Reagents and antibodies
Small interference RNAs (siRNAs) were from Ribobio (Guangzhou, China). Primers used for qPCR sequence were from Ribobio (Guangzhou, China), which are as follows: GAPDH (Forward: 5′-GGTCCTCAGTGTAGCCCAAG-3′, Reverse: 5′-AATGTGTC CGTCGTGGATCT-3′), E-cadherin (Forward: 5′-GTCTCTCTCACCACCTCCACA-3′, Reverse: 5′-CTCGGACACTTCCACTCTCTTT-3′), N-cadherin (Forward: 5′-TGCTA CTTTCCTTGCTTCTGAC-3′, Reverse: 5′-TAACACTTGAGGGGCATTGTC-3′), Vimentin (Forward: 5′-GAAGAGAACTTTGCCGTTGAAG-3′, Reverse: 5′-GAAGG TGACGAGCCATTTC-3′), Snail (Forward: 5′-TCGGAAGCCTAACTACACGA-3′, Reverse: 5′-AGATGAGCATTGGCAGCGAG-3′), Twist (Forward: 5′-GCAAGAAGT CGAGCGAAGAT-3′, Reverse: 5′-GCTCTGCAGCTCCTCGAA -3′).
Antibodies for GAPDH (60004-1-Ig), EGFR (18986-1-AP), AKT (10176-2-Ig), p-AKT (66444-1-Ig), N-cadherin (22018-1-AP), E-cadherin (20874-1-AP), Vimentin (10366-1-AP) were purchased from proteintech. Antibodies for p-EGFR (DF2969) were purchased from Affinity. Antibodies for CHRM3 (GTX111637) were purchased from Gene Tex. Reagents of 4-DAMP (1952-15-4), ACh (60-31-1) were purchased from Sigma Aldrich.
Cell lines and cell culture
Human ESCC cell lines, including TE1 and KYSE30, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Both of them were cultured in RPMI-1640 medium supplemented containing 10% fetal bovine serum (Clark, AUS). All cell lines were maintained in a humidified cell incubator at 37°C with an atmosphere of 5% CO2.
Clinical tissue microarray and Immunohistochemical staining
76 ESCC tissues and pair-matched para-carcinoma tissues were obtained from ESCC patients who underwent radical surgery at the Affiliated Huai’an No.1 People’s Hospital of Nanjing Medical University (Huai’an, China). Each of them was evaluated histologically by two experienced pathologists. This study was approved by the Committee for Ethical Review of Research involving Human Subjects of Nanjing Medical University and informed consent was obtained from all patients.Tissues were paraffin-embedded and cut into 4 μm think sections, which were then deparaffinized and rehydrated. All sections were incubated with anti-VAChT antibodies at 4°C in the refrigerator overnight. After incubating with HRP-conjugated secondary antibody (Dako, Glostrup, Denmark), the sections were counterstained with Mayer’s hematoxylin. The neural density calculation was independently evaluated by two pathologists.
Sequences of siRNA used for knockdown of CHRM3 were previously described  and synthesized by OBiO Technology (Shanghai, China). Cells were transfected with siRNAs using Lipofectamine 3000 (Invitrogen, USA), according to the manufacturer’s protocol. The interference efficiency was determined by western blotting after transfection for 48 h.
Cells were seeded into 6-well plate and scratches across the cell layers were made by the 200 μL pipet. Floating cells were washed 2 times with PBS. Photos were taken under microscope at 0, 24, 48, 72 and 96 h. The wound healing of each area was measured by ImageJ.
Migration and invasion assay
Cell migration and invasion ability were analyzed using transwell chambers (8-μm pore size, Corning Costar, Cambridge, MA, USA). 100 μL resuspended cells (0.8×105 per mL) were inoculated in each transwell, then cultured in 600 μL complete medium for 72 h. After 72 h incubation, cells were washed with PBS. Then penetrated cells were fixed by 4% paraformaldehyde for 25 minutes and stained with 600 μL 0.1% crystal violet solution for another 25 minutes. Pictures were collected under microscope by randomly selecting five fields of vision and the migration ability of cells was analyzed. Unlike migration, in invasion assay, the upper-chamber needed to add 100 μL matrigel gel (diluted with medium without FBS) in advance.
Real-time quantitative PCR (qRT-PCR)
Total RNA was extracted using Trizol reagent (Thermo, USA) as the manufacturer’s instructions. The cDNA was reversed with FastQuant RT Kit (Tiangen, Beijing, China). Then, PCR reaction was performed in triplicate with SuperReal PreMix Plus (Tiangen, Beijing, China) on the Real-Time PCR Detection System (Roche, California, USA). The formula 2-△△CT was used for relative expression, GAPDH as negative control. Western blot and Immunoprecipitation assay
Cells were dissolved in lysis buffer (RIPA, KeyGEN, China) and the concentrations were examined by BCA Protein Assay kit (KeyGEN, China). For each sample,30 μg total protein was applied for 10% or 7.5% SDS-PAGE. The proteins were transferred to PVDF membranes (Roche, Mannheim, Germany). The membranes were sealed with 5% BSA and incubated with primary antibodies in 4°C overnight and corresponding secondary antibodies at normal atmospheric temperature for 1 h the next day. Protein bands were visualized with ECL substrate (Affinity). For CO-IP, Protein A/G PLUS-Agarose (SANTA CRUZ BIOTECHNOLOGY, INC) was used. Protein bands were visualized with Automatic chemiluminescence image analysis system (Bio-Rad, USA).
RNA-seq and data analysis
Cell gene expression profile (choline stimulation groups and controls) was detected by RNA screening analysis in Novogenen Company (Tianjin, China). Raw data statistical significance assessment was accomplished using a Welch t-test with Benjamini-Hochberg FDR (|fold change | > 1 and padj < 0.05 as significant). Significant difference analysis and function analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was executed, and |Z - score | > 2 is considered meaningful.
Dorsal root ganglia (DRG) co-cultured with ESCC cells
The DRGs were extracted according to the method previously described . The mice were carefully necked and expose the position of the spine. Cut off the diaphragm, viscera and ribs from the anterior side of the spine, then cut the femur with a bone shear, and finally make a transverse cut at the femur level to remove the entire spine. From the head to the tail, peel off the spine to find the lower dorsal root ganglion and expose it. Carefully lift the dorsal root ganglion upward, using fine spring shears to trim the small axon bundles and soft tissues around DGRs, then transfer them to precooled HBSS solution. ESCC cells in logarithmic growth phase were digested with trypsin, terminated by complete medium, centrifuged and resuscitated, then transferred to 6-well plate or 24-well plate. DRGs were planted in the upper or lower layer of transwell chamber as needed to establish a co-incubation model.
All the cell experiments above were performed in triplicate. All data were analyzed by SPSS26.0 (SPSS, Chicago, IL, USA) or GraphPad PrismV8 (GraphPad Prism, Inc., La Jolla, CA, USA) and presented in the form of mean ± standard deviation (SD). Student’s t-test or one-way ANOVA was used for statistical analysis and p < 0.05 was considered to be significant. Nerve density and the patient clinicopathological parameters was performed by χ2-test and Kaplan-Meier survival analysis with log-rank test.