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. 2014 Aug 25;5(5):417–424. doi: 10.1111/1759-7714.12112

New orthotopic implantation model of human esophageal squamous cell carcinoma in athymic nude mice

Shuai Song 1, Dong Chang 1, Yong Cui 1, Jian Hu 1, Min Gong 1, Kai Ma 2, Fang Ding 3, Zhi-Hua Liu 3, Tian-You Wang 1,✉
PMCID: PMC4704367  PMID: 26767033

Abstract

Background

Subcutaneous xenograft is a common method to establish animal models of human esophageal squamous cell carcinoma (ESCC). However, the growth microenvironment of transplanted tumors is different from primary tumors. Orthotopic implantation models can provide more biologically relevant context in which to study the disease. So far, an orthotopic implantation model of ESCC has rarely been reported.

Methods

The human ESCC cell line KYSE30 was transfected with pLVX-Luciferase plasmids. KYSE30-Luciferase cells were isolated and injected into the flanks of nude mice to develop a subcutaneous tumor. An orthotopic implantation model was established using the fragments derived from the subcutaneous tumor. Fluorescence imaging was used to observe the development of the orthotopic implanted tumor. Hematoxylin and eosin staining was performed to evaluate the invasion and metastasis of the tumor.

Results

KYSE30 cells were successfully transfected with pLVX-Luciferase plasmids. A primary tumor was developed in all mice. The mice experienced body weight loss. The implanted tumor infiltrated into the esophageal muscularis propria. However, neither distant organ nor lymph node metastasis was found. The progression of the primary tumor was monitored by in vivo fluorescence imaging.

Conclusion

The orthotopic implantation model can be established by sewing the fragments of human ESCC to the abdominal esophagus of a nude mouse. The progression of an orthotopic implantation tumor can be monitored in real time by in vivo fluorescence imaging.

Keywords: Esophageal squamous cell carcinoma, fluorescence imaging, invasion, orthotopic implantation

Introduction

Esophageal carcinoma occurs when cancer cells develop in the esophagus. Squamous cell carcinoma and adenocarcinoma are the two main forms of esophageal carcinoma. Approximately 90–95% of esophageal carcinomas worldwide are squamous cell carcinoma. Esophageal carcinoma is one of the most incurable cancers and often has poor outcomes.1 In the early stage, surgical resection is the most effective treatment. However, most patients are diagnosed at advanced stage, when the tumor has invaded the paraesophageal organs and migrated to distant organs, making a complete resection impossible. Other treatment strategies, such as chemoradiotherapy, have not been definitively verified to have curative effectiveness.2 It seems that there are no effective therapeutic methods for patients with advanced carcinomas. Therefore, gaining a better understanding of the mechanisms related to invasion and metastasis and, consequently, developing novel therapeutic strategies, are important solutions for patients with advanced tumors. A clinically relevant model of esophageal carcinoma makes it possible to achieve such goals.

Subcutaneous transplantation is a common and simple method to establish animal models of human cancers. However, metastasis is rarely observed in these types of models. Surgical orthotopic implantation (SOI) is an alternative method that refers to transplantation of histologically intact fragments of human cancer to the corresponding organ of immunodeficient rodents. Models of surgical orthotopic implantation have been established for numerous tumor entities, and serve as a bridge linking pre-clinical, clinical research, and drug development.3 So far, this type of model has been reported in colorectal,4 lung,5 stomach,6 bladder,7 melanoma,8 breast,9 thyroid,10 and head and neck carcinoma.11 All of these studies showed that orthotopic implantation is an ideal method to observe primary growth and metastatic spread. Thus, an orthotopic implantation animal model would be useful for studying esophageal cancer invasion and metastasis. Gros et al. established an orthotopic implantation model of esophageal adenocarcinoma, and via in vivo fluoresce imaging, found that the primary tumor could metastasize to the lymph nodes and distant organs.12 However, an analogical animal model of esophageal squamous cell carcinoma (ESCC) has not been reported.

