An improved model to study tumor cell autonomous metastasis programs using MTLn3 cells and the Rag2−/− γc−/− mouse

Clinical & Experimental Metastasis, May 2009

The occurrence of metastases is a critical determinant of the prognosis for breast cancer patients. Effective treatment of breast cancer metastases is hampered by a poor understanding of the mechanisms involved in the formation of these secondary tumor deposits. To study the processes of metastasis, valid in vivo tumor metastasis models are required. Here, we show that increased expression of the EGF receptor in the MTLn3 rat mammary tumor cell-line is essential for efficient lung metastasis formation in the Rag mouse model. EGFR expression resulted in delayed orthotopic tumor growth but at the same time strongly enhanced intravasation and lung metastasis. Previously, we demonstrated the critical role of NK cells in a lung metastasis model using MTLn3 cells in syngenic F344 rats. However, this model is incompatible with human EGFR. Using the highly metastatic EGFR-overexpressing MTLn3 cell-line, we report that only Rag2−/−γc−/− mice, which lack NK cells, allow efficient lung metastasis from primary tumors in the mammary gland. In contrast, in nude and SCID mice, the remaining innate immune cells reduce MTLn3 lung metastasis formation. Furthermore, we confirm this finding with the orthotopic transplantation of the 4T1 mouse mammary tumor cell-line. Thus, we have established an improved in vivo model using a Rag2−/− γc−/− mouse strain together with MTLn3 cells that have increased levels of the EGF receptor, which enables us to study EGFR-dependent tumor cell autonomous mechanisms underlying lung metastasis formation. This improved model can be used for drug target validation and development of new therapeutic strategies against breast cancer metastasis formation.

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An improved model to study tumor cell autonomous metastasis programs using MTLn3 cells and the Rag2−/− γc−/− mouse

Sylvia E. Le Devedec 0 Wies van Roosmalen 0 Naomi Maria 0 Max Grimbergen 0 Chantal Pont 0 Reshma Lalai 0 Bob van de Water 0 0 S. E. Le Devedec W. van Roosmalen N. Maria M. Grimbergen C. Pont R. Lalai B. van de Water (&) Division of Toxicology, Leiden Amsterdam Center for Drug Research (LACDR), Leiden University , P.O. Box 9502, 2300 RA Leiden, The Netherlands The occurrence of metastases is a critical determinant of the prognosis for breast cancer patients. Effective treatment of breast cancer metastases is hampered by a poor understanding of the mechanisms involved in the formation of these secondary tumor deposits. To study the processes of metastasis, valid in vivo tumor metastasis models are required. Here, we show that increased expression of the EGF receptor in the MTLn3 rat mammary tumor cell-line is essential for efficient lung metastasis formation in the Rag mouse model. EGFR expression resulted in delayed orthotopic tumor growth but at the same time strongly enhanced intravasation and lung metastasis. Previously, we demonstrated the critical role of NK cells in a lung metastasis model using MTLn3 cells in syngenic F344 rats. However, this model is incompatible with human EGFR. Using the highly metastatic EGFRoverexpressing MTLn3 cell-line, we report that only Rag2-/-cc-/- mice, which lack NK cells, allow efficient lung metastasis from primary tumors in the mammary gland. In contrast, in nude and SCID mice, the remaining innate immune cells reduce MTLn3 lung metastasis formation. Furthermore, we confirm this finding with the orthotopic transplantation of the 4T1 mouse mammary tumor cell-line. Thus, we have established an improved in vivo model using a Rag2-/- cc-/- mouse strain together with MTLn3 cells that have increased levels of the EGF receptor, which enables us to study EGFR-dependent tumor cell autonomous mechanisms underlying lung metastasis formation. This improved model can be used for drug target validation and development of new therapeutic strategies against breast cancer metastasis formation. - Breast cancer is the most common cause of cancer among women and the second leading cause of cancer deaths in Western countries. The metastatic spread of tumor cells from their primary site to distant organs in the body is the principal cause of mortality [1]. Thus, understanding metastasis is one of the most significant problems in cancer research [24]. A key challenge is to develop suitable animal models to enhance our understanding of the mechanisms that underlie metastatic progression and to evaluate treatments for metastatic diseases [5]. Currently available in vivo models of breast tumor progression and metastasis include transplantable models and genetically engineered mice that develop primary and metastatic cancers [58]. Transplantable tumor models include syngeneic models, in which the cancer cell line/ transplanted tissue is of the same genetic background as the animal, and xenograft models whereby human cancer cell lines or tissues are transplanted into immunocompromised hosts, such as nude and severe combined immunodeficient mice [5]. Breast xenograft tumors are produced by injecting breast cancer cells into the flank (subcutaneous) or preferably into the mammary fat pad (orthotopic) of a female animal. Subcutaneous xenograft mouse models are typically the standard for cancer drug screening in the pharmaceutical industry [9], but the use of orthotopic xenotransplantation models should be favored since tissue specific stromal cell interactions play a crucial role in the biology of cancer progression and metastasis. Metastasis is a consequence of multiple steps, including growth of a primary tumor, intravasation, arrest and growth in a secondary site [2, 3]. The study of metastasis requires both a relevant mouse model and an appropriate tumor cell line. On the basis of gene profiling and previous in vivo results, rat mammary adenocarcinoma MTLn3 cells have been identified as a suitable model to study breast cancer progression and treatment [10, 11]. The epidermal growth factor receptor (EGFR, also referred as ErbB1) is often overexpressed in breast cancer, resulting not only in uncontrolled cell proliferation [12, 13] but also in increased tumor cell motility and invasion [1416]. To study intravasation leading to metastasis formation, we therefore evaluated the effect of enhanced ErbB1 signaling in MTLn3 cells in the orthotopic Rag mouse breast cancer model. Efficient metastasis formation is dependent on tumor cell autonomous biological programs that define migration, intravasation survival and extravasation [14]. For xenotransplantation models, immune cell responses to foreign antigens on the injected tumor cells need to be avoided, necessitating the use immunocompromised hosts. The most widely used immunodeficient mice (nude and SCID) lack the adaptive immune response. However, these mice still harbor large numbers of cells of the innate immune system, including natural kil (...truncated)


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Sylvia E. Le Dévédec, Wies van Roosmalen, Naomi Maria, Max Grimbergen, Chantal Pont, Reshma Lalai, Bob van de Water. An improved model to study tumor cell autonomous metastasis programs using MTLn3 cells and the Rag2−/− γc−/− mouse, Clinical & Experimental Metastasis, 2009, pp. 673, Volume 26, Issue 7, DOI: 10.1007/s10585-009-9267-6