Disruption of TGF-ß signaling pathway is required to mediate effective killing of hepatocellular carcinoma by human iPSC-derived NK cells.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Transforming growth factor beta (TGF-ß) is highly expressed in the liver tumor microenvironment and is known to inhibit immune cell activity. Here, we used human induced pluripotent stem cells (iPSCs) to produce natural killer (NK) cells engineered to mediate improved anti-HCC activity. Specifically, we produced iPSC-NK cells with either knockout TGF-ß receptor 2 (TGFBR2-KO) or expression of a dominant negative (DN) form of the TGF-ß receptor 2 (TGFBR2-DN) combined with chimeric antigen receptors (CARs) that target either GPC3 or AFP. The TGFBR2-KO and TGFBR2-DN iPSC-NK cells are resistant to TGF-ß inhibition and improved anti-HCC activity. However, expression of anti-HCC CARs on iPSC-NK cells did not lead to effective anti-HCC activity unless there was also inhibition of TGF-ß activity. Our findings demonstrate that TGF-ß signaling blockade is required for effective NK cell function against HCC and potentially other malignancies that express high levels of TGF-ß.

Ultrasound-Responsive Nanodelivery System of GPC3-Targeting and Sonosensitizer for Visualized Hepatocellular Carcinoma Therapy.

Purpose: The incidence of hepatocellular carcinoma (HCC) is continuously increasing, and the mortality rate remains high. Thus, more effective strategies are needed to improve the treatment of HCC. Methods: In this study, we report the use of a visualized glypican-3 (GPC3)-targeting nanodelivery system (named GC-NBs) in combination with sonodynamic therapy (SDT) to enhance the therapeutic efficacy for treating HCC. The obtained nanodelivery system could actively target hepatocellular carcinoma cells and achieve ultrasound imaging through phase changes into nanobubbles under low-intensity ultrasound irradiation. Meanwhile, the released chlorine e6 (Ce6) after the nanobubbles collapse could lead to the generation of reactive oxygen species (ROS) under ultrasound irradiation to induce SDT. Results: Both in vitro and in vivo experiments have shown that GC-NBs can accumulate in tumour areas and achieve sonodynamic antitumour therapy under the navigation action of glypican-3-antibody (GPC3-Ab). Furthermore, in vitro and in vivo experiments did not show significant biological toxicity of the nanodelivery system. Moreover, GC-NBs can be imaged with ultrasound, providing personalized treatment monitoring. Conclusion: GC-NBs enable a visualized antitumour strategy from a targeted sonodynamic perspective by combining tumour-specific targeting and stimuli-responsive controlled release into a single system.

Pilot study of humanized glypican-3-targeted zirconium-89 immuno-positron emission tomography for hepatocellular carcinoma.

Purpose: Glypican-3 (GPC3)-targeted radioisotope immuno-positron emission tomography (immunoPET) may lead to earlier and more accurate diagnosis of hepatocellular carcinoma (HCC), thus facilitating curative treatment, decreasing early recurrence, and enhancing patient survival. We previously demonstrated reliable HCC detection using a zirconium-89-labeled murine anti-GPC3 antibody (89Zr-αGPC3M) for immunoPET. This study evaluated the efficacy of the humanized antibody successor (αGPC3H) to further clinical translation of a GPC3-based theranostic for HCC. Methods: In vitro αGPC3 binding to HepG2 cells was assessed by flow cytometry. In vivo 89Zr-αGPC3H and 89Zr-αGPC3M tumor uptake was evaluated by PET/CT and biodistribution studies in an orthotopic xenograft mouse model of HCC. Results: αGPC3H maintained binding to GPC3 in vitro and 89Zr-αGPC3H immunoPET identified liver tumors in vivo. PET/CT and biodistribution analyses demonstrated high 89Zr-αGPC3H tumor uptake and tumor-to-liver ratios, with no difference between groups. Conclusion: Humanized αGPC3 successfully targeted GPC3 in vitro and in vivo. 89Zr-αGPC3H immunoPET had comparable tumor detection to 89Zr-αGPC3M, with highly specific tumor uptake, making it a promising strategy to improve HCC detection.

