Generation of a lung squamous cell carcinoma three-dimensional culture model with keratinizing structures.

Tumor nests in lung squamous cell carcinoma (LUSC) have a hierarchical structure resembling squamous epithelium. The nests consist of basal-like cells on the periphery and layers of keratinocyte-like cells that differentiate towards the center of the nest, forming keratin pearls. Reproducing this spatial heterogeneity in in vitro models would be useful for understanding the biology of LUSC. Here, we established a three-dimensional (3D) culture model with a squamous epithelial structure using LUSC cell lines PLR327F-LD41 and MCC001F, established in-house. When PLR327F-LD41 cells were cultured in a mixture of Matrigel and collagen I, they generated 3D colonies (designated cancer organoids, or COs) with involucrin (IVL)-positive keratinizing cells in the center (IVLinner COs). COs with uniform size were generated by seeding PLR327F-LD41 cells in a form of small cell aggregates. Since Notch signaling induces the differentiation of squamous epithelium, we confirmed the effect of γ-secretase inhibitor in inhibiting Notch signaling in IVLinner COs. Surprisingly, γ-secretase inhibitor did not block induction of IVL-positive cells; however, cells residing between the CK5-positive basal-like layer and IVL-positive layer decreased significantly. Thus, our 3D culture model with uniform size and structure promises to be a useful tool for elucidating the biology of LUSC and for screening drug-candidates.

Microtubule nucleation in the cytoplasm of developing cortical neurons and its regulation by brain-derived neurotrophic factor.

The centrosomes of neurons do not nucleate microtubules (MTs). It is not well known where MTs are nucleated in neurons. We performed MT regrowth experiments in primary cultured cortical neurons from mice. After cold-nocodazole depolymerization of MTs in neurons at 3 days in vitro (DIV), we observed numerous short MTs that regenerated in the cytoplasm; they were roughly equal in length and randomly oriented. Gamma-tubulin and MZT2 were detected at one end of these MTs, indicating that the short MTs were nucleated by γ-tubulin-ring complexes; the Golgi apparatus was not the origin site of their nucleation. The ratio of MT-regenerating cells, the density of regenerated MTs, and their lengths decreased at 7 DIV. Pretreatment of neurons with protein-kinase inhibitors, K-252a, staurosporine, or H7 diminished the length of the regenerated MTs, while pretreatment of neurons with brain-derived neurotrophic factor increased the length of regenerated MTs. These results support the idea that short MTs with mixed orientations are transiently generated in the cytoplasm of differentiating neurons, and that they are possibly transported into dendrites to provide seeds for MTs with mixed polarity.