Fig. 1

Bronchial carcinoid cell line growth assessed as monolayers and as spheroids with serially passaging and assayed by clonogenic assay show evidence of TIC. a Monolayer cultures of parental (PA) H727 and H720 cell lines were passaged under non-adherent stem cell culture conditions to permit spheroid growth (SP) visualized under phase microscopy. b Spheroids growing in methyl cellulose medium progressively expanded from an initial size of 200 to 300 cells per spheroid after 5 days to ~ 6 x the volume after 20 days. Both cell lines were able to form spheroids of different sizes and efficiencies. c Numbers of spheroids formed per 1000 cells seeded comparing H727 to H720. d First generation spheroids as in (c) were dissociated and replated to form 2nd and 3rd generation spheroids with a significant increase in spheroid numbers. e Spheroid forming ability evaluated over 20 days comparing PA cells with 3rd generation spheroids for both H727 and H720 starting from an initial seeding of 100 cells. f Spheroids formed from PA and 3rd generation spheroids were dissociated and cell numbers enumerated. g To assess clonogenic capacity 3 × 10⁴ cells from PA and 3rd generation spheroids of H727 and H720 were seeded in methylcellulose medium in 35 mm dishes and cultured for 7 days. Spheroids were visualized under phase microscopy. h Numbers of spheroids formed enumerated per dish in triplicates