Glypican-3 Research Articles

Abstract 13972: Metabolic Reprogramming of Diabetic Mesenchymal Stromal Cells by Inhibiting Glypican 3 Enhances Their Cardioprotective Functions via Pyruvate Kinase Activation

Mesenchymal stromal cells (MSC) represent promising stem cell therapy for the treatment of cardiovascular diseases. Diabetes affects the functional capability of MSC and is an impediment to cell-based therapy. However, the impact of diabetes on MSC myocardial reparative activity, metabolic fingerprint, and the mechanism of dysfunction is still poorly understood. Therefore, our study aimed to understand the molecular basis of diabetes-induced MSC functions and their metabolic mechanism, and potential metabolic reprogramming of diabetic MSC to reverse these dysfunctions may represent a strategy to enhance cell-based therapeutics for myocardial repair in diabetic patients. The seahorse and 13 C glucose tracing studies identified defective energy metabolism in db/db-MSC. Our data also shows the diminished functional activity of db/db-MSCs to improve post-MI cardiac functions. Furthermore, we found that glypican-3 (GPC3), a heparan sulfate proteoglycan, is highly upregulated in db/db-MSC when compared to WT-MSC. GPC3 overexpression in WT-MSC showed abrogated metabolic fingerprints, lowered immunosuppression activity, and reduced angiogenic potential. Interestingly, GPC3 knockdown in WT-MSC showed an improved metabolic state, increased immunosuppression activity, and enhanced angiogenic potential. Furthermore, GPC3 knockdown in diabetic MSC restored their energy metabolism and their functions. Mechanistically, we revealed that GPC3 binds to pyruvate kinase (PK), which subsequently increases the rate of glycolysis and ATP production. In a mouse model of myocardial infarction (MI), intramyocardial transplantation of GPC3-metabolic reprogrammed db/db-MSC promoting neovascularization (at 28 days, post-MI) and lower cardiac fibrosis, leading to improved left ventricular function. These findings indicate that diabetes increased GPC3 expression and altered the metabolic rates in MSC which is detrimental to its function, and transplantation of GPC3-metabolic reprogrammed db/db-MSC into ischemic myocardium promoted their cardiac functions. Thus, inhibiting GPC3 may potentially be used to restore the metabolic state and functions of db/db-MSC for cell therapy applications.

GPC3 and PEG10 peptides associated with placental gp96 elicit specific T cell immunity against hepatocellular carcinoma

The placenta and tumors can exhibit a shared expression profile of proto-oncogenes. The basis of placenta-derived heat shock protein gp96, which induces prophylactic and therapeutic T cell responses against cancer including hepatocellular carcinoma (HCC), remains unknown. Here, we identified the associated long peptides from human placental gp96 using matrix-assisted laser desorption/ionization-time-of-flight and mass spectrometry and analyzed the achieved proteins through disease enrichment analysis. We found that placental gp96 binds to numerous peptides derived from 73 proteins that could be enriched in multiple cancer types. Epitope-harboring peptides from glypican 3 (GPC3) and paternally expressed gene 10 (PEG10) were the major antigens mediating anti-HCC T cell immunity. Molecular docking analysis showed that the GPC3- and PEG10-derived peptides, mainly obtained from the cytotrophoblast layer of the mature placenta, bind to the lumenal channel and client-bound domain of the gp96 dimer. Immunization with bone marrow-derived dendritic cells pulsed with recombinant gp96-GPC3 or recombinant gp96-PEG10 peptide complex induced specific T cell responses, and T cell transfusion led to pronounced growth inhibition of HCC tumors in nude mice. We demonstrated that the chaperone gp96 can capture antigenic peptides as an efficient approach for defining tumor rejection oncoantigens in the placenta and provide a basis for developing GPC3 and PEG10 peptide-based vaccines against HCC. This study provides insight into the underlying mechanism of the antitumor response mediated by embryonic antigens from fetal tissues, and this will incite more studies to identify potential tumor rejection antigens from placenta.

