PDAC microtumours are triggered by a cell-in-cell structure anchored to a microisland and are covered by membranous expression of α-tubulin. (a) Time-lapse images. At least nine PCI-55 cells were anchored to a microisland through cell-in-cell invasions. Time shown as hh:mm:ss (see Supplementary Movie 3). (b) Time-lapse images of PCI-55 cells cultured on the micro/nanoplate. Cell-in-cell structures on the micro/nanoplate are reversible. Time is shown as hh:mm (See Supplementary Movie 4). (c) When a mix of CFSE-labelled PCI-55 cells and unlabelled PCI-55 cells were sparsely cultured, they showed a cell-in-cell structure on the rough circular microisland. (d) Papillary PCI-55 microtumour formed by cell-in-cell structures. (e,f) Immunofluorescent staining for α-tubulin in the microtumours self-organised by CFSE-labelled PCI-55 cells. CFSEHigh-labelled PCI-55 cells penetrating CFSELow-labelled cells that formed a microtumour (e). Multinucleated giant PCI-55 cells containing at least six high CFSE-labelled PCI-55 cells (f,g). The surface of a papillary microtumour self-organised by PCI-55 cells. Bars: 30 µm. (h) Many PCI-55 cells in a mitotic phase were outside the surface of the microtumours. Z-stack full-focus images of immunofluorescent staining for α-tubulin in PCI-55 microtumours anchored to the micro/nanoplate. (i) Counts of mitotic cells in PCI-55 microtumours. (j,k) PCI-55 microtumours were treated with 1 µm nocodazole for 1 day. Time-lapse images of nocodazole-treated PCI-55 microtumour. Time shown as hh:mm (see Supplementary Movie 5) (j). 3D images (k). (l) Time-lapse images of live and dead cell assays. PDAC microtumours treated with 1 µm nocodazole. Live PCI-55 microtumours that had self-organised on the micro/nanoplate for 1 day were further cultured with medium containing Annexin V Alexa Flour 488 and EthD-1 (See Supplementary Movies 6). m, Immunofluorescent staining for α-tubulin in PCI-55 cells colonised on the peritoneum. CFSE-labelled PCI-55 cells were grafted intraperitoneally into SCID mice. After 3 days, the peritonea were extracted.