GPC3-targeted CAR-T cells expressing GLUT1 or AGK exhibit enhanced antitumor activity against hepatocellular carcinoma.

Chimeric antigen receptor-expressing T (CAR-T) cells induce robust antitumor responses in patients with hematologic malignancies. However, CAR-T cells exhibit only limited efficacy against solid tumors such as hepatocellular carcinoma (HCC), partially due to their limited expansion and persistence. CD8+ T cells, as key components of the adaptive immune response, play a central role in antitumor immunity. Aerobic glycolysis is the main metabolic feature of activated CD8+ T cells. In the tumor microenvironment, however, the uptake of large amounts of glucose by tumor cells and other immunosuppressive cells can impair the activation of T cells. Only when tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment have a glycolytic advantage might the effector function of T cells be activated. Glucose transporter type 1 (GLUT1) and acylglycerol kinase (AGK) can boost glycolytic metabolism and activate the effector function of CD8+ T cells, respectively. In this study, we generated GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK for the treatment of HCC. GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK specifically and effectively lysed GPC3-positive tumor cells in vitro in an antigen-dependent manner. Furthermore, GLUT1 or AGK overexpression protected CAR-T cells from apoptosis during repeated exposures to tumor cells. Compared with second-generation CAR-T cells, GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK exhibited greater CD8+ T-cell persistence in vivo and better antitumor effects in HCC allograft mouse models. Finally, we revealed that GLUT1 or AGK maintained anti-apoptosis ability in CD8+ T cells via activation of the PI3K/Akt pathway. This finding might identify a therapeutic strategy for advanced HCC.

Fucosylated glycan inhibition of human hepatocellular carcinoma cell migration through binding to chemokine receptors.

SMMC-7721 hepatocellular carcinoma cells (HCC) were incubated with fucosylated glycoproteins that had been isolated from retinoic acid-treated cells by affinity chromatography. HCC migration was significantly inhibited by AAL- and LCA-glycoproteins. Glycopeptides, obtained by digestion of the glycoproteins with trypsin and papain, were found to have a similar inhibitory effect on HCC migration as the corresponding glycoproteins. The inhibitory actions of the glycoproteins were almost abolished after digestion with alpha-L-1,3/4- or alpha-L-1,2-fucosidase. Induction of HCC migration with chemokines including interleukin-8 (IL-8), lymphotactin, monocyte chemoattractant protein-1, and stroma cell-derived factor-1 was examined and IL-8 was found to be the most potent. Interestingly, the isolated glycoproteins significantly inhibited HCC migration and F-actin aggregation induced by IL-8, whereas the glycans themselves did not induce F-actin assembly. From receptor binding analysis AAL-glycan was found to bind IL-8 receptors especially CXCR2 directly and such binding could be blocked by 3'- or 2'-fucosyllactose. After CXCR2 silence by target RNAi, the cells almost lost the response to AAL-glycan inhibition. Our findings suggest that fucosylation plays an important role in the interaction between IL-8 and its receptors inducing HCC migration.

Development of efficient RNA interference system using EGF-displaying phagemid particles.

AIM: To develop an efficient RNA interference system using phagemid particles displaying the epidermal growth factor (EGF) ligand. METHODS: pSilencer1.0-siEGFP and pSilencer4.1-siAkt plasmids were constructed by gene clone technology. The modified helper phage genome (plasmid) M13KO7EGFCT was used to package phagemids, such as pSilencer1.0-siEGFP and pSilencer4.1-siAkt. ELISA was used to quantify the titer of the progeny virus particles. Single-strand DNA was extracted and analyzed by agarose gel electrophoresis to evaluate the percentage of the phagemid particles. The expression level of the reporter gene enhanced green fluorescence protein (EGFP) was determined by transducing phagemid particles packaging pSilencer1.0-siEGFP into cells. The level of Akt gene expression in cells transduced phagemid particles packaging pSilencer4.1-siAkt was examined by Western blotting. Hydroxycamptothecin (HCPT) was used to enhance the gene transduction efficiency. RESULTS: RNAi vectors pSilencer1.0-siEGFP and pSilencer4.1-siAkt were successfully constructed. Phagemid-encoding siRNA can be packaged efficiently. After the cells were infected by EGF displaying phagemid particles in the presence of HCPT, the expression of the target gene EGFP or Akt was substantially downregulated. CONCLUSION: Cell-targeted phagemid particles are efficient siRNA delivery vectors in the presence of HCPT.

[Establishment of a NIH3T3 cell line stably expressing EGFRvIII extracellular domain and evaluation of its immunogenicity].

AIM: To establish a cell line stably expressing EGFRvIIIex (epidermal growth factor receptor variant III extracellular domain) and evaluate its immunogenicity. METHODS: NIH3T3 cells stably expressing EGFRvIIIex was obtained by screening NIH3T3 cells transfected with pLNCX2-EGFRvIIIex, a plasmid encoding EGFRvIIIex. The expression level of EGFRvIIIex was examined by immunohistochemical staining and Western blot. The cell clone with the highest expression of EGFRvIIIex named as 3T3-vIIIex was used to immunize BALB/c mice. The titer and specificity of murine antiserum was evaluated by ELISA, Western blot and immunofluorescent staining. RESULTS: A NIN3T3 stable cell line with high EGFRvIIIex expression was obtained. The titer of the antiserum against human EGFRvIIIex was about 10(-5). Western blot and immunofluorescent staining analysis revealed the high specificity of the antiserum. CONCLUSION: High titer of antiserum against EGFRvIIIex can be obtained by NIH3T3 cell lines stably expressing EGFRvIIIex.

Application of EGFP-EGF fusions to explore mechanism of endocytosis of epidermal growth factor.

AIM: To develop a simple method for monitoring protein localization of epidermal growth factor (EGF) in living cells. METHODS: Enhanced green fluorescent protein (EGFP) was used as an autofluorescent tag to label EGF ligands. SDS-PAGE and Western blot analysis were used to detect the expression of the EGFP-tagged EGF (EGFP-EGF) protein. The cell-binding and internalization activity of EGFP-EGF were analyzed by fluorescence-activated cell sorting (FACS) and confocal microscopy. RESULTS: EGFP-EGF protein was expressed in Escherichia coli and purified. A cell-binding assay demonstrated that the EGFP-EGF protein could bind efficiently to the cells expressing EGFR. The binding and internalization of EGFP-EGF can be visualized even at a very low concentration under confocal microscopy. The FACS-based assay for internalization activity indicated the accumulation of internalized EGFP-EGF over time. Furthermore, the results of the competition assay indicated its EGFR binding specificity. Using such a method, it does not need to label EGF with chemicals and avoid light in the experimental process. CONCLUSION: The fusion protein EGFP-EGF has several characters including high sensitivity, stability and convenience for manipulation, and is a powerful tool for the study of EGF endocytosis.

Inhibitory effects of N-(4-hydrophenyl) retinamide on liver cancer and malignant melanoma cells.

AIM: To investigate the effect of N-(4-hydrophenyl) retinamide (4-HPR), the derivative of retinoic acid, on inhibition of migration, invasion, cell growth, and induction of apoptosis in hepatocellular carcinoma cells (HCCs) and malignant melanoma cells. METHODS: 4-HPR was chemically synthesized. Cellular migration and invasion were assayed by Borden chamber experiment. Cell growth was assayed by MTT chromometry. Apoptosis effect was measured using Hoechst 32258 staining and flow cytometry. Gene transfection was performed with lipofectamine. RESULTS: We observed that the migration of HCC and melanoma cells was significantly suppressed by 4-HPR and the migration cells were reduced to 58+/-5.03 (control 201+/-27.2, P<0.05, n = 4) in SMMC 7721-k3 HCC, and to 254+/-25.04 (control 302+/-30.1, P<0.05, n = 4) in melanoma cells after 6-h incubation with 4-HPR. The invasion through reconstituted basement membrane was also significantly reduced by 4-HPR treatment to 11.2+/-3.3 in SMMC 7721-k3 HCC (control 27+/-13.1), and to 24.3+/-3.2 in melanoma cells (control 67.5+/-10.1, P<0.05, n = 3). Cell growth, especially in melanoma cells, was also significantly inhibited. Furthermore, 3 micromol/L of 4-HPR induced apoptosis in B16 melanoma cells (37.11+/-0.94%) more significantly than all-trans retinoic acid (P<0.05), but it failed to induce apoptosis in SMMC 7721-k3 HCC. The mechanism for 4-HPR-induced apoptosis was not clear, but we observed that 4-HPR could regulate p27(kip1), and overexpression of cerebroside sulfotransferase (CST) diminished the apoptosis induced by 4-HPR in melanoma cells. CONCLUSION: 4-HPR is a potent inhibitor of HCC migration and inducer of melanoma cell apoptosis. CST and p27(kip1) expression might be associated with 4-HPR-induced apoptosis.

Gal3ST-2 involved in tumor metastasis process by regulation of adhesion ability to selectins and expression of integrins.

In this study, significantly higher expression of Gal3ST-2 (Gal: 3-O-sulfotransferase-2) and 3'-sulfated glycoconjugates were observed in highly metastastic cancer cells and in larynx cancer tissues with lymph node metastasis than those in lowly metastatic cancer cells and larynx cancer tissues without metastasis (P<0.01, n=42). These results indicated that there was a marked correlation between the expression of Gal3ST-2 and tumor metastasis potential. After RNAi transfection, a striking morphological change of SMMC7721 hepatoma cells from polygon to shuttle shape and significant decrease in adhesion to sL-selectin and HUVEC were observed. Interestingly, the expression of integrin subunit alphaV was markedly downregulated and 3'-sulfated subunit alphaV almost disappeared in the transfectants, but integrin subunit beta3 almost had no change. These results suggested that Gal3ST-2 was involved in tumor metastasis process by regulation of adhesion ability to selectins and expression of integrins.

The expression of core fucosylated E-cadherin in cancer cells and lung cancer patients: prognostic implications.

It is well documented that the glycosylation of E-cadherin is correlated with cancer metastasis, but whether E-cadherin could be core fucosylated remains largely unknown. We found that E-cadherin was core fucosylated in highly metastatic lung cancer cells while absent in lowly metastatic lung cancer cells. Since alpha-1,6 Fucosyltransferase (alpha-1,6 FucT) is known to catalyze the reaction of core fucosylation, we investigated the biological function of core fucosylation on E-cadherin by alpha-1,6 FucT targeted RNAi and transfecting alpha-1,6 FucT expression vector. As a result, calcium dependent cell-cell adhesion mediated by E-cadherin was strengthened with the reduction of core fucosylation on E-cadherin after RNAi and was weakened with the elevated core fucosylation on E-cadherin after alpha-1,6 FucT over expression. Our data indicated that alpha-1,6 FucT could regulate E-cadherin mediated cell adhesion and thus play an important role in cancer development and progression. Computer modeling showed that core fucosylation on E-cadherin could significantly impair three-dimensional conformation of N-glycan on E-cadherin and produce conformational asymmetry so as to suppress the function of E-cadherin. Furthermore, the relationship between the expression of core fucosylated E-cadherin and clinicopathological background of lung cancer patients was explored in lung cancer tissue of patients. It turns out to demonstrate that core fucosylated E-cadherin could serve as a promising prognostic indicator for lung cancer patients.