Carcinoembryonic antigen as a marker of radioresistance in colorectal cancer: a potential role of macrophages

Patients with histologically proven pT3–4 or N1–2 adenocarcinomas of the rectum who were treated with curative-intent surgery and post-operative radiotherapy were reviewed in this study. Patients who did not receive the planned dose (45–55 Gy) of radiotherapy or exhibited distant metastasis were excluded from the study. Between July 1997 and January 2008, 104 patients were included in the study. The characteristics of patients are presented in Table 1. This study was approved by the Institutional Review Board of our hospital (99-3189B).

In general, the upper margin of the pelvic irradiation was delivered with 10- or 15-MV photons through 3-dimensional conformal radiotherapy (3D-CRT) or intensity-modulated radiotherapy (IMRT) techniques. Typically, the upper margin was 1.5 cm above the level of the sacral promontory. The posterior margin was 1.5–2 cm behind the anterior bony sacral margin. The dose was 45–55 Gy in 25 to 28 fractions that were administered within 5–6 weeks. Typically, the patients received 5-fluorouracil (5-FU)-based chemotherapy concurrently with the radiotherapy.

After the completion of the radiotherapy, follow-ups of the patients were performed and included physical examinations, laboratory tests, chest X-rays, colonoscopies, and computed tomography (CT) scans. Local recurrence (LR) and distant metastasis (DM) were confirmed by biopsy, physical examination, or imaging diagnosis. The overall survival (OS), LR, and DM rates were calculated via the Kaplan-Meier method, and the differences were examined with the log rank test. The interval to the last follow-up was defined by the last administration of radiotherapy. Multivariate analysis was performed using the Cox proportional hazard model with a forward stepwise procedure for OS, LR, and DM. The relative risks of these variables are represented by the hazard ratios (HRs) with the 95% confidence intervals (CIs). All variables, including age (< 57 and ≥57 years), stage, CEA level (< 10 and ≥10 ng/mL), tumor location, and operation, were treated as categorical data. The statistics were performed on a personal computer using the SPSS 17.0 software (SPSS Inc., Chicago, IL) for MS Windows®.

THP-1 (TIB-202), SW48 (CCL-231), SW620 (CCL-227), DLD-1 (CCL-221), LS1034 (CRL-2158), HCT116 (CCL-247), LS174T (CL-188), LS180 (CL-187), and Caco2 (HTB-37) cells were purchased from the American Type Culture Collection. The cells were grown in the suggested media with 4.5 g/l glucose and supplemented with 10% fetal bovine serum (Gibco Life Technologies, Grand Island, NY, USA) and antibiotics at 37°C in 5% CO₂. The media were changed every 3 days.

Harvested differentiated THP-1 cells were seeded at a density of 2×10⁶ cells in 25 T flasks. The THP-1 cells were differentiated to the attached state (M0) with 12-myristate 13-acetate (PMA; Sigma) at 50 ng/ml for 6 h. The attached cells generated M2-polarized macrophages following treatment with PMA plus 20 ng/ml IL-4 and 20 ng/ml IL-13 (R&D Systems) for another 66 h. The M1 polarization medium was treated with PMA plus 10 ng/ml LPS (Sigma) and 20 ng/ml IFN-γ (R&D systems) for 66 h.

Differentiated THP-1 cells were washed with PBS three times. To detect M1 markers, the cells were incubated with CCR7-APC (MACS) monoclonal antibody for 1 h. To detect the M2 markers, the cells were incubated with primary CD163 (Santa Cruz) monoclonal antibody for 1 h. After 1 h, the cells were washed with PBS and then incubated with FITC-conjugated goat anti-mouse secondary antibody for 1 h. The samples were analyzed by flow cytometry on an FLSR II flow cytometry system (BD).

In addition to IL-4 and IL-13, human CEA (300 ng/mL) (Abcam) was also added for 66 h during M2 differentiation. We collected CEA-stimulated M2 conditioned medium (CM) for 1: 1 mixture of culture of colon cancer cells. After 24 h, cancer cells were irradiated with 8 Gy then seeded in 96 well plate for 72 h. The cells were counted using a Cell Proliferation Reagent WST-1 (Roche Molecular Biochemicals, Mannheim, Germany). The analysis method was applied according to the instructions supplied by the manufacturer. Briefly, WST-1 (20μl) was added to well for 30 min the OD (450 nm) was measured for counting of cell number. No CEA-stimulated M2 CM, CEA + cancer cell-free medium, and cancer cell-free medium were also added to culture medium of colon cancer cells for comparison. OD after irradiation was compared between M2 CM and M2 CM + CEA group using paired-t test. The corresponding OD was also compared between cancer cell-free medium and cancer cell-free medium +CEA group.

The total RNAs were run on an Agilent RNA 6000 Nanochip (Agilent Technologies, Palo Alto, CA) using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) to detect the RNA quality and to determine the concentration of RNA using a NanoDrop spectrophotometer (Thermo Science, Wilmington, DE). Only samples with A260/A280 ratios of 1.9 to 2.1 and RNA integrity numbers (RINs) over 7.0 were used for the subsequent cRNA amplification analysis. A total of 500 ng of RNA was used for the synthesis of the first strand cDNA and the in vitro transcription (IVT) of the cRNA using an Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX) according to the manufacturer’s directions. Briefly, a single 20-μl aliquot of RNA and reverse transcription master mix was incubated for 2 h at 42°C to synthesize the first strand cDNA. Eighty microliters of second strand master mix was added, and the solution was incubated for 2 h at 16°C to synthesize the second strand cDNA. After incubation with 7.5 μl IVT master mix for 14 h at 37°C and purification, the size distribution of the synthesized biotin-labeled cRNA was evaluated with an Agilent 2100 Bioanalyzer. Gene expression was analyzed using the Sentrix HumanHT-12 v4 Expression BeadChip (Illumina, San Diego, CA) to generate the expression profiles of more than 47,000 probes with 750 ng of labeled cRNA for each sample according to the manufacturer’s protocol. After hybridization for 20 h at 58°C, washing and fixation, the HumanHT-12 Expression BeadChip was detected with Cy3-streptavidin (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and quantitated using an Illumina BeadStation 500GX (Illumina, San Diego, CA). The expression intensity measures were obtained and extracted with the gene expression module version 1.9.0 of the Illumina GenomeStudio V2011.1 software (Illumina, San Diego, CA) for further bioinformatics analysis.

The univariate and multivariate analyses are presented in Tables 2, 3 and 4. The 5-year OS rates were 64.5% and 35.9% (p = 0.004) in the patients with pretreatment CEA levels of < 10 and ≥ 10 ng/mL (Fig. 1), respectively. The DM rates were 40.6% and 73.1% (p = 0.024) in the patients with pretreatment CEA levels of < 10 and ≥ 10 ng/mL (Fig. 2), respectively. The LR rates were 6.6% and 33.9% (p = 0.002) in the patients with pretreatment CEA levels of < 10 and ≥ 10 ng/mL (Fig. 3), respectively. The multivariate analyses revealed that pretreatment CEA levels of < 10 and ≥ 10 ng/mL were the only independent predictive factors for OS (p = 0.005; HR: 2.229; 95% CI 1.272–3.906), DM (p = 0.026; HR: 1.923; 95% CI 1.080–3.423), and LR (p = 0.004; HR: 5.340; 95% CI 1.682–16.955).

Different morphologies were noted between the M0, M1, and M2 cells (Fig. 4). We used CCR7 and CD163 as markers of M1 and M2 cells, respectively. We noted a dose-response relationship of CEA with M2 differentiation (Fig. 5). We tested the CEA levels of the supernatants from different colon cancer cell lines (SW48, SW620, DLD-1, HCT116, LS1034, LS174T, and Caco2). We noted 18.392 ng/mL in LS1034 cells and 8.357 ng/mL in LS174T. Levels lower than 1 ng/mL were noted in the other cell lines (Table 5). Hence, we used the LS1034 cells for radiosensitivity testing.

In the CEA-stimulated M0 macrophages (Fig. 6 and Table 6), four mRNAs that exhibited at least 5-fold down-regulation compared with the vehicle-stimulated M0 cells were noted. Five mRNAs, including MMP12, GDF15, and JAG1, exhibited at least 5-fold up-regulation. In the CEA-stimulated M1 macrophages (Fig. 7 and Table 7), three mRNAs were at least 2-fold down-regulated compared with the vehicle-stimulated M1 cells. Six mRNAs exhibited at least 2-fold up-regulation. In the CEA-stimulated M2 macrophages, 15 mRNAs exhibited at least 2-fold down-regulation compared with the non-stimulated M2 cells. Twenty-four mRNAs exhibited at least 2-fold up-regulation. The GADD45G mRNA achieved a 4-fold up-regulation of expression. GADD45G is a stress response gene to radiotherapy.

The conditioned media from M2 macrophages or medium only was applied to the LS1034, SW620, LS180 cells prior to irradiation. We compared the OD for cell proliferation between the CEA-stimulated and vehicle-stimulated cells after irradiation. We found a higher OD in the CEA-stimulated group with M2 conditioned medium but not medium only group (Fig. 8).