Modeling metastasis biology and therapy in real time in the mouse lung

Abstract

Pulmonary metastasis remains the leading ca use of death for cancer patients. Opportunities to improve treatment outcomes for patients require new methods to study and view the biology of metastatic progression. Here, we describe an ex vivo pulmonary metastasis assay (PuMA) in which the metastatic progression of GFPexpressing cancer cells, from a single cell to the formation of multicellular colonies, in the mouse lung microenvironment was assessed in real time for up to 21 days. The biological validity of this assay was confirmed by its prediction of the in vivo behavior of a variety of high-and low-metastatic human and mouse cancer cell lines and the discrimination of tumor microenvironments in the lung that were most permissive to metastasis. Using this approach, we provide what we believe to be new insights into the importance of tumor cell interactions with the stromal components of the lung microenvironment. Finally, the translational utility of this assay was demonstrated through its use in the evaluation of therapeutics at discrete time points during metastatic progression. We believe that this assay system is uniquely capable of advancing our understanding of both metastasis biology and therapeutic strategies. IntroductionPulmonary metastasis remains a leading cause of death for cancer patients (1, 2). Opportunities to improve outcomes for these patients require a greater understanding of the biology of metastasis. In addition, there is a need to evaluate novel therapeutics, in a timely manner, that specifically target metastases and metastatic progression. Simple in vitro assay systems are not sufficient to model the complex interaction between cancer cells and the surrounding microenvironment that is necessary for metastasis (3). Accordingly, in vivo models of metastasis, largely in mice, have been necessary. For the most part, these models provide end points of metastatic outcome (i.e., yes or no metastasis) and time to late-stage metastatic events.A "black box" exists during which metastatic progression from single cells to gross metastatic lesions at a secondary site occurs. Recent attempts to shed light on this process have included imaging strategies that allow some of the steps of metastatic progression to be followed in vivo (4). However, these approaches often involve sophisticated and expensive imaging techniques that are time consuming and do not easily allow serial assessment of early metastatic progression at secondary sites, particularly in the lung and at the single-cell level. Challenges associated with studying metastasis have resulted in limited opportunities to include the assessment of novel treatment agents against metastatic end points (5). Therefore, an unmet need in the field of cancer research is a simple assay in which the process of metastatic progression at a secondary site can be reproduced and studied over time.

TGF-β Signaling in Myeloid Cells Is Required for Tumor Metastasis

“…Mice were euthanized 5 minutes after injection, and the lungs were infused with agarose as described (59). Lung sections were sliced (1–2mm thick) and placed on Gelfoam (Pfizer-Pharmacia & Upjohn Co.) for culture.…”

“…LEICA-DM IRB fluorescent inverted microscope (Leica) and Retiga-EXi Fast 1394 Mono Cooled CCD camera (QImaging) were used to capture GFP positive cells at ×10 magnification. The area was quantified by OpenLab software (Improvision)(59). For the effect of IFN-γ deficient myeloid cells on metastasis, the Ly-6G cells were sorted from IFN-γ ko mice bearing B16 melanoma (C57/Bl6 background).…”

PIK3CAH1047R Accelerates and Enhances KRASG12D-Driven Lung Tumorigenesis

“…Mouse lungs were inflated with 2%(w/v) LMT-agarose (Sigma) at 42°C, then excised and chilled in tissue culture media (24). Once the agarose solidified, 150μm-thick sections were generated using a vibratome.…”

Breast cancer molecular subtypes and the metastatic microenvironment: Review of literature

“…Protein array analysis of organ-CM identified several secreted metastasis-promoting factors from the lung and the innovative 3D ex vivo pulmonary metastasis assay (PuMA) confirmed the lung as an effective metastatic microenvironment [51] .…”