Tumour induction in BALB/c mice for imaging studies: An improved protocol

Abstract Dealing with nude mice, which lack thymus and therefore are sensitive to unsterile conditions, needs special care and laboratory conditions. For preclinical studies, especially tumour imaging purposes, in which therapeutic properties of drugs or therapeutic compounds are not studied, mice with normal immune system can be a favourable alternative if they carry tumours of interest. In the current study, we introduce an optimized protocol for induction of human tumours in BALB/c mice for preclinical studies. Immune system of BALB/c mice was suppressed by administration of cyclosporine A (CsA), ketoconazole and cyclophosphamide. The tumours of MDA‐MB‐231, A‐431 and U‐87‐MG human cancer cells were induced by subcutaneous injection of the cells to the immunosuppressed mice. Tumour size was calculated weekly. Histopathological and metastatic analyses were performed using haematoxylin and eosin staining. The combination of the three drugs was found to suppress immune system and decrease the numbers of white blood cells, including lymphocytes. At the eighth week, tumours with a dimension of approximately 1400 mm3 developed. Large atypical nuclei with scant cytoplasm were found to exist using histopathological analysis. No metastasis was observed in the tumour‐bearing mice. A combination of CsA, ketoconazole and cyclophosphamide can be used to suppress the immune system in BALB/c mice and induce tumours with significant size.


| INTRODUC TI ON
Immune cells in laboratory animals with normal immune system invade tumour cells and prevent tumour growth and development, making it impossible to establish tumours with adequate size for in vivo studies. For most in vivo studies, the use of nude mice is unavoidable-where the animal immune system should be inactive to ensure that the drug (or compound) of interest can affect tumour cells. For imaging purposes, in which tumour growth suppression is not the goal of study, laboratory animals with normal immune system will be of great interest, if they carry tumours of the cells of interest. In such cases, results may be more consistent to real conditions when compared to those obtained with nude mice. Compared to nude mice, animals with normal immune system are not highly sensitive to unsterile conditions and do not need strict care during study. Considering such limitations of nude mice, numerous researchers have proposed various immunosuppression regimens for the development of drug-induced immunosuppression.
Cyclosporine A (CsA) is an immunosuppressive peptide drug produced by the fungus Tolypocladium inflatum. It is a lipophilic molecule, which can readily cross the blood-brain barrier. 1 By blocking the nuclear factor of activated T cells (NFAT), a cytokine transcription factor, CsA primarily inhibits the activation of T lymphocyte proliferation and cytokine production. Additionally, CsA affects growth and activation of B lymphocyte, regulates cell homeostasis, and prevents neutrophil and macrophage migration into the inflammatory zone. Because of these immunosuppressive effects, CsA is used to study immune response in inflammatory, autoimmune and infectious diseases. 2,3 Since 1978, CsA has been frequently used both clinically and experimentally. 4 Many clinical researchers reported tumour xenograft development by modifying the CsA dose, administration route, combination with another drug and duration of treatment regimens. [5][6][7][8][9] CsA is metabolized in the liver by cytochrome P450. CsA metabolism can be decelerated by cytochrome inhibitors like diltiazem or ketoconazole. 10 The risk for opportunistic invasive fungal infections is decreased when CsA and ketoconazole are combined. 11 In our previous work, we induced tumours of A-431, U-87-MG human cancer cell tumours in BALB/c mice, 9 using the combination of CsA, ketoconazole and cyclophosphamide. In the current study, we describe the improved protocol in detail.

| Animals
BALB/c female mice weighing 20 ± 2 g (4-6 weeks old) were used in this study. The mice were initially kept in individually sized standard cages with a relative humidity of 60 ± 5% and a constant temperature of 25 ± 2°C. Access to autoclaved water and rodent food was available to all mice at all times. All procedures for anaesthesia/euthana-

| Immunosuppression
Healthy BALB/c female mice were divided into 10 groups (eight mice in each group). Groups 1-3 were administered 20 mg/kg CsA; Groups 4-6 were administered 30 mg/kg CsA; and Groups 7-9 were administered 40 mg/kg CsA by the intraperitoneal route on seven subsequent days. Groups 1, 4 and 7 were administered 5 mg/ kg ketoconazole; Groups 2, 5, and 8 were administered 10 mg/kg ketoconazole; and Groups 3, 6 and 9 were administered 15 mg/kg ketoconazole by the oral route every day for 7 days. Groups 10 and 11 were administered 30 mg/kg CsA and 10 mg/kg ketoconazole, respectively. No treatment was given to Group 12 (control).
Autoclaved water and rodent food pellets were provided for the mice. The animals were given Co-amoxiclav (0.1 μg/mL) by drinking water during the experiment. After completion of the study, haematology was carried out to determine total white blood cell (WBC) and lymphocyte count to confirm immunosuppression. In groups of mice with the highest levels of immunosuppression, CP was subcutaneously given at a dose of 30 mg/kg on 1 day before tumour cell injection.

| Total WBC and lymphocyte count
Under ketamine-xylazine anaesthesia, blood samples were taken from the submandibular vein of each animal and placed in a heparinized 1.5-mL microcentrifuge tube. Then, total WBC and lymphocyte counts were performed in an automated haematology analyser (Sysmex XP-300).

