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Tumor Biology

Human Myeloid and Lymphoid Malignancies in the Non-Obese Diabetic/Severe Combined Immunodeficiency Mouse Model: Frequency of Apoptotic Cells in Solid Tumors and Efficiency and Speed of Engraftment Correlate with Vascular Endothelial Growth Factor Production

Lisa Fusetti, Giancarlo Pruneri, Alberto Gobbi, Cristina Rabascio, Nadia Carboni, Fedro Peccatori, Giovanni Martinelli and Francesco Bertolini
DOI:  Published May 2000

Abstract

Recent studies have suggested that non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice transplanted with human hematological malignancies show higher levels of engraftment compared with other strains. We used this model to compare xenotransplantability of human leukemia and lymphoma cell lines and to investigate angiogenesis in hematopoietic malignancies. Ten of 12 evaluated cell lines were able to engraft NOD/SCID mice within 120 days. A strong correlation was observed between the amount of vascular endothelial growth factor (VEGF) produced in vitro by cultured cells and the efficiency of tumor engraftment (r = 0.808; P = 0.001), and an inverse correlation was found between VEGF production and the time of tumor engraftment (r = −0.792; P = 0.006) and between VEGF production and the frequency of apoptotic/dead cells in solid tumors (r = −0.892; P = 0.007). Moreover, VEGF production correlated with the frequency of endothelial (CD31+/CD34+) cells in solid tumors (r = 0.897; P = 0.001). Taken together with in vitro data presented here and indicating that the VEGF antagonist Flt-1/Fc chimera inhibits leukemia and lymphoma cell proliferation, our findings support a role for tumor-derived VEGF in leukemia and lymphoma progression. Furthermore, the present study confirms previous observations indicating that VEGF expression may play a crucial role in xenotransplantability of human solid malignancies in SCID mice. The NOD/SCID model is promising for future evaluations of antiangiogenic drugs, alone or in combination with established chemo- or immunotherapy regimens.

INTRODUCTION

Animal models are increasingly used to evaluate the growth and behavior of human tissues and malignancies in xenograft studies. Mice bearing the Nude or the SCID 2 mutation have been considered for decades the standard model to evaluate human malignancies in vivo, but these mouse strains have some residual immunity that somewhat limits the posttransplant growth of human hematopoietic malignancies (1 , 2) . Recent studies from independent laboratories have suggested that the NOD/SCID mouse strain is more promising as a tool for human leukemia/lymphoma xenotransplantation (1, 2, 3, 4) . Bonnet and Dick (1) demonstrated high levels of AML engraftment in sublethally irradiated NOD/SCID mouse transplanted i.v. and reported significantly superior engraftment in NOD/SCID compared with SCID mice. Another study (2) compared s.c. xenograft transplant of lymphoid malignancies in Nude, recombination activating gene 1-deficient, SCID, and NOD/SCID mice, and only the NOD/SCID model engrafted 100% of tumors. Moreover, in contrast to results seen in Nude, recombination activating gene 1-deficient, and SCID mice, the NOD/SCID model showed no further benefit from the addition of radiation or radiation and anti-natural killer cell antibodies to increase immunosuppression before xenotransplantation. Along this line we have evaluated in NOD/SCID mice the engraftment potential of 12 hematopoietic neoplastic cell lines, including nine established myeloid and lymphoid lines (U-937, K-562, Jurkat, CEM, MOLT-4, Karpas 299, Namalwa, HS Sultan, and L-363) and three in-house generated lines (QD1-EIO and BC1-EIO myeloid lines and the RAP1-EIO lymphoid line).

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.

MATERIALS AND METHODS

Cell Lines, VEGF, and bFGF Production in Vitro.

