Ann Thorac Surg 2000;69:1691-1695
© 2000 The Society of Thoracic Surgeons
Original articles: General thoracic
Artificial lymphogenous metastatic model using orthotopic implantation of human lung cancer
Hisashi Ishikura, MDa,
Kazuya Kondo, MD, PhDa,
Takanori Miyoshi, MD, PhDa,
Hidetaka Kinoshita, MDa,
Toshiyuki Hirose, MDa,
Yasumasa Monden, MD, PhDa
a Second Department of Surgery, School of Medicine, Tokushima University, Tokushima, Japan
Address reprint requests to Dr Kondo, Second Department of Surgery, School of Medicine, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima City, Tokushima 770-8503, Japan
e-mail: kondo{at}clin.med.tokushima-u.ac.jp
 |
Abstract
|
|---|
Background and Methods. We established a new, patientlike orthotopic model of lung cancer metastasis with human non–small cell lung cancer cell lines. In this report, we describe the progressive stages of development of lymphogenous mediastinal metastasis in the Ma44-3 cell line from day 3 to day 15 after implantation in severe combined immunodeficiency mice and the process of lymphogenous metastasis.
Results. All mice killed after day 12 had perivascular and peribronchial tumor growth. Micrometastasis to the mediastinum was first observed on day 5. On days 5 through 9, 10 of 13 mice had metastasis to the mediastinum, and all mice had one by day 12. When perivascular and peribronchial tumor growth was present by day 5, metastasis to the mediastinum developed in all mice.
Conclusions. This study demonstrates the lymphogenous spread of human lung cancer in severe combined immunodeficiency mouse using an orthotopic implantation model. Our model was thought to be an artificial lymphogenous metastasis model, owing to forced tumor inoculation into lymphatic vessels.
|
Introduction
|
|---|
Lung cancer is now the most common cause of death from cancer worldwide in both men and women. It is the general consensus that the poor prognosis of lung cancer reflects the aggressive biologic nature of the cancer and the ineffectiveness of early detection procedures and treatment. In particular, metastasis to mediastinal lymph nodes or other organs produces poor prognosis in lung cancer.
An orthotopic implantation model of human lung cancer was first developed by McLemore and associates [1, 2]. Since then, a number of orthotopic implantation models have been developed for human lung cancer [3–6]. However, there has as of now been no lymphogenous metastatic orthotopically implanted model for lung cancer, and the detailed mechanism of lymphogenous metastasis remains unknown.
We established a new, patientlike model of lung cancer metastasis by orthotopic implantation using human non–small cell lung cancer cell lines [7]. According to our results, the Ma44 lung cancer cell line formed tumors in the lung at a high rate (17 of 25, 68%), and many of them metastasized to mediastinal lymph nodes (13 of 17, 76%). Our results showed the following advantages of this model: (1) the implantation procedure is simple, (2) the procedure can provide models in a short time (2 weeks), (3) the procedure is not limited to the Ma44 cell line, and (4) the metastatic form was similar to that of human lung cancer.
In our laboratory, four cell line clones (Ma44-1, -2, -3, and -4) were isolated from the Ma44 parent cell line by the limiting dilution method. Although Ma44-3 cells did not produce tumors in the lung after intravenous injection, they metastasized to the mediastinum at a high rate after direct injection into the lung.
In this report, we describe the progressive stages of development of lymphogenous mediastinal metastases using the Ma44-3 cell line from day 3 to day 15 after intrapulmonary implantation, and describe the process of lymphogenous metastasis in detail: (1) anchoring in the lung, (2) flowing in the lymphatic vessels around the blood vessels and bronchi, and (3) reaching the mediastinum.
|
Material and methods
|
|---|
Animals and human non–small cell lung carcinoma cell line
Male severe combined immunodeficiency mice (6 weeks of age) with a CB-17 genetic background were purchased from CLEA Japan Inc (Tokyo, Japan).
Cultured human squamous cell carcinoma cells (Ma44) were kindly supplied by the Osaka Prefectual Habikino Hospital, Osaka, Japan. The Ma44 cell line was established from the primary lesion of a 68-year-old man with squamous cell lung carcinoma, pT2N0M0, stage IB. A curative operation was performed on January 16, 1993. This cell line grows attached to a substrate.
