Purpose: Platelet-activating factor (PAF), a phospholipid mediator of inflammation, has been recently detected on tumor cells but its effect in tumor development is largely undefined.

Experimental Design: To address its potential role in tumor biology, we inhibited intratumor PAF activity by engineering tumor cell lines to express plasma PAF-acetylhydrolase (PAF-AH), the major PAF-inactivating enzyme, and studied their behavior in vitro and in vivo.

Results: When transfected with PAF-AH, KS-Imm human Kaposi’s sarcoma cells implanted in SCID mice and B16F10 mouse melanoma cells implanted in syngenic C57Bl/6J mice showed significantly reduced vascularization and growth allowing longer survival compared with control tumors. The amounts of bioactive PAF extracted from PAF-AH-transfected tumors were significantly reduced. In vitro, expression of PAF-AH did not influence cell proliferation, whereas it inhibited PAF-dependent cell motility in Kaposi’s sarcoma cells that express PAF-receptor but not in melanoma cells that did not express it. On the other hand, PAF-induced endothelial tubulogenesis in Matrigel was inhibited by incubation with supernatant from PAF-AH-transfected melanoma cells, indicating that PAF-AH inhibits in vitro neoangiogenesis.

Conclusions: We demonstrated that in situ PAF inactivation affects tumor vascularization and growth through inhibition of neoangiogenesis and, in the case of cells expressing PAF receptor, also tumor cell motility.

PAF3 (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is a mediator of several vascular and inflammatory processes (1, 2). Originally described as an inducer of platelet degranulation and aggregation, PAF was subsequently found to possess multiple biological activities on different cell types related to the expression of a specific receptor (3). Its physiological activity is restrained by the existence of a powerful enzymatic PAF-inactivating system, including both intracellular and plasmatic enzymes able to catalyze the hydrolysis of the sn-2 ester bond of PAF (4). The recent molecular identification of some of these enzymes allowed investigation on their physiological functions. The PAF-AH is a single polypeptide released mostly by activated macrophages (5), and its inactivation by mutation is responsible for an increased incidence and severity of asthma and vascular diseases (2). The beneficial therapeutic effect of administration of recombinant PAF-AH has been recently evaluated in animal models of inflammation such as pleurisy (5), paw edema (5), and acute pancreatitis (6).

A relevant part of the role of PAF is because of its effect on endothelial cells. It has long been known that endothelial cells may either synthesize PAF or promptly respond to its stimulation by redistributing their cytoskeleton with an increase in vascular permeability and expression of adhesion molecules for leukocyte interaction (2). Recently, PAF has been found to be synthesized by endothelial cells upon stimulation with VEGF (7), tumor necrosis factor α (8), HGF (9), or HIV-1 Tat (10) and to mediate their motogenic effect. These observations may be of particular relevance in the study of tumor growth and vascularization because PAF is detected within tumor lesions such as those of KS (11) and breast cancer (12). Moreover, several neoplastic cell lines produce PAF in culture or respond to its stimulation (13, 14, 15) and transgenic mice overexpressing the PAF-R typically develop melanocytic tumors (16). These data strongly suggest a role for this phospholipid in tumor biology, but a direct evidence is currently lacking. In this study, we engineered tumor cells to directly express plasma PAF-AH within tumor environment to study the role of PAF in tumor development.

Cell Lines and Transfectants.

The spontaneously immortalized iatrogenic KS-Imm (17) and mouse melanoma B16F10 cell lines (American Type Culture Collection, Manassas, VA) were transfected with pRC/CMV vector-expressing mouse PAF-AH (18) or with the empty vector. Transfectants were generated by electroporation (Gene Pulser; Bio-Rad Laboratories, Richmond, CA), selected in 1 mg/ml G418 (Boehringer Mannheim Corp., Indianapolis, IN), and tested by reverse transcription-PCR by using mouse and human PAF-AH mRNA-specific primers: 5′-TTTTCACTGGCAAGACACATCTTCTTTTGACTTC-3′ (forward) and 5′-CGTCAAAGTTCTGGTGCCTGAGCCCTTGATTGTA-3′ (reverse). PAF-AH activity was tested by a colorimetric assay based on hydrolysis of 2-thio PAF (Cayman Chemical, Ann Arbor MI). Primers for human PAF-R were described previously (19). Primers for mouse PAF-R were 5′-TTTTGGGTGTCATCACTTATA-3′ (forward) and 5′-GGCTGCGTGAGCAGCGTGTGG-3′ (reverse).

In Vitro Cell Migration Assay.

Cell migration was analyzed as described previously (19). Briefly, 105 cell/well were plated and rested for 12 h with medium containing 1% FCS, then washed three times with PBS and incubated with RPMI and the agonists. Cell migration was studied under a Nikon Diaphot inverted microscope in a plexiglass Nikon NP-2 incubator at 37°C. Image analysis of at least 30 cells/sample was performed by digital saving of images at 30 min of interval with a MicroImage analysis system (Casti Imaging srl, Venice, Italy). Migration tracks were generated by marking the position of nucleus of individual cells on each image. Values are given as mean ± SD.

In Vitro Endothelial Tubulogenesis Assay.

In vitro formation of tubular structures was studied on growth factor-reduced Matrigel-diluted 1:1 in ice-cold DMEM as described previously (20). Briefly, 5 × 104/well HMECs (21) were seeded onto Matrigel-coated wells in 10% FCS/DMEM. After cells had attached, the FCS-containing media were removed, and 0.5 ml of supernatant containing the stimuli were added.

Determination of Cellular Growth Rates in Vitro.

Each transfectants was cultured in 96-well flat-bottomed microtiter plates (Falcon Labware, Oxnard, CA) at a concentration of 5 × 104 cells/well in DMEM/10% FCS. After 24 h of culture, cells were incubated at 37°C in serum-free DMEM containing 250 μg/ml 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (Sigma) and read at 620 nm in an ELISA reader. All cultures were done in triplicate.

Evaluation of Tumor Growth in Vivo.

For in vivo experiments, cells were gently detached from plates with EDTA, washed, counted, and resuspended in saline. A total of 107 KS-Imm cell transfectants in a total volume of 150 μl was injected s.c. into the left back of 8-week-old female SCID mice (Charles River; Calco, Lecco, Italy) via a 26-gauge needle. Likewise, 5 × 106 B16F10 transfectants were injected s.c. into 18–25-g female C57Bl/6J mice (Charles River). Mice were kept in individual cages and sacrificed at the end point or prior, according to the internal ethical procedures for animal experimentation. Tumor size was documented by measuring two perpendicular diameters in millimeters using a caliper. Tissues were fixed in formaldehyde and a portion was immediately weighted and submitted for PAF extraction. Endothelial cells in the neoformed vessels were stained with fluorescein-labeled Griffonia simplicifolia lectin (Sigma; Ref. 22) and von Wille-brand factor antiserum (Sigma). Quantification of the vessel area was performed by digital analysis of 20 fields/slide of tumors, and vessels structures were counted only if showing a patented lumen with red cells and/or leukocytes. For in situ detection of apoptotic cells, tissue sections were subjected to TUNEL assay (ApoTag Oncor, Gaithersburg, MD) and counterstained with 1 μg/ml propidium iodide (Sigma).

Extraction and Quantification of PAF.

Lipids were extracted from tumor specimen homogenates by chloroform/methanol/water acidified to pH 3.0 to pH 3.5 with formic acid as described previously (12). Purified PAF was quantified by bioassay on washed rabbits platelets (23). PAF bioactivity was characterized by comparison with synthetic PAF according to the following criteria: (a) induction of platelet aggregation by a pathway independent of both ADP- and arachidonic acid/thromboxane A2-mediated pathway; (b) specificity of platelet aggregation as inferred from the inhibitory effect of PAF-R antagonist WEB 2170 (3 μm; Boehringer, Ingelheim am Rheim, Germany); and (c) chromatographic behavior on silica gel 60F254 TLC plates (Merck, Darmstadt, Germany) and on μPorasil high-performance liquid chromatography columns (Millipore; Waters, Milford, MA) and physicochemical characteristics such as inactivation by base-catalyzed methanolysis or phosholipase A2 (Sigma, St. Louis, MO) treatment and resistance to phospholipase A1 (Sigma) or treatment with weak base and acids (23).