[Preparation of bacterial outer membrane vesicles with anti-GPC3 single-chain antibody and identification of their targeting effects on hepatocellular carcinoma].

This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti-glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo. The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD600=0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 ℃. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs (P=0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.

Prediction of glypican-3 expression in hepatocellular carcinoma using multisequence magnetic resonance imaging-based histology nomograms.

Background: Hepatocellular carcinoma (HCC) is often associated with the overexpression of multiple proteins and genes. For instance, patients with HCC and a high expression of the glypican-3 (GPC3) gene have a poor prognosis, and noninvasive assessment of GPC3 expression before surgery is helpful for clinical decision-making. Therefore, our primary aim in this study was to develop and validate multisequence magnetic resonance imaging (MRI) radiomics nomograms for predicting the expression of GPC3 in individuals diagnosed with HCC. Methods: We conducted a retrospective analysis of 143 patients with HCC, including 123 cases from our hospital and 20 cases from The Cancer Genome Atlas (TCGA) or The Cancer Imaging Archive (TCIA) public databases. We used preoperative multisequence MRI images of the patients for the radiomics analysis. We extracted and screened the imaging histologic features using fivefold cross-validation, Pearson correlation coefficient, and the least absolute shrinkage and selection operator (LASSO) analysis method. We used logistic regression (LR) to construct a radiomics model, developed nomograms based on the radiomics scores and clinical parameters, and evaluated the predictive performance of the nomograms using receiver operating characteristic (ROC) curves, calibration curves, and decision curves. Results: Our multivariate analysis results revealed that tumor morphology (P=0.015) and microvascular (P=0.007) infiltration could serve as independent predictors of GPC3 expression in patients with HCC. The nomograms integrating multisequence radiomics radiomics score, tumor morphology, and microvascular invasion had an area under the curve (AUC) value of 0.989. This approach was superior to both the radiomics model (AUC 0.979) and the clinical model (AUC 0.793). The sensitivity, specificity, and accuracy of 0.944, 0.800, and 0.913 for the test set, respectively, and the model's calibration curve demonstrated good consistency (Brier score =0.029). The decision curve analysis (DCA) indicated that the nomogram had a higher net clinical benefit for predicting the expression of GPC3. External validation of the model's prediction yielded an AUC value of 0.826. Conclusions: Our study findings highlight the close association of multisequence MRI imaging and radiomic features with GPC3 expression. Incorporating clinical parameters into nomograms can offer valuable preoperative insights into tailoring personalized treatment plans for patients diagnosed with HCC.

Two Cases of Gastric Adenocarcinoma with Enteroblastic Differentiation which Demonstrated Rapid Progressive Courses.

We herein report two extremely rare cases of gastric adenocarcinoma with enteroblastic differentiation (GAED) that underscore the aggressive nature of GAED. Case 1: ESD was scheduled for early-stage gastric cancer, however, the tumor increased in size drastically and the morphology changed to type "0-I + IIc" in one month. Surgery was performed and the patient was diagnosed with GAED. Case 2: ESD was performed for early-stage gastric cancer, and the pathological findings revealed GAED. The horizontal margin was positive for clear cells in the muscularis mucosa. Additional surgery was performed; however, recurrence occurred one year later. Therefore, the treatment strategies should be carefully considered for GAED.

Development of GPC3-CAR-NK cells and optimization as a therapy for HCC.

