Cell line and maintenance
Murine colon carcinoma cell line (CT26) was obtained from American Tissue Culture Collection (ATCC, Manassas, VA). Cells were cultured in endotoxin-free RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin and 100 μg/ml streptomycin (all tissue culture reagents were from Sigma-Aldrich, St Louis, MO). Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2 and passaged as described previously .
CT26 spheroids were generated by the hanging drop method . Five hundred cancer cells suspended in 40 μl of medium (RPMI with 10% FBS and antibiotics) were dispensed into each well of a 48-well culture tray. Trays were then inverted and incubated for 7 days. The number of cancer cells per spheroid was determined by disruption of individual 3D-tissue structures with PBS-EDTA (4 mM, 10 min) and cell counting using a Neubauer chamber. Same procedure was used prior to in vitro cancer cell adhesion assays and in vivo cancer cell injections in mice.
Isolation and primary culture of hepatic sinusoidal endothelium (HSE) cells
SyngeneicBalb/c mice (male, 6–8 weeks old) were obtained from Harlan Iberica (Barcelona, Spain). Animal housing, their care, and experimental conditions were conducted in conformity with institutional guidelines that are in compliance with the relevant national and international laws and policies (EEC Council Directive 86/609, OJ L 358. 1, Dec. 12, 1987; and NIH Guide for care and use of laboratory animals. NIH publication 85–23, 1985). HSE cells were separated from these mice, identified, and cultured as previously described . Briefly, hepatic tissue digestion was performed by sequential perfusion of pronase and collagenase, and DNase. Sinusoidal cells were separated in a 17.5% (wt/vol) metrizamide gradient and incubated in glutaraldehyde-treated human albumin-coated dishes for 30 minutes, as a selective adherence step for Kupffer cell depletion. Non-adherent sinusoidal cells were re-plated on type I collagen-coated 24-well plates, at 1 × 106 cells/ml/well, and 2 hours later were washed. HSE cell purity of resulting adherent sinusoidal cells was around 95% as checked by previously used identification parameters: positive endocytosis (acetylated low density lipoprotein, ovalbumin); negative phagocytosis (1 μm latex particles) and CD45 antigen expression; positive lectin binding-site expression (wheat germ and viscum album agglutinins); and negative vitamin A storage (revealed by 328 nm of UV fluorescence). Cultures of HSE cells were established and maintained in pyrogen-free RPMI (Sigma-Aldrich, St Louis, MO) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, St Louis, MO), at 37°C in a humidified atmosphere with 5% CO2.
Tumor cell adhesion assay to endothelial cells
CT26 cells were labeled with 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein-acetoxymethylester (BCECF-AM) solution (Invitrogen Co, Carsbad, CA). Next, 2 × 105cells/well CT26 cells grown in monolayer or as spheroids were disrupted with PBS-EDTA (4 mM, 10 min), stained with trypan blue for assessment of cell viability and added to 24-well-plate cultured HSE cells and, 30 minutes later, wells were washed three times with fresh medium. The number of adhering cells was determined using a quantitative method based on a previously described fluorescence measurement system .
SyngeneicBalb/c mice (male, 6–8 weeks old) were obtained from Harlan Iberica (Barcelona, Spain). Hepatic metastases were produced through the intrasplenic injection into anesthetized mice (0.078 mg/kg ketamine and 6.24 mg/kg xilacin) of 1.8 × 105 viable CT26 cells (obtained from monolayer- or 3D-spheroid-cultures) suspended in 0.1 ml of Hanks' Balanced salt solution (HBBS). Mice were cervically-dislocated on the 15th day after the injection of cancer cells and livers were removed. Livers were fixed by immersion in Zinc solution for 24 hours at room temperature and, then, paraffin-embedded. A minimum of nine 4-μm thick tissue sections of liver (three groups, separated 1 mm) were stained with H&E. An integrated image analysis system (Olympus Microimage 4.0 capture kit) connected to an Olympus BX51TF microscope was used to quantify the number, average diameter, and position coordinates of metastases. Percentage of liver volume occupied by metastases and metastases density (foci number/100 mm3) were also determined .
