fGOs at 7 days from last passage were removed from Matrigel® by incubation in Cell Recovery Solution for 45 min at 4 °C. This treatment released the organoids from the Matrigel® without damaging the structure of the organoids, allowing whole mount immunofluorescence staining. Organoids were collected, washed in PBS, and transferred to 1% BSA pre-coated 1.5 mL Eppendorf tubes, and resuspended in 4% PFA (PFA; Sigma-Aldrich, 100496) for 20 min in rotation. PFA was removed and quenched with 0.1 M NH4Cl (Sigma-Aldrich, 254134) for 1 h in rotation to decrease aldehyde-related autofluorescence. Quenched organoids were stored in PBS with 1% penicillin–streptomycin until staining.

fGAs were first fixed in 4% PFA for 30 min, washed with PBS, and residual PFA quenched with 0.1 M ammonium chloride (NH4Cl; Sigma-Aldrich, A9434) for 60 min to quench unreacted aldehyde groups and reduce autofluorescence. Samples were then dehydrated overnight in 30% sucrose (Sigma-Aldrich, S9378), before embedding in Polyfreeze Tissue Freezing Medium (Polysciences, 25113) over dry ice, and then sectioned at 7 μm on a Bright OTF cryostat. Sections were stored at − 20°C until staining. After defrosting, sections were re-fixed to the slide with 5 min of 4% PFA, quenched with 15 min of 0.1 M NH4Cl, and washed with PBS.

fGOs and fGAs were blocked and permeabilised with 0.5% Triton X-100 (Sigma-Aldrich, 100496) with 1% BSA in PBS for 2 h (fGOs) or 1h (fGA sections) at room temperature.

Primary antibodies were diluted in blocking buffer and incubated with sections or organoids for 24 h at 4 °C in rotation. Samples were then extensively washed in 0.5% Triton X-100 in PBS. Secondary antibodies were diluted and applied to samples as for primary antibodies and incubated overnight at 4 °C. fGOs in suspension were transferred to a glass-bottomed Petri dish immediately prior to imaging. fGAs’ sections were mounted in non-DAPI mounting medium (Biorad, BUF058A).

Primary antibodies: Chromogranin A (Abcam, ab15160), Mucin 5AC (Invitrogen, ma5-12178), Mucin 6 (Abcam, ab216017), Ki67 (Abcam, ab15580), Integrin beta-4 (INTB4) (Abcam, ab110167) all at 1 in 100 dilution. Ezrin (EZR) (thermos Fisher, PA5-29358) at dilution 1:200.

Secondary antibodies were AlexaFluor® anti-mouse 488 (Thermo Fisher, A11001), AlexaFluor® anti-rabbit 568 (Thermo Fisher, A11011), anti-Rat 594 (Thermo Fisher, A11007) all at 1 in 200 dilution. AlexaFluor® Phalloidin 647 (Thermo Fisher, A22287) was used at 1:100 dilution. Hoechst 33342 (Thermo Fisher, H1399) at 10 μg/mL.

Whole mount stainings were acquired on the Zeiss LSM 710 confocal microscope. Overview images were acquired using the Zeiss Plan-Apochromat 10x, NA 0.45 mm, dry (air) objective (working distance: 2 mm) with tile scanning. Brightfield images of organoids in culture were acquired using on a Zeiss Axio Observer A1 inverted widefield microscope with a Colibri 5 LED light source and Axiocam MRm camera system.

fGAs were removed from the culture well and excess collagen I hydrogel was trimmed from the edges with a scalpel under a dissecting microscope. fGAs were then minced with a scalpel in a petri dish on ice prior to transfer to lysis buffer. RNA extraction was done using Qiagen RNeasy Micro (74004) kit. RNA was extracted following the manufacturers recommendations. RNA quantified by spectrophotometry using the NanoDrop 1000 (Thermo Fisher Scientific) and immediately reverse transcribed to cDNA or stored at − 80 °C. Reverse transcription was performed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) according to the manufacturer’s instructions.

Quantitative real-time PCR was performed using the Applied Biosystems™ TaqMan® gene expression assays (all from Thermo Fisher Scientific) according to manufacturer’s instructions in 96-well PCR plates. The TaqMan® FAM-labelled probes were used according to the manufacturer’s instructions. The following probes (all from Thermo Fisher) were used: GAPDH (glyceraldehyde 3-phosphate dehydrogenase), LGR5 (leucine-rich repeat-containing G-protein coupled receptor 5), AXIN2 (axin-like protein), MUC5AC (mucin 5AC), MUC6 (mucin 6), PGA5 (pepsinogen A5), SST (somatostatin), GAST (gastrin), CHGA (chromogranin A). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as endogenous control in each well with the VIC reporter. The plate was loaded on a StepOnePlus Real Time PCR System running StepOne Version 2.3 software (Applied Biosystems, 4376600) for reactions. Data were downloaded to Microsoft Excel for Mac (Microsoft, Version 16.55) for analysis. RT-PCR data were analysed using the ΔΔCt method. Relative fold change was graphically represented as individual data points and mean ± standard error of the mean (SEM), using GraphPad Prism for Mac (Version 10).