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 11.

CT26 spheroids were generated by the hanging drop method 12. 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.

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 13. 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.

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 14.

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 15^th 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 14.

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 1516.

Endothelial cell migration was analyzed with a modified Boyden chamber, as previously described 3. 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 7^th 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.

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 18^th 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).

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 19^th 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.

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-IgG_2a 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.

Well-rounded compact 3D-spheroids with a homogeneous size distribution were formed in CT26 cancer cell-containing drops suspended from the inverted surface of 48-well microtiter plates. The efficiency level was nearly 100% –i.e., one spheroid per drop and well–, which is in contrast to the low-efficient 3D-growth capability of other cancer cell lines 12. CT26 spheroids exhibited a highly organized 3D-tissue-like structure where aggregated cancer cells evidenced high proliferation activity until day 7, when the plateau phase of the growth curve was reached by CT26 spheroids, while the percentage of Ki67-expressing cells markedly decreased (Figure 1). The absence of pimonidazole staining in 7-day cultured CT26 spheroids suggests that CT26 spheroids were not affected by hypoxia at this stage of in vitro growth (data not shown). However, the concentration of VEGF significantly (P < 0.01) increased in the supernatant of 3D-cultured CT26 cell spheroids compared to the level in monolayer-cultured CT26 cells (Figure 2A). This was particularly visible in the supernatants obtained on the 7^th day of spheroidal growth, when VEGF secretion increased by 2-fold, and led to a significant (p < 0.01) increase by 2-fold in the migration of primary cultured HSE cells, as compared to the migration induced by the conditioned medium from an equivalent number of monolayer-cultured cells (Figure 2B).

As shown by flow cytometry, integrin LFA-1-expressing cell fraction also increased by 3-fold in CT26 cancer cells obtained from spheroids (Figure 2C). Immuno-histochemical detection of LFA-1 confirmed that a majority of spheroid-cultured CT26 cells expressed the integrin, both at the peripheral and internal areas of the spheroid (Figure 2E–F). Consistent with this feature, the percentage of CT26 cell adhesion to primary cultured HSE cells significantly (p < 0.01) increased by 6-fold in 3D-spheroid-cultured CT26 cells compared to monolayer-cultured cells; and the addition of 1 μg/ml anti-murine LFA-1 antibody completely abrogated the adhesion of 3D-spheroid-cultured CT26 cells, but not of monolayer-cultured cells, to HSE cells (Figure 2D). Moreover, the majority of smallest avascular CT26 hepatic micrometastases (50–300 μm in diameter) of non-hypoxic character, and that did not extend beyond hepatic lobule limits, were already populated by LFA-1 integrin-expressing CT26 cells (Figure 2G).

In a preliminary study we reported that soluble ICAM-1 increases by two-fold in the supernatant of tumor-activated HSE cells and that, in turn, soluble ICAM-1 increases CT26 cell secretion of VEGF 16. Herein, both monolayer- and spheroid-cultured CT26 cells received 200 ng/ml recombinant human soluble ICAM-1 for 18 hours and VEGF concentration was determined by ELISA in their supernatants. In these conditions, VEGF secretion potential further increased by 30% and 90% in monolayer- and 3D-spheroid-cultured CT26 cells, respectively, given soluble ICAM-1 (Figure 3A). This remarkable VEGF secretion-stimulating activity of soluble ICAM-1 on 3D-spheroid cultured CT26 cells was consistent with LFA-1-expressing cell number augmentation by 3-fold in 3D-cultured cells compared to monolayer-cultured cells shown in Figure 2A. Because COX-2 contributes to colon carcinoma cell production of VEGF 17, in some experiments, both untreated and soluble ICAM-1-treated monolayer- and 3D-cultured CT26 cells received 1 μg/ml COX-2 inhibitor Celecoxib for 18 hours. VEGF levels did not significantly change in basal condition-cultured CT26 from both 3D-spheroid and monolayer cultures. However, Celecoxib completely abrogated VEGF secretion induced by soluble ICAM-1 on both monolayer- and spheroid-cultured CT26 cells, indicating that VEGF secretion-stimulating activity of soluble ICAM-1 was COX-2-dependent (Figures 3A and 3B).

Nineteen days after subcutaneous injection of monolayer- and 3D-cultured CT26 cells, the number of CD31-expressing cells was determined in developed tumors. As shown in Figure 4, a marked recruitment of angiogenic cells occurred at the periphery of subcutaneous tumors generated by CT26 cells derived from both in vitro growth conditions. However, only those tumors produced by 3D-cultured CT26 cells were efficiently neovascularized and had angiogenic tracts in the deepest areas of the tumor. Overall, CD31-expressing cell densities per unit area were 1.89 ± 0.56 and 0.66 ± 0.25 in tumors from 3D- and monolayer-cultured CT26 cells, respectively (n = 15 mice from 3 independent experiments having 5 mice per group; differences were statistically significant by the Student's two-tailed, unpaired t test, p < 0.01).

Intrasplenic injection of CT26 cancer cells revealed that hepatic metastasis development significantly (P < 0.01) increased in mice receiving 3D-spheroid-cultured CT26 cells, as compared to mice given monolayer-cultured CT26 cells (Figure 5A and Figure 5B). This was particularly evident when comparing the metastasis volume indices produced by well-established metastases of medium and big size. Consistent with the proangiogenic activation of spheroid-growing CT26 cancer cells, both endothelial cell (as CD31-expressing cells) and alpha-smooth muscle actin (SMA)-expressing cell numbers significantly (p < 0.01) increased in metastatic nodules produced by 3D-cultured CT26 cells as compared to those developed by monolayer-cultured CT26 cells (Figures 5C–F). As previously described3, intrametastatic SMA cells were mainly hepatic sinusoidal stellate cell-derived myofibroblasts acting as vascular coverage pericytes of neoangiogenic tumor vessels. Moreover, consistent with the metastatic volume augmentation evidenced in the livers of 3D-spheroid-cultured CT26 cell-injected mice, the average proliferating cell number per unit area of metastatic tissue also significantly increased by 35%, in 3D-spheroid-cultured CT26 cell-injected mice, as detected by immuno-histochemistry using anti-ki67 antibodies (Figures 5G–H).