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 (Fig. 1A-C).
Tissue samples
All samples were obtained from the Chinese PLA General Hospital Neurosurgery. A total of 90 paraffin tissue samples and 100 frozen tissue samples were included in this study. Paraffin tissue samples were subjected to IHC, 10 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.
Key Reagents
Key Reagents were shown in Supplementary Table S1.
RNA sequencing (RNA-seq)
Total RNA was extracted using TRIzol reagent (15596-018, Invitrogen, USA) and then we analyzed its concentration and purity by the RNA Nano 6000 Assay Kit of Bioanalyzer 2100 System (Agilent Technologies, USA). Global mRNA was purified by Oligo dT-Coated Magnetic Beads and then cleaved into small mRNA fragments. The mRNA-Seq sample preparation kit (Illumina, USA) was used to create the final cDNA library according to the instructions. NovaSeq 6000 200-cycle SP flow cell (Illumina, USA) was used to perform the paired-end sequencing (2X100). EdgeR software was used to examine differential expression of mRNA. 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 Paraffin sections (4 mm-thick) were deparaffinized in xylene and rehydrated through graded ethanol (100% I, 100% II, 90%, and 80%). For antigen retrieval, the sections were heated at 100°C in a microwave in 1× citrate buffer, pH 6.0 or an EDTA solution, pH 9.0 for 2.5 min. To block the endogenous peroxidase activity, the slides were immersed in 3% hydrogen peroxide (H2O2) in methanol for 15 min and then washed three times with 1× phosphate-buffered saline (PBS) for 5 min. The slides were then incubated with the primary antibodies to CTSK (ab207086, Abcam, USA), overnight at 4°C in a humidified chamber, and then washed with PBS. Biotinylated goat anti-polyvalent (ab6789, Abcam, USA), and streptavidin peroxidase were then added and incubated for 10 min at 20–25°C, in succession. After washing with PBS, the slides were treated with 3,3’-diaminobenzidine (DAB) substrate (ab64264, Abcam, USA), for 15 min at 20–25°C. The slides were then washed two times with water, lightly counterstained with hematoxylin, and finally, mounted with coverslips and DPX after dehydration through an ethanol gradient (80%, 90%, 100% II, and 100% II). 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 Eco™ real-time PCR system (Illumina, USA). PCR primers of mRNAs in our study were shown in Supplementary Table S2.
cell culture
GH3 cells were maintained in Ham’s F-12K (F12K) Nutrient Mixture (21127030, Gibco, USA) with 2.5% fetal bovine serum (FBS) and 15% horse serum (HS). GT1-1 cells were maintained in Dulbecco’s modified Eagle medium (DMEM, 11995065, Gibco, USA) with 8% FBS and 2% HS. 293T cells were maintained in DMEM with 10% FBS. MC3T3-E1 cells were maintained in minimum essential medium Alpha (Mem-α, 41061029, Gibco, USA) without acid containing 10% FBS. All culture medium used was supplemented with penicillin (100 U/mL) and streptomycin (100 mg/mL). All cells were maintained at 37°C in a 5% CO2 incubator.
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 S3. 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. Cells (2×103/well) were plated in 96-well plates at 37°C with 3 duplicated wells in each group. Cell numbers were counted at 0h, 12h, 24h, 48h, 72h, and 96h after transfection to determine the cell growth curve. For colony formation assays, cells (5×102/well) were seeded in the 6 cm dishes and incubate at 37°C in humidified incubator. When growing to a certain extent, cells were fixed with formaldehyde and stained with crystal violet. Cell colonies more than 50 cells were counted and imaged. For EdU assays, BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (C0071L, Beyotime, China) was used. Cells (1×104/well) were plated in 24-well plates and treated with EdU reagent for 1h. After washed, fixed, permeabilized, and blocked, cells were incubated with the Click Reaction Mixture for 30 min at room temperature darkly. Then the cells were incubated with Hoechst 33342 for 10 min and observed under a fluorescence microscope (ECLIPSE Ni, Nikon, Japan).
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 and induced by osteogenic induction medium containing L-ascorbic acid, β-glycerophosphatewhich, and dexamethasone. The culture medium was replaced every other day. Seven days later, cells were washed and lysed with Triton-NH4OH lysis Buffer. Then supernatant was removed and bone matrix was retained. GH3 cells (1×104/well) were seeded on the surface of bone matrix. Culture medium including 20% FBS was added to the lower chamber. Forty-eight hours later take out the transwell chamber and discard the culture medium. Then non-migrated cells were removed and migrated cells attached on the lower surface of the membrane were fixed with formaldehyde and stained with crystal violet. Three fields of each group were selected randomly to observe and count the migrated cells under a microscope.
Western blot
Radio immunoprecipitation assay (RIPA) buffer (P0013B, Beyotime, China) with Phenylmethanesulfonyl fluoride (PMSF), protease inhibitor cocktail, and sodium orthovanadate was used to extract protein. Enhanced BCA Protein Assay Kit (P0010, Beyotime, China) was used to quantified protein concentration. Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) was used to separate proteins and then proteins were transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk in Tris Buffered Saline with Tween-20 (TBST) for 40 min at room temperature. Primary antibodies CTSK, S6K, p-S6K, 4E-BP1, p-4E-BP1, UCH-L1, TLR4, and GAPDH were incubated overnight at 4°C. HRP labelled secondary antibodies (1:5000) (ab64264, Abcam, USA) were incubated for 2 hours at 37°C. For exposure, excellent chemiluminescent (ECL) substrate Detection Kit (NCI5079, Thermo, USA), developing powder (500048-0001, Sangon Biotech, China), and fixing powder (500047-0001, Sangon Biotech, China) were used.
Co-immunoprecipitation (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.
Immunofluorescence (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.
bone marrow macrophage (BMM) isolation Triple co-culture assay and TRAP staining
Whole bone marrow cells were collected from the femora and tibiae of 6–8 weeks C57BL/6 mice and resuspended in ACK Lysis Buffer (T10105, Saint-Bio, China). Supernatant were discarded and cell were resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (R8758, Sigma-Aldrich, USA) containing 10% FBS, 1% penicillin/streptomycin, 100 µl M-CSF. Then cells were cultured on 60-mm culture dishes at a density of 3 × 106 cells/well in the presence of M-CSF. The culture medium was replaced every other day and subsequent experiments were conducted on the sixth day. 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 Scanning electron microscope (SEM).
To explore the inhibitory function of CTSK inhibitor on tumor growth and bone invasion in vivo, the mice (n = 10) 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).
Enzyme-linked immunosorbent (ELISA) sassy
A C-telopeptide of type Ⅰ collagen (CTX-Ⅰ) 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/Kruskal Wallis 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.