3D-spheroids of various diameters were fixed in 4% paraformaldehide solution and paraffin-embedded, or OCT-embedded and frozen in liquid nitrogen. On the other hand, zinc-fixed livers and primary tumors from subcutaneously-injected mice were also paraffin-embedded. Four micron-thick paraffin sections were obtained from both spheroids and tissue samples and were reacted with 1:50 dilutions of rabbit anti-mouse alpha-smooth muscle actin monoclonal antibody (ASMA) (Zymed, San Francisco, CA), rat anti-mouse CD31 monoclonal antibody (Becton Dickinson, Madrid, Spain), or rat-anti-mouse LFA-1 monoclonal antibody (Acris Antibodies, Hiddenhousen, Germany), or with 1:25 dilutions of rat anti-mouse Ki67 (Dako, Denmark). Their appropriate secondary antibodies were anti-rabbit antibody (dilution 1:100, Dako, Denmark) and rabbit anti-rat antibody (dilution 1:100, Dako, Denmark), respectively. Immuno-labeled cells were detected with an avidin-biotin-phosphatase kit (Vectastain ABC-AP kit, Vector laboratories, Burlingame, CA) according to manufacturer's instructions. Sections were analyzed by quantitative image analysis to determine the number of Ki67-expressing CT26 cells, and the intrametastatic densities of ASMA-expressing cells and CD31-positive capillary cross-sections, as previously described [15, 16].
Cell migration assay
Endothelial cell migration was analyzed with a modified Boyden chamber, as previously described . HSE cells (2.5 × 105) were incubated on 0.01% type I collagen-coated inserts with 8 μm-pores and placed on top of 2 cm2 wells (Becton Dickinson, Madrid, Spain) containing RPMI or conditioned media from either monolayer-or 3D-cultured CT26 cells. After 48 hours, migrated cells were stained with H&E and counted in ×40 high-power fields per membrane.
Conditioned media from CT26 cancer cells were prepared as follows: 5 × 106monolayer-cultured CT26 cancer cells and 143 spheroids on the 7th day of culture (assuming that one single spheroid has 35,000 cells) were incubated in 10 ml of serum-free RPMI 1640 medium, in a 75-cm2T-flask, for 12 hours. Supernatants were then collected, 25% fresh serum-free medium supplemented, and 0.22 μm-filtered prior to being used.
Measurement of VEGF concentration
VEGF concentration was measured using an ELISA kit based on specific murine VEGF monoclonal antibody as suggested by the manufacturer (R&D Systems, Abingdon, UK). Tested supernatants were obtained on the 18th hour of incubation of monolayer- and 3D-spheroid-cultured CT26 cells. For both culture conditions, the concentration of VEGF was expressed as a function of the total number of cultured cells. In some experiments, CT26 cells received 1 μg/ml celecoxib (kindly supplied by Jaime Masferrer, Pfizer, Chesterfield, MO) 30 minutes prior to CT26 treatment with 200 ng/ml recombinant human soluble ICAM-1 (R&D Systems, Abingdon, UK).
Subcutaneous injection of spheroid- and monolayer-cultured CT26 cells
Balb/c mice received one single subcutaneous injection (using 16 G-syringe) of 0.1 ml serum-free culture medium containing either one CT26 cell spheroid- or an equivalent number of monolayer-cultured CT26 cells (around 35,000 cells for 7-day cultured spheroids). Primary tumors were removed on day 19th after tumor cell injection and fixed in Zinc solution for immuno-histochemical analysis of CD31-expressing neoangiogenic tracts using an integrated image analysis system (Olympus Micro image 4.0 capture kit) connected to an Olympus BX51TF microscope.
Flow cytometric analysis
CT26 cells were first incubated for 30 min at 4°C with 1 μg/106 cells of rat anti-mouse LFA-1 antibody (Acris Antibodies, Hiddenhousen, Germany) followed by conjugated alexa-IgG2a anti-rat antibody labeling (Invitrogen Co, Carsbad, CA). Cells were then analyzed by flow cytometry using a FACS Vantage SE flow cytometer (Becton Dickinson, Madrid, Spain) by using a wavelength analysis (green: 530 nm) after excitation with 488-nm light. Dead cells (<10%) were excluded from the analysis using Viaprobe (Becton Dickinson, Spain).
Data were expressed as means ± SD. Statistical analysis was performed by SPPS statistical software for Microsoft Windows, release 6.0 (Professional Statistic, Chicago, IL). Homogeneity of the variance was tested using the Levene test. If the variances were homogeneous, data were analyzed by using one-way ANOVA test with Bonferroni's correction analysis for multiple comparisons when more than two groups were analyzed. For data sets with non-homogeneous variances, ANOVA test with Tamhane'sposthoc was applied. Individual comparisons were made with Student's two-tailed, unpaired t test (program Statview 512; Abacus Concepts, Inc., for Macintosh). The criterion for significance was p < 0.01 for all comparisons.