Introduction
With the development of experimental zoology, the quality of animal tumor simulation models has attracted considerable interest. The orthotopic tumor transplantation model is commonly used because of its objective demonstration of the development and progression of malignant tumors; this model has been widely used in studies involving tumor growth, metastasis, and drug screening [
1]. An orthotopic tumor transplantation model has high success and metastasis rates, and the transplanted tumor is prone to metastasis, which resembles tumor response in the human body [
2]. In biology, orthotopic tumor transplantation model is superior to heterotopic transplantation model. As such, many researchers have established orthotopic transplantation models.
The orthotopic tumor transplantation model in nude mice, which exhibit a similar behavior to the natural growth of human tumors, has been a globally accepted technical and theoretical tumor model. Successful animal tumor models can simulate metastasis that occurs in the human body. Thus, an effective evaluation of the model can provide more efficient therapy to treat human diseases [
3]. In this study, BALB/c mice were used as experimental animals because of difficulty in feeding nude mice, slow growth of human ovarian cancer in mice, and adherence to the 3R principle of animal experimentations. Different methods were then used to establish orthotopic tumor transplantation models. The results of different models were compared to determine an effective method to establish an orthotopic tumor transplantation model.
Materials and methods
Materials
Experimental animal and cell line
Forty female BALB/c mice, weighing 17 g to 19 g, were purchased from Vital River Laboratories (Beijing, China). These mice were housed in the animal facility of the Institute of Biophysics, Chinese Academy of Sciences. The experimental breast cancer cell line 4T1-LUC was a generous gift from Dr. Wei Liang of the Laboratory of Protein and Peptide Drugs, the Institute of Biophysics, Chinese Academy of Sciences. The 4T1-LUC cells were immediately cultivated and stably transfected with luciferase gene, which can be luminescently detected in the body after the model was successfully established. This study used 4T1-LUC cells of mouse origin because of the difficulty in cultivating human cells in mice with normal immunity.
Experimental drugs and reagents
Ampicillin sodium (5 mg/vial) was prepared with 0.9% saline and used in mice for postoperative anti-infection. A 10 mg/ml sodium pentobarbital solution was prepared and administered in mice for preoperative anesthesia. Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ, USA), aliquoted, and stored at -20°C.
Main supplies and equipment
Centrifuge tubes (50 ml) and cell strainer were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Nikon SMA800 was used as surgical dissection microscope, and IVIS Spectrum (Xenogen Caliper, Life Sciences, USA) was used as small-animal in vivo luminescence imager.
Experimental methods and procedures
BALB/c mice were weighed and randomly assigned into groups. The experiment was initiated after one week of acclimation. The tumor was transplanted from three different sources: suspension of cultured cells; suspension of cells ground from subcutaneously-implanted tumor; and tumor piece dissected from subcutaneously-implanted tumor. The results of the three different transplantation methods were compared.
Transplantation with suspension of cultured tumor cells
Preparation of cell suspensions
Mouse mammary carcinoma cell line 4T1-LUC was cultured with RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Confluent cells in the logarithmic growth phase were collected, counted, and resuspended into a density of 1 × 107 cells/ml suspension. The frozen Matrigel aliquots were thawed in a water bath, mixed thoroughly with cell suspension at a ratio of 1:1, and placed in an ice bath until use.
Orthotopic implantation of cell suspensions
The mice were fasted for 12 h before operation but with free access to water. They were anesthetized by intraperitoneally injecting 10 mg/ml sodium pentobarbital (10 µl/g body weight). The anesthetized animals were placed in a left lateral position and fixed on an operating table. An approximately 1 cm of incision was made vertically near the spine between the right costal arch and the femur. The abdominal cavity was opened according to an anatomical level to expose the ipsilateral ovary and the fallopian tube. The mouse was then placed under a dissecting microscope. The ovary was carefully fixed using ophthalmic forceps, and the prepared cell suspension (15 µl to 20 µl) was gradually injected into the ovary. The needle remained inserted for several seconds to ensure the gel formation of Matrigel at constant temperature and optimally fix the cells inside the ovary. After verifying the absence of active bleeding, we carefully returned the ovary to the abdominal cavity; the abdomen was then sealed layer by layer with sterile medical silk.The postoperative mice were returned to their cages for continued feeding and proper insulation. Each mouse received daily intramuscular injection of 200 μl of anti-infective ampicillin sodium for five consecutive days.
Orthotopic implantation of suspension of cells ground from subcutaneously-transplanted tumors in mice
The cultured cells were first implanted under mouse skin. After adaptation, growth, and tumor formation, the subcutaneous tumor mass was removed and ground into single cell suspension, which was then mixed with Matrigel and injected into the mouse ovary.
