Nitazoxanide protects against experimental ulcerative colitis through improving intestinal barrier and inhibiting inflammation.

Ulcerative colitis is a chronic disease with colonic mucosa injury. Nitazoxanide is an antiprotozoal drug in clinic. Nitazoxanide and its metabolite tizoxanide have been demonstrated to activate AMPK and inhibit inflammation, therefore, the aim of the present study is to investigate the effect of nitazoxanide on dextran sulfate sodium (DSS)-induced colitis and the underlying mechanism. Oral administration of nitazoxanide ameliorated the symptoms of mice with DSS-induced colitis, as evidenced by improving the increased disease activity index (DAI), the decreased body weight, and the shortened colon length. Oral administration of nitazoxanide ameliorated DSS-induced intestinal barrier dysfunction and reduced IL-6 and IL-17 expression in colon tissues. Mechanistically, nitazoxanide and its metabolite tizoxanide treatment activated AMPK and inhibited JAK2/STAT3 signals. Nitazoxanide and tizoxanide treatment increased caudal type homeobox 2 (CDX2) expression, increased alkaline phosphatase (ALP) activity and promoted tight junctions in Caco-2 cells. Nitazoxanide and tizoxanide treatment restored the decreased zonula occludens-1(ZO-1) and occludin protein levels induced by LPS or IL-6 in Caco-2 cells. On the other hand, nitazoxanide and tizoxanide regulated macrophage bias toward M2 polarization, as evidenced by the increased arginase-1expression in bone marrow-derived macrophages (BMDM). Nitazoxanide and tizoxanide reduced the increased IL-6, iNOS and CCL2 pro-inflammatory gene expressions and inhibited JAK2/STAT3 activation in BMDM induced by LPS. In conclusion, nitazoxanide protects against DSS-induced ulcerative colitis in mice through improving intestinal barrier and inhibiting inflammation and the underlying mechanism involves AMPK activation and JAK2/STAT3 inhibition.

Stimuli-Responsive Polymer-Based Nanosystems for Cancer Theranostics.

Stimuli-responsive polymers can respond to internal stimuli, such as reactive oxygen species (ROS), glutathione (GSH), and pH, biological stimuli, such as enzymes, and external stimuli, such as lasers and ultrasound, etc., by changing their hydrophobicity/hydrophilicity, degradability, ionizability, etc., and thus have been widely used in biomedical applications. Due to the characteristics of the tumor microenvironment (TME), stimuli-responsive polymers that cater specifically to the TME have been extensively used to prepare smart nanovehicles for the targeted delivery of therapeutic and diagnostic agents to tumor tissues. Compared to conventional drug delivery nanosystems, TME-responsive nanosystems have many advantages, such as high sensitivity, broad applicability among different tumors, functional versatility, and improved biosafety. In recent years, a great deal of research has been devoted to engineering efficient stimuli-responsive polymeric nanosystems, and significant improvement has been made to both cancer diagnosis and therapy. In this review, we summarize some recent research advances involving the use of stimuli-responsive polymer nanocarriers in drug delivery, tumor imaging, therapy, and theranostics. Various chemical stimuli will be described in the context of stimuli-responsive nanosystems. Accordingly, the functional chemical groups responsible for the responsiveness and the strategies to incorporate these groups into the polymer will be discussed in detail. With the research on this topic expending at a fast pace, some innovative concepts, such as sequential and cascade drug release, NIR-II imaging, and multifunctional formulations, have emerged as popular strategies for enhanced performance, which will also be included here with up-to-date illustrations. We hope that this review will offer valuable insights for the selection and optimization of stimuli-responsive polymers to help accelerate their future applications in cancer diagnosis and treatment.

Novel Therapeutic Strategies Exploiting the Unique Properties of Neuroendocrine Neoplasms.

