Collection of keloid samples
Eight freshly excised keloid scar samples were collected from six patients (three males and three females, age ranging 12–52 years) who had not received any preoperative treatment with informed consents. These keloid scars were originally located at chest or ear sites of patients.
Isolation and culturing of fibroblasts from normal skin and keloid biopsy, respectively
The keloid tissues were aseptically excised and collected in 50mL centrifuge tubes from the chest and ear sites of patients under local anesthesia with informed consent to isolate keloid-derived fibroblasts (KFs), followed by washing for three times in phosphate-buffered saline (PBS). Epidermal tissues were removed and the remained dermis tissue was cut into small pieces with a scalpel and incubated in 0.3% collagenase in Dulbecco's modified Eagle's medium (DMEM) at 37°C with shaking for 6h. Subsequently, the treated tissues were squashed to release detached cells and filtered with 100µm cell strainers. The obtained cell suspension was centrifuged at 1,200rpm for 5 minutes and then cell pellets were resuspended for cell culture in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin. Isolated cells were grown until reaching 80% confluence, then washed, trypsinized in 0.25% Trypsin-EDTA and collected by centrifugation as described above and designated as passage 0 (P0), frozen and stored in liquid nitrogen. Some of the P0 cells were plated for following cell culture and experiments, and the next passaged cells were regarded as P1. The third to fifth passaging of cells (P3-P5) were used in the following experiments. Human normal skin fibroblasts (NFs) were isolated and cultured from human foreskin tissue that was obtained with informed consent following the above-described procedure for KF isolation and culture.
Cell lines and cell culture
All cell culture agents were obtained from Gibco (Life Technologies, Gaithersburg, MD, USA) unless otherwise stated. Human KFs and NFs were self-isolated, 293T, human adipose tissue-derived mesenchymal stem cells (ADSCs), bone-marrow-derived mesenchymal stem cells (BMSCs) were obtained from Fuheng Biology (Shanghai, China). All cell lines were grown and cultured in DMEM medium with the supplement of 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2 at 37°C.
Chemical reagents
Sunitinib (su 11248) was purchased from MedChemExpress (China), dissolved in DMSO to make 10mM stock solution, and stored at -20°C under light-proof condition until use. In any of the experiments described below, the final concentration of DMSO did not exceed 0.1% (v/v), which is not toxic to keloid tissue-derived fibroblasts (KFs). Triamcinolone acetonide (TAC) was obtained from APExBIO, (China), dissolved in DMSO at 14.3mg/mL as the stock solution, and used as a positive control for keloid treatment.
Cell proliferation assay
In vitro cellular viability and proliferation assays were carried out using the Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) to examine the growth and proliferative potential of KFs and NFs or to test the cytotoxicity of sunitinib on KFs and normal lines including NFs, 293T, ADSC and BMSC. Passage 3–5 of NFs and KFs were tested. Cells were seeded in 96-well plates (2× 103/well in 100µl of complete culture medium) and allowed to settle down for overnight. Subsequently, cells were subjected to CCK-8 test, or were starved in serum-free medium for 24h and then treated with vehicle or sunitinib at 2.0–6.0µM for 24h, followed by CCK-8 assay.
Assays were carried out in triplicate and repeated for at least 3 times. The optical density (OD) values were measured at 450 nm using a microplate reader (model 2300, PerkinElmer, Boston, MA, USA), and the survival cell percentage is calculated using the equation: cellular viability (%) = (ODsample-ODblank) / (ODcontrol-ODblank) × 100. Data were presented as mean ± SD (n = 3).
Cellular 3D migration/invasion assay
A cellular 3D migration/invasion assay was performed to examine the 3D migration potential/invasiveness of cells using Matrigel (BD Biosciences, UK) and transwell sets with permeable polycarbonate membrane (8.0 µm pore size) (Corning Inc., UK) based on previous description [19] but with some modifications. Briefly, 40µl Matrigel was added into each transwell insert, and 2 × 104 cells were seeded on the top of Matrigel reconstructed matrix, followed by supplementation of 10% FBS containing DMEM with or without drugs above Matrigel and serum-free DMEM in the lower chamber. Cell were allowed to migrate into the Matrigel matrix and invade downwards to the bottom porous membrane for 3 days. Subsequently Matrigel matrix was removed and cells that have migrated through the matrix and attached to membrane were regarded as invaded cells, which were fixed with 4% paraformaldehyde and stained with crystalline violet, and finally imaged and quantitated. Each treatment was carried out in triplicate.
