Cell lines
Hepatocellular carcinoma cell lines Hep3B2.1-7 (Hep3B), Huh7, and MHCC97H, human embryonic kidney cells (HEK) 293 T were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), and were tested for mycoplasma contamination.
The human lung microvascular endothelial cells (HMVEC-L) were purchased from Cell Application, Inc. (Cat. #540-05a, Cell Application, Inc., San Diego, CA) and cultured with the microvascular endothelial cell growth medium (Cat. #111-500, Cell Application, Inc., San Diego, CA) according to the manufacturer’s instructions.
The human lung pericytes were isolated from an adjacent normal tissue in lung cancer patient as previously described [21, 22]. Briefly, tissue was minced and digested in HBSS buffer containing collagenase I (300 U/ml) and dispase (5 U/ml). The suspension was filtered (70 μm), centrifuged (350×g, 10 min), and washed with DMEM + 10% FBS. The dissociated cells were plated on 0.2% gelatin-coated dishes in human Pericyte Medium (ScienCell Research Laboratories, Carlsbad, CA). After expansion, cells were negatively selected by CD45, CD31, and CD326 magnetic beads (Miltenyi Biotec Inc. San Diego, CA) to deplete leukocytes, endothelial cells, and epithelial cells, respectively. Pericytes were collected for labeling with PE-conjugated anti-PDGFR-β (clone REA363; Miltenyi) and anti-PE magnetic microbeads (Miltenyi biotec, Germany) and passed through a magnetized column. Retained pericytes (PDGFR-β+) were cultured in human pericyte medium (ScienCell Research Laboratories, Carlsbad, CA).
The enhanced green fluorescent protein (EGFP+)-HCC cells were generated by lentiviral infection using GFP expressing lentiviral plasmid and selected for 10 days using 400 μg/mL neomycin. Furthermore, the EGFP+-HCC cells stably expressing ARHGEF37 cDNA or ARHGEF37 shRNA were established by lentiviral infection using pSin-EF2-ARHGEF37 or pSuper-retro-ARHGEF37 shRNA and selected for 10 days with 0.5 mg/mL puromycin.
Tissue specimens and immunohistochemistry (IHC)
All clinical HCC specimens, including 250 paraffin-embedded surgical non-metastatic HCC tissues and 70 intrahepatic-metastatic and 45 extrahepatic-metastatic biopsy specimens, and 10 freshly collected HCC samples, including 5 surgical HCC tissues and 5 metastatic biopsy specimens, used in the current basic research study were histopathologically and clinically diagnosed. This basic research study complied with all relevant ethical regulations involving human participants. The clinical information regarding the samples is summarized in Table S1–S4. Prior patient consent and approval were obtained from the Institutional Research Ethics Committee of the Jiangmen Central Hospital and the First affiliated hospital of Sun Yat-sen University (Approval number: 2022-51).
IHC staining for protein expression of ARHGEF37 in HCC samples further quantitatively analyzed using the mean optical density (MOD), defined as the mean of immunostaining intensities per positive pixels in a specimens according previously described [23,24,25,26,27,28]. Briefly, the stained sections were evaluated at × 400 magnification by using the SAMBA 4000 computerized image analysis system assisted with Immuno 4.0 quantitative program (Image Products International, Chantilly, VA), and 10 representative staining fields of each section were analyzed to verify the MOD, which represents the strength of staining signals as measured per positive pixels. A negative control with each batch of staining was used for background subtraction in the quantitative analysis. The MOD data were statistically analyzed by using the t test to compare the average MOD difference between different groups of tissues, and P < 0.05 was considered significant.