BACKGROUND: Over the last few decades of treatment, the outcomes for at least some subsets of neuroendocrine neoplasms (NENs) have improved. However, the identification of new vulnerabilities for this heterogeneous group of cancers remains a priority. METHODS: Using two libraries of compounds selected for potential repurposing, we identified the inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) and histone deacetylases (HDAC) as the agents with the highest activity. We validated the hits in an expanded set of neuroendocrine cell lines and examined the mechanisms of action. RESULTS: In Kelly, NH-6, and NCI-H82, which are two neuroblastoma and one small cell lung cancer cell lines, respectively, metabolic studies suggested that cell death following NAMPT inhibition is the result of a reduction in basal oxidative phosphorylation and energy production. NAMPT is the rate-limiting enzyme in the production of NAD+, and in the three cell lines, NAMPT inhibition led to a marked reduction in the ATP and NAD+ levels and the catalytic activity of the citric acid cycle. Moreover, comparative analysis of the mRNA expression in drug-sensitive and -insensitive cell lines found less dependency of the latter on oxidative phosphorylation for their energy requirement. Further, the analysis of HDAC and NAMPT inhibitors administered in combination found marked activity using low sub-lethal concentrations of both agents, suggesting a synergistic effect. CONCLUSION: These data suggest NAMPT inhibitors alone or in combination with HDAC inhibitors could be particularly effective in the treatment of neuroendocrine neoplasms.

International Association for the Study of Lung Cancer Study of Reproducibility in Assessment of Pathologic Response in Resected Lung Cancers After Neoadjuvant Therapy.

INTRODUCTION: Pathologic response has been proposed as an early clinical trial end point of survival after neoadjuvant treatment in clinical trials of NSCLC. The International Association for the Study of Lung Cancer (IASLC) published recommendations for pathologic evaluation of resected lung cancers after neoadjuvant therapy. The aim of this study was to assess pathologic response interobserver reproducibility using IASLC criteria. METHODS: An international panel of 11 pulmonary pathologists reviewed hematoxylin and eosin-stained slides from the lung tumors of resected NSCLC from 84 patients who received neoadjuvant immune checkpoint inhibitors in six clinical trials. Pathologic response was assessed for percent viable tumor, necrosis, and stroma. For each slide, tumor bed area was measured microscopically, and pre-embedded formulas calculated unweighted and weighted major pathologic response (MPR) averages to reflect variable tumor bed proportion. RESULTS: Unanimous agreement among pathologists for MPR was observed in 68 patients (81%), and inter-rater agreement (IRA) was 0.84 (95% confidence interval [CI]: 0.76-0.92) and 0.86 (95% CI: 0.79-0.93) for unweighted and weighted averages, respectively. Overall, unweighted and weighted methods did not reveal significant differences in the classification of MPR. The highest concordance by both methods was observed for cases with more than 95% viable tumor (IRA = 0.98, 95% CI: 0.96-1) and 0% viable tumor (IRA = 0.94, 95% CI: 0.89-0.98). The most common reasons for discrepancies included interpretations of tumor bed, presence of prominent stromal inflammation, distinction between reactive and neoplastic pneumocytes, and assessment of invasive mucinous adenocarcinoma. CONCLUSIONS: Our study revealed excellent reliability in cases with no residual viable tumor and good reliability for MPR with the IASLC recommended less than or equal to 10% cutoff for viable tumor after neoadjuvant therapy.

Boron supply restores aluminum-blocked auxin transport by the modulation of PIN2 trafficking in the root apical transition zone.

The supply of boron (B) alleviates the toxic effects of aluminum (Al) on root growth; however, the mechanistic basis of this process remains elusive. This study filled this knowledge gap, demonstrating that boron modifies auxin distribution and transport in Al-exposed Arabidopsis roots. In B-deprived roots, treatment with Al induced an increase in auxin content in the root apical meristem zone (MZ) and transition zone (TZ), whereas in the elongation zone (EZ) the auxin content was decreased beyond the level required for adequate growth. These distribution patterns are explained by the fact that basipetal auxin transport from the TZ to the EZ was disrupted by Al-inhibited PIN-FORMED 2 (PIN2) endocytosis. Experiments involving the modulation of protein biosynthesis by cycloheximide (CHX) and transcriptional regulation by cordycepin (COR) demonstrated that the Al-induced increase of PIN2 membrane proteins was dependent upon the inhibition of PIN2 endocytosis, rather than on the transcriptional regulation of the PIN2 gene. Experiments reporting on the profiling of Al3+ and PIN2 proteins revealed that the inhibition of endocytosis of PIN2 proteins was the result of Al-induced limitation of the fluidity of the plasma membrane. The supply of B mediated the turnover of PIN2 endosomes conjugated with indole-3-acetic acid (IAA), and thus restored the Al-induced inhibition of IAA transport through the TZ to the EZ. Overall, the reported results demonstrate that boron supply mediates PIN2 endosome-based auxin transport to alleviate Al toxicity in plant roots.

