Hedgehog on the Move: Glypican-Regulated Transport and Gradient Formation in Drosophila.

Glypicans (Glps) are a family of heparan sulphate proteoglycans that are attached to the outer plasma membrane leaflet of the producing cell by a glycosylphosphatidylinositol anchor. Glps are involved in the regulation of many signalling pathways, including those that regulate the activities of Wnts, Hedgehog (Hh), Fibroblast Growth Factors (FGFs), and Bone Morphogenetic Proteins (BMPs), among others. In the Hh-signalling pathway, Glps have been shown to be essential for ligand transport and the formation of Hh gradients over long distances, for the maintenance of Hh levels in the extracellular matrix, and for unimpaired ligand reception in distant recipient cells. Recently, two mechanistic models have been proposed to explain how Hh can form the signalling gradient and how Glps may contribute to it. In this review, we describe the structure, biochemistry, and metabolism of Glps and their interactions with different components of the Hh-signalling pathway that are important for the release, transport, and reception of Hh.

Peptide Binder to Glypican-3 as a Theranostic Agent for Hepatocellular Carcinoma.

Glypican-3 (GPC3) is a membrane-associated glycoprotein that is significantly upregulated in hepatocellular carcinomas (HCC) with minimal to no expression in normal tissues. The differential expression of GPC3 between tumor and normal tissues provides an opportunity for targeted radiopharmaceutical therapy to treat HCC, a leading cause of cancer-related deaths worldwide. Methods: DOTA-RYZ-GPC3 (RAYZ-8009) comprises a novel macrocyclic peptide binder to GPC3, a linker, and a chelator that can be complexed with different radioisotopes. The binding affinity was determined by surface plasma resonance and radioligand binding assays. Target-mediated cellular internalization was radiometrically measured at multiple time points. In vivo biodistribution, monotherapy, and combination treatments with 177Lu or 225Ac were performed on HCC xenografts. Results: RAYZ-8009 showed high binding affinity to GPC3 protein of human, mouse, canine, and cynomolgus monkey origins and no binding to other glypican family members. Potent cellular binding was confirmed in GPC3-positive HepG2 cells and was not affected by isotope switching. RAYZ-8009 achieved efficient internalization on binding to HepG2 cells. Biodistribution study of 177Lu-RAYZ-8009 showed sustained tumor uptake and fast renal clearance, with minimal or no uptake in other normal tissues. Tumor-specific uptake was also demonstrated in orthotopic HCC tumors, with no uptake in surrounding liver tissue. Therapeutically, significant and durable tumor regression and survival benefit were achieved with 177Lu- and 225Ac-labeled RAYZ-8009, as single agents and in combination with lenvatinib, in GPC3-positive HCC xenografts. Conclusion: Preclinical in vitro and in vivo data demonstrate the potential of RAYZ-8009 as a theranostic agent for the treatment of patients with GPC3-positive HCC.

Glypican-1-targeted antibody-drug conjugate inhibits the growth of glypican-1-positive glioblastoma.

Glioblastoma is the deadliest form of brain tumor. The presence of the blood-brain barrier (BBB) significantly hinders chemotherapy, necessitating the development of innovative treatment options for this tumor. This report presents the in vitro and in vivo efficacy of an antibody-drug conjugate (ADC) that targets glypican-1 (GPC1) in glioblastoma. The GPC1-ADC was created by conjugating a humanized anti-GPC1 antibody (clone T2) with monomethyl auristatin E (MMAE) via maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl linkers. Immunohistochemical staining analysis of a glioblastoma tissue microarray revealed that GPC1 expression was elevated in more than half of the cases. GPC1-ADC, when bound to GPC1, was efficiently and rapidly internalized in glioblastoma cell lines. It inhibited the growth of GPC1-positive glioma cell lines by inducing cell cycle arrest in the G2/M phase and triggering apoptosis in vitro. We established a heterotopic xenograft model by subcutaneously implanting KALS-1 and administered GPC1-ADC intravenously. GPC1-ADC significantly inhibited tumor growth and increased the number of mitotic cells. We also established an orthotopic xenograft model by intracranially implanting luciferase-transfected KS-1-Luc#19. After injecting Evans blue and resecting brain tissues, dye leakage was observed in the implantation area, confirming BBB disruption. We administered GPC1-ADC intravenously and measured the luciferase activity using an in vivo imaging system. GPC1-ADC significantly inhibited tumor growth and extended survival. In conclusion, GPC1-ADC demonstrated potent intracranial activity against GPC1-positive glioblastoma in an orthotopic xenograft model. These results indicate that GPC1-ADC could represent a groundbreaking new therapy for treating glioblastoma beyond the BBB.

Extracellular Vesicular Analysis of Glypican 1 mRNA and Protein for Pancreatic Cancer Diagnosis and Prognosis.

Detecting pancreatic duct adenocarcinoma (PDAC) in its early stages and predicting late-stage patient prognosis undergoing chemotherapy is challenging. This work shows that the activation of specific oncogenes leads to elevated expression of mRNAs and their corresponding proteins in extracellular vesicles (EVs) circulating in blood. Utilizing an immune lipoplex nanoparticle (ILN) biochip assay, these findings demonstrate that glypican 1 (GPC1) mRNA expression in the exosomes-rich (Exo) EV subpopulation and GPC1 membrane protein (mProtein) expression in the microvesicles-rich (MV) EV subpopulation, particularly the tumor associated microvesicles (tMV), served as a viable biomarker for PDAC. A combined analysis effectively discriminated early-stage PDAC patients from benign pancreatic diseases and healthy donors in sizable clinical from multiple hospitals. Furthermore, among late-stage PDAC patients undergoing chemotherapy, lower GPC1 tMV-mProtein and Exo-mRNA expression before treatment correlated significantly with prolonged overall survival. These findings underscore the potential of vesicular GPC1 expression for early PDAC screenings and chemotherapy prognosis.

Circadian gene Per3 promotes astroblastoma progression through the P53/BCL2/BAX signalling pathway.

The key circadian genes, Period1(Per1), Period2(Per2), and Period3(Per3), constitute the mammalian Period gene family. The abnormal expression of Per1 and Per2 is closely related to tumor development, but there are few reports on Per3 and tumorigenesis. This study was conducted to determine whether the abnormal expression of Per3 could influence the progression of astroblastoma. The results indicated that the expression level of Per3 was increased in astroblastoma cells, and the high expression of Per3 was correlated with the poor overall survival time of glioma patients. The role of Per3 in astroblastoma cells was then investigated using two approaches: interference and overexpression. The interference of Per3 inhibited astroblastoma cell proliferation by inducing the cell cycle at the S phase. The interference of Per3 inhibited the migration and invasion of astroblastoma cells, while promoted the astroblastoma cell apoptosis and the expression of the apoptosis genes Cleaved-CASP3, P53, and BAX. The overexpression of Per3 promoted proliferation by affecting the S phase distribution of the astroblastoma cell cycle. The overexpression of Per3 promoted the migration and invasion of astroblastoma cells, while inhibited the astroblastoma cell apoptosis and the expression of apoptosis genes Cleaved-CASP3, P53, and BAX. RNA-seq analysis showed that the interference of Per3 in astrocytoma cells resulted in significant changes in the expression levels of 764 genes. Among the differentially expressed genes enriched in apoptosis-related pathways, the interference of Per3 resulted in significant upregulation of MARCKSL1 expression, in contrast to significant downregulation of SFRP4, EPB41L3, and GPC5 expression. Taken together, our results suggest that Per3 appears to be a pro-cancer gene by altering the proliferation, migration, invasion, and apoptosis of astroblastoma cells. As a result, the Per3 gene may be a promising therapeutic target in the treatment of astroblastoma.

Soluble vascular endothelial glycocalyx proteoglycans as potential therapeutic targets in inflammatory diseases.

Reducing the activity of cytokines and leukocyte extravasation is an emerging therapeutic strategy to limit tissue-damaging inflammatory responses and restore immune homeostasis in inflammatory diseases. Proteoglycans embedded in the vascular endothelial glycocalyx, which regulate the activity of cytokines to restrict the inflammatory response in physiological conditions, are proteolytically cleaved in inflammatory diseases. Here we critically review the potential of proteolytically shed, soluble vascular endothelial glycocalyx proteoglycans to modulate pathological inflammatory responses. Soluble forms of the proteoglycans syndecan-1, syndecan-3 and biglycan exert beneficial anti-inflammatory effects by the removal of chemokines, suppression of proinflammatory cytokine expression and leukocyte migration, and induction of autophagy of proinflammatory M1 macrophages. By contrast, soluble versikine and decorin enhance proinflammatory responses by increasing inflammatory cytokine synthesis and leukocyte migration. Endogenous syndecan-2 and mimecan exert proinflammatory effects, syndecan-4 and perlecan mediate beneficial anti-inflammatory effects and glypican regulates Hh and Wnt signaling pathways involved in systemic inflammatory responses. Taken together, targeting the vascular endothelial glycocalyx-derived, soluble syndecan-1, syndecan-2, syndecan-3, syndecan-4, biglycan, versikine, mimecan, perlecan, glypican and decorin might be a potential therapeutic strategy to suppress overstimulated cytokine and leukocyte responses in inflammatory diseases.

Differential heparan sulfate dependency of the Drosophila glypicans.

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.

The role and mechanism of HIF-1α-mediated glypican-3 secretion in hypoxia-induced tumor progression in hepatocellular carcinoma.

OBJECTIVE: To explore the expression and secretion mechanism of glypican-3 (GPC3) in hepatocellular carcinoma (HCC) cells under hypoxic conditions, and its role in tumor progression. METHODS: Huh7 cells with and without the knockdown of hypoxia-inducible factor 1-alpha (HIF-1α) were cultured under 1% O2 for varying durations to induce hypoxia. The expression levels of GPC3, HSP70, CD63, STX11 and SYT7 in the cytoplasm and exosomes of Huh7 cells were evaluated by western blotting and immunofluorescence. GPC3 protein expression was further measured in cells treated with GW4869 under hypoxic conditions. Huh7 cells and human umbilical vein endothelial cells (HUVECs) were cultured with the exosomes extracted from the control and GPC3-knockdown cells, the cell proliferation, migration, epithelial-mesenchymal transition (EMT), invasion, and in vitro angiogenesis were analyzed. Tumor xenografts were established to assess the role of GPC3-deficient exosomes in tumor growth. RESULTS: Hypoxic culture conditions downregulated GPC3, STX11 and SYT7 protein levels in the Huh7 cells and upregulated GPC3 mRNA, and also increased GPC3 protein expression in the exosomes. HIF-1α knockdown, as well as treatment with GW4869, upregulated GPC3 protein in the Huh7 cells grown under 1% O2, but downregulated exosomal GPC3. Furthermore, exosomes derived from the GPC3-knockdown cells inhibited the proliferation and migration of Huh7 cells, decreased the expression of N-cadherin, vimentin, ß-catenin, c-Myc and cyclin D1, and increased that of E-cadherin. Likewise, the GPC3-deficient exosomes also suppressed the invasion and tube formation ability of the HUVECs compared to that of control cells. Consistent with the in vitro results, the GPC3-deficient exosomes also repressed tumor growth in vivo. CONCLUSION: Hypoxia promoted secretion of exosomal GPC3 through the activation of HIF-1α. GPC3-deficient exosomes inhibited the proliferation, migration and EMT of HCC cells via the Wnt/ß-catenin signaling pathway, and suppressed the angiogenic potential of HUVECs. This provided a novel understanding of the role of exosomal GPC3 in HCC progression.

Extracellular carriers control lipid-dependent secretion, delivery, and activity of WNT morphogens.