In vivo fluorescence imaging is a new technology utilized in biomedicine in recent years. Compared with traditional monitoring methods of carcinoma, it allows specific visualization of primary tumor growth, tumor cell motility, invasion, metastasis, and angiogenesis in real time.13 In addition, non-invasion and dynamic observation is available using in vivo fluorescence imaging. Luciferase is a common signal molecule that can be used to mark the tumor cells, and is wildly applied in in vivo fluorescence imaging.14

In this study, we aimed to establish a reliable orthotopic implantation model of ESCC and monitor the growth, invasion, and metastasis of this tumor by in vivo fluorescence imaging.

Methods

Cell lines and animals

In this study, we used the human ESCC line KYSE30, provided by Dr Shimada of Kyoto University. KYSE30 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator.

Female Nu/nu mice at eight weeks of age were purchased from Vital Co. Ltd (Beijing, China) and kept under a specific pathogen free condition. All mouse experiments were carried out in accordance with the guidelines of the Beijing Medical Experimental Care Commission.

Transfection of KYSE30 cells with pLVX-Luciferase lentiviral plasmids

The pLVX-Luciferase plasmids were gifted by Dr Zhihua Liu of the Chinese Academy of Medical Science. KYSE30 cells were transfected with pLVX-Luciferase plasmids by using Lipofectamine 2000 (Invitrogen, Camarillo, CA, USA) according to the manufacturers' instructions. G418 was added after transfection to select Luciferase-positive cells. The dose of G418 was increased in a stepwise manner. The isolated KYSE30-Luciferase cells were then cultured in RPMI 1640 with 10% FBS and antibiotics at 37°C in a 5% CO2 incubator. Luciferase activity of these cells was measured with the dual luciferase reporter assay system (Promega).

Orthotopic implantation model

The KYSE30-Luciferase cells were harvested after trypsinization and suspended at 1 × 107/mL in phosphate buffered saline (PBS). A total of 1 × 106 KYSE30-Luc cells in a 100 μL suspension were subcutaneously injected into the flanks of nude mice with a 1-mL syringe. When the tumor growth reached 0.5-0.8 cm, the mouse was anaesthetized by intraperitoneal injection of pelltobarbitalum natricum (300 mg/kg) and the subcutaneous tumor was resected aseptically. Three 1 mm3 fragments were derived from the tumor and kept in ice. These fragments were orthotopically implanted into the abdominal esophagus of the recipient mouse. The recipient mouse was anaesthetized as described above. A small transverse incision of the skin was made in the left upper abdomen, and then the muscles and peritoneum were separated by a sharp dissection to open the abdomen cavity. Smooth forceps held the greater curvature of the stomach and the abdominal esophagus was exposed by inserting a sterile cotton ball to raise the liver. The esophagus serosa was rubbed with a toothed forceps to create an inflammatory reaction to help tumor fragments adhere with the esophagus. Three 1 mm3 tumor fragments were sewn in this lesion with an 8-0 absorbable suture (Johnson & Johnson Medical Shanghai Ltd). The incision of the abdominal wall was then closed with a 6-0 absorbable suture (Johnson & Johnson Medical Shanghai Ltd). Before closure of the abdominal wall, 1 mL of 0.9% sodium chloride was installed into the peritoneal cavity. Orthotopic implantation was carried out in six nude mice. The changes in body weight and survival were monitored every other day. Fluorescence imaging was performed every week. Mice were sacrificed and autopsies were performed when their general performance status decreased. Open fluorescence imaging and histological analysis were conducted after autopsy. An additional six mice without implantation were selected as controls. These mice grew naturally without any process and were weighed at the time the experimental mice died.

