Fig 2: Organ-like structures of the NBOs and LCOs. a Bright-field microscopy images of a NBO, NBO-90. NBO-90 was cultured in MBM and MBM + WNA (W; Wnt3A, N; Noggin, A; A83-01). Scale bars, 200 μm. b H&E staining image of NBO-90. Scale bar, 100 μm. c–e Immunofluorescence images showing the expression of pancytokeratin, p63, MUC1, KRT5, KRT7, CC10, and cilia co-stained with acetylated α-tubulin (Ac-Tub) and Arl13b (The enlarged images in yellow boxes showed cilia co-stained with Ac-Tub and Arl13b). Nuclei (blue) were stained with DAPI. Scale bar, 20 μm

Fig 3: LCOs recapitulate the characteristics of the original tissues. a–d H&E-stained and IHC-stained images of LCOs and their original LC tissues. The enlarged images in blue boxes of b showed cytoplasmic keratinisation of individual squamous carcinoma cells. Scale bars, 100 µm. e H&E and IHC-stained images of an adenosquamous carcinoma organoid (upper panel). Individual organoids expressed either adenocarcinoma marker (CK7) or squamous cell carcinoma markers (CK5/6 or p63). Some single organoids showed mixed cell pattern composed of CK7+ and CK7- cells, CK5/6+ and CK5/6- cells, and p63+ and p63- cells. Scale bar, 100 µm. Bright field microscopy and immunofluorescence (IF) images of an adenosqumaous carcinoma organoid originated from a single cell (lower panel). The immunofluorescence staining was performed at Day 18 after seeding. The organoid from a single cell was composed of p63+/CK7- cells and p63-/CK7+ cells. The red arrow indicated a single cell seeded in a micro-well. Scale bar in bright-field microscopy images, 100 µm. Scale bar in IF images, 20 µm