After informed consent was obtained, the QD1-EIO cell line was established from the BM of a 45-year-old female M4 AML patient. Cells display a CD3−, CD14+, CD15+, CD19−, GlyA− phenotype and a t(9;11) karyotype. The BC1-EIO cell line was established from the BM of a female 69-year-old M2 AML patient. Cells display a CD3−, CD13+, CD14−, CD19−, CD33+, CD34+, GlyA−, CD117+ phenotype and a t(8;21) karyotype. The RAP1-EIO cell line was established from the BM of a male 61-year-old patient who had a diagnosis of T-cell-rich B-cell non-Hodgkin’s lymphoma. Cells display a CD3−, CD10+, CD13−, CD19+, GlyA−, sm/cy kappa+, sm/cy lambda− phenotype and a t(14;18) karyotype after passage in the mouse.

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 × 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 × 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 2μ g/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 × 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 (×40 and ×100) to detect the area with the highest MVD (hot spot). Three microscopic fields were then examined in this area at× 250 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 × 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.

RESULTS

In Vitro Studies.

As reported in Fig. 1 ⇓ 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.

Fig. 1.
Fig. 1.

Representative RT-PCR analysis of VEGF (four different splice variants of sizes of 516, 648, 720, and 771 bp, indicated by asterisks), Flt-1 (inner primer pair, 196 bp, indicated by an asterisk), and KDR (inner primer pair, 213 bp, indicated by an asterisk) transcripts in K-562 and QD1-EIO cell lines. HUVECs were also evaluated as a positive control for VEGF receptors.

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Table 1

Expression and production of VEGF, related receptors, and bFGF in hematopoietic cell lines

Studies on the expression of VEGF receptors by flow cytometry (Fig. 2) ⇓ 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 ⇓ ).

Fig. 2.
Fig. 2.

Representative dot plots indicating the expression of VEGF receptors in MOLT-4 and Jurkat cells incubated with biotinylated VEGF (or biotinylated negative reagent) and avidin-FITC. A, forward and side scatters of cell suspensions and analysis gates. B, negative controls. C, the frequency of cells expressing VEGF receptors. VEGF specificity is confirmed by the reduction of signal intensity in the presence of blocking anti-VEGF antibody (D). Apoptotic and dead cells are depicted by 7AAD staining.

Fig. 3.
Fig. 3.

In vitro inhibition of cell proliferation in the presence of the VEGF antagonist Flt-1/Fc chimera. A, representative dose-dependent inhibition of MOLT-4 cell proliferation. B, inhibition of cell proliferation in different cell lines after 5-h culture in the presence of 2 μg/ml Flt-1/Fc chimera and 10 or 1% FBS. n = 6, results are expressed as means; bars, 1 SD. C, correlation between VEGF production and inhibition of cell proliferation in different cell lines. Solid line, regression; dotted line, 95% confidence limits.

In Vivo Studies.

As shown in Table 2 ⇓ , 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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Table 2

Neoplastic cell engraftment efficiency in NOD/SCID mice, solid tumor, and BM vascularization

All of the solid tumors found in transplanted animals were vascularized, because vessels were found near engrafted cells. MVD ranged from 13 ± 2 in MOLT-4 tumors to 33 ± 2 in HS Sultan tumors. FC evaluation of solid tumors dissolved to single cells (Table 2) ⇓ 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) ⇓ .

Fig. 4.
Fig. 4.

FC evaluation of tumor and BM vascularization in NOD/SCID mice. Left, negative controls; other panels show representative engrafted NOD/SCID mice transplanted with 10 × 106 MOLT-4 (central A and B) and U-937 (right A and B). A, the percentage of vWF+ 7AAD− cells in solid tumors dissolved at the single-cell level. B, the percentage of CD31+/CD34+ cells in flushed BM after exclusion of 7AAD+ cells with a gate. As shown by the percentage of positive cells per quadrant, mice engrafted with U-937 cells had tumors that were more vascularized and had a higher frequency of BM endothelial cells. Cell lines MOLT-4 and U-937 were evaluated as a control in C and D.

Fig. 5.
Fig. 5.