Hepatocellular carcinoma (HCC) is a highly malignant tumor characterized by insidious onset and rapid progression, with limited treatment choices. One treatment modality, chimeric antigen receptor (CAR)-modified natural killer (NK) cell immunotherapy, has shown promise for various cancers. In this study, we developed two GPC3-specific CAR-NK-92 cell lines (GPC3-CAR-NK) and explored their antitumor efficacy for the treatment of HCC. Significant levels of cytokine production and in vitro cytotoxicity were produced following co-culture of GPC3+ HCC cells with the developed GPC3-CAR-NK cells. GC33-G2D-NK cells with NK cell-specific signaling domains showed better activation and killing abilities than GC33-CD28-NK cells containing T cell-specific signaling domains. Moreover, GC33-G2D-NK cells efficiently eliminated tumors in cell-derived xenograft and patient-derived xenograft mouse models. In an abdominal metastasis model, intraperitoneally delivered GC33-G2D-NK cells showed better antitumor ability than intravenously injected cells. Finally, the combination of microwave ablation with GC33-G2D-NK cell administration showed greater CAR-NK infiltration and tumor regression in ablated tumors than monotherapy alone. These findings indicate that administration of GPC3-CAR-NK cells may be a potential strategy for the treatment of HCC, and regional delivery or their combination with microwave ablation may optimize their efficacy against HCC and may have translational value.

Fetal gut cell-like differentiation in esophageal adenocarcinoma defines a rare tumor subtype with therapeutically relevant claudin-6 positivity and SWI/SNF gene alteration.

Esophageal adenocarcinoma (EAC) is one of the deadliest tumor entities worldwide, with a 5-year survival rate of less than 25%. Unlike other tumor entities, personalized therapy options are rare, partly due to the lack of knowledge about specific subgroups. In this publication, we demonstrate a subgroup of patients with EAC in a large screening cohort of 826 patients, characterized by specific morphological and immunohistochemical features. This subgroup represents approximately 0.7% (6/826) of the total cohort. Morphological features of this subgroup show a striking clear cytoplasm of the tumour cells and the parallel existence of rare growth patterns like yolk sac-like differentiation and enteroblastic differentiation. Immunohistochemistry reveals expression of the fetal gut cell-like proteins Sal-like protein 4 (SALL4), claudin-6, and glypican 3. Interestingly, we find a correlation with alterations of SWI/SNF-complex associated genes, which are supposed to serve as tumor suppressor genes in various tumour entities. Our results suggest a possible implication of rare tumour subtypes in the WHO classification for EACs according to the classification for gastric cancer. Furthermore, claudin-6 positive tumors have shown promising efficacy of CAR T cell therapy in the recently published BNT-211-01 trial (NCT04503278). This represents a personalized therapeutic option for this tumor subtype.

A dual-signal output electrochemical aptasensor for glypican-3 ultrasensitive detection based on reduced graphene oxide-cuprous oxide nanozyme catalytic amplification strategy.

Glypican-3 (GPC3) is an essential reference target for hepatocellular carcinoma detection, follow-up and prediction. Herein, a dual-signal electrochemical aptasensor based on reduced graphene oxide-cuprous oxide (RGO-Cu2O) nanozyme was developed for GPC3 detection. The RGO-Cu2O nanoenzyme displayed excellent electron transport effect, large specific surface area and outstanding peroxidase-like ability. The differential pulse voltammetry (DPV) signal of Cu2O oxidation fraction and the chronoamperometry (i-t) signal of H2O2 decomposition catalyzed by RGO-Cu2O nanozyme were used as dual-signal detection. Under optimal conditions, the log-linear response ranges were 0.1 to 500.0 ng/mL with the limit of detection 0.064 ng/mL for DPV technique, and 0.1-50.0 ng/mL for i-t technique (detection limit of 0.0177 ng/mL). The electrochemical aptasensor has remarkably analytical performance with wide response range, low detection limit, excellent repeatability and specificity, good recovery in human serum samples. The two output signals of one sample achieve self-calibration of the results, effectively avoiding the occurrence of possible leakage and misdiagnosis of a single detection signal, suggesting that it will be a promising method in the early biomarker detection.

Extraction-free, immuno-RPA-CRISPR/Cas13a-based one-pot detection of glypican-3 directly from extracellular vesicles.

