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Chair of Nephrology, Department of Internal Medicine, University of Torino, Torino, Italy;
Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; and
Molecular Pathology Unit, Department of Pathology, Harvard Medical School and Massachusetts General Hospital, Charlestown, MA 02129
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of the present study was to investigate both in vitro and in vivo the importance of CD40 in growth and vascularization of KS.
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Materials and Methods |
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HUVEC were isolated and cultured as previously described (11). The spontaneously immortalized iatrogenic KS cell line (12) was cultured in DMEM/10% FBS. Cells were transfected with PSV-2 vector (Invitrogen, San Diego, CA) containing the neomycin resistance gene or with PSV-2 vector and cDM8 expressing a soluble CD40 (sCD40)-Ig fusion protein as previously described (13). In addition, CHO cells (American Type Culture Collection (ATCC), Manassas, VA) were stably transfected with cDNA encoding soluble CD154-CD8 fusion protein (sCD154) (14), and serum-free supernatant was collected. Transfectants were generated by electroporation (Gene Pulser; Bio-Rad Laboratories, Richmond, CA) at 250 V and 960 µF in 4-mm electroporation cuvettes. Clones were selected in 1 mg/ml G418 (Boehringer Mannheim, Indianapolis, IN) and tested for soluble fusion protein expression, as previously described (15).
CD40 and CD154 expression
For cytofluorometric analysis, cells were detached from plates with EDTA, washed, resuspended in PBS, and incubated at 4°C for 30 min with RPMI containing 10 µg/ml anti-CD40 mAb (PharMingen, San Diego, CA) or control isotype-matched IgG. As a second step reagent, FITC-conjugated anti-mouse IgG (Sigma) was used. Cells were analyzed on a FACS (Becton Dickinson, Mountain View, CA).
For Western blot analysis of the expression of CD40 and CD154 by KS cells, cells were lysed at 4°C for 1 h in a lysis buffer (50 mM Tris-HCl, pH 8.3, containing 1% Triton X-100, 10 µM PMSF, 10 µM/ml leupeptin, and 100 U/ml aprotinin). As control, J111 cells (ATCC) transfected with the full-length human CD40 cDNA (16) were used. After centrifugation of the lysates at 15,000 x g, the supernatants were quantitated for protein content by the Bradford method. Aliquots containing 100 µg of protein per lane were subjected to SDS/10% PAGE under reducing conditions and electroblotted onto nitrocellulose membrane filters. The blots were blocked with 5% nonfat milk in 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, plus 0.1% Tween (TBS-T). The membranes were subsequently incubated overnight at 4°C with polyclonal rabbit Ab against human CD40 or human CD154 (Santa Cruz Biotechnology, Santa Cruz, CA) at a concentration of 500 ng/ml. After extensive washing with TBS-T, the blots were incubated for 1 h at room temperature with peroxidase-conjugated protein A (200 ng/ml; Amersham, Buckingamshire, U.K.), washed with TBS-T, developed with ECL detection reagents (Amersham) for 1 min, and exposed to X-Omat film (Eastman Kodak, Rochester, NY). Detection of CD154 was also performed by RT-PCR. Total RNA was extracted from cells by guanidinium thiocyanate phenol-chloroform and precipitated with isopropanol. One microgram of RNA was treated with 6 U of RNase-free DNase for 1 h at 37°C and then for 5 min at 94°C: cDNA was obtained by using random hexamer primers (Perkin-Elmer/Cetus, Norwalk, CT). Reverse transcription was conducted at 42°C for 60 min; in addition to 1 µg of RNA, the reaction mixture (20 µl) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1 mM dNTPs, 20 U ribonuclease inhibitor, and 50 U Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer/Cetus). cDNA was then subjected to 35 cycles of amplification by the PCR in an automated DNA thermal cycler (Perkin-Elmer/Cetus) by using CD154 mRNA-specific primer pairs: forward, 5'-TGTTCAGAGTTTGAGTAAGCC-3'; reverse, 5'-AGGTTGGACAAGATAGAAGAT-3'.
The PCR mixture (50 µl) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 20 pmol of (+) and (-) primers, and 2 U thermostable DNA polymerase (Perkin-Elmer/Cetus). Times and temperatures for denaturation, annealing, and extension were 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C, respectively. Amplification product was analyzed in 2% agarose gels containing 0.5 µg/ml of ethidium bromide. As control, CHO cells untransfected or transfected with sCD154-specific cDNA were used.
