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Ann Thorac Surg 2000;69:1010-1015
© 2000 The Society of Thoracic Surgeons
a Departments of General Surgery and Thoracic Surgery, Christian-Albrechts-University Hospital, Kiel, Germany
b Pathology, Christian-Albrechts-University Hospital, Kiel, Germany
Address reprint requests to Dr Boehle, Department of General Surgery and Thoracic Surgery, Christian-Albrechts-University Hospital Kiel, Arnold-Heller-Str 7, D-24105 Kiel, Germany
e-mail: boehle{at}allg-thorax-chir.uni-kiel.de
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Abstract |
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Methods. Tumor cells, originated from a human adenocarcinoma, a squamous cell carcinoma, and an undifferentiated large cell carcinoma, were xenotransplanted heterotopically by subcutaneous and intravenous injection and compared with orthotopic intrapleural and intrapulmonary xenotransplantation by a facilitated engraftment procedure into SCID bg mice.
Results. Subcutaneous injection of tumor cells resulted in a 100% engraftment rate with establishment of solid tumors without clinically relevant metastases. Intravenous injection had poor engraftment rates by hematogenous spread. Depending on the cell line, a 80% to 100% engraftment rate in orthotopic xenotransplantation was achieved, resulting in a consistent pattern of mediastinal and bilateral pulmonary metastases.
Conclusions. The facilitated orthotopic xenotransplantation of human lung cancer is easy to perform and results in a reproducible in vivo model that closely resembles the clinical features of advanced human lung cancer. Consequently, this model appears suitable for in vivo evaluation of novel cancer therapies in preclinical tests.
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Introduction |
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There is a strong need for improvement of therapy for the advanced stages of lung cancer, as the majority of patients are diagnosed at an irresectable stage of disease. Therefore, it is essential that clinically relevant in vivo models are established.
Subcutaneous induction of tumor growth has been described by several authors, but these tumors rarely metastasize and do not adequately reflect the clinical situation [4]. The intravenous injection of tumor cells induces a pattern of hematogenous metastases in solid organs [5], but a defined primary tumor as a source for metastatic spread is missing. The superiority of orthotopic implantation of tumor cells over ectopic implanta-tion was originally proposed by Paget [6], stating that an organ site-specific microenvironment is essential for optimal tumor growth and progression in vivo. To achieve intrapulmonary growth of metastasizing lung tumors, various techniques have been described, such as injection of tumor cells intrabronchially [7], implantation of tissue onto the parietal or visceral pleura [8], injection of cells into the pleural cavity [9], or intrapulmonary injection using fluoroscopic guidance [10]. The purpose of our study was to establish a reproducible animal model that closely resembles the clinical features of different types of advanced human lung cancer. Therefore, different techniques of cell inoculation were evaluated with respect to their effectiveness of tumor induction and metastatic growth properties. Orthotopic application of tumor cells was technically facilitated, to make this model feasible for even large-scale evaluation of novel lung cancer therapies.
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Material and methods |
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KNS-62 and Colo-699 were cultured as monolayers in 75-cm2 flasks using RPMI 1640 medium (Life Technologies Ltd, Paisley, Scotland), supplemented with 10% fetal calf serum, 1% sodium-pyruvate, and 1% L-glutamine. BEN was maintained in Dulbecco’s modified Eagle’s medium (Life Technologies Ltd) supplemented with 10% fetal calf serum, 1% sodium-pyruvate, and 1% L-glutamine. Cells were maintained at 37°C in a humidified incubator gassed with 5% CO2 and 95% air. When cells grew to approximately 80% confluence, they were subcultured or harvested using trypsin-EDTA (Life Technologies Ltd). Cells to be implanted were washed in phosphate-buffered saline (PBS) buffer (Life Technologies Ltd), resuspended in serum-free culture medium, counted in a hemocytometer, and equilibrated at a density of 2 x 106 cells per 50-µL injection volume. Cell viability was tested by trypan blue staining. All cell cultures were proven to be free of mycoplasma infection by reverse-transcriptase polymerase chain reaction (RT-PCR) (Takara Shuzo Co, Ltd, Kyoto, Japan) of supernatant from densely growing cells.
Experimental animals
Pathogen-free female SCID bg mice (Harlan Winkelmann, Borchen, Germany) were xenotransplanted at the age of 4 to 5 weeks either subcutaneously, intravenously, intrapleurally, or intrapulmonary with one of the described cell lines at a dose of 2 x 106 cells in 50 µL serum-free culture medium. Mice were maintained in sterile polycarbonate microisolator cages under pathogen-free conditions, fed autoclaved food and water ad libitum, and handled under stringent sterile conditions in a laminar flow hood. Mice were acclimated for 1 week before the start of this study. All animals have received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health.
