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Tumor Biology |
Angiogenesis Laboratory, Research Institute for Growth and Development (GROW), Department of Internal Medicine [A. E. M. D., S. T. v. d. N., J. C. A. B-t. S., J. W.] and Department of Pathology [A. W. G.], University Hospital Maastricht and Maastricht University 6229 HX Maastricht, and Laboratory for Microcirculation, Department of Physiology, Cardiovascular Research Institute Maastricht [M. G. A. o. E., M. J. E. K., V. V. T. H.], 6202 AZ Maastricht, the Netherlands
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-stimulated leukocyte-vessel wall interactions were studied in ear skin microvessels of nude mice bearing small human LS174T colon carcinomas and in C57Bl/6 mice bearing murine B16F10 melanomas. Leukocyte-vessel wall interactions were studied both within and outside small tumors growing in the ear, and in ear microvessels of mice with a large tumor growing on their flank. Tumor-free mice were used as controls. Compared with values measured at the edge of the ear and in the contralateral ear, leukocyte adhesion was found to be diminished significantly in vessels inside the ear tumor in both mouse models. This reduction disappeared with increasing distance from the tumor. Surprisingly, the level of leukocyte adhesion in ear venules of mice with a large flank tumor was also reduced significantly. Leukocyte rolling, i.e., the step preceding adhesion, was not influenced by the presence of a tumor in nude mice, but was down-regulated in immune-competent C57Bl/6 mice. Treatment of mice bearing a small ear tumor with a humanized antivascular endothelial growth factor antibody prevented the down-regulation of leukocyte-vessel wall interactions inside the tumor vessels compared with the nontreated group. Fluorescence-activated cell sorter analysis showed that isolated tumor ECs have suppressed levels of intercellular adhesion molecule 1 as compared with ECs from normal mouse tissues. In cultured b.END5 cells the tumor necrosis factor -induced up-regulation of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 was reduced in ECs that were preincubated with basic fibroblast growth factor or vascular endothelial growth factor. The current results may have an impact on the effectiveness of clinical immunotherapeutic treatment protocols, because immune effector cells may not be able to enter tumor tissue. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Angiogenesis, the formation of new blood vessels from pre-existing ones, is a prerequisite for outgrowth and metastasis of tumors (8 , 9) . To achieve this, tumors secrete high levels of angiogenic factors such as VEGF and fibroblast growth factors. We have shown previously that these factors are responsible for down-regulating endothelial adhesion molecules such as ICAM-1/-2 and CD34 (4 , 10) . Angiogenic factors also inhibit inflammatory cytokine induced expression of VCAM-1 and E-selectin (11 , 12) . In addition, it has been established both in vitro (13) and in vivo (14) that suppressed adhesion molecule expression by angiogenic factors results in diminished leukocyte-vessel wall interactions.
The present study aimed to investigate the regulation of adhesion molecule expression and leukocyte-vessel wall interactions in vivo by the presence of a tumor. To this end, a mouse model was developed that allows the investigation of leukocyte-vessel wall interactions, both inside and outside a tumor. The effects of a small tumor in the ear were compared with those of a larger tumor on the flank. Intravital microscopy was used to quantify the effects of the tumor on leukocyte-vessel wall interactions and on local fluid dynamic conditions in ear skin microvessels. To this purpose, a xenograft model was used in which a human tumor (LS174T) was grown in nude mice. These experiments were repeated using immune-competent mice (C57Bl/6) with a mouse tumor (B16F10). In addition, ex vivo and in vitro experiments were performed to detect ICAM-1 and VCAM-1 expression on ECs isolated from both tumor and healthy tissues (obtained from Swiss/nude mice and C57Bl/6 mice) and on mouse b.END5 brain endothelioma cells using flow cytometry.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
Tumor cells (LS174T) were cultured in DMEM (Life Technologies, Inc., Paisley, Scotland) containing 10% inactivated FCS (Bio Whittaker, Verviers, Belgium), glutamine, and antibiotics, and harvested using 0.25% trypsin. Mouse B16F10 melanoma cells (kindly provided by Dr. Isaiah J. Fidler, Houston, TX) were cultured similarly using Hank’s MEM (Life Technologies, Inc.) containing 5% FCS and antibiotics, and harvested using 0.25% trypsin. Mouse b.END5 brain endothelioma cells (ECACC, Salisbury, United Kingdom) were cultured in DMEM (Life Technologies, Inc.) containing 10% FCS.