In vitro cell migration
A total of 105 cells/well were plated and rested for 12 h with medium M199 containing 1% FCS, then washed three times with PBS and incubated with RPMI and the agonist. Cell division did not start to any significant degree during the experiments. Cell migration was studied over a 20-h period under a Nikon Diaphot inverted microscope with a x10 phase-contrast objective in an attached, hermetically sealed plexiglass Nikon NP-2 incubator at 37°C. Cell migration was recorded using a JVC-1CCD video camera. Image analysis was performed with a MicroImage analysis system (Cast Imaging srl, Venice, Italy) and an IBM-compatible system equipped with a video card (Targa 2000; Truevision, Santa Clara, CA). Image analysis was performed by digital saving of images at 30 min of interval. Migration tracks were generated by marking the position of nucleus of individual cells on each image. The net migratory speed (velocity straight line) was calculated by the MicroImage software based on the straight line distance between the starting and ending points divided by the time of observation. Migration of at least 30 cells was analyzed for each experimental condition. Values are given as means ± SD.
Apoptosis assays
Three assays of apoptosis were performed in this study. In the sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT)-based assay (17), cells were cultured in 96-well flat-bottom microtiter plates (Falcon Labware, Oxnard, CA) at a concentration of 5 x 104 cells/well in DMEM in the presence or absence of FBS. At different periods of time, cells were washed and incubated in serum-free DMEM containing 250 µg/ml XTT at 37°C. Cell growth was monitored by determination of the absorption values at 620 nm in an automated ELISA reader. In selected experiments, cells were incubated in serum-free medium in the presence of an agonist anti-human CD40 mAb (clone SC3; PharMingen) (18), or irrelevant isotype-matched mAb (PharMingen). Such assay was used also to evaluate cell proliferation.
The second assay was described by Kroesen et al. (19). Briefly, KS cells were labeled overnight with 3,3'-dioctadecylloxacarbocyanine (DiOC18; Molecular Probes, Eugene, OR), washed, and incubated at 37°C for 24 h with the stimuli. At the end of the incubation, a 3.75 mM solution of the membrane-impermeant nucleic acid counterstain propidium iodide (PI) is added to label any cells with compromised plasma membrane and cells are analyzed under FACS.
Third assay is based on PI staining of cells followed by flow cytometry analysis, as described (20). Briefly, 106 cells were incubated for 4 h at 4°C in 2 ml hypotonic solution containing 50 µg/ml PI, 0.1% sodium citrate, 0.1% Triton X-100, and 20 µg/ml DNase-free RNase A. Cells with subdiploid DNA content (sub-G0/G1 peak) were considered apoptotic cells. All cultures were done in triplicate.
For in situ detection of apoptotic cells, tissue sections were subjected to TUNEL assay (ApoTag Oncor, Gaithersburg, MD). Tissue from rat-regressing mammary glands obtained at the fourth day after weaning was used as positive control for the technique. Sections were counterstained with 1 µg/ml PI in PBS for 30 s, mounted with antifade mounting medium (Vector Laboratories, Burlingame, CA), and examined.
Evaluation of tumor growth in vivo
For in vivo experiments, cells were gently detached from plates with EDTA, washed with PBS, counted in a microcytometer chamber, and resuspended in saline. A total of 107 cells, in a total volume of 150 µl, was injected s.c. into the left back of SCID mice (Charles River, Wilmington, MA) via a 26-gauge needle and using a 1-ml syringe. Tumor size was documented by measuring two perpendicular diameters in millimeters using a caliper. Animals were sacrificed at 2 mo endpoint and subjected to autopsy. All organs were examined macroscopically for evidence of tumor growth. Tissue containing visible tumor growth was fixed in formaldehyde for light microscopy and immunohistochemical studies.
Immunofluorescence studies
For tissue staining, 5-µm paraffin-embedded tissue sections were stained with 10 µg/ml of goat anti-mouse CD154 (Santa Cruz Biotechnology) or control isotype-matched Ab (PharMingen) for 45 min at room temperature. The slides were washed in PBS, incubated with fluorescein-labeled rabbit anti-goat or goat anti-rabbit IgG affinity-purified Ab (Sigma, St. Louis, MO) for 30 min at room temperature, washed, mounted with antifade mounting medium (Vector Laboratories), and examined. For evaluation of neovascular structures within the tumor, sections were stained with FITC-conjugated Griffonia semplicifolia lectin (Sigma) (21).
Murine angiogenesis assay
Female C57 mice were used at 6–8 wk of age. Angiogenesis was assayed as growth of blood vessels from s.c. tissue into a solid gel of basement membrane, Matrigel (Becton Dickinson Labware, Bedford, MA), containing the test sample (22). Matrigel (8.13 mg/ml), in liquid form at 4°C, was mixed with 40 µg/ml of agonist rat anti-mouse CD40 (clone 3/23; Serotec, Oxford, U.K.) (23) or of control purified rat IgG (Sigma) and injected (0.25 ml) into the abdominal s.c. tissue of mice, along the peritoneal midline. Basic fibroblast growth factor (bFGF; 10 ng/ml) was used as positive control. Matrigel rapidly forms a solid gel at body temperature, trapping the factors to allow slow release and prolonged exposure to surrounding tissues. The Matrigel used was extracted according to the procedure described by Taub et al. (24), which has been previously shown to efficiently deplete Matrigel of angiogenic cytokines (25, 26). At various times, mice were subsequently killed and gels were recovered and processed for histology. Typically, the overlying skin was removed, and gels were cut out by retaining the peritoneal lining for support. Part of tissue was fixed in 10% buffered Formalin and embedded in paraffin. Sections cut at 3 µm and stained with hematoxylin and eosin were studied by light microscopy. Other sections, obtained from frozen tissue cut with a cryostat, were stained for von Willebrand factor by immunofluorescence microscopy, performed as previously described (11). Vessel area and the total Matrigel area were planimetrically assessed from stained sections, as described by Kibbey et al. (27). Were considered vessels only those structures possessing a patent lumen and containing RBC. Results were expressed as percentage ± SD of the vessel area to the total Matrigel area.
Statistical analysis
All data are expressed as mean SD. Statistical analysis was performed by ANOVA with Dunnett’s comparison test where appropriated.
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Results |
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). To further confirm
this result, Western blot analysis was performed (inset,
Fig. 1). As a positive control, J111 cells, which do not express CD40,
were transfected with the human full-length CD40 cDNA. Lysates of KS
cell and CD40-transfected J111 cells showed a 48-kDa band that was
absent in parental J111 cells (inset, Fig. 1). Absence of
CD154 expression was determined both by Western blot analysis and
RT-PCR (data not shown).
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A). The effect of
anti-CD40 mAb was dose dependent (Fig. 2B). No effect
was observed with control irrelevant mAb. Enhancement of cell motility
was also observed after stimulation of KS cells with sCD154 (Fig. 2A). Similar experiments were performed on HUVECs. As shown
by Fig. 3, transient cell contraction was
evident 30 min after CD40 ligation. A significantly enhanced
endothelial cell migration started at 1 h and remained sustained
for the whole period of observation (Figs. 3 and 4A). The effect of
anti-CD40 mAb was dose dependent (Fig. 4B). A similar
motogenic effect was triggered by sCD154, but not by vehicle alone, or
control mAb (Fig. 4A).
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). To test for an antiapoptotic effect
of CD40 activation, vincristine was chosen as apoptosis-inducing agent
for KS cells because it is currently adopted in the chemotherapy of
this tumor. The results show that CD40 ligation significantly reduced
the apoptotic effect of vincristine on these cells (Figs. 5A and
6A). A dose response to the
antiapoptotic effect of anti-CD40 mAb was observed. The
antiapoptotic effect on KS cells treated with 0.25 µg/ml for 48
h revealed by the XTT-based assay (n = 3 experiments) was
absent at 0.1 µg/ml anti-CD40 mAb, detectable with 1 µg/ml
anti-CD40 mAb (40 ± 7% inhibition of apoptosis), and was
maximal with 10 µg/ml (88 ± 12% inhibition of apoptosis). In
addition, CD40 engagement on endothelial cells inhibited apoptosis
induced by serum withdrawal, as shown by Figs. 5B and
6B. The antiapoptotic effect on serum-deprived HUVEC at
48 h studied by the XTT-based assay (n = 3
experiments) was undetectable at 0.1 µg/ml anti-CD40 mAb,
detectable with 1 µg/ml anti-CD40 mAb (52 ± 13%
inhibition of apoptosis), and was maximal with 10 µg/ml (74 ±
9% inhibition of apoptosis).
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). KS-sCD40 and KS-neo cells were compared for tumor
formation in s.c. tissue of SCID mice. A total of
107 cells were injected into SCID mice and
sacrificed 2 mo later. At autopsy, KS-sCD40-derived tumors showed a
marked reduction in size with respect to KS-neo-derived counterparts
(Fig. 7A). Histologic
examination revealed that KS-neo-derived tumors had few or no necrotic
areas, and only few apoptotic cells were observed (Fig. 8, A and C). In
contrast, KS-sCD40 tumors showed several areas of focal necrosis (Fig. 8B). In situ staining of apoptotic cells by the TUNEL assay
revealed a mantle zone of apoptotic cells surrounding necrotic
areas (Fig. 8D). Several apoptotic cells were also present
within histologically intact neoplastic tissue (Fig. 8D).
