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

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.

Keywords: Alveolar epithelium; Cancer; Co-culture; Fibroblast; Hydrogel; Matrix metalloproteinases.

Fig. 1

Cross-sectional schematic of the co-culture set-up. (i) Epithelial cells (red) were incubated on an orbital shaker with photodegradable microspheres (orange) containing RGDS peptides to allow for cellular attachment to the surface of the microsphere. (ii) Pre-cysts were co-encapsulated with a single cell suspension of fibroblasts (green) in a poly(ethylene glycol) (PEG) hydrogel (blue) containing pendant RGDS for cell adhesion and an enzymatically-degradable peptide crosslinker to allow for local matrix remodeling. (iii) One day after encapsulation, cytocompatible 365 nm light at ~10 mW/cm² for 15 minutes was applied to completely erode the microsphere templates, leaving a shell of epithelial cells surrounding a liquid-filled lumen. (iv) Cells were cultured for 1-5 days before being analyzed for proliferation, migration, or protease activity. In this confocal image slice, normal fibroblasts labeled with Cell Tracker Green were co-cultured with primary epithelial cells, which were subsequently stained for an alveolar epithelial type 1 (ATI) phenotype marker. Green = Cell Tracker (fibroblasts), Blue = DAPI (nuclei), Red = T1α (ATI).

Fig. 2

Click-iT Plus EdU proliferation assay. (A) Example confocal image slices used for automated nuclei counts. Each object found in the blue and red channels was measured for mean intensity in the green channel, and objects above a certain threshold were counted as fibroblasts. (i) Blue = DAPI (all nuclei). (ii) Red = EdU (proliferating nuclei). (iii) Green = Cell Tracker (fibroblasts). (iv) Merged image of previous three channels. (B) Plots depict percent of nuclei positive for EdU at the two time points in monoculture and both co-cultures, separated by cell type. Results are presented as means ± SEM of three biological replicates of each condition. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Normal fibroblast migration analysis. (A) Example images of fibroblasts (green) in co-culture with epithelial cysts (red) used in cell tracking. (i) Max z-projection of 400 μm confocal stack. (ii) 3D rendering of 200 μm confocal stack using Volocity (Perkin Elmer). (B) Plots show fraction migrating, fraction migrating toward cyst, migration speed, and directionality (Distance-To-Origin/Total Distance) of migrating normal fibroblasts (CCL-210) in monoculture and co-culture with both epithelial cell types. Data in fraction migrating plot represent means ± SEM of cells migrating per cyst. Data in the remaining plots represent means ± SEM of all migrating cells. ***p < 0.001

Fig. 4

(A) Metabolic activity on days 1 and 2 after encapsulation as measured by resorufin fluorescence normalized to cell count for each cell type cultured alone and in co-culture. (B) MMP activity on days 1 and 2 after encapsulation as measured by an MMP-sensitive fluorescent sensor peptide normalized to cell count for each cell type cultured alone and in co-culture. Dotted lines represent the weighted average of the two cell types cultured alone, which would be the expected value for the co-culture barring any crosstalk. Data represents means ± SEM for 3 biological replicates of each condition. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Click-iT Plus EdU proliferation assay. Plot depicts percent of A549 cell nuclei positive for EdU on day 4 in monoculture and the A549/CCL-210 co-cultures, separated by gel and media type. The nondegradable gels contained a peptide crosslinker insensitive to MMP cleavage. GM6001 was added at 10 μM, and the DMSO control media contained 0.05% DMSO. The degradable bars refer to the original experiment in MMP-degradable gels with regular growth media. Results are presented as means ± SEM of three biological replicates of each condition. *p < 0.05