Dimethyl sulfoxide (DMSO), potassium phosphate, trypan blue, propidium iodide (PI), Triton X-100, Tris-HCl and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), 1.5 mM L-glutamine, 10% fetal bovine serum (FBS), trypsin-EDTA, penicillin G, and streptomycin were obtained from Gibco BRL (Grand Island, NY, USA). Antibodies against β-actin (GTX109639, 1:5,000), protein kinase B (Akt; GTX128414, 1:500), phospho-Akt (GTX128414, 1:500), extracellular regulated kinases (ERK1/2; GTX59618, 1:500), phospho-ERK1/2 (GTX59568, 1:500), Bcl-2-associated death promoter (BAD; GTX130108, 1:1,000), phospho-BAD (GTX50136, 1:500), insulin-like growth factor 1 receptor (IGF1R; GTX83064, 1:1,000), phospho-IGF1R (GTX55013, 1:500), epidermal growth factor receptor (EGFR; GTX100448, 1:5,000), phospho-EGFR (GTX61353, 1:500), caspase-3 (GTX110543, 1:1,000), caspase-9 (GTX112888, 1:1,000), and all peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

CAL-27 and SCC-25, human tongue squamous cell carcinoma cell lines, were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cisplatin-resistant CAL-27 (CAR) cells were kindly provided by Dr Jai-Sing Yang. CAR cells were generated from the CAL-27 cell line by sub-culturing in increasing cisplatin concentrations from 10 to 80 µM by 10 cycles of 1 passage, as previously described (16). All cells were cultured in 75 cm² tissue culture flasks with 90% DMEM with 1.5 mM L-glutamine adjusted to 1.5 mg/l sodium bicarbonate, supplemented with 10% FBS and 1% penicillin-streptomycin (100 U/ml penicillin G and 100 mg/ml streptomycin), and maintained at 37°C in a 5% CO₂ atmosphere.

This study was approved by the Institutional Review Board of Taipei Veterans General Hospital (VGHIRB no. 2016-06-005A). The board is organized and operates according to the International Conference on Harmonisation (ICH)/WHO Good Clinical Practice (GCP) and applicable laws and regulations. Briefly, lipo-aspirate was obtained from the liposuction waste of three volunteers. The fat was immersed in phosphate-buffered saline (PBS) solution and centrifuged to remove excessive fluid. The washed fat tissue was mixed with DMEM, collagenase (1 mg/ml), N-acetylcysteine (2 mM), and l-ascorbic acid-2 phosphate (0.2 mM) and cultured at 37°C for 1 h. After centrifugation to remove excessive collagenase, the pellet was cultured with DMEM (10% FBS), N-acetylcysteine (2 mM), and l-ascorbic acid-2 phosphate (0.2 mM) under 5% CO₂ overnight. The flask was washed with PBS to remove unattached cells, and the medium was changed every other day until confluence. Cells were sub-cultured and characterized by flow cytometry after positive surface staining for CD29 and CD90, but not for CD11B, CD31 or CD45. Before experimental use, ASCs were tested for their ability to differentiate into various mesenchymal lineages, including adipocytes, osteoblasts and chondrocytes (data not shown).

To prepare ASC-CM, ASCs (1×10⁶) were cultured in T75 flasks for 2–3 days. Culture medium was removed at cell confluence, and the cells were washed twice with PBS. Fresh DMEM supplemented with 10% FBS was added and incubation was carried out for 1 day. After incubation, the culture medium was collected and centrifuged to remove the cells.

To evaluate the cytotoxic effect of cisplatin on CAL-27, SCC-25, and CAR cells, an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used. Briefly, the cells were placed in a 96-well cell culture plate at an initial concentration of 5×10⁴ cells/ml, treated with cisplatin at different concentrations (5, 10, 15 and 20 µM) or with 0.1% DMSO for 24 h, and cultured in DMEM or ASC-CM. Each concentration was repeated three times. After a 24-h incubation period, MTT solution (5 mg/ml, 100 µl/well) was added to the cells for 4 h. Subsequently, the growth medium was removed, and the formazan crystals formed by oxidation of the MTT solution were dissolved with DMSO in isopropanol and measured spectrophotometrically at 490 nm. The cell survival ratio was expressed as a percentage of the control. Viability assays were performed in triplicate from three independent experiments.

CAL-27 cells were cultured in 6-well plates at a density of 2.5×10⁵ cells/well/ml before being treated with 20 µM of cisplatin or with 0.1% DMSO for 24 h, and cultured in DMEM or ASC-CM. After treatment for 24 h, morphological changes were determined by a phase-contrast microscope.

CAL-27 and CAR cells were placed in a 6-well tissue culture plate for 24 h and grown to 80–90% confluence. Individual wells were scratched with a micro-pipette tip to create a denuded zone of constant width. Cells were then cultured in serum-low DMEM or ASC-CM for 24 h. Cells were photographed using phase-contrast microscopy (×100). Wound healing assay were performed in triplicate from three independent experiments.

CAL-27 cells (1×10⁵) were plated in 24-well plates and cultured in DMEM or ASC-CM with 20 µM cisplatin for 24 h. Cells were harvested and then fixed in 70% ethanol at 24°C overnight. Cells were incubated with PI buffer. The distribution of the cell cycle and the sub-G1 population (apoptotic cells) were detected using a flow cytometric method. BD Paint-A-Gate™ was used for analysis of the flow cytometry (BD Biosciences, San Jose, CA, USA).

