The OC-3-VGH cell line derived from epithelial ovarian cancer tissue was supplied by Shanghai Guoyuan Biotechnology Co., Ltd. (Shanghai, China). The drugs (¹³¹I-CA215 and CA215 antibodies) were supplied by Shanghai Guoyuan Biotechnology Co., Ltd. Trypsin, RPMI-1640, Trypan blue and a clean bench were purchased from Genetimes Technology, Inc. (Shanghai, China), Life Technologies (Carlsbad, CA, USA), Zhejiang Tianhang Biological Technology Co., Ltd. (Hangzhou, China), Shanghai Shisheng Cell Biology Technology Co., Ltd. (Shanghai, China), and Shanghai Boxun Industry and Commerce Co., Ltd. (Shanghai, China), respectively.

Female BALB/c mice aged 6–8 weeks were purchased from the Shanghai Laboratory Animal Center of Chinese Academy Science (Certificate no. SCXK 2003–0002; Shanghai, China). Animals were housed in a laminar flow rack and fed with sterilized standard diets. Water and food were allowed ad libitum. The animal housing was well-ventilated at a temperature of 25±2°C and a relative humidity of 40–70% under a 12-h light/dark cycle. The study was approved by the Shanghai Institute of Planned Parenthood Research (Shanghai, China) and the Animal Care and Use Committee, respectively.

The OC-3-VGH cells were removed from frozen storage in liquid nitrogen and placed into a water bath at 37°C. The freezing tube was removed following thawing and the surface of the tube was wiped with an alcohol cotton ball for sterilization. The cells were transfered to the culture flask with added medium on a clean bench. Fetal calf serum was added into the culture medium at a concentration of 10%. The revivified OC-3-VGH cell line was washed twice in a 10-fold volume of RPMI-1640 by low-speed centrifugation. The supernatant was discarded. The cancer cells were resuspended in 10 ml RPMI-1640, and then incubated in a humidified incubator at 37°C and 5% CO₂. The medium was changed the next day, and the cancer cells were cultured for 2–3 passages prior to inoculation.

Ovarian cancer cells at the logarithmic growth phase were treated with 0.25% trypsin for dissociation for a short time, and then suspended in fresh culture medium. The suspension was centrifuged at 89.52 × g for 5 min. The cell pellets were resuspended in phosphate-buffered saline checked with Trypan blue for cell viability (≥90%) and adjusted to 2×10⁷ cells/ml. Each female BALB/c mouse was inoculated hypodermically to the cervical spinal region (near the armpit) with 0.2 ml cell suspension. The formation of a skin mound indicated successful inoculation.

The tumor was removed from the tumor-bearing mice under aseptic conditions. Well-grown tumor tissue without liquefactive necrosis was used for the experiments. The tumor tissue was minced into 1×1×1 mm sections and transplanted into one side of the armpit (close to the galactophore) of other nude mice using a no. 20 trocar needle (Beijing Baichuan Tongxin Medical Equipment Co., Ltd., Beijing, China) within 1 h.

Following transplantation, when the tumors reached a volume of 100–300 mm³, a total of 42 tumor-bearing nude mice were divided randomly into seven groups (each n=6). These comprised the negative control (NC) group treated with isovolumetric normal saline, the positive control (PC) group treated with 60 mg/kg cyclophosphamide, the high-dose CA215 antibody group (HA; 10 mg/kg), low-dose CA215 antibody group (LA; 2 mg/kg), high-dose ¹³¹I-CA215 antibody group (¹³¹I-HA; 10 mg/kg + 125 μCi), medium-dose ¹³¹I-CA215 antibody group (¹³¹I-MA; 6 mg/kg + 75 μCi;) and low-dose ¹³¹I-CA215 antibody group (¹³¹I-LA; 2 mg/kg + 5 μCi). All test drugs were injected intraperitoneally every day for 14 days in an isotopic laboratory. The weight of each mouse and the tumor volume were measured on days 0, 4, 8, 12 and 15, and the survival time of the mice was also recorded.

With the formation of tumor nodules, the tumor volumes were calculated by measuring the tumor length (a) and width (b), and by using the formula V = 1/2 × a × b² to determine the growth curve. The relative tumor volume (RTV) was calculated by RTV = V_t/V₀, where V₀ is the tumor volume prior to drug administration and Vt is the tumor volume measured at a given time following drug administration. Antitumor activity was evaluated by the relative tumor increase rate (T/C) using the following formula: T/C (%) = TRTV/CRTV × 100, where TRTV is the RTV of the treatment group and CRTV is the RTV of the negative control group. The therapeutic efficiency was evaluated based on the following criteria: T/C >40% indicated no therapeutic effect, whereas T/C ≥40% with P<0.05 indicated a positive therapeutic effect.