WNT morphogens trigger signaling pathways fundamental for embryogenesis, regeneration, and cancer. WNTs are modified with palmitoleate, which is critical for binding Frizzled (FZD) receptors and activating signaling. However, it is unknown how WNTs are released and spread from cells, given their strong lipid-dependent membrane attachment. We demonstrate that secreted FZD-related proteins and WNT inhibitory factor 1 are WNT carriers, potently releasing lipidated WNTs and forming active soluble complexes. WNT release occurs by direct handoff from the membrane protein WNTLESS to the carriers. In turn, carriers donate WNTs to glypicans and FZDs involved in WNT reception and to the NOTUM hydrolase, which antagonizes WNTs by lipid moiety removal. WNT transfer from carriers to FZDs is greatly facilitated by glypicans that serve as essential co-receptors in Wnt signaling. Thus, an extracellular network of carriers dynamically controls secretion, posttranslational regulation, and delivery of WNT morphogens, with important practical implications for regenerative medicine.

Current methods for the detection of glypican-3.

Glypican-3 (GPC3) is a heparan sulfate proteoglycan (HSPG) that binds to the cell membrane via glycosylphosphatidylinositol (GPI), widely expressed in human embryos, and is undetectable in healthy adult liver but overexpressed in human hepatocellular carcinoma (HCC). Therefore, accurate and sensitive detection of GPC3 is critical for disease diagnosis. In recent years, a series of methods have been developed for the highly sensitive detection of GPC3, but there is a lack of reviews on recent advances in GPC3-related assays. In this review, we provide the recent advances in GPC3 detection and GPC3 concentration detection, mainly in terms of various optical sensor-based assays and electrochemical assays, and also provide new insights into the challenges and future directions of the field.

Biological and clinical significance of the glypican-3 gene in human lung adenocarcinoma: An in silico analysis.

Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan, has long been found to be dysregulated in human lung adenocarcinomas (LUADs). Nevertheless, the function, mutational profile, epigenetic regulation, co-expression profile, and clinicopathological significance of the GPC3 gene in LUAD progression are not well understood. In this study, we analyzed cancer microarray datasets from publicly available databases using bioinformatics tools to elucidate the above parameters. We observed significant downregulation of GPC3 in LUAD tissues compared to their normal counterparts, and this downregulation was associated with shorter overall survival (OS) and relapse-free survival (RFS). Nevertheless, no significant differences in the methylation pattern of GPC3 were observed between LUAD and normal tissues, although lower promoter methylation was observed in male patients. GPC3 expression was also found to correlate significantly with infiltration of B cells, CD8+, CD4+, macrophages, neutrophils, and dendritic cells in LUAD. In addition, a total of 11 missense mutations were identified in LUAD patients, and ~1.4% to 2.2% of LUAD patients had copy number amplifications in GPC3. Seventeen genes, mainly involved in dopamine receptor-mediated signaling pathways, were frequently co-expressed with GPC3. We also found 11 TFs and 7 miRNAs interacting with GPC3 and contributing to disease progression. Finally, we identified 3 potential inhibitors of GPC3 in human LUAD, namely heparitin, gemcitabine and arbutin. In conclusion, GPC3 may play an important role in the development of LUAD and could serve as a promising biomarker in LUAD.

Glutamine synthetase and hepatocellular carcinoma.

Glutamine synthetase (GS) is an enzyme that converts ammonia and glutamate to glutamine using adenosine triphosphate. GS is expressed in the brain, kidney, and liver tissues under normal physiological conditions. GS is involved in abnormal lipid metabolism of the liver by catalyzing de novo synthesis of glutamine, thereby inducing liver inflammation. Metabolic dysfunction-associated steatotic liver diseases (MASLD), such as Metabolic Associated Fatty Liver Disease and Metabolic Associated Steato Hepatitis, are considered risk factors for HCC. GS may also be involved in the development and progression of hepatocellular carcinoma (HCC) through other signaling pathways, including the rapamycin (mTOR) and Wnt/ß-catenin signaling pathways. Furthermore, the correct combination of HSP70, GPC3, and GS can improve the accuracy and precision of HCC diagnosis. However, the prognostic value of GS in different HCC populations remains controversial. The expression of GS affects the sensitivity of HCC cells to radiotherapy and chemotherapy. In addition, immunotherapy has been approved for the treatment of advanced HCC. This article delves into the development and application of GS in HCC, laying a theoretical foundation for the subsequent exploration of GS as a potential target for treating HCC.

