Extracellular glypican-1 affects tumor progression and prognosis in esophageal cancer.

INTRODUCTION: Cells are covered with a glycan surface layer that is referred to as the glycocalyx (GCX). It has been reported that the formation of the GCX is promoted on cancer cells and is associated with tumor growth and metastasis. Heparan sulfate proteoglycan glypican-1 (GPC1) is a core protein of the GCX that is overexpressed in esophageal squamous cell carcinoma (ESCC) and is involved in the development and progression of cancer cells. The purpose of the present study is to analyze the utility of GPC1 as a new biomarker ralated to glycocalyx that reflects therapeutic effect and prognosis of ESCC. METHODS: We measured the concentration of GPC1 protein in preoperative plasma from advanced esophageal cancer patients and examined its relationships with clinicopathological factors and therapeutic efficacy, and the effects of extracellular GPC1 were investigated. RESULTS: The following clinical factors were significantly correlated with the preoperative high GPC1 concentration: male, tumor size ≥30 mm, venous invasion, pT factor ≥2, pStage ≥3, residual tumor, and distant metastatic recurrence. Both overall and recurrence-free survival were significantly worse in the high GPC1 group. Extracellular GPC1 protein concentration reflected intracellular GPC1 expression. Furthermore, we examined the effects of extracellular recombinant human (rh)GPC1 on ESCC cells, and found that extracellular rhGPC1 affects cell motility, including migration and invasion. CONCLUSIONS: These results demonstrated the utility of extracellular GPC1 as a biomarker, which can be assayed from a less invasive blood sample-based liquid biopsy. Extracellular GPC1 protein plays a role in both tumor cell motility and cancer progression. Thus, plasma GPC1 is a useful biomarker for esophageal cancer progression and may be a potential candidate of therapeutic target.

IRTIDP: A simple integrated real-time isolation and detection platform for small extracellular vesicles Glypican-1 in pancreatic cancer patients.

Glypican-1 (GPC-1) protein-positive small extracellular vesicles (GPC-1+-sEV) have been proposed as potential biomarkers for early diagnosis of pancreatic cancer. In this study, we present an integrated real-time isolation and detection platform (IRTIDP) to capture and analyze GPC-1+-sEV directly from sera of pancreatic cancer patients. First, CD63 antibody-modified metal-organic framework (MOF) materials were utilized to enrich sEVs with a capture efficiency of 93.93 %. Second, a SERS probe was constructed by Raman reporter 4-MBA and GPC-1 antibody modified SERS active silver nanoparticles (AgNPs), which formed a sandwich complex structure of "MOFs@GPC-1+-sEV@AgNPs-4-MBA" with MOFs-enriched sEVs. The IRTSDP can complete the capture and detection process within 35 min, with a detection limit for 1 GPC-1+-sEV/µL, and linear range between 105∼109 GPC-1+-sEV/mL. Furthermore, this approach has been applied to quantify serum sEV GPC-1 in clinical pancreatic cancer patients. Based on the SERS intensity analysis, pancreatic cancer patients can be distinguished from pancreatic cystadenoma patients and healthy individuals effectively using this innovative platform that provides highly specific and sensitive means for early diagnosis of pancreatic cancer as well as other tumor types.

Evaluation of antitumor potential of an anti-glypican-1 monoclonal antibody in preclinical lung cancer models reveals a distinct mechanism of action.

Aim: The main objective of this study was to investigate the antitumor effect of a mouse anti-human glypican-1 (GPC1) monoclonal antibody (mAb) on non-small cell lung carcinoma (NSCLC) and associated molecular mechanisms. Methods: The anti-proliferative and anti-migratory activities of anti-GPC1 mAb were examined in A549 and H460 NSCLC cells and LL97A lung fibroblasts. The inhibitory effect of anti-GPC1 mAb on tumor growth was evaluated in an orthotopic lung tumor model. Results: The in vitro study showed that anti-GPC1 mAb profoundly inhibited the anchorage-independent growth of A549 and H460 NSCLC cells and exhibited relatively high cytotoxic activities towards LL97A lung fibroblasts, A549/LL97A and H460/LL97A coculture spheroids. Moreover, anti-GPC1 mAb significantly decreased the expression of phospho-Src (p-Src; Tyr416), p-Akt (Ser473) and ß-catenin in the co-cultured LL97A lung fibroblasts, and the expression of phospho-mitogen-activated protein kinase kinase (p-MEK; Ser217/221) and phospho-90 kDa ribosomal s6 kinase (p-p90RSK; Ser380) in co-cultured A549 cells. When anti-GPC1 mAb was administered to tumor-bearing mice, the inhibitory effect of anti-GPC1 mAb on the orthotopic lung tumor growth was not statistically significant. Nonetheless, results of Western blot analysis showed significant decrease in the phosphorylation of fibroblast growth factor receptor 1 (FGFR1) at Tyr766, Src at Tyr416, extracellular signal-regulated kinase (ERK) at Thr202/Tyr204, 90 kDa ribosomal S6 kinase (RSK) at Ser380, glycogen synthase kinases 3α (GSK3α) at Ser21 and GSK3ß at Ser9 in tumor tissues. These data implicate that anti-GPC1 mAb treatment impairs the interaction between tumor cells and tumor associated fibroblasts by attenuating the paracrine FGFR signal transduction. Conclusions: The relatively potent cytotoxicity of anti-GPC1 mAb in lung fibroblasts and its potential inhibitory effect on the paracrine FGFR signal transduction warrant further studies on the combined use of this mAb with targeted therapeutics to improve therapeutic outcomes in lung cancer.

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.

Glypican-1 may be a plasma biomarker for predicting the rupture of small intracranial aneurysms.

Currently, there are no effective methods for predicting the rupture of asymptomatic small intracranial aneurysms (IA) (<7 mm). In this study the aim was to identify early warning biomarkers in peripheral plasma for predicting IA rupture. Four experimental groups were included: ruptured intracranial aneurysm (RIA), unruptured intracranial aneurysm (UIA), traumatic subarachnoid hemorrhage control (tSAHC), and healthy control (HC) groups. Plasma proteomics of these four groups were detected using iTRAQ combined LC-MS/MS. Differentially expressed proteins (DEPs) were identified in RIA, UIA, tSAHC compared with HC. Target proteins associated with aneurysm rupture were obtained by comparing the DEPs of the RIA and UIA groups after filtering out the DEPs of the tSAHC group. The plasma concentrations of target proteins were validated using enzyme-linked immunosorbent assay (ELISA). The iTRAQ analysis showed a significant increase in plasma GPC1 concentration in the RIA group compared to the UIA group, which was further validated among the IA patients. Logistic regression analysis identified GPC1 as an independent risk factor for predicting aneurysm rupture. The ROC curve indicated that the GPC1 plasma cut-off value for predicting aneurysms rupture was 4.99 ng/ml. GPC1 may be an early warning biomarker for predicting the rupture of small intracranial aneurysms. SIGNIFICANCE: The current management approach for asymptomatic small intracranial aneurysms (<7 mm) is limited to conservative observation and surgical intervention. However, the decision-making process regarding these options poses a dilemma due to weighing their respective advantages and disadvantages. Currently, there is a lack of effective diagnostic methods to predict the rupture of small aneurysms. Therefore, our aim is to identify early warning biomarkers in peripheral plasma that can serve as quantitative detection markers for predicting intracranial aneurysm rupture. In this study, four experimental populations were established: small ruptured intracranial aneurysm (sRIA) group, small unruptured intracranial aneurysm (sUIA) group, traumatic subarachnoid hemorrhage control (tSAHC) group, and healthy control (HC) group. The tSAH group was the control group of spontaneous subarachnoid hemorrhage caused by ruptured aneurysm. Compared with patients with UIA, aneurysm tissue and plasma GPC1 in patients with RIA is significantly higher, and GPC1 may be an early warning biomarker for predicting the rupture of intracranial small aneurysms.

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.

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.

Immunohistochemical Evaluation of the Expression of Specific Membrane Antigens in Patients with Pancreatic Ductal Adenocarcinoma.