We obtained Ma44-3 by the limiting dilution method in our laboratory. Ma44-3 cells were cultured in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum (Bio Whittaker, Walkersville, MD). The cells were maintained at 37°C in a humidified incubator with 5% CO2 in air and harvested for implantation at 70% to 80% confluence using 1 mmol/L ethylenediaminetetraacetic acid (Wako Pure Chemical Industries Ltd, Osaka, Japan) in phosphate-buffered saline (Nissui Pharmaceutical Co Ltd, Tokyo, Japan). The cells were washed in RPMI-1640 medium and resuspended to a final concentration of 2.0 x 106 cells/mL in RPMI-1640 containing 0.1% bovine serum albumin (Boehringer Mannheim, Mannheim, Germany).
Surgical orthotopic implantation of human lung cancer cells in severe combined immunodeficiency mice
The mice were fully anesthetized by ether inhalation. The experimental mice were placed in the right lateral decubitus position with the four limbs restrained. A 1-cm transverse incision was made on the left lateral skin just below the inferior border of the scapula of the severe combined immunodeficiency mouse. Muscles were separated from the ribs by sharp dissection, and intercostal muscles were exposed. The left lung was visible through the intercostal muscles. A 30-gauge needle was inserted approximately 5 mm into the lung through the intercostal muscle, and an inoculum of 2.0 x 106 tumor cells/mL with 400 µg/mL Matrigel (Collaborative Biomedical Products, Bedford, Canada) was then dispersed into the left lung in a final volume of 10 µL (2.0 x 104 cells) medium. The procedure required approximately 1 minute for completion and was easily performed. The skin incision was closed with 3-0 silk.
Analysis of tumor growth and metastasis
Twenty-five mice were inoculated in the lung and were killed on day 3, 5, 7, 9, 12, or 15 after tumor cell implantation by ether inhalation and cervical dislocation. Major organs (bilateral lungs, heart, liver, kidneys, adrenal glands, and mediastinal tissues) were removed, fixed in 10% formalin, and embedded in paraffin. Five-micrometer histologic sections were made from the lung and mediastinal tissues at 300-µm intervals. Paraffin sections stained with hematoxylin and eosin were examined with a microscope.
|
Result
|
|---|
Table 1 shows the growth and metastasis of orthotopically implanted human lung cancer cell line Ma44-3. There were no surgical deaths caused by implantation. No metastasis was observed in the contralateral lung, thymus, liver, kidneys, or adrenal glands.
Local tumor growth
Until day 7 we could not detect the tumor macroscopically in the lungs of any of the 13 mice, but we could find cancer cells microscopically in 6 of 13 mice (Fig 1). Beginning on day 9, the tumor in the lung could be observed macroscopically (Fig 2). Although the size of the local tumors in the lung tended to enlarge gradually, the number of tumors at the site of injection was almost constant independent of the passage of time.
View larger version (65K):
[in this window]
[in a new window]
|
Fig 1. Histopathology of implanted Ma44-3 cells locally grown in the left lung on day 3. (A) Low-power field (x20). (B) High-power field (x200).
|
|
View larger version (74K):
[in this window]
[in a new window]
|
Fig 2. (A) Local tumor growth in the left lung and mediastinal metastasis on day 12. (B) Illustration of tissue in A.
|
|
Perivascular and peribronchial tumor growth
Cancer cells grew around the blood vessels and bronchus of the lung at a distance from the injection point (Fig 3). On day 3, 1 of 4 mice (25%) had perivascular and peribronchial tumor growth. Seven of 13 mice (54%) killed between days 5 and 9 had such tumor, and all mice (100%) killed after day 12 had one. The incidence of perivascular and peribronchial tumor growth in the lung became higher as time passed.
View larger version (120K):
[in this window]
[in a new window]
|
Fig 3. (A) Histopathology of perivascular tumor growth on day 5 (x20). (B) The epithelium of the blood vessel was intact (x200).
|
|
Metastasis to mediastinum
Metastasis to the mediastinum was never observed on day 3 even microscopically, and micrometastasis to the mediastinum was observed on day 5 for the first time, when a nest of tumor cells was formed in the mediastinum (Fig 4). On days 5 through 9, 10 of 13 mice (77%) had metastasis to the mediastinum, and all mice (100%) had one by day 12. The frequency of metastasis to the mediastinum became higher as time passed. Fifteen of 16 mice with perivascular or peribronchial tumor growth proceeded to develop metastasis to the mediastinum, but one mouse with local and peribronchial growth on day 3 did not (Table 1).
