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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2397-2406
NEOPLASIA
Bidirectional induction of the cognate receptor-ligand  4/VCAM-1
pair defines a novel mechanism of tumor intravasation
Laura Bogetto,
Elena Gabriele,
Roberta Cariati,
Riccardo Dolcetti,
Paola Spessotto,
Claudio Doglioni,
Mauro Boiocchi,
Roberto Perris, and
Alfonso Colombatti
From the Divisione di Oncologia Sperimentale 2 and Divisione di
Oncologia Sperimentale 1, CRO, Aviano, Italy; the Department of
Pathology, City Hospital, Belluno, Italy; the Dipartimento di Biologia
Evolutiva e Funzionale, Università degli Studi di Parma, Parma,
Italy; and the Dipartimento di Scienze e Tecnologie Biomediche,
Università degli Studi di Udine, Udine, Italy.
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Abstract |
Engagement of cell surface adhesion receptors with extracellular
constituents and with cellular counter-receptors is crucial for the
extravasation of blood-borne neoplastic cells and their seeding at
distant sites; however, the early events of tumor dissemination ie, the intravasation step(s)have been largely neglected. A role for the
 4 7 integrin was hypothesized to explain the high leukemogenicity exhibited by one (NQ22) among several T-cell lymphomas studied. To
clarify the mechanisms of early aggressivity, the behavior of highly
and poorly leukemogenic cell lines were compared in vitro.
Cocultivation of physically separated leukemic cells with resting
endothelial cells resulted in the up-regulation of VCAM-1 expression.
NQ22 cells expressed mRNA of different cytokines that up-regulate
VCAM-1 and at higher levels than cells of a nonaggressive lymphoma, and
they migrated more efficiently through an activated endothelial cell
layer. With the use of neutralizing antibodies against interferon- ,
granulocyte macrophage colony-stimulating factor, and tumor necrosis
factor (TNF)-, it was determined that TNF- is one of the soluble
factors released by NQ22 cells involved in the up-regulation of VCAM-1.
The finding that vascular cells within the early local growth were
strongly positive for VCAM-1 indicated that NQ22 cells could activate
endothelial cells also in vivo. Finally, cocultivation of
preleukemic 4 NQ22 cells with TNF--activated
endothelial cells induced the expression of 4 integrins on the
former cells. Reciprocal up-regulation and engagement of 4/VCAM-1
pairs determined the sequential transmigration and intravasation steps,
and similar mechanisms might affect the aggressivity of human T
lymphoblastic lymphomas.
(Blood. 2000;95:2397-2406)
© 2000 by The American Society of Hematology.
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Introduction |
Tumor progression is a complex biologic process for
which cells must possess a series of traits that enable them to
complete the multiple and sequential steps involved: detachment and
emigration from the primary site, invasion of surrounding tissues,
entrance into blood or lymphatic vessels (intravasation), escape from
the microvasculature (extravasation), seeding, and metastatic growth at
distant target sites.1,2 The interactions of migratory lymphoma cells with extracellular matrix (ECM) components, with stromal
cell elements, and with the endothelial cell surfaces are mediated by
the engagement of specific and multiple adhesion molecules expressed on
the disseminating cells.3 In addition, soluble cell
membrane- and ECM-bound chemotactic factors contribute to this complex
phenomenon. Numerous studies in several model systems have been
conducted in the attempt to clarify the mechanisms regulating the
various steps in lymphoma dissemination,4 and these have
revealed that transmigration, locomotion, and localization to target
organs resemble, at least in part, the physiology of leukocyte adhesion
and homing.5,6
Although much has been learned concerning the role of the endothelium
to allow blood-borne neoplastic cells to seed at distant sites, the
early events leading to intravasation of malignant lymphoma cells have
only occasionally been addressed. Elucidation of this issue is,
however, of relevance in light of the finding that intravasation and
the subsequent leukemic evolution of malignant lymphocytes adversely
affect the clinical outcome of patients with non-Hodgkin's lymphoma.
In particular, the switch from a sessile to a migratory condition after
intravasation is often detected at the onset of human T-lymphoblastic
lymphoma (T-LBL). The dissemination process represents a good example
of early intravasation and could be considered a model system for the
more generalized metastatic process of malignant cells. We have
previously developed and partially characterized an experimental model,
suitable for unraveling of this phenomenon, which consists of a series
of AKR T-lymphoma cell lines with different leukemic
potential.7-9 After subcutaneous (SC) inoculation, one of
these lines (NQ22) showed a unique leukemic phenotype with early
peripheral blood invasion and widespread organ dissemination. A similar
behavior was detected in a spontaneous T-lymphoma in an SJL
mouse.9 In contrast to several of our other poorly
leukemogenic and locally growing lymphomas, NQ22 cells were the only
ones displaying de novo expression of the  47 integrin complexes
on their surfaces concomitantly with in vivo spreading.8,9
Furthermore, using a less leukemogenic NQ22 variant, a direct
relationship was disclosed between the leukemic phenotype exhibited by
the cells and the in vivo induction, among a number of other cell
adhesion molecules investigated, such as Pgp-1/CD44, Mel 14, LFA-1,
ICAM-1, and CD26,8 only of the 47 integrin
complex.9 Interestingly, a similar correlation was found in
a series of pediatric T-LBL in which the early bone marrow infiltration
correlated with the expression of the 47 integrin on leukemic T
blasts.9
The 41 and 47 integrins are expressed on most leukocytes
and are well established to function as receptors for the vascular cell
adhesion molecule 1 (VCAM-1), the mucosal addressin cell adhesion
molecule 1 (MAdCAM-1), and fibronectin (FN).10-14 By virtue of their ability to interact with FN and with endothelial cells, 41 and 47 are believed to play an important role in
leukocyte-endothelial interactions.15-18 VCAM-1 has a
crucial role in supporting the capture and the immobilization of normal
and neoplastic leukocytes in the bloodstream. Furthermore, the
expression of VCAM-1 is induced by various stimuli including
lipopolysaccharide and a number of cytokines: IL-1, IL-4, IFN-,
tumor necrosis factor (TNF)-, granulocyte macrophage-colony
stimulating factor (GM-CSF).19-25 In their extravascular
condition, lymphoma cells interact with proteins of the ECM, with FN
representing a prototype constituent of ECM, that could be considered
inducers and regulators of the migration and transendothelial processes
leading to massive dissemination to distant sites.
