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

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.

Keywords: 3D co-culture; invasion; macrophage; mammosphere; motility.

Figure 1

CSF-1 expression by PyVmT cell line derived tumors. CSF-1 expression was measured by microarray. A. Spontaneous PyVmT tumors - PyVmT T; Py230 well differentiated luminal tumors - Py230 T; and Py8119 aggressive EMT tumors - Py8119 T. Data are means ± SEM from 6 tumors per group and were analyzed by one-way ANOVA and Holm-Sidak’s multiple comparisons test. B. Representative sections of PyVmT spontaneous tumor and Py230 and Py8119 cell line-derived tumors stained with anti-F4/80, a macrophage marker (brown), and counterstained with hematoxylin (blue).

Figure 2

Py8119 mammosphere formation. Py8119 cells were plated at 2.5 × 10⁴ cells/ml in 6-well ultra-low attachment plates and grown for 20–30 d before infiltration with BMM. Developing mammospheres were fed with supplemented Ham’s F12K medium every 7 d. Scale bar = 100 µm.

Figure 3

BMMs infiltrate Py8119 mammospheres. A. Py8119 mammospheres were infiltrated with BMM (red) for 3 d. After fixation and staining for F-actin (green), and DAPI (blue), mammospheres were analyzed by Confocal microscopy. Scale bar = 100 µm. B. Using > 3 mammospheres, the degree of infiltration relative to mammosphere area was quantified.

Figure 4

BMMs promote tumor cell invasion into Matrigel. A. Py8119 mammospheres, either infiltrated with BMM or not, were embedded into Matrigel and the area of invasive cells was measured after 7 d. Scale bar = 100 µm. B. Quantification of changes in mammosphere area, including surrounding invasive cells is shown, **P< 0.01. C. Phase contrast image (10x magnification) of a live culture BMM-infiltrated mammosphere embedded in Matrigel with streams of invading cells. Scale bar = 100 µm. D. Flow cytometric quantification of mammosphere invasion into Matrigel was carried out. Invasive EGFP⁺ Py8119 tumor cells were quantified by flow cytometry after gating for single cells and, in the absence of BMM, only EGFP^- was seen (first peak). In contrast, large numbers of EGFP⁺ tumor cells were seen invading the Matrigel from BMM-infiltrated mammospheres (second peak).

Figure 5

Inhibition of BMM motility blocks tumor cell invasion. A. Representative images of macrophage-infiltrated Py8119 mammospheres embedded in Matrigel in the presence of DMSO, CSF-1R inhibitor (GW2580; 2 µM) or PI3K p110δ inhibitor (GS-1101; 2 µM). After 7 d, the area of invasive cells was quantified for at least 3 mammospheres per condition. Scale bar = 100 µm. B. Quantification of tumor cell invasion. ***P< 0.001, **P< 0.01.