A three-dimensional co-culture system to investigate macrophage-dependent tumor cell invasion.

Amy R Dwyer,Andrea L Holme,Lesley G Ellies,Fiona J Pixley

doi:10.14440/jbm.2016.132

Amy R Dwyer, Andrea L Holme + Show 2 more

Open Access

https://doi.org/10.14440/jbm.2016.132

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Abstract

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 in vitro co-culture assay that demonstrates a central role for macrophage motility in the promotion of tumor cell invasion.

Highlights

While 2D cell culture monolayers facilitate examination of intracellular signaling cascades and cell behavior, this approach largely ignores the more complex structural organization present in the normal physiological setting
As tumor associated macrophages (TAMs) are key players in the regulation of breast cancer metastasis [7,8], the generation of mammospheres derived from breast cancer cells and subsequent infiltration of the spheroids by macrophages is an attractive model to examine mechanisms by which tumor-associated macrophage (TAM) assist in tumor progression to invasion and metastasis
We have developed a mouse mammary tumor cell-mouse bone marrow-derived macrophages (BMM) co-culture model to examine the role of BMM motility in encouraging tumor cell invasion in a more physiologically relevant context

Summary

BACKGROUND

While 2D cell culture monolayers facilitate examination of intracellular signaling cascades and cell behavior, this approach largely ignores the more complex structural organization present in the normal physiological setting. # 0030702115) 99 24-well ultra-low attachment cell culture dish 99 Py8119 cell culture medium (Growth medium): To F-12K nutrient medium add 5% FCS, 50 μg/ml gentamicin, 2.5 μg/ ml fungizone and 0.1% (v/v) MITO serum extender, and store at 4oC for 4–6 weeks. When mammospheres are ready to infiltrate with BMM, gently transfer them in a wide bore tip to a 24-well ultra-low attachment dish with one spheroid per well containing 1 ml growth medium to ensure each mammosphere is exposed to equal numbers of BMM. Try to minimize handling and, when necessary, use a wide bore tip

Co-culture with BMM

ANTICIPATED RESULTS

Matrigel is not polymerizing