Experimental Protocol and Intravital Microscopy.
In vivo experiments were performed in four groups of Swiss/nude mice. After anesthetizing these mice briefly (i.p.) using a mixture of ketamine (0.1 mg/g b.w. Nimatek; Ad Usem Veterinarium, Cuijk, the Netherlands) and xylazine (0.05 mg/g b.w. in nude mice and 0.02 mg/g b.w. in C57Bl/6 mice, Sedamun; Ad Usem Veterinarium), the first group (ear tumor group; n = 6) received 10 µl of a dense tumor cell suspension (106 cells) intradermally in the dorsal side of the left ear. Within 3–5 days the ear tumor grew to a diameter of 
2 mm, at which time point the experiment was performed. In the second group (flank tumor group; n = 4) 100 µl of a tumor cell suspension (106 cells) was injected s.c. into the flank. In this group experiments were performed after 2–3 weeks, at which time the flank tumor had reached a diameter of 1.5 cm. The third group served as control (n = 4). These mice were not injected with tumor cells, but were otherwise treated similarly. Two additional mice bearing a small LS174T ear tumor (n = 2) were treated with HuMV833 (a humanized anti-VEGF antibody, 3.0 mg/kg, i.p.; kindly provided by Protein Design Labs, Fremont, CA) on the days 5 and 7 after injection of the LS174T cells in the left ear. On day 8, intravital microscopy was performed. The experiments were repeated with three groups of C57B/6 mice carrying B16F10 murine melanoma tumors: an ear tumor group (n = 7), a flank tumor group (n = 4), and a control group (n = 4).
Four h before the start of the intravital microscopic observations, Swiss/nude mice received 750 ng of recombinant murine TNF- (R&D Systems, Abingdon, United Kingdom) whereas C57Bl/6 mice received 500 ng of TNF-. Two additional Swiss/nude mice as well as two C57Bl/6 mice were investigated that did not receive TNF-, to serve as control for the TNF- effects in the present setup.
After TNF- administration (3.5 h), mice were anesthetized by s.c. administration of the ketamine/xylazine mixture (see above). Body temperature was kept at 37°C by an infrared heating lamp. To enable intravital microscopic observation of leukocytes, 10–20 µl of a Rhodamine 6G solution (1 mg/ml in 0.9% NaCl solution) was injected into the tail vein when needed. In all of the mice, venules (10–40 µm) in both ears were visualized using a Leitz intravital microscope adapted for telescopic imaging (15)
. Images were recorded on videotape for offline analysis.
To enable quantification of systemic leukocyte counts, 20 µl of blood were sampled from the vena cava at the end of every experiment and added to Türks solution (Merck) in a 1:10 dilution. Leukocytes were counted and differentiated as PMN or MMN in a counting chamber (Clay Adams, Parsippany, NJ).
Experimental Parameters.
Venular diameters were determined with a home-built image-shearing device (16)
. Centerline blood flow velocity was measured by frame-to-frame analysis, using the fastest passing fluorescent leukocyte as a marker. The level of leukocyte rolling was determined by counting the number of rolling cells passing a vessel segment per min. Leukocytes were considered as rolling when their velocity along the vessel wall was at least an order of magnitude lower than that of the free-flowing blood cells. The level of leukocyte adhesion was assessed in a 100-µm segment of the venule and expressed as number of cells per endothelial surface area (assuming the cross-section of the vessels to be circular). Leukocytes were considered adherent when they remained stationary for at least 30 s. We also examined the total number of interacting leukocytes at a particular moment; this parameter includes not only the number of adhering and rolling leukocytes, but also the velocity of the latter. This parameter was determined by counting in a frozen video frame the total number of rolling and adhering leukocytes in a 100-µm vessel segment; at each time point, this count was performed in 4 randomly chosen video frames, and the data were averaged. In the tumor-bearing ears, leukocyte-vessel wall interactions were quantified in venules in three different regions: (a) within the tumor; (b) adjacent to the tumor (within a radius of 2.5 mm around the tumor); and (c) at the edge of the ear. In ears without a tumor the venules were chosen at random.