Tumor cell implantation
Subcutaneous injection was performed dorsally into the right flank. Intravenous injection was performed into the tail vein while animals were kept in a restrainer. For intrapleural and intrapulmonary injections, animals were anesthetized by intraperitoneal injection of Avertin 20 µL/g body weight. A left posterolateral thoracic incision was made and the thoracic wall exposed by blunt dissection of the muscles, leaving the parietal pleura intact. The tip of a 1.2-cm 27-gauge needle was advanced under visual control through the translucent pleura at the third intercostal space at the dorsal midaxillary line into the pleural cavity or below the visceral pleura, where the tumor cell inoculum was released. Occasional pneumothorax was evacuated by a 2-mL syringe and a 27-gauge needle. Complete reexpansion of the lung was verified by an increase in respiratory rate and visualization of the lung through the chest wall.
Clinical and postmortem evaluation
Animals were followed daily after surgery and monitored for signs of wound healing disturbances, decreased physical activity, and visible tumor growth. Animals were weighed weekly. When animals became moribund, they were sacrificed by CO2 inhalation; otherwise, animals were killed after 6 weeks.
Primary tumors and detectable metastases were harvested immediately for microscopic examination. Tumor width and length were noted in order to calculate tumor volume using the formula for a prolate ellipsoid (width2 x length x 0.52). The lungs, heart, mediastinal lymph nodes, liver, kidneys, spleen, adrenal glands, and mesenteric and retroperitoneal lymph nodes were processed for macroscopic and histological examination. Tissues were fixed in formaline and stained with hematoxylin and eosin using standard procedures.
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Results |
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Histopathologic examination confirmed for KNS-62 the growth of a solid squamous lung carcinoma with a G2 differentiation (Fig 1). The tumors appeared to have a relative sharp border and displayed moderate invasion into the surrounding tissue. Colo-699 injection induced huge tumors reflecting a poorly differentiated (G3) adenocarcinoma of the lung (Fig 2). In comparison with KNS-62, these tumors grew locally more aggressively, infiltrating the surrounding tissue. BEN cell injection induced growth of solid tumors, histologically confirmed as a poorly differentiated large cell lung carcinoma (G3) that showed a marked invasion into the surrounding tissue (Fig 3). After 6 weeks of subcutaneous growth, mean tumor weight was 611 mg (±466 SD) for BEN-induced tumors, representing 4.6% body weight on average. KNS-62-induced tumors grew to an average weight of 1,010 mg (±729 SD), consistent with 5.8% of the body weight on average. Colo-699 tumors weighed on average 3,008 mg (±926 SD), consistent with 13.4% body weight. Tumors grew exponentially, reflecting the different cell doubling times in vitro, ranging from 38 hours for Colo-699 to 50 hours for BEN.
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There was no procedure-related mortality.
Intravenous injection of tumor cells
Intravenous injection of 2 x 106 KNS-62 cells induced metastatic growth of solid metastases in the lungs of two animals, representing an engraftment rate of 34%. Lesions were confirmed histologically by microscopic examination as metastases of a squamous cell carcinoma. Intravenous injection of 2 x 106 Colo-699 cells induced metastatic growth of a poorly differentiated adenocarcinoma in the liver, kidney, and mediastinal lymph nodes in one animal and metastatic growth in the pancreas, abdominal, and mediastinal lymph nodes of another animal, consistent with a take rate of 34%. All metastases were confirmed histologically as adenocarcinomas. Intravenous injection of 2 x 106 BEN cells was not efficient to induce any metastatic tumor growth; take rate was 0%. There was no procedure-related mortality.
Intrapleural application of tumor cells
Intrapleural application of 2 x 106 KNS-62 cells induced the growth of a primary tumor at the injection site in the upper left pleural cavity with subsequent infiltration of the visceral pleura. There was secondary metastatic growth in the ipsilateral lung, the mediastinal lymph nodes, and the contralateral lung and right pleural cavity by lymphogenic spread. Engraftment rate for intrapleural application of KNS-62 was 100%; metastatic tumor growth by lymphogenic pathways was 100%. In two animals (40%), malignant pleural effusions were observed. Median survival time of mice was 27.3 ± 2 days. One animal died 24 hours postoperatively due to intrathoracic bleeding. Intrapleural application of Colo-699 resulted in 100% engraftment rate of a primary tumor at the injection site. All animals developed lymphogenic metastatic spread to the ipsilateral lung, mediastinal lymph nodes, as well as the contralateral lung (100%). Malignant pleural effusion was observed in 20% of the animals. In comparison with KNS-62, there was a higher number of metastases, leading to confluent conglomerates with subsequent destruction of the lung. Animals were killed due to respiratory insufficiency; median survival was 26.8 ± 3.2 days. One animal died 12 hours postoperatively due to intrathoracic bleeding.