View larger version (104K):
[in this window]
[in a new window]
|
Fig 4. (A) Histopathology of mediastinal micrometastasis (arrow) in the mediastinal fat tissue on day 5 (x20). (B) High-power field (x200).
|
|
|
Comment
|
|---|
Several authors have previously reported models of spontaneous metastasis to lymph nodes using subcutaneous implantation of cancer cells [8, 9] or by orthotopic implantation [10–17]. However, there has not as yet been an optimal animal model for lymphogenous metastasis of human lung cancer. The present study demonstrates the lymphogenous spread of human lung cancer in severe combined immunodeficiency mouse using an orthotopic implantation model. Our model was thought to be an artificial lymphogenous metastasis model, owing to forced tumor inoculation into lymphatic vessels. Metastatic models in which tumor cells are injected intravenously are termed hematogenous or experimental metastatic models, whereas the model reported here will be termed lymphogenous metastatic model. The closer correspondence of our model to natural metastasis suggests that it would be useful for elucidation of the mechanism of lymphogenous metastasis.
In this study, the rate of tumor formation in the inoculated site was 0% to 60% regardless of time. Perivascular or peribronchial tumors and mediastinal metastatic lesions were observed more frequently with the passage of time after inoculation, and all mice with perivascular or peribronchial tumors by day 5 proceeded to have metastasis of the mediastinum. We propose that the tumor cells flow immediately into the lymphatic vessel at the inoculation site, regardless of the anchoring of the tumor cells, and spread in the perivascular, peribronchial, or mediastinal lymphatic vessels, and then some cells stay in lymphatic vessels and proliferate there.
The lymphatics in the lung form a perivascular and peribronchial network directed toward the hilum. In our study, we can observe the phenomenon of the lymphatic spread of cancer cells similar to that which occurs in human primary lung cancer. Therefore, in our model the metastatic tumors develop in the perivascular or peribronchial space, in which lymph vessels are rich.
Although on day 3 mediastinal metastasis was not observed, by day 5 all 15 mice with peribronchial or perivascular lesions showed metastasis to the mediastinum. Therefore, we presume that day 3 is near the midpoint of the course of events by which cells proceed from the lung to the mediastinum.
The rate of tumor formation at the inoculated site was low (0% to 60%). Many previous reports suggested that the organ site-specific environment is an important factor for the growth of implanted human cancer cells in nude and severe combined immunodeficiency mice [18]. Friedman and colleagues [19, 20] reported that coinjection with tumor cells and Matrigel enhanced the local growth rate of tumors subcutaneously implanted in athymic mice. In our preliminary study, inoculation of tumor cells with 10 mg/mL Matrigel enhanced the local growth rate compared with 400 µg/mL (7 of 8 [88%] versus 9 of 25 [36%]). These data in our laboratory suggest that Matrigel enhances local tumor formation in the lung dose dependently.
In summary, we have demonstrated the lymphogenous spread of human lung cancer cells orthotopically implanted in severe combined immunodeficiency mice, and we hope this model will be useful for elucidating the mechanism of lymphogenous metastasis.
|
Acknowledgments
|
|---|
We are grateful to Kiyomi Aihara for preparing the slides for microscopy.