Plasmids, lentiviral infection, and transfection
The human ARHGEF37 cDNA and the CDC42 cDNA were PCR-amplified from ARHGEF37 plasmid, purchased from GeneCopoeia (EX-Y4343-M35, GeneCopoeia, China), and CDC42 plasmid, purchased from ViGene Biosciences (OENM-001791, WZ Biosciences Inc., China), and subcloned into pSin-EF2 vector. The serially truncated fragments of the human ARHGEF37 were amplified by PCR and cloned into the pcDNA3.1 vector, which including F1 (residues 1-250), F2 (residues 250-675), F3 (residues 1-500), F4 (residues 120-675). Short hairpin RNAs (shRNAs) targeting ARHGEF37 were cloned into the pSuper-retro vector. All primers and oligonucleotides used in plasmid construction are listed in Table S5 and S6. Transfection of small interfering RNAs (siRNAs) or plasmids was performed using the Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
In vivo metastasis assays
Male BALB/c-nu mice (5–6 weeks of age, 18–20 g) were purchased and housed in specific pathogen-free facilities on a 12 h light/dark cycle. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University and the approval number was SYSU-IACUC-2022-000322. For tail intravenous injection, the indicated luciferase-expressing cells (5 × 105) were resuspended in 0.1 mL phosphate-buffered saline (PBS) and inoculated into the lateral tail vein of nude mice. To determine the effect of ZCL278 treatment on tumor metastasis, mice were intraperitoneally injected with dimethyl sulfoxide or ZCL278 (20 mg/kg of body weight) every other day for 28 days. We used an In Vivo Imaging System (Xenogen, Alameda, CA, USA) to monitor the metastasis of these transduced cells in mice at the indicated time points. At the end of observation, the mice were killed by cervical dislocation and their lungs were collected to count surface metastases. Extracted lungs were embedded in paraffin and subjected to hematoxylin and eosin staining was performed on sections from paraffin-embedded samples for histological evidence of the tumor phenotype.
Extravasation analysis in vivo
The in vivo extravasation assays were based on previously described methodology [29,30,31]. Briefly, at 4 or 24 h after intravenous injection of the indicated EGFP+-HCC cells, the mice were anesthetized with sodium pentobarbital (50 mg/kg), and then longitudinally cut the skin and subcutaneous tissues from the abdomen to the chest region with surgical scissors. The pleural cavity was opened with surgical scissors and forceps to fully expose the heart and lungs, and the superior vena cava (SVC) and inferior vena cava (IVC) were tied with sterile surgical sutures to prevent the backflow of perfusion solution during lung perfusion. Furthermore, the left ventricle wall was cut with surgical scissors to open a fissure (about 2-4 mm) to drain the perfusion solution from the lungs. Then the right ventricle was washed with 1x PBS to remove the drained solution with constant suction during lung perfusion until the lungs turn from a reddish color to completely pale, and lungs were perfused and fixed with 2% paraformaldehyde. Lung sections were acquired and immediately imaged using fluorescence microscopy to monitor the location and relative amounts of these transduced cells in mice at the indicated time points.
RNA extraction and real-time PCR
Total RNA was extracted from the indicated cells using the Trizol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Real-time PCR was performed using the Eastep® qPCR Master Mix Kit (Promega, Madison, USA) under the following condition, which involved a pre-heating step at 95 °C for 2 min, 40 cycles at 95 °C for 15 s, 60 °C for 30 s. The qPCR primers were designed with the assistance of the Primer Express v 2.0 software (Applied BioSystems, Foster City, CA, USA). Expression data were normalized to the geometric mean of housekeeping gene GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) to control the variability in expression levels and calculated as 2- [(Ct of gene) – (Ct of GAPDH)], where Ct represents the cycle threshold for each transcript [32]. All primers are listed in Table S6.
Chemical reagents
ZCL278 was purchased from Selleck Chemicals (Houston, TX, USA). Human recombinant Cdc42 protein was purchased from Abcam (Cambridge, MA, USA).
Immunoblotting (IB) analysis
Immunoblotting analysis was performed according to a standard protocol with the following antibodies: anti-ARHGEF37 (HPA053487) and anti-Flag (F3165) (Sigma-Aldrich, St. Louis, MO, USA); anti-phosphorylated (p)-PAK1) (phospho S204) (ab79503) and anti-PAK1 (ab131522) antibodies (Abcam, Cambridge, MA, USA); anti-Cdc42 (CST#2466S), anti-N-cadherin (CST#13116S), and anti-Connexin-43 (Cx43) (CST#3512S) antibodies (Cell Signaling technology, Danvers, MA, USA); anti Cdc42-GTP (26905), anti Ras-GTP (26903), and anti RhoA-GTP (26904) antibodies (NewEast Biosciences; King of Prussia, PA, USA), and anti-GAPDH (60004-1-Ig) antibodies (Proteintech, Rosemont, IL, USA).
