Primary NPC tissues and normal adjacent tissues were collected from the patients during biopsy in Eye, Ear, Nose and Throat Hospital, Fudan University. Tissues were transferred on ice from the outpatient clinic to laboratory immediately when the biopsy examination of NPC patient was finished. Every sample was divided into two parts, and half was for pathologicalhistology and half for 3D culture. The cultures were discarded once the pathologicalhistology showed negative outcome. Consent forms were signed by all patients and the experimental procedures were approved by the Institutional Research Ethics Committee of Eye, Ear, Nose and Throat Hospital, Fudan University (Permit Number: 2019081).
Culture medium preparation
The medium was sterilized through 0.22μm filter (Millipore, #SLGP033RB) before use. The culture medium was based on advanced DMEM/F12 (ThermoFisher, #11320033) with epidermal growth factor (EGF, 50 ng/ml, ThermoFisher, #PHG0311), basic fibroblast growth factor (bFGF, 20 ng/ml, (ThermoFisher, #PHG0369), B27 (2%, Thermo Fisher, #17504044), N2 (1%, ThermoFisher, #17502001), HEPES (1 mM, ThermoFisher, #15630080), Penicillin-Streptomycin (P/S, 1%, ThermoFisher, #10378016) and GlutmaxTM supplement (1%, ThermoFisher, #35050061). In addition, chemical cocktail was supplemented into the culture medium to support the favorable outgrowth of NPC organoids, including R-spondin-1 (200 ng/ml, R&D, #4645-RS), Noggin (100 ng/ml, PeproTech, #250-38), Wnt3a (50 ng/ml, R&D, #5036-WN), Y27632 (10 μM, Sigma-Aldrich, #Y0503), A83-01 (500 nM, Sigma-Aldrich, #SML0788), N-acetylcysteine (1.25 mM, Sigma-Aldrich, #A9165) and nicotinamide (10 mM Sigma-Aldrich, #N0636). The medium was stored at 4℃ and worked for one week.
Primary nasopharyngeal carcinoma organoid culture
All the dissection apparatus was sterilized by autoclaving at 121℃ for 20min. Matrigel Matrix GFR (Corning, #356231) was kept on ice until ready to use. All procedures were conducted on ice to keep the maximal viability of cultured cells. The samples were washed in ice-cold phosphate-buffered saline (PBS) for twice and sheared into small pieces. Tissues were transferred into red blood cell lysis buffer (Miltenyi Biotec, #130094183) to remove the blood cell debris and then washed in PBS. Tissue pieces were transferred to petri dishes and minced using an eye scissor into 1–2 mm3 small pieces. Approximately 1~1.5 mL 0.25% trypsin-EDTA was added into tissue pieces and incubated at 37℃ for 20~30 minutes to make single cell preparations. Trypsinization was stopped by addition of 0.5mL defined trypsin inhibitor (DTI, Sigma Aldrich, #T7659), and cells were centrifuged at 1,500 r.p.m. for 3 min. The supernatant was aspirated and discarded without disrupting the tumor pieces and pelleted cells at the bottom of the tube. While keeping on ice, the pellet was washed and resuspended in 1mL culture medium. The single cell suspensions were filtered by 70-µm and 40-µm nylon meshes (BD Falcon, #352350 and #352340) before seeding into Ultra-low-attached 24-well plate (Corning, #3473). Approximately 10000 cells in 0.7mL culture medium was seeded in each well. Cultures were supplemented with 3-5% (V/V) Matrigel Matrix GFR. The medium was changed every 3-4 days based on the organoid density.
Organoid passaging and Cisplatin treatment
On Day14 post culture, organoids were collected by centrifuging at 1,200 r.p.m. Cell pellet were incubated with 0.25% trypsin-EDTA for 10 min at 37℃. Single cell suspension was prepared by separating the cell pellet using 1ml microsyringe. Cell suspension in culture medium was seeded in low attachment 24-well plate at density of 5000 cells in each well. For cisplatin treatment, organoids were treated with 5μM cisplatin (Sigma-Aldrich, #C2210000) for 24 hours.
Organoids were collected and fixed in 4% paraformaldehyde for 15 minutes on ice. After being washed in PBS, organoids were equilibrated sequentially in 10%, 20%, and 30% sucrose, and embedded in gelatin by freezing in liquid nitrogen. The frozen organoids were cut into 20μm coronal sections on a Cryostat (Leica CM1950).
Immunofluorescence staining was performed according to a standard protocol. The organoid sections were rinsed in PBS for three times and blocked for 1 hour at room temperature in PBS containing 5% bovine serum albumin (BSA) and 0.3% Triton X-100, and followed by incubation with primary antibodies overnight at 4°C in a humidified chamber. Afterwards, the sections were rinsed three times in PBS followed by incubation with second antibodies at room temperature for 1 hour. Nucleus were counterstained with DAPI (1μg/mL, ThermoFisher, #D3571). The primary antibodies used included rabbit anti-Sox2 (#ab92494, 1:100; Abcam Inc), mouse anti-Sox2(#sc-365823, 1:100; Santa Cruz Technology), mouse anti- Ki67 (#550609, 1:100; BD Biosciences), rabbit anti-P63 (#ab63881, 1:200; Abcam Inc), goat anti-CD54 (#BAF796, 1:500; R&D), rabbit anti-Vimentin (#ab45939, 1:100; Abcam Inc), rabbit anti-E-cadherin (#ab40772, 1:500; Abcam Inc), goat anti- E-cadherin (#AF748, 1:100; R&D), mouse anti-EGFR (#sc-120, 1:200; Santa Cruz Biotechnology), mouse anti-CK34βE12 (#m0630, 1:100; DAKO)，rabbit anti-cleaved Caspase-3 (#9664, 1:100; Cell Signaling Technology). All secondary antibodies were purchased from ThermoFisher and diluted as 1:300, including Alexa Fluor 488 donkey anti-mouse (#A21202), donkey anti-goat (#A11055), donkey anti-rabbit (#A21206), Alexa Fluor 568 donkey anti-rabbit (#A10042), Alexa Fluor 594 donkey anti-rabbit (#A21207), donkey anti-goat (#A11058), Alexa Fluor 633 donkey anti-goat (#A21082) and Alexa Fluor 647 donkey anti-rabbit (#A31573). Fluorescent images were captured using Leica TCS SP8 with LAS AF Lite software.
Organoids size was measured by SPOT software. Measurement was carried out by someone who blinded to the experimental conditions design to eliminate bias. Data were presented as mean ± SEM from at least three independent experiments. The statistical difference was determined by unpaired t test using Graphpad Prism software and the p value less than 0.05 was considered as significant difference.