Statistical Analysis.

Statistical analysis was performed using SPSS, Inc. (Chicago, IL). Univariate survival analysis was performed by Kaplan-Meier method with log-rank test. Student t test was performed where indicated.

To address the role of PAF in tumor growth and vascularization, we selected an highly angiogenic KS cell line (KS-Imm) shown to form vascularized tumors in SCID mice (23, 24, 25). KS-Imm cells were found to produce PAF under basal condition and to increase its production after stimulation (19). Moreover, KS-Imm cells express the PAF-R and display a motogenic response to PAF stimulation. PAF synthesis has been shown to be required for motility induced by VEGF and HIV-1 Tat (19).

To evaluate the effect of PAF inactivation in vivo, we engineered KS-Imm cells to stably express mouse plasma PAF-AH, which was absent in parental cells as detected by PCR or by PAF-AH enzymatic assay (data not shown). In parallel, KS-Imm cells transfected with the empty vector containing the neomycin resistance gene (KS-Imm/neo) were developed. No significant difference in the in vitro growth rate among the transfectant isolates was observed (data not shown).

KS-Imm transfectants were injected s.c. into SCID mice, and tumor growth was monitored. Tumors derived from KS-Imm/PAF-AH cells after an initial growth showed a significant reduction in size with respect to KS-Imm/neo-derived counterparts at the end point of 1 month postimplantation (Table 1). Histological analysis of the tumor vascularization revealed a microvascular density significantly lower in KS-Imm/PAF-AH-derived than in KS-Imm/neo-derived tumors (Table 1 and Fig. 1, A and C). Additionally, KS-Imm/PAF-AH-derived tumors were characterized by large areas of necrosis and apoptosis (Fig. 1,D). The amount of PAF extracted from KS-Imm/PAF-AH-derived tumors was significantly lower than that from KS-Imm/neo counterpart demonstrating effective intratumor PAF inactivation (Table 1). As shown in Fig. 2, a survival experiment demonstrated that mice bearing KS-Imm/neo-derived tumors did not survive longer than 52 days, whereas survival was 100% for mice injected with KS-Imm/PAF-AH cells at the completion of this study (3 months; Fig. 3 A). Similar results were obtained when the in vivo experiments were repeated with two other transfectant isolates of KS-Imm/PAF-AH cells and KS-Imm/neo cells (3 mice/group) to dispel the possibility of a clone-specific effect (data not shown).

It is conceivable that the observed results derive from inhibition of angiogenesis as well as of tumor cell invasiveness because KS-Imm cells themselves showed a marked motogenic response to PAF stimulation. In our system, the basal spontaneous motility of both transfectant lines was similar (Fig. 2 A). However, incubation with PAF (5 ng/ml) markedly enhanced motility of KS-Imm/neo but not KS-Imm/PAF-AH cells. The motility of KS-Imm/neo and KS-Imm/PAF-AH was not different under incubation with human basic fibroblast growth factor (30 ng/ml) plus porcine heparin (30 ng/ml), an agonist complex that induces motility independently from PAF synthesis (26). Therefore, PAF-AH gene transfer into KS-Imm cells specifically modifies PAF-dependent cell migration.