Intrapleural application of 2 x 106 BEN cells resulted in only 34% induction rate of a primary tumor at the injection site. In one of these animals, ipsilateral metastatic tumor growth was observed. There were no procedure-related deaths or disease-related deaths within the 6 weeks of the study course in this group. Overall procedure-related mortality for intrapleural application was 11%.
Intrapulmonary injection of tumor cells
Intrapulmonary engraftment of KNS-62 and Colo-699 cells induced a homogenous pattern of local growth of the primary tumor at the injection site in the left upper lung with subsequent lymphogenic metastatic spread to the ipsilateral and contralateral lung and the mediastinal lymph nodes.
Engraftment rates for KNS-62 and Colo-699 were 100%, with 100% metastatic spread to mediastinum and both pleural cavities. In the KNS-62 group median survival was 30.2 ± 4.9 days; in the Colo-699 group, median survival was 20.1 ± 1.5 days. Sixty-eight percent of these animals suffered from malignant pleural effusions, in contrast to 40% of the KNS-62 group. In the BEN-engrafted group, take rates were 80% (n = 4); all of these animals had metastatic spread to the mediastinal lymph nodes. In one animal, a lymph node metastasis in the abdominal cavity was detected. None of these animals developed respiratory insufficiency during the 6 postoperative weeks. One animal died 2 hours postoperatively due to intrapulmonary bleeding. Overall procedure-related mortality was 5.5% for intrapulmonary injection. Data are summarized in Table 1.
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Comment |
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This is the first report utilizing SCID bg mice for the establishment of a human lung cancer xenotransplant model. This inbred double-mutant model carries the SCID mutation, which results in a lack of both T- and B-lymphocytes, and also carries the beige mutation, which results in T-cell and macrophage defects as well as selective impairment of natural killer cell functions. In addition, SCID bg mice are known to express a much lower frequency (2%) of the "leaky" phenotype (ie, immunocompetent) compared with the scid/scid genotype (> 5%). Furthermore, the age-correlated increase of animals spontaneously producing oligoclonal serum immunoglobulins, as seen in scid/scid genotypes, is not seen in mice with the scid/scid, bg/bg genotype [11]. This makes SCID bg mice a superior graft recipient in comparison with conventionally used SCID or nude mice. The propagation of human lung tumor xenografts over several generations of murine recipients by continued transplantation has been reported to result in a loss of human metastasis suppressor genes and a gain of murine DNA in transplanted tumor cells [12]. This phenomenon was observed after the fourth generation, whereas in earlier generations no alterations in the DNA of transplanted cells were observed. Therefore, tumor induction by xenotransplantation of a monoclonal human cell suspension from an in vitro culture provides a reduced probability of an exchange of donor and recipient’s DNA compared with propagation by continued transplantation of tumor tissue over several generations of recipients.
High engraftment rates with a short delay of tumor establishment are required to make the in vivo model feasible for research purposes. Carcinogen induction of murine tumors appears not to be appropriate for this goal. Disadvantages are a long time period of 6 months for tumor induction and the yield of a variety of different histological tumor cell types, which might not be directly relevant to human lung cancer, thereby limiting their usefulness for cell biology and drug testing studies [13].
Subcutaneous implantation of tumors has been associated with low take rates, and the tumor often appeared to be encapsulated and failed to infiltrate or metastasize [3, 4, 14]. In our study engraftment rates for subcutaneous implantation were 100% for all three cell lines, but only in one case could a pulmonary lesion be detected microscopically, so that despite high engraftment rates, this model does not resemble a suitable model for the clinical situation of advanced lung cancer. This observation is in accordance with the results of McLemore and coworkers [15], where orthotopically implanted tumors were almost universally fatal (92%), and those implanted subcutaneously had a mortality rate of 5%. According to Kubota [16], orthotopically transplanted human small cell lung carcinoma displayed a different chemosensitivity pattern compared with the subcutaneously transplanted tumor, suggesting different pharmacodynamics between the orthotopic lung tumor and the ectopic, subcutaneous tumor site. This finding strongly suggests the use of orthotopic models for the evaluation of treatment modalities of human lung cancer.
Intravenous injection of tumor cells leads to the formation of a hematogenous spread of metastases, but this reflects only poorly the clinical pattern of human lung carcinoma, because a defined primary tumor resulting in secondary metastatic tumor growth is missing. After intravenous injection of 106 human lung tumor cells in SCID mice, Reddy and associates [17] observed only a 50% pulmonary engraftment rate, and an engraftment rate as low as 37% was observed by Nagamachi and associates [9], in contrast to a 100% engraftment rate after subcutaneous injection, which is in accordance with our observations, where only 0% to 34% engraftment rates after intravenous injection could be achieved. This observation may be explained by the "seed and soil theory," originally proposed by Paget [6]. There may be additional differences in the hematogenous and lymphatic propensity of tumor cells to develop metastases, as we observed in our orthotopic model, a tumor spread exclusively via lymphatic pathways to the mediastinum, supraclavicular lymph nodes, and the contralateral lung.
