Abstract
Background/Aim. Orthotopic implantation of cancer allows metastasis to occur. The most patient-like metastatic orthotopic models are developed with surgical orthotopic implantation using intact tissue in order to preserve the natural tissue structure of the tumor which contains both cancer cells and stroma. Materials and Methods. In the present study, we performed a simple thoracotomy by making an intercostal incision between the fourth and fifth ribs on the left side of the chest of nude mice. Lung tumor fragments expressing red fluorescent protein were then implanted on the left lung. Results. It was possible to monitor tumor formation in the lung non-invasively by spectral imaging using the Maestro system with a liquid tunable filter. The model described here has high tumorigenicity in the lung (100%) and a low mortality rate (5%). Conclusion. This imageable nude mouse model using surgical orthotopic implantation of lung cancer will be useful for all types of longitudinal studies.
- Lung cancer
- surgical orthotopic implantation
- nude mice
- high tumor take rates
- noninvasive imaging
- spectral separation
The usual xenograft models for human lung cancer use tumor implanted subcutaneously into nude mice. However, it is very rare for subcutaneously-growing tumors to metastasize (1). Lung cancer cells have also been implanted under the renal capsule (2). However, these types of models are not sufficiently representative of the clinical situation. McLemore et al. implanted lung cancer cells via intrabronchial injection (3, 4); disaggregated fresh tumor specimens were also implanted intrabronchially by McLemore et al. The tumors grew intrabronchially much more extensively than when transplanted subcutaneously. Intrabronchial tumors have only low rates of metastasis, with only 1% metastasizing to the left lung, 2% to the trachea, 6% to the peritracheal area and only 3% to lymph nodes, liver, or spleen (4). McLemore et al. (3) also injected lung cancer cells into the pleural space resulting in local growth with low rates of metastasis.
Our laboratory has used histologically-intact tumor tissue for surgical orthotopic implantation (SOI). SOI can use surgical patient specimens and the resulting tumor exhibits the natural history of cancer of the human patients. These models are termed patient-derived orthotopic xenografts (PDOX) (5-19). SOI is also useful for implantation of cancer cell lines (1).
We have previously described lung cancer models where tumor tissue is implanted into the left lung by a thoracotomy procedure (20). The present report demonstrates a lung cancer model using SOI with a 100% tumor take rate, 95% survival and non-invasive longitudinal fluorescence imaging of lung tumor growth made possible by fluorescent protein expression and a liquid tunable filter for spectral separation.
Materials and Methods
Mice. Nude mice (AntiCancer, Inc., San Diego, CA, USA), 5-6 weeks old, were used in the study. Test animals were bred and maintained at AntiCancer Inc. in a high-efficiency particulate arrestance (HEPA)-filtered environment for the experiment. Cages, food and bedding were autoclaved. Animals were housed in a barrier facility on a HEPA-filtered rack under standard conditions of a 12-hour light/dark cycle. Animals were housed at no more than five individuals per cage. The animals were fed an autoclaved laboratory rodent diet.
All animal studies were conducted with an AntiCancer Institutional Animal Care and Use Committee protocol specifically approved for this study and in accordance with the principals and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals under Assurance Number A3873-1. In order to minimize any suffering of the animals, anesthesia and analgesics were used for all surgical experiments. Animals were anesthetized by isoflurane inhalation (Henry Schein, Melville, NY, USA). The response of animals during surgery was monitored to ensure adequate depth of anesthesia. Ibuprofen (7.5 mg/kg orally in drinking water every 24 hours for 7 days post-surgery) was used in order to provide analgesia postoperatively in the surgically-treated animals. The animals were observed on a daily basis and humanely sacrificed by CO2 inhalation when they met the following humane endpoint criteria: prostration, skin lesions, significant body weight loss, difficulty breathing, epistaxis, rotational motion and body-temperature drop.
Mouse cancer cell line. The Lewis lung carcinoma (LLC) red fluorescent protein (RFP)-expressing cell line was used (21-24). For RFP gene transduction, 70% confluent murine LLC cells were incubated with a 1:1 mixture of RFP retroviral supernatants of PT67 packaging cells (Clontech, Mountain View, CA, USA) and RPMI-1640 medium for 72 h. Fresh medium was replenished at this time. LLC cells were harvested with trypsin-EDTA 72 h post transduction and subcultured at a ratio of 1:15 into selective medium that contained 200 μg/ml G418 (Life Technologies, Grand Island, NY, USA). The level of G418 was increased to 1000 μg/ml stepwise in order to select the brightest fluorescent cells (22-24).
