2.1 Patients in the OSCC cohort
In this study, we retrospectively reviewed the archived files of patients with OSCC at the Dental Hospital, Yonsei University Medical Center, Seoul, Korea, from 1997 to 2011. Of the 268 patients reviewed, 116 patients with OSCC occurring in the maxillary and mandibular gingiva, as well as retromolar trigone where cancer could invade the nearby bone, were included. The mean follow-up period was 88.4 months. Of the 116 patients in the OSCC cohort, 72 (62.1%) had bone invasion, while no bone invasion was observed in the other 44 (37.9%) patients. The clinicopathologic characteristics of the patients in the OSCC cohort are shown in Table 1. The American Joint Committee on Cancer (AJCC) 8th Staging Manual was adopted for staging. Pathological diagnosis of the tissue samples was independently performed by two pathologists. This study was approved by the Yonsei University Health System, Severance Hospital, Institutional Review Board (IRB 2-2019-0050).
2.2 Immunochemical staining
Mouse monoclonal anti-human α-SMA antibody (Dako, Santa Clara, CA, USA) and rabbit monoclonal anti-human TP (Abcam, Cambridge, UK) were used as the primary antibodies. Immunochemical staining of the OSCC tissue sections and primary CAFs was performed as previously described (25). The REAL EnVision HRP Rabbit/Mouse Detection System (Dako) was used as the secondary antibody and visualized by chromogen 3,3′-diaminobenzidine.
The total histoscore for each protein was calculated based on the staining intensity and the percentage of positive cells. The patients were subdivided into two groups based on the total histoscore: low expression (total histoscore ≤ 100) and high expression (total histoscore > 100) groups.
2.3 CAF culture from OSCC tissues
Primary CAFs were prepared using tissue samples obtained from patients with OSCC, as described previously (25). The CAFs used in this study were at passage 5. This procedure was approved by the Institutional Review Board of Yonsei University Health System, Severance Hospital (IRB 2-2021-0094).
2.4 Proliferation assay
To investigate the effect of TP on the proliferation ability of CAFs, the number of cells in CAFs with or without recombinant human TP treatment (rhTP, R&D system, Minneapolis, MN, USA) was measured at each indicated time point using trypan blue staining. The experimental groups were treated with 20, 40, and 80 ng/mL of rhTP, respectively, for every 24 h. For each group, 1 × 104 cells per well were seeded in a six-well plate for examination.
2.5 Invasion assay
To investigate the effect of TP on the invasion ability of CAFs, a Matrigel invasion assay was performed as previously described (25). Prior to the invasion assay, the experimental group was treated with rhTP twice every 24 h. Cells were seeded at 1 × 105 cells per well in a 24-well cell culture insert with a pore size of 12 μm, and rhTP (40 ng/mL) containing media was added in the experimental group. A comparative investigation of the number of penetrating cells in CAFs with or without rhTP treatment was carried out.
2.6 Quantitative reverse transcription-polymerase chain reaction
Quantitative reverse transcription-polymerase chain reaction was performed using 2× SYBR Premix Ex Taq II (Tli RnaseH Plus) (RR82LR; Takara, Ann Arbor, MI, USA) in an Applied Biosystems Thermocycler (Foster City, CA, USA). TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) was used for total RNA extraction, and the target complementary DNA was synthesized using oligo (dT) primers. The target mRNA expression was normalized with glyceraldehyde-3-phosphate dehydrogenase expression. The primers used in this study are listed in Table 2.
2.7 Patient-derived xenograft mouse models
Fresh OSCC surgical tissue specimens with low or high TP+CAFs were obtained from two OSCC patients and then used for the construction of patient-derived xenograft (PDX) mouse models.
Each surgical specimen was cut into five cubical pieces with 3 mm sides and then subcutaneously implanted into the calvaria of female 5-week-old BALB/c athymic nude mice (Central Laboratory Animal Inc., Seoul, Korea). The mice were sacrificed 4 weeks after implantation of the OSCC tissues. The area of bone invasion was evaluated using micro-computed tomography (micro-CT) images with Skyscan 1173 (Bruker-microCT, Kontich, Belgium) and reconstructed using Nrecon software.
2.8 Statistical analysis
The association between clinicopathologic variables and protein expression and the status of desmoplasia was analyzed using the chi-squared test. The Mann–Whitney U-test was used to evaluate the influence of TP on the biological behaviors of CAFs. SPSS software version 26.0 (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis, and a p-value of less than 0.05 was considered statistically significant.