Using a thermo-responsive polymer, a collagen-conjugated poly-N-isopropyl acrylamide (PNIPAAm) as a substratum, we developed hetero-multicellular spheroids (MCS) composed of esophageal squamous carcinoma cells (TE10) and esophageal fibroblasts isolated from esophageal carcinoma tissue. PNIPAAm is insoluble in water above the lower critical solution temperature (LCST; about 32 degrees C) and becomes reversibly solubilized below the LCST. Taking advantage of this conversion, we prepared three types of hetero-MCS as follows: F/T-multicellular spheroids made by seeding of TE10 onto a preprepared monolayer of fibroblasts; T/F-multicellular spheroids made by seeding of fibroblasts onto a preprepared monolayer of TE10; Mixed-multicellular spheroid made from a monolayer of mixed fibroblasts and TE10. Histolo,oical and immunohistochemical examinations revealed that fibroblasts and TE10 cells were intermingled in 5-day-old multicellular spheroids but were divided into three zones in 1- or 2 week-cultured spheroids: into an external zone composed almost entirely of TE10 cells that were positive for epithelial membrane antigen (EMA), an intermediate zone composed of fibroblasts that were positive for vimentin, and a necrotic zone showing variable evidence of cell injury. This distribution was observed in all three types of the spheroids described above. These findings indicate that TE10 cells are able to migrate and cover the surface to make organizing spheroid, thus mimicking in vivo structures. We conclude that the three-dimensional culture system using a thermo-responsive polymer, which enables coculturing with different types of cells as a heteromilticellular spheroid, is a useful model to examine the interaction between carcinoma cells and their stroma cells as it occurs in vivo.

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