To study a tumor model where only endothelial cells could be influenced by the local inactivation of PAF, we extended this approach to B16F10 melanoma cells that were reported unable to produce PAF and did not express PAF-R (27). Melanoma cells stably expressing plasma PAF-AH (B16F10/PAF-AH) or control empty vector (B16F10/neo) were generated. No difference in proliferation rate was observed among the two lines (data not shown). In addition, PAF (up to 50 ng/ml) failed to induce proliferation (data not shown) or to increase cell motility of B16F10 (Fig. 2,B). Only transfection of human PAF-R was able to modify the response to PAF (Fig. 2,B). In addition, B16F10 cells were shown to be unable to produce PAF (data not shown; Ref. 27). In a 2-week end point study, 5 × 106 B16F10 cells/mouse were injected s.c. in syngeneic C57Bl/6J mice. Histological analysis showed reduced vascularization in melanoma tumors expressing PAF-AH (Fig. 1, E–H) and a slight but significant reduction of tumor size in comparison to their control counterparts (Table 1). PAF extracted from tumor tissue was almost undetectable for B16F10/PAF-AH but not for B16F10/neo tumors (Table 1). In the latter, endothelial cells migrating within the tumor are conceivably the main PAF target and source because only few inflammatory cells were detected.

In a second set of experiments, survival of melanoma tumor-bearing mice was analyzed. Mice bearing B16F10/PAF-AH tumors survived significantly longer that those bearing B16F10/neo tumors (Fig. 3,B). This experiment was repeated with similar results with two other different transfectant isolates (data not shown). All together, these experiments suggest that in B16F10 tumors the main effect of PAF-AH relays on partial inhibition of neoangiogenesis. To investigate this hypothesis, in vitro experiments were performed to evaluate whether PAF-AH production by B16F10/PAF-AH inhibits the formation of capillary-like tubular structures on Matrigel by HMEC that is typically induced by PAF (26). Supernatants from serum-free overnight cultured B16F10/PAF-AH and B16F10/neo cells were used based on the inability of these cells to produce endogenous PAF. In basal condition, both supernatants did not substantially modify endothelial behavior (Fig. 4, A and D). When the transfectant supernatants were preincubated for 5 min with 5 ng/ml PAF and then added to the endothelial cells, a marked inhibition of PAF-induced endothelial tube formation in Matrigel was observed after 5 h with B16F10/PAF-AH (Fig. 4,E) but not with B16F10/neo supernatant (Fig. 4,B), suggesting that inactivation of PAF inhibits in vitro angiogenesis. To dispel the possibility that other factors than PAF-AH may be responsible for the effect of B16F10/PAF-AH supernatant, the same experiment was repeated by replacing PAF with the same amount of C-PAF, which is resistant to PAF-AH hydrolysis. Incubation of 5 ng/ml C-PAF with B16F10/PAF-AH supernatant did not modify its ability to induce in vitro angiogenesis (Fig. 4, C and F).

Endothelial-stimulating factors may promote tumor growth and invasiveness by supporting neovascularization of tumor tissue as well as by directly influencing tumor cell behavior. PAF, a well-known vascular inflammatory agent, has been shown to induce angiogenesis through stimulation of endothelial migration. Response to PAF stimulation depends on the engagement of a specific cell surface receptor that is a member of a family of seven-transmembrane-spanning G-protein-linked receptors. Beside endothelium, several tumor cell types were shown to express PAF-R such as endometrial cancer cells (13), KS cells (19), breast cancer cells (12), colon carcinoma cells (28), and neuroblastoma cells (15). We have previously studied the effect of PAF stimulation in vitro on KS (19) and breast cancer cells (29) that produce PAF and express PAF-R. PAF was able to induce proliferation in the latter and to enhance motility in both tumor cell types. An indirect evidence for the role of PAF in tumor vascularization was obtained in an in vivo model where breast cancer cells were implanted s.c. in Matrigel containing a PAF-R antagonist (29). In this model, neoangiogenesis elicited by tumor cells was inhibited. However, direct evidence for the role of PAF in the development and vascularization of tumor was lacking. We addressed this issue by suppressing PAF activity in the intratumor environment of two different tumor cell types: human KS and murine melanoma. This effect was obtained by stably transfecting plasma PAF-AH into tumor cells, then implanting them in mice and observing the development of tumor lesions. This approach was selected because several problems account for the difficulty of blunting PAF activity in vivo for a sustained period of time. Indeed, the enzymes responsible for its synthesis are poorly characterized, and it is currently unfeasible to selectively control PAF production. In addition, although the use of chemical PAF-R antagonists has allowed in vivo studies in several models of vascular and inflammatory diseases (2), their short half-life and undetermined bioavailability within the neoplastic tissue make difficult their use. Inhibition studies may also be performed by enhancing PAF inactivation. Because it has been observed low diffusion inside tumors of PAF-AH derived from the circulation (30), we engineered tumor cells to directly express plasma PAF-AH within tumor environment. After this approach, PAF detection within PAF-AH-transfectant tumors was indeed efficiently reduced or even absent and resulted in reduction of tumor mass and prolonged survival in both tumor models. The effect of PAF-AH expression was more pronounced in a system where tumor cells responded to PAF and were themselves source of this phospholipid. However, retarded growth was obtained also when a tumor cell line that did not synthesize and express PAF-R was used. In this setting, it is likely that PAF-AH inactivated PAF derived from endothelial cells or infiltrating leukocytes. Tissue analysis revealed reduction of vessel density, thus suggesting that local PAF inactivation primarily resulted in impaired neoangiogenesis. The profound impact in tumor behavior obtained through local inactivation of PAF may be explained by the peculiar nature of this mediator. Indeed, PAF is considered a short-range inflammatory agent that physiologically trigger adhesion and migration of leukocytes tethered on the surface of activated endothelial cells (1, 2). This phospholipid is synthesized within minutes after stimulation by a number of inflammatory and pro-angiogenic mediators, such as tumor necrosis factor (8), HGF (9), and VEGF (7). Because its rapid inactivation is guaranteed by the presence of both plasmatic and intracellular PAF-AHs, it is conceivable that most of the activity of this phospholipid is exerted within defined areas of cell-to-cell contact where inactivators are excluded. This is also consistent with the fact that several cell types produce PAF only as cell associated and may therefore stimulate PAF-R-expressing cells only during cell surface interaction. In turn, PAF itself may induce the expression of several angiogenic factors or chemokines, including basic and acidic fibroblast growth factor, placental growth factor, VEGF and its specific receptor flk-1, HGF, KC, and macrophage inflammatory protein 2, thus sustaining and amplifying the inflammatory or angiogenic process (31).

In conclusion, these findings provide direct evidence that disruption of PAF-mediated signaling by local production of PAF-AH resulted in impairment of tumor growth and vascularization.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the Associazione Italiana per la Ricerca sul Cancro, “MURST Cofin 2001,” CNR-Targeted Project on Biotechnology, and by the Istituto Superiore di Sanità (Pathology, Clinic and Therapy of AIDS) (to G. C.).

3

The abbreviations used are: PAF, platelet-activating factor; PAF-AH, PAF-acetylhydrolase; PAF-R, platelet-activating factor receptor; VEGF, vascular endothelial growth factor; KS, Kaposi’s sarcoma; HMEC, human microvascular endothelial cell; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; C-PAF, carbamyl PAF; HGF, hepatocyte growth factor.

Fig. 1.

Histological analysis of KS-Imm- and B16F10-derived tumors examined 1 month and 2 weeks after s.c. inoculation, respectively. Control KS-Imm/neo tumors injected in SCID mice (A) displayed several canalized vessels (trichromic staining, ×200) containing RBCs and leukocytes (inset, ×600). TUNEL assay revealed absence of apoptotic cells within tumor tissue (B, ×200). In tumors derived from KS-Imm/PAF-AH cells (C, ×200), the number and size of vessels (inset) were markedly reduced and large necrotic areas were present. TUNEL assay revealed the presence of several apoptotic (yellow) cells (D, ×200). As shown in the bottom panel, inoculation of control B16F10/neo into C57Bl/6J mice lead to highly vascularized tumors (E, ×200) with large patented vessels (F, ×600). In contrast, B16F10/PAF-AH tumors presented large necrotic areas (G, ×200) with few small vessels (H, ×600).

Fig. 1.

