Transcriptomic profiling of GBM revealed three molecularly distinct subtypes: proneural (PN), mesenchymal (MES), and proliferative (Prolif), with the MES subtype exhibiting the most aggressive clinical behavior characterized by extensive necrosis and significantly reduced patient survival [23]. To delineate receptor-mediated pathogenic mechanisms underlying GBM progression, particularly in the MES subtype, we employed weighted gene co-expression network analysis (WGCNA). This systems biology approach identified HRH1 (histamine H1 receptor) as a top-ranked differentially expressed receptor gene showing strong network connectivity. Functional validation through HRH1 knockdown demonstrated potent inhibition of malignant phenotypes across multiple GBM models. While traditionally associated with allergic responses, recent findings position HRH1 as a novel modulator of the GBM tumor microenvironment (TME). For instance, elevated levels of histamine and HRH1 in the TME have been linked to T cell dysfunction [24]. Electronic medical records from the Lausanne University Hospital also showed that GBM patients who took antihistamines during treatment had better survival rates [25]. However, the specific role and underlying mechanisms of HRH1 in GBM progression remain unclear [26, 27]. In this study, we identified HRH1 as a novel therapeutic target for GBM treatment, and developed a powerful mAb drug. Protein kinase C (PKC)-dependent modulation of the RAF/MEK/ERK signaling pathway was demonstrated to be the underlying mechanism.
Human GBM tissues were collected from patients with a pathological diagnosis of IDH-wildtype glioblastoma at the Affiliated Hospital of Wenzhou Medical University. The study protocol received approval (KY2021-R129) from the hospital's Clinical Research Ethics Committee. All patients provided informed consent to provide the samples, and none of them received chemotherapy, radiation, or immunotherapy prior to tumor resection.
The GBM cell lines (U251, U87, LN229, and KNS89) were obtained from the American Type Culture Collection or the Japanese Collection of Research Bioresources, with their authenticity verified by short tandem repeat profiling and mycoplasma testing. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (11995040, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (10099141, Gibco, USA) and 1% penicillin/streptomycin (15140122, Gibco, USA). The culture conditions of HEK-293T cells and normal human astrocytes (NHA) were the same as above. Primary GBM stem cells (GSCs) (CD133+/ SOX2+) were isolated from human tumor specimens as described in our previous report [17], and cultured in DMEM/F12 (11320033, gibco, USA) with 1×B27 (17504044, Gibco, USA), 1×Insulin (51500056, Gibco, USA), 20 ng/mL progesterone (GC12408, GLPBIO, USA), 20 ng/mL EGF (100-47, PeproTech, USA), 20 ng/mL FGF (AF-100-18B, PeproTech, USA), and 1% penicillin/streptomycin. Primary GBM cells were extracted and cultured following established protocols from prior publications [28], and passages 2-3 were utilized for experimentation.
The 97-mer oligonucleotide (Table S1), synthesized by Sangon Biotech (China), was PCR-amplified and subsequently digested with restriction endonucleases (NEB, USA) before being ligated into an mCherry-tagged lentiviral vector. The construct was then transformed into E. coli for plasmid amplification and sequence verification [29, 30]. The CRISPR/Cas9 plasmid was designed and constructed following the protocol established by the Zhang Laboratory (Table S2) [31]. The Flag-tagged KRAS-G12C mutant overexpression plasmid and the USP10-expressing plasmid were provided by Youze Bio Co., Ltd. (China). For lentivirus production, the recombinant plasmid was co-transfected with packaging plasmids (pMDL, VSV-G, and pRSV) into HEK-293T cells using PEI. Following 16-hour incubation, the medium was replaced with fresh DMEM supplemented with 10% FBS. Viral supernatants were collected 48 hours post-transfection, filtered through 0.22 µm membranes, and used to infect GBM cell lines (U251, U87, KNS89, and LN229) at a multiplicity of infection (MOI) of 5. Infection efficiencies were quantified by fluorescence microscopy (Fig. S1A, B).
