Collection of clinical samples

OSF and their adjacent normal mucosa (ANM) tissues were collected from the surgical specimens of OSCC patients with betel nut chewing history (n = 10), according to IRB–approved guidelines at the Ethics Committee of School and Hospital of Stomatology at Wuhan University (IRB-ID:2021A18). Clinical information of relevant samples has been described in supplementary table 1. The pathological diagnosis of OSF samples was processed by 2 experienced pathologists according to the Pindborg and Sirsat criteria: excessive collagen deposition in the connective tissues below the oral mucosal epithelium, local inflammation in the lamina propria or deep connective tissues, obliterated vessels and degenerative changes in the muscles2,43.

Organoid culture

OSF and ANM tissues were minced into small pieces and dissociated into single cells for organoid culture, the composition of basal organoid culture medium was prepared as we recently described44,45. Advanced DMEM/F12 was supplemented with 1 x N2 (07152, Stemcell technology, CA), 1 x B27 (7511, Stemcell technology, CA), 10 μmol/L Y-27632 (HY-10071, MCE, USA), 0.5 μmol/L A83-01 (HY-10432, MCE, USA), 10 μmol/L forskolin (HY-15371, MCE, USA) and 10 ng/mL EGF (236-EG-200, R&D, USA)44,45. For organoid passage assay, 125 ng/mL Noggin (6057, R&D system, USA), 125 ng/mL R-spondin (4645, R&D system, USA) and 2 μg/mL Wnt3A (5036, R&D system, USA) were added to the basal medium. The growth dynamic of organoid was recorded under a fluorescence microscope (Leica Microsystems, GER).

Fibroblasts and endothelial cells culture

OSF and ANM tissues were minced into small pieces for explant culture as described previously46,47. Briefly, the explants were digested with 0.25% trypsin containing EDTA (25200-056, Gibco, USA) for 30 min. After centrifugation, the supernatant was removed and the digested explants were then seeded to a T25 culture flask. The flask was inverted overnight at 37°C in a humidified atmosphere containing 5% CO2. After the explants were attached to the culture flask, MEM ALPHA (C12571500BT, Gibco, USA) containing 10% foetal bovine serum (FBS, 10099141, Gibco, USA) were added to the flask for further culture and expansion. The human umbilical vein endothelial cells (HUVECs) cell line was purchased from Sciencell (DFSC-EC-01, Sciencell, USA) and were cultured in Endothelial Cell Medium (ECM; 1001, Sciencell, USA). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

Construction of Fibroblast-attached Organoid (FAO)

Briefly, 105 epithelial cells and 3 × 105 fibroblast were suspended in ultralow-attached plates (ULA) for 24–48 h to generate cell clusters, which were then embedded in Matrigel (356237, Corning, USA) to construct FAO, as we recently described26. To promote the efficiency of generating OSF- and ANM-derived FAO, the basal organoid culture medium was supplemented with 125 ng/mL Noggin (6057, R&D system, USA) and 125 ng/mL R-spondin (4645, R&D system, USA). The morphology and growth dynamic of FAOs was recorded under a fluorescence microscope (Leica Microsystems, GER). To validate the spatial location of epithelial cells and fibroblasts in FAOs, the cells were respectively labelled with mCherry or GFP. Then, an automated microlens-enhanced spinning disc confocal microscope (Opera PhenixTM High Content Screening System, PerkinElmer) was utilized to capture the fluorescence signal of cells at distinct plane30. The reconstruction of superimposed images for FAOs was processed by the Harmony software v.4.9 (PerkinElmer) according to the manufacture’s instruction.

Fibroblast-colony formation assay

Single fibroblast was suspended in ultralow-attached plate (3471, Corning) for 24 h to generate clusters. Then, the clusters were seeded in matrigel (356237, Corning, USA). Morphological characteristics were observed under a fluorescence microscope (Leica Microsystems, GER). The extension length of the cluster was measured to evaluate the fibroblast activation as described previously48.

Co-culture of fibroblast and HUVEC

Single fibroblast (labeled with GFP) and HUVEC (labeled with mCherry) were mixed at a 3:1 ratio and suspended in ultralow-attached plates (ULA) for 24 h to generate fibroblast-HUVEC clusters. The cluster with diameter >40 μm was selected for embedding in matrigel (356237, Corning, USA), and the morphology of clusters was recorded under an inverted microscope (Leica Microsystems, GER). The number of cherry-labeled cells with sprouting >10 μm were counted to evaluate the activation of HUVEC. The length of GFF-labeled cells with extension >40 μm were measured to evaluate the activation of fibroblast. The sprouting of HUVEC in the co-culture model was analyzed by Opera PhenixTM High Content Screening System.

Generation of vascularized FAO (v-FAO)

Briefly, 105 epithelial cells and 3 × 105 fibroblast and 105 HUVEC were suspended in ultralow-attached plates (ULA) for 24 h to generate cell clusters, which were then embedded in matrigel (356237, Corning, USA) to generate v-FAO. To promote the efficiency of generating v-FAO, the basal organoid culture medium was supplemented with 33.3% MEM ALPHA (C12571500BT, Gibco, USA) and 33.3% EGM-2 BulletKit (CC-3162, Lonza, CH). To analysis the phenotype transition of cells, the fibroblasts were labeled with GFP, while the HUVEC were labeled with mCherry. The growth dynamic of v-FAO, morphological activation of fibroblasts, and sprouting of HUVEC was recored using an inverted microscope (Leica Microsystems, GER).

Immunofluorescence assay

Serial freezing section of FAO and v-FAO was processed as we recently described44,49. The freezing section at the core of FAO/v-FAO was employed to analysis the cell composite and protein expression. The following primary antibodies were used for immunofluorescence: THBS1 (ab1823, Abcam, UK), CD31 (3528, CST, USA), Collagen III (ab184993, Abcam, UK), Pan-Keratin (4545, CST, USA), COL1A1 (72026, CST, USA), COL3A1 (66887, CST, USA).

Immunoblotting assay

The following primary antibodies were used for immunoblotting according to our previously reported protocol: THBS1 (18204-1-AP, Proteintech, China), Pan-Keratin (4545 S, CST, USA), Smad2 (5339, CST, USA), Phospho-Smad2 (p-Smad2, 3108, CST, USA), Smad3 (9523, CST, USA), Phospho-Smad3 (p-Smad3, 9520, CST, USA) α-SMA (19245 S, CST, USA), caspase-3, cleaved caspase-3 (ab32042, Abcam, UK).

Chromatin immunoprecipitation (ChIP) assay

The ChIP assay was conducted according to the manufacturer’s instruction of the ChIP kit (Thermo Fisher Scientific, USA). Briefly, 2×106 cells in each group were harvest for the assay. The protein-DNA complexes were cross-linked by 1% formaldehyde then quenched by glycine, after which the cells were lysed in lysis buffer. Then the lysates containing chromatin were sonicated to shear DNA. The supernatant after centrifugation was diluted in IP dilution buffer for immunoprecipitation. After incubated with antibodies or IgG at 4 °C overnight, ChIP Grade Protein A/G Magnetic Beads were used to bind chromatin DNA. The cross-links were reversed after the immunoprecipitation complexes were eluted, and then the DNA was purified and subjected for qPCR assay. The primers sequence used in the qPCR assay were listed in Table S2.

Masson staining

Tissues of SD rats were embedded with paraffin and then mounted on slides. Masson staining was employed to evaluate the collagen deposition according to the he manufacturer’s instructions (G1006, Servicebio, China).

Enzyme-linked immunosorbent assay (ELISA)

To measure TGF-β1 released by organoids or corresponding fibroblasts, cells were seeded into 6-well plates and stimulated with arecoline at the indicated concentrations (10 μg/mL) or not for 48 h. Then, the supernatant was harvested and the concentrations of TGF-β1 were measured using a human TGF-β1 ELISA kit (EHC107b.96, Neo Bioscience Technology, China) according to the manufacturer’s instructions. To confirm the THBS1 secretion of fibroblast with THBS1 knockdown or overexpression, the supernatant was harvested and the concentrations of THBS1 were measured using a Human THBS1/TSP1 ELISA Kit (EK0899, Raybiotech, USA).

Tube formation assay

HUVECs were cultured in Endothelial Cell Medium (1001, Sciencell, USA). When reached 80% confluence, the HUVECs were seeded onto 96 well plates embedded with matrigel in the presence of VEGFA, hrTHBS1, fibroblast CM and Anti-CD36, respectively. Tube formation was visualized under inverted microscope (Leica Microsystems, GER) at 12 h after incubation. Total tube length was measured for analysis.

Animal experiments

For animal experiments, 28 female Sprague-Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and maintained according to the protocol approved by the Ethics Committee of the Hospital of Stomatology at Wuhan University (S07921060J). Sprague-Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. In total, 28 SD rats were randomly divided into four groups (i.e. A, B, C, D). The submucosal injection method on the right cheek was used in this experiment. Arecoline (S2614, Selleck, USA), LSKL (HY-P0299, MCE, USA) and SLLK (HY-P0301, MCE, USA) were dissolved in DMSO (A3672, Applichem, GER) to 10 mg/mL for storage and were diluted to 2 mg/mL in 0.9% normal saline (NS) immediately before injection. To establish the OSF lesion (n = 7), the rats in group A were injected with 20% DMSO and the rats in group B were injected with arecoline. As for drug administration assay (n = 7), the rats in group C were injected with SLLK and the rats in group D were injected with LSKL. All groups were injected twice a week and with 100 μL each time. The mouth opening of animals were measured once a week. After 12 weeks, the rats were humanely euthanized, and the buccal tissue was attained for further research.