Sw620

The human tumor cell lines SW480 (primary colon adenocarcinoma), SW620 (metastatic colon adenocarcinoma), MDA-MB-231 (metastatic breast adenocarcinoma) and U87-MG (glioblastoma) were purchased from Sigma-Aldrich. Cells were grown in two different Dulbecco’s modified essential media (DMEM high glucose for SW480 and SW620 and DMEM low glucose for MDA-MB-231 and U87-MG) (Sigma-Aldrich) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin solution (Gibco) at 37 °C with 5% CO₂ in humidified air.
To activate TLR4 cells were incubated with [1 µg/ml] LPS (E. coli 055:B5 LPS, Sigma-Aldrich) for 24 hours, washed three times with PBS and then culture medium was replaced with fresh medium supplemented with 10% certified exosomes-free serum (Gibco). After 24 hours, supernatants were collected and stored at −20 °C until use, while cells were harvested, and their proliferation/viability was determined by the trypan blue exclusion assay.
Cell homogenates were analyzed for TLR4 expression by immunoblotting and the human anti-TLR4 (1:500, Cell Signaling) and anti-β-actin (1:10,000, Sigma-Aldrich) were used as primary antibodies.
All methods of analysis were carried out in accordance with the relevant guidelines and regulation with appropriate quality control.

The following cell lines were used in the study: SW480, SW620, DLD-1, HT29, HCT116, LoVo (all human colon cancer), MC38 (mouse colon cancer), OE33 (human esophageal cancer), and HeLa (human cervical cancer). All the cell lines were purchased from American Type Culture Collection (ATCC), except for MC38 and OE33 which were purchased from Kerafast and Sigma, respectively. Early passage SW480, SW620, DLD-1, HT29, HCT116, and OE33 cell lines were cultured in Roswell Park Memorial Institute (RPMI-1640) media (R8758; Sigma). Early passage MC38, and HeLa cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media (D2429; Sigma). All media were supplemented with 10% fetal bovine serum (FBS, 16000-044; Thermo Fisher), and 100 IU/ml penicillin/streptomycin (15140-122; Thermo Fisher). All cells were cultured under sterile conditions and maintained in a 37 °C incubator with 5% CO₂, regularly tested for mycoplasma and genetically authenticated by CRUK Cancer Centre Genomics Facility, Leeds, UK. Cell lines were passaged upon reaching ∼80–90% confluency and their morphology was regularly checked to ensure the absence of cross contamination between cell lines or mycoplasma contamination. To determine cell number, the automated cell counter NucleoCounter NC-100 (Chemometec) or manual hemocytometers were used.

Human CRC cell lines (LoVo, HCT‐116, SW620, SW480, Caco‐2, and HT‐29) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The culture mediums were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific), and prepared for specific cell lines as follows: McCoy's 5A culture medium (Sigma‐Aldrich, USA) for HCT‐116 and HT‐29 cell line, L‐15 culture medium (Thermo Fisher Scientific) for SW620 and SW480 cell line, F12K (Sigma‐Aldrich), and MEM (Thermo Fisher Scientific) culture medium for LoVo and Caco‐2 cell line, respectively. All the cell lines were cultured in a humidified atmosphere containing 5% CO₂ at 37°C.
Short hairpin RNA (shRNA) was utilized to stably downregulate NDC80 expression in CRC cells. The sequences of shRNA and its negative control (NC) are designed as follows: shRNA:5′‐CATTCTTGACCAGAAATTA‐3′; NC:5′‐TTCTCCGAACGTGTCACGT‐3′. The lentivirus product was purchased from Genechem and transfection procedure was performed in 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific) as described previously 13. Then, CRC cells in logarithmic phase were infected with lentivirus carrying shRNA or NC based on multiplicity of infection (MOI). The interference efficacy of shRNA was examined by Quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot.