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[Cancer Research 64, 2039-2046, March 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Human T-Cell Lymphotropic Virus Type I-Infected Cells Extravasate through the Endothelial Barrier by a Local Angiogenesis-Like Mechanism

Ali Bazarbachi1, Raghida Abou Merhi1, Antoine Gessain2, Rabih Talhouk3, Hilda El-Khoury4, Rihab Nasr1, Olivier Gout5, Rita Sulahian3, Fadia Homaidan6, Hugues de Thé7, Olivier Hermine8 and Marwan E. El-Sabban4

1 Department of Internal Medicine, 3 Department of Biology, 4 Department of Human Morphology, and 6 Department of Physiology, American University of Beirut, Lebanon; 2 Unité d’Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Paris, France; 5 Fondation Rotschild, Paris, France; 7 Unité Propre de Recherche 9051, Centre National de la Recherche Scientifique, Laboratoire associé No. 11 du comité de Paris de la Ligue contre le Cancer, conventionné par l’Université Paris VII, Hôpital St. Louis, Paris, France; 8 Department of Clinical Hematology, Necker Hospital, Paris, France


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extravasation of tumor cells through the endothelial barrier is a critical step in cancer metastasis. Human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia/lymphoma (ATL) is an aggressive disease characterized by visceral invasion. We show that ATL and HTLV-I-associated myelopathy patients exhibit high plasma levels of functional vascular endothelial growth factor and basic fibroblast growth factor. The viral oncoprotein Tax transactivates the promoter of the gap-junction protein connexin-43 and enhances gap-junction-mediated heterocellular communication with endothelial cells. The interaction of HTLV-I-transformed cells with endothelial cells induces the gelatinase activity of matrix metalloproteinase (MMP)-2 and MMP-9 in endothelial cells and down-regulates the tissue inhibitor of MMP. This leads to subendothelial basement membrane degradation followed by endothelial cell retraction, allowing neoplastic lymphocyte extravasation. We propose a model that offers a mechanistic explanation for extravasation of HTLV-I-infected cells: after specific adhesion to endothelia of target organs, tumor cells induce a local and transient angiogenesis-like mechanism through paracrine stimulation and direct cell-cell communication with endothelial cells. This culminates in a breach of the endothelial barrier function, allowing cancer cell invasion. This local and transient angiogenesis-like sequence that may facilitate visceral invasion in ATL represents a potential target for ATL therapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metastasis is the colonization of selective, secondary organ sites by dissemination of tumor cells from their primary location (reviewed in Refs. 1, 2, 3 ). Early steps in the metastatic cascade include the shedding of cancer cells from the primary neoplasm, the invasion of extracellular matrices, and the intravasation of tumor cells into lymph and/or blood vessels (reviewed in Refs. 4, 5, 6 ). On circulation, many cancer cells are eradicated by physical forces exerted on them during passage through the microvasculature of secondary organs (reviewed in Ref. 7 ) and by host defense mechanisms. The few surviving tumor cells are thought to colonize selective organ sites by recognizing organ-specific adhesion molecules expressed on the surfaces of vascular endothelial cells (1, 2, 3 , 8, 9, 10) . This step is usually followed by cancer cell extravasation and penetration into the secondary organ. However, little is known about the mechanism by which cancer cells traverse this endothelial barrier.

Angiogenesis is critical for the growth of solid tumors (reviewed in Refs. 11, 12, 13, 14 ) or hematological malignancies (15, 16, 17) and is induced by angiogenic factors such as vascular endothelium growth factor (VEGF) and basic fibroblast growth factor (bFGF; Refs. 18 , 19 ) secreted by the tumor and/or stromal cells. Angiogenesis involves activation of endothelial matrix metalloproteinases (MMPs) involved in the degradation of subendothelial basement membrane, which is a prerequisite for directed migration and proliferation of endothelial cells (reviewed in Ref. 20 ).

Human T-cell lymphotropic virus type I (HTLV-I) is the causative agent of adult T-cell leukemia/lymphoma (ATL; Ref. 21 ) and tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM; Ref. 22 ). ATL is an aggressive malignancy of mature activated T-cells characterized by frequent visceral invasion by tumor cells (reviewed in Ref. 23 ). On the other hand, TSP/HAM is characterized by central nervous system invasion by HTLV-I-infected lymphocytes (24) . The invasive nature of HTLV-I-associated diseases strongly suggests a possible interaction between HTLV-I-infected cells and endothelial cells. In that sense, we have recently shown that HTLV-I-positive cells communicate with endothelial cells through both paracrine stimulation and direct gap-junction-mediated heterocellular communication (25) . Indeed, ATL-derived cells specifically secrete high levels of the angiogenic factors VEGF and bFGF, induce endothelial tube formation in vitro, and establish functional gap-junction-mediated communication with endothelial cells. These gap junctions are clusters of transmembrane channels composed of structurally related proteins known as connexins (26) , which play an important role in tissue organization and cellular differentiation (27, 28, 29) .

In this report we demonstrate that plasma from ATL and TSP/HAM patients contains very high levels of VEGF or bFGF, which suffice to induce angiogenesis. We also show that the HTLV-I oncoprotein Tax transactivates the connexin-43 promoter and enhances the formation of functional gap junctions with endothelial cells. Interaction of HTLV-I-transformed cells with endothelial cells induces the production of functional MMPs by endothelial cells, resulting in the degradation of subendothelial basement membrane and allowing extravasation of transformed lymphocytes between endothelial cells. Using ATL as a model, we propose that after adhesion of tumor cells to endothelia of the target organ, tumor cells induce an angiogenesis-like mechanism that leads to a breach in the endothelial barrier and allows cancer cells to invade.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Antibodies.
HuT-102 and MT-2 cells are HTLV-I-transformed CD4+ T-cell lines that constitutively express HTLV-I virus. The HTLV-I-negative CD4+ T-cell lines CEM, Molt-4, and Jurkatt were used as control cells. Cells were grown in RPMI 1640 containing 10% heat-inactivated FCS (Life Technologies, Inc., Paisley, United Kingdom) and antibiotics. A cell concentration of 2 x 105 cells/ml was chosen for seeding for all experiments unless otherwise stated. The human cell lines ECV-304, HeLa, and MCF-7 were grown in the same medium. Human aortic endothelial cells (HAECs; Clonetics, San Diego, CA) were grown in medium provided by the supplier at an initial concentration of 4 x 105 cells/ml.

Rabbit polyclonal antibodies to MMP tissue inhibitor-1 were obtained from Serotec Ltd. (Oxford, United Kingdom). Mouse monoclonal anti-Tax (168-A51) was obtained from the NIH AIDS Research and Reagent Program. Rabbit antiactin (Santa Cruz Biotechnology, San Jose, CA) antibodies were used to assess protein loading.

RNA Isolation and Reverse Transcription-PCR.
cDNA was synthesized from 500 ng of total cellular RNA and isolated by use of Trizol reagent (Life Technologies, Inc.) with reverse transcriptase (Super script II; Life Technologies, Inc.). The cDNA was then amplified by PCR using Taq DNA polymerase (Life Technologies, Inc.). We used PCR primers that recognize all VEGF isoforms, MMP-2, MMP-9, and MMP-14, and ß-actin-specific primers. Amplification of the housekeeping gene ß-actin was used to verify RNA quality and reverse transcription-PCR techniques. The PCR conditions used to amplify VEGF, MMP-2, MMP-9, MMP-14, and ß-actin consisted of a precycle of 95°C for 3 min followed by 35 cycles consisting of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. A final extension at 72°C for 7 min was then performed. The primers used were as follows: ß-actin forward primer, 5'-CGC CTG CGC CTG GTC GTC GAC A-3'; ß-actin reverse primer, 5'-GTC ACG CAC CGA TTT CCC GCT-3'; VEGF forward primer, 5'-TCG GGC CTC CGG AAA CCA TGA-3'; VEGF reverse primer, 5'-CCT GGT GAG AGA TCT GGT TC-3'; MMP-2 forward primer, 5'-GTG CTG AAG GAC ACA CTA AAG AAG A-3'; MMP-2 reverse primer, 5'-TTG CCA TCC TTC TCA AAG TTG TAG G-3'; MMP-9 forward primer, 5'-CAC TGT CCA CCC CTC AGA GC-3'; MMP-9 reverse primer, 5'-GCC ACT TGT CGG CGA TAA GG-3'; MMP-14 forward primer, 5'-CGC TAC GCC ATC CAG GGT CTC AAA-3'; and MMP-14 reverse primer, 5'-CGG TCA TCA TCG GGC AGC ACA AAA-3'. PCR products were electrophoresed in 1.5% agarose gels and visualized with ethidium bromide staining.

Western Blot Analysis.
Approximately 107 cells were solubilized at 4°C in lysis buffer consisting of 0.125 M Tris-Cl (pH 6.8), 2% SDS, 2.5% ß-mercaptoethanol, and 10% glycerol. Samples were loaded onto a 12% SDS-polyacrylamide gel, subjected to electrophoresis, and transferred to nitrocellulose membranes. After blocking of the membrane in 5% skim milk in Tris-buffered saline containing 0.05% Tween 20, the blots were incubated with specific antibodies. The blots were washed, and protein bands were visualized by chemiluminescence (Amersham, Buckinghamshire, United Kingdom).

ELISA.
Blood was obtained, after informed consent, from 17 ATL patients (11 with acute ATL, 3 with lymphoma, 2 with chronic ATL, and 1 with smouldering ATL), 29 TSP/HAM patients, and 9 HTLV-I seronegative healthy control individuals. Plasma was prepared by centrifugation of blood collected in citrate-containing tubes and stored at -80°C. VEGF and bFGF protein levels in patient plasmas or in the cell-free supernatants were determined by ELISA (R&D Systems, Minneapolis, MN) as recommended by the manufacturer.

Luciferase Assays.
Connexin-43-Luc construct in pGL-2 (provided by Dr. Stephen Lye, McGill University, Montreal, Canada) was cotransfected into HeLa cells with the internal control Renilla luciferase and either pSG5-Tax or empty vector using Lipofectamine plus (Life Technologies, Inc.) according to the manufacturer’s recommendations. Luciferase activity was quantified 24 h later by the Luciferase Assay System (Promega, Madison, WI). Values were normalized with the Renilla activity.

Matrigel-Induced Capillary Tube Formation.
Endothelial cells (4 x 105) were seeded in 12-well plates precoated with 200 µl/well growth factor-reduced Matrigel (Becton Dickinson, San Jose, CA) and cultured for 18 h. Induction of endothelial cell tube formation was assessed after incubation with test plasma at 37°C for 48 h. Function-blocking anti-VEGF antibodies (10 ng/ml; Santa Cruz Biotechnology) or MMP inhibitor (5 µM 1,10-phenanthroline; Sigma, St. Louis, MO) were added concomitantly to the plasma where indicated. Cells were then stained with 0.5 µg/ml Hoechst (Molecular Probes, Eugene, OR) for 10 min and observed by fluorescence microscopy.

Functional Assay of Adhesion and Communication.
HeLa cells were transiently transfected with pSG5-Tax or empty vector using Lipofectamine plus (Life Technologies, Inc.) according to the manufacturer’s recommendations. Transfected HeLa cells or HTLV-I-positive cells were labeled with the membrane-permeable dye calcein-AM (Molecular Probes) as described previously (25) . Confluent monolayers of endothelial cells grown in 35-mm Petri dishes were cocultured for 1 h at 37°C with calcein-labeled cells. Unbound cells were removed by a single wash. Endothelial cells and adhered HuT-102 or HeLa cells were then released from the growth surface with trypsin/EDTA in HBSS, washed once with HBSS, fixed in 4% formaldehyde in PBS, and analyzed by flow cytometry (FACScan; Becton Dickinson). Alternatively, confluent monolayers of endothelial cells grown in coverslip-fitted 35-mm Petri dishes were cocultured with calcein-labeled HuT-102 cells and observed under fluorescence microscopy at different time intervals.

Gelatin Zymography.
Endothelial cells, cocultured with HTLV-I-positive or -negative cells or with their cell-free supernatant for 72 h, were washed three times in PBS before their protein extracts were analyzed by zymography. The gap-junction inhibitor 18-{alpha}-G (Sigma; 50 µM) was added to the cell coculture when indicated. Proteins (20 µg) from cell extracts in 2x sample buffer [0.25 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and 0.01% bromphenol blue] were electrophoresed on 10% polyacrylamide gel copolymerized with 1 mg/ml gelatin (Sigma), washed twice for 30 min with 2.5% Triton X-100 (Sigma), and then incubated overnight in substrate buffer [150 mM NaCl, 5 mM CaCl2, 50 mM Tris-Cl (pH 8)] at 37°C with gentle shaking. The gels were then stained with 0.25% Coomassie Brilliant Blue R-250 in 45% methanol-10% acetic acid-45% water for 4 h at 37°C with gentle shaking and destained in doubly distilled water.

Invasion Assay.
Six-well tissue culture plates were fitted with inserts (8 µm pore size) coated with growth factor-reduced Engelbreth Holm-Swarm (Matrigel, 125 ng/cm2). Endothelial cells were grown in the inserts to confluency and then seeded with calcein-labeled tumor cells. After 24 h of coculture, inserts were removed, and the endothelial cell layer was gently removed with a cotton swab. The membrane of the insert was then removed, mounted on a microscopic slide, and examined by fluorescence microscopy. Fluorescent tumor cells that successfully invaded through the endothelial layer were photographed and counted. At least four fields at x20 magnification were counted per experiment (n = 3), averaged, and reported as the mean ± SD. Statistical comparison was performed with a t test with unequal variances.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
High Plasma Levels of Functional Angiogenic Factors in HTLV-I-Associated Diseases.
We evaluated plasma levels of VEGF and bFGF in 17 ATL patients, 29 TSP/HAM patients, and 9 HTLV-I seronegative healthy controls. Patients with HTLV-I-associated diseases exhibited significantly elevated levels of VEGF (mean ± SD, 243 ± 277 pg/ml) and bFGF (17 ± 14 pg/ml) compared with HTLV-I seronegative healthy controls (mean VEGF, 29 ± 3 pg/ml; bFGF below assay detection limit in 8 donors, and 10 pg/ml in 1 donor; P = 0.025 and 0.001, respectively; Fig. 1ACitation ). We observed no significant difference in mean plasma VEGF levels between ATL (234 ± 241 pg/ml) and TSP/HAM patients (248 ± 299 pg/ml; P = 0.871) or in mean plasma bFGF levels (ATL patients, 22 ± 16 pg/ml; TSP/HAM patients, 14 ± 12 pg/ml; P = 0.074), although ATL patients had slightly, but nonsignificantly higher bFGF plasma levels compared with TSP/HAM patients.



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  		Fig. 1. High plasma levels of functional angiogenic factors in patients with adult T-cell leukemia/lymphoma (ATL) and tropical spastic paraparesis/human T-cell lymphotropic virus type I associated myelopathy (TSP/HAM). A, ELISA measurement of plasma vascular endothelium growth factor (VEGF) and basic fibroblast growth factor (bFGF) in 17 ATL patients (mean VEGF, 234 ± 241 pg/ml; mean bFGF, 22 ± 16 pg/ml), 29 TSP/HAM patients (mean VEGF, 248 ± 299 pg/ml; mean bFGF, 14 ± 12 pg/ml), and 9 human T-cell lymphotropic virus type I (HTLV-I) seronegative healthy controls (Ctrl; mean VEGF, 29 ± 3 pg/ml; bFGF undetectable in 8 donors; 10 pg/ml in 1 donor). The right panels show the mean ± SD; the left panels show individual patients values. The amounts of VEGF and bFGF proteins in the samples were calculated by use of a reference curve established from serial dilutions of recombinant human VEGF and bFGF proteins. All experiments were performed in triplicate and repeated a minimum of two times. B, Hoechst nuclear staining of endothelial cells [human aortic endothelial cells (HAEC) or ECV-304 cells] plated on growth factor-reduced Matrigel in the presence of plasma from one HTLV-I seronegative healthy control, two ATL patients, and one TSP/HAM patient (top panels). Where indicated, anti-VEGF blocking antibodies were added to plasma from one ATL patient (bottom panels).

 
The biological activity of elevated plasma levels of angiogenic factors in patients with HTLV-I-associated diseases was evaluated on endothelial cells in vitro. In the presence of eight different ATL plasma samples and six different TSP/HAM plasma samples, HAEC and ECV-304 cells formed a network of capillary-like tubules with multicentric junctions (Fig. 1BCitation and not shown). These experiments demonstrate that the circulating angiogenic factors in the plasma of patients with HTLV-I-associated diseases are sufficient to induce biological function. Minimal or no effect was observed with plasma from HTLV-I seronegative healthy controls (Fig. 1B)Citation . Addition of function blocking anti-VEGF antibody attenuated (two patients) or abolished (four patients) the angiogenic activity of plasma from four tested ATL and two tested TSP/HAM patients (Fig. 1BCitation and not shown), implicating VEGF in the induction of angiogenesis by plasma from HTLV-I-infected patients.

Tax Expression Increases Adhesion and Gap-Junction Communication with Endothelial Cells.
Coculture experiments between leukemic cells and the endothelium revealed that HTLV-I-transformed cells adhere and communicate with endothelial cells, in agreement with our previous report (25) . Coculture of HAECs with calcein-labeled HuT-102 cells produced a progressive transfer of fluorescence from the leukemic cells to the HAECs (Fig. 2A)Citation . Indeed, cocultures at HuT-102:HAEC ratios 1:1 and 2:1 for 1 h produced an increase in mean fluorescence intensity in the HAECs to 37 and 92, respectively (Fig. 2ACitation , graphs c and d). The addition of the gap-junction inhibitor heptanol sharply reduced endothelial cell fluorescence from 92 to 44 (compare graphs d and e in Fig. 2ACitation ), confirming the specificity of the assay. Similar results were observed in coculture experiments between calcein-labeled HuT-102 cells and ECV-304 cells, in which a progressive transfer of fluorescence from the leukemic cells to the endothelial cells was observed (Fig. 2BCitation , compare panels f and g to e). This transfer was significantly inhibited by the addition of 50 µM of the gap-junction inhibitor 18-{alpha}-G (Fig. 2BCitation , compare panels h and g).



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  		Fig. 2. Human T-cell lymphotropic virus type I (HTLV-I)-positive cells establish functional gap junctions with endothelial cells: adhesion and dye transfer from HTLV-I-transformed cells to endothelial cells. A, flow cytometric analyses of cocultures of unlabeled human aortic endothelial cells (HAEC; a) with calcein-labeled HTLV-I-positive cells (HuT-102*; b). The relative height of the peak for HuT-102 cells compared with that of HAECs is a measure of cell adhesion. The increase in the mean fluorescent intensity (MFI) of endothelial cells reflects dye transfer through gap junctions from the leukemic cells. Endothelial cells were cocultured for 1 h with HuT-102* at ratios of 1:1 (c); 1:2 (d); or 1:2 (e) in the presence of the gap-junction inhibitor heptanol (1 mM). Histogram analysis of mean fluorescent intensity of HAECs under these different conditions is presented. B, phase-contrast (a–d) and fluorescence microscopy (e–h) of cocultures of unlabeled endothelial cells (ECV-304) with the calcein-labeled HTLV-I-positive cells (HuT-102) at initiation of coculture (a and e), 30 min of coculture (b and f), 1 h of coculture (c and g), and 1 h of coculture with the gap-junction inhibitor 18-{alpha}-G (d and h). Panel d represents an overlay of phase-contrast and fluorescence microscopy for a better identification of the two cell types. In panels f–h, intensely labeled cells represent calcein-labeled tumor cells, whereas endothelial cells can be seen in the background. Fluorescent endothelial cells (panels g and h) on coculture with tumor cells indicate progressive dye transfer through gap junctions.