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a). In contrast, KS-sCD40
tumors showed few vessels with poor branching (Fig. 9b).
Similar results were obtained when the in vivo experiments were
repeated with two other transfectant isolates of KS-sCD40 and KS-neo
cells (three mice/group) to dispel the possibility of a clone-specific
effect (not shown).
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c) and on endothelial cells of
tumor vessels (Fig. 9d). Platelet clumps were identified by
immunofluorescence positive staining for P-selectin (data not
shown).
The in vivo angiogenic effect of CD40 engagement was studied in the
murine model of Matrigel s.c. implantation. Fig. 7B shows
the quantitative morphometric analysis of neo-angiogenesis induced
within Matrigel 6 days after implantation by agonist rat anti-CD40
mAb, irrelevant rat IgG as negative control, and bFGF as positive
control. Anti-CD40 mAb induced a significant angiogenic effect, as
shown by the presence of canalized vessels and microaneurismatic
structures (Fig. 8F) containing RBC and leukocytes within
the Matrigel. Sections of the gel were stained with anti-von
Willebrand factor Abs to confirm the presence of endothelial cells in
association with the vessels (data not shown). Angiogenesis was absent
in mice injected with Matrigel containing irrelevant rat IgG.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two mechanisms may account for the results observed in this study. First, as shown by in vitro experiments, CD40 engagement activates cell motility of both KS and HUVEC. This suggests that CD40 activation may enhance tumor cell invasion of tissues and endothelial cell organization to form a network of neo-formed vessels, thus allowing tumor expansion. Second, we observed a protective effect of CD40 stimulation on apoptosis in both KS and HUVEC, in vitro. This is consistent with the in vivo observations that disruption of the CD40-CD154 interaction results in extensive KS tumor necrosis as well as apoptosis. Indeed, in vivo lack of CD40-mediated survival signals may render tumor and endothelial cells sensitive to proapoptotic stimuli. It is conceivable that the two mentioned mechanisms may synergize in vivo. In fact, reduced neo-vascularization in a rapidly growing tumor may expose central areas to ischemia. The consequences of such an event may be enhanced by lack of survival signals, such as the ones derived from CD40 activation. To identify potential physiologic sources of CD154 in vivo, we performed in situ analysis of CD154 expression. Several studies identified a number of cellular sources of CD154, including lymphocytes, monocytes, basophils, platelets, endothelial and smooth muscle cells, and tumor cells (9). In addition, biologically active sCD154 may also be released and act in an autocrine or paracrine way (28, 29). In our experimental conditions, contribution of CD154-expressing lymphocytes to intratumor CD40 stimulation was absent, as SCID mice were used as tumor recipients. In contrast, we observed several tumor vessels positive for CD154 as well as platelet aggregates. Expression of CD154 by endothelial cells has been recently reported in atheromatous plaques (30). Moreover, activated platelets may express CD154 that is able to interact with CD40 on endothelial cells (31). In support of this notion, recent studies have indicated a potential role for platelets in delivering stimulatory signals for tumor cells (32).
In conclusion, this study identifies a novel and potentially relevant role for CD40 expressed on tumor and endothelial cells that favors the development of KS, and may contribute to the understanding of the biological role of CD40-CD154 axis in tumor biology.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Giovanni Camussi, Cattedra di Nefrologia, Dipartimento di Medicina Interna, Corso Dogliotti 14, 10126, Torino, Italy. E-mail address:
3 Abbreviations used in this paper: KS, Kaposi’s sarcoma; bFGF, basic fibroblast growth factor; DiOC18, 3,3'-dioctadecylloxacarbocyanine; PI, propidium iodide; s, soluble; XTT, sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate.
Received for publication June 16, 1999. Accepted for publication September 14, 1999.
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is required for kidney tubulogenesis in Matrigel cultures in serum-free medium. Proc. Natl. Acad. Sci. USA 87:4002.This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), sCD154 (dilution 1/4) (
), or control supernatant
(dilution 1/4) (
). B, The dose-response effect of
anti-CD40 mAb (open columns) or control mAb (filled columns) on KS
cells incubated for 2 h at 37°C. Results are expressed as
means ± SD of three separate experiments.
), sCD154 (dilution 1/4) (