Histological analysis of KS-Imm- and B16F10-derived tumors examined 1 month and 2 weeks after s.c. inoculation, respectively. Control KS-Imm/neo tumors injected in SCID mice (A) displayed several canalized vessels (trichromic staining, ×200) containing RBCs and leukocytes (inset, ×600). TUNEL assay revealed absence of apoptotic cells within tumor tissue (B, ×200). In tumors derived from KS-Imm/PAF-AH cells (C, ×200), the number and size of vessels (inset) were markedly reduced and large necrotic areas were present. TUNEL assay revealed the presence of several apoptotic (yellow) cells (D, ×200). As shown in the bottom panel, inoculation of control B16F10/neo into C57Bl/6J mice lead to highly vascularized tumors (E, ×200) with large patented vessels (F, ×600). In contrast, B16F10/PAF-AH tumors presented large necrotic areas (G, ×200) with few small vessels (H, ×600).

Close modal
Fig. 2.

Motility of tumor cell transfectants. As shown by migration tracks of representative KS-Imm/neo and KS-Imm/PAF-AH cells (A) and by their speed averages (B), the basal spontaneous motility of KS-Imm/neo and KS-Imm/PAF-AH cell lines was similar. Incubation with PAF (5 ng/ml) markedly enhanced motility of KS-Imm/neo but not KS-Imm/PAF-AH cells. No difference was observed under incubation with 30 ng/ml basic fibroblast growth factor (bFGF) plus 30 ng/ml porcine heparin, an agonist complex that induces motility independently from PAF synthesis. C, motility of B16F10/neo and B16F10/PAF-AH cell lines was unchanged after stimulation with PAF. Increased motility was observed only when melanoma cells where stably transfected with PAF-receptor (B16F10/PAF-R).

Fig. 2.

Motility of tumor cell transfectants. As shown by migration tracks of representative KS-Imm/neo and KS-Imm/PAF-AH cells (A) and by their speed averages (B), the basal spontaneous motility of KS-Imm/neo and KS-Imm/PAF-AH cell lines was similar. Incubation with PAF (5 ng/ml) markedly enhanced motility of KS-Imm/neo but not KS-Imm/PAF-AH cells. No difference was observed under incubation with 30 ng/ml basic fibroblast growth factor (bFGF) plus 30 ng/ml porcine heparin, an agonist complex that induces motility independently from PAF synthesis. C, motility of B16F10/neo and B16F10/PAF-AH cell lines was unchanged after stimulation with PAF. Increased motility was observed only when melanoma cells where stably transfected with PAF-receptor (B16F10/PAF-R).

Close modal
Fig. 3.

Survival of mice-bearing tumors induced by KS-Imm or B16F10 transfectants. A, SCID mice (6/each group) were injected s.c. with KS-Imm/neo (———) and KS-Imm/PAF-AH cells (····) as described in the “Materials and Methods” section and monitored for 3 months. All of the mice injected with KS-Imm/neo cells were dead within 52 days as a consequence of tumor progression, whereas at the end of the experiment (90 days) survival was 100% for mice injected with KS-Imm/PAF-AH cells. B, the survival of C57Bl/6J mice (6/each group) injected s.c. with B16F10/neo (———) was significantly shorter than the one of mice injected with B16F10/PAF-AH cells (····). Kaplan-Meyer analysis between the KS-Imm/neo and KS-Imm/PAF-AH group (log-rank test: 11.34; P < 0.01) and between the B16F10/neo and the B16F10/PAF-AH group (log-rank test: 11.91; P < 0.01) was performed.

Fig. 3.

Survival of mice-bearing tumors induced by KS-Imm or B16F10 transfectants. A, SCID mice (6/each group) were injected s.c. with KS-Imm/neo (———) and KS-Imm/PAF-AH cells (····) as described in the “Materials and Methods” section and monitored for 3 months. All of the mice injected with KS-Imm/neo cells were dead within 52 days as a consequence of tumor progression, whereas at the end of the experiment (90 days) survival was 100% for mice injected with KS-Imm/PAF-AH cells. B, the survival of C57Bl/6J mice (6/each group) injected s.c. with B16F10/neo (———) was significantly shorter than the one of mice injected with B16F10/PAF-AH cells (····). Kaplan-Meyer analysis between the KS-Imm/neo and KS-Imm/PAF-AH group (log-rank test: 11.34; P < 0.01) and between the B16F10/neo and the B16F10/PAF-AH group (log-rank test: 11.91; P < 0.01) was performed.