In the current study we hypothesized that the aggressive behavior of
highly leukemogenic NQ22 cells, enabling their efficient and rapid
migration from the inoculation site, could result from interactions
with ECM and endothelial cells. NQ22 cells recognized and bound to
VCAM-1 and up-regulated the expression of VCAM-1 on endothelial cell
lines grown in vitro. This in vitro activity, at least partly dependent
on TNF- released from NQ22 cells, was reflected in the strong VCAM-1
positivity of endothelial cells in the SC inoculation site of mice
injected with NQ22 cells but not with cells of other T-lymphoma cell
lines. Thus, the engagement of 4/VCAM-1 pair(s) appear(s) to be
involved in the controlled and sequential transmigration and
intravasation of the highly malignant NQ22 lymphoma cells.
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Materials and methods |
Antibodies and proteins
The monoclonal antibodies (mAbs) used in this study were the
following: L3T4 (CD4) and Lyt-2 (CD8) were purchased from Becton Dickinson (Milan, Italy); DATK-32 (47)16 was obtained
from Dr E. C. Butcher (Stanford University Medical Center, Stanford, CA); 2E7 (4IEL),26 R1-2
(4),13 NFR5 (5H10) (5), and 429 (CD31/VCAM-1) were
purchased from PharMingen (San Diego, CA); GoH3
(61)27 was a gift of Dr A. Sonnenberg (The
Netherlands Cancer Institute, Amsterdam, The Netherlands); M/K-2.7
(VCAM-1),15 FIB504 (7),16 M298
(7),17 M1/70 (M2),28 and PS/2
(4)15 were from ATCC (Rockville, MD); KM16
(1)29 was from Dr P. Kinkade (Oklahoma Medical Research
Foundation, Oklahoma City, OK); 9EG7 (1)30 was from Dr
D. Vestweber, Max Plank Institute for Immunobiology, Freiburg, Germany.
A rabbit polyclonal antibody against the cytoplasmic portion of mouse
1 chain was provided by Dr G. Tarone (University of Turin, Turin,
Italy). FN was purified from human plasma according to the procedures
of Vuento and Vaheri.31
Cell lines and subcutaneous inoculations
The origin, establishment, growth behavior, and optimal culture
conditions of MCF-247 virus-induced AKR T-cell lymphoma-derived cell
lines NQ9, NQ22, NQ29, NQ35, and NQ36 have been described previously.7,8 The murine endothelioma cell lines H-end73 and H-end80, produced by immortalization of primary murine heart microvasculature endothelial cells with the polyoma virus, were a
generous gift of Dr F. Bussolino (University of Turin, Turin, Italy)
and were maintained in Dulbecco's minimum essential
medium (DMEM) containing 10% fetal calf serum.
AKR/J mice of both sexes maintained in our laboratory were injected in
the flank with 105 cells of each line suspended in 0.1 mL
sterile phosphate-buffered saline. Injected animals were monitored
daily, and tumors were allowed to grow until the death of the
recipients.7,8 For the various types of experiments
described, only neoplastic cell suspensions in which the
DATK32-positive cells exceeded 90% were used.
Immunohistochemistry and flow cytometry
Fresh tissue samples from subcutaneous neoplastic masses were either
embedded in OCT compound (Miles Laboratories, Naperville, IL),
snap-frozen in liquid nitrogen-cooled isopentane, and stored at
 70°C until the time of processing or directly
cryosectioned. Sections 5-µm thick were stained with the
antibodies indicated and then by anti-rat IgG and peroxidase ABC-Elite
complex (Vector Laboratories, Burlingame, CA). For
double-immunolabeling, the peroxidase ABC method developed in black
with diaminobenzidine-nickel was used in the first reaction, followed
by an alkaline phosphatase-biotin-streptavidin method developed in
red with Vector-Red (Vector Laboratories). Other sections were
incubated unfixed with the anti-VCAM-1 429 monoclonal antibody and
examined under the confocal laser scanning microscope (Diaphot 200, Nikon; MRC-1024, Bio-Rad Laboratories, Hercules, CA) using the
computerized serial optical sectioning function provided within the
Lasersharp software (Bio-Rad).
Cell surface expression of putative adhesion receptors on cultured
cells, on lymphoma cells grown subcutaneously, and on leukemic cells at
dissemination sites was assessed by flow cytometry. Data were acquired
using a FACScan flow cytometer and analyzed using PAINT-A-GATEPlus (Becton Dickinson) gated to exclude cell
debris and nonviable elements.
Radiolabeling of cell surface proteins and immunoprecipitation
NQ22 cells from terminal stage leukemic mice were surface labeled
with 125I as described.35 Different mAbs were
added to cell lysates along with protein G-Sepharose, and the mixture
was incubated overnight at 4°C under gentle shaking. For sequential
immunoprecipitations, the cell lysates were incubated repeatedly with 1 antibody until no radioactivity was detected on the immunoprecipitates,
and then the cell extract supernatants were sequentially incubated with the other antibodies. Samples were analyzed by SDS-PAGE on a 7% polyacrylamide gel, and dried gels were processed for autoradiography using MP films (Amersham, Buckinghamshire, UK).
RNA extraction and RT-PCR
Total cellular RNA was extracted from sorted cells by acid
guanidinium thiocyanate-phenol-chloroform.32 cDNA was
synthesized from 10 ng RNA using 20 U AMV reverse transcriptase
(Promega, Madison, WI), 1 mmol/L dNTPs (each), 20 U RNAsin
(Promega), and hexanucleotides (12.5 pmol). The following
oligonucleotide primers were used: IL-1 (sense:
5'-GCAACTGTTCCTGAACTCA-3'; antisense: 5'-CTCGGAGCCTGTAGTGCAG-3'); IFN- (sense:
5'-AACGCTACACACTGCATCTTGG-3'; antisense:
5'-GACTTCAAAGAGTCTGAGG-3'); TNF- (sense:
5'-CTCACACTCAGATCATCTTCTC-3'; antisense:
5'-GGCTACAGGCTTGTCACTCGA-3'); GM-CSF (sense:
5'-TTCCTGGGCATTGTGGTC-3'; antisense:
5'-TGGATTCAGAGCTGG CCTGG-33); HPRT (sense:
5'-GTTGGATACAGGCCAGACTTTGTTG-3'; antisense:5'-GATTCAACTTGCGCTCATCTTAGGC-3') (40 cycles,
annealing temperature 52°C). The amplified products were diluted
500-fold and then subjected to a nested polymerase chain reaction (PCR) using the following sense primers: IL-1
5'-ATTGTGGCTGTGGAGAA-3'; IFN-
5'-GGAGGAACTGGCAAAAGGA-3'; TNF-
5'-TCGAGTGACAAGCCTGTAGCC-3'; GM-CSF
5'-CACGTTGAATGAAGAGGT-3' (40 cycles, annealing temperature 53°C). The amplification products of this second reaction
were resolved by agarose gel and visualized by ethidium bromide and ultraviolet light.
Preparation of Fab fragments
The anti-4 chain mAb PS/2 was purified from the 50% ammonium
sulfate precipitate by loading this fraction on a protein G-Sepharose column (mAb Trap; Pharmacia, Uppsala, Sweden) and eluting the mAb with
50 mmol/L glycine-HCl, pH 2.7. The purified mAb was dialyzed for 16 hours at 4°C against phosphate-buffered saline. The Fab fragments
were isolated by digestion of the purified antibody (1.5 mg/mL) with
immobilized ficin (Immunopure preparation kit; Pierce Chemical,
Rockford, IL) according to the manufacturer's instructions.
Purification of the digested antibody was performed by loading the
mixture on a protein G-Sepharose columns. The effluent contained only
the Fab fragment as indicated by separation on a 10% polyacrylamide
SDS gel under reducing conditions.
Cell adhesion assays
Cell adhesion to molecular substrates.
The cell adhesion assay was a centrifugation-based protocol originally
introduced by Lotz et al33 and applied by us with the use
of 35S-methionine-labeled34,35 and of
calcein-labeled cells36 to measure cell adhesion to
molecular and cellular substrates, respectively. For cell adhesion
assays PVC 96-well plates (Microtiter flexible Falcon; Becton
Dickinson) were coated for 16 hours at 4°C with 50 µL appropriate
concentration of the substrate molecule in bicarbonate buffer, pH 9.6. The percentage of attached cells was calculated from those values.
Assays were repeated at least 3 times, and average values that did not
deviate more than 10% from the mean were plotted. In experiments aimed
at examining the effects of competing antibodies, the various
antibodies were added directly to the wells just before adding the
cells under investigation.
Cell adhesion to cellular substrates.
H-end80 cells were brought to confluence into PVC 96-well plates
(Microtiter flexible Falcon); cells were then stimulated for 16 hours
with 10 ng/mL TNF-, rinsed with DMEM containing 0.3%
polyvinylpyrrolidone and incubated with calcein-labeled NQ cells. The
plates were centrifuged for 5 minutes at 500 rpm and incubated at
37°C for 20 minutes. At the end of the incubation, nonadherent
cells were removed by gently rinsing with DMEM containing 0.3%
polyvinylpyrrolidone, and the extent of adherent cells was measured on
a microplate fluorometer (SPECTRAFluor Plus; TECAN, Salzburg, Austria)
at 485 nm excitation and 535 nm emission. The percentage of bound cells
was determined by dividing the fluorescence of adherent cells by the
fluorescence of the input cells.
Coculture of lymphoma and endothelial cells
H-end73 and H-end80 cells were grown in Transwell culture inserts
(Costar, Cambridge, MA) measuring 2.4 cm in diameter bearing polycarbonate membranes with 3- to 5-µm diameter pores until they reached confluence. Lymphoma cells grown in vitro or cells isolated from in vivo growing tumors were plated directly onto the endothelial monolayers and cocultured for different time intervals. Alternatively, lymphoma cells were physically separated from the endothelial cell
layers by a tissue culture insert bearing membranes with 0.02-µm
diameter pores (Nunc, Roskilde, Denmark). At the end of the coculture
period, lymphoma cells were removed by extensive washing, and the
endothelial cells were recovered after trypsin-EDTA detachment. These
were then processed for FACS analysis to determine the levels of VCAM-1
cell surface expression. Endothelial cells grown in the presence of 10 ng/mL TNF- for 16 hours were adopted as a positive control for
up-regulated VCAM-1 expression.
Neutralization of cytokine bioactivity
Cytokines released by NQ22 cells during the cocultivation assay with
endothelial cells were neutralized by the addition, at the start of the
assay, of saturating amounts of polyclonal antibodies against mouse
IFN-, GM-CSF, and TNF- (R&D Systems, Abingdon, UK). The final
concentration of antibodies added was 10 µg/mL irrespective of
whether a single antibody or a mixture of 3 different antibodies was
used. Normal goat IgG was used as negative control. After 7 hours of
coculture, H-end80 cells were processed for FACS analysis as above.
Cell migration
The ability of lymphoma cells to transmigrate through an endothelial
cell layer was examined using Transwell (Costar) culture chambers
bearing polycarbonate membranes with 3- to 5-µm diameter pores. At
various time points, the number of cells migrated to the lower chamber
was determined, and the mean ± SD was calculated in 9 microscope
fields. In this series of experiments, not more than approximately 8%
to 12% of the input cells were able to transmigrate within the
timeframe allocated for the transmigration to occur.
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Results |
Integrin expression on NQ22 cells
In a previous immunophenotypic study, we had shown that the highly
leukemogenic NQ22 cell line displayed a remarkable induction of the
47 integrin after in vivo inoculation.9 For the
current study, a comparative analysis of the integrin expression of
NQ22 and of poorly leukemogenic cells confirmed the in vivo
up-regulation of 4, 7, and 47 of NQ22. In addition, the
expression of the 1 integrin chain, Mac-1, and the 6 integrin
chain were also induced in NQ22 cells; the 5 integrin chain was
weakly expressed, whereas the v and
4IEL chains were not detected at all. Instead, the poorly leukemogenic cells did not show the induction of
the integrin chains 4, 1, 7, Mac-1, and 6 as detected
in NQ22 cells (data not shown).
