Animals
Seven- to 10-week-old male BALB/C and 7- to 8-week-old female C57BL/6 mice from our own animal facilities were used. All animal procedures were in accordance with the ethical principles adopted by the Brazilian College of Animal Experimentation and approved by the Ethical Committee for Animal Research of the Institute of Biomedical Sciences, University of São Paulo.
Tumours
The murine melanoma cell line B16F10 was a kind gift from I. Fidler (M.D. Anderson, Texas, USA) and was cultured in RPMI 1640 medium (Cultilab, Campinas, Brazil), pH 7.4 supplemented with 10% fetal bovine serum (FBS, Cultilab, Campinas, Brazil), in the absence of antibiotics, at 37°C and 5% CO2. Ehrlich ascitis tumour (EAT) cells were grown as described elsewhere [12].
Evaluation of tumour growth
Ehrlich ascitis tumour (103-105 cells) was injected intraperitoneally (i.p.) in BALB/c mice. At different times after tumour implantation, aliquots of the ascitis fluid were recovered using a 21-gauge needle to measure cell number and mediator/cytokine levels. B16F10 melanoma cells (5 × 105) were injected subcutaneously (s.c.) in C57BL/6 mice and tumour growth was determined via measurement of the diameter of the solid tumour mass, from which the volume was estimated using the formula for a spheroid: V = 0.52 × (largest axis) × (smallest axis) 2.
Treatments
The PAF-R antagonist WEB2170 (5 mg/Kg; Boehringer Ingelheim, Germany) was injected i.p. 30 minutes before tumour implantation and injections were repeated every 24 h until the end of the experiment. In order to evaluate the possible benefit of the combined therapy with WEB2170, treatment with the chemotherapeutic drug DTIC was chosen at a suboptimal regimen based on previously published studies [13, 14]. In our study 40 μg DTIC (Sigma Chem. Co.) was given i.p. to each mouse every 3 days after tumour implantation. To determine the impact of these treatments on the survival of B16F10 melanoma-bearing mice, the treatment protocols were extended to 35 days. The Kaplan-Meier method and the log-rank test were used to estimate and compare the different groups.
Quantification of VEGF, PGE2 and nitrite in the ascitis fluid
At day 10 of EAT growth, BALB/C mice were sacrificed and the ascitis fluid was recovered. The suspension was centrifuged (180 × g for 10 min) and VEGF levels were determined in the supernatant by ELISA using goat polyclonal antiserum against mouse VEGF (Santa Cruz Biotechnology, Inc.). PGE2 levels were also determined by ELISA using a commercially available kit (Cayman Chem. Co.). NO levels were determined according to the method described by Bartholomew (1984) [15]. Briefly, aliquots of 150 μL of the ascitis fluid were treated with 15 μL of 10% ZnSO4 solution for 10 minutes at 4°C. Three μL of 2.5 N NaOH were added and the ascitis fluid was incubated at 4°C for 10 minutes. The NO3
- content was converted to NO2
- through addition of 15 μL of 0.5 M NaH2PO4, 15 μL of 2.4 M ammonium formiate and 10 μL of a suspension of E. coli to the ascitis fluid. The bacterium E. coli expresses nitrate reductase, which catalyses the conversion of nitrate to nitrite. Samples were further incubated at 37°C for 2 h, centrifuged (18000 × g for 5 min at 20°C), and 100 μL of Griess Reagent (0.1% N-naphthyl-ethyl-indiamine, 1% sulphanilamide, 2.5% orthophosphoric acid - Sigma Co.) were added to the same volume of the supernatant. NO levels were quantified in a spectrophotometer at 540 nm.
Characterization of apoptotic and necrotic cells in the ascitis fluid
BALB/C mice were inoculated i.p. with 1 × 103 EAT cells and sacrificed after 7 or 10 days. The peritoneal cavity was injected with 3 mL of PBS to recover the ascitis. The fluid was centrifuged (180 × g for 10 min) and the cell pellet resuspended in 1 ml of RPMI 1640 with 10% fetal calf serum. Trypan Blue dye (0.2%) was added to aliquots of cellular suspensions and the percentage of cells that had taken up the dye (necrotic cells) was determined microscopically. The percentage of apoptotic cells was determined by flow cytometry (FACScalibur, Becton Dickinson, San Jose, CA) after incubation of 3 to 5 × 105 cells with propidium iodide (20 μg/mL in 0.1% sodium citrate and 0.1% Triton X-100) at 4°C for 2 h in the dark.
