The mouse Sarcoma 180 cell line was obtained from the Korean Cell Line Bank (Seoul, Korea) and grown in a humidified incubator at 37℃ under 5% CO₂ and 95% air in Dulbecco's modified Eagle's medium (DMEM, Thermo Scientific, Waltham, MA, USA) containing 10% fetal bovine serum, 2mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Sarcoma 180 cells were maintained in an ascetic form by serial transplantation every 13 days in Korl:ICR mice.

Protocols for the animal experiments were carefully reviewed for ethical and scientific care procedures and approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC; Approval PNU-2015-0916). Six-week-old male ICR mice were obtained from three difference sources. Korl:ICR mice were kindly provided by the Department of Laboratory Animal Resources in the National Institute of Food and Drug Safety Evaluation (NIFDS, Cheongju, Korea). The other two stocks of ICR mice were provided from vendors located in the United States (A:ICR) and Japan (B:ICR). All mice were provided with ad libitum access to water and a standard irradiated chow diet (Samtako, Osan, Korea) consisting of moisture (12.5%), crude protein (25.43%), crude fat (6.06%), crude fiber (3.9%), crude ash (5.31%), calcium (1.14%), and phosphorus (0.99%). During the experiment, mice were maintained in a specific pathogen-free (SPF) state under a strict light cycle (lights on at 08:00 A.M. and off at 08:00 P.M.) at 23℃±2℃ and 50%±10% relative humidity. The mice were housed in the Pusan National University-Laboratory Animal Resources Center, which is accredited by the Korea Ministry of Food and Drug Safety (MFDS; Accredited Unit 000231) and AAALAC International (Accredited Unit 001525).

Antitumor activity of Cis in the three ICR stocks was evaluated by using methods described in previous studies [1112], with some modifications. Briefly, Sarcoma 180 cells (5×10⁶ cells) collected from ascetic tumors in Krol:ICR mice were subcutaneously injected into the groin region of six-week-old Korl:ICR, A:ICR, and B:ICR mice (n=28/group; Figure 1A). Subsequently, Sarcoma 180 tumor cell-bearing ICR mice in each stock were randomly divided into one of four groups. The first group (No group, n=7) did not receive any treatment, the second group (Vehicle-treated group, n=7) received a constant volume of 1x PBS every 2 days for 6 days. The other two groups were intraperitoneally received either a low concentration of Cis (1.5 mg/kg; L-Cis-treated group, n=7) or a high concentration of Cis (3 mg/kg; H-Cistreated group, n=7) every 2 days for 6 days. At 24 h after final treatment, all animals in each group were euthanized by placing them in a chamber filled with CO₂ gas, after which tumor and blood samples were collected (Figure 1B).

The weights of tumors dissected from Sarcoma 180 tumor-bearing ICR mice were determined by using an electronic balance (Mettler Toledo, Greifensee, Switzerland) according to the Korean MFDS guideline. After measuring tumor length and width by using calipers (Matusutoyo, Tokyo, Japan), tumor volume was calculated using following formula: Tumor volume=AB²/2, where A is the length of tumor, and B is the width of tumor.

Tumor tissues were excised from Sarcoma 180 tumor-bearing ICR mice, fixed in 10% formalin, embedded in paraffin wax, routinely processed, and then sectioned into 4 µm thick slices. The tumor sections were then stained with hematoxylin and eosin (H&E), after which they were examined by light microscopy (Leica Microsystems, Wetzlar, Germany) to detect alterations in histological structure.

After fasting for 8 h prior to euthanasia, whole blood from each mouse in all groups was collected from their abdominal veins and incubated for 30 min at room temperature in a serum separating tube (BD Container, Franklin Lakes, NJ, USA). Serum was obtained by centrifugation at 1,500×g and analyzed to determine alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), and creatinine (CRE) levels by using an Automatic Biochemical Analyzer (BS-120, Mindray, China). All assays were conducted in duplicate using fresh serum.

One-way ANOVA (SPSS for Windows, release 10.10, standard version; IBM, Chicago, IL, USA) was used to identify significant differences among No, Vehicle-treated, and Cis-treated groups as well as among Korl:ICR, A:ICR, and B:ICR. All values are reported as means±standard deviations (SD) and a P value of <0.05 was considered significant.

To compare the antitumor activity responses to Cis treatment in ICR mice stocks obtained from three different sources, alterations in tumor size and volume were measured in Sarcoma 180 tumor-bearing Korl:ICR, A:ICR, and B:ICR mice treated with low or high concentrations of Cis. After 6 days of Cis treatment, skin tumor size gradually decreased in both L-Cis- and H-Cis-treated groups. The size decrease was greater in the L-Cis and H-Cis-treated groups than that in the Vehicle-treated group. Moreover, the size decreases were similar in the three ICR stocks (Figure 2A). Furthermore, tumor volumes collected from Sarcoma 180 tumor-bearing ICR mice were smaller in the L-Cis- and H-Cis-treated groups than in the Vehicle-treated group. In both L-Cisand H-Cis-treated groups of Korl:ICR, A:ICR, and B:ICR mice, the decreases in tumor volume were similar, although the greatest decreases were observed in the HCis- treated group (Figure 2B). Therefore, the results indicate that Korl:ICR, A:ICR, and B:ICR exhibit similar patterns of antitumor activity to Cis treatment in Sarcoma 180 cells-induced tumors.

To examine whether the antitumor activity of Cis is accompanied by histopathological alterations in the tumor tissue of ICR stocks from three different sources, alterations in the histological structure of tumor were observed in Korl:ICR, A:ICR, and B:ICR mice injected with Sarcoma 180 cells. In the Vehicle-treated group, solid tumors were well formed in the area of intradermal fat and dermis compared with the No group. Any significant difference was not observed in tumor formation of Korl:ICR, A:ICR, and B:ICR mice after the injection of Sarcoma 180 cells (Figure 3). In the L-Cis- and H-Cis-treated groups, the necrotizing area and the number of dead cells were significantly greater than those in the Vehicle-treated group. These patterns on the tumor tissue were constantly observed in three stocks of ICR mice from different sources, although there are non-significant difference between L-Cis- and H-Cis-treated group. (Figure 3). The above results suggest that similar alterations in tumor histopathological structure were induced by Cis treatment in the three stocks of ICR mice.

Finally, we compared the toxic responses in the liver and kidney of the three ICR mice stocks to Cis treatment. Alterations in the level of serum biomarkers indicating liver and kidney toxicity were measured in the blood serum of Sarcoma 180 tumor-bearing Korl:ICR, A:ICR, and B:ICR mice treated with Cis. Significant differences in toxic responses were detected in two liver toxicity markers and one kidney toxicity marker. In the H-Cistreated group, a significant difference on the level of AST and ALT were detected among three ICR stocks, while the level of ALP in the three ICR stocks were maintained at a consistent level (Figure 4A). In contrast, the BUN level varied among the three ICR stocks in the L-Cis- and H-Cis-treated groups. Specifically, high BUN levels were detected in B:ICR mice treated with both L-Cis and H-Cis and in Korl:ICR mice treated with H-Cis compared with A:ICR mice (Figure 4B). Overall, the above results indicate that there are few difference on the toxic responses to the antitumor drug Cis in Sarcoma 180 tumor-bearing ICR stocks from three different sources.