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

Abstract

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.

Animal models for exploring the pharmacokinetics of breast cancer therapies

“…In accomplishing the goal of bridging the chasm between the bench and the clinic, the ideal animal model best mimics breast cancer metastasis and provides endpoints that are of significance in human breast cancer [4,5,7,8,11-14]. The first challenge for many researchers is determining what models meet these twin objectives for the purposes of testing their hypothesis.…”

“…Many have argued for orthotopic implantation directly into the mammary fat pad under direct vision (ODV) so that cancer cells can benefit from the microenvironment of the organ of origin [8,12-14,21, 41-44 ], the majority use either ectopic subcutaneous injection (SQ) [4,7,10,11,14, 45 , 46 ] or percutaneous injection blindly in the area of the nipple in attempt to implant the cells into the mammary fat pad (OP) [19]. Although advocates of ODV have argued that tumor microenvironment matters in drug development and have even cited multiple examples of SQ limiting the viability of cell lines, and despite evidence that ODV promotes progression and metastasis more efficiently than SQ [4,11-13,21, 42-44,46-53 ], until recently research has not focused on critically examining these models [4,5,7,10-13, 33 , 36 , 52-68 ]. In fact, over the last 20 years while the above evidence has been reported in favor of ODV, there has been a shift away from ODV towards SQ and OP [19], even though there is evidence that orthotopic models correlate with the bioavailability of therapeutics in the host's organ of origin [4,13] and that ectopic implantation in other cancers produced false positive results that were later contradicted by orthotopic implantation[ 64,65,69 ] and human clinical trials [7, 59,69-77 ].…”

“…Although increasing resources have been dedicated to developing increasingly sophisticated interventions, it has been unclear how well these animal models serve the purpose of adequately predicting the promise of curing breast cancer in humans. Until recently investigators have not integrated tumor gene profiling into a critical evaluation of breast cancer metastatic animal models [4,5,7,8,11-14,18,19]. …”

“…First, the focus has been to study this disease in a mouse system that mimics human cancer progression [4,8,10-14]. Second, the approach has been to determine the efficacy of novel therapeutics in mice with endpoints that are clinically significant to human cancer [4,5,7,8,11-14]. Applying these two main priorities, the literature has evolved multiple methods of modeling metastatic breast cancer: transgenic mice, implantation of human xenografts into immune-deficient mice, implantation of mouse derived breast cancer into immune competent syngeneic mice, orthotopic implantation, ectopic subcutaneous implantation, and intravenous implantation methods.…”

In Vivo Tomographic Imaging of Deep-Seated Cancer Using Fluorescence Lifetime Contrast

“…3e) showed a 10-fold increase in the injected mice compared to that observed from naive animals. Twenty four hours after injection, the fluorescence yield rapidly drops as the MTLn3-iRFP720 emboli are cleared by the mouse’s innate immune system(32). Continued imaging illustrates the progress of experimental MTLn3-iRFP720 metastasis formation as those cells that were able to extravasate into the lungs continue to survive and grow.…”