D9542

FFPE Sections (4-μm thick) were mounted and routinely stained with H&E for histopathological examination (Supplementary Fig. S1a). Multiplex immunofluorescence was applied to identify the expression patterns of key transcriptomic regulators of SCLC, including ASCL1 (Abcam, ab211327), NEUROD1 (Abcam, ab60704) and POU2F3 (Novus Biologicals, NBP1–83966). Multiplex immunofluorescence staining was performed using a PANO 7-plex IHC kit (Panovue, Cat# 0004100100), as previously described.⁶⁵ In brief, the FFPE sections were subjected to deparaffinization, rehydration, and antigen retrieval according to the protocol supplied by the manufacturer. After blocking, the sections were incubated with a primary antibody and then a secondary antibody (polymer HRP-anti-mouse/Rabbit IgG). Other primary antibodies were sequentially applied by repeating the previous procedures. Nuclei were stained with DAPI (Sigma-Aldrich, D9542) after all the human antigens had been labeled. Multispectral images were obtained by scanning the stained slides with the Mantra System (PerkinElmer, Waltham, Massachusetts, US) and analyzed using inForm image analysis software (PerkinElmer, Waltham, Massachusetts, US) (Fig. 4c).

For histological analysis, the bladders of xenograft mice were fixed with 4% paraformaldehyde for 24 h. After cryoprotection in 30% sucrose for 24 h, each bladder was sliced into 20 μm sections using a cryostat (Leica, Lussloch, Germany) and stained with hematoxylin and eosin (H&E). For immunofluorescence (IF) staining, bladders were stained with antibodies against ID2 (NBP-88630; NovusBio), TFCP2L1 (OAAB09732; Aviva Systems Biology), CDK1 (ab131450; Abcam, Cambridge, MA, USA), CD44 (ab78960; Abcam), and cytokeratin 14 (KRT14; ab7800; Abcam). Alexa Fluor 488-conjugated (A11001 and A11008) anti-mouse and anti-rabbit antibodies or an Alexa Fluor 546-conjugated anti-rabbit antibody (A11010) were used as secondary antibodies (Molecular Probes). Nuclei were counterstained with 4′,6-diamino-2-phenylindole (DAPI; D9542; Sigma–Aldrich). Three representative areas per slide were randomly selected for each animal. The stained samples were imaged using an inverted fluorescence microscope (EVOS® FL Color Imaging System, Life Technologies).

Briefly, 3 × 10⁴ cells were seeded on rounded cover slips in 24-well plates and treated the next day as indicated. Cell monolayers were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Blocking was performed for 2 h at room temperature with 1% BSA in PBS with 0.05% Tween 20 (PBST). Cells were incubated with primary antibodies overnight at 4 °C and washed 3 times for 5 min with 0.1% PBST. Primary antibodies were directed against αSMA (Abcam, Cambridge ab7817, UK), FN1 (Sigma Aldrich, F7387), and pNFκB Ser536 (Cell Signaling, Danvers, MA 3031, USA). Secondary antibodies were conjugated with AlexaFlour-488 and AlexaFlour-547 fluorescent dyes (Thermo Fisher Scientific, Waltham, MA, USA, A-11034 and A-11029, respectively) and incubated with the cells for 1 h at room temperature. Nuclei were counterstained with DAPI (Sigma Aldrich, D-9542). After mounting with DABCO-Mowiol, the samples were examined and photographed using an epifluorescence microscope (Olympus Provis AX70, Tokyo, Japan). Quantification of αSMA-positive cells and the FN1 fluorescence area was performed in ImageJ. The percentage of positive cells and FN1 fluorescence were calculated based on the total number of DAPI-positive nuclei and the total image area, respectively. An average of four sections per patient were quantified.

Human embryonic lungs were fixed on ice for 1-3 h depending their size in 4% (w/v) paraformaldehyde in 1× PBS, washed in 15, 20 and 30% (w/v) sucrose solutions in PBS for 1 h each at room temperatures before incubating in a 1:1 mix of optimal cutting temperature compound (OCT; Tissue-tek):30% sucrose overnight at 4°C. Lungs were embedded in 100% OCT and stored at −70°C before sectioning. Human embryonic lung cryosections (12 μm) were rinsed with PBS and permeabilised with 0.2% Triton-X/PBS (washing solution). Normal donkey serum (5%; Stratech, 017-000-121-JIR) in washing solution containing 0.5% (w/v) bovine serum albumin (BSA) was used for blocking at room temperature for 1 h. Primary antibodies (Table S2) in blocking solution were incubated at 4°C overnight. After three washes in washing solution, secondary antibodies (Table S3) in 0.2% Triton-X/0.5% BSA/PBS were incubated at 4°C overnight. After three washes, DAPI (100 ng/ml, Sigma, D9542) was added for 30 min at room temperature. Samples were mounted in Fluoromount Aqueous Mounting Medium (Sigma, F4680). Confocal z stacks of single planes were acquired using Leica SP8 at an optical resolution of 1024×1024 at 40×. Images were processed using ImageJ (version 2.1.0).

GFP expression analysis of in vitro cultured cells was performed by flow cytometry: Cells were trypsinized, diluted in a 1% paraformaldehyde‐2% FBS solution, stained with DAPI (D9542, Sigma), and analyzed with FACS flow cytometer (CyAn™, DAKO).