Primary breast tumor induced extracellular matrix remodeling in premetastatic lungs

The premetastatic niche hypothesis proposes an active priming of the metastatic site by factors secreted from the primary tumor prior to the arrival of the first cancer cells. We investigated several extracellular matrix (ECM) structural proteins, ECM degrading enzymes, and ECM processing proteins involved in the ECM remodeling of the premetastatic niche. Our in vitro model consisted of lung fibroblasts, which were exposed to factors secreted by nonmalignant breast epithelial cells, nonmetastatic breast cancer cells, or metastatic breast cancer cells. We assessed ECM remodeling in vivo in premetastatic lungs of female mice growing orthotopic primary breast tumor xenografts, as compared to lungs of control mice without tumors. Premetastatic lungs contained significantly upregulated Collagen (Col) Col4A5, matrix metalloproteinases (MMPs) MMP9 and MMP14, and decreased levels of MMP13 and lysyl oxidase (LOX) as compared to control lungs. These in vivo findings were consistent with several of our in vitro cell culture findings, which showed elevated Col14A1, Col4A5, glypican-1 (GPC1) and decreased Col5A1 and Col15A1 for ECM structural proteins, increased MMP2, MMP3, and MMP14 for ECM degrading enzymes, and decreased LOX, LOXL2, and prolyl 4-hydroxylase alpha-1 (P4HA1) for ECM processing proteins in lung fibroblasts conditioned with metastatic breast cancer cell media as compared to control. Taken together, our data show that premetastatic priming of lungs by primary breast tumors resulted in significant ECM remodeling which could facilitate metastasis by increasing interstitial fibrillar collagens and ECM stiffness (Col14A1), disruptions of basement membranes (Col4A5), and formation of leaky blood vessels (MMP2, MMP3, MMP9, and MMP14) to promote metastasis.

GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500pg/mL) and CCL19 at a high level (15000pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56days after intravenous infusion. In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

Circulating glypican-4 is a new predictor of all-cause mortality in patients with heart failure

BackgroundHeart failure confers a high burden of morbidity and mortality. However, risk prediction in heart failure patients still is limited. Blood-based biomarkers hold promise to improve clinical risk assessment. Recently we have identified circulating glypican-4 (GPC4) as a significant predictor of mortality in coronary angiography patients and patients with peripheral artery disease. The impact of serum GPC4 on mortality in patients with heart failure is unknown and is addressed in this prospective cohort study. MethodsWe prospectively recorded all-cause mortality in 288 patients with heart failure. GPC4 levels were measured using an enzyme-linked immunosorbent assay at baseline. ResultsDuring the 24-month follow-up period, 28.1% (n = 81) of the patients died. Serum GPC4 significantly predicted all-cause mortality (hazard ratio (HR) per doublingof GPC4 = 3.57 [2.31–5.53]; P < 0.001). Subgroup analysis showed that GPC4 was significantly associated with all-cause mortality in patients with reduced ejection fraction (HR per doubling = 3.25 [1.75–6.04]; P < 0.001) as well as in those with preserved ejection fraction (HR per doubling = 3.07 [1.22–7.70]; P = 0.017). The association between serum GPC4 and all-cause mortality remained significant in multivariable Cox regression analysis correcting for traditional risk factors (P = 0.035). Results from C-statistics indicated an additional prognostic value of GPC4 relative to NT-proBNP for the prediction of two-year all-cause mortality (P = 0.030). ConclusionCirculating GPC4 independently predicts all-cause mortality in patients with heart failure.