(1) Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. The lack of validated disease biomarkers makes timely diagnosis challenging in most cases. Cell membrane and surface proteins play a crucial role in several routes of oncogenesis. The aim of this study was to evaluate the expression of six membrane antigens on PDAC (CA 19-9, mucin 1 and 4 (MUC1, MUC4), mesothelin (MSLN), Annexin A10 (ANXA10), Glypican-1 (GPC-1)) and their correlation with oncologic outcomes. (2) Methods: Immunohistochemical staining for CA 19.9, MUC1, MUC4, MSLN, ANXA10, and GPC-1 of surgical samples of 50 consecutive patients with PDAC was performed. Antigen expression for tumor, ductal, and acinar tissues was classified according to the histo-score (H-score) by two pathologists. (3) Results: Recurrence rate was 47% and 18 patients (36%) deceased (median follow-up 21.5 months). Immunostaining for CA 19-9 and MUC1 showed a significantly higher expression in the neoplastic tissue compared to non-tumor ductal and acinar tissues (p < 0.001). MUC4, MSLN, ANXA10, and GPC-1 were selectively expressed in the neoplastic tissue (p < 0.001). A CA 19-9 H-score value >270 was independently associated with a worse overall survival (p = 0.05) and disease-free survival (p = 0.05). (4) Conclusions: CA 19-9 and MUC1 are highly expressed in PDAC cells. The histological expression of CA 19-9 may predict prognosis. MUC4, MSLN, ANXA10, and GPC-1 are selectively expressed by neoplastic tissue and may represent a potential histological biomarker of disease.

Silencing Glypican-1 enhances the antitumor effects of Pictilisib via downregulating PI3K/Akt/ERK signaling in chemo-resistant esophageal adenocarcinoma.

Poorly differentiated esophageal adenocarcinoma (PDEAC) has a dismal prognosis. Glypican-1(GPC-1) is known to be upregulated in several cancer types in contrast to healthy tissues, rendering it as a biomarker. Nevertheless, the potential therapeutic targeting of GPC-1 has not been explored in PDEAC. There is accumulating evidence that GPC-1, via upregulation of PI3K/Akt/ERK signaling, plays a crucial role in the progression and chemoresistance in cancer. Pictilisib, a class I pan PI3K inhibitor, has shown promising antitumor results in clinical trials, however, has not gained widespread success due to acquired drug resistance. This study investigated the role of GPC-1 in chemo-resistant PDEAC and appraises the impact of targeted silencing of GPC-1 on the antitumor effects of Pictilisib in PDEAC cell lines. Immunohistochemistry assays in PDEAC tissue specimens demonstrated a pronounced intensity of staining with GPC-1. Upregulation of GPC-1 was found to be correlated with advanced stage and poor prognosis. In-vitro studies examined the influence of GPC-1 knockdown and Pictilisib, both as individual agents and in combination, on cytotoxicity, cell cycle distribution, apoptosis, and gene expression profiles. Silencing GPC-1 alone showed significantly reduced cell viability, migration, colony formation, epithelial-mesenchymal transition, and stemness in PDEAC cells. Significantly, knockdown of GPC-1 combined with low-dose Pictilisib led to enhancement of cytotoxicity, cell cycle arrest, and apoptosis in ESO-26 and OE-33 cells. In the xenograft mouse model, the combination of Pictilisib and GPC-1 knockdown exhibited synergy. These findings suggest that GPC-1 represents a promising target to augment chemosensitivity in esophageal adenocarcinoma.

Targeting Glypican-1 Reverses Resistance to 5-Fluorouracil in Esophageal Adenocarcinoma Cells.

BACKGROUND/AIM: The primary mode of therapy for individuals with locally advanced esophageal adenocarcinoma (EAC) is neoadjuvant chemotherapy, commonly 5-Fluorouracil (5-FU). However, approximately 30% of these patients develop resistance to therapy. Glypican-1 (GPC-1) has been identified as one of the key drivers of chemoresistance in cancer; however, its role in EAC cells has not been explored. The objective of the present study was to evaluate the role of GPC-1 in chemoresistance to 5-FU in EAC cells. MATERIALS AND METHODS: Cell viability to 5-FU was measured with CCK-8 assay, and GPC-1 expression was validated using western blot. 5-FU resistant cell lines were generated. The effect of lentivirus-mediated GPC-1 knockdown on FLO-1 cell viability, cell cycle, and apoptosis was evaluated. RESULTS: 5-FU resistant EAC cells showed increased GPC-1 expression and knockdown of GPC-1 increased cell death and apoptosis. Importantly, the knockdown of GPC-1 enhanced the antitumor effects of 5-FU in vitro via down-regulating AKT/ERK/ß-catenin signaling. CONCLUSION: Silencing GPC-1 has the potential to augment the efficacy of 5-FU chemotherapy in resistant EAC tumors.

Glypican1: A potential cancer biomarker for nanotargeted therapy.

Glypicans (GPCs) are generally involved in cellular signaling, growth and proliferation. Previous studies reported their roles in cancer proliferation. GPC1 is a co-receptor for a variety of growth-related ligands, thereby stimulating the tumor microenvironment by promoting angiogenesis and epithelial-mesenchymal transition (EMT). This work reviews GPC1-biomarker-assisted drug discovery by the application of nanostructured materials, creating nanotheragnostics for targeted delivery and application in liquid biopsies. The review includes details of GPC1 as a potential biomarker in cancer progression as well as a potential candidate for nano-mediated drug discovery.

Nano-particulate Toll-like Receptor 9 Agonist Potentiates the Antitumor Activity of Anti-Glypican-1 Antibody.

BACKGROUND/AIM: Monoclonal antibodies (mAbs) that target tumor antigens have recently been developed. Their antitumor activity is mainly achieved through antibody-dependent cellular cytotoxicity (ADCC) via effector cells such as tumor-infiltrated macrophages and natural killer (NK) cells. CpG oligodeoxynucleotides (ODNs) have potent antitumor activity and are considered to increase the tumor infiltration of macrophages and NK cells; however, a completely solubilized novel CpG-schizophyllan (SPG) complex, K3-SPG, displays more potent antitumor activity. We recently reported the significant antitumor activity of anti-glypican-1 (GPC1) mAb against GPC1-positive esophageal squamous cell carcinoma (ESCC) via ADCC. The aim of this study was to evaluate the potential synergistic antitumor activity of anti-GPC1 mAb and K3-SPG and elucidate the underlying mechanisms using a xenograft model of GPC1-positive human ESCC cells. MATERIALS AND METHODS: The established human esophageal cancer cell line TE14 was subcutaneously injected into SCID mice. Xenograft mice were treated with anti-GPC1 mAb, K3-SPG, or their combination. Antitumor activity was evaluated by measuring the tumor volume. For FACS analysis, agents were administrated, and tumors were resected 1 day after the final treatment. RESULTS: Anti-GPC1 mAb or K3-SPG monotherapy showed dose-dependent antitumor activity, and combination therapy with anti-GPC1 mAb and K3-SPG showed antitumor activity (p=0.0859). Flow cytometry revealed significantly increased numbers of macrophages (p=0.0133) and of the ratio of activated NK cells/total NK cells (p=0.0058) following K3-SPG or combination therapy. CONCLUSION: Combination therapy with K3-SPG and anti-GPC1 mAb or another antitumor mAb may represent a new cancer treatment option acting via ADCC.

Attenuation of cancer proliferation by suppression of glypican-1 and its pleiotropic effects in neoplastic behavior.

Glypicans (GPC1-6) are associated with tumorigenic processes and their involvement in neoplastic behavior has been discussed in different cancer types. Here, a cancer-wide GPC expression study, using clinical cancer patient data in The Cancer Genome Atlas, reveals net upregulation of GPC1 and GPC2 in primary solid tumors, whereas GPC3, GPC5 and GPC6 display lowered expression pattern compared to normal tissues. Focusing on GPC1, survival analyses of the clinical cancer patient data reveal statistically significant correlation between high expression of GPC1 and poor prognosis in 10 particular cancer types i.e., bladder urothelial carcinoma, brain lower grade glioma, liver hepatocellular carcinoma, colon adenocarcinoma, kidney renal clear cell carcinoma, lung adenocarcinoma, mesothelioma, ovarian serous cystadenocarcinoma, uterine corpus endometrial carcinoma and uveal melanoma. In vitro studies targeting GPC1 expression by CRISPR/Cas9 or siRNA or treatment with an anti-GPC1 antibody resulted in attenuation of proliferation of cancer cells from bladder carcinoma, glioma and hepatocellular carcinoma patients (T24, U87 and HepG2 cells). Further, overexpression of GPC1 exhibited a significant and negative correlation between GPC1 expression and proliferation of T24 cells. Attempt to reveal the mechanism through which downregulation of GPC1 leads to attenuation of tumor growth using systematic Ingenuity Pathway Analysis indicate that suppression of GPC1 results in ECM-mediated inhibition of specific pro-cancer signaling pathways involving TGF-ß and p38 MAPK. Identified differential expression and pleiotropic effects of GPCs in specific cancer types emphasize their potential of as novel diagnostic tools and prognostic factors and open doors for future GPC targeted therapy.

