Inhibition of PCSK9 enhances the anti-hepatocellular carcinoma effects of TCR-T cells and anti-PD-1 immunotherapy.

T cells play important roles in antitumor immunity. However, given that the hepatocellular carcinoma (HCC) tumor microenvironment confers resistance to T cell-based immunotherapies, novel strategies to boost T cell-mediated antitumor efficacy are urgently needed for the treatment of HCC. Here, we show that high proprotein convertase subtilisin/kexin type9 (PCSK9) expression was negatively associated with HCC patient's overall survival and markers of CD8+ T cells. Pharmacological inhibition of PCSK9 enhanced tumor-specific killing and downregulated PD-1 expression of AFP-specific TCR-T. Inhibition of PCSK9 significantly enhances the anti-HCC efficacy of TCR-T cells and anti-PD-1 immunotherapy in vivo. Moreover, PCSK9 inhibitor suppressed HCC growth dependent on CD8+ T cells. Mechanically, pharmacological inhibition of PCSK9 promoted low-density lipoprotein receptor (LDLR)-mediated activation of mTORC1 signaling in CD8+ T cells. LDLR deficiency was shown to impair cellular mTORC1 signaling and the anti-HCC function of CD8 T cells. On the basis of our findings in this study, we propose a potential metabolic intervention strategy that could be used to enhance the antitumor effects of immunotherapy for HCC.

Dexamethasone Long-Term Controlled Release from Injectable Dual-Network Hydrogels with Porous Microspheres Immunomodulation Promotes Bone Regeneration.

Long-lasting, controlled-release, and minimally invasive injectable platforms that provide a stable blood concentration to promote bone regeneration are less well developed. Using hexagonal mesoporous silica (HMS) loaded with dexamethasone (DEX) and poly(lactic-co-glycolic acid) (PLGA), we prepared porous DEX/HMS/PLGA microspheres (PDHP). In contrast to HMS/PLGA microspheres (HP), porous HMS/PLGA microspheres (PHP), DEX/PLGA microspheres (DP), and DEX/HMS/PLGA microspheres (DHP), PDHP showed notable immuno-coordinated osteogenic capabilities and were best at promoting bone mesenchymal stem cell proliferation and osteogenic differentiation. PDHP were combined with methacrylated silk (SilMA) and sodium alginate (SA) to form an injectable photocurable dual-network hydrogel platform that could continuously release the drug for more than 4 months. By adjusting the content of the microspheres in the hydrogel, a zero-order release hydrogel platform was obtained in vitro for 48 days. When the microsphere content was 1%, the hydrogel platform exhibited the best biocompatibility and osteogenic effects. The expression levels of the osteogenic gene alkaline phosphatases, BMP-2 and OPN were 10 to 15 times higher in the 1% group than in the 0% group, respectively. In addition, the 1% microsphere hydrogel strongly stimulated macrophage polarization to the M2 phenotype, establishing an immunological milieu that supports bone regrowth. The aforementioned outcomes were also observed in vivo. The most successful method for correcting cranial bone abnormalities in SD rats was to use a hydrogel called SilMA/SA containing 1% drug-loaded porous microspheres (PDHP/SS). The angiogenic and osteogenic effects of this treatment were also noticeably greater in the PDHP/SS group than in the control and blank groups. In addition, PDHP/SS polarized M2 macrophages and suppressed M1 macrophages in vivo, which reduced the local immune-inflammatory response, promoted angiogenesis, and cooperatively aided in situ bone healing. This work highlights the potential application of an advanced hydrogel platform for long-term, on-demand, controlled release for bone tissue engineering.

A novel engineered IL-21 receptor arms T-cell receptor-engineered T cells (TCR-T cells) against hepatocellular carcinoma.