Induction of apoptosis in murine thymocytes
Murine thymocytes were obtained through surgical extraction of the thymus from BALB/C mice. After thymus dissociation, the thymocyte suspension was centrifuged (400 × g, 4°C, 7 min), the pellet was resuspended in RPMI medium and cell viability was determined using Trypan Blue vital dye. After this, thymocytes were γ-irradiated (450 rad) and incubated at 37°C for 18 hours. Apoptotic cells were detected by annexin V staining and the percentage of apoptotic cells was measured by flow cytometry (FACS, Becton-Dickinson, CA), as described elsewhere [5]. To evaluate the effect of apoptotic thymocytes on EAT growth, BALB/C mice were inoculated with 6 × 106 viable or apoptotic thymocytes into the peritoneal cavity 2 h before the inoculation of 1 × 105 EAT cells. Animals were sacrificed after 5 days and aliquots of the ascitis fluid were recovered to estimate EAT growth via direct cell counting in a Neubauer chamber under a light microscope.
Histological, immunohistochemistry and morphometric analysis of melanoma specimens
Tumours derived from B16F10 melanoma cells were excised and processed for immunohistochemistry to detect the expression of galectin-3 (1:32, ATCC), PAF-R (1:80; Cayman), COX-2 (1:100; Santa Cruz) and activated caspase-3 (1:600; Cell Signaling). Reactions were performed as described elsewhere [16, 17]. Images of different areas of each tumour specimen were acquired with a digital camera DXM 1200F (Nikon) and quantitative analysis was performed using Eclipse Net software (Nikon). For percent area measurements, grids were projected on tissue sections and the number of grid intersections that overlaid a cell with a specific type (identified by immunohistochemistry reaction) or functional blood vessels (identified by routine H&E staining and by the identification of blood cells) was counted. Data were expressed as percent area occupied by vessels or cells expressing a given marker (COX-2-expressing cells, galectin-3-expressing cells, and activated caspase-3-expressing cells). The total number of cells positively stained by each marker was determined by counting multiple grids randomly placed on tissue sections at medium power field magnification (40×). Spleens were also processed for immunohistochemistry to detect cells expressing galectin-3 in the white pulp.
Preparation of cell suspensions from tumours
Tumours were excised and kept in RPMI 1640 at 4°C for immediate processing. All collected tumours were washed in ice-cold PBS, finely minced and transferred to 60 mm-diameter Petri dishes containing 1 mL MTH (HBSS, 0.5 U/mL DNAse in 15 mM HEPES) supplemented with 0.15 Wunsch units/mL of Liberase Blendzyme 3 (Roche). Fragments were incubated for 30 minutes at 37°C under constant agitation (80 rpm) for mechanical dissociation. Cell suspensions were then filtered using nylon membranes (41 μm pores). The filtrate was centrifuged at 4°C (1200 rpm for 5 min), washed in cold PBS and resuspended in 3 mL of red blood cell lysis solution (Amersham Biotech). After 3 min, another 3 mL of RPMI 1640 was added to the suspension, which was then centrifuged as above and washed in PBS. Cell number was then determined by direct counting using a Neubauer chamber.
Flow cytometry
For flow cytometry experiments, a million cells were separated for each reaction tube or well. Cells were washed with PBS-BSA (PBS containing 0.5% BSA) and then incubated with an Fc blocker antibody for 15 min at 4°C. Cells were then centrifuged (1200 rpm for 5 min at 4°C), washed with PBS-BSA and incubated with 0.3 μg/mL of a rabbit polyclonal anti-PAF-R antibody (Cayman Chemical, USA) for 60 min on ice. After washing, cells were incubated with a FITC-labeled anti-rabbit IgG for 30 min on ice in the dark. After washing, the cells were then suspended in 2% formaldehyde in PBS and kept in the dark until acquisition and analysis in a flow cytometer (FACScalibur, Becton Dickinson, San Jose, CA, USA). Histogram analysis was done using the CellQuest software. The control samples were incubated with the Fc blocker and with the FITC-labeled secondary antibody.
Statistical analysis
Data were analysed using ANOVA followed by Tukey or Bonferroni post-hoc tests. Survival was analysed by the Kaplan-Meier method and survival curves were compared using the log-rank test. Differences were considered significant when p < 0.05.