Relationship of tumor marker CA125 and ovarian tumor stem cells: preliminary identification

A total of 40 female SCID (Severe combined immunodeficiency) mice of 6 to 8 weeks old was purchased from Shanghai SLRC Laboratory Animal Center (NO. 0014393 and NO. 0015117). Their weight ranged from 15 g to 18 g. 1 week later, they received intraperitoneal injection of Etoposide (10 mg/kg dose, diluted into 200 ml with HBSS, Hank’s balanced salt solution), and meanwhile received a small piece of Estrogen sustained release tablet in the hypoderm of the neck, which was placed through trocar and was to lower the immunity and shorten the period of neoplasia. And 6 days later, they were inoculated with cell suspension under 4% Chloral Hydrate (0.1 ml/g dose) anesthesia. The animal experiments were approved by the animal care and use committee in HeBei University.

Human ovarian cancer specimens were obtained within 1 hour of surgical resection (frozen disease results in epithelial ovarian carcinoma) and placed in cellular suspension under aseptic conditions. Patients were diagnosed as ovarian cancer by preoperative CT scan. This study was approved by the ethics committee and patients signed informed consent.

The first antibody OC125 (ab1107) was purchased from Abcam company. The second antibody labeled by allophycocyanin (APC) was purchased from Unitech Company. Anti-CD140b labeled by phycoerythrin (PE phycoerythrin 28D4) was purchased from American Pharmingen Company. The Fluorescein isothiocyanate (FITC)-labeled anti-CD31 (11–0319) and FITC-labeled anti-mouse class I major histocompatibility complex (MHC) antibody (11–5998, H-2Kd/H-2Dd) were purchased from American eBioscience Company. FITC-labeled lineage-specific antibodies (340546; CD3, CD16, CD19, CD20, CD14, CD56) were purchased from American BD Company. Fetal bovine serum, trypsin and EDTA were purchased from American GIBCO Company. And the Metrigel was purchased from American BD Biosciences Company. PI dye was awarded by Institute of Pharmaceutical Technology of the First Affiliated Hospital of Guangzhou Medical College, and other analytical reagents were purchased from Guangzhou WeiJia Technology Limited.

Flow cell sorter (BECKMAN) (Beckman Coulter, Inc. American), Flow cytometry (for cells detection of BD FACS Ariat™) (BD Biosciences, American), Centrifuge machine (Anke TDL-40B), superspeed refrigerated centrifuge and confocal microscopy (LSM 510META) (ZEISS, German) and fluorescence microscopy (Axioplanz, ZEISS, German).

A total of 40 SCID mice were raised in the SPF (specific pathogen free) laboratory of Zhongshan Ophthalmic Hospital Center and the animal occupancy permit number was SKXY Guangdong 2005–0058. Mice were anesthetized with 4% chloral hydrate (0.1 mL/1 g).

The fresh human ovarian cancer specimens were cut into small blocks under aseptic conditions and applied with pancreatin containing 0.02% ethylene diamine tetra-acetic acid (EDTA) and incubated at 37°C for 15–20 min, while blown with a 10 mL straw every 3–4 min to mix the tissue blocks and pancreatin thoroughly. After that DMEM containing 20% fetal bovine serum was added to stop digestion, the cells were rinsed twice with HBSS, and filtered with a 200 wells filter. Next, the tissues were collected and applied with mixture of HBSS and matrigel (1:1).

