Elsevier

Biomaterials

Volume 155, February 2018, Pages 124-134
Biomaterials

Epithelial-mesenchymal crosstalk influences cellular behavior in a 3D alveolus-fibroblast model system

https://doi.org/10.1016/j.biomaterials.2017.11.008Get rights and content

Abstract

Interactions between lung epithelium and interstitial fibroblasts are increasingly recognized as playing a major role in the progression of several lung pathologies, including cancer. Three-dimensional in vitro co-culture systems offer tissue-relevant platforms to study the signaling interplay between diseased and healthy cell types. Such systems provide a controlled environment in which to probe the mechanisms involved in epithelial-mesenchymal crosstalk. To recapitulate the native alveolar tissue architecture, we employed a cyst templating technique to culture alveolar epithelial cells on photodegradable microspheres and subsequently encapsulated the cell-laden spheres within poly (ethylene glycol) (PEG) hydrogels containing dispersed pulmonary fibroblasts. A fibroblast cell line (CCL-210) was co-cultured with either healthy mouse alveolar epithelial primary cells or a cancerous alveolar epithelial cell line (A549) to probe the influence of tumor-stromal interactions on proliferation, migration, and matrix remodeling. In 3D co-culture, cancerous epithelial cells and fibroblasts had higher proliferation rates. When examining fibroblast motility, the fibroblasts migrated faster when co-cultured with cancerous A549 cells. Finally, a fluorescent peptide reporter for matrix metalloproteinase (MMP) activity revealed increased MMP activity when A549s and fibroblasts were co-cultured. When MMP activity was inhibited or when cells were cultured in gels with a non-degradable crosslinker, fibroblast migration was dramatically suppressed, and the increase in cancer cell proliferation in co-culture was abrogated. Together, this evidence supports the idea that there is an exchange between the alveolar epithelium and surrounding fibroblasts during cancer progression that depends on MMP activity and points to potential signaling routes that merit further investigation to determine targets for cancer treatment.

Graphical abstract

Cancerous alveolar epithelial cells were co-cultured in a 3D cyst-like model with healthy pulmonary fibroblasts for analysis of cell behavior in comparison to monoculture.

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Introduction

Lung cancer kills more people in the U.S. than any other type of cancer, underscoring the need for better treatments. To better understand and effectively treat lung cancer, the complexity of the tumor microenvironment, needs to be considered. In particular, the influence of the physical cues together with biochemical cues between cells in the lungs merits further investigation.

Given the three-dimensional architecture of alveolar tissue and tumor masses, more physiologically relevant models must employ ECM mimics that support the growth and culture of 3D multicellular tissue structures. While many techniques exist to form dense tumor spheroids (e.g., the hanging drop method) [1], [2], the cyst-like alveolus structure is notoriously difficult to achieve and manipulate in vitro with primary alveolar epithelial cells, especially in synthetic ECM mimics. Recently, we demonstrated the use of photolabile microspheres as templates for patterning hollow, spherical model alveoli within peptide-modified poly (ethylene glycol) (PEG) hydrogels [3]. These hydrogels capture several key features of the native ECM (e.g., high water content; lung tissue appropriate elasticity; enzymatically degradable crosslinkers that enable local remodeling by cell-secreted proteases; introduction of integrin binding sites, such as the fibronectin-derived RGDS sequence), with the added advantage of precise user control over matrix properties (e.g., elastic modulus, scaffold geometry, tethered biochemical cues) [4]. To complement this approach, our lab has also developed a PEG crosslinker that cleaves upon exposure to selected light wavelengths (365–420 nm) under cytocompatible conditions. These materials have been used to synthesize microspheres of discrete size ranges that are completely degradable upon exposure to light, and have applications for templating of multicellular cyst-like structures (50–200 μm) [3], [5], [6]. In the work presented here, our cyst templating technique was used to create model epithelial alveoli that were subsequently encapsulated in a PEG hydrogel laden with pulmonary fibroblasts.

This approach allowed the culture of two distinct lung cell types in a platform that captures physiological aspects, such as soft matrix modulus and 3D architecture, of the lung tissue to study the effects of paracrine signaling. In cancer, paracrine signaling has been shown to be a key regulator in tumor formation and invasion. As in many carcinomas, there appears to be a reciprocal exchange of signals between pulmonary fibroblasts and epithelial-derived lung cancer cells. For example, alveolar epithelium-derived adenocarcinoma cells increase α-SMA and matrix metalloproteinase production in fibroblasts [7], [8]. Fibroblasts also signal to back to epithelial cells. Cancer-associated fibroblasts (CAFs) increase epithelial tumor proliferation, migration, and drug resistance [9], [10]. Although in vitro co-culture models have proven to be useful tools for studying such crosstalk between cell types, the intricacies of epithelial-mesenchymal crosstalk during disease progression warrants further investigation in 3D platforms that better mimic the in vivo physical cues surrounding these cells [7], [11], [12], [13], [14], [15], [16], [17], [18], [19].

Here, the results report on two types of epithelial cells: primary mouse alveolar epithelial cells to represent a healthy epithelium and an adenocarcinoma cell line (A549) to represent lung tumor cellular structures. These epithelial cells were co-cultured with a pulmonary fibroblast cell line (CCL-210) using the 3D cyst templating technique. The co-cultured cells were analyzed for signs of disease progression by measuring for proliferation, migration, and MMP activity. These measurements are especially relevant in a 3D context in which cells need to interact with and degrade their matrix to migrate and proliferate. Our goal was to test whether a diseased epithelium would influence the surrounding fibroblasts by increasing their proliferation and migration, and whether these changes relate to overall MMP activity. Interestingly, our results suggest a more complex feedback loop between diseased and healthy cells, in which cancer cell proliferation is increased in the presence of healthy fibroblasts.

Section snippets

Microsphere synthesis

Photodegradable microspheres [6] were formed by inverse suspension polymerization via base-catalyzed Michael addition of a photodegradable diacrylate (PEGdiPDA; Mn∼4070 Da) with a poly (ethylene glycol) tetrathiol (PEG4SH; Mn∼5000 Da). The PEGdiPDA was synthesized as previously described [20], and PEG4SH was purchased from JenKem Technology. An aqueous phase consisting of 6.9 wt% PEGdiPDA, 4.2 wt% PEG4SH, CRGDS peptide (1.5 mM final concentration), 300 mM triethanolamine (Sigma-Aldrich) in pH

Results

Using a previously developed cyst-forming technique [3], a 3D co-culture system for lung cells was created with alveolar epithelial cysts embedded in a synthetic polymer hydrogel and surrounded by low-density pulmonary fibroblasts (Fig. 1). This in vitro culture system enabled us to probe cellular behavior in response to co-culture with a diseased cell type in the appropriate structural context in an attempt to elucidate the role of epithelial-mesenchymal crosstalk in disease progression. To

Discussion

Epithelial-mesenchymal crosstalk is a key regulator during lung development and normal wound healing processes, and growing evidence suggests that altered paracrine signaling between the alveolar epithelium and interstitial fibroblasts may lead to disease progression in multiple pathologies [8], [9], [34], [43], [44], [45]. To study these interactions, in vitro co-culture systems, and particularly biomaterial matrices, have evolved to serve as valuable tools for controlling the cell types

Conclusions

The 3D in vitro co-culture system used in this study provided an innovative platform for studying the interactions between alveolar epithelial cysts and dispersed pulmonary fibroblasts and investigating cell functions related to disease progression. The results presented here support the growing body of evidence in the literature that crosstalk between the alveolar epithelium and interstitial fibroblasts influences their behavior in terms of proliferation, migration, and protease activity.

Acknowledgements

The authors would like to thank Sharon Ryan for teaching us the ATII mouse cell isolation procedure, Kyle Kyburz for discussions concerning live cell tracking experiments and analysis, Emi Tokuda for providing the MMP sensor peptide and many helpful discussions, and Chun Yang for providing the non-degradable peptide crosslinker. Funding for this work was provided by the Howard Hughes Medical Institute, the National Science Foundation (CTS1236662), and the NIH Biophysics training grant (T32

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