Cell culture
HepG2 and SK-Hep-1 cells (human liver cancer cell lines) were purchased from the American Type Culture Collection (ATCC), and cells were grown with high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin and streptomycin (pen/strep, 10,000 U mL− 1; Gibco) solution. All cell culture were maintained in a humidified incubator with 5% CO2 at 37 ℃ (Nuaire) and subcultured at 70–80% confluency every 2 to 3 days.
Bloodstream-Like microfluidic circulatory system
A peristaltic pump (Longer, China) was employed for producing a pulsatile flow in silicon tubes (ID 0.5mm) to build the bloodstream-Like microfluidic circulatory system. FSS values were determined by Poiseuille's equation [4c] of τ = 4Qη/πR3, where τ represents the FSS values (dyne/cm2), Q denotes the fluid flow rate (cm3/s), η is the dynamic viscosity of fluids, and R is the radius of the tube.
Cell viability assay
The viability of cancer cells was achieved by both Live/Dead cell staining and CCK-8 methods. Specifically, Calcein-AM (1 µL) and propidium iodide (PI, 1 µL) solutions (Beyotime) were added to PBS (1 mL) to prepare the working solution for Live/Dead cell staining. Following the removal of cell culture medium and three washes with PBS, the working solution was added directly to culture plates containing cancer cells for incubation at 37°C for half an hour. Dyed samples were then imaged under a confocal laser scanning microscope (Nikon A1HD25) with dead cells stained in red and live cells stained in green. Since dead cells were in the form of cell debris after circulation, CCK-8 assay was further performed to quantify the cell viability. Briefly, CCK-8 solution (Dojindo) was added to 96-well plates containing cancer cells and incubated at 37°C for 1 hour. Cell viability was determined by measuring the optical density with a microplate reader (BioTek Synergy™ H1) at a wavelength of 450 nm.
Single-cell tracking assay
The migration of circulated cells was explored by single-cell tracking assay. Specifically, cancer cells with different treatments were seeded into collagen-coated 12-well plates (30,000 cells/per well). After cell adhesion, the well plates were placed under a real-time tracking microscope (LS720, Etaluma, USA). Cell positions were automatically recorded using a phase contrast microscopy with a 10X objective (Nikon) with 15 min interval for 24 h. Cell tracking was performed manually for all selected viable cells using ImageJ software. Migration parameters velocity (accumulated distance/time) and tack length (total length of displacements within the track) [20] were determined by Chemotaxis and Migration tool (ibidi).
Colony formation assay
HepG2 (2000 cells per well) and SK-Hep-1 (500 cells per well) cells were seeded in 6-well plates for 10 and 7 days, respectively, to allow colony formation. After washing the cells with PBS, the colonies were fixed with ice-cold 70% ethanol solution for 10 min and then stained by 0.2% crystal violet for additional 10 min. Colonies containing over fifty cells were counted under microscopy for calculating colony number per well.
Wound healing assay
The wound healing assay was conducted by employing ibidi Culture-Inserts (ibidi®) following the manufacturer's instructions. A cell suspension of 5×105 cells in 200 µL was added into ibidi Culture-Inserts in 24-well plates. When the confluent cell monolayer was formed, the ibidi Culture-Inserts were removed, creating a defined cell-free gap of a width at 500 µm. Then, the wound closure was monitored and imaged at 0, 6,12, 24 hours with a real-time tracking microscope (LS720, Etaluma, USA). The wound area at indicated time points was subtracted from the initial wound area (0 h), and the resulting area was normalized to the initial wound area to determine cell migration ability. Wound area was defined using Image J software. Each sample was assessed in three fields for replicates.
Cell apoptosis assay
HepG2 cells with downregulation of TLR4 or TPPP3 were cultured in ultra-low attachment 6-well plates at a density of 1х106 for suspension culture. After a 12-hour incubation peroid, cells were harvested, washed with PBS, and then resuspended in Annexin binding buffer before staining by an Annexin-V-Alexa Fluor 647 apoptosis detection kit (Invitrogen) for 15 min in dark. Finally, the stained cells were immediately examined using a flow cytometry machine (CytoFLEX, Beckman) to assess the apoptosis rate. The data obtained were analyzed with FlowJo software (BD Biosciences).