Strategies to improve T cell therapy efficacy in solid tumors such as hepatocellular carcinoma (HCC) are urgently needed. The common cytokine receptor γ chain (γc) family cytokines such as IL-2, IL-7, IL-15 and IL-21 play fundamental roles in T cell development, differentiation and effector phases. This study aims to determine the combination effects of IL-21 in T cell therapy against HCC and investigate optimized strategies to utilize the effect of IL-21 signal in T cell therapy. The antitumor function of AFP-specific T cell receptor-engineered T cells (TCR-T) was augmented by exogenous IL-21 in vitro and in vivo. IL-21 enhanced proliferation capacity, promoted memory differentiation, downregulated PD-1 expression and alleviated apoptosis in TCR-T after activation. A novel engineered IL-21 receptor was established, and TCR-T armed with the novel engineered IL-21 receptors (IL-21R-TCR-T) showed upregulated phosphorylated STAT3 expression without exogenous IL-21 ligand. IL-21R-TCR-T showed better proliferation upon activation and superior antitumor function in vitro and in vivo. IL-21R-TCR-T exhibited a less differentiated, exhausted and apoptotic phenotype than conventional TCR-T upon repetitive tumor antigen stimulation. The novel IL-21 receptor in our study programs powerful TCR-T and can avoid side effects induced by IL-21 systemic utilization. The novel IL-21 receptor creates new opportunities for next-generation TCR-T against HCC.

Atovaquone enhances antitumor efficacy of TCR-T therapy by augmentation of ROS-induced ferroptosis in hepatocellular carcinoma.

T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for tumor treatment, yet hindered by tumor immune evasion resulting in poor therapeutic efficacy. The introduction of ferroptosis-targeted inducers offers a potential solution, as they empower T cells to induce ferroptosis and exert influence over the tumor microenvironment. Atovaquone (ATO) stands as a prospective pharmaceutical candidate with the potential to target ferroptosis, effectively provoking an excessive generation and accumulation of reactive oxygen species (ROS). In this study, we evaluated the effectiveness of a combination therapy comprising ATO and TCR-T cells against hepatocellular carcinoma (HCC), both in vitro and in vivo. The results of lactate dehydrogenase and cytokine assays demonstrated that ATO enhanced cytotoxicity mediated by AFP-specific TCR-T cells and promoted the release of IFN-γ in vitro. Additionally, in an established HCC xenograft mouse model, the combined therapy with low-dose ATO and TCR-T cells exhibited heightened efficacy in suppressing tumor growth, with no apparent adverse effects, comparable to the results achieved through monotherapy. The RNA-seq data unveiled a significant activation of the ferroptosis-related pathway in the combination therapy group in comparison to the TCR-T cells group. Mechanistically, the synergy between ATO and TCR-T cells augmented the release of IFN-γ by TCR-T cells, while concurrently elevating the intracellular and mitochondrial levels of ROS, expanding the labile iron pool, and impairing the integrity of the mitochondrial membrane in HepG2 cells. This multifaceted interaction culminated in the potentiation of ferroptosis within the tumor, primarily induced by an excess of ROS. In summary, the co-administration of ATO and TCR-T cells in HCC exhibited heightened vulnerability to ferroptosis. This heightened susceptibility led to the inhibition of tumor growth and the stimulation of an anti-tumor immune response. These findings suggest that repurposing atovaquone for adoptive cell therapy combination therapy holds the potential to enhance treatment outcomes in HCC.

[Retracted] TRIM16 suppresses the progression of prostate tumors by inhibiting the Snail signaling pathway.

Following the publication of this paper, it was drawn to the Editor's attention by concerned readers that the western blotting data shown in Figs. 4C and 7B and D, the scratch­wound assay images shown in Figs. 5A and 6A, and certain of the cell migration and invasion assay data shown in Figs. 5B and 6B were strikingly similar to data that had previously appeared in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to International Journal of Molecular Medicine, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they accepted the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 38: 1734­1742, 2016; DOI: 10.3892/ijmm.2016.2774].

Preparation of a 3D printable high-performance GelMA hydrogel loading with magnetic cobalt ferrite nanoparticles.

Osteosarcoma remains a worldwide concern due to the poor effectiveness of available therapies in the clinic. Therefore, it is necessary to find a safe and effective therapy to realize the complete resection of osteosarcoma and reconstruction of the bone defect. Magnetic hyperthermia based on magnetic nanoparticles can kill tumor cells by raising the temperature without causing the side effects of conventional cancer treatments. This research aims to design a high-performance magnetic hydrogel composed of gelatin methacrylate and highly magnetic cobalt ferrite (CFO) nanoparticles for osteosarcoma treatment. Specifically, CFO is surface functionalized with methacrylate groups (MeCFO). The surface modified CFO has good biocompatibility and stable solution dispersion ability. Afterward, MeCFO nanoparticles are incorporated into GelMA to fabricate a three-dimensional (3D) printable MeCFO/GelMA magnetic hydrogel and then photocross-linked by UV radiation. MeCFO/GelMA hydrogel has high porosity and swelling ability, indicating that the hydrogel possesses more space and good hydrophily for cell survival. The rheological results showed that the hydrogel has shear thinning property, which is suitable as a bioprinting ink to produce desired structures by a 3D printer. Furthermore, 50 µg/mL MeCFO not only decreases the cell activity of osteosarcoma cells but also promotes the osteogenic differentiation of mBMSCs. The results of the CCK-8 assay and live/dead staining showed that MeCFO/GelMA hydrogel had good cytocompatibility. These results indicated that MeCFO/GelMA hydrogel with potential antitumor and bone reconstruction functions is a promising therapeutic strategy after osteosarcoma resection.

Analysis of L Antigen Family Member 3 as a Potential Biomarker and Therapeutic Target Associated With the Progression of Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is the third cause of cancer-related deaths worldwide. L antigen family member 3 (LAGE3) is a prognostic biomarker and associated with progression in a variety of tumors. However, little has been reported about the role and potential mechanism of LAGE3 in HCC. Methods: The clinical value and function of LAGE3 in HCC were obtained from multiple online databases. The potential functions and pathways of LAGE3 in HCC were analysed by R package of "clusterProfiler". LAGE3 knockdown cells were constructed in HepG2, HuH7 and MHCC97H cell lines, respectively. The biological roles of LAGE3 were examined by in vitro and in vivo experiments. Results: LAGE3 was upregulated in HCC tissues compared with normal tissues, and high expression of LAGE3 was significantly associated with several clinical characteristics and indicated a worse prognosis of HCC. The co-expressed genes of LAGE3 could be enriched in the mTOR signaling pathway in HCC. LAGE3 was upregulated in HCC cell lines. Functionally, knocking down LAGE3 expression not only increased apoptosis and inhibited growth rate, cell death mediated by T cells, colony formation, migration and invasion ability of HCC cell lines in vitro, but also reduced the progression of HCC in the subcutaneous xenotransplanted tumor model. Conclusion: Our results suggested that LAGE3 served as an oncogenic factor of HCC and could be a potential biomarker and therapeutic target for HCC.

Developing a novel calcium magnesium silicate/graphene oxide incorporated silk fibroin porous scaffold with enhanced osteogenesis, angiogenesis and inhibited osteoclastogenesis.

Recently, biofunctional ions (Mg2+, Si4+, etc) and graphene derivatives are proved to be promising in stimulating bone formation. In this study, a novel inorganic/organic composite porous scaffold based on silk fibroin (SF), graphene oxide (GO), and calcium magnesium silicate (CMS) was developed for bone repair. The porous scaffolds obtained by lyophilization showed a little difference in pore structure while GO and CMS displayed a good interaction with SF matrix. The addition of CMS with good mineralization potential and sustainedly release ability of biofunctional ions (Ca2+, Mg2+and Si4+) increased the strength of SF scaffolds a little and facilitated the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) by upregulating bone formation-related genes (ALP, COL1, OC and Runx2). The further incorporation of GO in SF scaffolds enhanced the compressive strength and water retention, and also remarkably promoted the osteogenic differentiation of BMSCs. Besides, the angiogenesis of human umbilical vein endothelial cells was significantly promoted by CMS/GO/SF scaffold extract through the upregulation of angiogenesis genes (eNOs and bFGF). Moreover, the osteoclastic formation ability of RAW264.7 cells was suppressed by the released ions from CMS/GO/SF scaffold through the down-regulation of CAK, MMP9 and TRAP. The promoted osteogenesis, angiogenesis and inhibited osteoclastogenesis functions of CMS/GO/SF composite scaffold may enable it as a novel therapy for bone repair and regeneration.

Advances in Filament Structure of 3D Bioprinted Biodegradable Bone Repair Scaffolds.

Conventional bone repair scaffolds can no longer meet the high standards and requirements of clinical applications in terms of preparation process and service performance. Studies have shown that the diversity of filament structures of implantable scaffolds is closely related to their overall properties (mechanical properties, degradation properties, and biological properties). To better elucidate the characteristics and advantages of different filament structures, this paper retrieves and summarizes the state of the art in the filament structure of the three-dimensional (3D) bioprinted biodegradable bone repair scaffolds, mainly including single-layer structure, double-layer structure, hollow structure, core-shell structure and bionic structures. The eximious performance of the novel scaffolds was discussed from different aspects (material composition, ink configuration, printing parameters, etc.). Besides, the additional functions of the current bone repair scaffold, such as chondrogenesis, angiogenesis, anti-bacteria, and anti-tumor, were also concluded. Finally, the paper prospects the future material selection, structural design, functional development, and performance optimization of bone repair scaffolds.