 
We investigated the role of HTLV-I viral genes in the expression of functional gap junctions by ATL-derived cells. Coculture for 1 h at a cellular ratio of 1:1 of endothelial cells with HeLa cells transfected with the viral transactivator Tax produced a significant up-regulation of both cell-cell adhesion and gap-junction-mediated heterocellular communication between the two cell types (Fig. 3A)Citation . The endothelial cell mean fluorescence intensity significantly increased on coculture with HeLa-Tax cells compared with coculture with HeLa cells, indicating that Tax expression in Tax-transfected cells (Fig. 3B)Citation leads to enhancement in the functional gap-junction activity. These results directly implicate viral gene expression in the formation of functional gap junctions with endothelial cells. Because HTLV-I-transformed cells were shown to express the gap-junction protein connexin-43 (25) , we investigated the role of Tax expression on the transcriptional activity of the connexin-43 gene. HeLa cells were cotransfected with Tax, and a reporter plasmid in which the promoter region of the gap-junction protein connexin-43 controls the luciferase cDNA. Tax expression induced a 6-fold increase in connexin-43 promoter activity as measured by the luciferase assay (Fig. 3C)Citation . Furthermore, Tax expression led to a significant increase in the number of VEGF transcripts in Tax-transfected Molt-4 cells (Fig. 3D)Citation and a significant increase in VEGF secretion in Tax-transfected HeLa cells (Fig. 3E)Citation , confirming our previous findings that HTLV-I-positive cells synthesize and secrete VEGF, presumably as a result of Tax-induced transcriptional activation of the VEGF gene (25) .



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  		Fig. 3. Tax increases adhesion and gap-junction communication with endothelial cells and transactivates the connexin-43 promoter. A, adhesion and dye transfer from Tax-transfected HeLa cells to endothelial cells (ECV-304). Flow cytometry analyses of cocultures of unlabelled endothelial cells (EC) with calcein-labeled (*) HeLa cells transfected with Tax (HeLa Tax) or empty vector (HeLa {phi}). The relative height of the peak for HeLa cells compared with that for the endothelial cells is a measure of cell adhesion. The increase in the mean fluorescence intensity (MFI) of endothelial cells reflects dye transfer through gap junctions from HeLa cells. Endothelial cells were cocultured for 1 h with calcein-labeled HeLa cells at a ratio of 1:1. The bottom histogram is an overlay to demonstrate the shift in endothelial cell MFI on coculture with calcein-labeled HeLa-Tax. The graph represents the mean and SD (bars) MFI from two independent experiments with endothelial cells cocultured with HeLa-Tax* or HeLa-{phi}*. B, Western blot analysis using anti-Tax antibodies. C, connexin 43-Luc (Cx43-Luc) construct in pGL-2 corresponding to the luciferase gene under the control of the connexin-43 promoter was cotransfected into HeLa cells with the internal control Renilla luciferase and either pSGTax or empty vector. Luciferase activity was quantified 24 h later and normalized with the Renilla luciferase activity. D, Molt-4 cells transfected with Tax (Molt-4 Tax) or empty vector were analyzed for vascular endothelium growth factor (VEGF) mRNA expression by reverse transcription-PCR using 100 ng of total RNA. E, HeLa cells were transfected with pCMV-Tax or empty vector. Levels of vascular endothelium growth factor (VEGF) in the cell supernatant were measured with a commercial ELISA.

 
HTLV-I-Transformed Cells Induce MMP Activity by Endothelial Cells.
HTLV-I-transformed cells interact with endothelial cells through both paracrine stimulation and direct gap-junction-mediated communication (25) . We tested the effect of this dual interaction on endothelial cells function by assessing the production of MMP transcripts and gelatinase activity by endothelial cells after exposure to HTLV-I-transformed cells or their cell-free supernatants. Confluent ECV-304 cells in culture produce low amounts of MMP-2 and MMP-9 transcripts (Fig. 4ACitation and not shown).