Identification of heterochirality-mediated stereochemical interactions in peptide architectures.

In this work, we illustrate a strategy for constructing heterochiral peptide architectures with distinct structural, mechanical and thermal characteristics. A series of nanotube structures based on diphenylalanine (FF) and its chiral derivatives were examined. Pronounced effects relating to heterochirality on mechanostability and thermal stability can be identified. The homochiral peptide FF and its enantiomer ff formed nanotubes with high thermal and mechanical stabilities (Young's modulus: 20.3 ± 5.9 GPa for FF and 21.2 ± 4.7 GPa for ff). In contrast, heterochiral nanotubes formed by Ff and fF manifest superstructures along the axial direction with differed thermal and mechanical strength (Young's modulus: 7.3 ± 2.4 GPa for Ff and 8.3 ± 2.1 GPa for fF). Combining their single-crystal XRD structure and in silico results, it was demonstrated that the spatial orientations of aromatic moieties were subtly changed by heterochirality of peptide building blocks, which led to intramolecular face-to-face interactions. As the result, both intermolecular axial and interchannel interactions in heterochiral nanotubes were weakened as reflected in the strikingly deteriorated mechanical and thermal stabilities. Conversely, two aromatic side chains of the homochiral peptides were staggered and formed interdigitated steric zippers, which served as strong glues that secured the robustness of nanotubes in both axial and radial orientation. Furthermore, the generality of the heterochiral-mediated stereochemical effects was demonstrated in other "FF class" dipeptides, including fluorinated Ff, FW and FL. Our results unequivocally revealed the relationship between amino acid chirality, peptide molecule packing, and physical stabilities of "FF class" dipeptide self-assembled materials and provide valuable molecular insights into chirality-mediated stereochemical interactions in determining the properties of peptide architectures.

Preferential Gs protein coupling of the galanin Gal1 receptor in the µ-opioid-Gal1 receptor heterotetramer.

Recent studies have proposed that heteromers of µ-opioid receptors (MORs) and galanin Gal1 receptors (Gal1Rs) localized in the mesencephalon mediate the dopaminergic effects of opioids. The present study reports converging evidence, using a peptide-interfering approach combined with biophysical and biochemical techniques, including total internal reflection fluorescence microscopy, for a predominant homodimeric structure of MOR and Gal1R when expressed individually, and for their preference to form functional heterotetramers when co-expressed. Results show that a heteromerization-dependent change in the Gal1R homodimeric interface leads to a switch in G-protein coupling from inhibitory Gi to stimulatory Gs proteins. The MOR-Gal1R heterotetramer, which is thus bound to Gs via the Gal1R homodimer and Gi via the MOR homodimer, provides the framework for a canonical Gs-Gi antagonist interaction at the adenylyl cyclase level. These novel results shed light on the intense debate about the oligomeric quaternary structure of G protein-coupled receptors, their predilection for heteromer formation, and the resulting functional significance.

Glycosylation increases the anti-QS as well as anti-biofilm and anti-adhesion ability of the cyclo (L-Trp-L-Ser) against Pseudomonas aeruginosa.

