Fig. 1

Schematic of coculture strategy with intimate and distant communication for the generation of CAFs. a Intimate communication: normal human fibroblasts destined to become CAFs are seeded onto inverted permeable microporous inserts consisting of a 10 μm-thick polyester membrane with 1 μm-pores. Following attachment, the inserts are inverted and placed into the wells of plates and cultured to confluency. Cancer cells or control fibroblasts grown separately in flasks are then seeded on the top side of the inserts with the fibroblasts growing intimately on the bottom side. The cocultured cells are fed every other day and maintained for the desired time. At the respective times following coculture and/or experimental treatments, the CAFs (bottom side of the inserts) and/or cancer cells (top side of the insert) are carefully isolated by trypsinization, yielding highly pure (99.8%) cell populations for analysis or propagation for subsequent studies. b Distant communication: Normal human fibroblasts are cultured on the bottom of the well housing the insert, while cancer cells are grown on the top side of the insert