Identification of PAFAH1B3 as Candidate Prognosis Marker and Potential Therapeutic Target for Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths worldwide. PAFAH1B3 plays an important role on occurrence and development in a variety tumor. However, the function of PAFAH1B3 in HCC remains unclear. METHODS: The TIMER, ONCOMINE, Human Protein Atlas (HPA), GEPIA, The Cancer Genome Atlas (TCGA), HCCDB, UALCAN and LinkedOmics database were used to analyze the prognostic value, co-expression genes and regulator networks of PAFAH1B3 in HCC. siRNA transfections and inhibitor of PAFAH1B3 P11 were used to verify the anti-tumor effect on HCC cell lines. Gene expression was detected by qRT-PCR. The functions of PAFAH1B3 downregulation in HCC cell lines were investigated using cell cycle analysis, apoptosis detection, CCK8 assay and transwell assay. Western blot was used to evaluate the role of PAFAH1B3 on metabolic pathways in HCC cells. RESULTS: Based on the data from databases, the expression of PAFAH1B3 was remarkably increased in HCC patients. High expression of PAFAH1B3 was associated with poorer overall survival (OS) and disease-free survival (DFS). And PAFAH1B3 was notably linked to age, sex, grade, stage, race, and TP53 mutational status. Then, the functional network analysis showed PAFAH1B3 may be involved in HCC through cell cycle, cell metabolism, spliceosome, and RNA transport. Furthermore, the mRNA expression of PAFAH1B3 was also increased in HCC cell lines. Flow cytometry analysis showed that PAFAH1B3 manipulated apoptosis and cell cycle regulation. CCK8 assay showed that PAFAH1B3 silencing or pharmacologic inhibitor of PAFAH1B3 inhibited the proliferation of HepG2, Huh7 and MHCC-97H cells. Transwell assay results showed that PAFAH1B3 silencing also significantly impaired the invasion and migratory ability of HCC cells. In addition, PAFAH1B3 silencing significantly downregulated the expression of glycolysis and lipid synthesis signaling pathways. CONCLUSION: Our findings suggested that PAFAH1B3 plays a critical role in progression of HCC. PAFAH1B3 as a prognosis marker and potential target for HCC has prospective clinical significance.

Alveolar bone repair of rhesus monkeys by using BMP-2 gene and mesenchymal stem cells loaded three-dimensional printed bioglass scaffold.

Over the past years, the study about bone tissue engineering in the field of regenerative medicine has been a main research topic. Using three-dimensional (3D) porous degradable scaffold complexed with mesenchymal stem cells (MSCs) and growth factor gene to improve bone tissue repair and regeneration has raised much interest. This study mainly evaluated the osteogenesis of alveolar bone defects of animal in the following experimental groups: sham-operated (SO), 3D printed bioglass (3D-BG), 3D-BG with BMP-2 gene loaded CS (3D-BG + BMP/CS) and 3D-BG with rhesus marrow bone MSCs and BMP/CS (3D-BG + BMP/CS + rBMSCs). Simulated human bone defect with critical size of 10 × 10 × 5 mm were established in quadrumana - rhesus monkeys, and in vivo osteogenesis was characterized by X-ray, micro-Computed Tomography (mCT) and history. Our results revealed that 3D-BG + rBMSCs + BMP/CS scaffold could improve bone healing best by showing its promote osteogenic properties in vivo. Considering the great bone repair capacity of 3D-BG + BMP/CS + rBMSCs in humanoid primate rhesus monkeys, it could be a promising therapeutic strategy for surgery trauma or accidents, especially for alveolar bones defects.

Identification of Potential Key Genes for Hepatitis B Virus-Associated Hepatocellular Carcinoma by Bioinformatics Analysis.