Pseudomonas aeruginosa infections pose a huge threat to cystic fibrosis patients, as well as those suffering from immunodeficiency. Antimicrobial resistance, especially multi-drug resistance, due to its ability to aggregate the compact biofilm, makes it more inefficient to treat this pathogen with traditional antibiotics. Biofilm and quorum sensing (QS) have become the alternative targets for treating P. aeruginosa infections. Previously, a cyclic dipeptide cyclo(L-Trp-L-Ser) has been identified as a QS inhibitor of P. aeruginosa. On the other hand, some monosaccharides have been proved lectin-targeting behavior and to mediate biofilm formation and adhesion of P. aeruginosa. We constructed novel cyclic dipeptide-carbohydrate conjugates as a low molecular weight dual-functional QS inhibitor, which can not only enhance its anti-QS activity but also enable good anti-biofilm and anti-adhesion ability. The IC50 of galactosylated c(WS) on biofilm formation and glass adhesion was 1/6 and 1/4 of that of the unmodified cyclic dipeptide, respectively. And the ability to eliminate the preformed biofilm was increased 10-fold. Furthermore, the carbohydrate conjugates can increase the germicidal efficiency of clinical antibiotic azithromycin when used synergistically. Our results provide a novel scaffold for developing anti-virulence adjuvants when taken with clinical antibiotics.

GPC1-Targeted Immunotoxins Inhibit Pancreatic Tumor Growth in Mice via Depletion of Short-lived GPC1 and Downregulation of Wnt Signaling.

Glypican-1 (GPC1) is a cell surface proteoglycan that is upregulated in multiple types of human cancers including pancreatic cancer. Here, we investigated whether GPC1 could be a target of antibody-toxin fusion proteins (i.e., immunotoxins) for treating pancreatic cancer. We constructed a panel of GPC1-targeted immunotoxins derived from a functional domain of Pseudomonas exotoxin A. An albumin-binding domain was also introduced into the anti-GPC1 immunotoxin to improve serum half-life. Small-molecule screening was performed to identify irinotecan that shows synergistic efficacy with the immunotoxin. We showed that GPC1 was internalized upon antibody binding. Anti-GPC1 immunotoxins alone inhibited tumor growth in a pancreatic cancer xenograft model. The immunotoxin treatment reduced active ß-catenin expression in tumor cells. Furthermore, immunotoxins containing an albumin-binding domain in combination with irinotecan caused pancreatic tumor regression. GPC1 expression was reduced by the immunotoxin treatment due to the degradation of the internalized GPC1 and its short cellular turnover rate. Our data indicate that the GPC1-targeted immunotoxin inhibits pancreatic tumor growth via degradation of internalized GPC1, downregulation of Wnt signaling, and inhibition of protein synthesis. The anti-GPC1 immunotoxin in combination with irinotecan thus provides a potential new treatment strategy for patients with pancreatic tumors.

Heterochirality-Mediated Cross-Strand Nested Hydrophobic Interaction Effects Manifested in Surface-Bound Peptide Assembly Structures.

Amino acid chirality has been envisioned as an important strategy to regulate structure and function of peptide self-assembled architectures. However, the molecular mechanism of chirality effects in peptide assemblies remains largely elusive. Here, the assembly structures of l-peptide polyphenylalanine F10 (FFFFFFFFFF) and block heterochiral peptide F5f5 (FFFFFfffff) composed of two FFFFF repeat blocks with opposite chirality were characterized at the single-molecule level by using scanning tunneling microscopy. Each peptide formed two distinctively different assembly structures on the HOPG surface, in which peptide chains took parallel and antiparallel ß-sheet conformations, respectively. The molecular-level observations revealed that the staggered arrangement of cross-strand side chains achieved in the antiparallel ß-sheet structure of the block heterochiral peptide facilitated intimate packing of side chains and maximized inter-residue van der Waals interactions, which led to more residues participating in assembly and greatly stabilized the ß-sheet structure of the surface-bound peptide assembly, but such cross-strand nested interactions were not accessible in the heterochiral parallel ß-sheet structure and the enantiomerically pure assembly structures. This work could contribute to the molecular insights of stereochemical interactions in peptide assemblies and feasibility of extending this block heterochirality pattern to other peptides with various lengths and amino acid compositions for structural regulations.

Armochaetoglasins J and K: new cytochalasans from Chaetomium globosum.

Two novel cytochalasans, armochaetoglasin J (1) and armochaetoglasin K (2), along with 14 known analogues (3-16) were isolated from Chaetomium globosum. Their structures were elucidated by HRESIMS, NMR spectroscopy, single-crystal X-ray crystallography, and ECD spectra. Armochaetoglasins J and K were found to be inactive against the HepG2, HT-29, K562, HL-60, and A549 cancer cell lines.

Controllable synthesis of coral-like hierarchical porous magnesium hydroxide with various surface area and pore volume for lead and cadmium ion adsorption.

A coral-like hierarchical porous magnesium hydroxide (HPMH) with various surface area and pore volume was controllably prepared using a simple one-step hydrothermal process, for which MgO, water and citric acid were applied. The citric acid (CA), as a structure-directing molecule, is a key factor in regulating the pore structure of HPMH products. With different additive dosages, the nanostructure, surface area and pore volume of HPMH products can be controllably regulated. The MH-CA20 product (prepared in the presence of 20 wt% CA) with high BET surface area (159 m2/g) and pore volume (0.75 cm3/g) was used to investigate the adsorption properties for Pb(II) and Cd(II) ions. The experimental adsorption capabilities of the MH-CA20 for Pb(II) and Cd(II) are respectively 4535 and 3530 mgg-1, very close to the maximum adsorption capabilities calculated by Langmuir equation (4545 and 3571 mgg-1). According to the adsorption kinetics and adsorption isotherm data, the adsorption process conforms to the Pseudo-second-order and Langmuir model, indicating that heavy metal ions conduct monolayer chemical adsorption mechanism. Since the preparation of HPMH is simple, low-cost and filtrate recycling, the process can easily be scaled up and could be a good candidate for application in tackling different wastewater.

Human GPR17 missense variants identified in metabolic disease patients have distinct downstream signaling profiles.

GPR17 is a G-protein-coupled receptor (GPCR) implicated in the regulation of glucose metabolism and energy homeostasis. Such evidence is primarily drawn from mouse knockout studies and suggests GPR17 as a potential novel therapeutic target for the treatment of metabolic diseases. However, links between human GPR17 genetic variants, downstream cellular signaling, and metabolic diseases have yet to be reported. Here, we analyzed GPR17 coding sequences from control and disease cohorts consisting of individuals with adverse clinical metabolic deficits including severe insulin resistance, hypercholesterolemia, and obesity. We identified 18 nonsynonymous GPR17 variants, including eight variants that were exclusive to the disease cohort. We characterized the protein expression levels, membrane localization, and downstream signaling profiles of nine GPR17 variants (F43L, V96M, V103M, D105N, A131T, G136S, R248Q, R301H, and G354V). These nine GPR17 variants had similar protein expression and subcellular localization as wild-type GPR17; however, they showed diverse downstream signaling profiles. GPR17-G136S lost the capacity for agonist-mediated cAMP, Ca2+, and ß-arrestin signaling. GPR17-V96M retained cAMP inhibition similar to GPR17-WT, but showed impaired Ca2+ and ß-arrestin signaling. GPR17-D105N displayed impaired cAMP and Ca2+ signaling, but unaffected agonist-stimulated ß-arrestin recruitment. The identification and functional profiling of naturally occurring human GPR17 variants from individuals with metabolic diseases revealed receptor variants with diverse signaling profiles, including differential signaling perturbations that resulted in GPCR signaling bias. Our findings provide a framework for structure-function relationship studies of GPR17 signaling and metabolic disease.

One new spirocyclic lactone and one new benzopyran derivative from Aspergillus terreus.

One new spirocyclic lactone, terreinlactone C (1), and one new benzopyran derivative, 2,2-dimethyl-3-hydroxychroman-6-aldehyde (2), were discovered from the fungus Aspergillus terreus. The chemical structures of compounds 1 and 2 were elucidated by detailedly analyzing NMR and HRESIMS data. Compound 1 is the first natural product with a 1-oxaspiro[4.5]decan-2-one ring system and a possible biogenetic pathway is proposed. Two compounds were tested for their cytotoxic activities against five human cancer cell lines.[Formula: see text].

[Adsorption of Cadmium and Arsenic by Corn Stalk Biochar Solidified Microorganism].

