Fifty-three colon carcinomas were collected after surgical resection performed at Shandong Provincial Hospital. None of the patients had received chemo-, radio- or immuno-therapy before resection. Paraffin wax-embedded surgically resected tumor sections were deparaffinized in xylene and rehydrated before analysis. The primary antibodies were purified rabbit polyclonal anti-human Fas and FasL-specific IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 0.1 mg/mL in a wash buffer. Antibody binding was localized using a biotinylated secondary antibody, avidin conjugated horseradish peroxidase, and diamiobenzidine substrate, contained within the ABC detection kit. The immunizing peptide (Santa Cruz Biotechnology), to which the antibody was raised, was included at 1 mg/mL during primary antibody incubation as a direct internal competitive control for antibody specificity.

In situ hybridization was performed on paraffin-embedded human colonic tumor sections (4 mm thick), mounted on aminopropylethoxysilane-treated slides. A digoxigenin-labeled RNA hybridization probe (344 bp) was used, corresponding to codons 96-210 of the human FasL cDNA sequence. The nucleotide sequence of the FasL probe showed insignificant homology to any other sequence in the EMBL DNA sequence database. Prehybridization and hybridization were performed as previously described[3]. In order to confirm the specificity of hybridization, a 10-fold excess of unlabeled riboprobe was added to the digoxigenin-labeled probe prior to hybridization.

To identify tumor-infiltrating lymphocytes (TILs), CD45 staining was performed on consecutive sections with a mouse anti-human CD45 monoclonal IgG (clone 2B11+PD7/26, Dako Corp., Carpinteria, CA, USA, dilution 1/70) according to the standard immunohistochemical methods as described above. Alkaline phosphatase conjugated anti-alkaline phosphatase complex (DO651, Dako Corp.) was used to display the color of CD45 staining. The membrane of the CD45-positive cells was red. Apoptosis of TILs was detected in situ in frozen sections of resected tissues using Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay (Boehringer Mannheim GmbH, Mannheim, Germany) applied to the histological sections. Visualization of the final reaction product was achieved by diaminobenzidine. TUNEL-positive TILs (with red membrane and brown nucleus) were counted at high power (×400) from fields of view chosen according to a systematic random sampling method. Approximately 1 000 TILs were counted and a TUNEL index (TI) was expressed as the percentage of TUNEL lymphocytes.

Colon cancer SW480 cells were grown in RPMI medium supplemented with penicillin G (100 U/mL), streptomycin (100 mg/mL) and 100 mL/L fetal bovine serum at 37 °C in humidified atmosphere containing 50 mL/L CO2 in air. Cell growth was determined by the 3-(4,5-methylthiazol-2yl)-2,5-diaphenyl-tertrazolium bromide (MTT) colorimetric assay. The assay was initiated by adding MTT solution to each well at a final concentration of 0.625 mg MTT/mL medium. After 4 h, the optical density was determined with an ELISA plate reader after removal of the medium and dissolving of the dye crystals in acidified isopropylalcohol.

Different concentrations of SW480 cells (0, 1 ×10⁵/mL, 2×10⁵/mL, 4×10⁵/mL) were plated in triplicate on 24-multiwell tissue culture dishes. After 24 h, 2×10⁴/mL Jurkat cells were seeded onto the plates. The wells with colon cancer cells alone (without overlying Jurkat cells) were maintained as additional controls. After 48 h, the non-adherent phase of the mixed culture was aspirated and assayed for viability using MTT assay. In experiments using recombinant FasFc (ZB4) as a competitor, similar experimental conditions were used with the following exception. MTT assay was used to evaluate the amount of Jurkat cells at the co-culture time of 24, 48, 72, and 96 h.

After being co-cultured with SW480 for 48 h, the Jurkat cells were collected and added with Hoechst 33258 (Sigma) on a slide glass. The specimens were examined under a Nikon fluorescence microscope with B2-type filter (magnification ×200). When apoptotic bodies or chromatin condensations were observed, the observation was defined as apoptosis. Apoptotic cells were assessed morphologically by staining with Hoechst 33258 using cells fixed with Clarke fixative (ethanol:acetic acid = 3:1). Apoptotic index (AI) was defined as follows: AI = 100×apoptotic cells/200 cells.

All experiments for cell cultures were performed at least in triplicate. Data were expressed as mean±SD. Statistical analysis was performed by one-way variance (ANOVA), and the comparisons between groups were performed by independent sample t-test. The regressive relation between two variables was performed by linear regression. A two-tailed P value less than 0.05 was considered statistically significant.

Of 53 cases of colon carcinomas, 23 cases (43.4%) expressed Fas which was significantly lower as compared to that in normal colonic mucosa (73.3%) (P<0.01), and 45 cases (84.9%) of colon carcinomas expressed FasL, whereas only 2 cases (3.75%) in normal mucosa expressed FasL. Intensity and extent of positive staining varied within individual tumors. Neoplastic areas with positive and negative staining for FasL frequently co-existed within the same tumor. The extent of FasL expression varied significantly between different cancer stages and differentiations (data not shown, Figures 1A and B). FasL expression also existed on TILs in all cases. TILs were smaller in volume than cancer cells, which could distinguish the two kinds of FasL-expressing cells.

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Figure 1 Immunohistochemical staining of Fas and FasL expression in colon of FasL in colon cancer cells; and C: high expression of FasL in lymph node cancer cells. A: Low expression of Fas in colon cancer cells; B: high expression metastases from colon cancer cells.

In situ hybridization was performed on paraffin-embedded human colonic tumor sections in order to detect the localization of FasL mRNA expression. FasL mRNA was detected in the colon cancer cells and in TILs (Figure 2). The location of FasL mRNA and expression of FasL protein coincided.

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Figure 2 Localization of FasL mRNA by in situ hybridization.

To further study the role of FasL expressed in cancer cells in tissues, 23 cases of lymph node metastases from colorectal carcinomas were obtained to detect the expression of FasL immunohistochemically. All cases of lymph node metastases showed FasL immunoreactivity in greater than 60% of the metastatic cells (Figure 1C).

To evaluate apoptosis of TILs, we detected the presence of apoptotic cells in tissue sections using CD45 staining (WBC common antigen) and TUNEL techniques. Tonsil sections were included as positive controls, stained strongly, and omission of the primary antibody abolished staining. To quantify the extent of apoptosis of TILs, the percentage of TUNEL-positive and CD45-positive cells was calculated. The apoptotic rate of TILs in FasL-positive staining regions of tumor cells was significantly higher than that in FasL-negative region of tumor cells (54.84±2.79% vs 25.73±1.98%, P<0.01, Figure 3). In vivo FasL expression in colon cancer cells was found to be associated with increased cell death of TILs.

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Figure 3 Detection of TILs. They were displayed by double staining (CD45 staining and TUNEL).

Jurkat cells grown in the absence of human colon cancer cells showed a marked proliferation after 48 h. However, when co-cultured over a mono-layer of the FasL-expressing colon cancer cell line SW480, we observed a marked decrease in Jurkat cells while using MTT assay (Figure 4). The Jurkat cell killing was largely inhibited by pre-incubating the colon cancer mono-layer with a low concentration of recombinant Fas neutral antibody ZB4, which is capable of binding to FasL and blocking Fas/FasL interaction.

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Figure 4 Jurkat cells growth curve after being co-cultured with SW480. 1: Control group; 2: 0.5×105/mL SW480 planting group; 3: 1×105/mL SW480 planting group; and 4: 2×105/mL SW480 planting group.

We used fluorescence microscopy to observe the shape of apoptotic Jurkat cells by the untreated SW480 cells. Condensation of chromatin, nuclear fragmentation and typical apoptotic bodies were observed in the tested group (Figure 5). The fluorescence microscopy results revealed that the apoptotic rates of Jurkat cells induced by SW480 cells after being co-cultured had a significant difference with the natural apoptotic rate of Jurkat cells (Table 1)

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Figure 5 Detection of apoptotic Jurkat cells by fluorescence microscopy. A: Negative control of Jurkat cells; B: apoptotic Jurkat cells after being co-cultured with SW480 cells.