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A pulmonary metastatic model of human non-small cell lung carcinoma cells in SCID mice
International Congress Series 1255 (2003) 239 – 242
A pulmonary metastatic model of human non-small cell lung carcinoma cells in SCID mice Eiji Tanaka a, Jun-ichi Yamashita b, Naoko Hayashi a, Michio Ogawa a,* a
Department of Surgery II, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 860-8556, Japan b Department of Surgery, Aichi Medical School, Aichi, Japan Received 27 February 2003; accepted 28 March 2003
Abstract We found that EBC-1, a human non-small cell lung cancer (NSCLC) cell line, formed multiple metastases in the lung after subcutaneous inoculation into severe combined immunodeficiency (SCID) mice. SCID mice were injected in the flank with 106 EBC-1 cells. Metastases in the lung were counted in histologic sections and stained immunohistochemically for neutrophil elastase (NE). Solid tumors were palpable in the flank at 3 weeks and grew steadily until 10 weeks. EBC-1 cells formed multiple metastases in the lung at 7 weeks; their numbers increased steadily until 12 weeks in all mice. Immunoreactivity for NE was intense in the metastatic tumor cells. As a marker for circulating tumor cells, human h-actin mRNA was detected in blood by reverse transcriptase – polymerase chain reaction (RT-PCR). Blood samples obtained at 3 weeks after tumor inoculation contained human h-actin mRNA. 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 screening potential new drugs, including specific NE inhibitors, for effectiveness against pulmonary metastasis of NSCLC. D 2003 Elsevier B.V. All rights reserved. Keywords: Non-small cell lung cancer; EBC-1 cell; Pulmonary metastasis; SCID mice; Neutrophil elastase-like molecule in cancer
1. Background Neoplasms metastasize as a result of a complex series of events [1]. One initial step is the degradation of basement membranes and the extracellular matrix (ECM), followed by * Corresponding author. Tel.: +81-96-373-5211; fax: +81-96-371-4378. E-mail address: [email protected] (M. Ogawa). 0531-5131/03 D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)00648-4
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E. Tanaka et al. / International Congress Series 1255 (2003) 239–242
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 polymorphonuclear 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 extracellular matrix 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 in patients with NSCLC [11,12]. Furthermore, a specific NE inhibitor, ONO-5046Na, completely suppressed growth of EBC-1 cells transplanted into severe combined immunodeficiency (SCID) mice [13]. These findings indicated that the enzyme designated as neutrophil elastase-like molecule in cancer (NELMIC), may play an active role in progression of NSCLC. During an investigation into in vivo effects of ONO-5046Na on growth of EBC1 cells transplanted subcutaneously into SCID mice, we found that this cell line formed multiple metastatic foci in the lung. In this paper, 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.
2. 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).
3. 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 the liver, kidney or brain. As shown in Table 1, the number of microscopic lung metastases increased steadily throughout the 12-week observation period (24 F 10 foci/ lung at 7 weeks vs. 144 F 35 foci/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 F 99 mg at 7 weeks vs. 1044 F 353 mg at 12 weeks, p < 0.001).
4. Immunohistochemical staining Paraffin-embedded sections of lung were deparaffinized and subjected to immunohistochemical staining with an antihuman neutrophil elastase mouse monoclonal antibody. Intense neutrophil elastase staining was uniformly observed in metastatic EBC-1 tumor cells in lungs examined 12 weeks after EBC-1 inoculation. Immunoreactivity was
E. Tanaka et al. / International Congress Series 1255 (2003) 239–242
241
Table 1 Tumorigenicity and lung metastasis Weeks after primary pulmonary tumor inoculation tumorigenicitya
Lung weight metastasisb (mg)
1 2 3 4 5 6 7 8 9 10 11 12
201 F 39 190 F 62 194 F 56 211 F 47 199 F 49 206 F 31 315 F 99 521 F 212d 602 F 231d 813 F 345e 897 F 302e 1044 F 353e
0/12 0/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12
(4.2 F 1.9) (10.4 F 3.1) (12.7 F 3.1) (16.6 F 2.8) (27.3 F 4.5) (35.9 F 3.7) (47.5 F 7.7) (59.0 F 9.3) (57.4 F 11.2) (58.9 F 9.4)
0/12 0/12 0/12 0/12 0/12 0/12 12/12 12/12 12/12 12/12 12/12 12/12
(24 F 10) (65 F 23)c (98 F 31)c (123 F 26)c (144 F 42)c (144 F 35)c
a Data represent the number of mice with a palpable mass in the flank/number of mice evaluated. Calculated tumor volumes (in cm3, mean F S.D.) are given in parentheses. b Data represent the number of mice with spontaneous lung metastasis/number of mice evaluated. Numbers of microscopic lung metastatic foci (mean F S.D.) then are given in parentheses. c p < 0.001 vs. 7 weeks. d p < 0.02 vs. 7 weeks. e p < 0.001 vs. 7 weeks.
cytoplasmic, with a granular appearance. Control sections with no primary antibodies showed no staining.