Real-Time Quantitative RT-PCR.
To detect VEGF and bFGF gene transcription, RNA was extracted from frozen LS174T tumor tissue using a RNeasy mini kit (Qiagen, Valencia, CA) and from proliferating LS174T cells using TRIzol reagent (Invitrogen, Breda, the Netherlands). Subsequently, a real-time quantitative RT-PCR was performed to quantitate transcription levels of VEGF and bFGF as described by Meulemans et al.4
Adhesion Molecule Expression on Isolated ECs and b.END5.
To culture mouse microvascular ECs, normal (lung, heart, and kidney) and tumor tissues obtained from both Swiss/nude and C57Bl/6 mice were mechanically and enzymatically digested during 1 h at 37°C, using a mixture of 1 mg/ml collagenase and 2.5 IU/ml dispase (Life Technologies, Inc.). After 30 min of incubation, 75 µg DNase (Sigma Chemical Co.) was added for another 30 min. The single-cell suspensions were allowed to adhere for 3 h to gelatin-coated tissue culture flasks (Costar, Corning, NY). The remaining adherent cell population consisted of 2–15% ECs as determined by CD31 expression. Cells were cultured for 3 days in DMEM (with 1000 mg/liter glucose) containing 20% FCS. In one experiment, cells obtained from normal C57Bl/6 tissues were cultured for 3 days in the presence of 0, 10, 25, 50, or 100 ng/ml bFGF (Sanvertech, Heerhugowaard, the Netherlands). b.END5 cells were cultured for 3 days with 50 ng/ml bFGF or 50 ng/ml VEGF. When applied, TNF- was added 6 h before harvesting. Finally, the expression of ICAM-1 and VCAM-1 was determined flow cytometrically (see below).
FACS Analysis of Endothelial and Leukocyte Adhesion Molecules.
Cells were harvested and fixed for 30 min in 1% paraformaldehyde (Merck) at room temperature. Afterward, cells were resuspended in 20 µl of appropriately diluted rat anti-ICAM-1 monoclonal antibody (R&D Systems) or rat anti-VCAM-1 monoclonal antibody (PharMingen, San Diego, CA) and incubated for 1 h on ice. Subsequently, the cells were incubated for 1 h with goat-antirat IgG conjugated to FITC (Pickcell Laboratories, Amsterdam, the Netherlands). Finally, the cells were incubated with phycoerythrin-conjugated rat antimouse CD31 (Dako, Glostrup, Denmark). Stained cells were analyzed on a FACScalibur flow cytometer. Data analysis was performed using Cellquest software (Becton Dickinson, Mountain View, CA).
Mouse blood leukocytes were isolated by Ficoll density gradient centrifugation (Amersham, Uppsala, Sweden). Cells were fixed for 30 min in 1% paraformaldehyde (Merck) at room temperature. Cells were incubated with the supernatant of the following hybridoma cell lines: R1-2 (rat antimouse Vla-4), Mel14 (rat antimouse L-selectin), M17.4 (rat antimouse LFA-1 chain), or M18.2 (rat antimouse LFA-1 ß chain; all obtained from PharMingen) for 1 h at 4°C. Subsequently, the cells were incubated with FITC-conjugated goat antirat IgG (Pickcell Laboratories) for 1 h at 4°C.
Statistics.
Because of their nonsymmetrical distribution, data obtained by intravital microscopic experiments are presented as medians with interquartile ranges (i.e., the spread from the 25th to 75th percentile). Data obtained by FACS analysis are presented as means with corresponding SE. Differences between two independent data groups were tested with the Mann-Whitney U test. Differences between paired data groups were tested with the Friedman test (more than two groups), followed by a multiple-comparison procedure. Correlation between variables was determined using Spearman’s correlation test. In all of the tests the level of significance was set at 0.05.