Representative dot plots indicating the frequency of apoptotic/dead cells in tumors from engrafted animals. As indicated by the percentage of positive cells per quadrant, two-color FC evaluation of 7AAD and appropriate tumor markers (human CD45 for U-937 and CEM, GlyA for K-562, CD15 for QD1-EIO, CD2 for MOLT-4, CD19 for Namalwa, and RAP1-EIO cells) demonstrated that the frequency of apoptotic/dead cells in solid tumors was higher in mice engrafted with CEM and MOLT-4 cells (P < 0.01). Right top, frequency of neoplastic cells in the BM of animals engrafted with U-937 and QD1-EIO cells.

Fig. 6.
Fig. 6.

Frequency of apoptotic/dead cells in solid tumors and BM vascularization. The frequency of apoptotic cells (some of which are depicted by arrows) was markedly reduced in Namalwa (a) compared with CEM (b) tumors. An increased frequency of sinusoids vessels (asterisks) was found in the BM of a NOD/SCID mouse engrafted with U-937 cells, which generated ascites (c). Some sprouts-like vessels are indicated by arrows in the BM of a mouse engrafted with QD1-EIO cells (d).

Remarkably, a strong correlation was found between VEGF in vitro production and the efficiency of tumor engraftment (r = 0.808; P = 0.001; Fig. 7A ⇓ ), 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 ⇓ ).

Fig. 7.
Fig. 7.

Correlation between VEGF production and the neoplastic cell line engraftment efficiency. (A), the time of engraftment (B), the frequency of apoptotic/dead cells (C), and the frequency of CD31+/CD34+ cells in solid tumors (D). Solid line, regression; dotted line, 95% confidence limits.

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Table 3

P (by Spearman rank test) of correlation between efficiency of engraftment, speed of engraftment, and frequency of apoptotic/dead cells in solid tumors and different cell line variables

DISCUSSION

In the past 2 years, it has been suggested that angiogenesis may play a role in myelodysplastic syndromes, leukemia, lymphoma, and myeloma (6, 7, 8, 9, 10, 11 ,, 16) . In this context, we evaluated the expression of angiogenic growth factors VEGF and bFGF in a number of hematopoietic malignant cell lines and used, for the first time, the NOD/SCID mouse model to investigate neovascularization in human myeloid and lymphoid malignancies generated by these cells. In most of the cell lines evaluated in this study, VEGF (but not bFGF) was found to be generated at concentrations that are within its known range of biological activity. Furthermore, all of the cell lines expressed at least one of the VEGF-related receptors KDR and Flt-1. Thus, we investigated the presence of an autocrine pathway between VEGF and related receptors in hematological malignancies by means of cell culture in the presence of the Flt-1/Fc chimera already known to inhibit VEGF-dependent HUVEC proliferation. Using this approach, VEGF deprivation from the culture medium was associated with 5–44% inhibition of cell proliferation. It should be noted that a strong inverse correlation was found between cell line VEGF production and the extent of inhibition of cell proliferation. For this reason, it seems possible to speculate that, in the future, tools for VEGF deprivation more efficient than the Flt-1/Fc chimera might be able to induce a more extensive inhibition of leukemia/lymphoma cell proliferation.

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) .

Acknowledgments

We thank Francesco Pezzella, Davide Soligo, and Domenico Delia for critical reading of the manuscript, and we thank Giuseppina Giardina and Maria Teresa Sciurpi for precious technical assistance.

Footnotes

  • 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.

  • ↵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 August 12, 1999.
  • Accepted March 6, 2000.