Administration of a glypican-3 peptide increases the infiltration and cytotoxicity of CD8+ T cells against testicular yolk sac tumor, associated with enhancing the intratumoral cGAS/STING signaling.

BACKGROUND: Glypican-3 (GPC3) is highly expressed in testicular yolk sac tumor (TYST). GPC3 has been evaluated as a cancer vaccine for some types of tumors, but little is known on the effects of GPC3 peptide-based therapy on TYST. Here, we evaluated the antitumor effect of GPC3144-152 on TYST and its potential mechanisms. METHODS: GPC3144-152 -specific CD8+ T cells were induced by vaccine immunization and examined by ELISPOT. The CD8+ T cells were purified for testing their cytotoxicity in vitro against TYST cells by CCK-8 and TUNEL assays and in vivo against tumor growth. The influence of GPC3144-152 loading and/or cGAS silencing on the tumor growth, apoptosis and cGAS/STING signaling was tested by immunohistochemistry, immunofluorescence, flow cytometry, and Western blot. RESULTS: Vaccination with GPC3144-152 induced tumor-specific CD8+ T cells that secreted high levels of IFN-γ and granzyme B, and had potent cytotoxicity against TYST in a dose-dependent manner. Adoptive transfer of CD8+ T cells and treatment with GPC3144-152 significantly inhibited the growth of TYST tumors, but less effective for cGAS-silenced TYST tumors in vivo. Treatment with GPC3144-152 enhanced the infiltration of CD8+ T cells into the tumor environment and their cytotoxicity against TYST tumors in vivo by up-regulating granzyme B and IFN-ß expression, but down-regulating GPC3 expression in the tumors. Co-culture of CD8+ T cells with TYST in the presence of exogenous GPC3144-152 enhanced peptide-specific CD8+ T-cell cytotoxicity in vitro, accompanied by enhancing cGAS, γH2AX, TBK1, and IRF3 phosphorylation in TYST cells, but less effective in cGAS-silenced TYST cells. CONCLUSIONS: These data indicated that GPC3 peptide-specific CD8+ T cells had potent antitumor activity against TYST tumor, particularly for combined treatment with the peptide, which was partially dependent on the intratumoral cGAS/STNG signaling. GPC3 peptide vaccine may be valuable for the combination treatment of TYST.

A novel complement-fixing IgM antibody targeting GPC1 as a useful immunotherapeutic strategy for the treatment of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a very low survival rate at 5 years. The use of chemotherapeutic agents results in only modest prolongation of survival and is generally associated with the occurrence of toxicity effects. Antibody-based immunotherapy has been proposed for the treatment of PDAC, but its efficacy has so far proved limited. The proteoglycan glypican-1 (GPC1) may be a useful immunotherapeutic target because it is highly expressed on the surface of PDAC cells, whereas it is not expressed or is expressed at very low levels in benign neoplastic lesions, chronic pancreatitis, and normal adult tissues. Here, we developed and characterized a specific mouse IgM antibody (AT101) targeting GPC1. METHODS: We developed a mouse monoclonal antibody of the IgM class directed against an epitope of GPC1 in close proximity to the cell membrane. For this purpose, a 46 amino acid long peptide of the C-terminal region was used to immunize mice by an in-vivo electroporation protocol followed by serum titer and hybridoma formation. RESULTS: The ability of AT101 to bind the GPC1 protein was demonstrated by ELISA, and by flow cytometry and immunofluorescence analysis in the GPC1-expressing "PDAC-like" BXPC3 cell line. In-vivo experiments in the BXPC3 xenograft model showed that AT101 was able to bind GPC1 on the cell surface and accumulate in the BXPC3 tumor masses. Ex-vivo analyses of BXPC3 tumor masses showed that AT101 was able to recruit immunological effectors (complement system components, NK cells, macrophages) to the tumor site and damage PDAC tumor tissue. In-vivo treatment with AT101 reduced tumor growth and prolonged survival of mice with BXPC3 tumor (p < 0.0001). CONCLUSIONS: These results indicate that AT101, an IgM specific for an epitope of GPC1 close to PDAC cell surface, is a promising immunotherapeutic agent for GPC1-expressing PDAC, being able to selectively activate the complement system and recruit effector cells in the tumor microenvironment, thus allowing to reduce tumor mass growth and improve survival in treated mice.

The Role of microRNAs in Regulating Cancer Cell Response to Oxaliplatin-Containing Regimens.

Oxaliplatin (cyclohexane-1,2-diamine; oxalate; platinum [2+]) is a third-generation chemotherapeutic drug with anticancer effects. Oxaliplatin has a role in the treatment of several cancers. It is one of the few drugs which can eliminate the neoplastic cells of colorectal cancer. Also, it has an influential role in breast cancer, lung cancer, bladder cancer, prostate cancer, and gastric cancer. Although oxaliplatin has many beneficial effects in cancer treatment, resistance to this drug is in the way to cure neoplastic cells and reduce treatment efficacy. microRNAs are a subtype of small noncoding RNAs with ∼22 nucleotides that exist among species. They have diverse roles in physiological processes, including cellular proliferation and cell death. Moreover, miRNAs have essential roles in resistance to cancer treatment and can strengthen sensitivity to chemotherapeutic drugs and regimens. In colorectal cancer, the co-treatment of oxaliplatin with anti-miR-19a can partially reverse the oxaliplatin resistance through the upregulation of phosphatase and tensin homolog (PTEN). Moreover, by preventing the spread of gastric cancer cells and downregulating glypican-3 (GPC3), MiR-4510 may modify immunosuppressive signals in the tumor microenvironment. Treatment with oxaliplatin may develop into a specialized therapeutic drug for patients with miR-4510 inhibition and glypican-3-expressing gastric cancer. Eventually, miR-122 upregulation or Wnt/ß-catenin signaling suppression boosted the death of HCC cells and made them more sensitive to oxaliplatin. Herein, we have reviewed the role of microRNAs in regulating cancer cells' response to oxaliplatin, with particular attention to gastrointestinal cancers. We also discussed the role of these noncoding RNAs in the pathophysiology of oxaliplatin-induced neuropathic pain.

The Pulmonary Endothelial Glycocalyx Modifications in Glypican 1 Knockout Mice Do Not Affect Lung Endothelial Function in Physiological Conditions.

The endothelial glycocalyx is a dynamic signaling surface layer that is involved in the maintenance of cellular homeostasis. The glycocalyx has a very diverse composition, with glycoproteins, proteoglycans, and glycosaminoglycans interacting with each other to form a mesh-like structure. Due to its highly interactive nature, little is known about the relative contribution of each glycocalyx constituent to its overall function. Investigating the individual roles of the glycocalyx components to cellular functions and system physiology is challenging, as the genetic manipulation of animals that target specific glycocalyx components may result in the development of a modified glycocalyx. Thus, it is crucial that genetically modified animal models for glycocalyx components are characterized and validated before the development of mechanistic studies. Among the glycocalyx components, glypican 1, which acts through eNOS-dependent mechanisms, has recently emerged as a player in cardiovascular diseases. Whether glypican 1 regulates eNOS in physiological conditions is unclear. Herein, we assessed how the deletion of glypican 1 affects the development of the pulmonary endothelial glycocalyx and the impact on eNOS activity and endothelial function. Male and female 5-9-week-old wild-type and glypican 1 knockout mice were used. Transmission electron microscopy, immunofluorescence, and immunoblotting assessed the glycocalyx structure and composition. eNOS activation and content were assessed by immunoblotting; nitric oxide production was assessed by the Griess reaction. The pulmonary phenotype was evaluated by histological signs of lung injury, in vivo measurement of lung mechanics, and pulmonary ventilation. Glypican 1 knockout mice showed a modified glycocalyx with increased glycocalyx thickness and heparan sulfate content and decreased expression of syndecan 4. These alterations were associated with decreased phosphorylation of eNOS at S1177. The production of nitric oxides was not affected by the deletion of glypican 1, and the endothelial barrier was preserved in glypican 1 knockout mice. Pulmonary compliance was decreased, and pulmonary ventilation was unaltered in glypican 1 knockout mice. Collectively, these data indicate that the deletion of glypican 1 may result in the modification of the glycocalyx without affecting basal lung endothelial function, validating this mouse model as a tool for mechanistic studies that investigate the role of glypican 1 in lung endothelial function.

Immuno-PET and Targeted α-Therapy Using Anti-Glypican-1 Antibody Labeled with 89Zr or 211At: A Theranostic Approach for Pancreatic Ductal Adenocarcinoma.

Glypican-1 (GPC1) is overexpressed in several solid cancers and is associated with tumor progression, whereas its expression is low in normal tissues. This study aimed to evaluate the potential of an anti-GPC1 monoclonal antibody (GPC1 mAb) labeled with 89Zr or 211At as a theranostic target in pancreatic ductal adenocarcinoma. Methods: GPC1 mAb clone 01a033 was labeled with 89Zr or 211At with a deferoxamine or decaborane linker, respectively. The internalization ability of GPC1 mAb was evaluated by fluorescence conjugation using a confocal microscope. PANC-1 xenograft mice (n = 6) were intravenously administered [89Zr]GPC1 mAb (0.91 ± 0.10 MBq), and PET/CT scanning was performed for 7 d. Uptake specificity was confirmed through a comparative study using GPC1-positive (BxPC-3) and GPC1-negative (BxPC-3 GPC1-knockout) xenografts (each n = 3) and a blocking study. DNA double-strand breaks were evaluated using the γH2AX antibody. The antitumor effect was evaluated by administering [211At]GPC1 mAb (∼100 kBq) to PANC-1 xenograft mice (n = 10). Results: GPC1 mAb clone 01a033 showed increased internalization ratios over time. One day after administration, a high accumulation of [89Zr]GPC1 mAb was observed in the PANC-1 xenograft (SUVmax, 3.85 ± 0.10), which gradually decreased until day 7 (SUVmax, 2.16 ± 0.30). The uptake in the BxPC-3 xenograft was significantly higher than in the BxPC-3 GPC1-knockout xenograft (SUVmax, 4.66 ± 0.40 and 2.36 ± 0.36, respectively; P = 0.05). The uptake was significantly inhibited in the blocking group compared with the nonblocking group (percentage injected dose per gram, 7.3 ± 1.3 and 12.4 ± 3.0, respectively; P = 0.05). DNA double-strand breaks were observed by adding 150 kBq of [211At]GPC1 and were significantly suppressed by the internalization inhibitor (dynasore), suggesting a substantial contribution of the internalization ability to the antitumor effect. Tumor growth suppression was observed in PANC-1 mice after the administration of [211At]GPC1 mAb. Internalization inhibitors (prochlorperazine) significantly inhibited the therapeutic effect of [211At]GPC1 mAb, suggesting an essential role in targeted α-therapy. Conclusion: [89Zr]GPC1 mAb PET showed high tumoral uptake in the early phase after administration, and targeted α-therapy using [211At]GPC1 mAb showed tumor growth suppression. GPC1 is a promising target for future applications for the precise diagnosis of pancreatic ductal adenocarcinoma and GPC1-targeted theranostics.

Interpretation of Farnesoid X Receptor Immunohistochemical Expression in Discriminating Hepatocellular Carcinoma from Its Non-Neoplastic Mimics as an Adjunct to Glypican 3.

AIMS: Differentiating hepatocellular carcinoma (HCC) and non-neoplastic lesions may be challenging. Immunohistochemistry (IHC) can help in the comparative morphologic evaluation of HCC and its mimics. Farnesoid X receptor (FXR) is a nuclear metabolic receptor essential for bile salts homeostasis and other biological functions of liver cells. Preliminary studies have shown that FXR can be useful for diagnosing HCC. This study aimed to assess the role of Farnesoid X receptor (FXR) combined with Glypican 3 (GPC3) in differentiation between HCC and non-neoplastic hepatic lesions. MATERIAL AND METHODS: Immunohistochemistry of GPC3 and FXR was performed in 38 cases of primary hepatic lesions using an automated immunohistochemical stainer. The study included 17 primary HCC cases and 21 non-neoplastic hepatic lesions (5 cases were focal nodular hyperplasia, 7 were regenerative nodules and 9 were dysplastic nodules). RESULTS: The percentage of positive GPC3 and low or negative FXR expression was significantly higher in HCC cases than non-neoplastic hepatic lesions (P value <0.001). The sensitivity and specificity of GPC3 in differentiating HCC from non-neoplastic hepatic lesions were 70.6% and 85.7%, respectively, while the sensitivity and specificity of FXR were 58.8% and 100%, respectively. CONCLUSION: The present work revealed that FXR could be combined with GPC3 in distinguishing between HCC and non-neoplastic hepatic lesions with improved specificity rather than using an individual marker.

Proteomic Investigation of Differential Interactomes of Glypican 1 and a Putative Disease-Modifying Variant of Ataxia.

In a currently 13-year-old girl of consanguineous Turkish parents, who developed unsteady gait and polyneuropathy at the ages of 3 and 6 years, respectively, we performed whole genome sequencing and identified a biallelic missense variant c.424C>T, p.R142W in glypican 1 (GPC1) as a putative disease-associated variant. Up to date, GPC1 has not been associated with a neuromuscular disorder, and we hypothesized that this variant, predicted as deleterious, may be causative for the disease. Using mass spectrometry-based proteomics, we investigated the interactome of GPC1 WT and the missense variant. We identified 198 proteins interacting with GPC1, of which 16 were altered for the missense variant. This included CANX as well as vacuolar ATPase (V-ATPase) and the mammalian target of rapamycin complex 1 (mTORC1) complex members, whose dysregulation could have a potential impact on disease severity in the patient. Importantly, these proteins are novel interaction partners of GPC1. At 10.5 years, the patient developed dilated cardiomyopathy and kyphoscoliosis, and Friedreich's ataxia (FRDA) was suspected. Given the unusually severe phenotype in a patient with FRDA carrying only 104 biallelic GAA repeat expansions in FXN, we currently speculate that disturbed GPC1 function may have exacerbated the disease phenotype. LC-MS/MS data are accessible in the ProteomeXchange Consortium (PXD040023).

SMOC-1 interacts with both BMP and glypican to regulate BMP signaling in C. elegans.

Secreted modular calcium-binding proteins (SMOCs) are conserved matricellular proteins found in organisms from Caenorhabditis elegans to humans. SMOC homologs characteristically contain 1 or 2 extracellular calcium-binding (EC) domain(s) and 1 or 2 thyroglobulin type-1 (TY) domain(s). SMOC proteins in Drosophila and Xenopus have been found to interact with cell surface heparan sulfate proteoglycans (HSPGs) to exert both positive and negative influences on the conserved bone morphogenetic protein (BMP) signaling pathway. In this study, we used a combination of biochemical, structural modeling, and molecular genetic approaches to dissect the functions of the sole SMOC protein in C. elegans. We showed that CeSMOC-1 binds to the heparin sulfate proteoglycan GPC3 homolog LON-2/glypican, as well as the mature domain of the BMP2/4 homolog DBL-1. Moreover, CeSMOC-1 can simultaneously bind LON-2/glypican and DBL-1/BMP. The interaction between CeSMOC-1 and LON-2/glypican is mediated specifically by the EC domain of CeSMOC-1, while the full interaction between CeSMOC-1 and DBL-1/BMP requires full-length CeSMOC-1. We provide both in vitro biochemical and in vivo functional evidence demonstrating that CeSMOC-1 functions both negatively in a LON-2/glypican-dependent manner and positively in a DBL-1/BMP-dependent manner to regulate BMP signaling. We further showed that in silico, Drosophila and vertebrate SMOC proteins can also bind to mature BMP dimers. Our work provides a mechanistic basis for how the evolutionarily conserved SMOC proteins regulate BMP signaling.