5. Human h -actin mRNA expression in blood To test whether evidence of circulating tumor cells could be detected in the blood of tumor-bearing SCID mice, we assayed blood samples for human h-actin (actin-H) mRNA
Fig. 1. Human h-actin mRNA expression by RT-PCR in blood samples sequentially obtained after EBC-1 inoculation. Colon 26 clone 20; a cell line from murine colon cancer. Positive control; EBC-1 cells. Negative control; mice without inoculation. Actin-H, human h-actin. Actin-MH, both human and murine h-actin. The results were similar in blood samples from all mice studied.
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by reverse transcriptase –polymerase chain reaction (RT-PCR). The integrity of RNA and cDNA was confirmed by the generation of a h-actin PCR product with primers which hybridize with both human and murine h-actin (actin-MH) [14,15]. As shown in Fig. 1, RT-PCR amplification detected no human h-actin mRNA in blood taken at 3 days, 1 week or 2 weeks after EBC-1 cell inoculation. However, blood samples obtained 3 weeks after tumor inoculation contained human h-actin mRNA in all mice studied.
6. Conclusions In conclusion, we report 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.
References [1] L.A. Liotta, W.G. Stetler-Stevenson, Tumor invasion and metastasis: an imbalance of positive and negative regulation, Cancer Res. 51 (1991) 5054s – 5059s. [2] L.M. Matrisian, The matrix-degrading metalloproteinases, Bioessays 14 (1992) 455 – 463. [3] P. Mignatti, E. Robbins, D.B. Rifkin, Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade, Cell 47 (1986) 487 – 498. [4] M. Nakajima, A.M. Chop, Tumor invasion and extracellular matrix degradative enzymes: regulation of activity by organ factors, Semin. Cancer Biol. 2 (1991) 115 – 127. [5] Z. Werb, S. Gordon, Elastase secretion by stimulated macrophages: characterization and regulation, J. Exp. Med. 142 (1975) 361 – 377. [6] R.J. Baugh, J. Travis, Human leukocyte granule elastase: rapid isolation and characterization, Biochemistry 15 (1976) 836 – 841. [7] A. Janoff, Elastase in tissue injury, Annu. Rev. Med. 36 (1985) 207 – 216. [8] A. Janoff, J. Schere, Mediators of inflammation in leukocyte lysosomes: IX. Elastolytic activity in granules of human polymorphonuclear leukocytes, J. Exp. Med. 128 (1968) 1137 – 1156. [9] C. Mainardi, S. Dixit, A. Kang, Degradation of type IV (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocyte granules, J. Biol. Chem. 255 (1980) 5435 – 5441. [10] J.A. McDonald, D.G. Kelley, Degradation of fibronectin by human leukocyte elastase: release of biologically active fragments, J. Biol. Chem. 255 (1980) 8848 – 8858. [11] J. Yamashita, K. Tashiro, S. Yoneda, K. Kawahara, T. Shirakusa, Local increase in polymorphonuclear leukocyte elastase is associated with tumor invasiveness in non-small cell lung cancer, Chest 109 (1996) 1328 – 1334. [12] J. Yamashita, M. Ogawa, M. Abe, N. Hayashi, Y. Kurusu, K. Kawahara, T. Shirakusa, Tumor neutrophil elastase is closely associated with the direct extension of non-small cell lung cancer into the aorta, Chest 111 (1997) 885 – 890. [13] M. Inada, J. Yamashita, M. Ogawa, Neutrophil elastase inhibitor (ONO-5046Na) inhibits the growth of human lung cancer cell lines transplanted into severe combined immunodeficiency (SCID) mice, Res. Commun. Mol. Pathol. Pharmacol. 97 (1997) 229 – 232. [14] N. Yoshida, E. Ishii, M. Nomizu, Y. Yamada, S. Mohri, N. Kinukawa, A. Matsuzaki, K. Oshima, T. Hara, S. Miyazaki, The laminin-derived peptide YIGSR (Tyr – Ile – Gly – Ser – Arg) inhibits human pre-B leukaemic cell growth and dissemination to organs in SCID mice, Br. J. Cancer 80 (12) (1999) 1898 – 1904. [15] S. Nakajima-Iijima, H. Hamada, P. Reddy, T. Kakunaga, Molecular structure of human cytoplasmic h-actin gene: interspecies homology of sequences in the introns, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 6133 – 6137.