| Tumour induction
In the present study, immunosuppressed female BALB/c mice were used. After shaving the body hairs of the mice, 100 μL FBS free culture medium containing cells (approximately 8 × 10 6 either A-431, U-87-MG or MDA-MB-231 cells) was subcutaneously administered to each mouse into the right hind flank region. Tumour growth was observed at the injection site. In order to estimate tumour volume by an external calliper, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were determined while mice were conscious. Tumour volume was calculated by the modified ellipsoidal formula: V = ½ (Length × Width 2 ). 12 Tumours were excised at the end of the study and used for histopathological study.

| Histopathological analysis
Tumours were removed from the animals at the end of the study and kept in 10% neutral buffered formalin. Haematoxylin and eosin was used to stain the tumour samples, which were cut into 5 μm sections. To confirm the presence of cancerous cells, optical microscopy (Olympus) was used to observe the slices.

| Statistical analysis
All data are presented as mean ± SD. One-way anova followed by post hoc Bonferroni correction was applied to determine the significance of differences among groups. p ≤ 0.05 was considered to be significant.

| Immunosuppression
Compared to the control mice, the combination of CsA and ketoconazole drugs significantly suppressed the immune system in BALB/c mice. Figure 1

| Histopathological image analysis
Histopathology experiments proved that a malignant tumour was present. Tumours were removed, sectioned and stained using the common haematoxylin and eosin method. Figure 4 displays slices F I G U R E 1 White blood cell (WBC) and lymphocyte (LYM) counts of different groups of mice following treatment with CsA and ketoconazole (n = 8). Groups 1-3 were administered 20 mg/kg CsA; Groups 4-6 were administered 30 mg/kg CsA; and Groups 7-9 were administered 40 mg/kg CsA by the intraperitoneal route for seven subsequent days. Groups 1, 4 and 7 were administered 5 mg/kg ketoconazole; Groups 2, 5 and 8 were administered 10 mg/ kg ketoconazole; and Groups 3, 6 and 9 were administered 15 mg/ kg ketoconazole by the oral route for seven subsequent days. Groups 10 and 11 were administered 30 mg/kg CsA and 10 mg/kg ketoconazole, respectively. No treatment was given to Group 12 (control). Asterisks indicate significant differences between groups (*p < 0.05).

| Histopathological findings of metastasis
No metastases were found in the liver and kidney using microscopic histopathologic evaluation in all the 12 groups (Figures 5 and 6).

| DISCUSS ION
Anti-cancer drugs are ussualy evaluated on animal xenograft models after in vitro studies. 13 The immunocompromised mice models, such as Nude (nu), severe-combined immunodeficient (SCID) and nude SCID gamma (NSG) strains, are widely used as hosts for cancer cell lines derived from human tumours. However, nude mice have significant disadvantages, including high cost, high mortality rate, high sensitivity, low tumorigenesis rate and difficult maintenance. 14 All anti-cancer agents that have received clinical approval have been assessed using traditional preclinical research models. The process of conducting xenograft studies can be extremely complicated, starting with the choice of the best animal model, selection of the tumorigenic cell line, administration strategy, dosing and tumour growth rate analysis. 15,16 To increase rates of tumour engraftment, ionizing irradiation to the whole body can also cause a more severe immune deficiency in mice. 17 The immune system can be ablated by a radiation dose of 4-6 Gy in athymic nude mice, while 2-3 Gy can be used on NSG mice (which are already radiation sensitive). 13 However, these procedures are very costly and have a high mortality rate.
Numerous immunosuppressive drugs, including CsA, tacrolimus, ketoconazole, CP, azathioprine, glucocorticoids and methotrexate, are used alone or in combination to prevent the rejection of human tumour xenografts in murine models. 1,5,18 CsA, a potent immunosuppressive drug, was first used to induce tumour xenograft development in C3H mice. 18 CsA selectively inhibits calcineurin; thereby, interleukin 2 and other cytokine transcription in T lymphocytes is impaired. Since more than 30 years ago, calcineurin inhibitors have been the cornerstone of immunosuppression in solid organ transplants. 19 There are a number of reports on immune system suppression using chemical compounds as a single agent or in a combination. Zeyghami: Conceptualization (equal); data curation (equal); formal F I G U R E 6 Histopathological analysis of kidney tissues (haematoxylin and eosin staining). Panel C (×400) shows higher magnification of photomicrographs in panel B (×200) and panel B is higher than panel A (×100). All groups showed the normal renal structure and no metastatic foci were seen. analysis (equal); methodology (equal); validation (equal); visualization (equal); writing -review and editing (equal). Yaghoub Safdari: Supervision (equal); writing -review and editing (equal).

ACK N O WLE D G E M ENTS
The authors thank the Deputy for Research and Technology of Golestan University of Medical Sciences for providing the animal imaging system.

FU N D I N G I N FO R M ATI O N
This project was supported by Golestan University of Medical Sciences (grant number: 110121).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors confirm that there are no conflicts of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
No dataset was generated during this study.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.