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  		Fig. 4. Human T-cell lymphotropic virus type I (HTLV-I)-transformed cells induce matrix metalloproteinase (MMP) activity by endothelial cells through paracrine stimulation and gap-junction-mediated communication. A, endothelial cells (ECV-304; EC) were cocultured with 72-h conditioned cell-free supernatant from HuT-102 or CEM cells and analyzed for MMP-9 and MMP-14 mRNA expression by reverse transcription-PCR using 100 ng of total RNA. B, endothelial cells were cocultured with 72-h conditioned cell-free supernatant from HuT-102, MT2, CEM, or Jurkatt cells and analyzed for MMP-2 and MMP-9 activity by zymogram. Controls are fetal bovine serum (Positive Ctrl), the third PBS wash after coculture (HuT-102 Wash), and extracts from endothelial cells incubated in culture medium alone (Negative Ctrl; see "Materials and Methods"). C, detection of endothelial cell-associated tissue inhibitor of MMP (TIMP-1) protein. Endothelial cells (EC) were cocultured with 72-h conditioned cell-free supernatant from HuT-102 or CEM cells. Total cell extracts were analyzed by Western blot using anti-TIMP-1-specific antibodies. D, endothelial cells were cocultured with supernatant-free HuT-102 or CEM cells at a cellular ratio of 1:2 and analyzed for MMP-2 and MMP-9 activity by zymogram. The gap-junction inhibitor 18-{alpha}-G was added at 50 µM where indicated. E, HuT-102 cells were cocultured with 100 µM 18-{alpha}-G and analyzed for vascular endothelium growth factor (VEGF) mRNA expression by reverse transcription-PCR. F and G, effect of HTLV-I-positive cells or cell-free supernatant on Matrigel-induced tube formation of endothelial cells in vitro. Human aortic endothelial cells (HAEC; F) or ECV-304 cells (G) were plated on growth factor-reduced Matrigel in the presence of culture medium, 72-h HuT-102 or CEM cell-free supernatant, or HuT-102 cells (cellular cocultures are shown after 1 and 72 h of incubation). Plates were photographed under fluorescence microscopy with Hoechst nuclear staining. H, effect of the metalloproteinase inhibitor 1,10-phenanthroline on Matrigel-induced tube formation of endothelial cells in vitro. Endothelial cells were plated on growth factor-reduced Matrigel in the presence of culture medium (left), 72-h HuT-102 cell-free supernatant (middle), and 72-h HuT-102 cell-free supernatant in the presence of 1,10-phenanthroline (right). Plates were photographed with light microscopy.

 
Paracrine Stimulation.
Endothelial MMP-9 transcript levels were significantly increased after incubation for with 72-h cell-free supernatant from HTLV-I-positive HuT-102 cells but not from HTLV-I-negative CEM cells (Fig. 4A)Citation . In contrast, transcript levels of endothelial MMP-2 were unaffected (not shown). However, endothelial MMP-14 transcript levels (Fig. 4A)Citation were up-regulated after incubation with 72-h cell-free supernatant from HuT-102 cells and significantly down-regulated after incubation with 72-h cell-free supernatant from HTLV-I-negative CEM cells. MMP-14 has been reported to convert proMMP-2 to active MMP-2 (30) . However, proMMP-2 was undetectable by zymography in unstimulated confluent ECV-304 cells, suggesting that cell-free supernatant from ATL-derived cells stimulates translation of preexisting MMP-2 mRNA. Furthermore, production of MMP tissue inhibitor-1 by endothelial cells was significantly down-regulated after incubation with 72-h supernatant from HuT-102 cells and CEM cells (Fig. 4C)Citation . Importantly, zymogram analysis (Fig. 4B)Citation of endothelial extracts clearly indicated that incubation of endothelial cells with 72-h cell-free supernatant from HTLV-I-transformed cells (HuT-102 and MT2) significantly up-regulated MMP-2 activity and, to a lesser extent, MMP-9 activity, whereas incubation with 72-h cell-free supernatant from HTLV-I-negative cells (CEM and Jurkatt cells) led to minimal induction of MMP-2 and MMP-9 activity. These results indicate that HTLV-I-transformed cells stimulate the production and/or activation of functional MMPs by endothelial cells through a paracrine mechanism.

Gap-Junction-Mediated Communication.
Similarly, coculture of endothelial cells with supernatant-free HTLV-I-transformed cells at a 1:2 cellular ratio also up-regulated MMP-2 and MMP-9 activity (Fig. 4D)Citation . This up-regulation was attenuated after treatment with the gap-junction inhibitor 18-{alpha}-G (50 µM), implicating gap-junction-mediated communication in the induction of MMP by endothelial cells (Fig. 4D)Citation . Importantly, the gap-junction inhibitor 18-{alpha}-G (50 µM) had no detectable toxicity on endothelial cells (not shown) and did not affect VEGF transcript level in HuT-102 cells (Fig. 4E)Citation .

Finally, the Matrigel assay clearly demonstrated that in the presence of a 2:1 cellular ratio of supernatant-free HTLV-I-transformed cells or after incubation with 72-h cell-free supernatant, ECV-304 and HAEC endothelial cells formed a network of capillary-like tubules with multicentric junctions, whereas cell-free supernatant from CEM cells had no effect (Fig. 4, F and G)Citation . This in vitro angiogenesis was completely inhibited when an MMP inhibitor, 1,10-phenanthroline (5 µM) was added (Fig. 4H)Citation . Altogether, our results indicate that interaction of HTLV-I-transformed cells with endothelial cells induces an angiogenesis-like mechanism characterized by the production of functional MMPs by endothelial cells, leading to the degradation of subendothelial basement membrane.

