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Clinical Cancer Research Vol. 6, 2037-2043, May 2000
© 2000 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Endoglin Is a Suitable Target for Efficient Imaging of Solid Tumors: In Vivo Evidence in a Canine Mammary Carcinoma Model1

Ester Fonsatti2, Antti P. Jekunen2, Kalevi J. A. Kairemo, Sandra Coral, Marjatta Snellman, Maria Rita Nicotra, Pier Giorgio Natali, Maresa Altomonte and Michele Maio3

Advanced Immunotherapy Unit, Centro di Riferimento Oncologico, Istituto Nazionale di Ricovero e Cura a Carattere Scientifico, Aviano 33081, Italy [E. F., S. C., M. A., M. M.]; Departments of Clinical Pharmacology [A. P. J.], Nuclear Medicine [K. J. A. K.], and Clinical Veterinary Sciences [M. S.], Helsinki University, Helsinki 00014, Finland; and Institute of Biomedical Technologies, Consiglio Nazionale delle Ricerche [M. R. N.], and Laboratory of Immunology, Istituto Regina Elena [P. G. N.], Rome 00158, Italy


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increasing evidence suggests that endoglin (CD105) is a new powerful marker of neovascularization in solid malignancies; thus, using breast cancer as a model, we investigated whether targeting of CD105 by monoclonal antibody (mAb) MAEND3 can be used for in vivo imaging of solid tumors. Immunohistochemistry and flow cytometry identified differential expression of CD105 on breast cancer and endothelial cells; in fact, neoplastic cells were weakly and rarely stained by mAb MAEND3, which in contrast, strongly and invariably stained blood vessel endothelia within the breast adenocarcinomas investigated and cultured endothelial cells. Moreover, in contrast to CD31, which currently represents the reference marker to assess angiogenetic activity, CD105 expression was highest in semiconfluent and actively proliferating endothelial cells, and it progressively decreased as cells reached tight confluency and low [3H]thymidine uptake. i.v. administration of 18 MBq of 125I-labeled mAb MAEND3 efficiently imaged spontaneous mammary adenocarcinomas in two dogs; the uptake of radiolabeled mAb was rapid and intense because tumor:background ratios of 8.2:1 and 9.3:1 were reached 8 h after mAb administration, in the absence of immediate and/or long-term clinical side effects. Altogether, our present data suggest that targeting of CD105 on tumor-associated blood vessels may represent a new strategy for in vivo imaging of solid malignancies, regardless of their histological origin.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endoglin (CD105) is a homodimeric cell membrane glycoprotein of Mr ~180,000 composed of disulfide-linked subunits of Mr 95,000 (1) , which has limited species specificity (2) . CD105 acts as an accessory protein that interacts with the ligand-binding receptors of multiple members of the transforming growth factor-ß superfamily (3) . Moreover, CD105 is critical for correct blood vessel development (4) , and mutations in its coding gene cause hereditary hemorrhagic telangiectasia type 1 (5) .

In normal human tissues, CD105 is weakly expressed on erythroid precursors, stromal cells, early fetal B cells (6 , 7) , and activated monocytes (8) , whereas it is strongly expressed on syncytiotrophoblasts of term placenta (8) and on vascular endothelial cells (1) . In several solid human malignancies of the different histotypes investigated, anti-CD105 mAb reacted exclusively with tumor endothelia (9, 10, 11) ; however, a weak staining of neoplastic cells for CD105 was observed in 30% of primary and metastatic cutaneous melanomas (12) and in 22 of 38 ovary carcinomas investigated (13) .

It has long been established that endothelial cells of tumor-associated neovasculature proliferate 20–2000 times more rapidly than endothelial cells of normal tissues (14 , 15) . Moreover, recent in vitro and in vivo studies suggested that CD105 may represent a proliferation-associated marker of endothelial cells. In fact, a higher expression of CD105 was identified on HUVECs4 with protein, RNA, and DNA levels consistent with cellular activation and proliferation (10) . In agreement with this observation, a correlation has been found between levels of CD105 expression and markers of cell proliferation (i.e., cyclin A and Ki-67) in tumor endothelia (16) . These findings, together with the demonstration that a greater intensity of staining for CD105 is detectable in blood vessel endothelia within neoplastic tissues, as compared with those within normal tissues (10 , 17, 18, 19) , indicate that CD105 is a powerful marker of neovascularization in solid malignancies. Consistent with this assumption, CD105 has been shown to represent an ideal marker to quantify tumor angiogenesis and to be an independent predictor of prognosis in patients with breast cancer (20) .