Identification and validation of microtubule depolymerizing agent, CYT997, as a potential drug candidate for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a typically fatal malignancy with limited treatment options and poor survival rates, despite recent FDA approvals of newer treatment options. We aim to address this unmet need by using a proprietary computational drug discovery platform that identifies drug candidates with the potential to advance rapidly and successfully through preclinical studies. We generated an in silico model of HCC biology to identify the top 10 small molecules with predicted efficacy. The most promising candidate, CYT997, was tested for its invitro effects on cell viability and cell death, colony formation, cell cycle changes, and cell migration/invasion in HCC cells. We used an HCC patient-derived xenograft (PDX) mouse model to assess its invivo efficacy. CYT997 was significantly more cytotoxic against HCC cells than against primary human hepatocytes, and sensitized HCC cells to sorafenib. It arrested cell cycle at the G2/M phase with associated up-regulations of p21, p-MEK1/2, p-ERK, and down-regulation of cyclin B1. Cell apoptosis and senescence-like morphology were also observed. CYT997 inhibited HCC cell migration and invasion, and down-regulated the expressions of acetylated tubulins, β-tubulin, glypican-3 (GPC3), β-catenin, and c-Myc. Invivo, CYT997 (20 mg/kg, three times weekly by oral gavage) significantly inhibited PDX growth, while being non-toxic to mice. Immunohistochemistry confirmed the down-regulation of GPC3, c-Myc, and Ki-67, supporting its anti-proliferative effect. CYT997 is a potentially efficacious and non-toxic drug candidate for HCC therapy. Its ability to down-regulate GPC3, β-catenin, and c-Myc highlights a novel mechanism of action.

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.

Artificial intelligence-based prediction of molecular and genetic markers for hepatitis C-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the main cause of mortality from cancer globally. This paper intends to classify public gene expression data of patients with Hepatitis C virus-related HCC (HCV+HCC) and chronic HCV without HCC (HCV alone) through the XGboost approach and to identify key genes that may be responsible for HCC. The current research is a retrospective case-control study. Public data from 17 patients with HCV+HCC and 35 patients with HCV-alone samples were used in this study. An XGboost model was established for the classification by 10-fold cross-validation. Accuracy (AC), balanced accuracy (BAC), sensitivity, specificity, positive predictive value, negative predictive value, and F1 score were utilized for performance assessment. AC, BAC, sensitivity, specificity, positive predictive value, negative predictive value, and F1 scores from the XGboost model were 98.1, 97.1, 100, 94.1, 97.2, 100, and 98.6%, respectively. According to the variable importance values from the XGboost, the HAO2, TOMM20, GPC3, and PSMB4 genes can be considered potential biomarkers for HCV-related HCC. A machine learning-based prediction method discovered genes that potentially serve as biomarkers for HCV-related HCC. After clinical confirmation of the acquired genes in the following medical study, their therapeutic use can be established. Additionally, more detailed clinical works are needed to substantiate the significant conclusions in the current study.

The Effects of Glypican-3 Deficiency on Radiosensitivity in Liver Cancer Cells

Glypican-3 (GPC-3), a heparan sulfate proteoglycan involved in cellular proliferation, modulates signaling of FGF/FGFR, IGF/IGFR, HGF/Met, Wnt/Frizzled, among others and correlates with survival. GPC-3 is overexpressed in the majority of hepatocellular carcinoma and hepatoblastoma, but not in normal hepatocytes. Accordingly, it is being investigated as a liver cancer-selective target for radiopharmaceutical imaging and therapy. However, the potential linkage between GPC-3 expression and radiosensitivity has not yet been defined. In this study, we investigated the effects of GPC-3 deficiency on radiosensitivity in liver cancer cell lines. CRISPR/Cas9 system was used to engineer GPC-3 knockout variants of liver cancer cell lines, HepG2 & Hep3B, both of which natively express GPC-3. Confirmation of knockout of GPC-3 was evaluated by RT-PCR, western blotting, flow cytometry, immunocytochemistry, and gDNA sequencing. Cell growth and migration were evaluated by BrdU insertion and wound-healing assays, respectively. In vitro radiosensitivity was examined by radiation-induced apoptosis/necrosis (Annexin V-APC and PI staining), cell cycle modification, γH2AX foci formation, and clonogenic assays (6 Gy). Wildtype and knockout lines were engrafted into athymic mice to assess tumor growth kinetics. RT-PCR, western blotting, flow cytometry, and immunocytochemistry all confirmed GPC-3 knockout in both HepG2 and Hep3B cell lines. Nucleotide deletion at exon 3 of the GPC-3 gene was confirmed by gDNA sequencing in HepG2ΔGPC3 and Hep3BΔGPC3. GPC-3 deficiency reduced liver cancer cell proliferation (HepG2ΔGPC3, p = 0.027, and Hep3BΔGPC3, p = 0.031) and migration (HepG2ΔGPC3: 1.5-fold, p<0.001, and Hep3BΔGPC3: 2.3-fold, p<0.001) significantly when compared with wild type. GPC-3 deficiency reduced cell survival and clonogenicity (HepG2ΔGPC3: DEF = 1.23, Hep3BΔGPC3: DEF = 1.23) in liver cancer cells exposed to irradiation (6 Gy). The delayed repair of double-stranded DNA damage was observed in irradiated GPC-3 deficient liver cancer cells. Tumor growth was dramatically delayed by GPC-3 deficiency. Tumor weight measured at 50 (Hep3B) and 60 (HepG2) days after liver cancer cell inoculation corroborated these effects. Knockout lines of HepG2 and Hep3B exhibited decreased cell proliferation, migration, and in vivo tumor growth compared to wildtype. GPC-3 deficiency was associated with increased sensitivity to radiation therapy. Studies identifying the pathways through which this radiosensitivity is mediated are ongoing.

Radiomics nomogram for prediction of glypican-3 positive hepatocellular carcinoma based on hepatobiliary phase imaging.

The hepatobiliary-specific phase can help in early detection of changes in lesion tissue density, internal structure, and microcirculatory perfusion at the microscopic level and has important clinical value in hepatocellular carcinoma (HCC). Therefore, this study aimed to construct a preoperative nomogram for predicting the positive expression of glypican-3 (GPC3) based on gadoxetic acid-enhanced (Gd-EOB-DTPA) MRI hepatobiliary phase (HBP) radiomics, imaging and clinical feature. We retrospectively included 137 patients with HCC who underwent Gd-EOB-DTPA-enhanced MRI and subsequent liver resection or puncture biopsy at our hospital from January 2017 to December 2021 as training cohort. Subsequently collected from January 2022 to June 2023 as a validation cohort of 49 patients, Radiomic features were extracted from the entire tumor region during the HBP using 3D Slicer software and screened using a t-test and least absolute shrinkage selection operator algorithm (LASSO). Then, these features were used to construct a radiomics score (Radscore) for each patient, which was combined with clinical factors and imaging features of the HBP to construct a logistic regression model and subsequent nomogram model. The clinicoradiologic, radiomics and nomogram models performance was assessed by the area under the curve (AUC), calibration, and decision curve analysis (DCA). In the validation cohort,the nomogram performance was assessed by the area under the curve (AUC). In the training cohort, a total of 1688 radiomics features were extracted from each patient. Next, radiomics with ICCs<0.75 were excluded, 1587 features were judged as stable using intra- and inter-class correlation coefficients (ICCs), 26 features were subsequently screened using the t-test, and 11 radiomics features were finally screened using LASSO. The nomogram combining Radscore, age, serum alpha-fetoprotein (AFP) >400ng/mL, and non-smooth tumor margin (AUC=0.888, sensitivity 77.7%, specificity 91.2%) was superior to the radiomics (AUC=0.822, sensitivity 81.6%, specificity 70.6%) and clinicoradiologic (AUC=0.746, sensitivity 76.7%, specificity 64.7%) models, with good consistency in calibration curves. DCA also showed that the nomogram had the highest net clinical benefit for predicting GPC3 expression.In the validation cohort, the ROC curve results showed predicted GPC3-positive expression nomogram model AUC, sensitivity, and specificity of 0.800, 58.5%, and 100.0%, respectively. HBP radiomics features are closely associated with GPC3-positive expression, and combined clinicoradiologic factors and radiomics features nomogram may provide an effective way to non-invasively and individually screen patients with GPC3-positive HCC.

Combined Use of Serum N-terminal Pro-B-Type Natriuretic Peptide and Glypican-6 in the Diagnosis of Heart Failure.