Close modal
Fig. 4.

Effect of B16F10/neo and B16F10/PAF-AH supernatants on PAF-mediated endothelial tubulogenesis in vitro. Micrographs (A–F) and capillary connection counts (G) of endothelial cells (HMEC) seeded on Matrigel-coated wells and incubated with transfectant supernatants (sup) with or without preincubation for 5 min with 5 ng/ml PAF or C-PAF. After 5 h, scarce tubulogenesis was observed in cells incubated with B16F10/neo or B16F10/PAF-AH alone. Addition of PAF induced tubulogenesis in the presence of B16F10/neo but not B16F10/PAF-AH supernatant. In contrast, a similar amount of tubulogenesis was observed by replacing PAF with C-PAF, a PAF-AH-resistant homologue.

Fig. 4.

Effect of B16F10/neo and B16F10/PAF-AH supernatants on PAF-mediated endothelial tubulogenesis in vitro. Micrographs (A–F) and capillary connection counts (G) of endothelial cells (HMEC) seeded on Matrigel-coated wells and incubated with transfectant supernatants (sup) with or without preincubation for 5 min with 5 ng/ml PAF or C-PAF. After 5 h, scarce tubulogenesis was observed in cells incubated with B16F10/neo or B16F10/PAF-AH alone. Addition of PAF induced tubulogenesis in the presence of B16F10/neo but not B16F10/PAF-AH supernatant. In contrast, a similar amount of tubulogenesis was observed by replacing PAF with C-PAF, a PAF-AH-resistant homologue.

Close modal
Table 1

Size, vascularization, and PAF content of experimental tumors

Cell line-derived tumorsaMean tumor diameter (mm)Intratumor vessel area (%)Intratumor PAF content (pg/mg)
KS-Imm/neo 15.05 ± 4.26 11.24 ± 2.93 34.61 ± 11.43 
KS-Imm/PAF-AH 4.94 ± 2.70b 2.60 ± 2.42b 3.15 ± 2.52c 
B16F10/neo 16.72 ± 3.24 6.45 ± 2.66 11.25 ± 3.40 
B16F10/PAF-AH 9.25 ± 1.72b 3.04 ± 1.20b 1.46 ± 1.05c 
Cell line-derived tumorsaMean tumor diameter (mm)Intratumor vessel area (%)Intratumor PAF content (pg/mg)
KS-Imm/neo 15.05 ± 4.26 11.24 ± 2.93 34.61 ± 11.43 
KS-Imm/PAF-AH 4.94 ± 2.70b 2.60 ± 2.42b 3.15 ± 2.52c 
B16F10/neo 16.72 ± 3.24 6.45 ± 2.66 11.25 ± 3.40 
B16F10/PAF-AH 9.25 ± 1.72b 3.04 ± 1.20b 1.46 ± 1.05c 
a

KS-Imm/neo- and KS-Imm/PAF-AH-derived tumors were examined 1 month after s.c. inoculation in SCID mice (6 mice/group). B16F10/neo- and B16F10/PAF-AH-derived tumors were examined 2 weeks after s.c. inoculation in C57B1/6J mice (6 mice/group). Statistical analysis was performed for KS-Imm/neo versus KS-Imm/PAF-AH tumors and for B16F10/neo versus B16F10/PAF-AH tumors in each result category (Student t test:

b

, P < 0.05;

c

, P < 0.01).

KS-Imm cells were obtained from Dr. Adriana Albini (Istituto Nazionale per la Ricerca Sul Cancro, Genova, Italy). We thank Fabrizio Fop for statistical analysis of data.

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