Integrin complexes identified by flow cytometry were further
characterized immunochemically by precipitation of the iodinated complexes present on NQ22 cells from leukemic spleens (90% DATK-32 positive cells by FACS analysis) with several mAbs. As shown in Figure
1A, SDS-PAGE under nonreducing conditions
of such immunoprecipitates revealed 2 bands of approximately 150 and
110 kd (anti-4 mAb) and of approximately 150 and 100 kd (anti-7
and anti-47), respectively (Figure 1), demonstrating that both
41 and 47 integrins were expressed on highly leukemogenic
NQ22 cells. The direct physical association between the 4 and the
1 chains and the relative amounts of 47 and 41 was
demonstrated by sequential immunoprecipitation: thus, NQ22 cells were
first depleted of the 47 integrins (Figure 1B, lane 1); then the
lysate supernatant was incubated with the anti-4 mAb R1-2, and, in
this case, a strong 150 kd polypeptide band associated with a 110 kd
polypeptide band was immunoprecipitated (lane 2). Finally, the cell
lysate supernatant was immunoprecipitated with a polyclonal anti-1
antibody (lane 3) revealing the persistence of other 1 integrin
complexes, likely 51 and 61.
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| Fig 1.
Immunoprecipitation of integrins from NQ22 cells.
Approximately 2.5 × 107 cells were radiolabeled
with 125I and extracted in lysis buffer. (A) Aliquots of
the cell lysate were immunoprecipitated using the following antibodies:
lane 1, anti-4 (R1-2); lane 2, anti-7 (M298); lane 3, anti-47 (DATK-32); lane 4, anti-61 (GoH3). (B) Sequential
immunoprecipitations. NQ22 cell lysates were first depleted with 3 rounds of immunoprecipitation with anti-7 (lane 1), followed by 3 rounds with anti-4 (lane 2), and finally with the polyclonal
anti-1 antibody (lane 3). Samples were then analyzed by SDS-PAGE on
a 7% gel under nonreducing conditions. The migration of standard
molecular weight markers is shown in the center. The migration of
integrin and chains is also indicated.
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Adhesion of highly and poorly malignant cells to FN and to
endothelial cells
Both pre-leukemic (ie, in vitro grown) and highly leukemogenic NQ22
cells that had infiltrated the spleen and lymph nodes bound strongly to
FN, the latter with a slightly lower efficiency compared to the cells
maintained in culture (Figure 2A). Cell adhesion to FN of several other in vitro grown cell lines, which are
characterized by a prevalent local SC growth when transplanted in vivo,
was variable: NQ9 cells displayed high binding to FN, whereas NQ29,
NQ35, and NQ36 displayed intermediate or low levels of cell adhesion.
Cell attachment of NQ22 cells to FN was mediated by its cell-binding
domain comprising the RGD and the primary synergistic site within the
3Fn-9 repeat. In fact, the inhibition of cell adhesion by 80% and
40%, respectively, was attained with the addition of antibody 333, which binds near the RGD site,37 and antibody HFN-7 against
the synergistic site (data not shown). Furthermore, cell adhesion to FN
was not inhibited by anti-4 (PS/2) or anti-47 (DATK-32) mAbs
(data not shown), suggesting that the 41 and 47 integrins
contributed poorly to the overall interaction with FN and that binding
was primarily mediated by other integrins (ie, 51).
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| Fig 2.
Cell adhesion.
(A) Cell adhesion of lymphoma cell lines to bovine serum albumin or FN
coated at 10 µg/mL was carried out in the presence of 0.3 mmol/L
Mg2+ or 20 µmol/L Mn2+. The values reported
represent the average of 3 experiments. (B) Cell adhesion to
endothelial cells. Confluent H-end80 monolayers were incubated with
media alone or with media containing TNF- for 16 hours and rinsed
with DMEM. NQ22 and NQ29 cells (2 × 105) in DMEM
were added for 20 minutes at 37°C under static conditions. At the
end of the incubation period, the nonadherent cells were removed by
gentle rinsing with DMEM and the percentage of bound cells was
determined as detailed in "Materials and methods." (C) NQ22 and
NQ29 cell adhesion to purified VCAM-1 or bovine serum albumin coated at
10 µg/mL. For the inhibition of cell attachment to H-end80 cells and
to purified VCAM-1, the different antibodies were added at 5 µg/mL
just before the lymphoma cells were plated. The antibodies used were
the following: DATK-32 (47); M1/70 (Mac-1/M2); PS/2 (4);
M298 (7); and 429 (VCAM-1).
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To assess whether adhesion to endothelial cells might represent a
peculiar property of NQ22 cells in comparison to other lymphomas, we
used endothelial cell lines. Although these cells are unique and likely
lack a normal counterpart in vivo, they expressed several endothelial
cell markers (data not shown) and proved suitable and highly
reproducible. Very low binding was detected to nonactivated H-end80
cells (Figure 2B). Instead, NQ22 cells isolated from infiltrated spleen
bound to TNF--activated H-end80 cells. Blocking studies with
anti-4, -7, -47, and -Mac-1 mAbs showed that only the PS/2
anti-4 antibody abrogated cell adhesion (Figure 2B). In contrast,
the overall effect of anti-7, -47, and -Mac-1 antibodies was
negligible (Figure 2B). These results indicated a strong predominance of the 41 integrin in determining the adhesion of NQ22 cells to
the activated endothelial cells. To demonstrate that the 4-integrin cognate ligand on TNF--activated endothelial cells was VCAM-1, endothelial cells were preincubated with a blocking anti-VCAM-1 mAb,
and, in this case, NQ22 cell adhesion was to a great extent abrogated.
Furthermore, we investigated the ability of NQ22 cells to adhere to
surfaces coated with recombinant VCAM-1. NQ22 cells bound pronouncedly
to VCAM-1 (75%), and this binding was completely inhibited by
anti-4 mAb (Figure 2C); the contribution of the 47 integrin to
cell adhesion appeared to be much lower (approximately 30%).
Therefore, as reported in other experimental systems involving lymphocytes expressing both 41 and 47,11
41-dependent binding to VCAM-1 was predominant also for NQ22
cells. NQ29 cells showed no binding to VCAM-1 (Figure 2B) even if
plated at higher concentrations of the ligand (data not shown).
Induction of VCAM-1 on endothelial cells
The in situ expression of VCAM-1 at local sites of the initial
neoplastic growth of NQ22 and NQ29 cells was examined, and it was found
that cells intensely positive for VCAM-1 were present as fully formed
vessels and as single cells likely of endothelial or stromal origin if
the infiltrating tumor cells were NQ22 (Figures 3A, 3C); instead, weak positivity or a
nonactivated pattern of staining for VCAM-1 in small locally growing
NQ29 tumors was detected (Figure 3D). As reported previously for large
tumor masses,9 few vascular spaces were engulfed by NQ22
cells highlighted by their strong positivity for the 47 marker;
neoplastic cells could be seen protruding into the lumen and crossing
the endothelial layer (Figures 3B, 3C). These morphologic and
immunochemical findings suggested that the interactions between NQ22
leukemic cells and endothelial cells might indeed play an important and
necessary role in the early leukemic evolution of this lymphoma. To try to recapitulate this phenomenon in vitro, leukemic cells were cultured
for different lengths of time over a monolayer of endothelial cell
lines (H-end73 and H-end80). These cells were rapidly induced to
express high levels of VCAM-1 by LPS (Figure
4A). The same result was obtained if either
H-end73 or H-end80 cells were cocultured with NQ22 cells; in this case
VCAM-1 expression peaked at approximately16 hours and decreased to near
basal levels at 24 hours. On the contrary, NQ29 cells were not able
significantly to up-regulate VCAM-1 expression on endothelial cells.
The continuous presence of NQ22 cells was not necessary for the
induction of VCAM-1 on H-end80 cells, as shown by the fact that if NQ22
cells were removed after 1 to 3 hours of coculture, H-end80 cells still
showed a significantly increased VCAM-1 expression at 7 hours.
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| Fig 3.
Immunohistochemical staining of SC tumor masses.
Immunolabeling of cryostat sections of masses at their initial growth
was performed with anti-47 and anti-VCAM-1 (A, C) or with
anti-47 and anti-CD31 (B) mAbs on NQ22 cells or with anti-VCAM-1
alone on NQ29 cells (D). Brown, 47; red, CD31 and VCAM-1.
Magnification, × 200 (A, B) and × 400 (C, D).
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| Fig 4.
Induction of VCAM-1 on murine endothelial cells.
(A) Time-course. H-end73 ( ,  ) and H-end80 ( )
cells were cocultured for different lengths of time with NQ22 (,
 ) or NQ29 (, ) cell suspensions obtained from frankly leukemic
spleen (NQ22) or from a large SC tumor mass (NQ29), and the percentage
of VCAM-1 positive cells was assessed by flow cytometry analysis using
anti-VCAM-1 mAb 429. H-end80 stimulated with 100 ng/mL
lipopolysaccharide ( ) for 3 hours was used as positive control and
similarly processed for flow cytometry analysis. (B) Induction of
VCAM-1 by different cell types. H-end80 cells were cocultured for 7 hours with cell suspensions obtained from in vitro-grown NQ22 cells
(NQ22 vitro), NQ22 cells isolated from an infiltrated spleen (NQ22
spleen), or a subcutaneous tumor mass (NQ22 SC mass) and from a
subcutaneous mass of NQ29 cells (NQ29 SC mass). The expression of
VCAM-1 was assessed with mAb 429. Control, isotype-matched unrelated
primary antibody was added; basal, basal expression of VCAM-1 in
H-end80 cells.
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To assess more accurately whether the ability to up-regulate VCAM-1 in
endothelial cells was an intrinsic property of NQ22 cells, coculture
experiments were carried out between H-end80 and NQ22 leukemic cells
from different sources. As shown in Figure 4B, though NQ29 cells from
an SC mass did not up-regulate VCAM-1 within the timeframe of this
experiment, in vitro cultured NQ22 cells weakly induced its expression,
cells from a locally growing SC NQ22 mass had an intermediate capacity
of induction, and NQ22 cells obtained from dissemination sites
up-regulated VCAM-1 expression to levels comparable to those attained
with TNF-. Separation of the 2 cell populations with a membrane
(0.02-µm pores) resulted in a much lower induction of VCAM-1 (Figure
5C) than TNF--treated cells (Figure 5A).
These types of filters allow the diffusion of soluble mediators but not
cell-cell contacts and cell transmigration. However, the distance
between the 2 cell types in the above experiment was such
(approximately 1 mm) that the inductive cues eventually exerted by
diffusible factors might not reach the target endothelial cells at
functionally effective levels. Thus, a different experimental setup was
developed to maintain the cocultured cells still physically separated,
but by a much shorter distance (0.06 mm). This was achieved by first
growing the H-end80 cells on the lower side of an inverted Transwell
(Costar) and then positioning it in the culture wells in the correct
upright position with the endothelial cells facing downward. In this
case the up-regulation of VCAM-1, achieved after the coculture of NQ22
cells added in the upper chamber, reached intermediate levels (Figure
5D) and was nearly comparable to the up-regulation attained by
cocultivating the 2 cell types (Figure 5B), suggesting that soluble
factors actively released by NQ22 cells likely contribute to the
induction of VCAM-1 in endothelial cells.
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| Fig 5.
Induction of VCAM-1 can be inhibited by a nitrocellulose
membrane.
VCAM-1 expression was evaluated on H-end80 cells cocultured for 7 hours
with NQ22 cells (B); with NQ22 cells separated by a tissue culture
insert with an anopore membrane bearing 0.02-µm diameter pores in
which the 2 cell types were separated by a distance of approximately1
mm (C); with NQ22 cells separated by a tissue culture insert with an
anopore membrane bearing 0.02-µm diameter pores in which the 2 cell
types were separated by a distance of approximately 0.06 mm (D). Other
endothelial cells were grown for 16 hours in the presence of 10 ng/mL
TNF- (A). NQ22 cells were isolated from an infiltrated spleen;
basal, basal expression of VCAM-1 in H-end80 cells.
|
|
That physical cell-cell interaction between endothelial and NQ22 cells
was not a prerequisite for VCAM-1 up-regulation was further
investigated: monovalent Fab fragments of the blocking PS/2 mAb (Figure
6A, inset), prepared to avoid integrin
cross-linking and further activation of NQ22 cells, were used to block
NQ22 cell adhesion to TNF--stimulated H-end80 cells; Fab was almost as efficient as bivalent IgG (Figure 6A). In a parallel experiment nonactivated H-end80 cells were cocultured for 7 hours with NQ22 cells
in the presence of saturating amounts of Fab fragments of PS/2 antibody. In this case, though effective in blocking the cell-cell interaction, Fab fragments did not prevent the
up-regulation of VCAM-1 induced by the coculture (Figure 6B).