Co-immunoprecipitation (Co-IP) assay
Cells grown in 100-mm culture dishes were lysed using 500 μL of lysis buffer [25 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 1% NP-40, 1 mmol/L EDTA, 2% glycerol, and 1 mmol/L phenylmethylsulfonyl fluoride]. After being maintained on ice for 30 minutes, the lysates were clarified by microcentrifugation at 12,000 rpm for 10 minutes. To preclear the supernatants, the lysates were incubated with 20 μL of agarose beads (Calbiochem, San Diego, CA, USA) for 1 h with rotation at 4 °C. After centrifugation at 2000 rpm for 1 min, the supernatants were incubated with 20 μL of antibody-cross-linked protein G-agarose beads overnight at 4 °C. The agarose beads were then washed six times using wash buffer [25 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 0.5% NP-40, 1 mmol/L EDTA, 2% glycerol, 1 mmol/L phenylmethylsulfonyl fluoride]. After removing all the liquid, the pelleted beads were resuspended in 30 μL of 1 M glycine (pH 3), after which, 10 μL 4 × sample buffer was added, the samples were denatured, and then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis for immunoblotting analysis.
Far-Western analysis
Far-Immunoblotting was performed by using the proteins immunoprecipitated by anti-Flag antibody and human recombinant Cdc42 protein. Briefly, the proteins were separated by SDS-PAGE, and were transferred onto a PVDF membrane. Membranes were then preincubated in 10% skimmed milk for 1 hour at 4 °C. As indicated, recombinant Cdc42 protein was added at 3 μg/mL and incubated at 4 °C for 18 hours. After extensive washing six times with TBST, the membrane was subjected to immunoblotting analysis using indicated antibodies.
Pericyte–epithelial cell (EC) co-culture model
HMVEC-L cells and pericytes were incubated for 30 min at 37 °C with 8 μM Cell-Tracker Green CMFDA and Cell-Tracker Red CMPTX dyes (Thermo Fisher Scientific, Waltham, MA), separately. The cells were then washed using 1× PBS, and incubated with fresh complete cell culture medium for an additional 24 h. In the 2D co-culture model, HMVEC-Ls and pericytes were seeded in 8-well Lab-Tek chamber slides at densities of 20,000 and 6600 cells/well (ratio HMVEC-Ls-pericytes 3:1), respectively. Cell monolayers were processed for N-cadherin (CST#13116S, Cell Signaling technology, Danvers, MA) or Cx43 (CST#3512S, Cell Signaling technology, Danvers, MA) immunofluorescence staining 24 h later. The secondary antibodies were donkey anti-rabbit IgG (H + L) conjugated with Alexa Fluor 647 (A-31573, Thermo Fisher Scientific, Waltham, MA). Cell nuclei were counterstained and mounted with antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, MA). The images were captured using the AxioVision Rel.4.6 computerized image analysis system (Carl Zeiss, Jena, Germany).
Monolayer adhesion assay
The monolayer adhesion assay was performed in a parallel plate flow chamber kit (Cat#31-001, GlycoTech, Gaithersburg, MD, USA) as previously described [33, 34]. The kit contains 4 parts, including: (1) base plate with an entrance and exit port through which cells and media are perfused, (2) a glass or plastic slide plate on which the substrate or cellular monolayer is placed, (3) a gasket that controls the chamber diameter, and (4) a vacuum outlet so that the apparatus can be held in place. HMVEC-L cells were cultured on 24-well and allowed to grow to full confluence. Then, the monolayers were stained with 8 μM Cell-Tracker Red CMPTX dyes (Thermo Fisher Scientific, Waltham, MA) and inserted into a flow chamber. The indicated HCC cells were resuspended (1 × 105 cells per mL) in serum-free culture medium with 0.88 mM fibrinogen and 2.5 mM CaCl2 and loaded into a syringe pump. The dynamic adhesion assays were performed at a shear stress in the range of 0.1–4.0 dynes cm− 2 according to the manufacturer’s manual. The tumor cells adhering to the monolayers were imaged under a fluorescence microscope and the average number of adherent tumor cells in five random fields at × 200 magnification was calculated.
