Data collection

A comprehensive cancer genomics program, The Cancer Genome Atlas (TCGA) has conducted molecular characterizations of 33 primary cancer types. Using UALCAN (https://ualcan.path.uab.edu/analysis.html), exploration of FOXC1 expression in esophageal squamous cell carcinoma was conducted utilizing data extracted from the TCGA database.

Human esophageal squamous cell carcinoma cell lines, such as TE-1, ECA-109, KYSE-30, and KYSE-150, were procured from the Institute of Biological Sciences of the Chinese Academy of Sciences in Shanghai. Subsequently, routine mycoplasma contamination testing was conducted. Cells were maintained at 37C with 5% CO2, cultured in DMEM medium (GIBCO) containing 1% penicillin-streptomycin and 10% Fetal Bovine Serum (GIBCO).

Cells were seeded into 6-well plates for transient knockdown transfection, followed by the transfection of 100pmol siRNA-FOXC1 (GenePharma, Shanghai, China) using HighGene (ABclonal, Wuhan, China) ECAh well, following the manufacturers guidance. Cells were seeded into 6-well plates for transient gene overexpression transfection, followed by the transfection of the plasmids expressing CBX7 or IGF-1R were purchased from Genechem (Shanghai, China) using HighGene (ABclonal, Wuhan, China).

At 48h post-transfection, transfection efficiency was assessed using RT-qPCR and western blot. For stable transfection, ECA-109 and KYSE-150 cells were transfected with lentivirus vectors encoding either FOXC1-targeting shRNA or non-targeting control shRNA, following the manufacturers instructions (Genechem, Shanghai, China). Briefly, cells were seeded in 6-well plates, and when the cell density reached 30%, a medium containing viral fluid at an MOI of 10, without serum, was added. This medium was replaced with a complete medium 24h later. After lentiviral infection, ECA-109 cells and KYSE-150 cells underwent a two-week selection process with 1g/mL puromycin to obtain stable clones. Transfection efficiency for each vector was evaluated through a western blot.

In the cell migration experiment, 1 105 cells were re-suspended in 200L serum-free DMEM medium and added to the upper compartment, and 500 l DMEM medium containing 10%FBS was added to the lower compartment to induce the migration of cells. In the cell invasion experiment, the cells re-suspended in 200L serum-free DMEM medium were added to the upper chamber coated with Matrigel matrix (Corning, 356234), and the rest procedures were performed the same as the cell migration experiment. Fixation with 4% paraformaldehyde and staining with crystal violet dye were conducted after a 24-h incubation period. Subsequently, IMAGEJ software was employed for cell number quantification.

We introduced a seeding density of 2000 cells per well into 96-well plates and established an arrangement of 10 sub-wells. After the cells were fully attached to the plate, CCK8 reagent (10 l per well) was added at 0,24,48,72,96h, respectively. Subjected to incubation in the absence of light for an hour, the microplate reader was employed to analyze the absorbance at 450nM. Three repetitions of the experiments were executed, followed by the final statistical analysis performed using GraphPad Prism 8.0.

6-well plates were used for cell inoculation, with ECAh well receiving 1 103 cells, and subsequent culture was carried out in DMEM medium containing 10% FBS and 1% Penicillin-Streptomycin Solution. After a 14-day incubation period, cell fixation was performed using 4% paraformaldehyde, followed by staining with 0.1% crystal violet dye. The colony count was determined using ImageJ software.

The ECA-109 cells and KYSE-150 cells were plated into ultra-low six-well plates (Corning) at 1 103 cells/well. The cells were cultured in serum-free DMEM/F12(Gibco) with 2% B27(Invitrogen)20ng/mL EGF(PeproTech)20ng/mL bFGF(PeproTech) for 14 days. The size of the tumor spheroids was observed under a light microscope and the count of spheres with a diameter greater than 100M was counted.

ECA-109 cells and KYSE-150 cells were incubated in 6-well plates in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 1M cisplatin. After incubation for 24h, the cells were gathered, and an Annexin V-FITC apoptosis analysis kit (Elabscience Biotechnology) was utilized to assess the percentage of apoptotic cells, following the step-by-step instructions in the user manual. Results were represented as the mean of % cell death of at least three independent replicates.

1 106 ECA-109 cells and 1 106 KYSE-150 cells were incubated with CD44 antibody(R&D Systems)for 10min at room temperature, and washed twice twice after that. The FACS was performed using the Beckman CytoFLEX and the percentage of CD44+ cells was analyzed.