The mechanisms underlying the enrichment and action of glypican-1-positive exosomes in colorectal cancer cells.

BACKGROUND: Glypican-1 (GPC1) is overexpressed in several tumors, and GPC1+ exosomes have shown the potential to predict early colorectal cancer (CRC). However, the mechanisms underlying the enrichment and action of GPC1+ exosomes in CRC remain unknown. METHODS: The expression of slit guidance ligand 2 (SLIT2), hypoxia-inducible factor (HIF)-1α/2α, and GPC1 in clinical CRC tissues was detected using immunohistochemistry and western blot. Exosomes were isolated from the supernatants of CRC cell cultures. The effects of SLIT2, hypoxia, heparin, and phospholipase C (PLC) on exosomal GPC1 expression and GPC1+ exosome enrichment in CRC cells were analyzed with western blot and flow cytometry. CRC cell proliferation was assessed with MTT and colony formation assays. Co-immunoprecipitation was used to detect the binding of GPC1 and SLIT2 in SW480 cells. Nude mice were subcutaneously inoculated with SW480 cells with different treatments. The Wnt signaling was detected. RESULTS: SLIT2 was poorly expressed and GPC1, HIF-1α, and HIF-2α were highly expressed in human CRC tissues. SLIT2 in CRC cells inhibited GPC1+ exosome enrichment and exosomal GPC1 expression. PLC and heparin increased GPC1+ exosome enrichment in CRC cells in a concentration-dependent manner. Hypoxia increased the enrichment of GPC1+ exosomes in CRC cells depending on HIF-2α expression. GPC1+ exosomes stimulated CRC cell proliferation and xenograft tumor growth through activation of Wnt signaling. CONCLUSIONS: GPC1+ exosome enrichment is related to PLC and heparin. Hypoxia increases the enrichment of GPC1+ exosomes in CRC cells by activating HIF-2α and downregulating SLIT2. GPC1+ exosomes further drive CRC progression by activating Wnt signaling.

Interplay between APP and glypican-1 processing and α-synuclein aggregation in undifferentiated and differentiated human neural progenitor cells.

In Parkinson's disease, there is an accumulation of α-synuclein (SYN) aggregates in neurons, which is promoted by neuroinflammation. In neural cells, cytokine-induced SYN aggregation is modulated by heparan sulfate (HS) derived from glypican-1 (GPC1) by amyloid precursor protein (APP) and nitric oxide (NO)-dependent cleavage. We have explored possible interplay between APP, GPC1, and SYN in undifferentiated and differentiated neural progenitor cells (NPCs) by modulating APP and GPC1 processing. Effects were monitored by immunofluorescence microscopy and slot immunoblotting using antibodies recognizing APP degradation products, HS released from GPC1, and SYN aggregates (filamentous SYN [SYNfil]). Suppression of HS release from GPC1 by inhibition of ß-secretase or by NO deprivation resulted in no or slight increase in SYNfil aggregation. Stimulation of HS release by ascorbate did not further increase SYNfil staining. Interleukin-6 (IL-6) induced increased APP and GPC1 processing and SYNfil formation, which was reduced when ß-secretase was inhibited and when HS release was impeded by NO deprivation. Ascorbate restored APP and GPC1 processing but did not affect SYNfil formation. Ascorbate-dependent differentiation of NPC resulted in the expression of tyrosine hydroxylase (TH) which colocalized with SYNfil. Suppression of APP processing by inhibition of ß-secretase greatly disturbed the differentiation process. IL-6 induced coclustering of APP-degradation products, TH, HS, and SYNfil, which could be reversed by stimulation of HS release from GPC1 by excess ascorbate. We suggest that continuous release of HS from GPC1 moderates SYN aggregation and supports differentiation of NPC to dopaminergic neurons.

Pharmacokinetics and Biodistribution of 89Zr-Miltuximab and Its Antibody Fragments as Glypican-1 Targeting Immuno-PET Agents in Glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain cancer, accounting for about 85% of all primary central nervous system (CNS) tumors. With standard treatment strategies like surgery, radiation, and chemotherapy, the median survival time of patients with GBM is only 12-15 months from diagnosis. The poor prognosis of GBM is due to a very high tumor recurrence rate following initial treatment, indicating a dire need for improved diagnostic and therapeutic alternatives for this disease. Antibody-based immunotheranostics holds great promise in treating GBM, combining the theranostic applications of radioisotopes and target-specificity of antibodies. In this study, we developed and validated antibody-based positron emission tomography (PET) tracers targeting the heparan sulfate proteoglycan, glypican-1 (GPC-1), for noninvasive detection of disease using diagnostic molecular imaging. GPC-1 is overexpressed in multiple solid tumor types, including GBM, and is a promising biomarker for novel immunotheranostics. Here, we investigate zirconium-89 (89Zr)-conjugated Miltuximab (a clinical stage anti-GPC-1 monoclonal antibody developed by GlyTherix, Ltd.) and engineered fragments for their potential as immuno-PET tracers to detect GPC-1positive GBM tumors in preclinical models. We explore the effects of molecular size, avidity, and Fc-domain on the pharmacokinetics and biodistribution in vivo, by comparing in parallel the full-length antibody (Miltuximab), Fab'2, Fab, and single-chain variable fragment (scFv) formats. High radiolabeling efficiency (>95%) was demonstrated by all the formats and the stability post-radiolabeling was higher for larger constructs of Miltuximab and the Fab. Receptor-mediated internalization of all 89Zr-labeled formats was observed in a human GBM cell line in vitro, while full-length Miltuximab demonstrated the highest tumor retention (5.7 ± 0.94% ID/g, day-9 postinjection (p.i.)) and overall better tumor-to-background ratios than the smaller Fc-less formats. Results from in vivo PET image quantification and ex vivo scintillation counting were highly correlated. Altogether, 89Zr-DFO-Miltuximab appears to be an effective immuno-PET imaging agent for detecting GPC-1positive tumors such as GBM and the current results support utility of the Fc containing whole mAb format over smaller antibody fragments for this target.

Glypican 1 promotes proliferation and migration in esophagogastric adenocarcinoma via activating AKT/GSK/ß-catenin pathway.

Background: Glypican 1 (GPC1) is a heparan sulphate proteoglycan cell membrane protein. It is implicated in driving cancers of the breast, brain, pancreas, and prostate; however, its role in esophagogastric cancer (EGAC) remains unexplored. The aim of the study was to investigate and elucidate the molecular mechanistic of GPC1 in human EGAC. Methods: Thirty tissue and 120 microarray sections of EGAC were evaluated with Anti-GPC1 immunohistochemistry. Loss and gain of GPC1 function were performed using lentivirus transfection in EGAC cell lines. Mechanistically, AKT/GSK/ß-catenin pathway was evaluated using AKT inhibitor MK-2206 and Wnt/ß-catenin stimulant LiCl. Results: GPC1 overexpression was found in 102 cases (68%). Overexpression of GPC1 correlated with lymph node metastasis, poor differentiation and decreased overall survival. Lentivirus mediated GPC1 knockdown resulted in decreased cell proliferation, migration, invasion, and colony formation. Knockdown caused G0/G1 cell cycle arrest, increased apoptosis, and reduced epithelial mesenchymal transition (EMT). GPC1 mediated its effects by activation of AKT/GSK/ß-catenin pathway. Conclusions: This is the first descriptive study to decipher the role of GPC1 in EGAC. Our results suggest that GPC1 regulates cell proliferation and growth and may serve as an attractive oncotarget in EGAC.

Recombinant thrombomodulin may protect cardiac capillary endothelial glycocalyx through promoting Glypican-1 expression under experimental endotoxemia.

Introduction: Myocardial dysfunction occurs in patients with sepsis due to vascular endothelial injury. Recombinant human thrombomodulin (rhTM) attenuates vascular endothelial injuries through endothelial glycocalyx (eGC) protection. Hypothesis: We hypothesized that rhTM attenuates myocardial dysfunction via the inhibition of vascular endothelial injury during sepsis. Methods: Ten-week-old male C57BL6 mice were injected intraperitoneally with 20 mg/kg of lipopolysaccharide (LPS). In rhTM-treated mice, rhTM was injected intraperitoneally at 3 and 24 h after LPS injection. Saline was injected intraperitoneally as control. To assess for eGC injury, intensity score was measured 48 h after the LPS injection. To confirm vascular endothelial injuries, ultrastructural analysis was performed using scanning (SEM) and transmission electron microscopy (TEM). Results: The survival rate of the rhTM group at 48 h after LPS injection was significantly higher than that of the control group (68% vs. 17%, p < 0.05). The serum level of troponin I in the rhTM group was lower than that in the control (2.2 ± 0.4 ng/dL vs 9.4 ± 1.1 ng/dL, p < 0.05). The expression of interleukin-6 (IL-6) was attenuated in the rhTM-treated group than in the control (65.3 ± 15.3 ng/mL vs 226.3 ± 19.4 ng/mL, p < 0.05). The serum concentration of syndecan-1, a marker of glycocalyx damage, was significantly decreased 48 h post-administration of LPS in the rhTM-treated group than in the control group. In ultrastructural analysis using SEM and TEM, eGC peeled off from the surface of the capillary lumen in the control. Conversely, the eGC injury was attenuated in the rhTM group. Gene set enrichment analysis revealed that osteomodulin, osteoglycin proline/arginine-rich end leucine-rich repeat protein, and glypican-1, which are proteoglycans, were preserved by rhTM treatment. Their protein expression was retained in endothelial cells. Conclusion: rhTM attenuates sepsis-induced myocardial dysfunction via eGC protection.

Serum exosomal and serum glypican-1 are associated with early recurrence of pancreatic ductal adenocarcinoma.

Background: The diagnostic performance and prognostic value of serum exosomal glypican 1 (GPC-1) in pancreatic ductal adenocarcinoma (PDAC) remain controversial. In this study, we detected serum exosomal GPC-1 using enzyme-linked immunosorbent assay (ELISA) and determined whether it serves as a predictor of diagnosis and recurrence for early-stage PDAC. Methods: Serum samples were obtained from patients with 50 PDAC, 6 benign pancreatic tumor (BPT), or 9 chronic pancreatitis (CP) and 50 healthy controls (HCs). Serum exosomes were isolated using an exosome isolation kit. Exosomal and serum GPC-1 levels were measured using ELISA. The freeze-thaw process was carried out to analyze the stability of GPC-1. Receiver operating characteristic (ROC) analysis was employed to assess the diagnostic value of GPC-1. Kaplan-Meier and multivariate Cox analyses were used to evaluate the prognostic value of GPC-1. Results: The average concentrations of serum exosomal and serum GPC-1 were 1.5 and 0.8 ng/ml, respectively. GPC-1 expression levels were stable under repeated freezing and thawing (d1-5 freeze-thaw cycles vs. d0 P > 0.05). Serum exosomal and serum GPC-1 were significantly elevated in patients with PDAC compared with HCs (P < 0.0001) but were slightly higher compared with that in patients with CP and BPT (P > 0.05). The expression levels of exosomal and serum GPC-1 were elevated 5 days after surgery in patients with PDAC, CP, and BPT (P < 0.05). Patients with high levels of exosomal and serum GPC-1 had a shorter relapse-free survival (RFS) (P = 0.006, and P = 0.010). Multivariate analyses showed that serum exosomal and serum GPC-1 were independent prognostic indicators for early RFS (P = 0.008, and P = 0.041). Conclusion: ELISA is an effective and sensitive method to detect exosomal and serum GPC-1. The detection of GPC-1 was stable under repeated freezing and thawing cycles and could distinguish early-stage PDAC from HCs but not CP and BPT. Exosomal and serum GPC-1 may be good independent predictors of early recurrence in early-stage PDAC.