The induction of orthotopic tumor engraftment using solid tumor tissue samples has been described by several authors by suturing tumor specimens onto the parietal pleura or the visceral pleura. Engraftment rates ranged from 31% to 100%; metastatic spread was observed in 50% to 80% of the successfully xenotransplanted tumors. Median survival ranged from 29 days to over 6 months. But in these models, metastatic spread appeared to be mainly regional, as Astoul and associates [18] observed, none of the engrafted tumors metastasized to the contralateral lung, and Cuenca and associates [19] observed that distant lung metastases occurred in only 22% of engrafted tumors. Besides the limited growth of contralateral lung metastases, as observed in humans with advanced lung cancer, the xenografting of solid tumor tissue implies the possibility of transplanting polyclonal tumors and immunocompetent cells from the donor. This makes the use of defined tumor cells from in vitro culture preferable, as a high number of animals with identical tumors can be obtained.
Intrapleural application of adeno and squamous cell carcinoma resulted in a 100% engraftment of a pleural tumor with a 100% secondary lymphogenic metastatic spread to mediastinal lymph nodes and the contralateral lung, which closely resembles the clinical pattern. The lymphatic spread of intrapleural tumor cells can be explained by previously described pleural stomas, which are considered to be a gate through which tumor cells from the pleural cavity can enter the lymphatic circulation. Intrapleural injection of BEN cells was only 34% successful in inducing tumor growth, which may be explained by the necessity for a specific microenvironment at the injection site, as intrapulmonary injection of BEN cells were 80% successful in inducing malignant growth.
The closest resemblance of the clinical pattern of advanced human lung cancer can be achieved by intrapulmonary engraftment of tumor cells, which was initially described by McLemore and coworkers [15]. The intrapulmonary application was achieved via a transtracheal instillation of tumor cells, inducing a fatal pulmonary malignancy. This procedure appears time consuming and requires advanced surgical skills, so it seems not to be favorable for a large-scale application. In addition, this model lacks the induction of a defined primary tumor, as the cell suspension is applied into the right main-stem bronchus, from where the cells’ course cannot be controlled. The cells may be initially propagated into different lobes by ventilation, resulting in a polytype primary tumor growth. However, most of the tumors propagated intrabronchially were localized in the right lung, with only 1% metastasizing to the left lung, 2% to the trachea, and only 3% forming distant metastases. Similar observations were reported by Howard and associates [20], where intrabronchially propagated tumors resulted in local growth in the right lung with metastases to mediastinal lymph nodes but no metastases to the ipsilateral or contralateral lung, both important sites of clinical metastases in human lung carcinoma.
A direct intrapulmonary injection appears advantageous and has recently been described by Hamide and associates [10]. After percutaneous intrapulmonary injection proved to be unsatisfactory, the technique was performed under fluoroscopic guidance for more accurate implantation. This approach was effective, but requires huge technical equipment, which makes large-scale application complicated.
One aim of our study was to simplify the technique of intrapulmonary cell inoculation. The left lung was chosen for implantation because the possible loss of lung function is relatively smaller than in right lung procedures, and the left lung consists of only one lobe, so tumors can develop easily after implantation.
The surgical exposure of the thoracic wall is easy to perform, and the injection needle can be guided under visual control through the translucent parietal pleura. Leaving the parietal pleura intact decreases the incidence of pneumothorax and postoperative respiratory complications. The procedure can be performed with a low procedure-related mortality, if the intrapulmonary injection is guided precisely to the pulmonary apex in order to prevent bleeding complications, as SCID bg mice have a prolonged bleeding time.
The intrapulmonary implantation of the aforementioned tumor cells can be performed easily and results in an engraftment rate for KNS-62 and Colo-699 of 100%, with 100% metastatic spread into mediastinal lymph nodes and both lungs and malignant pleural effusions in 40% to 60%, closely resembling the clinical features of advanced human lung cancer. For BEN, the take rates were 80%, with 100% metastatic spread to mediastinal lymph nodes, but due to a slower proliferation of this cell line, the onset of clinical symptoms was protracted.
This improved model of orthotopic induction of human lung carcinoma as described in this paper is reproducible with a fast tumor induction and a high incidence of metastases. Consequently, this model appears suitable for the evaluation of new therapeutic strategies against human lung cancer.
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Acknowledgments |
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