Fluorescence imaging

Mice were monitored by in vivo fluorescence imaging using an imaging system (IVIS Lumina, Caliper Life Science, Hopkinton, MA, USA) every week. The tumor growth in mice was monitored by bioluminescence. To improve the quality of the image, mice were anaesthetized by an intraperitoneal injection of pelltobarbitalum natricum (300 mg/kg). D-Luciferin, potassium salt was injected into the peritoneal cavity of nude mice as a substrate. At the time of sacrifice, open fluorescence imaging was carried out to determine whether there were tiny metastases. After whole body imaging, the primary tumor, as well as the lungs, liver, and lymph nodes, were dissected and examined for metastasis separately by ex vivo fluorescence imaging.

Histopathology examination

After open fluorescence imaging, the orthotopic primary tumor, as well as the lungs, liver, and lymph node were dissected and further examined for invasion and metastasis using histopathological examination. Tissues were fixed and stained by standard hematoxylin and eosin (H&E) staining. Examination was performed under light microscopy by two independent examiners.

Statistical analysis

Statistical analysis was carried out using SPSS software version 13.0. Normality of the variables was examined using a Kolmogorov-Smirnov test. A student's t test was used to evaluate the differences in non-categorical variables. All tests were two-sided, and P < 0.05 was considered statistically significant.

Results

Isolation of KYSE30-Luciferase cells

By using G418, the KYSE30 esophageal squamous carcinoma cells transfected with pLVX-Luciferase plasmids were selected. Furthermore, these cells displayed high luciferase activity, which demonstrated the successful transfection.

Orthotopic implantation tumor model

Five days after injection of KYSE30-Luciferase cells, all six mice developed visible subcutaneous tumors, and at 14 days the subcutaneous tumor reached 0.5–0.8 cm in size. The tumor was then harvested for orthotopic implantation. After implantation of the subcutaneous tumor fragment, all six mice developed orthotopic primary tumors. In the early tumor transplantation, the body weight increased because of the growth of the mice. With the growth of the orthotopic tumors, the body weight of the mice decreased as a result of the tumor oppression and the malignancies' consumption (Fig 1). The survival times, animal weight, and primary tumor weight are summarized in Table 1. Compared to the control mice without implantation, the mice bearing the orthotopic implanted tumor experienced a decrease in body weight by the time of sacrifice (20.2 g vs. 27.9 g, P < 0.01). The primary tumor weight of the mice ranged from 1.8 g to 4.6 g (average, 3.4). The shortest survival time was 37 days and the longest was 107 days (average, 84.2 days).

Figure 1.

Figure 1

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Trends of the body weight in the mice bearing the orthotopic implanted tumor. Inline graphic, mouse 1; Inline graphic, mouse 2; Inline graphic, mouse 3; Inline graphic, mouse 4; Inline graphic, mouse 5; Inline graphic, mouse 6.

Table 1.

Primary tumor growth of orthotopically implanted KYSE30-Luciferase human esophageal squamous cell carcinoma

Mouse Survival time (days) Weight (g) Primary tumor weight (g)
1 102 21.3 4.3
2 79 23.1 2.9
3 37 17.9 1.8
4 94 19.7 3.4
5 107 20.7 4.6
6 86 18.5 3.6
Average 84.2 20.2 3.4
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The first fluorescence imaging was performed at day seven after orthotopic implantation (Fig 2), and Luciferase fluorescence signals were detected in all mice (100%). The progressive growth of the primary tumors was consistently monitored by fluorescence imaging. Obviously, the luminescent intensity increased with the growth of transplanted tumors (Fig 3). No metastases were found either in in vivo fluorescence imaging or in open fluorescence imaging. Fluorescence signals were not detected in the dissected organs.

Figure 2.

Figure 2

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Whole body imaging of the orthotopic implantation model with the fluorescence imaging system (mouse 2). The first fluorescence signal was detected at seven days after orthotopic implantation to the abdominal esophagus. At the end point of the study, in vivo fluorescence imaging was performed, and no fluorescence signal was detected, except in the primary tumor location.

Figure 3.

Figure 3

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The luminescent intensity increased with the growth of the implanted tumor. Inline graphic, mouse 1; Inline graphic, mouse 2; Inline graphic, mouse 3; Inline graphic, mouse 4; Inline graphic, mouse 5; Inline graphic, mouse 6.