Male BALB/c nude mice (8 weeks, 20 g) provided by Shanghai Charles River Experimental Animal Limited Liability Company (Shanghai, China) were housed in a controlled environment with free access to food and water. Orthotopic GBM xenografts were established through intracranial transplantation of U87 cells, as reported previously [16]. Briefly, nude mice were anesthetized with isoflurane (2%) and placed on a stereotaxic apparatus (RWD Life Science, China). U87 cells with luciferase reporter gene (500,000/5 µL, three days post-infection) were injected through a 2 mm bone window (1 mm anterior to the sagittal suture, 2 mm right of the midline) into the striatum (3 mm below the dura). A PerkinElmer IVIS Lumina X5 in vivo imaging system was used to monitor xenografts every seven days (captured 10 min after luciferin injection with a 10-second exposure time) [32]. The survival time and body weight of these nude mice were documented for analysis. Twenty-eight days after transplantation, five mice per group were sacrificed to obtain brain samples.
All experimental procedures were approved by the Animal Ethics Committee of Wenzhou Medical University in strict accordance with the Guidelines for the Care and Use of Animals of the National Institutes of Health. Investigators randomly assigned the animals to each group (n = 5/group), followed by orthotopic U87 cell inoculation as previously described. Due to technical constraints of the intervention procedure, investigators were not blinded to group assignment during transplantation or administration; however, all endpoint measurements were performed by blinded personnel.
The total protein in cells was extracted with RIPA lysis buffer (89900, Thermo Fisher Scientific, USA) containing protease and phosphatase inhibitors (A32959, Thermo Fisher Scientific, USA). The concentration was determined using the bicinchoninic acid Protein Assay Kit (23227, Thermo Fisher Scientific, USA). Protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride membranes (IPVH00010, Millipore, USA). Membranes with protein bands were incubated with a buffer containing 5% nonfat powdered milk for 3 h to reduce non-specific bands and then incubated in primary antibodies (including HRH1 (DF4980), MARCKS (AF6250), p-MARCKS (AF3250), RAF-1 (AF6065), p-RAF-1 (AF3065), MEK1/2 (AF6385), p-MEK1/2 (AF8035), ERK1/2 (AF0155), p-ERK1/2 (AF1015), Flag-Tag(T0053), USP10 (DF8061) and GAPDH (AF7021) antibodies from Affinity (USA), 1:1000) and secondary antibodies (S0001, Affinity, USA, 1:5000). Invitrogen iBright FL1500 Imaging System and Image J software (v1.8.0) were used to capture and analyze western blots, respectively.
The total RNA in cells was extracted with TRIzol reagent (15596018, Thermo Fisher Scientific, USA) and then transcribed into cDNA using a Revert Aid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific, USA). The Specific primers were designed using Primer-BLAST and synthesized by Sangon Biotech (China, Table S3). Iraq Universal SYBR Green supermix (1725124, BIO-RAD, USA) was used to perform qRT-PCR. After the reaction, the calibrated target gene cycle threshold (Ct) value was inserted into the 2 formula to estimate the relative change in gene expression.
The brain tissue containing U87 xenografts was sampled and sectioned according to the protocol described previously [16]. The sections were incubated with KI67 primary antibody (ab15580, Abcam, USA, 1:1000) and then labeled with secondary antibody (ab150077, Abcam, USA, 1:1000) and DAPI (ab285390, Abcam, USA, 10 µg/mL). OLYMPUS confocal laser scanning microscope (JPN) and Image J software (v1.8.0) were used to capture and analyze IF staining, respectively. Quantification was performed on 45 random fields from a series of every 9th coronal brain section from five nude mice. Human brain tissue samples obtained under the approval of the ethics committee were subjected to HE and IHC staining as per the protocols [33, 34]. HRH1 antibody (13413-1-AP, 1:50) used in IHC was provided by Proteintech (USA).
Cell viability was assessed using the MTT assay (KGA312, KeyGen Biotech, China) according to the manufacturer's protocol. Briefly, U251, U87, LN229, KNS89, and NHA cell lines as well as GSCs, were seeded in 96-well plates at a density of 5 × 10³ cells/well and allowed to adhere for 24 h under standard culture conditions. Following treatment with viral vectors, pharmacological agents, or mAb, cells were incubated for specified time periods. The yellow 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) added to the cell culture medium was reduced to blue-purple formazan by the dehydrogenase in living cells and then dissolved with DMSO. Absorbance values at 490 nm were used to assess cell viability. The half-maximal inhibitory concentration (IC₅₀) values were calculated using GraphPad Prism software (v7.04).
GBM cells were scratched using a sterile 200 µL pipette tip to form a stable wound and washed with phosphate-buffered solution (PBS) to remove non-adherent cells. A medium containing 2% FBS was used to culture the adherent cells. The migration ability of live cells was observed under a microscope. Image J software (v1.8.0) was used to calculate the change in the wounding area. Wounding area (%) = A/A × 100%. A is the area of the wound measured immediately after scratching, and A is the area of the wound measured 48 or 60 h after scratching.
Matrigel was used to coat the polycarbonate membrane inside the transwell chamber (3422, Corning, USA) before assay. U251, U87, LN229, and KNS89 cells were re-suspended in a serum-free medium and seeded in the upper chamber at a density of 20,000/100 µL. The lower chamber was filled with a complete medium for inducing cell migration, and then incubated for 48 h. GBM cells will pass through the polycarbonate membrane and cling to the other side of the upper chamber. After removing non-migratory cells in the upper side of the upper chamber, down side cells were stained with crystal violet. Five fixed positions per chamber were captured to assess invasion ability.
To evaluate the self-renewal capacity of individual GSCs, a limiting dilution sphere formation assay was performed. Single-cell suspensions were plated at clonal density (2000 cells/well) in 6-well ultra-low attachment plates (3473, Corning, USA). The size and number of spheres were measured using an Olympus microscope (JPN) seven days post-treatment. Only well-defined, non-aggregated spheres exceeding 100 µm in diameter were counted to determine the sphere-forming efficiency (SFE), calculated as: SFE (%) = (Number of spheres formed/Number of single cells seeded) × 100.
HRH1-mAbs were produced by Abmart (China) using peptides with the following sequences: MSLPNSSCLLEDKM, CEGNKTTMASPQLMP, WNHFMQQTSVRREDKC, REDKCETDFYDVTW, and CNEHLHMFTI. To assess the blood-brain barrier (BBB) penetration and tumor-targeting specificity of HRH1-mAb, we prepared HRH1-mAb-FITC using the following protocol and administered it intravenously via tail vein injection into GBM xenograft models for evaluation. FITC conjugation was performed according to published protocols [35], with the brief procedure summarized as follows: antibodies in 0.1 M carbonate-bicarbonate buffer (pH 9.5) were conjugated with FITC-NHS ester (in anhydrous DMF) via NHS-amine bonding. After 1 h dark incubation at room temperature, free dye was removed by desalting. The FITC/Protein ratio (measured at 280/495 nm via NanoDrop) ranged from 1.8 to 2.1.
RNA sequencing data (raw count files) and corresponding clinical data for GBM samples were obtained from the Cancer Genome Atlas (TCGA) database. Read counts were normalized using the DESeq package in R to account for library size differences. For microarray datasets (Rembrandt and GSE4271), raw probe-level data (CEL files) were processed using the robust multi-array average (RMA) normalization algorithm implemented in the affy package in R, which included background correction, quantile normalization, and median polish summarization. Detailed protocols for the aforementioned preprocessing pipeline and HRH1-tumor immune microenvironment interaction analyses are documented in the Supplementary Methods [36].
All experiments were performed with three independent replicates, and statistical analysis was conducted using GraphPad Prism 7.04 (GraphPad Software, USA). The sample size was chosen based on prior studies and previously published literature. An unpaired t-test was used to compare differences between two groups. One-way analysis of variance was used to compare three or more groups. Log-rank (Mantel-Cox) test was used to compare Kaplan-Meier survival curves. All data were expressed as mean ± standard deviation, except for the expression of HRH1 in the GBM dataset, which is expressed as mean ± standard error. No data were excluded from the analysis of experiments. The WGCNA was performed via the R language (4.4.2). P < 0.05 was considered statistically significant in this study.