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RESULTS |
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LS174T human colon carcinoma tumor cells were injected in the ears of nude mice (Fig. 1, A and B)
, and after the outgrowth of small tumors, TNF--induced leukocyte-vessel wall interactions as well as local fluid dynamic parameters were examined in ear skin vessels. In the tumor-free control group, the median level of leukocytes interacting with the vascular endothelium after treatment with TNF- was 813 leukocytes/mm2. Inside the ear tumor, this level was significantly lower (382 leukocytes/mm2; P < 0.05). The total level of leukocyte-vessel wall interactions increased as a function of distance from the tumor [significant correlation of 0.35 (Rs); P < 0.01], being 728 leukocytes/mm2 in areas adjacent to the tumor and 953 leukocytes/mm2 at the outer edge of the ear. The latter level is comparable with the one measured in the contralateral ears (885 leukocytes/mm2) and with the level measured in the control group (Fig. 1C)
. Examples of intravital microscopic images of control and tumor vessels are presented in Fig. 1, D and E
. Without TNF- pretreatment, the total level of interacting leukocytes in ear skin venules was 318 cells/mm2 (data not shown), which indicates that the tumor microenvironment completely eradicated the TNF--induced inflammatory response. These results demonstrate a local effect of a small tumor on leukocyte interactions with the vascular endothelium.
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. Whereas the level of leukocyte adhesion in tumor vessels was drastically diminished to almost undetectable levels as compared with venules in the contralateral ear (375 leukocytes/mm2) and in the control group (331 leukocytes/mm2), such differences were not found at the level of leukocyte rolling (Fig. 2B)
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To investigate whether leukocyte-vessel wall interactions are similarly influenced in mice with an intact immune system, similar experiments were performed in the B16F10 mouse melanoma model in C57BL/6 mice. These experiments revealed comparable results, showing reductions in total level of interacting and adhering leukocytes in tumor vessels as compared with vessels outside the tumor [significant correlation of 0.55 (Rs) between leukocyte-vessel wall interactions and distance to the tumor; P < 0.01] and in tumor-free control mice (Fig. 3, A and B)
. Interestingly, in the immunocompetent mice we did find a significant inhibition in the level of rolling leukocytes inside the tumor (3 leukocytes/min) as compared with the edge of the ear (15 leukocytes/min; P < 0.05; Fig. 3C
).
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1500 mm3), noninfiltrating, and nonmetastasizing tumor within 3 weeks. Analysis of the ear venules of this group lead to the surprising finding of a significantly suppressed level of leukocyte-vessel wall interactions (509 leukocytes/mm2; P < 0.05) as compared with the control group without a tumor (813 leukocytes/mm2; P < 0.05; Fig. 4A
). Again, comparable with the situation in the small ear tumors, this reduced level of leukocyte-vessel wall interactions can be attributed to a diminished level of leukocyte adhesion (Fig. 4B)
. The level of leukocyte rolling was not found different from control (Fig. 4C)
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). This decline of leukocyte-vessel wall interactions can also be attributed to a decreased level of leukocyte adhesion as compared with the control group (Fig. 5B)
. No significant differences were found in leukocyte rolling (Fig. 5C)
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, LFA-1ß, VLA-4 or L-selectin between these two groups (data not shown), suggesting that the decreased level of interacting leukocytes we observe inside an ear tumor is not because of altered adhesion molecule expression on leukocytes.
TNF- induced a decrease in the number of circulating leukocytes from 2.4 x 106 leukocytes/ml to 0.9 x 106 leukocytes/ml (P = 0.05) in nude mice. This is probably because TNF- induces leukocyte adhesion systemically. This effect was evident for both MMNs and PMNs, but was most prominent for MMNs (80% reduction versus 55% for PMNs; P < 0.01). In contrast to a small ear tumor (1.3 x 106 leukocytes/ml), the presence of a large flank tumor tended to mask these TNF- induced changes (1.8 x 106 leukocytes/ml), also suggesting a systemic effect of the tumor.
No significant differences in blood flow velocities were detected between the vessels of the various experimental groups of both Swiss/nude and C57Bl/6 mice (Table 1)
, except for deviating centerline velocities in the vessels inside the tumor in both mouse models. However, no correlation between centerline velocity values and any of the leukocyte-vessel wall interaction related parameters could be found in any of the groups. Therefore, the observed differences in leukocyte-vessel wall interactions cannot be explained by differences in fluid dynamic parameters.
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. Whereas ICAM-1 levels in the tumor were low (mean fluorescence intensity of 2 in LS174T tumors and 1.5 in B16F10 tumors), in normal tissues these values ranged between 40 and 57. To investigate whether angiogenic factors produced by the tumor are responsible for this down-regulation of ICAM-1, normal tissue ECs were cultured for 3 days in the presence of various concentrations of bFGF (Fig. 6C)
. bFGF was observed to down-regulate ICAM-1 on freshly isolated ECs in a dose-dependent manner. In separate experiments, the mouse EC line b.END5 was cultured with or without bFGF or VEGF, and in presence or absence of TNF- (Fig. 6D)
. In the absence of TNF-, bFGF was observed to down-regulate ICAM-1 expression (P < 0.05). On the contrary, TNF- was able to significantly up-regulate both ICAM-1 (P < 0.01) and VCAM-1 (P < 0.01). The TNF--induced up-regulation of ICAM-1 and VCAM-1 was reduced in cells that were preincubated with bFGF (ICAM-1: P < 0.05; VCAM-1: P = 0.07) or VEGF (ICAM-1: P = 0.06; VCAM-1: P = 0.06). These results suggest angiogenic factors as regulators of leukocyte-vessel wall interactions.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced expression of ICAM-1 and VCAM-1 on cultured mouse ECs strongly suggests that tumor-derived growth factors are involved. A reduced level of leukocyte-vessel wall interactions is likely to result in impaired extravasation and infiltration of leukocytes into the tumor. Through this mechanism a tumor has the opportunity to escape the immune system. We developed a new tumor model in which leukocyte-vessel wall interactions in and outside a tumor can be measured noninvasively, allowing longitudinal analysis during tumor development and/or in the course of an anticancer treatment. Several other investigators have also used animal models to examine leukocyte-vessel wall interactions in the presence of a tumor (17, 18, 19, 20, 21, 22, 23) . However, these studies used invasive techniques such as dorsal skin fold chambers, cranial windows, or skin flaps. These techniques are more time consuming, carry a higher risk of failure, and may induce an inflammatory reaction by surgical procedures at the site of interest. In the present study, athymic nude mice were selected to enable implantation of a tumor of human origin. These mice have large, transparent, hairless ears, which is a clear advantage for intravital microscopic measurements. The use of athymic mice to study immune functions may be problematic, attributable to limited cognate immunity because of the lack of mature T lymphocytes. However, innate immunity (i.e., granulocytes, monocytes, and natural killer cells) and B lymphocytes are present and functional in this model. Nevertheless, we repeated the experiments using syngeneic B16F10 melanoma cells in immune-competent C57Bl/6 mice. This model was chosen because of the availability of a large body of literature on immunological antitumor responses in these mice (23, 24, 25, 26) .
It has been demonstrated previously that the expression of adhesion molecules is suppressed in human tumor ECs (6
, 27
, 28)
. Furthermore, we have shown earlier in vitro (4)
and in vivo (14)
that this is most probably because of differences in metabolic state and phenotype of tumor EC by exposure to tumor-derived angiogenic factors such as bFGF and VEGF. Other studies have shown that bFGF reduces monocyte and natural killer cell adhesion, and migration by down-regulating adhesion molecules on HUVECs (7
, 29) . Here, we show that in our mouse models tumor ECs also have suppressed ICAM-1 expression. In addition, TNF--induced up-regulation of ICAM-1 and VCAM-1 was reduced in cultured b.END5 cells (mouse endothelioma cells) pretreated with either bFGF or VEGF. Because we also show that LS174T tumor cells contain mRNA for VEGF, whereas it is known from literature that B16F10 tumor cells express high levels of bFGF and VEGF (30
, 31)
, our data strongly suggest that the down-regulation of leukocyte-vessel wall interactions in tumor vessels, as observed in the present study, is a result of exposure of ECs to these angiogenic factors. This hypothesis is supported by our observation that treatment of mice bearing a small ear tumor with humanized anti-VEGF antibody prevents the down-regulation of leukocyte-vessel wall interactions inside the tumor.