BACKGROUND: Glypican-3 (GPC3) is a heparan sulfate proteoglycan (HSPG) that binds to the cell membrane via glycosylphosphatidylinositol (GPI). It is not found in healthy adult liver but is overexpressed in human hepatocellular carcinoma (HCC). The protein marker GPC3 on extracellular vesicles (GPC3+ EVs) is also useful for HCC detection. Nevertheless, the absence of practical and dependable quantitative techniques to evaluate EVs proteins prevents their clinical implementation. RESULTS: Here, using an immuno-recombinase polymerase amplification (immuno-RPA) process and dual amplification of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, we firstly create an extraction-free one-pot immuno-RPA-CRISPR (opiCRISPR) for the direct and extremely sensitive detection of EVs proteins. The EVs protein-targeted detection probe is amplified by RPA to generate a long repetitive sequence containing multiple CRISPR RNA (crRNA) targeting barcodes, and the signal is further amplified by the CRISPR-Cas13a side-chain cleavage activity to generate a fluorescent signal. The results show that circulating extracellular vesicle GPC3 (eGPC3) levels are a reliable marker for GPC3 expression in tumor, opening up new avenues for tumor diagnosis. SIGNIFICANCE AND NOVELTY: We created an eGPC3 assay based on the CRISPR-Cas13a system, and successfully study the significance of extracellular vesicle GPC3 markers in hepatocellular carcinoma.

Combination of serum alpha-fetoprotein, PIVKA-â ¡ and glypican-3 in diagnosis of hepatocellular carcinoma: a meta-analysis. / è¡€æ¸ å­¦æ ‡å¿—ç‰©ç”²èƒŽè›‹ç™½ã€PIVKA-â ¡å’Œç£·è„‚é °è‚Œé†‡è›‹ç™½èšç³–3联合诊断肝癌的meta分析.

OBJECTIVES: 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). METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

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

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

Smart glypican-3-targeting peptide-chlorin e6 conjugates for targeted photodynamic therapy of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly aggressive and lethal malignancy with poor prognosis, necessitating the urgent development of effective treatments. Targeted photodynamic therapy (PDT) offers a promising way to selectively eradicate tumor cells without affecting normal cells. Inspired by promising features of peptide-drug conjugates (PDCs) in targeted cancer therapy, herein a novel glypican-3 (GPC3)-targeting PDC-PDT strategy was developed for the precise PDT treatment of HCC. The GPC3-targeting photosensitizer conjugates were developed by attaching GPC3-targeting peptides to chlorin e6. Conjugate 8b demonstrated the ability to penetrate HCC cells via GPC3-mediated entry process, exhibiting remarkable tumor-targeting capacity, superior antitumor efficacy, and minimal toxicity towards normal cells. Notably, conjugate 8b achieved complete tumor elimination upon light illumination in a HepG2 xenograft model without harm to normal tissues. Overall, this innovative GPC3-targeting conjugation strategy demonstrates considerable promise for clinical applications for the treatment of HCC.

Colostomy-site carcinoma with primitive phenotype in a rectal cancer patient after achieving pathological complete response with neoadjuvant chemoradiotherapy.

Herein, we report a rare case of a carcinoma with primitive phenotype (enteroblastic and/or hepatoid differentiation) occurring at a colostomy site. The patient was an elderly male who underwent neoadjuvant chemoradiotherapy for rectal cancer, followed by abdominoperineal resection. A biopsy specimen for the rectal carcinoma before neoadjuvant chemoradiotherapy was conventional tubular adenocarcinoma. Moreover, a pathological complete response was confirmed in the proctectomy specimen. However, a colostomy-site tumor appeared 6 months after the proctectomy, and it was resected 1 year after the initial proctectomy. The colostomy-site tumor comprised solid to focal glandular growth of atypical polygonal cells with clear to pale eosinophilic cytoplasm and was immunohistochemically positive for cytokeratin, spalt-like transcription factor 4, glypican-3, caudal type homeobox 2, and special AT-rich sequence-binding protein 2. Thus, the tumor was diagnosed as poorly differentiated adenocarcinoma with primitive phenotype, with suggested origin from the colorectal epithelium. Additionally, a multilocular cystic lesion comprising various types of epithelia was found adjacent to the tumor, suggestive of metaplasia or heterotopia. Changes in the histology and immunophenotype, and the findings of an adjacent cystic lesion suggest a metachronous tumor rather than a recurrence of the primary tumor.