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| Fig 6.
Inhibition of cell adhesion by anti-4 Fab fragments
does not inhibit VCAM-1 induction on endothelial cells.
(A) Confluent H-end80 monolayers were incubated with media alone or
with media containing 10 ng/mL TNF- for 16 hours and rinsed with
DMEM. NQ22 cells (2 × 105) in DMEM were added for
20 minutes at 37°C under static conditions either in the absence or
in the presence of PS/2 anti-integrin 4 subunit IgG (10 µg/mL) or
Fab fragment (5 or 15 µg/mL). At the end of the incubation period,
the nonadherent cells were removed as detailed in "Materials and
methods." The purity of the IgG and of the Fab fragment, analyzed by
SDS-PAGE on a 10% gel under reducing conditions, is shown in the
inset, lane 1 and lane 2, respectively. (B) Expression of VCAM-1 on
H-end80 cells was evaluated after 7 hours of cocultivation with NQ22
cells obtained from infiltrated spleen either in the presence or in the
absence of 50 µg/mL Fab fragment.
|
|
Expression of cytokines by NQ22 and NQ29 cells
Because it has been reported that a number of cytokines, including
IL-1, IFN-, TNF-, IL-4, and GM-CSF, elicit a profound and
relatively rapid up-regulation of VCAM-1 on several cell
types,19-25 we investigated by RT-PCR whether the
differential expression of mRNA of several cytokines might be a
mechanism by which NQ22 cells up-regulate VCAM-1 on endothelial cells.
Although TNF- appeared to be expressed in all cell types analyzed
(in NQ22 cells, it was already detectable after the first amplification
step; data not shown), the expression of IFN- was detected only in NQ22 cells grown in vitro and in highly leukemogenic NQ22 cells isolated from infiltrated spleen; furthermore, IL-1 and GM-CSF were
detected only in cells transplanted in vivo (Figure
7). These data indicated that a set of
cytokines (TNF-, IFN-, IL-1, and GM-CSF), expressed
exclusively or predominantly in NQ22 cells, contributed to the
up-regulation of VCAM-1 on local vessels on SC inoculation. To
investigate this possibility further, a cocultivation between H-end80
and NQ22 cells was set up in the presence of saturating amounts of
neutralizing antibodies against different mouse cytokines. Although the
addition of normal goat IgG or of anti IFN- or of anti GM-CSF had no
effect (76% VCAM-1 positive cells versus 71% when NQ22 cells alone
were cocultured), the presence of anti TNF- antibodies during the
coculture prevented to a large extent (40% positive cells) the
up-regulation of VCAM-1 on H-end80 cells (Figure 8A). If the 3 antibodies were added
together, keeping the total amount of immune IgG added still at 10 µg/mL, a slightly reduced inhibition was noticed (47% positive
cells) (Figure 8B), very likely because of the lower amount of anti
TNF- in the coculture system.
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| Fig 7.
Differential expression of cytokines in NQ22 and NQ29
lymphoma cells.
Total RNA was extracted from 107 NQ22 and NQ29 cells grown
in vitro, from a spleen heavily infiltrated with NQ22 cells, from NQ22
cells cocultured with H-end80 cells, and from NQ29 cells from a
subcutaneous tumor mass. RNA was reverse transcribed and amplified with
primers specific for IL-1, IFN-, TNF-, GM-CSF, and HPRT as
detailed in "Materials and methods." Aliquots of the nested PCRs
were separated by 2% agarose gel and visualized by ethidium bromide
and ultraviolet light. Only the relevant part of the gel is shown.
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|
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| Fig 8.
Inhibition of VCAM-1 induction by TNF--neutralizing
antibodies.
A polyclonal antibody (10 µg/mL) against TNF- (A) or a mixture of
polyclonal antibodies (3.3 µg/mL each antibody) against IFN-,
GM-CSF, and TNF- (B) were added to H-end80 cells during the
coculture with NQ22 cells. The expression of VCAM-1 was assessed with
mAb 429; basal, basal expression of VCAM-1 in H-end80 cells.
|
|
Migration of NQ22 cells in vitro
The transendothelial migratory capability of NQ22 cells was compared
to that of NQ29 cells. For this assay the Transwell (Costar) system was
used with H-end80 cells grown to confluence in the upper side of the
filter in either the presence or the absence of TNF-. An increasing
number of NQ22 cells was detected with time, migrating across the
activated endothelial cell layer to reach the lower chamber (Figure
9A). Significantly fewer NQ22 cells were
detected in the lower chamber of each Transwell (Costar) in
which nonactivated H-end80 cells were seeded, though a moderate increase with time was evident (compare the values at 3, 6, and 12 hours in Figure 9A). In contrast, few NQ29 cells transmigrated irrespective of whether the endothelial cell layer had been activated with TNF-, and only at 24 hours (data not shown) a certain degree of
equivalent migration was detected through both activated and nonactivated H-end80 cells.
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| Fig 9.
NQ22 cells migrate more efficiently than NQ29 cells.
Transendothelial migration. H-end80 cells were grown to confluence on
the upper side of uncoated wells of Transwell culture
chambers and either were left untreated or were treated for 16 hours
with 10 ng/mL TNF- . NQ22 cells ( , ) obtained
from heavily infiltrated spleen and NQ29 cells from a subcutaneous mass
( , ) (2 × 105) were seeded in the upper
chamber of the Transwell, and cells that migrated through untreated
(, ) or through TNF--treated (, ) H-end80 cells to the
lower chamber were harvested and counted at the indicated time-points.
The values shown represent the mean of 3 optical fields.
|
|
VCAM-1 is induced on the apical and basolateral sides of endothelial
cells
The adhesion to and the transmigration through an endothelial cell
layer displayed by NQ22 cells in vitro provided some clues about how
these cells could intravasate in vivo and then invade the bloodstream.