References

  1. ↵
    Bonnet D., Dick J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med., 3: 730-737, 1997.
    OpenUrlCrossRefPubMed
  2. ↵
    Hudson W. A., Li Q., Le C., Kersey J. H. Xenotransplantation of human lymphoid malignancies is optimized in mice with multiple immunologic defects. Leukemia (Baltimore), 12: 2029-2033, 1998.
    OpenUrlCrossRefPubMed
  3. ↵
    Baersch G., Mollers T., Hotte A., Dockhorn-Dworniczak B., Rube C., Ritter J., Jurgens H., Vormoor J. Good engraftment of B-cell precursor ALL in NOD-SCID mice. Klin. Paediatr., 209: 178-185, 1997.
    OpenUrl
  4. ↵
    Steele J. P., Clutterbuck R. D., Powles R. L., Mitchell P. L., Horton C., Morilla R., Catovsky D., Millar J. L. Growth of human T-cell lineage acute leukemia in severe combined immunodeficiency (SCID) mice and non-obese diabetic SCID mice. Blood, 90: 2015-2019, 1997.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other diseases. Nat. Med., 1: 27-31, 1995.
    OpenUrlCrossRefPubMed
  6. ↵
    Perez-Atayde A. R., Sallan S. E., Tedrow U., Connors S., Allred E., Folkman J. Spectrum of tumor angiogenesis in the bone marrow of children with acute lymphoblastic leukemia. Am. J. Pathol., 150: 815-821, 1997.
    OpenUrlPubMed
  7. ↵
    Pruneri G., Bertolini F., Soligo D., Carboni N., Cortelezzi A., Ferrucci P. F., Buffa R., Lambertenghi-Deliliers G., Pezzella F. Angiogenesis in myelodysplastic syndromes. Br. J. Cancer, 81: 1398-1401, 1999.
    OpenUrlCrossRefPubMed
  8. ↵
    Foss H. D., Araujo I., Demel G., Klotzbach H., Hummel M., Stein H. Expression of vascular endothelial growth factor in lymphomas and Castleman’s disease. J. Pathol., 183: 44-50, 1997.
    OpenUrlCrossRefPubMed
  9. ↵
    Vacca A., Ribatti D., Presta M., Minischetti M., Iurlaro M., Ria R., Albini A., Bussolino F., Dammacco F. Bone marrow neovascularization, plasma cell angiogenic potential, and matrix metalloproteinase-2 secretion parallel progression of human multiple myeloma. Blood, 93: 3064-3073, 1999.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Fiedler W., Graeven U., Ergun S., Verago S., Kilic N., Stockschlader M., Hossfeld D. K. Vascular endothelial growth factor, a possible paracrine factor in human acute myeloid leukemia. Blood, 89: 1870-1875, 1997.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Bellamy W. T., Richter L., Frutiger Y., Grogan T. M. Expression of vascular endothelial growth factor and its receptors in hematopoietic malignancies. Cancer Res., 59: 728-733, 1999.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Ferrara N., Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr. Rev., 18: 4-25, 1997.
    OpenUrlCrossRefPubMed
  13. ↵
    Gabrilovich D. I., Chen H. L., Girgis K. R., Cunningham H. T., Meny G. M., Nadaf S., Kavanaugh D., Carbone D. P. Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat. Med., 2: 1096-1103, 1996.
    OpenUrlCrossRefPubMed
  14. ↵
    Gabrilovich D., Ishida T., Oyama T., Ran S., Kravtsov V., Nadaf S., Carbone D. P. Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo. Blood, 92: 4150-4166, 1998.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Tokunaga T., Nakamura M., Oshika Y., Ohnishi Y., Ueyama Y. Is xenotransplantability of human colon cancers in SCID mice affected by angiogenic factors?. J. Natl. Cancer Inst., 90: 400-401, 1998.
    OpenUrlFREE Full Text
  16. ↵
    Bertolini F., Paolucci M., Peccatori F., Cinieri S., Agazzi A., Ferrucci P. F., Cocorocchio E., Goldhirsch A., Martinelli G. Angiogenic growth factors and endostatin in non-Hodgkin’s lymphoma. Br. J. Haematol., 106: 504-509, 1999.
    OpenUrlCrossRefPubMed
  17. ↵