The nude mice were sacrificed by decapitation on day 15, and the tumors were dissected and weighed. The TIR was calculated by comparing the weights of the transplanted tumors of the treatment group with those in the negative control group, using the following formula: TIR (%) = (1 - mean weight of the transplanted tumor of the treatment group/mean weight of the transplanted tumor of the negative control group) × 100.

Data are expressed as the mean ± standard deviation. Data were analyzed using SPSS version 11.0 for Windows (SPSS, Inc., Chicago, IL, USA). Multiple test groups in the antitumor experiments were compared with one-way analysis of variance using the post hoc Tukey test. Values of P<0.05 and P<0.01 were considered statistically significant.

The revivified cells were observed to be attached, stretched and roughly spherical on the first day after cell recovery. Cells grew actively the following day, and covered the bottom of the bottle, requiring passage on the third or fourth day (Fig. 1).

Approximately seven days after the 2×10⁷/ml ovarian cancer cell suspension was inoculated subcutaneously into the nude mice, a protuberance was observed on the back and neck of all nude mice (Fig. 2A). The tumor formation rate was 100%. The tumor growth curve indicated that the tumor grew exponentially with time. The tumor growth velocity was considerably enhanced following the 35th day. Compared with the tumor volume on the seventh day, a significant increase in volume occurred after 42 days (P<0.05; Table I).

Two months after inoculation, the tumor was dissected and found to be gray-white. The tumor tissues were embedded in parrafin (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), cut into sections (4 μm thick) and processed by hematoxylin and eosin staining (Zhuhai Baso Biotechnology Co., Ltd., Zhuhai, China). Histological examination using an inverted microscope (Motic AE31; Speed Fair Co., Ltd., Richmond, Canada) revealed that the tumor cells were large in volume, and the nuclei were large and strongly stained. A large amount of nuclear fission occurred and atypia was significant. No evident normal ovarian tissue was visible (Fig. 2B). In other pathological sections of the mouse organs, including the liver, lung, kidney and axillary lymph nodes, the structure was clear and the tissues were complete. No tumor cells were observed, indicating that the transfer to these organs did not occur.

All tumor-bearing mice survived during the administration period, and their weight increased to different degrees (Table II). Compared with that prior to the administration (day 0), at post administration (day 15) the weights of the mice in the NC, PC and ¹³¹I-LA groups showed significant increases (P<0.01). The weight in the LA and ¹³¹I-MA groups also increased and the differences were statistically significant (P<0.05). The weights of the nude mice of all groups on day 15 showed no statistically significant differences (P>0.05).

Prior to administration (day 0), the tumor volume of each drug-treated group showed no statistically significant difference compared with that of the NC group (P>0.05). On the second day after administration, the tumor volume in the PC, HA, ¹³¹I-HA and ¹³¹I-MA groups was clearly reduced compared with that of the NC group (P<0.05). The tumor growth rate in the PC and all treatment groups was slow, and the RTV in the PC, HA, LA, ¹³¹I-HA and ¹³¹I-MA groups was significantly reduced compared with that of the NC group (P<0.05 or P<0.01). The results are shown in Table III. On day 15, the T/C of each treatment group was greatly reduced. The T/C values of the HA, ¹³¹I-HA and ¹³¹I-MA groups were 54, 30, and 48%, respectively. With the exception of the ¹³¹I-LA group (P>0.05), the remaining groups showed a significant difference (P<0.01). Although the T/C of the ¹³¹I-LA group was higher than that of the LA group on day 15, the decline of the T/C in the LA group at each time point was not significant (P>0.05).

The results of the in vivo antitumor experiment demonstrated that, compared with the NC group, the PC, HA and LA groups exhibited inhibition of the growth of the solid OC-3-VGH tumors (P<0.05). An increase of the monoclonal antibody dose enhanced the antitumor effect. The tumor inhibition rate of HA was 33.59% (higher than that of the PC group), indicating that the tumor growth was significantly inhibited (P<0.01). ¹³¹I-HA, ¹³¹I-MA and ¹³¹I-LA treatment also inhibited the growth of solid OC-3-VGH tumors compared with those in the NC group. In addition, with the increase of the monoclonal antibody dose and ¹³¹I content, the antitumor effect was enhanced. Among the groups, the tumor inhibition rates in the ¹³¹I-HA and ¹³¹I-MA groups were 64.89 and 45.80%, respectively, indicating a significant inhibition of tumor growth (P<0.01) and were higher than those in the PC and HA groups. Compared with the HA group, the ¹³¹I-HA group exhibited an improved tumor inhibition effect and the difference was statistically significant (P<0.05). Compared with the LA group, the ¹³¹I-LA group exhibited improved tumor inhibition effects, but the difference was not statistically significant (P>0.05). The results are shown in Fig. 3.