To determine whether VCAM-1 was expressed also on the basolateral side
of endothelial cells, nonactivated (which display a constitutive weak
expression for VCAM-1) and activated H-end80 cells were grown to
confluence and were stained with anti VCAM-1 antibodies.
Permeabilization with saponin allowed the measurement of an increase in
the fluorescence intensity compared to nonpermeabilized cells (data not
shown). A confocal analysis of sections through a local site of
neoplastic growth of NQ22 cells stained with VCAM-1 antibodies indeed
showed that this ligand was expressed on the apical and the basolateral
sides in vivo (Figure 10).
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| Fig 10.
VCAM-1 is expressed on both the apical and the
basolateral surfaces of endothelial cells in vivo.
Cryostat sections of NQ22 masses at their initial SC growth were fixed
in acetone-methanol and stained with anti VCAM-1 mAb M/K-2 and
visualized with fluorescein-conjugated goat antimouse IgG. (A) Detail
of the 2-dimensional reconstruction. (B) Detail of the tridimensional
reconstruction of a series of sections taken in 0.13-µm steps through
a tissue section in which 2 blood vessels are clearly evident.
|
|
De novo expression of 4 integrins on pre-leukemic NQ22 cells
The data generated in the previous set of analyses outline a
possible sequence of events that might explain the aggressive behavior
of NQ22 cells: these cells spontaneously release, or are induced to
release, TNF- and perhaps a number of other cytokines that
up-regulate the expression of VCAM-1 on endothelial cells of nearby
small vessels and then reach the basolateral surface of a by-now
activated endothelial cell layer, bind to these cells by an
4/VCAM-1-dependent mechanism, and finally transmigrate through the
endothelium into the bloodstream. However, in vitro grown NQ22 cells do
not express 4 integrins; thus, 1 fundamental element of this
multistep process is still missing. To investigate whether activated
endothelial cells might induce the expression of 4 integrins in NQ22
cells, TNF- stimulated and nonstimulated H-end80 cells were
cocultured with in vitro grown NQ22 or NQ29 cells for 24 hours, and the
expression of the 4 subunit was measured with the PS/2 antibody.
Nonactivated H-end80 cells were ineffective with both NQ22 and NQ29
cells; on the contrary, pretreatment with TNF- resulted in de novo
expression of 4 integrins in a low percentage (2% to 3%) of NQ22
cells. Fewer NQ29 cells were induced than NQ22 cells (Figure
11).
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| Fig 11.
Activated endothelium can up-regulate the expression of
4 integrins on NQ22 cells.
H-end80 cells grown to confluence were left untreated (upper panels) or
were treated for 16 hours with 10 ng/mL TNF- (lower panels) and then
were cocultured for 24 hours with NQ22 or NQ29 cells grown in vitro. At
the end of the incubation period, NQ cells were collected, the
expression of 4 integrin subunit was determined with the use of PS/2
mAb, and the reactivity was examined by confocal fluorescence
microscopy with pseudocolor enhancement. NQ22 cells from heavily
infiltrated spleen (NQ22 vivo) were used as positive control. Selected
fields with a relatively higher percentage of positive cells were
chosen for display for NQ22 and NQ29 cells (A), whereas the percentage
of positive cells is graphically depicted (B).
|
|
| |
Discussion |
Malignant lymphomas may undergo transition from a nodal to an
intravascular state, leading to the extravasation, dissemination, and
accumulation of the malignant cells in other nodal and extranodal tissues. Several studies have analyzed the extravasation phenomena of
these cells, and, in some cases, functional similarities with the
process of leukocyte extravasation have been identified. However, little attention had been paid to the regulation of the early steps of
this "metastatic" behavior apart from a recent study on carcinoma
cells.38 To contribute to the understanding of the
principles governing intravasation, we have used a murine model system
based on the lymphoma cell line NQ22 that disseminates after a
subcutaneous injection.7-9 The current study indicates that
4 integrins (41 to a greater extent than 47) contribute mechanistically and in a substantial way to the process of leukemic dissemination of the NQ22 lymphoma cells. First, coculture of activated
endothelium with the in vitro grown pre-leukemic
4NQ22 cells induced 4 integrin expression on
these cells, allowing an 4/VCAM-1-dependent cell-cell interaction.
Second, the mRNA of cytokines known to up-regulate the expression of
VCAM-1, such as TNF-, IFN-, IL-1, and GM-CSF, were
differentially expressed in the aggressive NQ22 cells compared to the
locally growing NQ29 cells. Consistent with this finding, only
cocultivation of the highly leukemogenic NQ22 cells with nonactivated
endothelial cells was followed by the up-regulation of VCAM-1 on these
latter cells. Third, the NQ22-dependent up-regulation of VCAM-1 was
prevented to a large extent by TNF--neutralizing antibodies. Fourth,
leukemogenic NQ22 cells migrated in vitro more efficiently through an
activated endothelial cell layer than nonleukemogenic NQ29 cells.
Fifth, the endothelial cells of vascular spaces at SC sites of the
initial neoplastic growth of NQ22 cells expressed high levels of
VCAM-1. Finally, the demonstration that activated endothelial cells
grown in vitro, as well as at local sites of NQ22 initial growth, also expressed VCAM-1 on the basolateral surface indicated a mechanism by
which the 4/VCAM-1-dependent interaction between incoming NQ22 cells
and the basolateral surface of vascular endothelial cells could be a
prelude to the transendothelial migration occurring during the
intravasation step. The possibility that similar mechanisms may also be
used by intravasating aggressive experimental T lymphomas, such as those of the SJL mouse strain and by pediatric
T-LBL9 and even by neoplastic cells of other cell
lineages, represents an attractive hypothesis that deserves further investigation.
