Patients and specimens
This retrospective study consisted of 192 colorectal adenocarcinomas with available histopathological data. Patients were diagnosed and treated in our institute from January 2001 to December 2004. The 80 distal normal colorectal tissues were randomly selected from the 192 cases of colorectal cancer as normal controls. Ethical approval for this study was not required by our institution as the experiments carried out did not relate to patients’ privacy, impairment, or treatment. The ages of the patients ranged from 22 to 83 years (median, 62 years; mean, 58.1 years). Of the patients, 120 were men and 72 were women. According to histological grading, 22 patients were at grade 1, 107 were at grade 2, and 63 were at grade 3. According to the clinical TNM stage revised by the International Union Against Cancer (UICC) in 2009, 47 patients were stage I, 70 patients were stage II, 65 patients were stage III, and 10 patients were stage IV. All patients were followed up for survival. By April 2011 (the time of data analysis), 116 patients had died and 76 patients were alive. The median survival time was 59 months.
Cell line and cell culture
The human colon cancer cell line, Colo205 (ATCC, Manassas, VA, USA), was cultured in RPMI 1640 medium (GIBCO-BRL, Gaithesberg, MD) containing 10% FBS (GIBCO-BRL, Gaithesberg, MD, USA) at 37°C in a humidified 5% CO2/95% air atmosphere.
Immunohistochemical staining of Lgr5 and Ki-67 was carried out as previously described. Sections (4 μM thick) were cut from paraffin blocks and mounted onto APES-coated glass slides. The sections were deparaffinized in xylene and dehydrated in a graded series of ethanol. Antigen retrieval was performed by heating in 0.01 M citrate buffer (pH 6.0) in a microwave oven for 2 min at 100°C. The slides were then immersed in 3% hydrogen peroxidase-methanol to inhibit endogenous peroxidase activity. After washing with phosphate-buffered saline (PBS), the slides were incubated with primary monoclonal rabbit antibody to human Lgr5 (Abcam, Cambridge, MA, USA) diluted 1:50 in blocking solution, and mouse monoclonal antibody to human Ki-67 (Zymed Laboratories, San Francisco, CA, USA) diluted 1:150 in blocking solution, at 4°C overnight. The sections were then washed in PBS and incubated with Polyperoxidase-anti-mouse/rabbit IgG (Zymed Laboratories, San Francisco, CA, USA) for 20 min. After washing with PBS, 3,3′-Diaminobenzidine was used as the chromogen. Finally, the sections were counterstained with hematoxylin. As a negative control, the primary antibody was replaced with normal rabbit serum.
Evaluation of score
All specimens were examined by two pathologists who did not possess knowledge of the clinical data. In case of discrepancies, a final score was established by re-assessment on a double-headed microscope. In scoring Lgr5 and Ki-67 expression, both the extent and intensity of the immunopositivity were considered, according to Zhao et al., Hao et al., and Fan et al.. The staining intensity was scored as follows: 0, negative; 1, weak; 2, moderate; 3, strong. The positivity was quantified according to the percentage of positive tumor cells: 0, <5%; 1, >5-25%; 2, >25-50%; 3, >50-75%; 4, >75%. The final score was determined by multiplying the intensity and the quantity scores, which yielded a range from 0 to 12. The expression of Lgr5 and Ki-67 were regarded as positive when the score was >5.
Microscopoic analysis for Hoechst 33342 extrusion
Cells at a concentration of 106 cells/mL were stained at 37°C for 90 min with 5 μg/mL Hoechst 33342 (Sigma, MO, USA) in 4 mL of Dulbecco’s modified Eagle’s medium containing 2% bovine serum albumin (BSA). Verapamil (Sigma, MO, USA) was also added to a parallel set of samples for 10 min before Hoechst staining to analyze the effect of inhibiting Hoechst extrusion[26, 27]. After incubation, the cells were washed with Hanks’ balanced salt solution (Invitrogen Corporation, Grand Island, NY, USA) and fixed with 10% formalin for 120 min. Smear slides were made with Auto Smear CF-120 (SAKURA). The cells with Hoechst 33342 extrusion, which were presented with a low blue fluorescence signal or were negatively stained, were analyzed and recorded using a BX51 fluorescent microscope (Olympus) with a cell counter. Each experiment was performed in triplicate and was repeated at least three times.
The cells were immersed in goat serum at room temperature for 15 min to block non-specific binding sites. The slides were then incubated with a primary antibody to Lgr5 (Abcam, Cambridge, MA, USA) overnight at 4°C. The cells were then washed with PBS and then incubated with a goat anti-rabbit TRITC conjugated secondary antibody (1:50 dilution; Zymed Laboratories, San Francisco, CA, USA) for 2 h at room temperature. Images were randomly taken at 40×10 magnification with a digital color camera, and the Hoechst 33342 pre-stained cells with positive or negative signals from 10 images containing at least 800 cells were counted and analyzed with image analysis software.
SPSS V.13.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. The Pearson Chi’s square test was used to examine the various clinicopathological characteristics of Lgr5 and Ki-67 expression. The Spearman’s correlation coefficient test was used to assess the relationship between Lgr5 and Ki-67 expression. Univariate survival analysis was conducted according to the Kaplan-Meier method, and the difference between the survival curves was analyzed with the log-rank test. Multivariate survival analysis was performed using the Cox proportional hazard model. Statistical significance was considered at a value of P <0.05.