    Conn G., Soderman D. D., Schaeffer M. T., Wile M., Hatcher V. B., Thomas K. A. Purification of a glycoprotein vascular endothelial cell mitogen from a rat glioma-derived cell line. Proc. Natl. Acad. Sci. USA, 87: 1323-1327, 1990.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Philpott N. J., Turner A. J. C., Scopes J., Westby M., Marsch J. C. W., Gordon-Smith E. C., Dalgleish A. G., Gibson F. M. The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques. Blood, 87: 2244-2251, 1996.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Fukumura D., Xavier R., Sugiura T., Chen Y., Park E., Lu N., Selig M., Nielsen G., Taksir T., Jain R. K., Seed B. Tumor induction of VEGF promoter activity in stromal cells. Cell, 94: 715-725, 1998.
    OpenUrlCrossRefPubMed
  20. ↵
    Grunstein J., Roberts W. G., Mathieu-Costello O., Hanahan D., Johnson R. S. Tumor-derived expression of vascular endothelial growth factor is a critical factor in tumor expansion and vascular function. Cancer Res., 59: 1592-1598, 1999.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Okada F., Rak J. W., Croix B. S., Lieubeau B., Kaya M., Roncari L., Shirasawa S., Sasazuki T., Kerbel R. S. Impact of oncogenes in tumor angiogenesis: mutant K-ras up-regulation of vascular endothelial growth factor/vascular permeability factor is necessary, but not sufficient for tumorigenicity of human colorectal carcinoma cells. Proc. Natl. Acad. Sci. USA, 95: 3609-3614, 1998.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Chin L., Tam A., Pomerantz J., Wong M., Holash J., Bardeeesy N., Shen Q., O’Hagan R., Pantginis J., Zhou H., Horner J. W., Cordon-Cardo C., Yancopoulos G. D., DePinho R. A. Essential role for oncogenic Ras in tumour maintenance. Nature (Lond.), 400: 468-472, 1999.
    OpenUrlCrossRefPubMed
  23. ↵
    Holash J., Masonpierre P. C., Compton D., Boland P., Alexander C. R., Zagzag D., Yancopoulos G. D., Wiegand S. J. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science (Washington DC), 284: 1994-1998, 1999.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Solanilla A., Grosset C., Lemercier C., Dupouy M., Mahon F. X., Schweitzer K., Reiffers J., Weksler B., Ripoche J. Expression of Flt3-ligand by the endothelial cell. Regulation by IL-1α, glucocorticoids, IFN-α, MIP-1α and TGFβ. Key role in the proliferation of primitive hematopoietic progenitors. Blood, 92: 580a 1998.
    OpenUrl
  25. ↵
    Namikawa R. SCID-hu mice as a model for the study of human malignancies Roncarolo M. G. Peault B. Namikawa R. eds. . Human Hematopoiesis in SCID Mice, : 157-170, Springer-Verlag New York 1995.
  26. ↵
    Bohem T., Folkman J., Browder T., O’Reilly M. S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature (Lond.), 390: 404-407, 1997.
    OpenUrlCrossRefPubMed
  27. ↵
    Harris A. L. Antiangiogenesis for cancer therapy. Lancet, 349: 13-15, 1997.
    OpenUrl
  28. ↵
    Kim K. J., Li B., Winter J., Armanini M., Gillett N., Philips H. S., Ferrara N. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature (Lond.), 362: 841-844, 1993.
    OpenUrlCrossRefPubMed
  29. ↵
    Asano M., Yukita A., Matsumoto T., Kondo S., Suzuki H. Inhibition of tumor growth and metastasis by an immunoneutralizing monoclonal antibody to human vascular endothelial growth factor/vascular permeability factor121. Cancer Res., 55: 5296-5301, 1995.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Fong T. A. T., Shawver L. K., Li S., Tang C., App H., Powell T. J., Kim Y. H., Schreck R., Wang Y., Risau W., Ullrich A., Hirth K. P., McMahon G. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res., 59: 99-106, 1999.
    OpenUrlAbstract/FREE Full Text
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May 2000
Volume 60, Issue 9

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Human Myeloid and Lymphoid Malignancies in the Non-Obese Diabetic/Severe Combined Immunodeficiency Mouse Model: Frequency of Apoptotic Cells in Solid Tumors and Efficiency and Speed of Engraftment Correlate with Vascular Endothelial Growth Factor Production
Lisa Fusetti, Giancarlo Pruneri, Alberto Gobbi, Cristina Rabascio, Nadia Carboni, Fedro Peccatori, Giovanni Martinelli and Francesco Bertolini
Cancer Res May 1 2000 (60) (9) 2527-2534;
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