Our finding that leukocyte-vessel wall interactions are decreased inside a tumor was confirmed in several previous studies (17, 18, 19, 20, 21, 22)
. However, in literature there is some dispute with regard to the effects of VEGF on the expression of adhesion molecules on ECs. Whereas the results of some studies are in agreement with our data and show a role for VEGF as an inhibitor of leukocyte-vessel wall interactions (32)
, the results of other studies seem to be in contrast with our findings (7
, 33) . These latter studies report an increase in adhesion molecule expression on HUVEC after VEGF stimulation. However, in these studies VEGF stimulation was always 
24 h. In a pilot study, we also observed an initial up-regulation of ICAM-1 expression after VEGF stimulation of HUVECs; in contrast, prolonged stimulation (which is also the case in the presence of a tumor) results in a down-regulation of ICAM-1 expression and leukocyte adhesion (data not shown). In one other study, performed by Detmar et al. (34)
, leukocyte-vessel wall interactions were found to be increased in skin vessels of VEGF-transgenic mice. However, this experimental approach in which the effects of a continuous overexpression of VEGF were investigated is completely different from the situation within tumors where VEGF [and also other (growth) factors] is produced locally in increasing quantities.
The present study is the first to show a systemic effect of a large tumor: leukocyte-vessel wall interactions appeared to be down-regulated in ear microvessels of mice bearing a large tumor on their flank. This systemic effect is less pronounced as compared with the situation within a tumor; leukocyte adhesion was only partially down-regulated, whereas inside a tumor the level of adhesion was reduced to zero. This may be because the circulating concentration of angiogenic factors in mice with a large flank tumor is lower than the local concentration inside a small ear tumor. The fact that the systemic leukocyte count was higher in TNF--treated mice with a large tumor on their flank than in TNF--treated control mice also suggests a systemic inhibitory effect of the flank tumor on leukocyte-vessel wall interactions. Because it is known for that some tumors produce soluble TNF--receptors (35
, 36)
, this might also be an explanation for the observed systemic effect of a large tumor on leukocyte-vessel wall interactions. However, we also show data (Fig. 6D)
on the direct effect of angiogenic factors on ECs, which exclude tumor cell interference in this process. Therefore, we believe that the observed effects are explained by an EC-mediated mechanism rather than a mechanism controlled by tumor-derived signals.
The current results indicate that in the nude mouse model, adhesion but not rolling is modulated by tumor derived factors. This suggests that ICAM-1 but not the selectins are sensitive to regulation by these factors, which would be a discrepancy with the human situation, where E- and P-selectin were found to be suppressed in tumor ECs (11 , 37) . It is more likely that the absence of effects on leukocyte rolling is because of selection of the nude mouse model, because we did observe a significant effect on leukocyte rolling inside the tumor in C57Bl/6 mice. We hypothesize that either tumor regulation of leukocyte rolling is confined to a functional T-cell population or that this effect is dependent on specific (combinations of) cytokines that are expressed by an intact immune system. However, the observed differences between the two tumor models on leukocyte rolling may also be because of other mouse strain-specific characteristics.
During its development, a growing tumor needs to undergo the so-called angiogenic switch, which is the starting point of the attraction of new blood vessels to grow beyond the size of 1–2 mm in diameter. It is possible that the small ear tumors studied in our experiments did not display a full angiogenic profile. Although this certainly would be an issue for spontaneous tumor models, in our model the injected cells already expressed VEGF and/or bFGF during culture. Evidence for the fact that we injected the cells in an advanced angiogenic state is provided by the fact that we observed the formation of a hematoma-like spot within 24 h after injection, which might be because of a permeability increase induced by VEGF and the presence of new vasculature already after 2–3 days (Fig. 1, A and B)
. Moreover, it was established that the small ear tumors are able to grow to the size of more advanced tumors (diameter >3 mm), indicating that angiogenic factors are being excreted by the tumor cells. Also, the results we obtained using real-time quantitative RT-PCR show that LS174T cells contain mRNA for VEGF in culture already. Therefore, we favor the view that our ear tumor model is relevant for the study of the early outgrowth of metastases rather than the outgrowth of a primary tumor.