A role for decorin in improving motor deficits after traumatic brain injury.

Traumatic brain injury (TBI) is the leading cause of death and disability due to injury worldwide. Extracellular matrix (ECM) remodeling is known to significantly contribute to TBI pathophysiology. Glycosaminoglycans, which are long-chain, variably sulfated polysaccharides abundant within the ECM, have previously been shown to be substantially altered after TBI. In this study, we sought to delineate the dynamics of glycosaminoglycan alterations after TBI and discover the precise biologic processes responsible for observed glycosaminoglycan changes after injury. We performed state-of-the art mass spectrometry on brain tissues isolated from mice after TBI or craniotomy-alone. We observed dynamic changes in glycosaminoglycans at Day 1 and 7 post-TBI, with heparan sulfate, chondroitin sulfate, and hyaluronan remaining significantly increased after a week vis-à-vis craniotomy-alone tissues. We did not observe appreciable changes in circulating glycosaminoglycans in mice after experimental TBI compared to craniotomy-alone nor in patients with TBI and severe polytrauma compared to control patients with mild injuries, suggesting increases in injury site glycosaminoglycans are driven by local synthesis. We subsequently performed an unbiased whole genome transcriptomics analysis on mouse brain tissues 7 days post-TBI and discovered a significant induction of hyaluronan synthase 2, glypican-3, and decorin. The functional role of decorin after injury was further examined through multimodal behavioral testing comparing wild-type and Dcn-/- mice. We discovered that genetic ablation of Dcn led to an overall negative effect of TBI on function, exacerbating motor impairments after TBI. Collectively, our results provide a spatiotemporal characterization of post-TBI glycosaminoglycan alterations in the brain ECM and support an important adaptive role for decorin upregulation after TBI.

A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis.

Introduction: Extracellular vesicles could serve as a non-invasive biomarker for early cancer detection. However, limited methods to quantitate cancer-derived vesicles in the native state remain a significant barrier to clinical translation. Aim: This research aims to develop a rapid, one-step immunoaffinity approach to quantify HCC exosomes directly from a small serum volume. Methods: HCC-derived exosomes in the serum were captured using fluorescent phycoerythrin (PE)-conjugated antibodies targeted to GPC3 and alpha-fetoprotein (AFP). Total and HCC-specific exosomes were then quantified in culture supernatant or patient-derived serums using fluorescence nanoparticle tracking analysis (F-NTA). The performance of HCC exosome quantification in the serum was compared with the tumor size determined by MRI. Results: Initially we tested the detection limits of the F-NTA using synthetic fluorescent and non-fluorescent beads. The assay showed an acceptable sensitivity with a detection range of 104-108 particles/mL. Additionally, the combination of immunocapture followed by size-exclusion column purification allows the isolation of smaller-size EVs and quantification by F-NTA. Our assay demonstrated that HCC cell culture releases a significantly higher quantity of GPC3 or GPC3+AFP positive EVs (100-200 particles/cell) compared to non-HCC culture (10-40 particles/cell) (p<0.01 and p<0.05 respectively). The F-NTA enables absolute counting of HCC-specific exosomes in the clinical samples with preserved biological immunoreactivity. The performance of F-NTA was clinically validated in serum from patients ± cirrhosis and with confirmed HCC. F-NTA quantification data show selective enrichment of AFP and GPC3 positive EVs in HCC serum compared to malignancy-free cirrhosis (AUC values for GPC3, AFP, and GPC3/AFP were found 0.79, 0.71, and 0.72 respectively). The MRI-confirmed patient cohort indicated that there was a positive correlation between total tumor size and GPC3-positive exosome concentration (r:0.78 and p<0.001). Conclusion: We developed an immunocapture assay that can be used for simultaneous isolation and quantification of HCC-derived exosomes from a small serum volume with high accuracy.

The expression of Ki-67 and Glypican -3 in hepatocellular carcinoma was evaluated by comparing DWI and 18F-FDG PET/CT.