An autopsy showed that the primary tumor at the site of the implantation was located next to the abdominal esophagus, adhered to the surface of stomach and liver (Fig 4). Swollen lymph nodes around the tumor were not observed. The primary tumor, lungs, and liver were then dissected and sent for histopathological examination. H&E staining under light microscopy confirmed the tumor infiltration of the esophageal muscularis propria (Fig 5). However, encroachment and metastases were not found in the lung and liver.

Figure 4.

Figure 4

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(a) The primary tumor adheres to the surface of stomach and liver and, (b) locates next to the abdominal esophagus.

Figure 5.

Figure 5

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Hematoxylin and eosin staining shows the infiltration of esophageal muscularis propria by implanted tumor.

Discussion

Establishing an appropriate animal model of human carcinoma that can mimic the clinical characteristics of the disease is useful for searching for an effective therapeutic method. Although subcutaneous tumor models are wildly used for preclinical screening and evaluation of new therapeutic treatments, substantial evidence has shed light on the disadvantage of these models.3,15,16 The therapeutic strategies proposed based on subcutaneous models often have no effect on human cancer.10 The tumor rarely invades adjacent organs or metastasizes to lymph node and distant organs.17 Orthotopic tumor models, which implant tumor cells or tissue into the anatomical origin equivalent within a host animal, could develop the local and metastatic behaviors occurring in human patients.12 So with the help of this model, we can investigate the development of the tumor. Orthotopic models have been established for various human cancers.4–12 However, an orthotopic implantation model for esophageal cancer has seldom been reported because of its anatomical location and subsequent technical difficulties. In our study, we successfully established the orthotopic implantation model of ESCC in the abdominal esophagus, and local tumor growth was found in 100% of cases. The primary tumor oppressed and invaded the abdominal esophagus. We speculate that the oppression can lead to dysphagia, thus, the mouse lost body weight, had a decrease in general performance status, and finally died. This process was analogous to the clinical course of human beings. Therefore, we think that our model is appropriate to study the process of ESCC.

The cervical esophagus and the abdominal esophagus are the most common sites used to establish an orthotopic model of esophageal carcinoma. One model of squamous cell carcinoma locating the cervical esophagus established in 2001 achieved a 99.5% tumor take and developed symptoms analogous to the human clinical course, such as respiratory distress and dysphagia.18 The other orthotopic model of cervical esophagus was established by injecting squamous cell carcinoma cells into the esophagus from the mouth, also representing the major features of esophageal cancer, such as a poor diet intake and body weight loss.19 However, both of the models could result in the compression of the trachea in early stage and the animal often died of suffocation. It is recognized that human esophageal carcinoma often grows in the middle or lower segments of the esophagus, therefore, death caused by compression and invasion of the trachea is rare. Compared with the cervical esophagus, the abdominal esophagus may be a more suitable location for establishing an orthotopic implantation model. An orthotopic model of abdominal esophagus was established by injecting ESCC cells into the submucosa of the esophagus, and, subsequently, primary tumor growth and stomach and liver invasion was found.20 Gros et al. established a model of esophageal adenocarcinoma using the same method.21 Although extensive peritoneal and metastatic spread was seen in 50% of the mice, none of animals developed primary tumor growth at the injection site. The reason for a lack of primary tumor growth may be the lack of tissue structure of the cell suspension, as orthotopic implantation of tumor fragments led to primary tumor growth on the esophagus in another study by the same authors.12

In the present study, KYSE-30 cells were successfully transfected with pLVX-Luciferase plasmids, and the stable KYSE-30 cells expressing Luciferase were used to establish the orthotopic implantation model of ESCC. By sewing human tumor tissue into the abdominal esophagus of the nude mice, we successfully established an orthotopic implantation model of ESCC. All animals developed a primary tumor, which invaded the abdominal esophagus. The progression of the primary tumor can be visualized accurately by in vivo fluorescence imaging. Histopathological examination revealed the infiltration of the esophageal muscularis propria. We believe that this model is suitable for studying the tumorigenesis of ESCC in an in vivo environment.

In the present study, the mice bearing the orthotopic implanted tumor experienced decreased body weight at the time of sacrifice. The survival time ranged from 37 to 107 days. The survival time may not only be related to the degree of compression by the primary tumor, but also associated with the increasing consumption of tumor growth. We estimate that the shorter survival time of the third mouse was because of earlier tumor oppression. In contrast, the tumor oppression occurred late in the fifth mouse. A gradual growth of the primary tumor, the aggravation of the compression, and the increased consumption of the malignancy eventually led to the death of the fifth mouse. In our model, the primary tumor of the abdominal esophagus adhered to the surface of the stomach and liver, but the tumor that infiltrated the serosa of the stomach and liver was not found in histopathological examination. We speculate that the adherence between the primary esophageal tumor and its adjacent organs was because of the inflammatory reaction of the serosa after surgery. Because the serosa of the stomach and liver were intact in histopathological examinations, we had not found the invasion of the primary tumor into the adjacent organs in our six ESCC model mice. Further study with more animal models should be conducted to investigate the invasion and metastasis of ESCC.

Both lymph node and distant organ metastasis were not detected, although primary tumor growth was substantial in our model, which was similar to the earlier tumor model of ESCC.20 Although metastases to the lymph node have been reported, the ratio of metastases was very low.18 The similar model of esophageal adenocarcinoma showed extensive metastasis, including lymph node and distant organs.12 The metastasis of esophageal carcinoma is associated with the characteristics of cancer cells. Establishing the model using new cell lines with high metastatic ability will be helpful to study the pathway and mechanism of ESCC metastases.

In the present study, we used in vivo fluorescence imaging to detect the progress of the primary tumor development in the implanted mice. In vivo fluorescence imaging is a newly developed technique and is now being increasingly used in small-animal research because of its quantitative sensitivity, biological safety, and relative ease of use.22 In the tumor implantation model, it is frequently utilized to monitor the growth, invasion, and metastasis of a tumor. In vivo fluorescence imaging has been used to track the tumor spread in SOI models of colon,23 pancreatic,24 and esophageal adenocarcinoma,12 based on the green fluorescent protein. The use of in vivo fluorescence imaging in transplanted animals has provided new insights into the real-time growth and metastatic behavior of tumors.

Previous studies have shown that the distance of the tumor from the abdominal wall limits fluorescence imaging. Small metastases, especially of liver, lung, or lymph node that could not be detected by whole body imaging because of their size or depth, could be viewed by open fluorescence imaging with selective imaging of specific organs.12 Open fluorescence imaging and dissected organ fluorescence imaging were performed in our study, but metastases were not detected. This result is consistent with the histological examination.

Conclusion

We established a novel orthotopic implantation model of abdominal esophagus with the esophageal squamous cell line KYSE30, and monitored the progression of the primary tumor by in vivo fluorescence imaging. Invasion of the esophagus was observed in all animals. Tumor infiltration of the esophageal muscularis propria was confirmed by histological examination. This model can provide an in vivo platform of real-time monitoring for the study of human ESCC.

Acknowledgments

We are grateful to Drs Yun Zhan and Sheng Li for their assistance with plasmid construction, and Xiao Liang for assistance with photos. This research was supported by the Scientific Research Foundation of Beijing Friendship Hospital (No. z20140512030019-yyqdkt2013-3). Author Contributions are as follows. Conceived and designed the experiments: Tian-You Wang, Dong Chang. Performed the experiments: Shuai Song, Yong Cui, Jian Hu. Analyzed the data: Shuai Song, Kai Ma, Min Gong. Contributed reagents/materials: Zhi-Hua Liu, Fang Ding. Wrote the paper: Shuai Song, Dong Chang, Tian-You Wang.

Disclosure

No authors report any conflict of interest.

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