In conclusion, the currently described tumor models introduce a noninvasive method for studying leukocyte-vessel wall interactions in vivo. We have demonstrated that leukocyte-vessel wall interactions are suppressed within a tumor but increase as a function of distance from the tumor. Moreover, in case of a large tumor burden there appears to be a systemic down-regulation of leukocyte interactions with the vessel wall. This effect is mediated by down-regulation of endothelial adhesion molecules, probably by angiogenic factors, leading to the escape of tumors from the immune system. Future studies will address the issue of the reversal of the observed phenomena by treatment with angiogenesis inhibitors and the improvement of (adoptive) immunotherapy by inhibitors of angiogenesis as a novel treatment strategy for cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Dutch Cancer Society (grant number UM2001-2529) and the Stichting Vanderes (grant number 42). 
2 To whom requests for reprints should be addressed, at Angiogenesis Laboratory, Department of Pathology, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, the Netherlands. Phone: 31-43-3876543; Fax: 31-43-3876613; E-mail: a.griffioen{at}intmed.unimaas.nl
3 The abbreviations used are: EC, endothelial cell; VEGF, vascular endothelial growth factor; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; b.w., body weight; TNF, tumor necrosis factor; PMN, polymorphonuclear; MMN, monomorphonuclear; RT-PCR, reverse transcription-PCR; bFGF, basic fibroblast growth factor; FACS, fluorescence-activated cell sorter; HUVEC, human umbilical vein endothelial cell.
4 Els V. Meulemans, et al., Expression of VEGF, bFGF and VEGF receptors in diffuse large B cell lymphoma, submitted for publication.
5 Internet address: http://www.fdg.unimaas.nl/angiogenesislab.
Received 9/26/02. Accepted 3/ 5/03.
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S. I. Zittermann and A. C. Issekutz Endothelial growth factors VEGF and bFGF differentially enhance monocyte and neutrophil recruitment to inflammation J. Leukoc. Biol., August 1, 2006; 80(2): 247 - 257. [Abstract] [Full Text] [PDF]
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A. E. M. Dirkx, M. G. A. oude Egbrink, K. Castermans, D. W. J. van der Schaft, V. L. J. L. Thijssen, R. P. M. Dings, L. Kwee, K. H. Mayo, J. Wagstaff, J. C. A. B. ter Steege, et al. Anti-angiogenesis therapy can overcome endothelial cell anergy and promote leukocyte-endothelium interactions and infiltration in tumors FASEB J, April 1, 2006; 20(6): 621 - 630. [Abstract] [Full Text] [PDF]
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 S. I. Zittermann and A. C. Issekutz Basic Fibroblast Growth Factor (bFGF, FGF-2) Potentiates Leukocyte Recruitment to Inflammation by Enhancing Endothelial Adhesion Molecule Expression Am. J. Pathol., March 1, 2006; 168(3): 835 - 846. [Abstract] [Full Text] [PDF]
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D. W.J. van der Schaft, F. Hillen, P. Pauwels, D. A. Kirschmann, K. Castermans, M. G.A. oude Egbrink, M. G.B. Tran, R. Sciot, E. Hauben, P. C.W. Hogendoorn, et al. Tumor Cell Plasticity in Ewing Sarcoma, an Alternative Circulatory System Stimulated by Hypoxia Cancer Res., December 15, 2005; 65(24): 11520 - 11528. [Abstract] [Full Text] [PDF]
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 J. C. A. B.-t. Steege, C. I. M. Baeten, V. L. J. L. Thijssen, S. A. Satijn, I. C. L. Verhoeven, H. F. P. Hillen, J. Wagstaff, and A. W. Griffioen Angiogenic Profile of Breast Carcinoma Determines Leukocyte Infiltration Clin. Cancer Res., November 1, 2004; 10(21): 7171 - 7178. [Abstract] [Full Text] [PDF]
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 A. W. Nap, A. W. Griffioen, G. A. J. Dunselman, J. C. A. Bouma-Ter Steege, V. L. J. L. Thijssen, J. L. H. Evers, and P. G. Groothuis Antiangiogenesis Therapy for Endometriosis J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1089 - 1095. [Abstract] [Full Text] [PDF]
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