JP2007274950A - Lung cancer-grafting model animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple technique with reproductivity for preparing a model animal for orthotopic grafted lung cancer. <P>SOLUTION: The invention relates to the method of preparing a non-human animal with taking lung cancer cells comprising a process preparing a non-human animal with reduced immune strength, a process exposing the tracheae of the non-human animal, and a process injecting the lung cancer cells into the tracheae. The invention relates to the non-human animal with taking lung cancer cells. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a lung cancer transplant model animal.

  Lung cancer is the most common malignant tumor in the world. In the United States, about 170,000 lung cancer cases were diagnosed in 2002, of which 80,000 were women and 90,000 were men (Non-patent Document 1). In Japan, lung cancer accounted for 12.7% of cancer-related deaths in women in 2001 and 22% of cancer-related deaths in men (2001). In addition, less than 14.9% of patients survived after lung cancer treatment. It is generally recognized that the prognosis of patients with lung cancer is unlikely to reflect that the biological nature of this cancer is invasive and that early detection procedures and treatments are ineffective.

  Efforts have been made to develop effective treatments for lung cancer from in vivo models. For example, an orthotopic transplantation model of human small cell lung cancer (SCLC) has been demonstrated to be sensitive to cisplatin and resistant to mitomycin C, reflecting the current state of the clinic. is doing. In contrast, xenografts of the same tumor implanted subcutaneously responded to mitomycin C, but not cisplatin, and therefore did not match the clinical behavior of SCLC (Non-patent Document 3). . As first described by Pagets' "seed and soil" theory (Non-Patent Document 4) and confirmed by other researchers (Non-Patent Documents 5 and 6), the organ microenvironment is May affect tumor cell phenotype and sensitivity to chemotherapy. Therefore, it is very important to establish a clinically relevant in vivo model that simulates clinical features for lung cancer research.

  Several orthotopic models of lung cancer have been developed in rodents. Examples include direct transplantation of human cancer tissue (Non-Patent Document 7), as well as rodent airways (Non-Patent Documents 8 and 9), pleural cavity (Non-Patent Documents 10 and 11), or after skin incision (Non-Patent Documents). 12) Or injection of tumor cells into the lung parenchyma after thoracotomy (Non-patent Documents 13 and 14). However, the transplantation techniques in these models are complex and there are limits to their widespread use. Much of the research and development of new diagnostic methods and therapeutic agents for lung cancer still relies on subcutaneous tumor models, but these models may not be clinically relevant.

American Cancer Society. Cancer facts and figures 2002. Atlanta GA: American Cancer Society, 2002. Vital statistics of Japan, Statistics and Information Dept., Minister ’s Secretarial, Ministry of Health, Labor and Welfare, 2001 Kuo TH, Kubota T, Watanabe M, Furukawa T, Kase S, Tanino H, Saikawa Y, Ishibiki K, Kitajima M, Hoffman RM.Site-specific chemosensitivity of human small-cell lung carcinoma growing orthotopically compared to subcutaneously in SCID mice: the importance of orthotopic models to obtain relevant drug evaluation data.Anticancer Res 1993; 13: 627-30 Paget S. The distribution of secondary growths in cancer of the breast.Cancer Metastasis Rev 1989; 8: 98-101 Wilmanns C, Fan D, O'Brian CA, Bucana CD, Fidler IJ. Orthotopic and ectopic organ environments differentially influence the sensitivity of murine colon carcinoma cells to doxorubicin and 5-fluorouracil. Int J Cancer 1992; 52: 98-104 Fidler IJ, Wilmanns C, Staroselsky A, Radinsky R, Dong Z, Fan D. Modulation of tumor cell response to chemotherapy by the organ environment.Cancer Metastasis Rev 1994; 13: 209-22 Hoffman RM, Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic.Invest New Drugs 1999; 17: 343-59 McLemore TL, Liu MC, Blacker PC, Gregg M, Alley MC, Abbott BJ, Shoemaker RH, Bohlman ME, Litterst CC, Hubbard WC, Brennan RH, McMahon JB, Fine DL, Eggleston JC, Mayo JG, and Boyd MR. intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice.Cancer Res 1987; 47: 5132-40 Howard RB, Chu H, Zeligman BE, Marcell T, Bunn PA, McLemore TL, Mulvin DW, Cowen ME, Johnston MR.Irradiated nude rat model for orthotopic human lung cancers.Cancer Res 1991; 51: 3274-80 McLemore TL, Eggleston JC, Shoemaker RH, Abbott BJ, Bohlman ME, Liu MC, Fine DL, Mayo JG, Boyd MR. 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: 2880-6 Kraus-Berthier L, Jan M, Guilbaud N, Naze M, Pierre A, Atassi G. Histology and sensitivity to anticancer drugs of two human non-small cell lung carcinomas implanted in the pleural cavity of nude mice.Clin Cancer Res 2000; 6 : 297-304 Doki Y, Murakami K, Yamaura T, Sugiyama S, Misaki T, Saiki I. Mediastinal lymph node metastasis model by orthotopic intrapulmonary implantation of Lewis lung carcinoma cells in mice.Br J Cancer 1999; 79: 1121-6 Wang HY, Ross HM, Ng B, Burt ME.Establishment of an experimental intrapulmonary tumor nodule model.Ann Thorac Surg 1997; 64: 216-9 Miyoshi T, Kondo K, Ishikura H, Kinoshita H, Matsumori Y, Monden Y. SCID mouse lymphogenous metastatic model of human lung cancer constructed using orthotopic inoculation of cancer cells. Anticancer Res 2000; 20: 161-3

  It is an object of the present invention to provide a simple and reproducible technique for producing orthotopic transplanted lung cancer model animals.

  We have established an orthotopic tumor transplantation model in nude mice that is very similar to the clinical features of human lung cancer. Human lung adenocarcinoma A549 cell line and squamous cell carcinoma SQ5 cell line were used. Tumor cells suspended in serum-free medium with or without 0.01 M ethylenediaminetetraacetic acid (EDTA) were co-administered to the main bronchus of anesthetized athymic nude mice (7-9 weeks old) Injected directly. In some experiments, lung cancer cells obtained from subcutaneously transplanted tumors were re-cultured and used for endotracheal transplantation. Tumor nodules formed in the lung were counted and confirmed by histological examination. When A549 cells were administered with EDTA, the engraftment rate was 70% (n = 10). The engraftment rates of A549 cells re-cultured without EDTA or A549 cells re-cultured with EDTA were 20% (n = 5) and 80% (n = 5), respectively. The survival rates when SQ5 cells were administered without EDTA or with EDTA were 50% (n = 4) and 67% (n = 6), respectively. When re-cultured SQ5 cells were administered with EDTA, the engraftment rate further increased to 100% (n = 6). A number of tumors formed mainly in the left and right upper lobes. When EDTA was co-administered, more tumor nodules occurred in the lungs. Histological findings revealed that A549 tumor nodules were mainly distributed in the alveoli. SQ5 solid tumor infiltrated the bronchiole and filled the alveoli. This reproducible orthotopic transplantation model resulted in tumor growth that simulated the clinical features of human lung cancer. The present invention has been completed based on these findings.

The gist of the present invention is as follows.
(1) A method for producing a non-human animal in which lung cancer cells are engrafted,
Preparing a non-human animal with reduced immunity,
Exposing the non-human animal trachea and injecting lung cancer cells into the trachea.
(2) The method according to (1), wherein lung cancer cells are mixed with ethylenediaminetetraacetic acid and injected into the trachea.
(3) The method according to (1) or (2), wherein the non-human animal with reduced immunity is a nude mouse.
(4) The method according to any one of (1) to (3), wherein the lung cancer cells are small cell lung cancer cells or non-small cell lung cancer cells.
(5) The method according to any one of (1) to (4), wherein the lung cancer cells are derived from human.
(6) The method according to any one of (1) to (5), wherein 10 ⁷ to 10 ⁸ lung cancer cells per non-human animal are injected into the trachea of the non-human animal.
(7) The method according to any one of (1) to (6), wherein a non-human animal in which lung cancer cells are engrafted in at least one organ selected from the group consisting of lung, trachea and bronchial tree.
(8) A non-human animal produced by the method according to any one of (1) to (7) and engrafted with lung cancer cells.

In accordance with the present invention, a new orthotopic transplanted human lung cancer model has been developed using a reproducible technique.
The model of the present invention is useful for studying the biological behavior and treatment of human lung cancer in vivo.

Hereinafter, embodiments of the present invention will be described in more detail.
The present invention is a method for producing a non-human animal in which lung cancer cells are engrafted, comprising the steps of preparing a non-human animal with reduced immunity, exposing the trachea of the non-human animal, and lung cancer cells. The method is provided comprising the step of injecting into the trachea.

  First, a non-human animal with reduced immunity is prepared. The non-human animal should have reduced immunity to such an extent that lung cancer cells can be engrafted. As non-human animals with reduced immunity, various immunodeficient animals (for example, nude mice, nude rats, skid mice, ALY mice, etc.) are commercially available and can be used. Furthermore, in order to reduce immunity, non-human animals may be irradiated with radiation such as X-rays.

  The prepared non-human animal is preferably an adult, for example, 7 to 9 weeks in the case of mice, and 8 to 10 weeks in the case of rats. The non-human animal may be bred in an environment in which infection is protected according to the degree of immunity reduction. Antibiotics may be administered to non-human animals for the purpose of infection protection.

Next, the trachea of the non-human animal is exposed. For example, a non-human animal with reduced immunity can be anesthetized, placed on its back, fixed on a table, and the trachea can be exposed by incising the neck skin with a scalpel or the like.
The lung cancer cells are then injected into the lumen of the exposed trachea. For example, lung cancer cells can be injected into the trachea using a syringe equipped with an injection needle. Lung cancer cells may be injected at any location in the trachea, but preferably at about 1/3 from the top.

Examples of lung cancer cells include small cell lung cancer cells, non-small cell lung cancer cells (eg, lung adenocarcinoma cells, lung squamous cell carcinoma cells, etc.). The lung cancer cells are preferably derived from humans. Examples of human-derived lung cancer cells include human small cell lung cancer cells, human non-small cell lung cancer cells (eg, lung adenocarcinoma cell lines A549PC14, PC9, lung squamous cell lines SQ5, Ma44, NCI-H226, etc.) Can do. The lung cancer cells may be collected from animal tumors or subcultured from them. Alternatively, a tumor may be collected from an animal transplanted subcutaneously with subcultured lung cancer cells and subcultured again.
10 ⁷ to 10 ⁸ lung cancer cells per non-human animal may be injected into the trachea of the non-human animal.

  Lung cancer cells may be suspended in a serum-free medium (eg, αMEM, RPMI1640, etc.) or PBS and then injected into the trachea of a non-human animal. When injecting a lung cancer cell suspension into a mouse, the volume of the lung cancer cell suspension may be 100 μl or less, preferably 30 μl to 50 μl. When injecting into other non-human animals, the volume of the lung cancer cell suspension may be converted from the above values based on the body weight of the animals. The lung cancer cell suspension used for the injection may have a lung cancer cell survival rate of 90% or more, and preferably has a lung cancer cell survival rate of more than 95%. The survival rate of lung cancer cells can be measured by Trypan blue dye exclusion. Briefly, mix cell fluid and Trypan blue solution. The final concentration of Trypan blue is 0.02%. Cells are observed with a microscope, and cells that are stained blue are dead cells. Count a total of 500 or more cells and calculate the viability of the cells.

Ethylenediaminetetraacetic acid may be added to the lung cancer cell suspension. Addition of ethylenediaminetetraacetic acid increases the survival rate of lung cancer cells in the recipient. Ethylenediaminetetraacetic acid may be added at a concentration of 0.01 to 0.02 M, preferably at a concentration of 0.01 M.
After injecting lung cancer cells into the trachea, if non-human animals are bred for an appropriate period, engraftment of lung cancer cells can be confirmed. Lung cancer cells can engraft in organs such as the lung (for example, the left lobe, upper lobe, middle lobe, lower lobe, posterior lobe), trachea (trachea at the site where lung cancer cells are injected), bronchial tree, and the like. For example, when lung cancer cells are injected into the trachea of a mouse, tumor engraftment in the lung tissue can be confirmed with the naked eye after 4 weeks or more. Lung cancer cell engraftment involves counting the number of tumor nodules by visual observation, embedding the intrathoracic organ in paraffin, slicing it into 5 μm thick sections, and staining these sections with hematoxylin-eosin. Can be observed.

The present invention also provides a non-human animal produced by the above method and engrafted with lung cancer cells. In the non-human animals of the present invention, lung cancer cells can grow at an appropriate site that reflects their original characteristics.
By using a non-human animal engrafted with lung cancer cells produced by the method of the present invention, development of a therapeutic drug for cancer, verification of therapeutic effect, verification of side effects due to cancer treatment of normal lung cancer cells, lung cancer It is possible to develop a reagent for detecting and imaging the above.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.