Briefly, CAL-27 cells were plated in a T75 flask at an initial concentration of 5×10⁶ cells, and cultured in DMEM or ASC-CM with 20 µM cisplatin for 24 h. The cells were harvested and total protein was collected. Samples were electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Invitrogen Life Technologies, Carlsbad, CA, USA). The membranes were blocked with PBST solution (0.1% Tween-20 in PBS) plus 5% powdered non-fat milk for 1 h, and incubated overnight at 4°C with the primary antibody diluted in blocking solution (0.1% Tween-20 in PBS plus 5% powdered non-fat milk). Subsequently, the membranes were washed with PBST three times for 10 min and incubated with the appropriate alkaline HRP-conjugated secondary IgG antibody (horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse) for 1 h at 24°C and then washed three times again. Bands were detected by enhanced chemiluminescence with ECL reagents (Amersham Pharmacia, Buckinghamshire, UK) and exposed on X-OMAT AR film (Eastman Kodak, Rochester, NY, USA). Auto-radiograms were scanned on a UMAX PowerLook Scanner (UMAX Technologies, Fremont, CA, USA) with Photoshop software (Adobe Systems, Seattle, WA, USA). Western blot analysis was performed in triplicate from three independent experiments.

All data are expressed as mean ± SEM from at least three separate experiments. Statistical calculations of the data were performed using a one-way ANOVA. P<0.05 was considered to indicate a statistically significant difference.

We conducted a retrospective review using data drawn from electronic medical records retained by our medical institution, Taipei Veterans General Hospital. Between January 2000 and December 2014, patients who underwent fat grafting for scar revision after head and neck cancer surgical resection were included in the data collection and analysis.

To determine the cytotoxic effects of cisplatin on SCC-25, CAL-27 and CAR cells, an MTT assay was performed. As shown in Fig. 1, cisplatin treatment significantly decreased the viability of the SCC-25 and CAL-27 cells in a concentration-dependent manner (P<0.05). Cisplatin was also found to induce the formation of apoptotic bodies in the SCC-25 and CAL-27 cells after 10–20 µM cisplatin challenge (data not shown). Importantly, there was no cytotoxic effect or morphological characteristic change in the cisplatin-treated CAR cells.

To determine the cell activity of human tongue cancer cells following ASC-CM treatment, viable cells were counted using a hemocytometer after cell culture in DMEM or ASC-CM for 24 h. As shown in Fig. 2A, no significant difference in viability of the SCC-25, CAL-27, and CAR cells was noted between the DMEM and ASC-CM treatment group. We subsequently performed a wound healing assay. As shown in Fig. 2B, our results also failed to reveal a significant difference in the migration ability of the cancer cells between cells cultured in DMEM and ASC-CM.

Phase-contrast microscopy was used to detect the morphological differences between DMEM (control) and ASC-CM groups. As shown in Fig. 3A, CAL-27 cells grew well and spread with a flattened morphology after a 24-h culture. The number of viable cells was decreased and some detached from the surface and contained some debris in the cisplatin-treated CAL-27 cells. However, an increased number of viable cells in the cisplatin-treated CAL-27 cells was observed in the presence of ASC-CM compared to the control group. To detect the growth inhibitory effect of ASC-CM on cisplatin-treated SCC-25 and CAL-27 cells, a cell survival assay by MTT was performed. As shown in Fig. 3, ASC-CM from three different patients increased viable cells in the cisplatin-treated SCC-25 (Fig. 3B) and CAL-27 cells (Fig. 3C) (P<0.05).

To further evaluate the apoptotic process in cisplatin-treated CAL-27 cells under ASC-CM, flow cytometry was used to exam the DNA content of the apoptotic population (sub-G1 phase). As shown in Fig. 4, ASC-CM significantly reduced the sub-G1 phase cell population in the cisplatin-treated CAL-27 cells (38.1±4.2% in DMEM and 28.4±0.4% in ASC-CM, P<0.05).

To elucidate the possible molecular signaling pathways in cisplatin-treated CAL-27 cells with ASC-CM treatment, the protein levels in the tyrosine kinase signaling and apoptotic pathways were evaluated by western blotting. As shown in Fig. 5, ASC-CM caused an increase in protein levels of pro-caspase-3, pro-caspase-9 (Fig. 5A), and phospho-BAD (Fig. 5B) in the cisplatin-treated CAL-27 cells. To further investigate cell signaling, upstream-associated proteins were also studied. ASC-CM caused an increase in protein expressions of phospho-IGF-1R, phospho-AKT, and phospho-ERK (Fig. 5C). These results suggest that ASC-CM attenuated cisplatin-triggered apoptosis in CAL-27 cells through the IGF-1R/AKT/ERK signaling pathway.

In the retrospective review, there were 10 patients with head and neck squamous cell carcinoma who underwent fat grafting for scar revision after head and neck cancer surgical resection between January 2000 and December 2014. Patient demographics, cancer type, cancer staging after initially surgical resection, follow-up period, and outcome are summarized in Table I. Patient 1 died of radiotherapy-induced sarcoma. There was no local recurrence in all patients during the follow-up period.