The complexity of the above experimental evidence and the notion that angiogenesis is a common feature of solid malignancies (21) prompted us to investigate whether targeting of CD105 can be used for in vivo imaging of solid tumors, using a spontaneous canine mammary adenocarcinoma model. To this end, we first analyzed the differential expression of CD105 on breast cancer cells and on endothelial cells both in vitro and in vivo; we then tested whether endothelial cell density and proliferation could differentially affect the expression of CD105 compared with that of CD31, which currently represents the "golden standard" for the assessment of angiogenetic activity (22) . The results of our studies indicate that targeting of CD105 on tumor endothelia by mAb MAEND3 (12) represents an efficient procedure to image solid malignancies and may have prospective diagnostic applications.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
mAb and Conventional Antisera.
The antihuman CD105 mAb MAEND3, which cross-reacts with rat endoglin,5 the anti-ICAM-1 (ICAM-1/CD54) mAb 84H10, the anti-LFA-3 (LFA-3/CD58) mAb TS2/9, and the anti-protectin (CD59) mAb MEM-43 were developed and characterized as described elsewhere (12 , 23, 24, 25) . The anti-HLA class I antigen mAb W6/32 was purchased from the American Type Culture Collection. The anti-PECAM-1 (PECAM-1/CD31) mAb TP1/15 was obtained through the V International Workshop on Human Leukocyte Differentiation Antigens. FITC-conjugated F(ab')2 fragments of rabbit antimouse immunoglobulins were purchased from Dako (Glostrup, Denmark); ChromePure mouse IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Cells and Serological and Proliferation Assays.
The human breast carcinoma cell lines SK-BR-3, DAL, and MCF7 (kindly provided by Dr. Paola Nisticò, Istituto Regina Elena, Rome, Italy) and the human EBV-B lymphoblastoid cell line JY, which lacks CD105 expression (12) , were grown in RPMI 1640 supplemented with 10% FCS and 2 mM L-glutamine.

Primary cultures of HUVECs were obtained as described previously (24) and grown in 199 medium (Flow Laboratories, McLean, VA) supplemented with 15% FCS, 2 mM L-glutamine, 24 IU/ml sodium heparin (Roche, Milan Italy), 100 µg/ml bovine endothelial cell growth supplement (Sigma Chemical Co., St. Louis, MO), 8% pooled human AB serum (Flow), and 100 µg/ml gentamicin (Sigma; complete 199 medium). All experiments were performed using HUVECs at third passage in culture.

HUVECs were seeded in T25 tissue culture flasks (Falcon, Lincoln Park, NJ) at 0.5, 1, and 2 x 106 cells/flask. After a 48-h incubation at 37°C, cells were harvested separately and divided in two aliquots; one aliquot was used to assess CD105, CD31, CD54, CD58, CD59, and HLA class I antigens expression by IIF as described previously (26) . The additional aliquot was used for proliferation assays; briefly, HUVECs (2.5 x 104 cells/well) were seeded in triplicate in 96-well U-bottomed plates (Falcon) in complete 199 medium and pulsed with 1 µCi/well of [3H]thymidine (Amersham International, Buckinghamshire, United Kingdom); after a 12-h incubation, plates were frozen and thawed three times, and then cells were harvested on glass fiber strips. [3H]Thymidine incorporation was measured by a MATRIX 96 Direct Beta Counter (Packard, Meridian, CT).

Indirect immunoperoxidase stain was performed using primary MAEND3 mAb at 20 µg/ml and with a commercially available avidin-biotin kit (Vector, Burlingame, CA), as described previously (12) .

RT-PCR Analysis.
Total RNA extraction and RT-PCR were performed as described previously (27) . Amplification of CD105 cDNA was performed at 94°C for 5 min, followed by 28 cycles of 94°C for 75 s, 58°C for 75 s, and 72°C for 2 min, with a final extension at 72°C for 15 min, using sense 5'-TGTCTCACTTCATGCCTCCAGCT-3' and antisense 5'-AGGCTGTCCATGTTGAGGCAGT-3' primers, which resulted in a specific 378-bp amplificate. The integrity of RNA and oligo(dT)-synthesized cDNA was confirmed by amplification of all cDNA samples with ß-actin sense 5'-GGCATCGTGATGGACTCCG-3' and antisense 5'-GCTGGAAGGTGGACAGCGA-3' primers for 21 cycles of 94°C for 1 min, 68°C for 2 min, and 72°C for 2 min, resulting in a specific 615-bp amplificate. Ten µl of each RT-PCR sample were run on a 2% agarose gel and visualized by ethidium bromide staining.

Tissue Samples.
Surgical biopsies of infiltrating ductal breast adenocarcinomas were obtained from patients who had undergone surgery and who had not received treatment in the previous 2 months. Normal breast samples were excised far from transformed tissues. Tissues samples were processed as described previously (12) .

Animals.
A 6-year-old intact female Beagle dog weighing 15 kg (identified as dog A) and an 8-year-old intact female mixed breed dog weighing 18 kg (identified as dog B), each bearing a palpable mammary tumor of about 3 and 1.5 cm, respectively, highly suspicious for malignancy, and with no additional clinical and radiological evidence of disease, were used for imaging studies. Surgical excision of mammary neoplasm was performed 10 days after imaging, and histological diagnosis of grade II ductal mammary adenocarcinoma was obtained for both tumors on formalin-fixed tissues. Dogs received 50-mg tablets of iodine every other day, 8 days before and after imaging.

Gamma Imaging.
Iodination of mAb MAEND3 was performed by the Iodogen 1,3,4,6-tetrachloro-3,6-diphenylglycoluril method (28) . Briefly, 1 mg of mAb MAEND3 and 50 MBq of 125I were mixed in a Iodogen-coated vial. To absorb free iodogen, AG1-X-8 resin was used, and the reaction was filtered through a 0.22 µm filter. The obtained specific activity was 37 MBq/mg.

Gamma imaging was performed with a Picker Prism 2000XP dual-head gamma camera connected to an Odyssey computer (Picker International, Highland Heights, OH). Dogs were sedated with i.m. administration of 40 µg/kg of medetomidine (Dormitor; Orion-Farmos, Turku, Finland), injected i.v. with 18 MBq of 125I-labeled anti-CD105 mAb MAEND3, placed in a prone position between collimators of the gamma camera, and imaged from the ventral side. A low energy ultra-high resolution collimator was used to record the 30 keV gamma energy peak with 30% window. The matrix size was 256 x 256 x 16, and a zoom factor of 1.887 was used, making the pixel size 0.84 mm x 0.84 mm. The tumor sites were analyzed using the region-of-interest technique, with a maximum size of the imaged field of 39.6 cm [height x 31.7 cm (width)]. The tumor:background ratios were calculated, and the background was the contralateral, nonaffected mammillary region. The study had the permission of the local Ethical Committee for animal experiments, and the animals were handled in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (NIH Publication 86-23, Revised 1985); an informed consent was obtained from the dog owners.

Statistical Analysis.
Data were analyzed by the Student’s paired t test using the StatWorks statistical package from Cricket Software, Inc. (Philadelphia, PA). Differences with P < 0.05 were considered statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Differential Expression of CD105 on Human Breast Cancer and Endothelial Cells.
IIF staining of breast cancer cell lines by the anti-CD105 mAb MAEND3 demonstrated a weak expression of CD105 on SK-BR-3 and DAL cells, whereas no expression of CD105 was detected on MCF7 cells (Fig. 1)Citation ; in contrast, mAb MAEND3 strongly stained HUVECs (Fig. 1)Citation . Comparable results were obtained with the anti-CD31 mAb TP1/15 (data not shown). In agreement with serological data, RT-PCR analyses detected CD105 mRNA in HUVECs, in SK-BR-3 and DAL breast carcinoma cells, but not in MCF7 cells (Fig. 2)Citation .



