Search Results (63)

Glypican-1 (GPC1) has emerged as a critical mediator of malignant tumor progression. GPC1 plays essential roles in regulating various signaling pathways involved in tumor cell proliferation, invasiveness, and tumorigenesis. Overexpression of GPC1 in tumors mediates oncogenic transformation, epithelial-to-mesenchymal transition, metastatic dissemination, and therapeutic resistance. Accordingly, GPC1-targeted therapeutic strategies have been investigated in clinical and preclinical studies. However, clinical efficacy has been limited. We previously developed an anti-GPC1 monoclonal antibody (mAb), G₁Mab-28 (mouse IgG₁, κ), which exhibits high affinity and specificity for GPC1. In the present study, we generated recombinant isotype-converted G₁Mab-28, including G₁Mab-28-mG_2a (mouse IgG_2a) and G₁Mab-28-hG₁ (human IgG₁). Both mAbs recognized GPC1-expressing human tumor cell lines, including lung squamous cell carcinoma PC-10 and pancreatic ductal adenocarcinoma PK-45H, by flow cytometry. Moreover, both mAbs exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against those cell lines. In mouse xenograft models, treatment with the mAbs resulted in potent antitumor efficacy against PC-10 and PK-45H tumors. Collectively, these findings support the therapeutic potential of G₁Mab-28 for the treatment of GPC1-positive tumors. Full article

Glypican-3 (GPC3)-targeted chimeric antigen receptor T (CAR-T) cell therapy is a promising approach for hepatocellular carcinoma (HCC), but marked interpatient variability and antigen heterogeneity limit its broader application. Here, we established a patient-derived organoid (PDO)-based platform to functionally evaluate autologous GPC3-targeted CAR-T cell activity in HCC. HCC PDOs preserved key histologic features and heterogeneous GPC3 expression patterns of the original tumors. In co-culture assays, CAR-T cell cytotoxicity was associated with GPC3 expression levels and was accompanied by IFN-γ and IL-2 release, supporting the feasibility of using PDOs for functional assessment of CAR-T cell sensitivity. We further found that matrix conditions strongly influenced organoid architecture, viral transduction, CAR-T cell infiltration, and killing efficiency, with lower Matrigel concentrations providing a more permissive setting for functional assessment. Importantly, in GPC3-low PDOs, pretreatment with the DNA methyltransferase inhibitor 5-azacytidine (5-AZA) reduced DNA methyltransferase 3 alpha (DNMT3A) expression, increased surface GPC3 expression, and significantly enhanced CAR-T-mediated cytotoxicity. Together, these findings provide proof-of-concept evidence supporting the use of HCC PDOs as a patient-derived platform for modeling selected determinants of GPC3-targeted CAR-T cell activity and for exploring combination strategies to improve therapeutic efficacy. Full article

Background/Objectives: Glypican-1 (GPC1) is a heparan sulfate proteoglycan that plays a critical role in regulating various signaling pathways and tumor development. Overexpression of GPC1 promotes tumor cell proliferation and invasiveness, and is associated with poor clinical outcomes. Therefore, anti-GPC1 monoclonal antibodies (mAbs) have been developed in various modalities for tumor therapy. Methods: We developed novel anti-GPC1 mAbs using a flow cytometry-based high-throughput screening approach, the Cell-Based Immunization and Screening (CBIS) method. Results: A clone G₁Mab-28 (IgG₁, κ) reacted with GPC1-overexpressed Chinese hamster ovary-K1 (CHO/GPC1), but not parental CHO-K1, in flow cytometry. Furthermore, G₁Mab-28 recognizes the endogenous GPC1-expressing human esophageal squamous cell carcinoma KYSE770 cell line. Furthermore, G₁Mab-28 specifically recognized only CHO/GPC1, but not the other GPC family-overexpressed CHO-K1. The dissociation constant values of G₁Mab-28 for CHO/GPC1 and KYSE770 were determined to be 3.3 × 10⁻⁸ M and 4.6 × 10⁻⁹ M, respectively. Moreover, G₁Mab-28 is suitable for Western blotting and immunohistochemistry. Conclusions: G₁Mab-28, established by the CBIS method, is versatile for basic research and is expected to contribute to antibody-based tumor therapy. Full article

Background: Tumor immune microenvironment (TIME) heterogeneity limits immunotherapy efficacy in hepatocellular carcinoma (HCC), underscoring the need for predictive biomarkers and therapeutic targets. We previously identified dual specificity phosphatase 4 (DUSP4) as a mediator of sorafenib resistance, but its immunomodulatory role remains unknown. Methods: Glypican-3 (GPC3)-specific chimeric antigen receptor (CAR) T-cell cytotoxicity assays were performed to assess the impact of DUSP4 on HCC immune susceptibility. A subcutaneous tumor model using Dusp4-overexpressing cells in female C57BL/6J mice was established to evaluate DUSP4-mediated microenvironment remodeling and anti-PD-L1 therapy efficacy. Bulk RNA sequencing of DUSP4-overexpressing HCC cells identified downstream pathways. Public datasets were interrogated to correlate DUSP4 expression with immune checkpoint blockade (ICB) response and immune infiltration in HCC. Results: DUSP4 overexpression significantly enhanced HCC cell susceptibility to CAR-T cell killing in vitro and potentiated anti-PD-L1 efficacy in vivo, accompanied by TIME remodeling. Mechanistically, RNA sequencing revealed DUSP4-mediated downregulation of the TGF-β signaling pathway, functionally confirmed using a neutralizing antibody that abrogated the enhanced CAR-T killing. Public datasets confirmed associations between DUSP4 expression and enhanced immune cytolytic activity with favorable prognostic outcomes in HCC. Conclusions: DUSP4 serves as a critical molecular nexus linking targeted therapy resistance to enhanced immunotherapy sensitivity. By attenuating the TGF-β signaling pathway, DUSP4 reprograms TIME toward an immunologically active state, thereby augmenting the efficacy of immunotherapy. These findings establish DUSP4 as a promising dynamic biomarker for guiding sequential therapy in HCC and highlight its potential as a novel therapeutic target to improve outcomes in solid tumor immunotherapy. Full article

Spinal cord injury (SCI) induces persistent locomotor deficits that are closely associated with maladaptive structural plasticity of spinal neuronal circuits. Although motor rehabilitation improves functional outcomes, the cellular substrates underlying rehabilitation-induced recovery remain incompletely understood, particularly in relation to activity-dependent neuromodulation strategies. Here, we investigated how treadmill-based motor training (TMT) and its combination with bioluminescent optogenetic (BL-OG) stimulation of Hb9 (homebox 9)-positive motoneurons and excitatory interneurons selectively modulate microarchitectural plasticity in the injured rat spinal cord. At the level of gross locomotor assessment, Basso, Beattie and Bresnahan (BBB) scores were comparable between the BL-OG and SCI+TMT groups. Although no statistically significant differences in the total score in rung ladder were observed at 28 days post-injury, animals in the BL-OG group showed a tendency toward a higher ratio of successful hindlimb placements, indicating improved step accuracy. BL-OG stimulation was associated with a slightly greater attenuation of SCI-induced spine abnormalities compared to TMT alone, with significant differences between the experimental groups detected specifically in laminae VIII and IX. These lamina-specific alterations in dendritic integration and dendritic spine composition were accompanied by preservation of wisteria floribunda agglutinin WFA-positive perineuronal net (PNN) architecture. Against this background, reduced glypican-4 (GPC-4) expression and attenuated WFA/GPC-4 colocalization were observed in the SCI+BL-OG group relative to SCI in laminae VII–IX, consistent with activity-dependent modulation of PNN-associated synaptic organization in Hb9-positive neuronal populations. Together, these findings indicate that motor rehabilitation and bioluminescent optogenetic stimulation engage distinct but partially overlapping mechanisms of activity-dependent microarchitectural remodeling, preferentially targeting synaptic and perineuronal net-associated substrates rather than inducing large-scale circuit reorganization. Further studies are warranted to elucidate the mechanisms underlying these distinct plasticity profiles. Full article

Background/Objectives: Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan, has been identified as a promising target for both the diagnosis and treatment of hepatocellular carcinoma (HCC). However, the diagnosis of GPC3 expression mainly depended on invasive procedures. This study aimed to investigate the potential of dual-energy computed tomography (DECT)-derived parameters for noninvasive prediction of GPC3 expression in HCC. Methods: This retrospective study included 79 HCC patients with confirmed GPC3 immunohistochemistry and pretreatment contrast-enhanced DECT. Qualitative imaging features and quantitative DECT parameters, including iodine density of HCC (ID_Ca), normalized iodine density (NID), slope of spectral attenuation curve (λ_HU), and effective atomic number (Z_eff), were evaluated in both arterial and portal venous phases. Univariate and multivariate logistic regression analyses were employed to identify independent predictors, and a combined model was subsequently constructed. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic efficiency of imaging parameters in predicting GPC3 expression. Interobserver agreement of DECT parameters was evaluated using intraclass correlation coefficients (ICC). Results: GPC3-positive HCCs demonstrated significantly higher arterial phase (AP) ID_Ca, NID, λ_HU, and Z_eff (all p ≤ 0.001) than GPC3-negative HCCs. Multivariate logistic regression analysis identified NID-AP [Odds ratio (OR) = 2.00, p = 0.010] and peritumoral enhancement (OR = 9.25, p = 0.046) as independent predictors. The model combining NID-AP and peritumoral enhancement achieved the best diagnostic performance (AUC = 0.781, sensitivity = 67.86%, specificity = 78.26%) for predicting GPC3 expression. All DECT-derived parameters showed excellent interobserver reproducibility (ICC > 0.75 for all). Conclusions: Parameters derived from DECT, especially combining NID-AP and peritumoral enhancement, may be a potential tool to noninvasively predict GPC3 expression in HCC. Full article

Heparan sulfates (HS) are polysaccharides abundantly expressed in the extracellular matrix and the glycocalyx of endothelial cells, having a putative role in vascular function. The role of HS in vascular reactivity remains unclear. Herein, we sought to determine whether HS regulate the vascular tone in physiological conditions. Using male, 6–8-weeks-old, CD1, C57BL/6, syndecan 1 (Sdc1^−/−) and glypican 1 (Gpc1^−/−) knockout mice, we investigated if the degradation of HS with heparinase III altered vascular reactivity to norepinephrine (NE), acetylcholine (ACh) and potassium chloride (KCl). Our findings indicate that HS are crucial players in the vascular response to NE and ACh in CD1, C57BL/6, and Sdc1^−/− but not in Gpc1^−/− mice. Both Sdc1^−/− and Gpc1^−/− showed increased compensatory expression of syndecan 2 and syndecan 4. However, while Sdc1^−/− showed decreased expression of glypican 1, Gpc1^−/− showed increased expression of syndecan 1 in aortic homogenates. The lack of response to the vascular reactivity effects of heparinase III in Gpc1^−/− suggests a differential role of HS to proteoglycan function in the regulation of the vascular tone. Our data demonstrate a physiological role for HS in the regulation of the vascular tone in physiological conditions. Full article

Neuroblastoma is a highly aggressive pediatric malignancy originating from neural crest progenitor cells, predominantly in the adrenal medulla. Amplification of the MYCN oncogene occurs in 20–30% of all neuroblastoma cases and approximately 50% of high-risk tumors, strongly correlating with poor prognosis, relapse, and multidrug resistance. MYCN-driven oncogenesis promotes tumor progression by suppressing apoptotic signaling and enhancing survival pathways, including autophagy—a key mechanism underlying resistance to chemotherapy and immunotherapy. This review examines current therapeutic strategies and resistance mechanisms in MYCN-amplified neuroblastoma, while introducing emerging approaches utilizing exosomes as precision drug delivery systems. Exosomes, nanoscale extracellular vesicles secreted by the tumor cells, exhibit natural tropism and can be engineered to selectively target neuroblastoma-specific biomarkers such as glypican-2 (GPC2), which is highly expressed in MYCN-amplified tumors. Leveraging this property, neuroblastoma-derived exosomes can be purified, modified, and loaded with small interfering RNA (siRNA) to silence MYCN expression, combined with chloroquine—an FDA-approved autophagy inhibitor—to simultaneously inhibit autophagy and induce apoptotic signaling. This dual-targeted approach aims to overcome drug resistance, reduce off-target toxicity, and enhance therapeutic efficacy through exosome-mediated specificity. Furthermore, gut dysbiosis has emerged as a critical factor influencing tumor progression and diminishing treatment efficacy in MYCN-amplified neuroblastoma. We propose integrating microbiota-derived exosomes engineered to deliver anti-inflammatory microRNAs (miRNAs) to the gut mucosa, restoring eubiosis and potentiating systemic anti-tumor responses. Collectively, exosome-based strategies represent a paradigm shift in formulating combination therapies, offering a multifaceted approach to target MYCN amplification, inhibit autophagy, induce apoptosis, and modulate the tumor-microbiome axis. These innovations hold significant promise for improving clinical outcomes in high-risk MYCN-amplified neuroblastoma patients. Full article

Background: Glypican-3 (GPC3) is frequently overexpressed in hepatocellular carcinoma (HCC) and serves as a circulating biomarker. Limited evidence exists regarding whether plasma or serum constitutes the optimal matrix for GPC3 measurement. This study aimed to investigate this gap. Methods: Between December 2024 and September 2025, 100 participants were prospectively enrolled, including 33 healthy controls, 29 individuals with chronic liver disease, and 38 patients with HCC. Paired serum and plasma samples were analyzed under fresh conditions and after storage for seven days at 4 °C and −70 °C. GPC3 concentrations were compared across groups. Subsequently, correlation and area under the receiver operating characteristic curve (AUROC) analyses were conducted. Results: In fresh samples of the controls, median plasma GPC3 levels were significantly higher than those in serum (82.36 pg/mL, IQR: 67.56–92.42 vs. 30.89 pg/mL, IQR: 20.36–41.12; p < 0.001). After seven days of storage, plasma GPC3 concentrations declined markedly at both 4 °C (41.73 pg/mL, IQR: 32.49–55.37; p < 0.001) and −70 °C (45.53 pg/mL, IQR: 25.30–55.65; p < 0.001), with no significant difference between the two storage conditions (p = 0.610). In contrast, serum GPC3 levels remained relatively stable across fresh, 4 °C (31.10 pg/mL, IQR: 16.84–38.60), and −70 °C (25.31 pg/mL, IQR: 14.36–40.74) conditions (p = 0.645). Both matrices under −70 °C storage effectively discriminated HCC from non-HCC cases, although serum demonstrated a significantly better diagnostic performance (AUROC: 0.836, 95% CI: 0.749–0.902 vs. 0.772, 95% CI: 0.677–0.850; p = 0.013). Conclusions: Although plasma offers operational convenience and higher baseline GPC3 levels, serum provides both greater stability and superior diagnostic accuracy under frozen conditions, thus supporting its use as the preferred specimen matrix in clinical and research applications. Full article

Background: Glypican-3 (GPC3) is overexpressed in most hepatocellular carcinoma (HCC) tissues but is absent in normal adult liver. We evaluated whether tumor GPC3 expression is associated with clinical outcomes in patients with advanced HCC treated with atezolizumab–bevacizumab (AB). Methods: We conducted a single-center retrospective cohort study of 139 patients with Barcelona Clinic Liver Cancer (BCLC) stage C HCC who received AB between January 2022 and August 2025. Tumor GPC3 expression was assessed by immunohistochemistry. The primary endpoints were overall survival (OS) and progression-free survival (PFS), and the secondary endpoint was objective response rate (ORR) according to modified Response Evaluation Criteria in Solid Tumors (mRECIST). Results: Baseline characteristics were largely balanced between GPC3-positive (n = 87) and GPC3-negative (n = 52) groups. Median OS was significantly shorter in patients with GPC3-positive tumors than in those with GPC3-negative tumors (p = 0.006). In multivariable analysis, GPC3 positivity remained independently associated with higher mortality (hazard ratio [HR] 1.77, 95% confidence interval [CI] 1.05–3.00; p = 0.033), along with Child–Pugh class B. PFS did not differ significantly between the groups (p = 0.712). ORR was lower in GPC3-positive tumors than in GPC3-negative tumors (approximately 17–18% vs. ~32%; p = 0.023). Membranous GPC3 localization was associated with inferior OS compared with cytoplasmic or absent expression (p = 0.025). Conclusions: Tumor GPC3 expression was associated with decreased OS and lower ORR among AB-treated patients with advanced HCC, suggesting potential clinical relevance and may help in risk stratification. Full article

Chimeric antigen receptor (CAR)-modified natural killer (NK) cells represent a promising immunotherapeutic approach for the treatment of oncological malignancies such as hepatocellular carcinoma (HCC). In this work, we have engineered primary human NK cells, re-directing them so they can specifically recognize Glypican-3 (GPC3), an immunotherapeutic target for HCC. In previous studies, we have demonstrated that IFN-α significantly enhances NK cells’ anti-tumor and anti-viral cytotoxicity. Fourth-generation self-inactivating lentiviral vectors were used to deliver a transgenic expression of IFN-α or its co-expression with IL-15 (which induces NK cells expansion, survival, and function), aiming to enhance CAR-GPC3 NK cells’ anti-tumor response against HCC. We optimized a protocol for efficient transduction of primary NK cells, demonstrating that CAR expression is maintained at high levels over time. Exposure of HCC ectopically expressing GPC3+ to CAR-GPC3-IL15 and CAR-GPC3-IL15-IFNα NK cells demonstrated significant in vitro cytotoxicity and cytokine production, dependent on GPC3 expression. To prevent undesired side effects of CAR-NK cell immunotherapy, co-delivery with a suicide gene is advised as a safety measure. Thus, a truncated epidermal growth factor receptor (tEGFR) was co-delivered with the anti-GPC3 CAR, which efficiently promoted the suicide of the CAR-NK used in this work. Our study demonstrates the efficacy of re-directed CAR-GPC3 primary NK cells, encouraging further preclinical and clinical translation studies and strengthening the potential of these cells as a novel treatment option for patients with HCC. Full article

Serum Glypican-3 (GPC3) levels in HCC patients are significantly higher than those in healthy individuals or patients with non-malignant liver diseases, making it a diagnostic marker for HCC. However, its diagnostic capability remains controversial due to its low sensitivity. The common marker AFP has limitations in terms of sensitivity and specificity, particularly in early-stage HCC. We sought to combine GPC3 detection with multi-biomarker panels to enhance sensitivity and specificity in early-stage HBV-, HCV-, and ALD-related liver cancer diagnosis. We applied receiver operating characteristic (ROC) analysis, which is used to evaluate the diagnostic performance of different biomarker tests, to develop comprehensive multi-biomarker panels that include GPC3, along with other biomarkers such as gender, age, AFP, AFP-L3%, and DCP, for assessment in the selected patients. We also applied univariate and multivariate logistic regression analysis to generate a specific diagnostic model for early HBV-induced HCC detection. We found that GPC3 levels in serum were significantly higher in HCC patients compared to CLD patients. We performed univariate and multivariate logistic regression analysis on the relevant indicators of early HCC to establish a new GDATA model for diagnosing early HCC. The new model included five indicators of early HCC: GPC3, DCP, AFP-L3%, TBIL and age. The diagnostic efficacy was better than that of GPC3, AFP, DCP and AFP-L3 alone. The diagnostic accuracy of the GDATA model for early HCC was significantly higher than that of the GALAD model or single indicators alone. The GDATA model thus provides a new promising diagnostic strategy for early HCC detection. Full article

Background/Objectives: The cell surface proteoglycan glypican-3 (GPC3) is reportedly overexpressed in hepatocellular carcinoma (HCC) tissues, but not in benign liver tissues, rendering this protein a potential target for radionuclide theranostic approaches. Peptides are generally a promising class of targeting molecules for the development of radioligands because they combine straightforward synthetic access with favorable pharmacokinetics. Among the published peptides with disclosed structures, one of the most promising radioligands is [¹⁸F]AlF-NOTA-TJ12P2, which has a reported comparably high binding affinity to GPC3 and a high hydrophilicity. In this study, we aimed to design novel GPC3-targeting radioligands based on the TJ12P2 peptidic scaffold. Methods: Peptides were synthesized on solid phase using an Fmoc protecting group strategy. For comparative investigations, the reference nanobody HN3 was expressed in E. coli, isolated and subsequently modified with NODA-GA or SulfoCy3. The binding of native peptides, scrambled variants and reference nanobodies to GPC3 was investigated by surface plasmon resonance (SPR) interaction analysis, and fluorescently labeled versions of peptides and nanobodies were used for fluorescence microscopy in HepG2 (GPC3+) or SK Hep1 (GPC3−) cells. The chelator-bearing peptides were radiolabeled with gallium-67 and their stability towards radiolysis and in human serum was investigated. The binding of radiolabeled peptides and nanobodies to HepG2 cells was assessed in real-time ligand binding experiments. Results: The synthesized native peptides did not exhibit binding towards GPC3 in SPR interaction analyses, and the observed response was comparable to that of the scrambled variants at equal concentrations. Additionally, no binding to or uptake of the fluorescent constructs into cells was observed with fluorescence microscopy regardless of cellular GPC3 expression level. In real-time radioligand binding experiments, very fast association and dissociation of the gallium-67 labeled peptides to GPC3 positive HepG2 cells was observed, suggesting either extremely fast binding kinetics or unspecific binding of the peptides. Conclusions: Taken together, these findings suggest that the peptide TJ12P2 lacks specific binding to GPC3 in vitro and might not serve as a basis for the development of radioligands targeting GPC3. Full article

Background/Objectives: Glypican-3 (GPC3) is a cell surface oncofetal protein that is highly expressed in hepatocellular carcinoma (HCC) but absent in normal liver tissue, making it an attractive target for molecularly targeted diagnosis and therapy. To support GPC3-targeted treatment strategies, there is a need for a non-invasive imaging tool capable of detecting GPC3-positive tumors. Methods: We conjugated a commercially available murine anti-GPC3 antibody (1G12), or a proprietary chimeric anti-GPC3 antibody (ET58) to the standard magnetic resonance imaging (MRI) contrast agent, gadolinium, via a DOTA chelator. The resulting probes, 1G12-DOTA-Gd or ET58-DOTA-Gd, respectively, were assessed for in vitro relaxivity and binding specificity to GPC3-positive HCC cells, as well as for in vivo imaging performance in mouse xenograft models bearing GPC3-positive or GPC3-negative HCC tumors. Conclusions: ET58-DOTA-Gd shows high specificity, imaging efficacy, and a favorable immunogenicity profile, thereby making it a promising candidate for clinical translation as a GPC3-targeted MRI probe. It holds potential as a non-invasive companion diagnostic for identifying GPC3-positive HCC patients who may benefit from GPC3-targeted therapies. Full article

Hepatocellular carcinoma (HCC) remains a global health challenge due to molecular heterogeneity and frequent delayed diagnosis. This comprehensive review synthesizes recent immunohistochemistry (IHC) advancements for HCC diagnosis, prognostication, and therapeutic prediction. We systematically evaluate conventional markers, such as hepatocyte paraffin 1 (HepPar1), arginase-1 (Arg-1), and glypican-3 (GPC3), as well as emerging biomarkers, detailing their diagnostic sensitivities and specificities in HCC with varied tumor differentiation. Prognostic immunostaining markers, such as Ki-67 proliferation index and vascular endothelial growth factor (VEGF) overexpression, correlate with reduced 5-year survival, while novel immune checkpoint IHC markers (PD-L1 and CTLA-4) predict response to immunotherapy, particularly in advanced HCC. This work provides evidence-based recommendations for optimizing IHC utilization in clinical practice while identifying knowledge gaps in biomarker validation and standardization. Full article