Cancer Investigation, Early Online:1–4, 2011 ISSN: 0735-7907 print / 1532-4192 online Copyright C  Informa Healthcare USA, Inc. DOI: 10.3109/07357907.2011.606247 Original Article Development of New Spontaneous Metastatic Heterotopic Model of Lewis Lung Carcinoma Imaged by GFP Expression Vladimir Bobek, 1,2 Katarina Kolostova, 1 Daniela Pinterova, 1 Michael Boubelik, 1 and Robert M. Hoffman 3,4 Department of Tumor Biology, Third Faculty of Medicine, Charles University, Prague, Czech Republic, 1 Department of Surgery, Third Faculty of Medicine, Charles University Prague, Czech Republic, 2 AntiCancer, Inc., San Diego, California, USA, 3 Department of Surgery, University of California, San Diego, California, USA 4 Many studies have demonstrated the importance of spontaneous metastases in cancer research. Until now, we still had only a few spontaneous metastatic models with high occurrence rate of metastasis in distant lymph and visceral tissues. We report a syngeneic heterotopic metastatic model using the Lewis lung cancer cell line with high metastatic ratio in C57BL/6 mice after transplantation by injection of cancer cells and without surgical intervention. Metastatic process was declared for each mouse in two groups – sacrificed 3 or 5 weeks after subcutaneous (s.c.) injection of the tumor cells into the dorsal side of the tail. The total number of metastases was counted as the sum of observed macrometastases. Our model produced produced a 100% rate of spontaneous lymphatic and visceral metastases after a simple injection transplantation into the heterotopic site. In mice with large primary tumors which are non-lethal, visceral and lymph macrometastases were observed. Tumor volume correlated linearly not only with the tumor growth time, but also with the number of metastases in lymph nodes and organs. This new metastatic model could be useful for studying the metastasis mechanism and for developing therapy for lymph and visceral metastases. Keywords: GFP (green fluorescent protein); Lewis lung cancer; Lymph node; Metastasis model; Metastasis; Spontaneous metastasis; Tumor INTRODUCTION Lung cancer is the most common cancer in the world. In men, the highest incidence rates are seen in Europe and North America. Lung cancer is a form of cancer which commonly forms lymph and hematogenous metastases. For the study of lung cancer metastases, a suitable metastasis model is needed. The oldest and perhaps one of the most common cell lines used in the lung cancer study is the Lewis lung cancer cell line (carcinoma). This cancer cell line was first isolated by his study was supported by grant KONTAKT ME 10045 by the Ministry of Education, Youth and Sports; and grant IGA NS 9976/3 by the Ministry of Health, Czech Republic. Correspondence to: Vladimir Bobek, MD, PhD, Department of Tumor Biology, hird Faculty of Medicine, Charles University, Ruska 87, 100 34 Prague, Czech Republic. email: vbobek@centrum.cz Margaret Lewis in 1951 from spontaneous epidermoid car- cinoma of the lung in mouse (1). This cancer has been used for metastatic and angiogenesis studies and for neoadjuvant chemotherapy (2–6). The Lewis lung cancer cell line does not usually metas- tasize spontaneously to visceral organs after subcutaneous transplantation. Metastases in the subcutaneous site have not been observed in most tumor transplantation experiments in mice. Injecting a tumor cell suspension into orthotopic sites occasionally allowed relevant metastases to occur (4). The tumors resulting from orthotopic transplantation of tumor cell suspension often showed relatively low rates of metasta- sis compared with the original tumors in the mice and com- pared with surgical orthotopical implantation (SOI) of tumor tissue (7, 8). SOI is a method that ensures a high rate of spon- taneous hematogenous and lymph metastasis, but demands complicated surgical tumor implantation. Here, we report a syngeneic heterotopic spontaneous metastatic model of the Lewis Lung carcinom with high metastatic ratio in C57BL/6 mice after transplan- tation of cancer cells by injection and without surgical intervention. MATERIALS AND METHODS Cell culture Cell lines used in this study have been described previ- ously (6). Except where mentioned, cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum and gentamicin to 70–80% confluence, as described previously (13). Subcutaneous tumor growth Three C57BL/6 mice, 6 weeks of age, were injected s.c. with single dose of 2 × 10 6 Lewis lung cancer cells. Cells were first harvested by trypsinization and washed three times with  Cancer Invest Downloaded from informahealthcare.com by 90.183.120.1 on 09/03/11 For personal use only.