HTLV-I-Transformed Cells Invade Endothelial Cells by an Angiogenesis-Like Mechanism.
We then set out to document, using time-lapse photography and video microscopy, extravasation process of HTLV-I-transformed cells in vitro. Confluent monolayers of HAEC or ECV-304 cells were seeded with HuT-102 cells and observed by light microscopy over a period of 8 h. Within 15–60 min of coculture, HuT-102 cells adhered to and established pseudopod junctions with the luminal aspects of endothelial cells [Fig. 5ACitation , top panel (ECV-304) and bottom panel (HAEC); 60 min, arrow]. Most observed HuT-102 cells eventually settled between two adjacent endothelial cells, and the effect of their interaction became noticeable. ATL-derived cells sent cytoplasmic processes in the area where a cleft between two adjacent endothelial cells appeared (Fig. 5ACitation , 120 min, top panel, arrow) indicative of possible extravasation of HTLV-I-transformed cells. Finally, the morphology of endothelial cells became significantly altered [Fig. 5ACitation , top panel (ECV-304) and bottom panel (HAEC); 240 min, arrow]. In contrast, CEM cells exhibited very low overall activity, remained loosely attached to endothelial cells and resulted in no dramatic changes in endothelial cell morphology (Fig. 5A)Citation . The inclusion of 50 µM 18-{alpha}-G throughout the coculture time used for video microscopy impeded most of the morphological alterations observed when HuT-102 cells were cocultured with endothelial cells (not shown). These observations were repeated with the breast cancer-derived cell line MCF-7, which secretes high levels of VEGF. Video microscopy revealed that within 60 min of coculture, MCF-7 cells adhered to and established pseudopod junctions with the luminal aspect of endothelial cells (Fig. 5ACitation , 60 min, arrow). Similar to HuT-102 cells, MCF-7 cells induced endothelial retraction with clear cytoskeletal rearrangement and the creation of a local cleft between two neighboring cells (Fig. 5ACitation , 240 min, white arrow). This "retraction" was not observed between endothelial cells that had no contact with cancer cells.



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  		Fig. 5. Interaction between human T-cell lymphotropic virus type I-transformed cells and endothelial cells. A, confluent monolayers of endothelial cells [ECV-304 or human aortic endothelial cells (HAEC)] were seeded with tumor cells (HuT-102, CEM, or MCF-7) and observed by light microscopy over a period of 8 h. Time sequence images are displayed. 30 min, tumor cells (black arrow) adhere to the luminal aspect of endothelial cells (white arrow); 60 min, HuT-102 and MCF-7 cells establish pseudopods junctions with endothelial cells (arrows); 120 min, HuT-102 and MCF-7 cells start sending cytoplasmic processes in the area where a cleft between two adjacent cells appeared (arrows); 240 min, the morphology of endothelial cells change (black arrows), and a cleft between two endothelial cells become visible (white arrow in MCF-7). B, invasion assay of tumor cells (MCF-7 and HuT-102) through endothelial cell layer. Invasion of HuT-102 cells is attenuated by the gap-junction inhibitor 18-{alpha}-G (18{alpha}G).

 
We assessed the ability of tumor cells to traverse the endothelial cell barrier by use of an invasion assay with a two-compartment model. This overcomes the limitation of video microscopy of cells plated directly on plastic dishes, where the tumor cells do not have a physical space to invade. Both HuT-102 and MCF-7 cells efficiently invaded the endothelial monolayer as judged by the number of fluorescent tumor cells in the abluminal surface of the 35-mm culture filter insert (mean ± SD, 37 ± 18 and 60 ± 20, respectively, after 24 h). Addition of 18-{alpha}-G (100 µM) led to a significant reduction in the number of HuT-102 cells (8 ± 3; P < 0.001) and MCF-7 (21 ± 5; P < 0.05) that invaded the insert. These data highlight the critical role of gap-junction-mediated communication in the invasive potential of ATL-derived and MCF-7 cells.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report we show that patients with HTLV-I-associated diseases have high plasma levels of functional angiogenic factors. The origin of VEGF and bFGF in the plasma of these patients remains to be determined. However, because HTLV-I-transformed cells specifically secrete high levels of these two angiogenic factors (25) , it is most likely that they are produced by the HTLV-I-infected cells, presumably as the result of Tax-dependent transcriptional activation of the VEGF gene (25) . Most HTLV-I-infected leukemic cells express fms-like tyrosine kinase 1, one of the receptors for VEGF (31) . Hence, elevated plasma VEGF levels may contribute to the proliferation of HTLV-I-infected cells through autocrine stimulation of growth and may represent a good target for anti-VEGF therapy.