Immobilization of bacteria on biochar can improve the performance of the soil complex polluted with cadmium (Cd) and arsenic (As). In this study, bacteria (Delftia sp. B9, B9), biochar (corn stalks biochar, CSB), and biochar-bacteria complexes (B-CSB) were used as adsorption materials to explore the adsorption characteristics of Cd and As. The effects of pH on the adsorption performance of Cd and As and the ion removal from the aqueous solution were investigated, and the adsorption behaviors were simulated using an isothermal adsorption model. The changes in Cd and As speciation with the addition of B9, CSB, and B-CSB to As and Cd-contaminated soil were explored. The results showed that the Cd-saturated adsorption capacities of B9, CSB, and B-CSB were 49.43, 82.68, and 75.38 mg ·g-1, respectively; the As-saturated adsorption capacities were 24.67, 42.92, and 34.03 mg ·g-1, respectively. The concentration of available Cd and As significantly decreased, whereas the residual fraction increased after the addition of B-CSB. B-CSB was shown to be an effective material for the remediation of soil complexes polluted with Cd and As.

Pyrazole-Based Lactate Dehydrogenase Inhibitors with Optimized Cell Activity and Pharmacokinetic Properties.

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement in vivo, possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, in vitro drug-target residence times, and in vivo PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition in vivo. The lead compounds, named NCATS-SM1440 (43) and NCATS-SM1441 (52), possess desirable attributes for further studying the effect of in vivo LDH inhibition.

Rheological behaviors of a novel exopolysaccharide produced by Sphingomonas WG and the potential application in enhanced oil recovery.

A novel exopolysaccharide, named WL gum, was obtained from the fermentation broth of Sphingomonas sp. WG. The effects of temperature and salinity on the rheological properties of WL gum solution and fermentation broth (WL-Fer) were systematically investigated and compared with another exopolysaccharide, welan gum (WG). The results showed that the network structures formed in WL solution were more compact than those of WG. WL solution and WL-Fer were not sensitive to high temperatures (80-120 °C) and only weakly affected by the cations (Na+, K+, and Ca2+). Moreover, Fe2+ and high temperature (100 °C) even enhanced the viscosity of WL-Fer. The results of flooding experiments demonstrated that the enhanced displacement efficiency of WL gum (14.55%) was similar to that of partially hydrolyzed polyacrylamide (HPAM, 13.36%) at 65 °C. And the enhanced displacement efficiency of WL-Fer was as high as 23.31%. It can be concluded that WL gum is a kind of potential and environmentally benign polymer that could be used in EOR, and the fermentation broth could be used directly after dilution.

Polysubstituted Phenyl Glucosides Produced by the Fungus Metarrhizium anisopliae.

Metarhizosides A-G (1-7), seven new polysubstituted phenyl glucosides, were isolated from the extracts of solid rice medium of a marine-derived fungus Metarrhizium anisopliae. Compounds 1-7 all contain a polysubstituted phenyl group and the sugar unit is identified as 4'-O-methyl-ß-D-glucopyranose. Their structures were elucidated by NMR spectroscopy and chemical method. These compounds were evaluated for anti-inflammatory activity by using LPS-stimulated murine macrophage RAW 264.7 cells and the cytotoxicities against four human cancer cell lines.

One-step synthesis of rhodamine-based Fe3+ fluorescent probes via Mannich reaction and its application in living cell imaging.

Four rhodamine-based fluorescent probes M1-M4 were synthesized in one step using Mannich reaction. The Mannich reaction based approach has the advantages of simplicity, good yield and excellent atomic economy. The structures were determined by 1H NMR, 13C NMR, IR and HRMS. The probe M3 as a representative compound was characterized by single-crystal X-ray analyses. The fluorescence and absorbance spectra research of the probes demonstrated that they could be used as Fe3+-selective fluorescent probes with good sensitivity, excellent linearity, and outstanding anti-interference in acetonitrile/Tris-HCl buffer solution (3:7, V/V; pH = 7.4). Moreover, confocal laser scanning microscopy experiments have proven that the probe M3 was successfully used for fluorescence imaging in MCF-7 cells.

Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice.

BACKGROUND AND AIMS: Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties. METHODS: We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgcnull (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgcnull (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice. RESULTS: Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active ß-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice. CONCLUSIONS: In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.