Cell cycle analysis
Passage 3 of KFs were cultured and treated with vehicle or 6 µM sunitinib for 24 h, then harvested by trypsinization and washed with cold PBS, followed by fixing with ice-cold 70% ethanol at 4°C for overnight. Subsequently, cells were pelleted by centrifugation at 2000 g for 8 min, washed for twice with PBS and resuspended in cell cycle assay staining buffer containing 50 µg/mL PI and 100 µg/mL RNase A (Solarbio life sciences, Beijing, China), incubated for 1h at 37°C. The stained cells were pelleted by centrifugation at 2000 g for 10 min, washed with PBS, resuspended in 0.5 mL PI and RNase A-free staining buffer, and detected by flow cytometry (FCM). Fluorescence staining signal intensity was measured at 480 nm excitation wavelength through a FL-2 filter. Finally, FCM data were analyzed by using the FlowJo 7.6 software (Becton Dickinson, Ashland, OR, USA).
Apoptosis assay
KFs were seeded in 12-well plates at a density of 1 × 105 cells/well, and allowed to settle down for overnight, then treated with vehicle or 6 µM sunitinib for 24 h. Then, the treated cells were harvested, washed and stained with FITC-Annexin V and propidium iodide (PI) (Bestbio, Shanghai, China) for 30 min, followed by apoptosis assessment using flow cytometry analysis (FACS Calibur; Becton Dickinson). Annexin V+ /PI - and Annexin V+ /PI + cells were considered to have undergone early and late apoptosis, respectively, and Annexin V- /PI - cells were regarded as live cells.
Real-time relative quantification PCR
Real time relative quantification PCR (RQ-PCR) was performed to study the effect of sunitinib treatment on cellular collagen I and III gene expression at mRNA levels in KFs. Total RNA was extracted from cultured cells with or without sunitinib treatment using Trizol reagent (Invitrogen, China). Then 1 µg of total RNA was used to prepare cDNA for each sample by using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas, Life Sciences, Canada). Subsequently, RQ-PCR was carried out with SYBR Premix ExTaq TM II reagent, 2µl cDNA per sample (0.1 µg) and collagen I and III specific primer sets (Supplementary Table-1), respectively. Reactions were carried out on an ABI PRISM ® 7900HT system (Takara Biotechnology, Japan) following the manufacturer’s instruction. GAPDH primer set (Supplementary Table-1) was used as an endogenous control and all PCR reactions were carried out in triplicate. The RQ-PCR data were analyzed using the 2−ΔΔCt method [20] and relative mRNA expression levels were compared between vehicle and sunitinib treatments in KFs.
Immunoblotting assay
Western blotting was carried out to check the expression of proteins of interest. To this purpose, cultured or treated cells were harvested and cellular proteins were extracted using Radio-Immunoprecipitation Assay (RIPA) buffer supplemented with the protease inhibitor cocktail (MedChemExpress, China). Protein quantitation was performed using the BCA assay. Then, 10 µg of cellular protein were separated by using 7.5% SDS-PAGE for both mTOR and p-mTOR, or by using 12% SDS-PAGE for all other examined proteins. When electrophoresis was finished, proteins in gel were transferred onto a PVDF membrane, followed by blocking the membrane with TBST buffer of 5% skimmed milk. Then, primary antibody incubation was performed for overnight at 4 ℃ with antibodies against the following proteins, respectively: GAPDH (dilution 1:1000, AF0911-100, Affinity Biosciences), collagen I (dilution 1:1000, AF0134-50, Affinity Biosciences), collagen III (dilution 1:1000, AF0136-50, Affinity Biosciences), Akt (dilution 1:2000, 4691S, cell signaling technology, Danvers, USA), p-Akt (dilution 1:2000, 4060S, cell signaling technology, Danvers, USA), PI3K (dilution 1:2000, AB86714, Abcam, Cambridge, UK), mTOR (dilution 1:1000, 2972S, cell signaling technology, Danvers, USA), and p-mTOR (dilution 1:1000, 5536S, cell signaling technology, Danvers, USA). Subsequently, the membrane was washed in TBST for 3 times, and then incubated with the horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit second antibodies (dilution 1:2500, Proteintech, Chicago, IL, USA) for 1h at room temperature. The membrane was then washed in TBST again for 3 times, and developed for chemiluminescence exposure using the ECL detection kit (Merck Millipore), imaged and photographed by BioImage Lab (Bio-Rad, Hercules, CA, USA). Immunoblotting results were analyzed for expression quantification of target proteins with the Image J software (National Institutes of Health, Bethesda, MA, USA) and normalized using GAPDH as protein loading internal control. The expression level of target protein was presented relative to Ctrl, the value for which was set to 1.0.