The aim of this study was to investigate the efficacy of serum glypican-6 (GPC-6) levels and the combination of N-terminal pro-B-type natriuretic peptide (NT-ProBNP)and GPC-6 in the diagnosis of heart failure (HF). In this prospective study, patients older than 18 years of age, admitted to the emergency department of our hospital between December 2021 and April 2022, diagnosed with heart failure (patient group),and healthy volunteers with similar sociodemographic characteristics (control group) were included. The disease severity classification of the patient group was made according to the 2021 ESC guidelines, using echocardiographic findings. Serum GPC-6 andNT-ProBNP levels were measured by the enzyme-linked immunosorbent assay (ELISA) method, which determines the antigen-antibody relationship. Optimal GPC-6 and NT-ProBNP levels for the diagnosis of HF were determined by receiver operating characteristic (ROC) analysis. The patients were divided into three groups according to these levels. Group 1 consisted of patients with both markers below the cutoff values, Group 2 consisted of patients with either of these markers above the cutoff values, and Group 3 consisted of patients with both markers above the cutoff values. The study included 65 heart failure patients and 20 healthy volunteers. When the patient and control groups were compared in terms of serum GPC-6 and serum NT-ProBNP levels, both parameters were evaluated as significantly higher in the patient group (p=0.038 and p<0.001; respectively). In the ROC analysis, it was determined that GPC-6 indicated HF with 58.46% sensitivity and 75% specificity for an optimal cutoff value of 390 pg/ml. In the ROC analysis, it was determined that serum NT-ProBNP indicated HF with 89.23% sensitivity and 70% specificity for an optimal cutoff value of 122 pg/ml. When the groups were compared according to the rate of HF, it was found to be higher in Group 3 compared to Group 2 (97.1% vs. 70.3%, p<0.002) and Group 1 (97.1% vs. 38.5%, p<0.001). This rate was seen to be significantly higher in Group 2 compared to Group 1 (70.3% vs. 38.5%, p=0.042). The combination of GPC-6 and NT-ProBNP may help diagnose HF patients admitted to the emergency department.

The Diagnostic Value Of Serum Glypican-3 In The Diagnosis Of Hepatocellular Carcinoma: A Systematic Review And Meta-Analysis

Background and aim: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. It is curable in the early stages but has a poor prognosis when advanced. Current diagnostic protocols for the early stages of HCC are inefficient. Serum Glypican-3 (GPC-3) is a promising serum tumour marker in diagnosing HCC, and its diagnostic value has been explored in various studies. The outcomes, however, continue to be inconsistent and controversial. This systematic review and meta-analysis aimed to investigate the diagnostic accuracy of serum GPC-3 in hepatocellular carcinoma. This study included recent papers not captured in previous meta-analyses. Method: PUBMED, MEDLINE, Cochrane Library, and CINAHL databases were systematically searched according to preferred reporting items for a systematic review and meta-analysis of diagnostic test accuracy studies guidelines. Studies on the diagnostic accuracy of serum GPC-3 in HCC were selected. The Quality assessment of diagnostic accuracy studies 2 (QUADAS-2) tools were used to assess the quality of selected studies. Sensitivity, specificity, and other diagnostic parameters were pooled using the random effect model. The Summary receiver operating characteristic (sROC) was used to summarise the diagnostic accuracy of Serum GPC-3. Results: Eleven studies were included in this systematic review. The pooled sensitivity, specificity, and 95% confidence intervals (CIs) were 0.60 (0.57-0.62) and 0.67(0.64-0.70), respectively. The pooled positive and negative likelihood ratios and 95% CIs were 2.11 (1.51-2.93) and 0.59 (0.51-0.68), respectively; the pooled diagnostic odds ratio and the 95% CI were 4.07 (2.37-6.98), the area under the sROC curve was 0.7194. Conclusion: This meta-analysis has shown that Serum GPC-3 has an acceptable diagnostic value in diagnosing HCC compared to benign chronic liver diseases. A higher-quality study with a larger sample size is necessary to thoroughly assess the diagnostic utility of serum GPC-3.

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.

Targeted Near-Infrared Fluorescence Imaging of Liver Cancer using Dual-Peptide-Functionalized Albumin Particles.

Fluorescence imaging is an emerging strategy for preoperative diagnosis and intraoperative resection. In particular, owing to their outstanding spatial resolution and deep-tissue penetration, imaging agents in the near-infrared (NIR)-II window (1000-1700 nm) have received intensive interest for biomedical applications. However, NIR II-based imaging agents for targeted visualization of hepatocellular carcinoma (HCC) have barely been barely developed. Here, we report the construction of structurally uniform, biocompatible human serum albumin (HSA)-based particles orthogonally modified with two functional peptides as a carrier for the delivery of NIR-II imaging agents to HCC cell-derived solid tumor in vivo. Cysteine conjugation combined with host-guest chemistry enables the orthogonal introduction of two functionally independent peptides to HSA-based nanoparticles. One of these peptides targets glypican-3 (GPC-3), a specific biomarker of HCC, and the other facilitates the escape of the nanoparticles from macrophagic phagocytosis. Series of cellular and in vivo assays were carried out to demonstrate the efficacy of the dual-peptide-functionalized HSA nanoparticles for targeted NIR-II fluorescence imaging of HCC.

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

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. 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). 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. 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.

RUNX-3-expressing CAR T cells targeting glypican-3 in patients with heavily pretreated advanced hepatocellular carcinoma: a phase I trial

Glypican-3 (GPC3) is a well-characterized hepatocellular carcinoma (HCC)-associated antigen and a promising target for HCC treatment. CT017 CAR T cells were engineered to co-express CAR-GPC3 and runt-related transcription factor 3 (RUNX3), which triggers CD8+ T-cell infiltration into the cancer microenvironment. This single-center, single-arm, open-label, phase I clinical study enrolled heavily pretreated patients with GPC3-positive HCC between August 2019 and December 2020 (NCT03980288). Patients were treated with CT017 CAR T cells at a dose of 250×106cells. The primary objective was to assess the safety and tolerability of this first-in-human product. Six patients received 7 infusions (one patient received 2 infusions) at the 250×106cells dose. Three patients received CT017 monotherapy, and three patients received CT017-tyrosine kinase inhibitor (TKI) combination therapy at the first infusion. One patient received CT017-TKI combination therapy at the second infusion after CT017 monotherapy. All patients experienced cytokine release syndrome (CRS), with 50% (3/6) at Grade 2, 50% (3/6) at Grade 3, and all events resolved after treatment. No immune effector cell-associated neurotoxicity syndrome was observed. Dose escalation was not performed due to the investigator's decision regarding safety. Of six evaluable patients, one achieved partial response and two had stable disease for a 16.7% objective response rate, 50% disease control rate, 3.5-month median progression-free survival, 3.2-month median duration of disease control, and 7.9-month median overall survival (OS) with 7.87-month median follow-up. The longest OS was 18.2 months after CT017 infusion. Current preliminary phase I data showed a manageable safety profile and promising antitumor activities of CT017 for patients with advanced HCC. These results need to be confirmed in a robust clinical trial. This study was funded by CARsgen Therapeutics Co., Ltd.

GPC3 gene expression and allelic discrimination of FZD7 gene in Egyptian patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide, and especially in Egypt. Early diagnosis of HCC greatly improves the survival and prognosis of patients. Low sensitivity and specificity of alpha-fetoprotein (AFP) has led to the demand for novel biomarkers of HCC. The aim of the present study was to evaluate the validity of frizzled-7 (FZD7) and glypican-3 (GPC3) gene expression as potential biomarkers for HCC early diagnosis, and to investigate the association between FZD7 rs2280509 polymorphism and HCC risk. Quantification of FZD7 and GPC3 gene expression by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay, and genotyping FZD 7 (rs2280509 SNP) gene polymorphism using RT-PCR. The current results revealed that FZD7 gene expression had a greater area under the curve (AUC) for identifying HCC than GPC3 gene expression and AFP levels. The combination of the three markers as a panel showed a better diagnostic performance with a greater AUC than any of the single markers alone (p < 0.05). The FZD7 rs2280509 polymorphism (CT) was found to be significantly associated with an increased risk of HCC. The CT genotype and T allele were significantly more prevalent in the HCC group compared to either the cirrhosis (p = 0.03) or control groups (p = 0.0009 and 0.002; respectively). FZD7 and GPC3 gene expressions have a complementary role in early HCC detection, with a greater diagnostic sensitivity and accuracy than AFP. In addition, FZD7 rs2280509 polymorphism is significantly associated with an increased risk of HCC in the Egyptian population.