Example 1
Materials and Methods Preparation for cell culture and delivery The human lung adenocarcinoma cell line A549 was obtained from the American Type Culture Collection cell line repository. Human lung squamous cell carcinoma cell line SQ5 was provided by Dr. Kubota of Ibaraki Medical University. Both cell lines contain α-MEM containing L-glutamine and supplemented with 10% heat-inactivated fetal calf serum (Equitech-Bio Inc., Kerrville, TX), 50 units / ml penicillin and 50 μg / ml streptomycin. Maintained in medium (Sigma, St. Louis, MO). Cells were maintained in exponential growth phase at 37 ° C. and 5% CO ₂ in a humidified incubator. Adhesive tumor cells were collected from the culture just before reaching confluence by a quick exposure to 0.25% trypsin / 0.02% EDTA. Trypsinization was stopped with medium containing 10% serum, cells were washed once and resuspended in serum-free medium. Trypan blue staining was used to assess cell viability and only single cell suspensions with viability greater than 95% were used for injection.
In some experiments, tumor cell suspensions were supplemented with ethylenediaminetetraacetic acid (EDTA, 0.01M, Sigma) just prior to intratracheal delivery. This concentration of EDTA was well tolerated by tumor cells and mice in our studies and in other investigators (March TH, Marron-Terada PG, Belinsky SA. Refinement of an orthotopic lung cancer model in the nude rat. Vet Pathol 2001; 38: 483-90).

Female BALB / c nu / nu mice (Charles River, Tokyo, Japan) without experimental animal pathogens were xenotransplanted intratracheally or subcutaneously at 7-9 weeks of age with A549 or SQ5 cell lines. Mice were maintained under conditions free of pathogens, fed autoclaved food and water, and handled under strictly aseptic conditions. Mice were acclimated for one week prior to the start of the study.

Tumor cell intratracheal and subcutaneously transplanted mice were anesthetized with isoflurane (Merck Whey, Osaka, Japan), placed on their back and gently fixed with tape. A 1 cm long abdominal incision was made from the cervical skin to expose the trachea. A 27-3 / 4 gauge needle (Terumo, Tokyo, Japan) is tilted at an angle of approximately 135 ° and used to inject tumor cell suspensions directly into the main bronchus at various volumes and concentrations (Figure 1). ). The incision was closed with one surgical clip and subjected to recovery time under a warm lamp until the mouse was fully awakened (Terry NH, Brinkley J, Doig AJ, Ma J, Patel N, White RA, Mahajan N, Kang) Y. Cellular kinetics of murine lung: model system to determine basis for radioprotection with keratinocyte growth factor. Int J Radiat Oncol Biol Phys 2004; 58: 435-44). For subcutaneous implantation, 100 μl of tumor suspension (1 × 10 ⁷ ) was injected directly into the flank of unanesthetized mice.

In vivo selection of cell lines for increased tumorigenicity In the subcutaneous injection technique described above, A549 cells and SQ5 cells were injected into the flank of nude mice. Mice were sacrificed when tumors were 6-10 mm (diameter) each. Tumors were collected aseptically, minced with scissors, and cultured for passages 1-2. The recultured cells were used for intratracheal injection.

Clinical evaluation and histopathology After injection of tumor cells, mice were observed daily and monitored for signs of impaired wound healing, evidence of tumor development, and reduced physical activity. Mice were weighed twice a week. Mice were either moribund or sacrificed 8 weeks later. Necropsy was performed on all animals. The intrathoracic organ was removed and fixed in neutral buffered formalin for at least 24 hours. The number of tumor nodules in each lung was counted by visual observation. These intrathoracic organs were then embedded in paraffin for histological examination. Sections 5 μm thick were stained with hematoxylin and eosin.

Growth curves and doubling time cells (1 × 10 ⁵ cells) were inoculated into 6 cm dishes. Each day, three samples were collected and counted in a Coulter counter (Beckman Coulter Inc., Fullerton, CA). Cell numbers were averaged for each time interval. The doubling time was calculated from cells in the exponential growth phase.

Results Appropriate volume and technique for intratracheal delivery of tumor suspension In our pilot experiment, mice were tumor cells at a concentration of 2x10 < ⁷ > to 2x10 < ⁸ > / ml. 30 μl to 50 μl of suspension was allowed for intratracheal delivery, but 100 μl of tumor cell suspension was hardly tolerated (data not shown). All the above experiments were performed with a 50 μl intratracheal injection volume.
In this tumor transplant model, the trachea was easily exposed and the tumor cell suspension was accurately injected into the main bronchus. Of all 50 mice, only one died during anesthesia. None of the mice died during or immediately after surgery. In mice, each surgical wound healed within 3 days and no infection occurred.

Up to 15 weeks after tumor-forming endotracheal tumor implantation of A549 cells, no visible tumors were formed when 6 × 10 ⁵ or 3 × 10 ⁶ A549 cells were injected (n = 5). ). In order to obtain high tumorigenicity, A549 cells obtained from in vivo subcutaneous passage in nude mice were selected. After they were collected from subcutaneous tumors and passaged once in vitro, high doses (1 × 10 ⁷ cells) of recultured A549 cells were intratracheally injected into nude mice. The incidence of orthotopic tumors was 20% (n = 5) 60 days after transplantation (Table 1). This low tumorigenicity in this experiment was suggested to be insufficient and insufficient to increase the dose of cancer cells.
EDTA was co-administered to create a better environment for tumor cell growth. This can slightly destroy the epithelial and / or surfactant layers of the lung parenchyma. A549 cells or re-cultured A549 cells (1 × 10 ⁷ cells) were intratracheally injected into 5-10 mice in each group with 0.01M EDTA. When EDTA was co-administered, tumor formation was induced in 70% (n = 10) in A549 cells and 80% (n = 5) in re-cultured A549 cells (Table 1). The average number of tumor nodules per lung was 2 in mice that received recultured A549 cells without EDTA and 16 in mice that received recultured A549 cells with EDTA (Table 2). . The mice lost weight 2-3 days before they became moribund. Body weight change was not a sensitive parameter for early detection of tumor growth in the lung.

Tumorogenicity of SQ5 cells When 1 × 10 ⁷ SQ5 cells were injected, the tumorigenicity after 42 days of transplantation was 50% (n = 4). When 0.01M EDTA was co-administered, tumorigenicity was 67% in SQ5 cells and tumorigenicity was 100% in re-cultured SQ5 cells (n = 6) (Table 1). When SQ5 cells were administered with EDTA, the average number of tumor nodules per lung increased from 2 to 4 (Table 2).
In this model, the lungs of mice transplanted with either A549 cells or SQ5 cells did not adhere to the pleural wall and no effusion was seen in the pleural cavity. Regardless of whether or not EDTA was co-administered, numerous tumor nodules were formed mainly in the left and upper right lobes (FIG. 2, Table 2).

From Histology Histological examination, A549 tumor cells mainly infiltrated in alveolar walls, was shown to be distributed to the alveoli (Figure 3A, B). The A549 nodular xenografts were numerous, varied in size, and proliferated mainly in the periphery of the lung lobe (Figure 3B). A549 tumors derived from adenocarcinoma showed a partially incomplete glandular structure (Figure 3C). Necrosis was observed in the central part of the tumor mass. Normal lung tissue and blood vessels were shrunken by the tumor mass (Figure 3C).
Histological studies confirmed that SQ5 tumors grew as solid lung squamous cell carcinomas that originally infiltrated the bronchiolar walls and terminal bronchioles (FIGS. 4A, B). The tumor appeared to have a relatively distinct boundary. The bronchiole was compressed by the tumor (Figure 4C). Necrosis at the center of the large mass was common (Figure 4C). The number of nodular xenografts was large, varied in size, and was scattered both in the central region and the periphery of the lung lobe. Histological examination also showed that SQ5 cells had metastasized and infiltrated the mediastinal lymph nodes (FIG. 4D).
Normal lung tissue by microscopic observation showed no difference regardless of whether EDTA was administered or not. Regardless of whether or not EDTA was administered, there was no difference in the distribution of tumors engrafted in lung tissue in mice transplanted with either A549 or SQ5.

Tumor cell line growth curves and doubling time re-cultured cells had higher tumorigenicity than the original cultured cells. Here, growth curves were prepared for the original cultured cells and re-cultured cells, and the proliferation of tumor cells in vitro was measured. The doubling time was calculated from exponentially growing cells. In both A549 and SQ5 cells, the growth curve of the original cultured cells was almost the same as that of the re-cultured cells (data not shown). The doubling times for A549 cells and re-cultured A549 cells were 19.2 ± 0.68 hours and 17.7 ± 0.33 hours (mean ± SE), respectively. The doubling times for SQ5 cells and re-cultured SQ5 cells were 20.8 ± 0.5 hours and 20.8 ± 0.13 hours (mean ± SE), respectively. In any cell line, the doubling time of the original cultured cells was not significantly different compared to the re-cultured cells.
Discussion The purpose of this study was to develop a reproducible orthotopic transplant model that simulates the clinical features of human lung cancer. This model was developed to facilitate orthotopic tumor induction and suitable for the evaluation of novel lung cancer biology and therapeutic agents.

  To date, several rodent orthotopic transplant models have been developed to study human lung cancer. Orthotopic models of human lung cancer were first developed by endobronchial techniques (McLemore TL, Liu MC, Blacker PC, Gregg M, Alley MC, Abbott BJ, Shoemaker RH, Bohlman ME, Litterst CC, Hubbard WC, Brennan RH, McMahon JB, Fine DL, Eggleston JC, Mayo JG, and Boyd MR. Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice. Cancer Res 1987; 47: 5132-40). Fractions of human lung cancer can also be transplanted orthotopically by surgery (Hoffman RM, Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic. Invest New Drugs 1999; 17: 343-59). The method resulted in high tumor growth at the local site and metastasis to the other lung, lymph nodes, and other clinically relevant sites (Hoffman RM, Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic.Invest New Drugs 1999; 17: 343-59; Wang X, Fu X, Kubota T, Hoffman RM.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: 1403-6). However, the primary limitation of these models is that the implantation procedure requires a high level of surgical technique. The mortality associated with surgery was approximately 5%. On the other hand, the technology required for our model is simple and reproducible. All procedures can be performed quickly (ie, surgical procedures can be completed within 1 minute per mouse) and are easily taught to others. Our method is also advantageous for large scale applications with little animal death associated with the procedure. In this mouse model, advance to animals that were necessary in the rat model (March TH, Marron-Terada PG, Belinsky SA. Refinement of an orthotopic lung cancer model in the nude rat. Vet Pathol 2001; 38: 483-90) Neither irradiation nor maintenance of animals with antibiotics was necessary.

  In our orthotopic transplant model, the SQ5 cell line had a higher tumor formation rate than the A549 cell line in the same mouse lung environment (Table 1). From this result, it was shown that this lung cancer model reflects the characteristics of the transplanted cancer cells. On the other hand, the microenvironment of the host tissue is also an important factor for tumor growth. Co-administration of EDTA increased the tumor formation rate in both cell lines (Table 1). These results show that March's study of delivering tumor cells from the trachea of nude rats (March TH, Marron-Terada PG, Belinsky SA. Refinement of an orthotopic lung cancer model in the nude rat. Vet Pathol 2001; 38: 483- 90). EDTA may disrupt calcium-dependent cadherin-mediated intercellular adhesion that is important for maintaining normal bronchial epithelial cell integrity (Smythe WR, Williams JP, Wheelock MJ, Johnson KR, Kaiser LR, Albelda SM) Cadherin and catenin expression in normal human bronchial epithelium and non-small cell lung cancer. Lung Cancer 1999; 24: 157-68; Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991; 251: 1451-5). EDTA can expose the parenchymal interstitial matrix, thus promoting binding and reducing the subsequent clearance of the transplanted cells. Data from our lung tumor model showed that tumor formation in a specific microenvironment of lung tissue depends on both tumor cell characteristics and host tissue environment.

  To develop an in vivo lung cancer model, intrathoracic and intrapulmonary injection of tumor cells was performed, with local tumor growth ranging from 5% to 100% and intrathoracic metastasis ranging from 0% to 100%. (Boehle AS, Dohrmann P, Leuschner I, Kalthoff H, Henne-Bruns D. An improved orthotopic xenotransplant procedure for human lung cancer in SCID bg mice. Ann Thorac Surg 2000; 69: 1010-5; Ishikura H, Kondo K, Miyoshi T, Kinoshita H, Hirose T, Monden Y. Artificial lymphogenous metastatic model using orthotopic implantation of human lung cancer.Ann Thorac Surg 2000; 69: 1691-5; Onn A, Isobe T, Itasaka S, Wu W, O'Reilly MS , Ki Hong W, Fidler IJ, Herbst RS. Development of an orthotopic model to study the biology and therapy of primary human lung cancer in nude mice. Clin Cancer Res 2003; 9: 5532-9). In the intrapulmonary model, tumor growth in the lung was found 7 days after transplantation by microscopic observation. In contrast, micrometastasis to the mediastinum was seen on the 5th day. Tumor growth in the anthropogenic environment was thought to be caused by high-pressure injection of tumor cells (Ishikura H, Kondo K, Miyoshi T, Kinoshita H, Hirose T, Monden Y. Artificial lymphogenous metastatic model using orthotopic implantation of human lung cancer Ann Thorac Surg 2000; 69: 1691-5). Matrigel is a basement membrane matrix preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma. Matrigel is commonly used to reduce tumor mortality by transplanting tumor cells into the pleural cavity and fixing the tumor cells at the injection site (Boehle AS, Dohrmann P, Leuschner I, Kalthoff H, Henne). -Bruns D. An improved orthotopic xenotransplant procedure for human lung cancer in SCID bg mice.Ann Thorac Surg 2000; 69: 1010-5; Ishikura H, Kondo K, Miyoshi T, Kinoshita H, Hirose T, Monden Y. Artificial lymphogenous metastatic model using orthotopic implantation of human lung cancer.Ann Thorac Surg 2000; 69: 1691-5; Onn A, Isobe T, Itasaka S, Wu W, O'Reilly MS, Ki Hong W, Fidler IJ, Herbst RS.Development of an orthotopic model to study the biology and therapy of primary human lung cancer in nude mice.Clin Cancer Res 2003; 9: 5532-9; Jin H, Yang R, Fong S, Totpal K, Lawrence D, Zheng Z, Ross J, Koeppen H, Schwall R, Ashkenazi A. Apo2 ligand / tumor necrosis factor-related apoptosis-inducing ligand cooperates with chemotherapy to inhibit orthotopic lung tum Cancer Res 2004; 64: 4900-5; Fukunaga M, Takamori S, Hayashi A, Shirouzu K, Kosai K. Adenoviral herpes simplex virus thymidine kinase gene therapy in an orthotopic lung cancer model.Ann Thorac Surg 2002 ; 73: 1740-6). However, studies have shown that co-injection with Matrigel increases the local tumor growth rate but also increases the drug resistance of lung cancer cell lines (Ishikura H, Kondo K, Miyoshi T, Kinoshita H, Hirose T, Monden Y. Artificial lymphogenous metastatic model using orthotopic implantation of human lung cancer.Ann Thorac Surg 2000; 69: 1691-5; Fridman R, Giaccone G, Kanemoto T, Martin GR, Gazdar AF, Mulshine JL. Reconstituted Proc Natl Acad Sci USA 1990; 87: 6698-702). basement membrane (matrigel) and laminin can enhance the tumorigenicity and the drug resistance of small cell lung cancer cell lines. EDTA is commonly used for tissue culture and is not expensive. Co-administration of EDTA resulted in a tumor formation rate of 80% to 100%, but did not damage tumor-bearing normal tissue and did not affect tumor distribution histologically (Tables 1 and 2 and FIGS. 3 and 4). These results indicated that co-administration of EDTA was effective in increasing tumor formation in vivo. Using it made this in vivo model suitable for research applications.

  Multifocal xenografts were formed in this orthotopic lung tumor model. Tumor nodules were more frequent in the right lung than in the left lung, and were in the order of upper lobe, lower lobe, and middle lobe from the most frequent. This is similar to the position of lung cancer in humans (MacFarlane JCW, Doughty BJ, Crosbie WA. Carcinoma of the lung: An analysis of 362 cases diagnosed and treated in one year. Br J Dis Chest 1962; 56: 57- 63; Lisa JR, Trinidad S, Rosenblatt MB.Site of origin, histogenesis, and cytostructure of bronchogenic carcinoma.Am J Clin Pathol 1965; 44: 375-84; Byers TE, Vena JE, Rzepka TF.Predilection of lung cancer for the upper lobes: an epidemiologic inquiry. J Natl Cancer Inst 1984; 72: 1271-5). Multifocal xenografts can also mimic human symptoms. Reports show that 25% to 40% of patients undergoing resection for stage I lung cancer develop a second primary tumor (Boice JD Jr, Fraumeni JF Jr. Second cancer following cancer of the respiratory system in Connecticut, 1935-1982. Natl Cancer Inst Monogr 1985; 68: 83-98; Harpole DH Jr, Herndon JE 2nd, Wolfe WG, Iglehart JD, Marks JR.A prognostic model of recurrence and death in stage I non-small cell lung cancer utilizing presentation, histopathology, and oncoprotein expression. Cancer Res 1995; 55: 51-6). Histological expression showed that tumor nodules of adenocarcinoma A549 were distributed mainly in the alveoli, indicating an incomplete glandular structure (FIG. 3). Tumor nodules of squamous cell carcinoma SQ5 mainly invaded bronchioles and terminal bronchioles (FIG. 4). These results indicated that in this orthotopic model, tumor cells proliferate at appropriate sites that reflect their original characteristics. In the lung tumor model transplanted via the right main bronchus, 1% contralateral metastasis was seen (McLemore TL, Liu MC, Blacker PC, Gregg M, Alley MC, Abbott BJ, Shoemaker RH, Bohlman ME, Litterst) CC, Hubbard WC, Brennan RH, McMahon JB, Fine DL, Eggleston JC, Mayo JG, and Boyd MR. Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice. Cancer Res 1987; 47: 5132-40). In our lung cancer model, numerous tumor nodules were formed in both the right and left lungs.

  Clinically, lymph node metastasis is known to be an important metastatic pathway of human lung cancer. The degree of lymph node metastasis is critical for staging, treatment designation, and assessment of patient prognosis (Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest 1997; 111: 1710-7). Metastasis of SQ5 cells to mediastinal lymph nodes was seen in one mouse (FIG. 4D). In this study, tissue slides were created primarily for lung tumor nodules and not for the mediastinum. This may be the cause of the low detection rate of mediastinal lymph node metastasis.

  The re-cultured A549 cells or SQ5 cells showed a higher tumor formation rate than the original cultured cells (Table 1). However, the growth curves and doubling times of both re-cultured cell populations did not show significant differences from their original cultured cells. Previous studies have reported that in order to grow human cells in animals, the high invasiveness and / or adherence of tumor cells plays an important role in tumor growth in the tissue microenvironment ( Kikkawa H, Miyamoto D, Imafuku H, Koike C, Suzuki Y, Okada S, Tsukada H, Irimura T, Oku N. Role of sialylglycoconjugate (s) in the initial phase of metastasis of liver-metastatic RAW117 lymphoma cells. Jpn J Cancer Res 1998; 89: 1296-305; Greene GF, Kitadai Y, Pettaway CA, von Eschenbach AC, Bucana CD, Fidler IJ. Correlation of metastasis-related gene expression with metastatic potential in human prostate carcinoma cells implanted in nude mice using an in in situ messenger RNA hybridization technique. Am J Pathol 1997; 150: 1571-82). Lau et al. Have shown that human lung tumor xenografts over several generations of mouse recipients by serial transplantation result in loss of human genes and acquisition of mouse DNA in the transplanted tumor (Lau DH, Lu D, Hammond WG, Schmid CW, Benfield JR. Loss of nm23 and Alu DNA in human lung cancer propagated in nude mice. Cancer Lett 1995; 97: 163-8). This phenomenon was observed after the 4th generation, but no DNA modification was observed in the transplanted cells in the early generations. The increase in tumor formation by recultured cells grown once in subcutaneously injected nude mice is thought to be due to the selection of tumor cells with a more invasive phenotype for the host tissue.

  In summary, the present invention provides a reproducible and simple orthotopic in vivo animal model for human lung cancer research. Numerous nodules grew in the tumor-specific environment of lung tissue simulating the clinical and pathological features of human lung cancer. The present invention provided a suitable in vivo model for studying human lung cancer biology and assessing the efficacy of promising therapeutic agents.

  The present invention provides a non-human animal in which lung cancer cells are engrafted. By using this animal, it becomes possible to develop cancer therapeutic agents, verify therapeutic effects, verify side effects of cancer treatment of normal tumor-bearing lung tissue, develop reagents for detecting and imaging lung cancer, and the like.

Intratracheal delivery of tumor cells to BALB / c nude mice. Using a 27-3 / 4 gauge needle, a 50 μl volume of cells was injected directly into the main bronchus. Nodule formation of squamous cell SQ5 tumor in lungs of BALB / c nude mice. SQ5 tumor growth was seen 60 days after co-administration to the main trachea of nude mice with 0.01M EDTA (a). Formalin-fixed lung tissue sample obtained from the same mouse as a. Tumors were located in the upper and middle lobes of the right lung (b). Lung histological examination with hematoxylin and eosin staining 63 days after intratracheal delivery of 1 × 10 ⁷ A549 cells with 0.01 M EDTA. Tumor cells infiltrated the alveolar wall (a). Tumor cells spread to the alveoli and tumor nodules were formed mainly at the periphery of lung tissue (b). The tumor mass showed an incomplete glandular structure with mixed necrotic cells (c). Lung histological examination with hematoxylin and eosin staining 60 days after intratracheal delivery of 1 × 10 ⁷ SQ5 cells with 0.01 M EDTA. Tumor cells infiltrated the bronchiole wall (a). Tumor cells entered the wall of the terminal bronchiole (b). A large tumor mass compressed the alveoli and bronchioles, with a necrotic mass in the center (c). Mediastinal lymph node metastasis (d). BV: blood vessel; BT: bronchi; N: necrosis.

Claims (8)

A method for producing a non-human animal in which lung cancer cells are engrafted, comprising:
Preparing a non-human animal with reduced immunity,
Exposing the non-human animal trachea and injecting lung cancer cells into the trachea. The method according to claim 1, wherein the lung cancer cells are mixed with ethylenediaminetetraacetic acid and injected into the trachea. The method according to claim 1 or 2, wherein the non-human animal with reduced immunity is a nude mouse. The method according to any one of claims 1 to 3, wherein the lung cancer cells are small cell lung cancer cells or non-small cell lung cancer cells. The method according to any one of claims 1 to 4, wherein the lung cancer cells are derived from a human. The method according to any one of claims 1 to 5, wherein 10 ⁷ to 10 ⁸ lung cancer cells per non-human animal are injected into the trachea of the non-human animal. The method according to any one of claims 1 to 6, wherein a non-human animal in which lung cancer cells are engrafted in at least one organ selected from the group consisting of lung, trachea and bronchial tree. A non-human animal produced by the method according to claim 1 and engrafted with lung cancer cells.