Severe combined immunodeficiency

Study objectives: To establish a clinically relevant animal model of pulmonary metastases of human non-small cell lung carcinoma (NSCLC) cells in severe combined immunodeficiency (SCID) mice, which can be used for repetitive investigations, so as to improve our understanding and management of the cellular and molecular mechanisms of human lung cancer metastases. Methods and results: SCID mice subcutaneously injected in the flank with 1 x [10.sup.6] EBC-1 cells derived from human lung squamous cell carcinoma were killed weekly for examination until 12 weeks after tumor inoculation. The biological characteristics of implanted tumors and their metastatic foci were investigated by hematoxylin-eosin staining and immunostaining for neutrophil elastase (NE). Three weeks after ectopic implantation, EBC-1 cell lines formed a tumor at the inoculation site and grew steadily to show a plateau at 10 weeks. EBC-1 cells formed multiple metastases in the lung 7 weeks after tumor inoculation; their numbers increased steadily until 12 weeks in all mice. Immunoreactivity for NE was intense in the metastatic tumor cells. Then, to establish the primary tumor amputation/pulmonary metastasis model and to evaluate how primary tumor amputation influences the development of pulmonary metastases at the cellular and molecular level, excision was performed before (3 weeks and 5 weeks after inoculation) and after (7 weeks and 9 weeks after inoculation) formation of lung metastases. When the primary tumor was excised 3 weeks after tumor inoculation, all mice had pulmonary metastasis at 12 weeks after inoculation. Blood samples obtained at 3 weeks after tumor inoculation contained human [beta]-actin messenger RNA, which represents circulating tumor cells.

Conclusion: Our NSCLC EBC-1 pulmonary metastasis model is reliable, technically simple, and predictably results in pulmonary metastasis from early hematogenous spread. This model may be useful for elucidating the mechanism of pulmonary metastasis in human lung cancer, and testing anti-metastatic efficacy of therapeutic agents in vivo.

Key words: EBC-1 cell; neutrophil elastase-like molecule in cancer; non-small cell lung cancer; pulmonary metastasis; severe combined immunodeficiency mice

Abbreviations: actin-H = human [beta]-actin; ECM = extracellular matrix; IL = interleukin; mRNA = messenger RNA; NE = neutrophil elastase; NELMIC = neutrophil elastase-like molecule in cancer; NSCLC = non-small cell lung cancer; PCR = polymerase chain reaction; RT-PCR = reverse transcriptase-polymerase chain reaction; SCID = severe combined immunodeficiency

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Neoplasms metastasize as a result of a complex series of events. (1) One initial step is degradation of basement membranes and extracellular matrix (ECM) followed by local tumor cell invasion into surrounding tissue. This process requires various degradative enzymes including proteases. (2-4) Neutrophil elastase (NE) is a neutral serine protease produced by polymorphonuelear leukocytes, monocytes, and macrophages. (5,6) This enzyme has broad substrate specificity under physiologic conditions, and excessive NE results in digestion of not only elastin, but also other ECM proteins such as laminin, fibronectin, proteoglycans, and type IV collagens. (7-10) We recently demonstrated that several cell lines from human non-small cell lung cancer (NSCLC), including EBC-1 cells, produce immunoreactive NE. (11) The amount of immunoreactive NE in tumor tissue is an independent prognostic indicator of patients with NSCLC. (11,12) Furthermore, a specific NE inhibitor, ONO-5046*Na, completely suppressed growth of EBC-1 cells transplanted into severe combined immunodeficiency (SCID) mice. (13) These findings indicated that this enzyme, designated as NE-like molecule in cancer (NELMIC), may play an active role in progression of NSCLC.

During an investigation into in vivo effects of ONO-5046-Na on growth of EBC-1 cells transplanted subcutaneously into SCID mice, we found that this cell line formed multiple metastatic foci in the lung. If an animal model of metastasis with features similar to those of lung cancer metastasis in humans is available, it will be useful to assess the efficacy of therapeutic interventions in lung cancer. In the present article, we report that this pulmonary metastatic animal model of human NSCLC is highly stable, requires little technical expertise, and generates pulmonary metastasis in a predictable fashion as a result of early hematogenous spread.

MATERIALS AND METHODS

Mice

Five-week-old female C.B-17 SCID/SCID mice were obtained from Kyudo (Kumamoto, Japan). The mice were given breeding diet CRF-1 (Oriental Kobo Kogyo; Tokyo, Japan) and water ad libitum. Mice were used at the age of 6 weeks and were maintained in a pathogen-free environment throughout the experiment.

EBC-1 Cell Line

EBC-1, a human lung squamous cell carcinoma line, was obtained from the Japanese Research Resources Bank (Tokyo, Japan). This cell line was maintained in RPMI-1640 (Life Technologies; Gaithersburg, MD) supplemented with 10% fetal calf serum at 37[degrees]C under a humidified atmosphere containing 5% C[O.sub.2]/95% air. EBC-1 cells secreted NELMIC as detected by a highly specific and sensitive enzyme immunoassay (Mochida Pharmaceutical; Tokyo, Japan). (11) Viability of cultured cells exceeded 90% as determined by the trypan blue dye-exclusion method.

Tumor Implantation

Cultured EBC-1 cells were dissociated from stock plates with trypsin, washed, and resuspended in 0.1 mL of phosphate-buffered saline solution. SCID mice were injected subcutaneously in the flank with 106 EBC-1 cells. Tumor sizes were determined once a week; tumor volume was calculated as volume = (length)/(width) (2).

Histologic Examination

Animals were killed weekly until 12 weeks after tumor inoculation and examined for the presence of metastasis. At autopsy, lung, liver, kidney, brain, and other organs were removed, weighed and fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections then were cut and stained with hematoxylin-eosin. Numbers of metastatic foci were counted under a microscope in sections prepared from tissue blocks obtained from organs at 1-mm intervals.

Immunohistochemistry for NELMIC

Paraffin-embedded, 4-[micro]m-thick sections of lung were deparaffinized and subjected to immunohistochemical staining with an antihuman NE mouse monoclonal antibody (DAKO; Glostrup, Denmark). Sections were placed in 0.3% [H.sub.2][O.sub.2] in methanol for 30 min at room temperature to block endogenous peroxidase activity. Primary antibody incubations were carried out at a dilution of 1:100 in a humid chamber overnight at 4[degrees]C. Bound antibody was detected with a biotinylated secondary antibody and streptavidin-peroxidase complex, applied for 1 h at room temperature. Sections were washed in 0.01 mol/L phosphate-buffered saline solution (pH 7.2) between incubation steps. Peroxidase activity was visualized with 0.01% diaminobenzidine tetrahydrochloride (Sigma; St Louis, MO) in 0.05 mol/L Tris-HC1 buffer (pH 7.6). Negative controls were adjacent sections processed without the primary antibody.

Surgical Treatment

We tried to establish the primary tumor amputation/pulmonary metastasis model and to evaluate how primary tumor amputation influences the development of pulmonary metastases at the cellular and molecular level. When EBC-1 primary tumors on the flank grew palpable, surgery was performed according to the following group descriptions (Fig 1): control group, no operation; group 1, excision of subcutaneous tumor with a 5-mm surgical margin at 3 weeks after inoculation; group 2, excision at 5 weeks; group 3, excision at 7 weeks; and group 4, excision at 9 weeks. Each group included 12 mice.

Detection of Human [beta]-Actin Messenger RNA in Blood

To test whether the evidence of circulating tumor cells could be detected in the blood of tumor-bearing SCID mice, we assayed blood samples for human [beta]-actin (actin-H) messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction (RT-PCR). The integrity of RNA and complementary DNA was confirmed by the generation of a [beta]-actin polymerase chain reaction (PCR) product with primers that hybridize with both human and murine [beta]-actin. (14,15) Samples were obtained 3 days after tumor inoculation and again at 1 week, 2 weeks, and 3 weeks after inoculation. Total RNA was extracted from EBC-1 cells and blood samples using Qiamp RNA Blood Mini Kits (Qiagen; Valencia, CA), and complementary DNA was synthesized using SuperScript Preamplification system for first-strand complementary DNA synthesis (Life Technologies) according to the protocols provided by these manufacturers. The PCR was performed in a 25-[micro]L reaction mixture containing 1 [micro]L complementary DNA template, containing PCR buffer, 25 mM deoxynucleoside triphosphates, 5 [micro]mol/L of each primer, and 0.25 U of Taq DNA Polymerase (Life Technologies). PCR condition for actin-H was set up as follows: i cycle of denaturing at 94[degrees]C for 4 min, followed by 35 cycles of 95[degrees]C for 30 s, 53[degrees]C for 30 s, and 72[degrees]C 30 s before a final extension at 72[degrees]C for 3 min.

Statistics

Tumor volume was expressed as the mean [+ or -] SD. The nonparametric Wilcoxon signed rank test was used to compare differences in number of pulmonary metastases and lung weight between groups.

RESULTS

Tumorigenicity of EBC-1 Cells in SCID Mice

Three weeks after subcutaneous inoculation of EBC-1 cells into the flank of SCID mice, palpable solid tumors started to grow in all 12 mice. Tumors grew steadily until 10 weeks, followed by a plateau until 12 weeks (Table 1).

Spontaneous Pulmonary Metastasis of EBC-1 Cells in SCID Mice

EBC-1 cells formed multiple metastatic foci in the lung 7 weeks after tumor inoculation in all mice studied (Table 1). In this model, no metastasis was detected in other organs such as liver, kidney, or brain. As shown in Table 1, the number of microscopic lung metastases increased steadily throughout the 12-week observation period (24 [+ or -] 10 foci per lung at 7 weeks vs 144 [+ or -] 35 foci per lung at 12 weeks, p < 0.001). Because tumor growth in the lungs was considerable, lung weight also significantly increased throughout the period studied (315 [+ or -] 99 mg at 7 weeks vs 1044 [+ or -] 353 mg at 12 weeks, p < 0.001).

Effects of Surgical Treatment on Pulmonary Metastasis of EBC-1 Cells in SCID Mice When the primary tumor in the flank of SCID mice was excised 3 weeks after tumor inoculation (group 1), all 12 mice had pulmonary metastasis at 12 weeks after inoculation, as also was true for mice without excision (Table 2). Similarly, when the operation was performed 5 weeks (group 2), 7 weeks (group 3), and 9 weeks (group 4) after the tumor inoculation, pulmonary metastasis was present at 12 weeks. No differences in numbers of pulmonary metastases were evident between these groups. However, lungs were significantly heavier in groups 3 and 4 than in control mice (p < 0.001).

Immunohistochemical Staining

As shown in Figure 2, intense NE staining was uniformly observed in metastatic EBC-1 tumor cells in lungs examined 12 weeks after EBC-1 inoculation. Immunoreactivity was cytoplasmic, with a granular appearance. Control sections with no primary antibody showed no staining.

[FIGURE 2 OMITTED]

actin-H mRNA Expression in Blood

As shown in Table 3 and Figure 3, RT-PCR amplification detected no actin-H mRNA in blood obtained at 3 days, 1 week, or 2 weeks after EBC-1 cell inoculation. However, blood samples obtained 3 weeks after tumor inoculation contained actin-H mRNA in all mice studied.

[FIGURE 3 OMITTED]

DISCUSSION

Malignancy is defined most often as neoplastic growth that tends to metastasize. An animal model of metastasis therefore has uses beyond research directly concerning metastasis, and such a model can contribute importantly to evaluation of potential new treatment strategies.

In the present study we demonstrated that the human NSCLC line, EBC-1, could be used as a transplantable pulmonary metastasis model of NSCLC in SCID mice. The model is predicable, involves few technical demands, and reliably produces pulmonary metastasis in a reproducible fashion; without exception, multiple metastatic foci developed 7 weeks after tumor inoculation. Metastasis also developed when the inoculated primary tumor was excised at 3 weeks (group 1) or at 5 weeks (group 2) after the inoculation; at this time, pulmonary metastasis had not yet appeared microscopically. RT-PCR amplification detected actin-H mRNA in peripheral blood samples obtained 3 weeks after the tumor inoculation, indicating that early hematogenous spread occurred in this model and circulating tumor cells might play a central role in the metastatic process.

Lung cancer has been the leading cause of cancer death in Japan since 1998. Despite considerable progress in surgical oncology, nearly 90% of patients die of this disease within 2 years of diagnosis; even after curative surgical resection in stage I NSCLC, the 5-year survival rate in Japan is only 65%. (16) Radiotherapy and cytotoxic chemotherapy, either alone or in combination, have shown only a minor influence on survival. (17) None of the treatment regimens have been shown to significantly increase patient survival beyond that with resection alone, and a great need exists for an effective therapy for this lethal disease.

NE can degrade the structural proteins of the ECM. Our previous study showed that many lung carcinoma cell lines including EBC-1 cells produce immunoreactive NE, which is designated as NELMIC when detected as a tumor cell product. (11) Furthermore, the concentration of NELMIC in human NSCLC tumor extracts is closely related to both direct extension and metastasis of NSCLC, and is a strong and independent predictor for short survival of patients with NSCLC. (11,12) Recently, a specific NE inhibitor, ONO-5046*Na, has been developed and is being preclinically tested in connection with treatment of various inflammatory diseases. However, investigation of whether this NE inhibitor can inhibit growth and metastasis of human lung carcinoma cells is a matter of great interest. In fact, in our preliminary study, ONO-5046*Na administered to SCID mice completely inhibited tumor growth of EBC-1 cells after inoculation. (13) An unexpected finding in those experiments was that ONO-5046*Na treatment delayed the growth of inoculated PC-3 cells as well; this line is unable to produce NELMIC. (13) Thus, mechanisms of ONO-5046*Na effects remain to be clarified, but use of NE inhibitor appears to show promise for preventing invasion and metastasis of lung cancer. We currently are determining optimal timing and dose of ONO-5046*Na in this EBC-1 pulmonary metastatic model.

The present study also demonstrated that resection of the primary tumor formed by EBC-1 cells at 7 weeks or 9 weeks after tumor inoculation--when pulmonary metastases already had developed--significantly increased lung weight reflecting increased pulmonary metastatic tumor burden at 12 weeks. Surgeons are concerned that surgical stress might enhance the growth of residual cancer cells, resulting in an increase in distant metastases and a poorer prognosis. Various lines of evidence suggest that surgical stress can enhance metastasis. One factor involved is interleukin (IL)-6, a well-known cytokine that enhances cancer cell motility. (18) IL-6 is produced in the operative field during surgery and enters the peripheral blood, producing a detectable elevation in serum concentration. (19) Thus, IL-6 might effect growth, invasiveness, and metastasis of residual cancer cells after a noncurative resection. However, surgical stress is partly responsible for enhancing tumor growth through a variety of mechanisms that are still not fully elucidated. We consider that further examination using this primary tumor amputation/ pulmonary metastasis model will be useful for analyzing the complex mechanisms that surgical stress enhances tumor growth.

In conclusion, we reported a reliable transplantable pulmonary metastasis model of NSCLC that produces NELMIC in SCID mice. This model may be useful in development and screening of new drugs, including specific NE inhibitors, for potential effectiveness against pulmonary metastasis of NSCLC.

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* From the Department of Surgrye II (Drs. Tanaka, Hayashi, and Ogawa) Kumamoto University School of Medicine Kumamoto Japan; and the Department of Surgery (Drs. Kato, Kondo, and Yamashita), Aichi Medical School, Aichi, Japan.

This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan.

Manuscript received February 1, 2002; revision accepted July 8, 2002.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).

Correspondence to: Michio Ogawa, MD, Department of Surgery II, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 860-8556, Japan; e-mail: mogawa@kaiju.medic. kumamoto-u.ac.jp

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