in leukemia and lymphoma progression. In fact, in our series of hematological malignancies, the prognostic significance of other variables including bFGF, c-myc, KDR, and EBV genome expression and cell line doubling time was markedly less relevant than that of VEGF production. Interestingly, our data confirm previous observations indicating that VEGF expression may play a crucial role in xenotransplantability of human colon cancer in SCID mice (15) .
Either tumor, endothelial, or stromal VEGF expression have been hypothesized to be critical for tumor angiogenesis. Fukumura et al. (19) have recently described transgenic mice expressing the GFP under the control of the promoter for VEGF. In these mice, spontaneous tumors induced by oncogene expression show strong stromal GFP expression. Conversely, GFP tumor expression was negligible, thus suggesting that the VEGF promoter might be activated by the tumor microenvironment. On the other hand, it must be noted that fibrosarcomas generated recently by Grunstein et al. (20) upon immortalization and H-ras transformation of VEGF-null murine fibroblasts showed dramatic decreases in vascular density and permeability and increases in tumor cell apoptosis. These and our data suggest that, at least in the fibrosarcoma and possibly in the leukemia/lymphoma murine models, tumorigenic VEGF expression may be a critical factor in tumor expansion and related angiogenesis. In fact, two recent papers have suggested that VEGF may be necessary (although not sufficient per se) for tumorigenicity of colorectal carcinoma (21) and melanoma (22) cells, and another study has described that VEGF, together with angiopoietins, play a cardinal role in the cooption and growth of tumor vessels (23) .
In this study, we used for the first time FC to evaluate angiogenesis in animal models of human malignancies. This assay is expected to generate quantitative data on the frequency of endothelial cells, whereas the measurement of MVD indicates the frequency of blood vessels. The strong correlation found between VEGF in vitro production and frequency of CD31+/CD34+ cells in solid tumors suggests that FC is promising for further quantitative studies on angiogenesis. Both FC and MVD studies indicated that angiogenesis was significantly reduced, albeit still present, in mice transplanted with MOLT-4 cells. It should be noted that this cell line, which produced very low amounts of VEGF, had a poor engraftment potential. Moreover, an increased frequency of apoptotic/dead cells was found in MOLT-4 tumors.
Another intriguing facet of VEGF-driven angiogenesis in hematological neoplastic diseases is the recent finding that VEGF-stimulated endothelial cells generate stem cell factor (11) , Flt3-ligand (24) , granulocyte/macrophage-colony stimulating factor (10) , and interleukin 6 (11) . These cytokines, in turn, may act as growth factors for myeloid and lymphoid malignant cells, thus suggesting possible paracrine machinery between hematopoietic malignant cells and newly generated endothelium. Concerning our animal model, it should be considered that some of these growth factors have cross-species activity (25) . For this reason, cytokine production by the mouse endothelial cells may be relevant in this paracrine loop.
Finally, recent data indicate that antiangiogenic therapy is highly promising in different experimental neoplastic diseases (5 , 26) . Thus, the NOD/SCID model of hematological malignancies may demonstrate to be a useful tool to study antiangiogenic drugs, alone or in combination with established chemo- or immunotherapy regimens. Along this line, we are currently evaluating in this model whether anti-VEGF therapies may be of help in inducing the regression of new tumor vessels and consequent tumor dormancy or stabilization (27) in leukemia and lymphoma animal models. In fact, some authors have indicated that reduction of VEGF levels by monoclonal antibodies (28 , 29) or drug-mediated inhibition of VEGF-related receptor KDR (30) may have therapeutic potential in animal models of other human malignancies. Moreover, we are also evaluating whether in this in vivo model tumor-derived VEGF effectively disturbs murine dendritic cell maturation and tumor immune control as suggested previously (13 , 14) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Hematology-Oncology Unit, European Institute of Oncology,
via Ripamonti 435, 20141 Milan, Italy. Phone: 39-02-57489535; Fax:
39-02-57489537; E-mail: francesco.bertolini{at}ieo.it 
2 The abbreviations used are: SCID, severe
combined immunodeficiency; NOD/SCID, non-obese diabetic/SCID; VEGF,
vascular endothelial growth factor; ALL, acute lymphocytic leukemia;
AML, acute myeloid leukemia; bFGF, basic fibroblast growth factor;
GlyA, glycophorin A; RT-PCR, reverse transcription-PCR; HUVEC, human
umbilical vein endothelial cell; BM, bone marrow; PB, peripheral blood;
FC, flow cytometry; PC, plasma cell; IHC, immunohistochemistry; vWF,
von Willebrand factor; MVD, microvessel density; 7AAD,
7-aminoactinomycin D; CML, chronic myeloid leukemia; GFP, green
fluorescent protein.
Received 8/12/99. Accepted 3/ 6/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, glucocorticoids, IFN-, MIP-1 and TGFß. Key role in the proliferation of primitive hematopoietic progenitors. Blood, 92: 580a 1998.
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