It has been reported that the use of the 51 integrin in the
recognition of the FN ligand predominates over the use of other integrins when more FN-binding integrins are expressed on lymphoid cells.39 Accordingly, we could not affect binding to FN by
the simple neutralization with anti-4 and anti-47 integrin
antibodies. In contrast, the binding of NQ22 cells to purified
recombinant VCAM-1 was fully inhibited by an anti-4 integrin chain
mAb and partly inhibited by an anti-47 integrin mAb. The
up-regulation of VCAM-1 observed after coculture of endothelial and
NQ22 cells resulted in the acquisition of a pro-adhesive state by
endothelial cells. This activity depended on the induction of VCAM-1
expression because the cell-cell adhesion of NQ22 to endothelial cells
was fully inhibited with the use of functional anti-4 and
anti-VCAM-1 mAbs. Although the use of mAbs against the 7 subunit and
the 47 complex suggested that 47 played only a minor role,
it is plausible that both 4 integrins are instrumental in this cell adhesion step.
The finding that endothelial cells cocultured with NQ22 cells
up-regulated VCAM-1 expression in vitro is consistent with the results
of the immunohistochemical analysis showing that VCAM-1 was induced on
the vessel wall at sites of early NQ22 local growth. On the contrary,
the inability of NQ29 cells to up-regulate VCAM-1 expression on
endothelial cells in vitro as well as in vivo was consistent with their
prevalent local growth. This difference disappeared if NQ22 and NQ29
cells were injected intravenously; in those instances, mice injected
with either type of lymphoma cells had equivalent life expectancies
(Dolcetti et al, unpublished data). The inherent ability of NQ22 cells
to up-regulate VCAM-1 expression was confirmed by the strong expression
of VCAM-1 on endothelial cells observed at distant sites of
dissemination whenever leukemic cells displayed perivascular
growth.9
Although the main focus of recent studies has been the extravasation
steps,4,40-46 other reports have suggested that the ability
of transformed cells to metastasize at distant organ sites can be
independent of the extravasation steps47 or can manifest itself at a stage subsequent to cell dissemination.18,48 In the current study, the pro-invasive role of 4 integrins appears of
primary importance in the early steps. In a different tumor cell model,
a high expression of transfected 41 integrin has instead resulted
in reduced invasion resulting from the homotypic intercellular adhesion
of 4+ cells in the early invasive stage of the
metastatic cascade before intravasation.49 Thus, the
contrasting involvement of 4 integrins observed in the various
experimental systems studied points to the importance of the cellular
context in determining the functional role of integrins. NQ22 cells did
not need any further activation to adhere to FN or to adhere to and
transmigrate across endothelial cells. In the context of NQ22 cells,
4 integrins, with the 41 playing a more fundamental role than
the 47 integrin, can be used for efficient dissemination.
Using the H-end73 and the H-end80 in vitro endothelial cell model
system explored herein, the need for a direct cell-cell contact did
not seem to be a prerequisite for the activation of endothelial cells
and the consequent up-regulation of VCAM-1 by NQ22 cells. Provided that
the 2 cell types were not too far apart, consistent up-regulation of
VCAM-1 was detected even when the cells were physically separated by a
tissue-culture insert. Instead, in other experimental systems, the
cell-cell contact seemed to be a prerequisite for cell surface
molecule up-regulation or for cytokine release.50,51
Because 4 integrins are not expressed on in vitro grown pre-leukemic
NQ22 and NQ29 cells, the de novo expression of 4 integrins induced
after the cocultivation with activated endothelial H-end80 cells was
more pronounced in NQ22 than in NQ29 cells. Their differential
expression of cytokines and the NQ22-dependent up-regulation of VCAM-1
on endothelial cells that is largely prevented by TNF- neutralizing
antibodies likely represent key elements in determining the aggressive
behavior of NQ22 cells. In addition, it was ascertained that NQ22 cells or their 4 integrins are particularly sensitive to activation by
still unknown signals generated by TNF--activated endothelial cells.
This latter might represent another important influence that impinges
on the intrinsic propensity of NQ22 cells to disseminate through the
tissues. Although there is not yet direct evidence, it is likely that
the action of soluble factors precedes the cell-cell interaction when
NQ22 cells establish close contact with endothelial cells. This was
suggested by the observation that in vitro grown NQ22 cells
constitutively express the mRNA for IFN-, GM-CSF, and TNF-. It
could then be hypothesized that after the induction of cell adhesion
molecules on NQ22 cells by the intimate interaction with endothelial
cells, NQ22 cells may adhere to activated, VCAM-1 positive endothelial
cells and produce increased amounts of cytokines. These would in turn
further activate the microvasculature in a continuous loop. While we
have demonstrated that VCAM-1 supports NQ22 cell adhesion to
endothelial cells, it is likely that additional or synergistic
mechanisms regulate the movement of migratory NQ22 cells toward the
vasculature. For instance, CD40L,52 the TNF receptor,53 and Fas54 have been found to
represent determining factors in the up-regulation of endothelial cell
adhesion molecules. Similarly, the disruption of the
vascular-endothelial cadherin complex, triggered by an efficient
adhesion of the leukocytes to the activated endothelium, is thought to
constitute a fundamental step in leukocyte transendothelial
migration.55 In this context, NQ22 cells that adhere
efficiently to H-end cells and transmigrate efficiently across
cytokine-activated endothelial cells in vitro are found in vascular
spaces at the site of inoculation shortly after injection.
In light of the possible application of our findings in a clinical
setting, it is worth considering that in addition to the important role
played by TNF-, the cytokines involved in the intravasation of NQ22
cells might be even more than the ones we have studied, thus making
particularly difficult any attempt to block VCAM-1 up-regulation
therapeutically. Nevertheless, 4 integrins and VCAM-1 may still be
considered appropriate molecular targets for the design of new
therapeutic strategies aimed at counteracting the leukemic evolution of
T-LBLs.
| |
Acknowledgments |
We thank Drs F. Bussolino, E. C. Butcher, P. J. Kilshaw, P. Kinkade, A. Sonnenberg, G. Tarone, and D. Vestweber for providing antibodies.
| |
Footnotes |
Submitted June 3, 1999; accepted December 21, 1999.
Supported by the Associazione Italiana Ricerca sul Cancro (A.C., R.D.)
and by the FSN 1994-1995 grant (A.C.) of the Ministero della
Sanità.
L.B. and E.G. contributed equally to this work.
Reprints: Alfonso Colombatti, Divisione di Oncologia
Sperimentale 2, CRO, 33081 Aviano, Italy; e-mail: acolombatti{at}ets.it.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction