Experimental research that is reported in the manuscript have been performed with the approval of the Animal Care and Welfare Committee of CIH-CAMS-PUMC (approval date: 20 June 2009; approval number: 20120002). All the experimental research on animals followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 85-23, revised 1985).
Female 6-week-old Balb/C mice (Animal Center of the Chinese Academy of Medical Sciences, Beijing, China) were kept under specific pathogen-free conditions.
Cell line and cell culture
All mouse tumor cell lines were cultured in RPMI 1640 medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% FBS, at 37°C in a humidified atmosphere containing 5% CO2. YAC-1: a mouse lymphoma cell line which is a specific target for NK cells. We used mouse TC-1 tumor cells derived from primary epithelial cells of C57BL/6 mice co-transformed with HPV-16 E6, E7 and c-Ha-ras oncogene (kind gift of Dr TC Wu, Johns Hopkins Medical Institutions, Baltimore, MD, USA); 4T1 (a mammary gland tumor cell line from Balb/C mice with high metastatic potency); and CT-26 (a colon tumor cell line from Balb/C mice) (both American Type Culture Collection (ATCC), Manassas, VA, USA).
DiI staining and cell sorting
Tumor cells were stained with DiI (Dil (1,1'-dioctadecyl 3,3,3',3'-tetramethyl-indocarbocyanine perchlorate); Invitrogen Corp., Carlsbad, CA, USA) in accordance with the protocol for attached cells . Cells were suspended at a density of 1 × 106/ml in 1640 culture medium, DiI solution was added at a concentration of 5 μl/ml and the cell suspension was incubated at 37°C for 20 minutes. After washing with phosphate-buffered solution (PBS) with 2% FBS, the cells were analyzed and sorted using a fluorescence-activated cell sorting (FACS) system (Vantage SE; Becton Dickinson, Franklin Lakes, NJ, USA).
To determine the heterogeneity of tumor cells with respect to cell-cycle length in vitro, DiI-labeled cells were allowed to grow for 8 days in complete RPMI 1640 culture medium under normal conditions, and were analyzed by flow cytometry on days 1, 3, 5, and 8. To determine the heterogeneity of tumor cells with respect to cell-cycle length in vivo, DiI-labeled cells were injected subcutaneously into the left groin of Balb/C mice (2 × 106 cells per mouse; four mice in total). Tumors were digested in complete RPMI medium containing 1 mg/ml type IV collagenase and 300 U/ml DNase I (Sigma AB, Göteborg. Sweden) incubated for 30 min at 37°C, and analyzed by flow cytometry on days 10, 15, and 25. When sorting, the slow-cycling cells were identified as a bright positive population.
Hoechst-Pyronin Y staining and cell sorting
Cells were detached from the cell-culture flask with 0.1% trypsin, and Trypan blue-nonstaining viable cells were counted and suspended at a density of 1 × 106/ml in DMEM culture medium. Then they were stained with the fluorescent dye Hoechst 33342 (Sigma AB) at a concentration of 5 μg/ml at 37°C for 45 minutes. At the end of this time, 1 μg/ml of Pyronin Y (PY) was added, and cells were incubated at 37°C for an additional 45 minutes as described previously . After washing with PBS plus 2% FBS, the cells were analyzed and sorted (FACS Vantage SE; Becton Dickinson). When sorting, cells residing in the G0/G1 peak that simultaneously stained weakly with PY were regarded as cells in G0 phase, and these were sorted and used for further studies .
Cells were detached from the cell-culture flask with 0.1% trypsin, and Trypan blue-nonstaining viable cells were counted, and suspended at a density of 1 × 106/ml in DMEM culture medium. Then they were stained with the fluorescent dye Hoechst 33342 (Sigma AB) at a concentration of 5 μg/ml (37°C for 90 min) as described previously . After washing with PBS plus 2% FBS, the cells were incubated with 2 μg/ml propidium iodide (PI) to exclude dead cells, then cell analysis was performed (FACS Vantage SE; Becton Dickinson).
Viable fast-cycling and slow-cycling tumor cells obtained using the DiI-based FACS, and viable G0 and non-G0 cells obtained by Hoechst-PY-based FACS, were stained with Trypan blue and counted. Then cells of every population were injected subcutaneously into the left groin of Balb/C mice at a gradient dose of 5000, 1000, or 500 cells. The mice were examined visually every day.
Chemotherapy resistant assay
To investigate the chemotherapy resistance of slow-cycling cells in vivo, DiI-labeled cells (1 × 106 per mice) were injected subcutaneously into Balb/C mice. When the tumors had grown to 10 × 10 mm in size, 5-fluorouracil (5-FU) 40 mg/kg was injected intraperitoneally every 3 days for a total of four injections. The vehicle control mice were injected with PBS, using the same regimen. After the final treatment, tumors were digested into a single-cell suspension as described above, and analyzed by flow cytometry the next day.
To determine the chemotherapy resistance of slow-cycling cells in vitro, the same numbers of DiI-labeled cells were seeded into a cell-culture flask (day 1), and grown for 24 hours, then treated with 5-FU (day 2) at a concentration of 1.5 μg/ml. On day 3, the medium was replaced with fresh medium without 5-FU, and the cells were grown under normal conditions for 24 hours. On day 4, 5-FU 1.5 μg/ml was added into the medium again, and cells were grown for a further 24 hours, then on day 5, the medium was again replaced with fresh medium without 5-FU, and cells were grown for another 24 hours. Finally, on day 6, cells were treated with trypsin and analyzed by flow cytometry. The control cells were treated in the same way but were never exposed to 5-FU.
To detect the inhibition of cell proliferation by 5-FU in vitro, DiI-labeled cells of test group and control group were seeded in triplicate into 96-well culture plate at 3,000 cells/well, then challenged with 5-FU 24 hours later in the same manner above. On day 6, 3-(4,5-dimethylthiazol-2-yl)- 2,5- diphenyltetrazolium bromide (MTT) method was performed as described previously .
In vitro lymphocyte proliferation assay
Mixed lymphocyte tumor cell culture (MLTC) was used to investigate the proliferation of spleen cells. Tumor-bearing mice were killed by broken neck and spleens were harvested. The spleen tissue was ground and suspended in PBS, then spleen cells were isolated using density gradient centrifugation (Ficoll-Hypaque, Haoyang Biological Manufacture, Tianjin, China) and stored as a single-cell suspension. CT-26 cells treated with 5-FU (FU-CT-26) or not (non-FU-CT-26) were exposed to mitomycin C (MMC) for 1.5 hours, then these cells were seeded in triplicate at a density of 1 × 104
cells per well in 96-well culture plates, along with spleen cells (1 × 105
cells per well), and incubated with interleukin (IL)-2 (100 U/ml) for 4 days at 37°C in a humidified 5% CO2
atmosphere. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to test the lymphocyte proliferation [9
], and the results were expressed as:
where A is the experimental absorbance from the spleen plus tumor cell co-cultures, B is the absorbance from the tumor cells alone, and C is the absorbance from the spleen cells alone.
ELISA for the production of interferon-gamma (IFN-γ)
CT-26 cells treated with 5-FU or not were exposed to MMC for 1.5 hours, then these cells (2 × 105 cells per well) were co-cultured separately with spleen cells (2 × 106 cells per well) from tumor-bearing mice at ratio of 1:10 in 400 μl complete RPMI 1640 containing IL-2 (100 U/ml) for 3 days. The supernatant was collected on day 4, and the concentration of IFN-γ was analyzed using a mouse IFN-γ ELISA kit (eBioscience Inc., San Diego, CA, USA) as described previously .
Flow cytometry and antibodies
The following anti-mouse monoclonal antibodies (mAbs) were used for flow cytometry: anti-H2-Kd-PE (phycoerythrin conjugated); anti-major histocompatibility complex (MHC) I-PE-Cy5; anti-CD80-FITC (fluorescein isothiocyanate) and anti-CD86-FITC (BioLegend, San Diego, CA, USA). Flow cytometry was performed using a flow cytometer (Epics XL; Beckman Coulter Inc., Brea, CA, USA) equipped with Expo32 software (Beckman Coulter).
In vitro cytotoxic assay
Mice (three mice per group, and three groups in total) were challenged with 3 × 105 CT-26 cells injected subcutaneously into the left groin (day 0), then separately immunized with subcutaneous injection of FU or non-FU-CT-26 cells (1 × 106) that had also been pretreated with MMC on days 3, 6, 9, 13, 18, and 25. 7 days after the final booster. Spleen cells from the immunized mice (FU or non-FU-CT-26 groups) were prepared as effector cells. Mice in the control group were treated in the same way, but using PBS for injection.
4T-1, YAC-1, and FU or non-FU CT-26 cells were used as target cells. As described previously , target cells were labeled with 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE; Sigma AB) for 10 minutes at 37°C using a final concentration of 2 μmol/L. After labeling, the cells were washed once and re-suspended in complete RPMI 1640. Effector and target cells were mixed to a final volume of 200 μl in complete RPMI 1640, with the ratio of effector:target being 50:1. The tubes were mixed and spun down at 120 × g for 2 minutes, then the samples were incubated at 37°C for 4 hours. At the end of incubation time, 2.5 μg PI (Sigma AB) was added for DNA labeling of dead cells. The samples were then incubated for 5 minutes and analyzed by flow cytometry within 60 minutes.
Vaccination treatment in a murine colon cancer model
To establish a colon cancer model, 3 × 105 CT-26 cells were subcutaneously inoculated at the left groin of Balb/C mice on day 0 (five mice per group, and five groups in total). Then tumor cells were injected combined with or not with granulocyte-macrophage colony-stimulating factor (GM-CSF, 1 ng per mouse) on days 3, 6, 9, 13, 18, 25. The FU and non-FU CT-26 cells used in the earlier vaccination were pretreated with MMC and injected subcutaneously at 1 × 106 per mouse. Control mice were treated with PBS. Tumor growth was monitored every 2-3 days by palpation, and tumor size was measured in two perpendicular tumor diameters, as described previously .
Statistical significance of difference between the two groups was determined by the Student paired t-test. The Kaplan-Meier plot for survival was assessed for significance using the log-rank test (SPSS software; version 12.0; SPSS Inc., Chicago, IL, USA). P < 0.05 was considered significant.