ATL is characterized by a particularly high frequency of cutaneous and visceral invasion by leukemic cells. A link between high plasma VEGF levels and extranodal involvement in ATL has been suggested (32) . This invasive potential of ATL cells dictates an interaction between leukemic cells and the endothelium. Here we show that ATL-derived cells communicate with endothelial cells through both angiogenic factor-mediated paracrine stimulation and direct gap-junction-mediated heterocellular communication. A role for Tax in this interaction is demonstrated by transcriptional induction of VEGF promoter (25) and connexin-43 promoter and by increased functional heterocellular communication on Tax transfection. This dual interaction between ATL-derived cells and endothelial cells induces the production of functional MMPs by endothelial cells. The gelatinase activity of MMPs leads to the degradation of subendothelial basement membrane and retraction of endothelial cells, allowing extravasation of ATL-derived cells. In TSP/HAM, a similar angiogenesis-like mechanism, triggered by HTLV-I-infected cells, may be responsible for passage of HTLV-I-infected cells through the blood-brain barrier into the central nervous system. Moreover, using the breast cancer-derived cell line MCF-7, which produces high levels of functional VEGF, we showed that these malignant cells also induce morphological modifications of endothelial cells (Fig. 5)Citation . Hence, tumor-cell-induced angiogenesis-like events may represent a general mechanism of tumor cell extravasation.

Several reports have implicated high plasma levels of angiogenic factors (32) , signaling through adhesion molecules, and production of MMPs by tumor cells (33 , 34) in extranodal organ invasion by tumor cells. However, this model does not explain how tumor cells extravasate through the endothelial barrier. Indeed, a generalized response of the endothelium to high plasma levels of VEGF and bFGF may produce detrimental consequences for many physiological functions and does not explain the preference of organ invasion in cancer (35 , 36) . In addition, MMPs produced by tumor cells on the endoluminal side of the endothelial cells will have no access to the subendothelial basement membrane and hence have little relevance in the context of extravasation. Their importance should be emphasized in tissue invasion once a tumor cell has extravasated. Finally, on the specific binding of a tumor cell to the endothelium, three mechanisms potentially influence endothelial cells: local reciprocal paracrine interaction, signaling through adhesion molecules, and the direct exchange of regulatory molecules through gap junctions (37) . Although many studies have been dedicated to elucidate the partners and players of adhesion events between endothelial cells and tumor cells, little is known about the other two mechanisms.

On the basis of our data and the fact that most tested malignant cells produce angiogenic factors, we propose a novel role for these angiogenic factors, at the site of cancer cell extravasation, in cancer metastasis (Fig. 6)Citation : (a) Adhesion of tumor cells to endothelial cells leads to signaling through adhesion molecules and/or through gap-junction-mediated direct cytoplasmic exchange of messages that influence endothelial barrier function. (b) After this interaction, tumor cells and endothelial cells will be at such proximity as to allow for paracrine reciprocal interaction. The binding of angiogenic factors, such as VEGF produced by tumor cells, to their receptors on the endothelial cell surfaces sets a mechanism for transient and localized angiogenesis into motion. (c) This mechanism includes activation of endothelial MMPs involved in the degradation of subendothelial basement membrane. (d) The result of this activation is translated into cytoskeletal rearrangement and de-adhesion of adjacent endothelial cells (38) . This is perceived by the adhered tumor cells as a breach of endothelial cell barrier. (e) Tumor cells will then send processes that will adhere to the subendothelial matrix, and extravasation ensues. The directed migration out of the vessel lumen (extravasation) of tumor cells is driven by chemokines and other degradation products of the extracellular matrix, which are known chemoattractant factors for many cancer cells, that result from the degradation of subendothelial basement membrane. This model, which offers a mechanistic explanation for tumor cell extravasation based on local and transient angiogenic reactions, supports a novel role for angiogenic factors and cell-cell communication in cancer metastasis and could explain the effectiveness of antiangiogenic treatment in preventing tumor cell dissemination and organ invasion (39) .



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  		Fig. 6. Proposed model for cancer cell extravasation.

 


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Kamal Badr for critical reading of this manuscript, and Najeeb Halabi for technical assistance in video microscopy.


   FOOTNOTES
 
Grant support: American University of Beirut Medical Practice Plan and University Research Board, the Lebanese National Research Council, the CNRS, ARECA, HERN, the Programme Franco-Libanais "Coopération pour l’Evaluation et le Développement de la Recherche," the Diana Tamari Sabbagh Research Fund, and the Eli-Lilly International Foundation.

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.

Requests for reprints: Marwan E. El-Sabban, Department of Human Morphologym, Faculty of Medicine, American University of Beirut, P.O. Box 11-0236, Beirut, Lebanon. Phone: 961 1 350 000, ext. 4765; Fax: 961 1 744 464; E-mail: me00{at}aub.edu.lb; or Ali Bazarbachi, Department of Internal Medicine, Faculty of Medicine, American University of Beirut, P.O. Box 113-6044, Beirut, Lebanon. Phone: 961 361 2434; Fax: 961 134 5325; E-mail: bazarbac{at}aub.edu.lb

Received 8/ 2/03. Revised 1/ 7/04. Accepted 1/ 8/04.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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