After conducting cell counting, cells were placed into a 5 mL tube, and rinsed with HBSS containing 2% heat inactivated calf serum(HICS)3 times; each rinse lasted for 5 min with a centrifugal speed of 350 g at 4°C and light-proof. Then applied with 100 uL HBSS (10⁶ cells/100 μL) containing 2% HICS. First antibodies such as PI, lineage-specific antibodies, CD140b, CD31, and OC125 were added with a dilute concentration of 10⁶cells/2 μL, incubated at 4°C and light-proof conditions for 1 h, rinsed with HBSS containing 2% HICS twice, followed by suspension with 100 μL HBSS/2% HICS (10⁶ cells /100 μL) and applied with 1–4 μL second antibody (anti-OC125) and incubated at 4°C and light-proof conditions for another 1 h, rinsed with HBSS/2% HICS twice and suspended with 1 mL HBSS/2% HICS. Antibodies, including first the antibodies OC125 and APC-labeled (red) second antibody, were used for labeling CA125 tumor cells, and PE-labeled (orange color) anti-CD140b was used to eliminate normal epithelial cells. FITC-labeled (green light) anti-CD31 was used to eliminate blood platelets, myelomonocytic and lymphocytes. The FITC-labeled lineage-specific antibodies included anti-CD2, −CD3 -CD10, −CD16, −CD14, −CD18, −CD19, −CD64. Antibodies with unspecified manufacturer were purchased from PharMingen Company and they were marked directly or indirectly with different fluorescence to facilitate the sorting. Among the lineage-specific antibodies (anti-CD2, −CD3 -CD10, −CD16, −CD14, −CD18, −CD19, −CD64, -CD140b, CD31), CD2 and CD3 were used for inhibiting T-lymphocytes reaction, CD10 and CD19 for B-lymphocytes, CD16 for NK cells, CD14 for monocyte, CD18 for granulocyte, CD31 for blood platelet, myelomonocyte and lymphocyte, CD64 for neutrophil granulocyte, CD140b for human normal fibroblasts, smooth muscle cells, glial cells, and chondrocytes. PI (Spontaneous red fluorescence, can be isolated from the APC) was used to label dead cells. A flow cell sorter (BECKMAN) was used for cell sorting, where forward scatting and side scattering were applied to remove overlapping cells. In the sorting process, cells labeled by green, orange, and spontaneous fluorescence were removed, while the red labeled cells were expressions of the CA125+/lineage-ovarian cancer cells and the remaining were CA125-/lineage-ovarian cancer cells.

A total of 1.52 × 10⁵ CA125+/lineage-cells and 11.1 × 10⁵ CA125-/lineage-cells were obtained. Negative cells were conducted with negative gradient dilution, of which 1.52 × 10⁵ cells were selected and the rest were discarded, and the selected cells were re-suspended with fetal bovine serum/Matrige (1:1). A total of 40 SCID mice was randomly divided into 4 groups: positive group (A and B groups), negative group (A and B groups), experimental control group and blank control group, each containing 10 mice. The positive group was vaccinated with CA125+/lineager-cells, of which A group (5 mice) and B group (5 mice) were vaccinated with 2 × 10⁴ and 1 × 10⁴ cells, respectively, with a vaccination dose of 10 μL each. The experimental control group was vaccinated with10 μL fetal bovine serum (1:1) and nothing was applied in the blank control group. All the mice were raised under the same conditions. The vaccination methods referred to reference [3], and were illustrated in Figure 1.

Each mouse was labeled with a random number according to a random table prior to vaccination and weighted every week. Every week, palpation was conducted in mice at the vaccination site, and the reduction of subcutaneous fat, touchable abdominal mass, abdominal bloating, rounded protuberance, activity reduction and other signs associated with tumor development were visually observed. About 3 months later, mice began to become thin and appeared to have reduced their physical activity. Six months later, the mice were executed by breaking their necks; bilateral ovaries and related organs were harvested and their abdominal cavities were dissected for to determine metastasis. The weight growth curve of mice are shown in Figure 2. Group A was positive group. Group B was negative group. Group C was the experimental control group. Group D was the control group. Group A B C curves first increased and then decreased, especially the curve of group A decreased significantly in the 5^th month. Group D curve had been a growing trend.

After 6-months of observation, the vaccinated ovary and the contralateral ovary of each mouse were cut into 2 parts, of which one part was fixed with 4% neutral formaldehyde liquid, followed by paraffin-embedded, general slice and H&E staining, as well as immunohistochemistry for detecting the CA125 expression (first antibody dilution: 1:50, second antibody dilution: 1:100, second antibody was the mouse monoclonal antibody against human ovarian cancer antigen (CA125), and OC125). The other part conducted with frozen section and labeled with fluorescent antibody: indirect APC-labeled mouse anti-human CA125, and FITC-labeled anti-mouse I type MHC molecular, or H-2K^d/H-2D^d (first antibody dilution: 1:50, second antibody dilution: 1:100, second antibody was the mouse monoclonal antibody against human ovarian cancer antigen (CA125) and OC125 without nucleus staining). A confocal laser scanning microscopy was used to observe the staining results and electronic microscopy was used to observe the morphology of tumor cells.

SAS software was used for statistical analysis. Single variance analysis and LSD/t test was used to calculate P values within each group and between the groups, where P < 0.0001 was considered statistically significant.