Cells were subjected to RNA isolation using Trizol (Vazyme) followed by reverse transcription into cDNA using the Reverse Transcriptase Kit (Abclonal). RT-qPCR was performed with the primers for FOXC1, CBX7, IGF- 1R, CD133, CD44, and -actin, and the fold change was calculated by the 2-Ct method. Cloud-Seq Biotech (Shanghai, China) conducted RNA high-throughput sequencing, wherein the removal of rRNAs was accomplished using the GenSeq rRNA Removal Kit (GenSeq, Inc.) with total RNA. After the removal of rRNA from the samples, library construction was carried out utilizing the GenSeq Low Input RNA Library Prep Kit (GenSeq, Inc.), following the prescribed protocol from the manufacturer. Quality control and quantification of the libraries were executed using the BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). The sequencing of the libraries transpired on an Illumina Novaseq instrument, employing 150bp paired-end reads. Primer sequences are listed in Table 1.

Proteins were extracted using RIPA lysate (Beyotime) supplemented with 1% PMSF (Beyotime) and 2% phosphatase inhibitor (Beyotime). Following electrophoretic separation through SDS-PAGE, the proteins were transferred onto PVDF membranes. After blocking with 5% skim milk, primary antibodies specific for FOXC1 (ab227977, Abcam,1:1000), CD44 (A19020, Abclonal,1:1000), CD133 (A0219, Abclonal,1:1000), CBX7 (ab178411, Abcam,1:1000), IGF-1R (ab182408, Abcam,1:1000), phosphor-IGF-1R (ab39398, Abcam,1:1000), Akt (4691, Cell Signaling Technology,1:1000), phospho-Akt (S473) (4060, Cell Signaling Technology,1:1000), ERK1/2 (ab184699, Abcam,1:1000), phospho -ERK1/2 (ab201015, Abcam,1:1000) primary antibodies overnight and -actin (AC026, Abclonal,1:10000) as internal reference were used for protein examination. .

Cultivated cells were fixed, chromatin sonicated, immunoprecipitated, and DNA purified according to ChIP-IT High Sensitivity kit (Active Motif) instructions, and the relative abundance of target DNA was analyzed by qPCR. Primer sequences are listed in Table 1.

Tissue samples for this study were sourced from individuals diagnosed with esophageal squamous cell carcinoma at Tongji Universitys Dongfang Hospital, totaling 79 patients. Following fixation in formalin and embedding in paraffin, tissue sections were sliced to a thickness of 4m. Subsequently, the sections underwent deparaffinization and hydration through immersion in xylene and graded alcohols. Heat-induced antigen retrieval was conducted in EDTA buffer (pH 8.0) for 15minutes, utilizing a microwave oven. To minimize nonspecific staining, blocking was carried out with 10% goat serum. Following this, specific primary antibodies, including FOXC1 (ab227977, Abcam, 1:200), CD44 (A19020, Abclonal, 1:200), and CD133 (A0219, Abclonal, 1:100), were applied to the sections and left to incubate overnight at 4C. The slides were then counterstained with light hematoxylin, subjected to dehydration, and covered with slips. The outcomes were evaluated by two pathologists independently, with no access to clinical data, and subsequent analyses encompassed TNM staging and survival assessment. The study was conducted with the written informed consent of the patients and approved by the Institutional Review Committee of East Hospital Affiliated with Tongji University in Shanghai.

The Animal Protection and Use Committee of Tongji University approved all animal experiments. Animal experimentation involved the utilization of 10 BALB/c nude mice, all of the female gender and aged 6 weeks.KYSE-150-FOXC1-LV and KYSE-150-NC-LV were injected subcutaneously into the right abdomen of two groups of mice, purchased from Gempharmatech Co., Ltd. The mice were euthanized, and the tumors were subsequently extracted after 4 weeks for size measurement and weighing. A portion of the tumor tissue was fixed with 10% paraformaldehyde and paraffin-embedded for subsequent immunohistochemical staining analysis, and the rest was used for protein and mRNA extraction.

The in vivo experiments were repeated three times and the final results were taken as the meanstandard deviation. Statistical comparison analysis was performed by GraphPad Prism 8.0. For survival analysis, the Kaplan-Meier method and log-rank test were employed, and statistical significance was established for P values less than 0.05.

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