Patients
Patients undergoing first surgery for resection of pituitary adenomas at the Chinese PLA General Hospital (Beijing, P.R. China) from January 2016 to June 2022 were included. All patients underwent preoperative head MRI, thin layer skull base CT scanning, and pathological examination and were divided into three groups: bone IPAs (BIPAs), IPAs but without bone invasion, and non-IPAs. The diagnosis of the tumor and its functional status was according to its clinical, biochemical, radiological, and pathological features, and was verified by immunohistochemical (IHC) staining for pituitary specific transcription factor 1 (PIT1), T-box pituitary transcription factor (TPIT), steroidogenic factor 1 (SH1), prolactin (PRL), growth hormone (GH), adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), follicular stimulating hormone (FSH), and luteinizing hormone (LH) according to the 2022 WHO classification of pituitary tumors20. The criteria of invasion included: (1) preoperative images showed that Knosp/Hardy grades were 3 or 4, (2) intraoperative inspection of destruction of sellar floor, clivus, anterior skull, or medial wall of cavernous sinus. The invasion was diagnosed only when both of the above criteria are met. The criteria of bone invasion included: (1) preoperative CT images showed severe destruction of the clivus or anterior skull, (2) intraoperative inspection of bone destruction, (3) pathological examination showed bone tissue infiltration5.
Tissue samples
All samples were obtained from the Chinese PLA General Hospital Neurosurgery. A total of 90 paraffin tissue samples and 105 frozen tissue samples were included in this study. Paraffin tissue samples were subjected to IHC, 15 frozen tissue samples were subjected to mRNA sequencing analysis, and 90 frozen tissue samples were subjected to RT-PCR.
Postoperative follow-up
Postoperative telephone or outpatient follow-up was conducted every 6 months until Februrary 2023. MR scans were used to evaluate tumor relapse. Tumor relapse is defined as tumor recurrence after gross-total resection (GTR) or an increase of residual tumor more than 2-mm in at least one dimension after subtotal resection (STR) according to contrast enhanced (CE) T1WI21.
RNA sequencing (RNA-seq)
The details about RNA-seq are given in the Supplementary Materials. Differential expression analysis of two groups was performed using the Mann Whitney U test with cutoff |log2(FC)|≧2 and P-value < 0.001.
IHC
IHC was performed as described previously.22 The intensity of expression was scored as 0 (negative staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). The final score was calculated as follows: (1×% of weak staining) + (2×% of moderate staining) + (3×% of strong staining). Taking 100% as the boundary line, patients could be separated into high expression and low expression groups.
RNA isolation and qRT-PCR
RNA was extracted from frozen tissue samples or cells using TRIzol reagent (15596-018, Invitrogen, USA) and then reverse transcribed using TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (AT341). Subsequently, we performed qRT-PCR using 2× SG Fast qPCR Master Mix (High Rox) (B639273) and EcoTM real-time PCR system (Illumina, USA). PCR primers of mRNAs in our study were shown in Supplementary Table S1.
cell culture
The GH3 cells, GT1-1 cells, 293T cells, and MC3T3-E1 cells were purchased from BNCC. All cells were maintained at 37°C in a 5% CO2 incubator. All culture medium used was supplemented with penicillin (100 U/mL) and streptomycin (100 mg/mL). More details about culture medium are given in the Supplementary Materials.
Transfection
The plasmid containing CTSK coding sequence (CDS) and lentiviruses containing CTSK CDS were purchased from Shanghai GenePharma Co., Ltd. The CTSK plasmid and siRNAs were transfected to GH3 and GT1-1 cells transiently by Lipo2000 (11668019, Thermo, USA). CTSK siRNA sequences used in our study were shown in Supplementary Table S2. GH3 cells were transfected with lentivirus containing CTSK CDS to get stable CTSK overexpression GH3 cells. Transfection efficiency was determined by western blot.
Cell proliferation assay
Cell proliferation was detected by CCK8 assay, colony formation assays, and EdU assays. For CCK8 assay, cell counting Kit-8 (CCK8, E606335-0500, BBI, China) was used. Cell numbers were counted at 0h, 12h, 24h, 48h, 72h, and 96h after transfection to determine the cell growth curve. For colony formation assays, Cell colonies more than 50 cells were counted. For EdU assays, BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (C0071L, Beyotime, China) was used. More details are given in the Supplementary Materials.
Transwell invasion assay
Transwell co-culture system (8.0-µm pore polyester membrane) (3422, Corning, USA) was used. MC3T3-E1 cells (1×104/well) were seeded in the upper chamber to prepare bone matrix. GH3 cells (1×104/well) were seeded on the surface of bone matrix. More details are given in the Supplementary Materials.
Western blot
protein extraction, quantification, separation, and transfer, immunoblot incubation, and antibody probing were described in the Supplementary Material. Primary antibodies included CTSK (ab207086, Abcam, USA), S6K (2708T, CST, USA), p-S6K (9208T, CST, USA), 4E-BP1 (9644T, CST, USA), p-4E-BP1 (2855T, CST, USA), UCH-L1 (13179T, CST, USA), TLR4 (38519S, CST, USA), and GAPDH (5174S, CST, USA).
Co-IP
CTSK-overexpression plasmid and TLR4-overexpression plasmid were co-transfected into 293T cells and MC3T3-E1 cells. After lysis by cold RIPA buffer and centrifugation for 30 min, the supernatant of were collected. anti-CTSK, anti-TLR4, and IgG were added to the supernatant and the mixture was incubated overnight at 4 °C. Protein A+G magnetic beads (P2179M, Beyotime, China) were then added and incubated for 2-4 h at 4 °C. After centrifugation for 3 min, agarose beads binding with antigen-antibody complex were isolated and washed with cold PBS for 3 times. The supernatant was collected for the following western blot analysis.
IF staining
MC3T3-E1 cells were cultured on coverslips and treated with Alexa Fluor-488 conjugated human CTSK protein (ab236553, Abcam, USA) for 24 h. The cells were fixed with 4% paraformaldehyde and treated with 0.25% Triton X-100. After blocking with 10% normal blocking serum, the cells were incubated with antibody against TLR4 (38519S, CST, USA) at 4oC overnight. The cells were then incubated with Alexa 594-labeled conjugated anti-rabbit secondary antibody (8889S, CST, USA) and observed under a confocal microscope (FV1000, Olympus, Germany).
Ubiquitination assay
The 293T cells were co-transfected with plasmids described in the figure legends for 48 h. The plasmids included pcDNA3.1-CTSK-HA, pcDNA3.1-Ub-His, pcDNA3.1-Raptor-Flag, pcDNA3.1-UCH-L1-Myc. RIPA buffer (P0013B, Beyotime, China) supplemented with PMSF (ST506, Beyotime, China), MG132 (S1748, Beyotime, China), and Ubiquitin Isopeptidase Inhibitor I (ab145967, Abcam, USA) was used to extract protein. For IP, Flag-tag antibody (14793S, CST, USA) was used. His-tag antibody (2365S, CST, USA) and HRP labelled secondary antibody (ab64264, Abcam, USA) was used to detect Raptor protein ubiquitination in whole cell lysates (WCL) and immune precipitates by western blot analysis.
BMM isolation Triple co-culture assay and TRAP staining
The steps of BMM isolation were described in the Supplementary Material. Triple co-culture assay was performed according to protocol described in previously published study23. BMM cells (2 × 104 cells/well) were seeded directly into 6-well plates, and MC3T3-E1 cells (3 × 104 cells/well) were seeded into Inserts (Millipore) in the 6-well co-culture plates. The next day, GH3 cells (3 × 104 cells/well) were added on top of the MC3T3-E1 cell layer in triplicate and treated with RANKL (50 ng/mL) and M-CSF (20 ng/mL). Six days later the BMM cells were washed and fixed, then TRAP staining was conducted using the TRAP stain kit (G1492, Solaribio, China) following the manufacturer’s instructions. TRAP-positive multinucleated cells with ≥3 nuclei were regarded as mature osteoclasts.
Xenograft tumor model
Female BALB/c-nu mice (6-8 weeks) were obtained from SPF (Beijing) Biotechnology Co., Ltd, China. CTSK-Con and CTSK overexpression GH3 cells (1×106) were injected subcutaneously into the top of the head of the mice (n=6), respectively. Tumor diameter was measured every three days. After 30 days, the mice were euthanized by spinal dislocation. The tumors and mouse skulls were isolated. The tumors were weighed, imaged and frozen for western blot and RT-PCR. The tumor volumes were calculated using the formula: e (length × width2)/224. The mouse skulls were for the subsequent examination by SEM. For intraosseous tumorigenesis, CTSK-Con and CTSK overexpression GH3 cells (1×106) were injected into the bone marrow cavity of left tibial of nude mice. The mice were euthanized on the thirtieth day and the left tibial were isolated for the subsequent examination by μCT.
To explore the inhibitory function of CTSK inhibitor on tumor growth and bone invasion in vivo, the mice (n=12) were injected with CTSK overexpression GH3 cells (1×106) subcutaneously the top of the head and divided into two groups: medication group given odanacatib (MK-0822, 5 mg/kg, dissolved in 2% DMSO/PBS) every other day by i.p. injection from the day of tumor cell inoculation and control group given DMSO. Tumor diameter was measured every three days. After 30 days, the mice were euthanized by spinal dislocation and the subsequent approach was the same as above.
SEM
The mouse skulls were fixed with glutaric dialdehyde solution (2.5%) overnight at 4 °C. Washed, fixed with osmiridic solution (1%) for 2h. Then sampled were dehydrated with gradient alcohol (50%, 70%, 80%, 90%, 95%, and 100%) and isopentyl acetate, dried using critical point drying (K850, Quorum, England), coated with gold powder in surface using ion sputtering instrument (MC1000, Hitachi, Japan) and examined using SEM (SU8100, Hitachi, Japan).
μCT
The left tibial of nude mice were fixed in 10% formalin for 2 days. Hiscan XM Micro CT (Suzhou Hiscan Information Technology Co., Ltd) was used. The X-Ray tube settings were 80 kV and 100 uA and images were acquired at 25um resolution. A 0.5° rotation step through a 360° angular range with 50 ms exposure per step was used. The images were reconstructed with Hiscan Reconstruct software (Version 3.0, Suzhou Hiscan Information Technology Co., Ltd) and analyzed with Hiscan Analyzer software (Version 3.0, Suzhou Hiscan Information Technology Co., Ltd).
ELISA sassy
A CTX-1 ELISA kit (E-EL-M3023, Elabscience, China) was used to examine the levels of CTX-1 in serum and supernatant according to the manufacturer’s instructions.
Statistical analysis
Continuous variables are expressed as mean with SD or median (interquartile range) and categorical data are expressed as frequency and percentage. Continuous variables were compared using the t test/one-way analysis of variance (ANOVA) (for data conforming to normal distribution and homogeneity of variance) or Wilcoxon test/KruskalWallis H test (for data not conforming to normal distribution or homogeneity of variance). Categorical variables were compared using the chi-square test. Statistical significance was determined by p < 0.05. Univariate and multivariate Cox regression model was conducted to analyze the risk factors for recurrence of pituitary adenomas. Kaplan–Meier method was conducted to generate the recurrence-free curves. Pearson analysis was used for correlation analysis of CTSK and tumor diameter. IBM SPSS statistics (version 25.0, SPSS, Inc, Chicago, Illinois) software were used to perform all analyses.