Immunofluorescence assay
Approximately 1x104 KFs and NFs per well were seeded in chambers slides, respectively, and allowed to grow for 3 days. Then medium was removed from wells and cells were washed with PBS for twice, fixed for overnight in 4% paraformaldehyde, followed by ki67 staining with a rabbit anti-human ki67 primary polyclonal antibody (Ab) (AF0198, Affinity Bioscience) and a second AF594- conjugated goat anti-rabbit polyclonal IgG (H + L) Ab (Affinity Bioscience). The stained cells were examined and imaged with a confocal microscope (LSM800, Zeiss, Jena, Germany).
In vivo study
In vivo study was performed using female Balb/c nude mice (4–6 weeks old) to evaluate the therapeutic efficacy of sunitinib on keloid scars. These mice were purchased from the SPF Biotechnology Company (Beijing, China). The animal study was approved by the Animal Ethics Committee of South China University of Technology (Approval ID: 2021315082129; Date: 15 August 2021). Animals were kept in pathogen-filtered conditions with filtered air, and sterilized water and food were provided all the time. To establish a keloid explant culture model, freshly excised keloid scars were cut into small fragments (ཞ5mm˟3mm˟3mm), and implanted under forelimb and hindlimb skin in mice, which were first anesthetized by intraperitoneal injection of 5% chloral hydrate. The keloids fragments were implanted approximately 1cm away from the skin cut sites and then the cut was closed using 5.0 suture. Three limb sites were implanted with keloid fragments in each mouse and 9 mice were tested in total, producing the test sample number of 9 for each group.
One week post implantation, mice were treated in three treatment groups: vehicle control, sunitinib and triamcinolone acetonide (TAC). As a positive therapeutic control, TAC was intralesionally injected at the dose of 2.0mg/0.1mL/kg, referring to previous report. And sunitinib was administrated by intralesional injection at 100.0mg/0.1mL/kg. Injection therapy was performed once a week and for four continuous weeks in total.
Keloid explants were measured every 3 days starting from day 6 post implantation until day 33, with a Vernier caliper, and the keloid scar volume (KV) was calculated as KV (mm3) = d2 × D/2, where d and D representing the shortest and the longest diameter, respectively. One animal from each group was culled 24h post the third reagent administration, and lesions, skins and organs were collected for immunohistochemistry analysis. All remained mice were sacrificed at day 33, and animal skins, remained lesions and organs were collected, fixed, embedded in wax and stored until use.
Immunohistochemistry analyses
For immunohistochemistry staining, paraffin-embedded keloid explants, mouse skin or organs were sectioned (ཞ3mm thick), deparaffinized using xylene and hydrated in a series of graded alcohol, and subsequently immersed in sodium citrate solution to retrieve tissue antigens at 100°C for 20 min. Then sections were treated with 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. The tissue slides were then incubated with 3% BSA solution (Servicebio, Beijing China) at room temperature for 15 min to reduce staining background. Specific primary antibodies were used to incubate with sections first, including collagen I Ab (ab138492; Abcam), collagen III antibody (22734-1-AP; Proteintech, Rosemont, IL, USA) and ki67 (1:600 dilution, AF0198, Affinity Biosciences) and then appropriate secondary antibodies were used for 1h incubation at room temperature. Nuclei were stained with hematoxylin. The stained tissues were developed to show labelling of targeted proteins using the UltraVisionQuanto Detection System HRP (Thermo Fisher Scientific Inc., Waltham, MA, USA) and DAB (Liquid DABþ Substrate Chromogen System, Dako, Copenhagen, Denmark). Finally, stained sections were imaged by Eclipse E600 microscope (Nikon, Tokyo, Japan).
Statistical analysis
All data were analyzed and presented as mean ± SD using the GraphPad Prism 9.0 Software (GraphPad Software Inc., La Jolla, CA, USA). Differences between two groups were analyzed by Student’s t-test and multiple group comparison was dealt with one-way ANOVA/Bonferroni post hoc correction. Probability values of significant differences are denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.