Subcutaneous xenografts. Tumor stocks of the LLC-RFP cells were established by subcutaneously injecting the LLC-RFP tumor cells into the right flank of nude mice at a cell density of 1×106/100 μl/mouse. Strong RFP expression of the implanted tumor tissue was confirmed by fluorescence imaging at 470 nm excitation before harvest.
Surgical orthotopic implantation (SOI). The stock tumors were harvested from the subcutaneous site and placed in RPMI-1640 medium. Necrotic tissues were removed and viable tissues, with bright RFP fluorescence, were cut into 1-1.5 mm3 pieces. The animals were anesthetized with isoflurane inhalation. The surgical area was sterilized with iodine and alcohol. A transverse incision approximately 1.5 cm long was made along the left chest wall of the nude mouse using a pair of surgical scissors. An intercostal incision was made between the third and the fourth ribs and the left lung was exposed (Figure 1A). Two LLC-RFP tumor fragments were transplanted to the surface of the lower level of the left lung with 8-0 surgical nylon sutures (Figure 1C-E). The chest wall and skin were closed with 6-0 surgical silk sutures (Figure 1). The left lung was re-inflated by intrathoracic puncture using a 3 cc syringe with a 25 G×1½ needle to draw out the remaining air in the chest cavity. All procedures of the operation described above were performed with a 7× magnification microscope (Olympus Corp., Tokyo, Japan under HEPA-filtered laminar flow hoods. The operation was completed within 1 minute in order to avoid mortality. A low mortality rate (1/20) was obtained.
Imaging. The Maestro™ fluorescence imaging system (Perkin-Elmer-CRi, Woburn, MA, USA) uses a liquid crystal tunable filter (LCTF) for spectral separation (25, 26). The LCTF is optically coupled to a CCD camera. Multispectral images are acquired, with images typically spaced every 10 nm throughout the desired spectral range (26, 27).
Statistical analysis. All statistical analyses were performed using SYSTAT 12.0 (SYSTAT, Inc., Chicago, IL, USA). The experimental data are expressed as the mean ± SD. The two-tailed Student's t-test was used for statistical analysis. The Kaplan–Meier test was used to analyze survival.
Results and Discussion
High tumor take rate with low mortality. A tumor-take rate of 100% (19/19) was obtained for SOI of LLC in nude mice. Only one mouse died from the procedure.
Imaging tumor growth on the lung. Statistically-significant growth was observed at day 21 after tumor implantation compared to day7 (p<0.05) (Figure 2). Non-invasive spectral-separation imaging clearly visualized tumor growth on the lung. The tumor grew exponentially over time (Figure 2). The tumor was removed on day 21 and processed for H&E staining (Figure 3), which shows the high malignancy of the LLC-RFP tumor.
Noninvasive imaging of lung cancer growth confers a great advantage. For example, it enables drug efficacy to be monitored multiple times without surgical procedures. It has been previously stated that fluorescent proteins are is not suitable for deep-tissue imaging (28, 29). However, the present report demonstrates that spectral separation imaging, especially with a tunable filter, enables very bright images of the LLC-RFP tumor on the lung to be non-invasively obtained, which is located deep in the body.
Imaging of tumor growth, progression and metastasis with fluorescent proteins in mouse models is a powerful technology. A limit to fluorescent protein imaging has previously been non-invasive deep-seated tumors, such as on the lung. In the present study, the Maestro spectral-separation fluorescence imaging system with a liquid crystal tunable filter enabled noninvasive detection of lung cancer growth (30).
Conclusion
The orthotopic implantation of lung cancer exhibited high tumorigenicity (100%) and a low mortality rate (5%). Bright RFP expression of the tumor and spectral-separation enables non-invasive imaging for long-term follow-up.
The LLC-RFP model has important advantages over luciferase models in that the strong RFP signal, which is approximately 1000 times stronger than that of luciferase (31), allows real-time imaging of metastasis in any organ, as can be seen in the present report where a deep-seated tumor in the lung was imaged non-invasively. Fluorescence imaging with fluorescent proteins is far superior to photon-counting with luciferase, whose signal is so weak only photon counts, without real images, is possible.
Footnotes
Dedication
This article is dedicated to the memory of A. R. Moossa, MD.
- Received April 8, 2015.
- Revision received April 28, 2015.
- Accepted May 1, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved