An in vivo Like Micro-Carcinoma Model

Abstract

We here present a novel micro-system which allows to reconstitute an in vivo lung carcinoma where the various constituting epithelial and/or stromal structural and/or cellular components can be incorporated at will. In contrast to various "organs on a chip" the model is based on the observation that in nature, epithelial cells are always supported by a connective tissue or stroma. The model is based on acellular micro-scaffolds of microscopic dimensions which enable seeded cells to obtain gases and nutrients through diffusion thus avoiding the need for vascularization. As a proof of concept, we show that in this model, Calu-3 cells can form a well-organized, continuous, polarized, one-layer epithelium lining the stromal derived alveolar cavities, and express a different pattern of tumor-related genes than when grown as standard monolayer cultures on plastic culture dishes. To our knowledge, this model, introduces for the first time a system where the function of carcinogenic cells can be tested in vitro in an environment that closely mimics the natural in vivo situation.

Keywords: carcinoma; decellularized scaffolds; tumor micro-culture; tumor microenvironment; tumor model.

Figure 1

General model. (A) Schematic diagram of the three-dimensional culture system. (B) Decellularized micro-scaffolds. (C) DAPI staining of micro-scaffolds seeded with Calu-3 cells. Scale bars (B) 1 mm, (C) 20 μm.

Figure 2

Calu-3 cells can organize themselves into a continuous one-layer epithelium lining the major micro-scaffold cavities. Cell viability by MTT assay at day 15 of (A) Calu-3 cells on micro-scaffolds and (B) Calu-3 cells and fibroblast on micro-scaffolds. Histological sections at day 15 of (C) Calu-3 cells seeded on micro-scaffolds. Scale bars (A,B) 1 mm, (C) 20 μm.

Figure 3

Calu-3 cells form a polarized single layer epithelium. Transmission electron microscopy of (A) epithelium formed by the Calu-3 cells, expanded around the alveolar cavity of the matrix. (B) Higher magnification of (A) demonstrates basal surface of the cells lined by a basement membrane (arrows) and cell contacts. (C) Higher magnification of (B) shows the apical surface of the cells covered by microvilli (arrows). Scale bars 10 μm (A), 2 μm (B), and 1 μm (C).

Figure 4

Significant changes tumor suppressors and oncogene expression patterns in Calu-3 cells seeded on micro-scaffolds. Gene expression of all genes tested in (A) Calu-3 seeded on micro-scaffolds in comparison with Calu-3 seeded on culture dishes and (B) Calu-3 co-cultured with fibroblasts seeded on micro-scaffolds in comparison with Calu-3 co-cultured with fibroblasts seeded on culture dishes. Selected (C) tumor suppressors and (D) oncogenes with significant differences in Calu-3 seeded on micro-scaffolds in comparison with Calu-3 seeded on culture dishes. Selected (E) tumor suppressors and (F) oncogenes with significant differences in Calu-3 co-cultured with fibroblasts seeded on micro-scaffolds in comparison with Calu-3 co-cultured with fibroblasts seeded on culture dishes. The line distinguishes between up- and down- regulated genes when compared to cells grown on culture dishes at four time points. Data represent mean ± SD. *p ≤ 0.05, **p ≤ 0.01 (t-test).