Glypican-3 Research Articles

Abstract 2585: First in human study with a novel peptide binder to glypican-3, demonstrates high specificity as a PET imaging agent in patients with hepatocellular carcinoma

Abstract Introduction: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. The American Association for the Study of Liver Disease recommends surveillance for high-risk individuals with abdominal ultrasound, triple phase contrast CT or MRI. However, these imaging techniques have limited sensitivity in classifying lesions smaller than 2 centimeters. The use of [18F]FDG Positron Emission Tomography (PET) is constrained due to insufficient uptake by HCC lesions over background liver uptake. Hence, there is an urgent need for more precise radiotracers for HCC. This study introduces the development of a novel radiotracer that binds to glypican-3 (GPC3), a cell membrane-anchored oncofetal protein overexpressed in HCC with minimal normal tissue expression. Methods: [68Ga]Ga-RYZ-GPC3 is composed of a small macrocyclic peptide binder with high affinity to GPC3 linked with a tetraxetan moiety capable of chelating a variety of radioisotopes including positron-emitting 68Ga and other radiometals. The unlabeled binder to GPC3 was synthesized by the sponsor and provided to three imaging centers in India as part of investigator-initiated trials reviewed and approved by each site’s institutional review board and ethics committee. Labeling with 68Ga and quality checks of the product was performed locally at each center. Patients with high clinical suspicion or with confirmed HCC who gave informed consent received injections of 1.5-5.5 mCi of [68Ga]Ga-RYZ-GPC3 and underwent PET-CT scanning at predetermined intervals. Some patients underwent companion [18F]FDG PET-CT scans in accordance with local guidelines. Results: A total of 64 patients underwent [68Ga]Ga-RYZ-GPC3 PET-CT scans. The median age within the cohort was 61 (41-83), 22% were female. Liver cirrhosis was present in 72% of patients, attributed to hepatitis B, hepatitis C or NASH in 17%, 13% and 20% of cases. Notably, 92% of patients exhibited at least one positive lesion on [68Ga]Ga-RYZ-GPC3 scans. The median SUVmax of GPC3-avid liver lesions was 15.5 (range 1.1 to 137.0) while the median liver SUVmean was 1.6 (0.3-7.9). Physiologic uptake was predominantly observed in the kidneys, the primary route of clearance, with a median SUVmean of 10.9 (2.2-20.7). Additionally, 20 patients underwent [18F]FDG PET-CT scan for comparison. The median SUVmax of lesions on 68Ga-RYZ-GPC3 vs [18F]FDG PET was 11.0 (1.1-137.0) vs 3.8 (1.2-12.2). Conclusion: Early human imaging results in patients with HCC indicate that [68Ga]Ga-RYZ-GPC3 has high tumor lesion specificity compared to normal liverand demonstrates the potential of [68Ga]Ga-RYZ-GPC3 to improve detection of HCC over conventional [18F]FDG PET-CT. In addition, the binder could be complexed with a therapeutic radioisotope as a novel targeted therapeutic option for HCC. Further investigation of this first-in-class binder to HCC is warranted. Citation Format: Chandresakhar Bal, Sanjana Ballal, Kumar Kallur, Anna Karmann, Ye Yuan, Jessica Rearden, Kathryn Shah, Charlotte Lorenz, Susan Moran, Ken Song, Ishita Sen. First in human study with a novel peptide binder to glypican-3, demonstrates high specificity as a PET imaging agent in patients with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2585.

Abstract 5248: Novel costimulatory domain improves CAR-monocyte activity against tumors

Abstract Myeloid cells, unlike other immune cells such as T cells or NK cells, are known residents in the solid tumor microenvironment (TME). In the absence of checkpoints and in proinflammatory conditions, myeloid cells (particularly M1 macrophages) are known to be capable of direct phagocytosis of tumor cells and can present tumor-associated antigens to the host immune system. However, the immunosuppressive conditions within the TME restrict and limit the anti-tumor response of tumor associated macrophages (TAMs), including their ability to recruit and activate other immune cells against the tumor. Engineered CAR-Monocytes can serve a unique function in cell therapy by bridging a key gap in the treatment of solid tumors. We are developing an autologous engineered CAR-M cell therapy product targeting Glypican-3 (GPC3) to treat hepatocellular carcinoma (HCC). The CAR serves a dual purpose as a homing ‘GPS’ signal for trafficking directly to the tumor site, and for directing phagocytosis specifically at targeted tumor cells. We have designed a proprietary CAR molecule (M83 CAR) which contains a macrophage-specific costimulatory domain that significantly increases the phagocytosis function of the CAR-M cells. Furthermore, we have demonstrated that the M83 CAR-M cells are not inhibited by the prevalent CD47 checkpoint (‘do not eat me’ signal expressed on tumor cells), which is known to restrict myeloid function in the TME. Effective phagocytosis of engineered CAR-M cells is central to subsequent mechanisms of actions that can elicit robust anti-tumor immunity against patient-specific neoantigens. However, the first barrier to overcome is effective infiltration of engineered CAR-M into the tumor from the periphery. We have demonstrated that our engineered CAR-Monocyte drug product can successfully home to the targeted tumor specifically, from the periphery. Subsequent in situ differentiation of CAR-monocytes into CAR-macrophages (M1) enables robust tumor cell phagocytosis, the central mechanism that can result in the following series of events: 1) proinflammatory shift in the TME milieu, 2) recruitment of innate and adaptive immune cells, 3) activation of T-cells against tumor neo-antigens. This is a unique outcome of the CAR-M mechanism of action that is not capitulated by CAR-T or CAR-NK cells. Leveraging and further enhancing CAR-M function could lead to the rejection of the tumor and its metastases, particularly in combination with other immune-modulating therapies. Citation Format: Kevin Carbajal, Warren Anderson, Kilsoo Jeon, Grace Reynolds, Prasanna Ekambaram, Beatrice Breckheimer, Anastasya Birger, Jessica Pafford, Matthew Esquivel, Adam Pecoraro, Sumiti Jain. Novel costimulatory domain improves CAR-monocyte activity against tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5248.

Abstract 4011: Locoregional GPC3 chimeric antigen receptor (CAR)-T cell therapy is associated with improved tumor control in hepatocellular carcinoma by enhancing CAR-T cell trafficking and functionality

Abstract Despite the rapid development of anti-cancer immunotherapy, the prognosis of hepatocellular carcinoma (HCC) patients is far from satisfactory. Glypican-3 (GPC3) CAR-T cell therapy has shown varied patient response. One of the potential reasons of low response rates may be due to the limited CAR-T cell infiltration. Therefore, locoregional GPC3 CAR-T therapy has been a promising option, with a hope of enhancing CAR-T cell tumor penetration. In this study, the therapeutic efficacy and underlying mechanisms of locoregional GPC3 CAR-T cell delivery were explored using HCC mouse xenografts. Orthotopic NOD-scid IL2Rgnull (NSG) HCC models were established by tumor fragment implantation into the liver. CAR-T cell injections were performed via the tail vein and portal vein to simulate systemic and locoregional administrations, respectively. Compared to the systemic treatment, locoregional therapy could better control the tumor growth, and was associated with improved liver function. In addition, the locoregional therapy group demonstrated a higher percentage of tumor-infiltrating CAR-T cells which showed an increased expression level of the cytotoxicity marker GZMB and a reduced level of the exhaustion marker LAG3. Moreover, significantly higher IFN-gamma level was found in the blood of the locoregional therapy group, indicating a better CAR-T cell functionality. In conclusion, we have demonstrated that locoregional administration of CAR-T cells is associated with increased CAR-T cell infiltration and better therapeutic efficacy. Additionally, CAR-T cells being injected locoregionally are correlated with enhanced functionality and reduced exhaustion phenotype. These findings highlight the potential of locoregional GPC3 CAR-T therapy as a promising treatment strategy for HCC patients. Citation Format: Jue Wang, Kinching Tsang, Jiale Qiu, Bo Feng. Locoregional GPC3 chimeric antigen receptor (CAR)-T cell therapy is associated with improved tumor control in hepatocellular carcinoma by enhancing CAR-T cell trafficking and functionality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4011.

Efficacy of radiofrequency ablation combined with sorafenib for treating liver cancer complicated with portal hypertension and prognostic factors.

Patients with liver cancer complicated by portal hypertension present complex challenges in treatment. To evaluate the efficacy of radiofrequency ablation in combination with sorafenib for improving liver function and its impact on the prognosis of patients with this condition. Data from 100 patients with liver cancer complicated with portal hypertension from May 2014 to March 2019 were analyzed and divided into a study group (n = 50) and a control group (n = 50) according to the treatment regimen. The research group received radiofrequency ablation (RFA) in combination with sorafenib, and the control group only received RFA. The short-term efficacy of both the research and control groups was observed. Liver function and portal hypertension were compared before and after treatment. Alpha-fetoprotein (AFP), glypican-3 (GPC-3), and AFP-L3 levels were compared between the two groups prior to and after treatment. The occurrence of adverse reactions in both groups was observed. The 3-year survival rate was compared between the two groups. Basic data were compared between the survival and non-surviving groups. To identify the independent risk factors for poor prognosis in patients with liver cancer complicated by portal hypertension, multivariate logistic regression analysis was employed. When comparing the two groups, the research group's total effective rate (82.00%) was significantly greater than that of the control group (56.00%; P < 0.05). Following treatment, alanine aminotransferase and aspartate aminotransferase levels increased, and portal vein pressure decreased in both groups. The degree of improvement for every index was substantially greater in the research group than in the control group (P < 0.05). Following treatment, the AFP, GPC-3, and AFP-L3 levels in both groups decreased, with the research group having significantly lower levels than the control group (P < 0.05). The incidence of diarrhea, rash, nausea and vomiting, and fatigue in the research group was significantly greater than that in the control group (P < 0.05). The 1-, 2-, and 3-year survival rates of the research group (94.00%, 84.00%, and 72.00%, respectively) were significantly greater than those of the control group (80.00%, 64.00%, and 40.00%, respectively; P < 0.05). Significant differences were observed between the survival group and the non-surviving group in terms of Child-Pugh grade, history of hepatitis, number of tumors, tumor size, use of sorafenib, stage of liver cancer, histological differentiation, history of splenectomy and other basic data (P < 0.05). Logistic regression analysis demonstrated that high Child-Pugh grade, tumor size (6-10 cm), history of hepatitis, no use of sorafenib, liver cancer stage IIIC, and previous splenectomy were independent risk factors for poor prognosis in patients with liver cancer complicated with portal hypertension (P < 0.05). Patients suffering from liver cancer complicated by portal hypertension benefit from the combination of RFA and sorafenib therapy because it effectively restores liver function and increases survival rates. The prognosis of patients suffering from liver cancer complicated by portal hypertension is strongly associated with factors such as high Child-Pugh grade, tumor size (6-10 cm), history of hepatitis, lack of sorafenib use, liver cancer at stage IIIC, and prior splenectomy.

Diagnostic and prognostic value of circulating exosomal glypican-1 in pancreatic cancer: a meta-analysis.

Pancreatic cancer (PC) is usually detected in the advanced stages. Liquid biopsy has become a revolutionary strategy for cancer diagnosis and prognosis prediction. This study aims to investigate the diagnostic and prognostic value of circulating exosomal glypican-1 (GPC-1) in PC. We systematically searched relevant studies. For diagnostic accuracy, pooled sensitivity and specificity and the area under the summary receiver operating characteristic curve (AUC) were calculated. Regarding prognostic value, hazard ratios (HRs) and 95% CIs for overall survival (OS) were summarized by using a random-effects model. We found 8 studies that examined the diagnostic value of circulating exosomal GPC-1 in PC, and 3 studies that investigated its prognostic value. Pooled sensitivity and specificity were 0.88 (95% CI, 0.65-0.97) and 0.86 (95% CI, 0.72-0.94). The AUC was 0.93 (95% CI, 0.90-0.95). Prognostic analysis showed that higher levels of circulating exosomal GPC-1 were associated with poorer OS in PC patients, and the combined HR for OS was 4.59 (random-effects model, 95% CI = 1.17-18.03, P = .022). The results of both studies were robust and neither had publication bias. Circulating exosomal GPC-1 may be used as a diagnostic and prognostic biomarker for PC. However, this result needs to be validated by further research using a larger sample size.

Glypican-4 serum levels are associated with cognitive dysfunction and vascular risk factors in Parkinson’s disease

Glypicans are biomarkers for various pathologies, including cardiovascular disease, cancer and diabetes. Increasing evidence suggests that glypicans also play a role in the context of neurodegenerative disorders. Initially described as supporting functionality of synapses via glutamate receptors during CNS development, Glypican 4 (GPC-4) also plays a role in the context of dementia via tau hyperphosphorylation in Alzheimer’s disease, which is also a co-pathology in Parkinson’s disease dementia. However, clinical evidence of circulating GPC-4 in Parkinson’s disease (PD) is missing so far. We therefore investigated GPC-4 in biofluids of PD patients. We analyzed GPC-4 levels in cerebrospinal fluid (CSF, n = 140), serum (n = 80), and tear fluid samples (n = 70) of PD patients and control subjects in a similar age range by ELISA (serum, CSF) and western blot (tear fluid). Expression of circulating GPC-4 was confirmed in all three biofluids, with highest levels in serum. Interestingly, GPC-4 levels were age-dependent, and multiple regression analysis revealed a significant association between GPC-4 serum levels and MoCA score, suggesting an involvement of GPC-4 in PD-associated cognitive decline. Furthermore, stratification of PD patients for vascular risk factors revealed a significant increase of GPC-4 serum levels in PD patients with vascular risk factors. Our results suggest GPC-4 as a clinical biomarker for vascular risk stratification in order to identify PD patients with increased risk of developing dementia.

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 invitro and invivo 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.

GenX caused liver injury and potential hepatocellular carcinoma of mice via drinking water even at environmental concentration

Hexafluoropropylene oxide dimer acid (GenX) is an alternative to perfluorooctanoic acid (PFOA), whose environmental concentration is close to its maximum allowable value established by the US Environmental Protection Agency, so its effects on human health are of great concern. The liver is one of the most crucial target organ for GenX, but whether GenX exposure induces liver cancer still unclear. In this research project, male C57 mice were disposed to GenX in drinking water at environmental concentrations (0.1 and 10 μg/L) and higher concentrations (1 and 100 mg/L) for 14 weeks to explore its effects on liver injury and potential carcinogenicity in mice. GenX was found to cause a dose-dependent increase in the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), and triglyceride (TG). As the content of GenX in drinking water increased, so did the concentrations of Glypican-3 (GPC-3) and detachment gamma-carboxyprothrombin (DCP), indicators of early hepatocellular cancer. GenX destroyed the boundaries and arrangements of hepatocytes, in which monocyte infiltration, balloon-like transformation, and obvious lipid vacuoles were observed between cells. Following exposure to GenX, Masson sections revealed a significant quantity of collagen deposition in the liver. Alpha-feto protein (AFP), vascular endothelial growth factor (VEGF), Ki67, matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) gene expression increased in a dose-dependent manner in the treatment group relative to the control group. In general, drinking water GenX exposure induced liver function impairment, elevated blood lipid level, caused liver pathological structure damage and liver fibrosis lesions, changed the liver inflammatory microenvironment, and increased the concentration of liver-related tumor indicator even in the environmental concentration, suggesting GenX is a potential carcinogen.

Hypoxia-Derived Exosomes Promote Lung Adenocarcinoma by Regulating HS3ST1-GPC4-Mediated Glycolysis.

The diagnosis of lung adenocarcinoma (LUAD) is often delayed due to the typically asymptomatic nature of the early-stage disease, causing advanced-stage LUAD diagnosis in most patients. Hypoxia is widely recognized as a driving force in cancer progression. Exosomes originating from hypoxic tumor cells promote tumorigenesis by influencing glycolysis, migration, invasion, and immune infiltration. Given these insights, our study aimed to explore the role of hypoxia-derived exosomal long non-coding RNA (lncRNA) OIP5-AS1 in LUAD cell lines and mouse models. Exosomes were meticulously isolated and authenticated based on their morphology and biomarkers. The interaction between heparan sulfate (glucosamine) 3-O-sulfotransferase 1 (HS3ST1) and Glypican 4 (GPC4) was examined using immunoprecipitation. The influence of the hypoxia-derived exosomal lncRNA OIP5-AS1 on glycolysis was assessed in LUAD cell lines. The effect of the hypoxia-derived exosomal lncRNA OIP5-AS1 on cell proliferation and metastasis was evaluated using colony formation, cell viability, cell cycle, and apoptosis analyses. Its effects on tumor size were confirmed in xenograft animal models. Our study revealed the mechanism of the hypoxia-derived exosomal lncRNA OIP5-AS1 in LUAD progression. We discovered that GPC4 promotes HS3ST1-mediated glycolysis and that the hypoxia-derived exosomal lncRNA OIP5-AS1 enhances glycolysis by regulating miR-200c-3p in LUAD cells. Notably, this lncRNA stimulates LUAD cell proliferation and metastasis and fosters LUAD tumor size via miR-200c-3p. Our findings underscore the potential role of the hypoxia-derived exosomal lncRNA OIP5-AS1 in LUAD progression. The hypoxia-derived exosomal lncRNA OIP5-AS1 promotes LUAD by regulating HS3ST1-GPC4-mediated glycolysis via miR-200c-3p.

Combination of serum alpha-fetoprotein, PIVKA-Ⅱ and glypican-3 in diagnosis of hepatocellular carcinoma: a meta-analysis.

To assess the value of serum alpha-fetoprotein (AFP), protein induced by vitamin K absence or antagonist-Ⅱ (PIVKA-Ⅱ) and glypican-3 (GPC-3) in the diagnosis of hepatocellular carcinoma (HCC). Studies of AFP, PIVKA-Ⅱ, GPC-3 or in combination for the diagnosis of HCC since 2002 were searched in PubMed, Web of Science and Embase databases. The literature was screened according to the inclusion and exclusion criteria, the quality of the included articles was evaluated by QUADAS checklist, and relevant data were extracted by Meta DiSc, Review Manager 5.4 and Stata 15.1. The diagnostic values of AFP, PIVKA-Ⅱ and GPC-3 alone or in combination for HCC were assessed with receiver operating characteristic (ROC) curve. A total of 32 articles were included in the study. Meta-analysis showed that when a single marker was used to diagnose HCC, the area under the ROC curve (AUC) of PIVKA-Ⅱ was the highest (0.88, 95%CI: 0.85-0.91), followed by GPC-3 and AFP. The AUC of combination of serum markers was higher than that of a single marker, and the AUC of PIVKA-Ⅱ combined with GPC-3 was the highest (0.90, 95%CI: 0.87-0.92). When a single marker was used for diagnosis, the sensitivity of PIVKA-Ⅱ and GPC-3 were relatively high (0.75 and 0.76), while the specificity of PIVKA-Ⅱ (0.88) and AFP (0.87) were higher than that of GPC-3 (0.81). The sensitivity of the combination of serum markers was higher than that of a single marker, while the specificity was not significantly improved. When a single marker is used to diagnose HCC, the diagnostic odds ratio (DOR) of PIVKA-Ⅱ was the highest (22, 95%CI: 13-36), followed by GPC-3 and AFP. The DOR of the combination of two markers in the diagnosis of HCC was higher than that of a single marker, and the DOR of AFP combined with GPC-3 was the highest (25, 95%CI: 9-67). The DOR of the combination of the three markers was significantly reduced to 10 (95%CI: 7-45). When a single marker is used, PIVKA-Ⅱ has a higher diagnostic value for HCC. The combination of two markers can significantly improve the diagnostic sensitivity, and AFP combined with PIVKA-Ⅱ is recommended for the diagnosis of HCC. The combination of all three markers failed to further improve the diagnostic value.

Simpson Golabi Behmel Syndrome: A New Case and Review of the Literature

Simpson Golabi Behmel Syndrome (SGBS) is a rare syndrome characterized clinically by multiple congenital anomalies, pre and postnatal overgrowth, characteristic craniofacial anomalies, macrocephaly, and organomegaly associated with abnormalities of the skeletal system. On the molecular level, there are genomic rearrangements involving point mutations of the glypican-3 (GPC3) gene at Xq26. The spectrum of signs and symptoms associated with SGBS is wide, ranging from very mild to fatal forms, especially in affected men. We report a rare case of a child affected by SGBS type 1, emphasizing the clinical, paraclinical, therapeutic and monitoring modalities of this possibly serious syndrome.

Increased GPC4 and clusterin associated with insulin resistance in patients with PCOS.

The aim of the study was to investigate the changes in serum glypican 4 (GPC4) and clusterin (CLU) levels in patients with polycystic ovary syndrome (PCOS) as well as their correlation with sex hormones and metabolic parameters. A total of 40 PCOS patients and 40 age-matched healthy women were selected. Serum GPC4 and CLU levels were compared between the PCOS and control groups, and binary logistic regression was used to analyze the relative risk of PCOS at different tertiles of serum GPC4 and CLU concentrations. Stepwise linear regression was used to identify the factors influencing serum GPC4 and CLU levels in PCOS patients. Serum GPC4 (1.82 ± 0.49 vs 1.30 ± 0.61 ng/mL, P < 0.001) and CLU (468.79 ± 92.85 vs 228.59 ± 82.42 µg/mL, P < 0.001) were significantly higher in PCOS patients than in healthy women after adjustment for body mass index (BMI). In the PCOS group, serum GPC4 was positively correlated with follicle-stimulating hormone, fasting plasma glucose (FPG), fasting insulin (FINS), homeostatic model assessment of insulin resistance (HOMA-IR), triglyceride, and CLU (P < 0.05), whereas serum CLU was positively correlated with BMI, FPG, FINS, and HOMA-IR (P < 0.05). Multiple stepwise linear regression analysis showed that HOMA-IR was independently associated with serum GPC4, and BMI and HOMA-IR were independently associated with CLU (P < 0.05). Serum GPC4 and CLU levels were significantly higher in PCOS patients than in healthy women, suggesting that GPC4 and CLU may be markers associated with insulin resistance in women with PCOS.

Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space.

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

Novel GPC3-targeting radiopharmaceutical therapy for hepatocellular carcinoma.

525 Background: Glypican-3 (GPC3) is a membrane-anchored oncofetal protein whose expression is largely absent in normal tissues. Significant upregulation of GPC3 protein has been observed in approximately 75% of hepatocellular carcinomas (HCC), and is associated with poor prognosis. The differential expression of GPC3 between tumor and normal tissues provides an opportunity for targeted radiopharmaceutical therapy (RPT) to treat HCC, a leading cause for cancer-related deaths worldwide. Methods: RAYZ-8009 comprises a novel macrocyclic peptide binder to GPC3, a linker, and a chelator that can be complexed with different radioisotopes. The affinity of peptide binders to GPC3 was determined by surface plasma resonance (SPR) 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 in HCC xenografts. Results: RAYZ-8009 showed high binding affinity (KD=0.7 nM) to human GPC3, with comparable affinity to GPC3 of human, mouse, canine and monkey origins, and no binding to other GPC family members. Potent cellular binding was confirmed in GPC3+ HepG2 cells, and was not affected by isotope switching. RAYZ-8009 achieved efficient internalization upon binding, with 42% internalized by 20 minutes in 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. Exquisite tumor-specific uptake was also demonstrated in orthotopic HCC tumors with no uptake in surrounding normal liver tissue. Therapeutically, significant and durable anti-tumor effect and survival benefit were achieved with 177Lu- and 225Ac-labeled RAYZ-8009, as monotherapy or in combination with lenvatinib, in GPC3+ HCC xenografts. Conclusions: Preclinical in vitro and in vivo data demonstrate the potential of RAYZ-8009 as a theranostic agent for the treatment of patients with GPC3+ HCC.

Extracellular Vesicular Analysis of Glypican 1 mRNAand 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.

Targeting glypicans through EGFR and JAK/STAT signaling axes drives breast cancer progression

AbstractExtracellular matrix (ECM) and its dynamic remodeling contribute to the progression of breast cancer, the most prevailing cancer type in women. Glypicans (GPCs) function as cell co‐receptors by facilitating the formation of ligand–receptor complexes. An important regulator in the context of breast cancer progression is the JAK/STAT signaling pathway that oversees the expression of genes associated with cancer cell characteristics. Epidermal growth factor receptor (EGFR) is a pivotal player in this process. The aim of this study is to examine the effect of the EGFR and JAK/STAT signaling pathways on GPCs expression in breast cancer cells with different estrogen receptor (ER) status, depicting different breast cancer subtypes. To this end, the ERα‐positive MCF‐7, and the ERβ‐positive MDA‐MB‐231 breast cancer cell lines were evaluated in terms of the impact of downstream inhibition of both pathways on the functional properties as well as the expression of GPCs 1‐6 genes. Notably, the downstream inhibition of both EGFR and JAK/STAT cascades mitigate cell proliferation and migration, while increasing cell adhesion on collagen type I in an ER‐independent manner. However, the inhibition exhibited a cell‐line‐dependent effect on GPC expression, as in MCF‐7 cells GPCs expression is mostly downregulated excepting GPC‐4 and GPC‐5. Conversely, in MDA‐MB‐231 cells, EGFR and JAK/STAT activation is essential for maintaining GPCs at low levels. Additionally, STRING analysis identified the small leucine‐rich PG decorin as a putative link between all GPCs and EGFR. Subsequently, a deeper understanding on the effect of EGFR and JAK/STAT signaling may shed light into the role and interplay between GPCs and decorin in breast cancer progression, thus contributing to novel therapeutic solutions.

Glypican-3 deficiency in liver cancer upregulates MAPK/ERK pathway but decreases cell proliferation.

Glypican-3 (GPC3) is overexpressed in hepatocellular carcinomas and hepatoblastomas and represents an important therapeutic target but the biologic importance of GPC3 in liver cancer is unclear. To date, there are limited data characterizing the biological implications of GPC3 knockout (KO) in liver cancers that intrinsically express this target. Here, we report on the development and characterization of GPC3-KO liver cancer cell lines and compare to them to parental lines. GPC3-KO variants were established in HepG2 and Hep3B liver cancer cell lines using a lentivirus-mediated CRISPR/Cas9 system. We assessed the effects of GPC3 deficiency on oncogenic properties in vitro and in murine xenograft models. Downstream cellular signaling pathway changes induced by GPC3 deficiency were examined by RNAseq and western blot. To confirm the usefulness of the models for GPC3-targeted drug development, we evaluated the target engagement of a GPC3-selective antibody, GC33, conjugated to the positron-emitting zirconium-89 (89Zr) in subcutaneous murine xenografts of wild type (WT) and KO liver cancer cell lines. Deletion of GPC3 significantly reduced liver cancer cell proliferation, migration, and invasion compared to the parental cell lines. Additionally, the tumor growth of GPC3-KO liver cancer xenografts was significantly slower compared with control xenografts. RNA sequencing analysis also showed GPC3-KO resulted in a reduction in the expression of genes associated with cell cycle regulation, invasion, and migration. Specifically, we observed the downregulation of components in the AKT/NFκB/WNT signaling pathways and of molecules related to cell cycle regulation with GPC3-KO. In contrast, pMAPK/ERK1/2 was upregulated, suggesting an adaptive compensatory response. KO lines demonstrated increased sensitivity to ERK (GDC09994), while AKT (MK2206) inhibition was more effective in WT lines. Using antibody-based positron emission tomography (immunoPET) imaging, we confirmed that 89Zr-GC33 accumulated exclusively in GPC3-expression xenografts but not in GPC3-KO xenografts with high tumor uptake and tumor-to-liver signal ratio. We show that GPC3-KO liver cancer cell lines exhibit decreased tumorigenicity and altered signaling pathways, including upregulated pMAPK/ERK1/2, compared to parental lines. Furthermore, we successfully distinguished between GPC3+ and GPC3- tumors using the GPC3-targeted immunoPET imaging agent, demonstrating the potential utility of these cell lines in facilitating GPC3-selective drug development.

Can Glypican-6 Levels Be Used to Determine Right Ventricular Remodeling After Non-ST Segment Elevation Myocardial Infarction?

Myocardial infarction is associated with right ventricular (RV) remodeling. Glypican-6 (GPC6), a member of the membrane proteoglycan family, plays a significant role in cardiac remodeling. This study aims to determine if GPC6 can predict RV remodeling after percutaneous coronary intervention (PCI) in patients with non-ST segment elevation myocardial infarction (NSTEMI). The study enrolled 164 consecutive patients with NSTEMI and controls. It compared baseline plasma GPC6 levels, echocardiography, and laboratory parameters between the RV remodeling and non-RV remodeling groups with NSTEMI. Echocardiographic data were measured at baseline and at six months. GPC6 levels were higher in the NSTEMI group 11.06 ng/mL (4.61-18.17) vs. 5.98 ng/mL (3.81-9.83) compared to the control group in the initial phase. RV remodeling, defined as a ≥ 20% increase in RV end-diastolic area (RV EDA), was observed in 23 patients (30%). After six months, RV EDA increased significantly from baseline 18.68 ± 1.20 cm2 vs. 24.91 ± 1.08 cm2, P < 0.001. GPC6 was a significant independent predictor of RV remodeling (hazard ratio [HR]: 1.546, 95% confidence interval [CI]: 1.056-2.245, P < 0.001). Receiver operating characteristic curve (ROC) analyses showed that GPC6 values > 15.5 ng/mL (area under the curve [AUC] = 0.828, sensitivity: 70%, specificity: 74%, P < 0.001) were strong predictors of RV remodeling. NSTEMI patients should be closely monitored for RV remodeling. GPC6 appears useful in detecting RV remodeling following NSTEMI in patients undergoing PCI.