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. 2013 Oct 4;8(10):e76373.
doi: 10.1371/journal.pone.0076373. eCollection 2013.

Understanding the impact of 2D and 3D fibroblast cultures on in vitro breast cancer models

Kyung Eun Sung  1 , Xiaojing Su, Erwin Berthier, Carolyn Pehlke, Andreas Friedl, David J Beebe
Affiliations

Understanding the impact of 2D and 3D fibroblast cultures on in vitro breast cancer models

Kyung Eun Sung et al. PLoS One. .

Abstract

The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs) cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com). We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models.

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Conflict of interest statement

Competing Interests: We have read the journal's policy and have the following conflicts. D. J. Beebe has equity in Bellbrook Labs which has licensed technology reported in this manuscript. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. 3D in vitro culture of HMF induces an increased transition of MCF-DCIS cells.
(A) Different morphologies of HMFs in 2D vs. 3D conditions. These images clearly show that HMFs in 3D have more fiber-like structures. The scale bar represents 60 µm. (B) Conceptual illustration of the difference of HMF behaviors in 2D and 3D. The conditioned medium collected from 3D culture of HMF (3D CM) stimulates invasive transition more than the conditioned medium collected from 2D culture of HMF (2D CM), and stimulates more invasive transition of MCF-DCIS cells in 3D. Outlines of MCF-DCIS clusters cultured in 3D mixed matrix with 3D CM and 2D CM. The clusters cultured with 3D CM produced more elongated clusters with aspect ratio (AR) 1.57. Scale bar is 100 μm. (C) Bar graph showing average aspect ratio of MCF-DCIS clusters cultured with control (serum free medium, mono), 2D HMF (co-cultured with HMFs in 2D), and 3D HMF (co-cultured with HMFs in 3D). ‡ represents p value of 0.048. (D) Bar graph showing data obtained from transwell invasion assays with conditioned media from 2D culture of HMF (2D HMF) and 3D culture of HMF (3D HMF). ‡ represents p value of 0.022.
Figure 2
Figure 2. HMFs in 3D produce more signaling molecules.
(A) Conceptual illustration showing HMFs in 3D produce more signaling molecules. (B) Bar graphs showing the mRNA expressions of HGF, MMP14, COX2, and CXCL12 in HMFs cultured in 2D and 3D conditions. ‡ represents a p value of less than 0.05. (C) Zymography showing the presence of increased active MMP2 in the 3D conditioned medium of HMFs. (D) Bead-based ELISA showing the concentrations of target proteins in conditioned media collected from 3D and 2D cultures of HMFs and MCF-DCIS cells.
Figure 3
Figure 3. Increased production of HGF by HMFs in 3D conditions.
(A) An ELISA assay showing the concentration of HGF [pg/ml] in different conditioned media collected from three 2D conditions (bare, collagen-coated, mixed matrix-coated), and two 3D conditions (collagen I only, mixed matrix). HMF cells in all 3D conditions significantly increased the secretion of HGF. ‡ represents a p value of less than 0.05. (B) Invasion of MCF-DCIS cells with HGF neutralizing antibody (anti HGF) using transwells. The HGF neutralizing antibody (0.5 μg/ml) is added to various 2D CM, 3D CM and BK CM (the conditioned medium collected from blank mixed gels). A serum free medium is used as a negative control and a 20% serum-containing medium is used as a positive control. ‡ represents p value of less than 0.05. (C) The aspect ratio of MCF-DCIS cells cultured in a 3D condition with 2D CM and 3D CM with or without the HGF neutralizing antibody. ‡ represents a p value of less than 0.05.
Figure 4
Figure 4. Microchannels used for 2D and 3D combined co-cultures of HMF and MCF-DCIS cells.
(A) 3D schematic and cross-section of the microchannels used for 2D and 3D combined co-culture of HMF and MCF-DCIS cells. (B) Illustrations of the loading process showing the simplicity of loading both in 2D and 3D conditions. (C) Visualization of the diffusion process in the microdevice using a numerical COMSOL simulation and a timelapse microscopy of AlexaFluor488-Dextran10kD dye. (D) Average fluorophore concentration in the inner chamber of the microdevice plotted through time.
Figure 5
Figure 5. MCF-DCIS clusters co-cultured with 3D HMF and with 2D HMF.
(A) MCF-DCIS clusters (red and outlines) co-cultured with 3D HMF and neutralizing HGF antibody at 0.5 μg/ml (3D HMF-HGF). SHG (yellow) shows changes in collagen architecture around MCF-DCIS cells. The addition of HGF neutralizing antibody significantly decreased the aspect ratio of MCF-DCIS cells and the mean intensity of SHG. ‡ represents p value less than 0.05. (B) MCF-DCIS clusters (red and outlines) co-cultured with 2D HMF and neutralizing HGF antibody at 0.5 μg/ml (2D HMF-HGF). Scale bar is 100 μm.
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