Hepatitis B virus-associated (HBV(+)) hepatocellular carcinoma (HCC) accounts for a large proportion of liver cancer with poor clinical outcomes and treatment options. However, the underlying molecular mechanisms are still poorly understood. To explore potential key genes in the development of HBV(+)HCC, four series of data (GSE14520, GSE94660, GSE25599, and GSE55092) derived from Gene Expression Omnibus database were analyzed. Totally, 84 upregulated and 46 downregulated common differentially expressed genes (DEGs) were discovered. Gene ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that these DEGs were mainly enriched in cell division and DNA replication biological processes, nucleoplasm and microtubule cellular components, protein-binding molecular functions, and cell cycle and DNA replication pathways. Through protein-protein interaction analysis, 10 hub DEGs with the highest degree of connectivity were indicated, including TOP2A, CDC20, MAD2L1, BUB1B, RFC4, CCNB1, CDKN3, CCNB2, TPX2, and FEN1. Kaplan-Meier analysis revealed that high expression of TOP2A and CDC20 was associated with poor overall survival, relapse-free survival, and high serum alpha-fetoprotein level in HBV(+)HCC. In conclusion, TOP2A and CDC20 were two potential key genes for HBV(+)HCC. Their value in the diagnosis and treatment of HBV(+)HCC requires further investigation.

Metformin combined with nelfinavir induces SIRT3/mROS-dependent autophagy in human cervical cancer cells and xenograft in nude mice.

The molecular mechanisms underlying the antineoplastic properties of metformin combined with nelfinavir remain elusive. To explore this question, transmission electron microscopy (TEM) was used to observe the combinatorial effect of inducing autophagosome formation in human cervical cancer cells. Western blotting respectively assayed protein expression of LC3I, LC3II, Beclin-1, Autophagy-related protein 7 (Atg7), Autophagy-related protein 3 (Atg3), NAD-dependent deacetylase sirtuin-3 (SIRT3) and major histocompatibility complex class I chain-related gene A (MICA). Lactate dehydrogenase (LDH) cytotoxicity assay evaluated natural killer (NK) cell cytotoxicity in the presence of metformin and nelfinavir in combination or each drug alone. Using tumor xenografts in a nude mouse model, antitumor efficacy of the drug combination was assessed. We found that the drug combination could induce autophagosome formation in human cervical cancer cells. The biomarker proteins of autophagy, including Beclin-1, Atg7 and Atg3, decreased, but the ratios of LC3I/II increased. We also found that this drug combination sensitizes human cervical cancer cells to NK cell-mediated lysis by increasing the protein of SIRT3 and MICA. Moreover, this drug combination markedly induced autophagy of SiHa xenografts in nude mice. Therefore, it can be concluded that metformin, in combination with nelfinavir, can induce SIRT3/mROS-dependent autophagy and sensitize NK cell-mediated lysis in human cervical cancer cells and cervical cancer cell xenografts in nude mice. Thus, our findings have revealed the detailed molecular mechanisms underlying the antitumor effects of metformin in combination with nelfinavir in cervical cancer.

TRIM16 suppresses the progression of prostate tumors by inhibiting the Snail signaling pathway.

Prostate carcinoma is a devastating disease which is characterized by insidious early symptoms, rapid progression and a poor prognosis. Tripartite motif-containing protein 16 (TRIM16) was identified as an estrogen- and antiestrogen-regulated gene in epithelial cells stably expressing estrogen receptors. The protein encoded by this gene contains two B-box domains and a coiled-coiled region that are characteristic of the B-box zinc finger protein family. Proteins belonging to this family have been reported to be involved in a variety of biological processes including cell growth, differentiation and pathogenesis. TRIM16 expression has been detected in most tissues. However, the funtions of this gene remain to be elucidated. In the present study, immunohistochemical staining revealed that the expression of TRIM16 was decreased in prostate adenocarcinoma compared with that in normal prostate tissues. The patients with high TRIM16-expressing tumors had a significantly greater survival than those with low TRIM16-expressing tumors. Western blot analysis showed that TRIM16 was downregulated in distant metastatic cancer tissues compared with that in non-distant metastatic cancer tissues. The overexpression of TRIM16 inhibited the migration and invasion of prostate cancer cells as well as inhibiting the epithelial-to-mesenchymal transition process, whereas TRIM16 depletion enhanced these processes. Moreover, TRIM16 inhibited the Snail signaling pathway. The silencing of Snail by small interfering RNA was performed in order to determine the role of Snail in the TRIM16-mediated tumor phenotype. Taken together, these findings suggest that TRIM16 may be an important molecular target which may aid in the design of novel therapeutic agents for prostate cancer.