Lentivirus infection
Three distinct siRNA duplexes of RND1 were purchased from Integrated DNA Technologies. Lipofectamine 3000 reagent (Thermo Fisher) was utilized. Each siRNA sequence was pre-mixed with Opti-MEM medium and incubated at room temperature for 15 min. The siRNA-lipid complexes were then mixed with cells and incubated for 2 days. After that, the knocking down effects were characterized via qRT-PCR. The siRNA sequences are shown in Table S1.
Lentiviruses carrying small hairpin RNA (shRNA) sequence targeting human TLR4 (Gene ID:7099) and TPPP3 (Gene ID:51673) were purchased from Ubigene (GuangZhou). The shRNA sequences are shown in Table S1. The plasmid encoding TLR4 (NM_138554.5) and TPPP3 (NM_015964.4) were synthesized using pcSLenti-EF1-mCherry-P2A-BSR-CMV-MCS-3xFLAG-WPRE (OBiO).
For infection, 2×105 of HepG2 cells were cultured in six-well plates. When the cell confluent was 30–50%, lentiviruses were added following by 24 h incubation. After that, the medium was refreshed, and blasticidin (1 mg/mL, Invivo Gen) or puromycin (2 µg/mL, InvivoGen) was added to select the stably transfected cell lines after 48 h of infection.
RNA-seq and data analysis
HepG2 cells with different treatment conditions, including adherent culture (Ad), suspension culture (Sus), and FSS, were harvested and lysed with Buffer RL (Takara). Each sample were prepared in three parallel duplicates. Then, all the samples were delivered to BGI Corporation (Shenzhen) for quality control, library preparation, and sequencing by a BGI SEQ-500 sequencer. Genes exhibiting a p-value < 0.05 and a fold change of ≥ 2 were considered as differentially expressed genes with statistical significance.
RNA extraction and qRT-PCR
Total RNA was extracted using TakaRa MiniBEST universal RNA extraction kit (Takara), and cDNA reverse transcription was then conducted with PrimeScript RT Master Mix (Takara). After that, the quantification of gene expression was carried out with SYBR Green Master Mix (Applied Biosystems) using a QuantStudio 12K Flex real-time PCR instrument (Thermo Fisher). All primer sequences can be found in Table S2.
Western blotting
To investigate the proteins expression, cell lysates were extracted by radio immunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor (Roche) and incubated on ice for 15 min during which the lysates were vortex every 2 min. Then, cell lysates were centrifuged at 12,000 g for 20 min, and the supernatants were harvested. The total protein concentration of cell lysates was quantified by a Bio-Rad colorimetric protein assay reagent. After that, protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore) by a semi-dry transfer apparatus (Bio-Rad). The PVDF membranes were blocked with QuickBlock (Beyotime) for 30 min and subsequently incubated at 4°C overnight with specific primary antibodies. The primary antibodies used were against TLR4 (A5258, 1:1000) and TPPP3 (A6775, 1:1000), both purchased from Abclonal. Antibodies against GAPDH (66009-1-Ig, 1:4000) and β-actin (66009-1-Ig, 1:4000) were obtained from Proteintech. After three washes with Tris-buffered saline with 0.1% Tween 20 (TBST), the PVDF membranes were further incubated with horseradish peroxidase-coupled secondary antibody (1:1000, Beyotime) at room temperature for 1 hour. Finally, the membranes were visualized using BeyoECL Plus (P0018S, Beyotime) and imaged with the ChemiDoc System (Bio-Rad).
Tail vein injection
One million of tumor cells (SK-O-Ctrl, SK-O-TLR4 and SK-O-TPPP3) with luciferase-expression were suspended in 200 µL of culture medium and introduced into the bloodstream of NOD/SCID mice aged from 6 to 8 weeks via the tail vein injection. Mice were imaged using IVIS Lumina system (Perkin Elmer) three weeks after injection. After the bioluminescence imaging at week 4, the mice were euthanized, and their organs were harvested for subsequent bioluminescence imaging. The Animal Care and Ethics Committee of City University of Hong Kong (A-0644) has granted approval for all animal experiments conducted in this study.
Statistical analysis
All data are reported as the means ± standard deviations (SD) obtained from at least three independent experiments. Statistical analysis was performed using Prism 9.5 software (GraphPad). Student's two-tailed t-test was used for two-group comparisons, while one-way analysis of variance (ANOVA) was used for multiple comparisons. The significance of differences was indicated as follows: ns for not significant, * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001.