Dwyer Amy R, Ellies Lesley G, Holme Andrea L, Pixley Fiona J
School of Medicine and Pharmacology, University of Western Australia, Perth Australia.
School of Anatomy, Physiology and Human Biology, University of Western Australia, Perth, Australia.
J Biol Methods. 2016 Jul 24;3(3):e49. doi: 10.14440/jbm.2016.132. eCollection 2016.
Macrophages infiltrate cancers and promote progression to invasion and metastasis. To directly examine tumor-associated macrophages (TAMs) and tumor cells interacting and co-migrating in a three-dimensional (3D) environment, we have developed a co-culture model that uses a PyVmT mouse mammary tumor-derived cell line and mouse bone marrow-derived macrophages (BMM). The Py8119 cell line was cloned from a spontaneous mammary tumor in a Tg(MMTV:LTR-PyVmT) C57Bl/6 mouse and these cells form 3-dimensional (3D) spheroids under conditions of low adhesion. Co-cultured BMM infiltrate the Py8119 mammospheres and embedding of the infiltrated mammospheres in Matrigel leads to subsequent invasion of both cell types into the surrounding matrix. This physiologically relevant co-culture model enables examination of two critical steps in the promotion of invasion and metastasis by BMM: 1) macrophage infiltration into the mammosphere and, 2) subsequent invasion of macrophages and tumor cells into the matrix. Our methodology allows for quantification of BMM infiltration rates into Py8119 mammospheres and demonstrates that subsequent tumor cell invasion is dependent upon the presence of infiltrated macrophages. This method is also effective for screening macrophage motility inhibitors. Thus, we have developed a robust 3D co-culture assay that demonstrates a central role for macrophage motility in the promotion of tumor cell invasion.
巨噬细胞浸润癌症并促进其向侵袭和转移进展。为了直接检测在三维（3D）环境中相互作用和共同迁移的肿瘤相关巨噬细胞（TAM）和肿瘤细胞，我们开发了一种共培养模型，该模型使用PyVmT小鼠乳腺肿瘤衍生细胞系和小鼠骨髓来源的巨噬细胞（BMM）。Py8119细胞系是从Tg(MMTV:LTR-PyVmT)C57Bl/6小鼠的自发性乳腺肿瘤中克隆而来的，这些细胞在低粘附条件下形成三维（3D）球体。共培养的BMM浸润Py8119乳腺球体，将浸润的乳腺球体包埋在基质胶中会导致两种细胞类型随后侵入周围基质。这种生理相关的共培养模型能够检测BMM促进侵袭和转移的两个关键步骤：1）巨噬细胞浸润到乳腺球体中，以及2）随后巨噬细胞和肿瘤细胞侵入基质。我们的方法允许量化BMM浸润Py8119乳腺球体的速率，并证明随后的肿瘤细胞侵袭取决于浸润巨噬细胞的存在。该方法对于筛选巨噬细胞运动抑制剂也有效。因此，我们开发了一种强大的3D共培养测定法，该方法证明了巨噬细胞运动在促进肿瘤细胞侵袭中的核心作用。