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  		Fig. 1. IIF analysis of CD105 expression on human breast cancer cells and HUVECs. Breast cancer cells and HUVECs (1 x 105) were sequentially incubated with the anti-CD105 mAb MAEND3 (····) or with isotype-matched mouse immunoglobulin (–—) and with FITC-conjugated F(ab')2 fragments of rabbit antimouse immunoglobulin. Then, cells (1 x 104, volume gated) were analyzed by flow cytometry.

 


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  		Fig. 2. RT-PCR analysis of CD105 expression on human breast cancer cells and HUVECs. Total RNA was extracted from breast cancer cells, JY cells, and HUVECs (2 x 106), and RT-PCR analysis was performed using CD105- or ß-actin-specific primers. PCR products were then separated on a 2% agarose gel. One hundred-bp markers were run in the flanking lanes of each gel.

 
Consistent with the in vitro results, mAb MAEND3 rarely and heterogeneously stained neoplastic cells in 5 of 30 infiltrating ductal breast adenocarcinomas investigated by immunohistochemistry; in contrast, blood vessel endothelia were strongly stained in all tumor samples analyzed. Representative data on the expression of CD105 in normal and malignant breast tissues are shown in Fig. 3Citation .



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  		Fig. 3. Immunohistochemical analysis of CD105 expression in human benign and malignant breast tissues. Tissue expression of CD105, as detected by indirect immunoperoxidase on 4-µm acetone-fixed cryostat sections of normal human mammary tissue (A) and primary infiltrating ductal breast adenocarcinomas (B–D), is shown. CD105 expression is confined to the capillaries (arrow) of normal interstitium (A), to the vessels (arrow) surrounding transformed epithelium (B), or to those intermingled with tumor cells nests (; C). Rarely, CD105 is heterogeneously expressed by tumor cells (arrow; D). Sections are counterstained with Mayer’s hematoxylin. A–D, x760.

 
Cell Density- and Proliferation-dependent Expression of CD105 and CD31 on HUVECs.
To investigate whether the expression of CD105 and CD31 on endothelial cells could be differentially affected by cell density and proliferation rate, HUVECs were seeded at scalar concentrations in T25 flasks. After a 48-h incubation at 37°C, cultures of HUVECs were subconfluent, confluent, and tightly confluent, respectively (data not shown), and their DNA synthesis decreased accordingly (Fig. 4B)Citation . The intensity of staining for CD105 was highest on subconfluent and actively proliferating HUVECs, and it progressively decreased on more confluent and slowly proliferating cells (Fig. 4)Citation . In contrast to CD105, the intensity of staining for CD31 was highest on tightly confluent and slowly proliferating HUVECs, and it gradually decreased on less confluent and actively proliferating cells (Fig. 4)Citation . The mean ± SD of mean fluorescence intensity values, obtained in three independent experiments, were 560 ± 83, 441 ± 107, and 329 ± 55 for CD105 expression on subconfluent, confluent, and tightly confluent cultures of HUVECs, respectively (Fig. 4)Citation , and were significantly (P < 0.05) different among them (Fig. 4)Citation . In contrast, the mean ± SD of mean fluorescence intensity values for CD31 expression were 204 ± 26, 241 ± 20, and 298 ± 19 on subconfluent, confluent, and tightly confluent cultures of HUVECs, respectively, and were significantly (P < 0.05) different on tightly confluent compared with subconfluent cells (Fig. 4)Citation .



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  		Fig. 4. Cell density- and proliferation-dependent expression of CD105 and CD31 on HUVECs. Subconfluent, confluent, and tightly confluent cultures of HUVECs were harvested and divided in two aliquots. A, cells were washed twice with HBSS and sequentially incubated with the anti-CD105 mAb MAEND3 ({blacksquare}), the anti-CD31 mAb TP1/15 (), the anti-HLA class I antigens mAb W6/32 ({square}), or the anti-CD54 mAb 84H10 () and with FITC-conjugated F(ab')2 fragments of rabbit antimouse immunoglobulin. Then, cells (1 x 104, volume gated) were analyzed by flow cytometry. Data represent the means of mean fluorescence intensity values obtained in three independent experiments; bars, SD. *, P < 0.05 versus confluent or tightly confluent HUVECs; **, P < 0.05 versus tightly confluent HUVECs; °, P < 0.05 versus tightly confluent HUVECs. B, cells were plated in triplicate in 96-well, U-bottomed plates, pulsed with 1 µCi/well of [3H]thymidine for 12 h, and harvested; incorporated radioactivity was measured by a beta counter. Data represent the means of [3H]thymidine uptake obtained in three independent experiments; bars, SD. *, P < 0.05 versus confluent or tightly confluent HUVECs; **, P < 0.05 versus tightly confluent HUVECs.