|
References
|
|---|
-
McLemore T.L., Liu M.C., Blacker P.C., et al. Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice. Cancer Res 1987;47:5132-5140.[Abstract/Free Full Text]
-
McLemore T.L., Eggleston J.C., Shoemaker R.H., et al. Comparison of intrapulmonary, percutaneous intrathoracic, and subcutaneous models for the propagation of human pulmonary and nonpulmonary cancer cell lines in athymic nude mice. Cancer Res 1988;48:2880-2886.[Abstract/Free Full Text]
-
Howard R.B., Chu H., Zeligman B.E., et al. Irradiated nude rat model for orthotopic human lung cancers. Cancer Res 1991;51:3274-3280.[Abstract/Free Full Text]
-
Kubota T. Metastatic models of human cancer xenografted in the nude mouse. J Cell Biochem 1994;56:4-8.[Medline]
-
Wang H.Y., Ross H.M., Ng B., Burt M.E. Establishment of an experimental intrapulmonary tumor nodule model. Ann Thorac Surg 1997;64:216-219.[Abstract/Free Full Text]
-
Wang X., Fu X., Kubota T., Hoffman R.M. A new patient-like metastatic model of human small-cell lung cancer constructed orthotopically with intact tissue via thoracotomy in nude mice. Anticancer Res 1992;12:1403-1406.[Medline]
-
Miyoshi T., Kondo K., Ishikura H., et al. SCID mouse lymphogenous metastatic model of human lung cancer constructed using orthotopic inoculation of cancer cells. Anticancer Res 2000;20:161-163.[Medline]
-
Takeda K., Hatakeyama K., Tsuchiya Y., Rikiishi H., Kumagai K. A correlation between GM-CSF gene expression and metastasis in murine tumors. Int J Cancer 1991;47:413-420.[Medline]
-
Vandendris M., Dumont P., Semal P., Heimann R., Atassi G. Investigation of a new murine model of regional lymph node metastasis. Clin Exp Metastasis 1985;3:7-19.[Medline]
-
Fu X.Y., Besterman J.M., Monosov A., Hoffman R.M. Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically intact patient specimens. Proc Natl Acad Sci USA 1991;88:9345-9349.[Abstract/Free Full Text]
-
Fu X., Guadagni F., Hoffman R.M. A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically with histologically intact patient specimens. Proc Natl Acad Sci USA 1992;89:5645-5649.[Abstract/Free Full Text]
-
Fujihara T., Sawada T., Hirakawa K., et al. Establishment of lymph node metastatic model for human gastric cancer in nude mice and analysis of factors associated with metastasis. Clin Exp Metastasis 1998;16:389-398.[Medline]
-
Furukawa T., Fu X., Kubota T., Watanabe M., Kitajima M., Hoffman R.M. Nude mouse metastatic models of human stomach cancer constructed using orthotopic implantation of histologically intact tissue. Cancer Res 1993;53:1204-1208.[Abstract/Free Full Text]
-
Rembrink K., Romijn J.C., van der Kwast T.H., Rubben H., Schroder F.H. Orthotopic implantation of human prostate cancer cell lines. Prostate 1997;31:168-174.[Medline]
-
Sato N., Gleave M.E., Bruchovsky N., Rennie P.S., Beraldi E., Sullivan L.D. A metastatic and androgen-sensitive human prostate cancer model using intraprostatic inoculation of LNCaP cells in SCID mice. Cancer Res 1997;57:1584-1589.[Abstract/Free Full Text]
-
Stephenson R.A., Dinney C.P., Gohji K., Ordonez N.G., Killion J.J., Fidler I.J. Metastatic model for human prostate cancer using orthotopic implantation in nude mice. J Natl Cancer Inst 1992;84:951-957.[Abstract/Free Full Text]
-
Vieweg J., Heston W.D., Gilboa E., Fair W.R. An experimental model simulating local recurrence and pelvic lymph node metastasis following orthotopic induction of prostate cancer. Prostate 1994;24:291-298.[Medline]
-
Fidler I.J. Modulation of the organ microenvironment for the treatment of cancer metastasis. J Natl Cancer Inst 1995;87:1588-1591.[Free Full Text]
-
Friedman R., Giaccone G., Kanemoto T., Martin G.R., Gazdar A.F., Mulshine J.L. Reconstituted basement membrane (Matrigel) and laminin can enhance the tumorigenicity and drug resistance of small cell lung cancer cell lines. Proc Natl Acad Sci USA 1990;87:6698-6702.[Abstract/Free Full Text]
-
Friedman R., Kibby M.C., Royce L.S., et al. Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with Matrigel. J Natl Cancer Inst 1991;83:769-774.[Abstract/Free Full Text]
Accepted for publication December 1, 1999.
This article has been cited by other articles:
|
|
|
|
 
H. Fujino, K. Kondo, H. Ishikura, H. Maki, H. Kinoshita, T. Miyoshi, Y. Takahashi, N. Sawada, H. Takizawa, T. Nagao, et al.
Matrix metalloproteinase inhibitor MMI-166 inhibits lymphogenous metastasis in an orthotopically implanted model of lung cancer
Mol. Cancer Ther.,
September 1, 2005;
4(9):
1409 - 1416.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Ishikura, K. Kondo, T. Miyoshi, H. Kinoshita, Y. Takahashi, H. Fujino, and Y. Monden
Suppression of Mediastinal Metastasis by Uracil-Tegafur or cis-Diamminedichloroplatinum(II) Using a Lymphogenous Metastatic Model in a Human Lung Cancer Cell Line
Clin. Cancer Res.,
December 1, 2001;
7(12):
4202 - 4208.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