Objective: The value of DWI and 18F-FDG PET/CT in evaluating the expression of Ki-67 and GPC-3 in HCC was compared. Materials and methods: Ninety-four patients with primary HCC confirmed by pathology were retrospectively divided into high- and low-Ki-67-expression groups and positive- and negative- GPC-3 groups. The ADC and SUVmax values of the lesions in both groups were measured. ROC curves were used to evaluate the identification efficiency of parameters with significant differences for each group of lesions, and AUCwas calculated. The combined ADC and SUVmax values were analyzed by binary logistic regression. The Delong test was used to compare the AUC values of the combined and single parameters. Pearson (in line with normal distribution) or Spearman (in line with abnormal distribution) correlation analysis was used to analyze the correlation. Results: The ADC value of the high-Ki-67-expression group was lower than that of the low-Ki-67-expression group (P<0.05), and the SUVmax value of the high-expression group was higher than that of the low-expression group (P<0.05). The ADC value of the positive-GPC-3 group was lower than that of the negative group (P<0.0.tive group (P<0.05). The combined ADC and SUVmax values in the GPC-3 group were better than those of a single parameter (P<0.05). There was a strong negative correlation between the SUVmax value and ADC value in the Ki-67 group (R=-0.578, P<0.001) and a weak negative correlation between the SUVmax value and ADC value in the GPC-3 group (R=-0.279, P=0.006). The SUVmax value was strongly positively correlated with the Ki-67 expression index (R=0.733, P<0.001), while the ADC value was strongly negatively correlated with the Ki-67 expression index (R=-0.687, P<0.001). Conclusion: DWI and 18F-FDG PET/CT can be used to evaluate the expression of Ki-67 and GPC-3 in HCC, and there is a certain correlation between the ADC value and SUVmax. Combined DWI and 18F-FDG PET/CT is superior to a single technique in evaluating the expression of GPC-3 in HCC patients. However, the combined model did not benefit the Ki-67 group.

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

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

Multiparametric MRI combined with clinical factors to predict glypican-3 expression of hepatocellular carcinoma.

Objectives: The present study aims at establishing a noninvasive and reliable model for the preoperative prediction of glypican 3 (GPC3)-positive hepatocellular carcinoma (HCC) based on multiparametric magnetic resonance imaging (MRI) and clinical indicators. Methods: As a retrospective study, the subjects included 158 patients from two institutions with surgically-confirmed single HCC who underwent preoperative MRI between 2020 and 2022. The patients, 102 from institution I and 56 from institution II, were assigned to the training and the validation sets, respectively. The association of the clinic-radiological variables with the GPC3 expression was investigated through performing univariable and multivariable logistic regression (LR) analyses. The synthetic minority over-sampling technique (SMOTE) was used to balance the minority group (GPC3-negative HCCs) in the training set, and diagnostic performance was assessed by the area under the curve (AUC) and accuracy. Next, a prediction nomogram was developed and validated for patients with GPC3-positive HCC. The performance of the nomogram was evaluated through examining its calibration and clinical utility. Results: Based on the results obtained from multivariable analyses, alpha-fetoprotein levels > 20 ng/mL, 75th percentile ADC value < 1.48 ×103 mm2/s and R2* value ≥ 38.6 sec-1 were found to be the significant independent predictors of GPC3-positive HCC. The SMOTE-LR model based on three features achieved the best predictive performance in the training (AUC, 0.909; accuracy, 83.7%) and validation sets (AUC, 0.829; accuracy, 82.1%) with a good calibration performance and clinical usefulness. Conclusions: The nomogram combining multiparametric MRI and clinical indicators is found to have satisfactory predictive efficacy for preoperative prediction of GPC3-positive HCC. Accordingly, the proposed method can promote individualized risk stratification and further treatment decisions of HCC patients.

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

BACKGROUND: 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. METHODS: 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 (500 pg/mL) and CCL19 at a high level (15000 pg /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. RESULTS: 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 56 days after intravenous infusion. CONCLUSIONS: 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.