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

ABSTRACT 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.

Clinicopathological characteristics of gastric adenocarcinoma with enteroblastic differentiation and gastric adenocarcinoma with enteroblastic marker expression.

Gastric adenocarcinoma (GA) with enteroblastic differentiation (GAED) is an aggressive carcinoma histologically characterized by a glycogen-rich clear cytoplasm and fetal gut-like structures. GAED shows the expression of at least one of the following enteroblastic markers (EMs): glypican-3 (GPC3), spalt-like transcription factor 4 (SALL4), and α-fetoprotein (AFP). Despite the absence of clear cytoplasm, we often encounter GA with EMs expression (GA with EM); however, the clinicopathological characteristics of GA with EM remain unclear. Immunohistochemical (IHC) expression of three EMs (AFP, GPC3, and SALL4) was examined on tissue microarray. According to the status of the clear cytoplasm of tumor cells, GAs showing IHC expression of EMs were classified as either GAED or GA with EM, and this analysis categorized 688 GAs into 94 GAEDs (13.7%), 58 GAs with EM (8.4%), and 536 conventional GAs (CGAs). Both GAED and GA with EM showed frequent lymphovascular invasion, lymph node metastasis, and liver metastasis compared to CGA. However, a higher frequency of venous invasion, but not of lymphatic invasion, was noted for GAED in comparison to CGA. GAED and GA with EM showed similar overall survival. GAED had significantly poorer prognosis than CGA; however, not for GA with EM. Furthermore, GA showing EM expression had a worse prognosis than CGA. Interestingly, GA showing EM-positive group was more aggressive than CGA group as they had frequent venous invasion and liver metastasis despite its smaller tumor size. GAED and GA with EM can be clinically classified as aggressive tumors but pathologically they seem to be slightly different.

Delta-radiomics models based on multi-phase contrast-enhanced magnetic resonance imaging can preoperatively predict glypican-3-positive hepatocellular carcinoma.

Objectives: The aim of this study is to investigate the value of multi-phase contrast-enhanced magnetic resonance imaging (CE-MRI) based on the delta radiomics model for identifying glypican-3 (GPC3)-positive hepatocellular carcinoma (HCC). Methods: One hundred and twenty-six patients with pathologically confirmed HCC (training cohort: n = 88 and validation cohort: n = 38) were retrospectively recruited. Basic information was obtained from medical records. Preoperative multi-phase CE-MRI images were reviewed, and the 3D volumes of interest (VOIs) of the whole tumor were delineated on non-contrast T1-weighted imaging (T1), arterial phase (AP), portal venous phase (PVP), delayed phase (DP), and hepatobiliary phase (HBP). One hundred and seven original radiomics features were extracted from each phase, and delta-radiomics features were calculated. After a two-step feature selection strategy, radiomics models were built using two classification algorithms. A nomogram was constructed by combining the best radiomics model and clinical risk factors. Results: Serum alpha-fetoprotein (AFP) (p = 0.013) was significantly related to GPC3-positive HCC. The optimal radiomics model is composed of eight delta-radiomics features with the AUC of 0.805 and 0.857 in the training and validation cohorts, respectively. The nomogram integrated the radiomics score, and AFP performed excellently (training cohort: AUC = 0.844 and validation cohort: AUC = 0.862). The calibration curve showed good agreement between the nomogram-predicted probabilities and GPC3 actual expression in both training and validation cohorts. Decision curve analysis further demonstrates the clinical practicality of the nomogram. Conclusion: Multi-phase CE-MRI based on the delta-radiomics model can non-invasively predict GPC3-positive HCC and can be a useful method for individualized diagnosis and treatment.