 
Cell density and proliferation rate of HUVECs did not affect the cell membrane expression of HLA class I antigens and of CD54; the mean ± SD of mean fluorescence intensity values were 62 ± 12, 67 ± 12, and 75 ± 15 for HLA class I antigens and 34 ± 8, 24 ± 11, and 22 ± 11 for CD54 on subconfluent, confluent, and tightly confluent cultures of HUVECs, respectively (Fig. 4)Citation . Moreover, the constitutive expression of neither CD58 nor CD59 were affected by cell density and proliferation rate of HUVECs (data not shown).

In Vivo Imaging of Canine Mammary Adenocarcinomas by 125I-Labeled anti-CD105 mAb MAEND3.
To investigate whether targeting of CD105 can be used for in vivo imaging of angiogenetic malignancies, two dogs with spontaneous mammary tumors were injected i.v. with 125I-labeled mAb MAEND3 and imaged after 8 h to detect early uptake of radiolabeled mAb at tumor site. Ten days after imaging procedures, mammary neoplasms were surgically excised and diagnosed as ductal mammary adenocarcinomas. Fig. 5Citation shows that the uptake of radiolabeled mAb in the tumor area of both dogs was rapid and intense; in fact, the tumor:background ratios were 8.2:1 and 9.3:1 for dogs A and B, respectively. Consistent with partial in vivo dehalogenation of radiolabeled mAb, radioactivity was also found in the urinary bladders of both dogs (Fig. 5)Citation . Stomach and heart were identified during the imaging of dog A (Fig. 5A)Citation ; these organs were not detected in dog B (Fig. 5B)Citation because they were outside the imaging field. Neither dog showed systemic side effects during 3 months of follow-up after the imaging procedures.



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  		Fig. 5. In vivo imaging of canine mammary adenocarcinomas by 125I-labeled anti-CD105 mAb MAEND3. Two dogs (A and B) with spontaneous mammary carcinomas were injected i.v. with 18 MBq of 125I-labeled anti-CD105 mAb MAEND3. After 8 h, dogs were placed in a prone position between collimators of a gamma camera and imaged for 45 min from the ventral side. Arrows, the uptake of radiolabeled mAb in the mammillary area with breast tumor. The calculated tumor:background ratios were 8.2:1 (dog A) and 9.3:1 (dog B). , urinary bladder (dogs A and B); •, stomach; and {blacktriangleup}, heart (dog A) were also visualized.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, using an in vivo model of spontaneous canine mammary adenocarcinoma, we report the first evidence that targeting of CD105, which has the highest expression on actively proliferating endothelial cells, could represent an effective strategy to image solid malignancies.

The observation that CD105 is weakly or not expressed on human breast cancer cells, both in vitro and in vivo, expands previous evidence demonstrating that neoplastic cells of solid tumors of different histotypes express negligible levels of CD105 (9, 10, 11, 12) . However, the strong expression of CD105 that we invariably found on endothelial cells, compared with breast cancer cells, suggested that CD105 may represent a useful target for in vivo imaging of breast cancer, regardless of its presence and level of expression on neoplastic cells.

The notion that endothelial cells actively proliferate in tumor blood vessels (14) and our demonstration that CD105 expression, contrary to that of CD31, is highest on rapidly proliferating endothelia, strongly suggested that CD105 is a better target than CD31 for in vivo imaging of vascularized tumors. Additionally, the finding that culture conditions of endothelial cells differentially affect CD105 and CD31 expression does not seem surprising. In fact, higher levels of CD105 on actively proliferating endothelia are likely required for the angiogenetic activity of transforming growth factor-ß (4) . On the other hand, higher levels of CD31 on tightly confluent endothelial cells are consistent with its function as a cell-cell adhesion molecule, predominantly localized at intercellular junctions (29) .

The clinically relevant finding of this study is that tumor imaging by the anti-CD105 mAb MAEND3 seems to represent a sensitive and rapid approach to identify malignant lesions through the targeting of tumor-associated angiogenesis. In fact, although imaged dogs were lying between the collimators of the gamma camera, the strong uptake of 125I-labeled mAb MAEND3 by breast tumors allowed us reach an optimal tumor:background ratio in both animals, and in a rather short time. Moreover, the lack of clinical side effects in imaged dogs during 3 months of follow-up provides preliminary evidence that targeting of CD105 on tumor-infiltrating blood vessels may represent a diagnostic procedure that could be safely used in the clinic.

At present, diagnostic imaging for primary, metastatic, and/or occult solid malignancies uses three main classes of compounds: mAb recognizing different tumor-associated antigens, ligands (e.g., hormones and substrates) for receptors that are expressed on neoplastic cells of specific histotypes (30) , and lipophilic (e.g., methoxy isobutyl isonitrile) and cationic (e.g., thallium-201) tracers (31) . The major practical disadvantages of the first approach derive from the substantial tumor specificity of different tumor-associated antigens, by their intra- and interlesional heterogeneity, and by the possible in vivo regulation of their expression by tumor microenvironmental factors (32) . As far as ligands for tumor-associated receptors, disadvantages are represented by the tumor-histotype specificity and by the levels of expression of their receptors (33) , whereas viability and biochemical and metabolic characteristics of neoplastic cells affect the uptake of lipophilic and cationic tracers (31 , 34) . Additionally, all of these compounds need to cross the blood-tissue barrier to reach neoplastic cells in sufficient amounts for efficient tumor imaging. Thus, at variance with currently used tumor targets, CD105 shows several potential major advantages including: (a) lack of tumor-histotype specificity; (b) independence from its expression on neoplastic cells; (c) prompt accessibility of malignant lesions through the blood stream; and (d) overexpression in the largest majority of solid malignancies because of tumor-associated neovascularization (21) .

It is well known that angiogenesis plays a crucial role in tumor growth (35) ; thus, mAb to endothelia-associated antigens (36, 37, 38) , as well as to CD105 (39 , 40) , have been used to target immunotoxins at tumor sites to lower blood supply to neoplastic cells. However, in light of the high uptake of mAb MAEND3 that we have obtained at tumor sites in vivo, an additional intriguing immunotherapeutic approach could rely on the ability of anti-CD105 mAb to activate complement cytotoxicity and/or antibody-dependent cellular cytotoxicity of tumor endothelia.

Additional preclinical in vivo studies (i.e., comparative uptake of a control irrelevant mAb, analysis of blood pool decay, immunoreactive fraction of circulating mAb, and extent of in vivo deiodination) are clearly required to extend the observations reported in this study; nevertheless, our present data indicate that CD105 may represent an optimal target for diagnostic purposes and possibly for therapeutic approaches in highly vascularized malignant diseases, regardless of their histological origin.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Eugenio Borsatti for critical readings of the manuscript and for useful comments.


   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 This work was supported by the Progetto Ricerca Finalizzata awarded by the Italian Ministry of Public Health and by the Associazione Italiana per la Ricerca sul Cancro. Back

2 E. Fonsatti and A. P. Jekunen contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Advanced Immunotherapy Unit, Centro di Riferimento Oncologico-Istituto Nazionale di Ricovero e Cura a Carattere Scientifico, Via Pedemontana Occidentale, 12, Aviano, Italy 33081. Phone: 39-0434-659342; Fax: 39-0434-659566; E-mail: mmaio{at}ets.it Back

4 The abbreviations used are: HUVEC, human umbilical vein endothelial cell; mAb, monoclonal antibody; ICAM-1, intercellular adhesion molecule-1; LFA-3, leukocyte function associated molecule-3; PECAM-1, platelet endothelial cell adhesion molecule-1; IIF, indirect immunofluorescence; RT-PCR, reverse transcription-PCR. Back

5 M. Maio, unpublished observations. Back

Received 10/ 4/99; revised 2/16/00; accepted 2/16/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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