Background

The 5-year survival rate for patients with advanced stage lung cancer is less than 15%, with the majority of patients die from metastatic diseases (Heist and Engelman, 2012; Whitsett, et al., 2013; D'Antonio et al., 2014; Steeg, 2016). Therefore, using mouse models to investigate the mechanisms of lung cancer invasion and metastasis could facilitate the development of novel strategy to control the metastatic process.

Metastasis is a multi-step process that includes local invasion, intravasation, and survival in the circulation, extravasation, and establishment of macrometastasis in the distant site. Both mouse xenografts and the genetically engineered mouse (GEM) models have been used to study lung cancer metastasis. The GEM models, mimicking the genetic signature of human lung cancer such as TP53, K-ras and LKB1 provide the immunocompetent system to study the mechanism of metastatic process and to investigate how novel anti-cancer agents affect the process (Dutt and Wong, 2006; Zheng et al., 2007). Patient-derived xenografts (PDX) models take tumor heterogeneity and the contribution of tumor microenvironment into the consideration and maintain the features of the primary patient tumor such as gene expression profiles and drug response (Morton and Houghton, 2007, Okada et al., 2019). Cell line-based xenografts either by subcutaneous or orthotopic injection or implantation of cells into mice are commonly used to study lung cancer metastasis. The orthotopic transplantation of tumor cells into mouse lung resembles most closely to human cancer in the aspect of tumor histology, tumor vasculature, gene expression, and responsiveness to drug treatment as well as the metastatic process (Khanna and Hunter, 2005) and this methodology has been reported previously (Justilien and Fields, 2013). Due to the complexity of the metastatic process, there is no single system that can model all of the steps of metastasis (Khanna and Hunter, 2005). The strength and weakness of different mouse models of metastasis were recently comprehensively reviewed (Gomez-Cuadrado et al., 2017). In this protocol, we used lung cancer cell line A549 as an example and utilized the methodology for generating PDX models to establish cell line-based xenograft and monitor tumor growth and spontaneous metastasis. Unlike the classical subcutaneous injection, we performed surgery on mouse to generate a wide subcutaneous pouch to increase the contact area of the cells and matrigel mixture with the tissue. In addition, the surgical incision is big enough to allow us to use the wide bore pipet tip to implant the cells and matrigel mixture smoothly and to prevent the destruction of the cells. This protocol ensures sufficient establishment of the mouse xenografts subcutaneously and allows us to monitor the formation of micrometastasis. This protocol can be adapted to study the mechanisms of metastatic process using the primary or established cell lines from other cancer types and to test the effect of the potential anti-cancer agents on tumor growth and metastatic capacity.