Trans-endothelium migration assay
HMVEC-L cells were seeded in 24-well Transwell inserts with a pore size of 8 μm and grown for 2 days until full confluence. The indicated ARHGEF37-dysregulated or control EGFP+-HCC cells (5 × 105) were added into the apical chamber. The basolateral chambers were filled with 600 μL culture medium with 10% fetal bovine serum, which was used to suspend the tumor cells in the apical chambers. After incubation for 24 h, the apical side of the apical chamber was scraped gently with cotton wool. Only the migrated EGFP+-HCC cells were detected by fluorescence microscopy and were counted from 10 random fields of × 200 magnification.
Invadopodia gelatin degradation assay
To assess the capability of HCC cells to form invadopodia and degrade the matrix, a QCM Gelatin Invadopodia Assay (ECM670, Merck Millipore, Darmstadt, Germany) was performed according to the manufacturers’ protocol. Briefly, slides were pre-coated with 0.2% w/v poly-L-lysine (20 min at room temperature), and then fixed with 0.5% glutaraldehyde (15 min at room temperature). Following incubation with a gelatin mixture (Fluorescein-labeled and unlabeled gelatin 1:5, 10 min at room temperature), the slides were sterilized with 70% ethanol. Then, cells were added onto labelled-gelatin-coated sides and cultured in the dark under standard tissue culture conditions for 24 h. After fixation with 3.7% formaldehyde, the cells were stained with TRITC-phalloidin and DAPI for 1 h, respectively. In invadopodia experiment, the number of HCC cells forming invadopodia was quantified in 15 microscope fields (63×) randomly and percentage of invadopodia forming cells was assessed. In ECM degradation experiment, 15 microscope fields (40×) imaged randomly and the percentage of degraded area was quantized using ImageJ software (National Institutes of Health) and normalized to the number of nuclei in that area, which was represented as “% degradation per cell”.
Cdc42-GTP pull-down assay
Cdc42 activation was examined using a Cdc42 Activation Assay Kit (ab211163, Abcam, Cambridge, MA) that uses GST-PAK1-PBD fusion protein agarose beads to selectively isolate and pull-down the active form of Cdc42 from endogenous lysates following the manufacturer’s instructions. GST-PAK1-PBD fusion protein is the p21 binding domain (PBD) of PAK1 that expressed as a GST-fusion protein and coupled to agarose beads. GST-PAK1-PBD fusion protein is used to bind the activated form of GTP-bound Cdc42, which can then be immunoprecipitated with glutathione resin. Briefly, cells were harvested in cell lysis buffer, and the indicated cell lysate was loaded with GDP or GTPγS and incubated with PAK1 p21-binding domain-Agarose beads as negative or positive controls, respectively. One mg of protein lysate in a total volume of 1 mL at 4 °C was immediately precipitated using 40 μL of PAK1 p21-binding domain beads for 60 minutes with rotation. After washing, the beads were resuspended and processed for immunoblotting.
Statistical analysis
Statistical tests for data analysis included log-rank test, Chi-square test One or Two-way analysis of variance (ANOVA) and Student’s two-tailed t test. Survival curves were plotted by the Kaplan–Meier method and compared by the log-rank test. The metastasis free survival was measured from the data of random assignment to the date of first evidence of recorded clinical metastasis-intrahepatic metastasis and/or extrahepatic metastasis confirmed by imaging or histologic evidence-was censored at the date of last follow-up. The cut-off values for high- and low- expression of ARHGEF37 proteins of interest were chosen based on a measurement of heterogeneity using the log-rank test with respect to metastasis free survival. The significance of various variables for survival was analyzed by univariate and multivariate Cox regression analyses. Statistical analyses were performed using the SPSS 18.0 statistical software package (SPSSInc., Chicago, Ill., USA) and GraphPad Prism 8 (GraphPad Inc., La Jolla, CA, USA). Data represent mean ± SD. P-values of 0.05 or less were considered statistically significant.