A pulmonary metastatic model of human non-small cell lung carcinoma cells in SCID mice Eiji Tanaka a, Jun-ichi Yamashita b, Naoko Hayashi a, Michio Ogawa a,* a
Department of Surgery II, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 860-8556, Japan b Department of Surgery, Aichi Medical School, Aichi, Japan Received 27 February 2003; accepted 28 March 2003
Abstract We found that EBC-1, a human non-small cell lung cancer (NSCLC) cell line, formed multiple metastases in the lung after subcutaneous inoculation into severe combined immunodeficiency (SCID) mice. SCID mice were injected in the flank with 106 EBC-1 cells. Metastases in the lung were counted in histologic sections and stained immunohistochemically for neutrophil elastase (NE). Solid tumors were palpable in the flank at 3 weeks and grew steadily until 10 weeks. EBC-1 cells formed multiple metastases in the lung at 7 weeks; their numbers increased steadily until 12 weeks in all mice. Immunoreactivity for NE was intense in the metastatic tumor cells. As a marker for circulating tumor cells, human h-actin mRNA was detected in blood by reverse transcriptase – polymerase chain reaction (RT-PCR). Blood samples obtained at 3 weeks after tumor inoculation contained human h-actin mRNA. 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 screening potential new drugs, including specific NE inhibitors, for effectiveness against pulmonary metastasis of NSCLC. D 2003 Elsevier B.V. All rights reserved. Keywords: Non-small cell lung cancer; EBC-1 cell; Pulmonary metastasis; SCID mice; Neutrophil elastase-like molecule in cancer
1. Background Neoplasms metastasize as a result of a complex series of events [1]. One initial step is the degradation of basement membranes and the extracellular matrix (ECM), followed by * Corresponding author. Tel.: +81-96-373-5211; fax: +81-96-371-4378. E-mail address: [email protected] (M. Ogawa). 0531-5131/03 D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)00648-4
240
E. Tanaka et al. / International Congress Series 1255 (2003) 239–242
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 polymorphonuclear 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 extracellular matrix 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 in patients with NSCLC [11,12]. Furthermore, a specific NE inhibitor, ONO-5046Na, completely suppressed growth of EBC-1 cells transplanted into severe combined immunodeficiency (SCID) mice [13]. These findings indicated that the enzyme designated as neutrophil elastase-like molecule in cancer (NELMIC), may play an active role in progression of NSCLC. During an investigation into in vivo effects of ONO-5046Na on growth of EBC1 cells transplanted subcutaneously into SCID mice, we found that this cell line formed multiple metastatic foci in the lung. In this paper, 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.
2. 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).
3. 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 the liver, kidney or brain. As shown in Table 1, the number of microscopic lung metastases increased steadily throughout the 12-week observation period (24 F 10 foci/ lung at 7 weeks vs. 144 F 35 foci/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 F 99 mg at 7 weeks vs. 1044 F 353 mg at 12 weeks, p < 0.001).
4. Immunohistochemical staining Paraffin-embedded sections of lung were deparaffinized and subjected to immunohistochemical staining with an antihuman neutrophil elastase mouse monoclonal antibody. Intense neutrophil elastase staining was uniformly observed in metastatic EBC-1 tumor cells in lungs examined 12 weeks after EBC-1 inoculation. Immunoreactivity was
E. Tanaka et al. / International Congress Series 1255 (2003) 239–242
241
Table 1 Tumorigenicity and lung metastasis Weeks after primary pulmonary tumor inoculation tumorigenicitya
Lung weight metastasisb (mg)
1 2 3 4 5 6 7 8 9 10 11 12
201 F 39 190 F 62 194 F 56 211 F 47 199 F 49 206 F 31 315 F 99 521 F 212d 602 F 231d 813 F 345e 897 F 302e 1044 F 353e
0/12 0/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12 12/12
(4.2 F 1.9) (10.4 F 3.1) (12.7 F 3.1) (16.6 F 2.8) (27.3 F 4.5) (35.9 F 3.7) (47.5 F 7.7) (59.0 F 9.3) (57.4 F 11.2) (58.9 F 9.4)
0/12 0/12 0/12 0/12 0/12 0/12 12/12 12/12 12/12 12/12 12/12 12/12
(24 F 10) (65 F 23)c (98 F 31)c (123 F 26)c (144 F 42)c (144 F 35)c
a Data represent the number of mice with a palpable mass in the flank/number of mice evaluated. Calculated tumor volumes (in cm3, mean F S.D.) are given in parentheses. b Data represent the number of mice with spontaneous lung metastasis/number of mice evaluated. Numbers of microscopic lung metastatic foci (mean F S.D.) then are given in parentheses. c p < 0.001 vs. 7 weeks. d p < 0.02 vs. 7 weeks. e p < 0.001 vs. 7 weeks.
cytoplasmic, with a granular appearance. Control sections with no primary antibodies showed no staining.
5. Human h -actin mRNA expression in blood To test whether evidence of circulating tumor cells could be detected in the blood of tumor-bearing SCID mice, we assayed blood samples for human h-actin (actin-H) mRNA
Fig. 1. Human h-actin mRNA expression by RT-PCR in blood samples sequentially obtained after EBC-1 inoculation. Colon 26 clone 20; a cell line from murine colon cancer. Positive control; EBC-1 cells. Negative control; mice without inoculation. Actin-H, human h-actin. Actin-MH, both human and murine h-actin. The results were similar in blood samples from all mice studied.
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E. Tanaka et al. / International Congress Series 1255 (2003) 239–242
by reverse transcriptase –polymerase chain reaction (RT-PCR). The integrity of RNA and cDNA was confirmed by the generation of a h-actin PCR product with primers which hybridize with both human and murine h-actin (actin-MH) [14,15]. As shown in Fig. 1, RT-PCR amplification detected no human h-actin mRNA in blood taken at 3 days, 1 week or 2 weeks after EBC-1 cell inoculation. However, blood samples obtained 3 weeks after tumor inoculation contained human h-actin mRNA in all mice studied.
6. Conclusions In conclusion, we report 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.
References [1] L.A. Liotta, W.G. Stetler-Stevenson, Tumor invasion and metastasis: an imbalance of positive and negative regulation, Cancer Res. 51 (1991) 5054s – 5059s. [2] L.M. Matrisian, The matrix-degrading metalloproteinases, Bioessays 14 (1992) 455 – 463. [3] P. Mignatti, E. Robbins, D.B. Rifkin, Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade, Cell 47 (1986) 487 – 498. [4] M. Nakajima, A.M. Chop, Tumor invasion and extracellular matrix degradative enzymes: regulation of activity by organ factors, Semin. Cancer Biol. 2 (1991) 115 – 127. [5] Z. Werb, S. Gordon, Elastase secretion by stimulated macrophages: characterization and regulation, J. Exp. Med. 142 (1975) 361 – 377. [6] R.J. Baugh, J. Travis, Human leukocyte granule elastase: rapid isolation and characterization, Biochemistry 15 (1976) 836 – 841. [7] A. Janoff, Elastase in tissue injury, Annu. Rev. Med. 36 (1985) 207 – 216. [8] A. Janoff, J. Schere, Mediators of inflammation in leukocyte lysosomes: IX. Elastolytic activity in granules of human polymorphonuclear leukocytes, J. Exp. Med. 128 (1968) 1137 – 1156. [9] C. Mainardi, S. Dixit, A. Kang, Degradation of type IV (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocyte granules, J. Biol. Chem. 255 (1980) 5435 – 5441. [10] J.A. McDonald, D.G. Kelley, Degradation of fibronectin by human leukocyte elastase: release of biologically active fragments, J. Biol. Chem. 255 (1980) 8848 – 8858. [11] J. Yamashita, K. Tashiro, S. Yoneda, K. Kawahara, T. Shirakusa, Local increase in polymorphonuclear leukocyte elastase is associated with tumor invasiveness in non-small cell lung cancer, Chest 109 (1996) 1328 – 1334. [12] J. Yamashita, M. Ogawa, M. Abe, N. Hayashi, Y. Kurusu, K. Kawahara, T. Shirakusa, Tumor neutrophil elastase is closely associated with the direct extension of non-small cell lung cancer into the aorta, Chest 111 (1997) 885 – 890. [13] M. Inada, J. Yamashita, M. Ogawa, Neutrophil elastase inhibitor (ONO-5046Na) inhibits the growth of human lung cancer cell lines transplanted into severe combined immunodeficiency (SCID) mice, Res. Commun. Mol. Pathol. Pharmacol. 97 (1997) 229 – 232. [14] N. Yoshida, E. Ishii, M. Nomizu, Y. Yamada, S. Mohri, N. Kinukawa, A. Matsuzaki, K. Oshima, T. Hara, S. Miyazaki, The laminin-derived peptide YIGSR (Tyr – Ile – Gly – Ser – Arg) inhibits human pre-B leukaemic cell growth and dissemination to organs in SCID mice, Br. J. Cancer 80 (12) (1999) 1898 – 1904. [15] S. Nakajima-Iijima, H. Hamada, P. Reddy, T. Kakunaga, Molecular structure of human cytoplasmic h-actin gene: interspecies homology of sequences in the introns, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 6133 – 6137.