Establishment of subcutaneously-transplanted tumor in mice
The 4T1-LUC cells in the logarithmic growth phase were harvested and prepared to produce single cell suspension at a density of 2.5 × 104 cells/ml. Three mice were injected with 0.3 ml of cell suspension under the skin of the neck, and feeding was continued routinely.
Orthotopic implantation of cell suspension from subcutaneously-transplanted tumors
After the subcutaneous tumors increased in diameter>1 cm (measured with Vernier caliper), the mice were sacrificed by cervical dislocation. The subcutaneous tumor masses were collected; the fibrous capsule and connective tissue were removed. The tumor tissue was cut into small pieces and rinsed with ice-cold phosphate buffer. The tumor pieces were then placed on a grinding sieve with small amount of culture medium and ground with grinding rods. The cell suspension was filtered through the sieve and collected. A corresponding amount of collected cells were then mixed thoroughly with Matrigel at a 1:1 ratio, placed in an ice bath, and injected into the right ovary of the mice as described previously. Afterward, the mice were returned to their cage for continued feeding.
Orthotopic implantation of tumor pieces from subcutaneously-transplanted tumors
Cultured 4T1-LUC cells were used to establish the subcutaneous tumor model as described previously. The mice were sacrificed after the tumors increased in diameter>1 cm; the subcutaneous tumor masses were collected. The fibrous capsule and connective tissue were removed. The tumor tissue was cut into 1 cm × 1 cm × 1 cm pieces and stored in ice-cold phosphate buffer. The anesthetized mice were then mounted on the operating table, and the abdomen was opened to expose the ovary. The animal was then placed under a dissecting microscope. A small incision was made on the ovary with a knife. The tumor piece was obtained with ophthalmic forceps and carefully inserted into the incision. The incision was then sutured with 8-0 absorbable suture, and the suture was passed through the implanted tumor piece to affix the tumor tissue inside the ovary. The abdomen was sealed layer by layer after bleeding completely stopped. The mice were then returned to their cages for continued feeding.
In vivo luminescence imaging of mice
BALB/c mouse tumor models were established using the three methods. All of the mice were observed with in vivo luminescence imaging one week after the surgery to monitor the growth of implanted tumor cells and tissue in the experimental mice. The luminescence imaging experiments were performed according to the following procedures.
Intraperitoneal injection of luciferase substrate
Each mouse was intraperitoneally injected with 200 μl of luciferase substrate D-luciferin (15 mg/ml) for in vivo luminescence imaging. After the substrate was injected, the mice were allowed to perform free-movement activities in their cages for 10 min to 15 min. The mice were then anesthetized inside the built-in anesthesia device of the in vivo luminescence imager. After the mice were placed in the device, the ether switch was turned on, and the flow of mixed ether and oxygen was adjusted to an appropriate amount. After anesthesia was successfully administered, the mice were moved to the imaging system and the position was adjusted for imaging.
In vivo luminescence imaging
After the mouse was successfully positioned, the door of the black-box was closed. Imaging began after the exposure time was adjusted according to the experiments. After imaging, we observed the varied strengths of luminescence in the mouse body. The different size and condition of the growth of implanted tumors generated various light-emitting cells of different amounts, resulting in different measured photon numbers. Tumors were observed as different colors according to the amount of light photons they emitted. One week after the surgery, in vivo imaging results showed that luminescent signals were visible in the right ovaries of the orthotopic tumor transplantation models. Strong luminescence was also observed in the neck of the mice subcutaneously implanted with 4T1-LUC2 cells.
Feeding was resumed after invivo luminescence imaging. The mice transplanted with cultured cell suspensions and ground cell suspension from subcutaneous tumor mass died the succeeding weeks. Anatomical analysis showed that the ipsilateral ovary with orthotopic tumor implant was enlarged, but apparent invasion and metastasis could not be observed with naked eyes. The mice with orthotopic implant of tumor pieces from the subcutaneous tumor mass remained alive, but palpable abdominal mass growth was observed. Reduced food intake, movement, and weight loss were observed because of the enlargement of the mass. On the basis of the palpation results of the abdominal mass, we subjected the mice to in vivo luminescence imaging at regular intervals to observe the growth and metastasis of tumor in the body.
Results
The mice survived after the models were established. The images from in vivo luminescence imaging indicated that the mice implanted with cultured cell suspension directly exhibited a strong luminescent signal in the whole abdominal cavity one week after the surgery (Fig. 1B). Tumor growth was not considered because of the short span of tumor inoculation time. Instead, luminescence was attributed to the leakage and dispersion of the cell suspension caused by the small size of ovary and overdispersion of the cell suspension. This leakage occurred during the operation, although the cell suspension was fixed with Matrigel. The model using ground subcutaneous tumor cell suspension showed only a small amount of scattered luminescent spots under in vivo imaging without strong luminescent signals (Fig. 1C). This result was possibly caused by low viability and poor cell growth because of tumor tissue damage from grinding. The mice belonging to the two previous tumor transplantation models died within 7 days to 10 days after the first in vivo imaging was performed. Autopsy did not show large tumors or evident metastases.
The mice with subcutaneous tumor injection showed a strong luminescent signal in neck one week after implantation (Fig. 1A). In the neck, evident mass could be observed with the naked eyes after another week of feeding. A strong luminescent signal was observed in the ipsilateral ovary transplanted with tumor pieces cut from the subcutaneously implanted tumor mass one week after surgery (Fig. 1D). The signal was observed in a limited area and exhibited a bright color, indicating the considerable growth of the implanted tumor piece in vivo.
The mice were continuously fed; the in vivo luminescence imaging was performed after various intervals according to the general conditions of the mice and the results of abdominal palpation, which showed a gradual increase in the luminescent signal in the mouse abdomen with time progression (Fig. 2A and 2B). At 50 days, a strong, diffused luminescent signal was observed in the whole abdominal cavity (Fig. 2C). After in vivo imaging of tumor-bearing mice was completed, D-luciferin was intraperitoneally injected into the abdomen of the tumor-bearing mice. The mice were then sacrificed by cervical dislocation and then dissected. Evident tumor mass (2 cm in diameter) was formed in the ipsilateral ovary that underwent orthotopic tumor implantation. No normal ovarian tissue was observed. The contralateral ovary appeared slightly swollen but showed normal morphology. Visible granular nodules were observed in several organs, including the liver, intestine, mesentery, and others. The mice were placed in the imager for instant luminescence imaging, and the tissue exhibited strong luminescent signals (Fig. 2D).
Discussion
Recently, animal models have been used to study tumors. The available pre-clinical ovarian cancer models were established in order to understand the biological mechanisms governing the development, progression, invasion, and metastasis of EOC (epithelial ovarian cancer) [
4]. The present study performed different approaches to establish ovarian cancer animal models to identify a method that more accurately simulates the human body environment, with least artificial effect. Matrigel, which was used in this study for tumor inoculation, consisted of proteins extracted from Engelbreth-Holm-Swarm murine sarcoma, mainly comprising laminin, type-IV collagen, entactin, heparan sulfate proteoglycan, fibroblast growth factor, tissue plasminogen activator, and other growth factors. Matrigel can simulate the structure and function of the basement membrane of epithelium
invivo, regulate the biological behavior of epithelial cells, promote cell proliferation [
5], and improve the growth rate of human tumor cells in nude mice. Studies have shown that it can promote the formation and proliferation rate of certain cancers as the original degree of tumor differentiation is maintained [
6]; thus, Matrigel can be used to establish tumor models
in vivo. Under isothermal conditions, Matrigel can rapidly coagulate to form a gel
in vivo and help fix the cell suspension and prevent spread. However, this study showed that Matrigel failed to prevent the artificial spread of tumor cells despite the direct injection of cell suspension with Matrigel into the ovary because the mice with orthotopic tumor transplant rapidly died during postoperative feeding. Anatomical observation did not reveal invasion, spread, and metastasis of the orthotopic tumor. In the mice directly implanted with tumor pieces cut from the subcutaneous tumor mass, considerable transplanted-tumor growth was recorded without artificial spread of the cancer cells. The invasion, metastasis, and other biological characteristics of the tumor during development resembled the processes inside the human body.
Upon the successful establishment of animal tumor models by transplanting luminescent cells, the strength of luminescent signal and implanted tumor cells could be monitored in real-time without sacrificing the animals; as such, the effects of different treatment methods can be objectively evaluated [
7]. Without trauma or animal death, this method of monitoring the whole range of growth, invasion, and metastasis of the implanted tumor cells in mice can accurately assess tumor cell activity [
8-
10]. The present study evaluated three methods used to establish mouse tumor models for
in vivo luminescence imaging. Among these methods, the orthotopic transplantation model of ovarian cancer in nude mice, in which tumor pieces cut from pre-established subcutaneous tumor mass were used, is optimal. The study also provided a basis for future experiments involving the transplantation of actual human tumor cells to nude mice.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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