Cell microparticles loaded with tumor antigen and resiquimod reprogram tumor-associated macrophages and promote stem-like CD8+ T cells to boost anti-PD-1 therapy.

The durable response rate to immune checkpoint blockade such as anti-programmed cell death-1 (PD-1) antibody remains relatively low in hepatocellular carcinoma (HCC), mainly depending on an immunosuppressive microenvironment with limited number of CD8+ T cells, especially stem-like CD8+ T cells, in tumor tissues. Here we develop engineered microparticles (MPs) derived from alpha-fetoprotein (AFP)-overexpressing macrophages to load resiquimod (R848@M2pep-MPsAFP) for enhanced anti-PD-1 therapy in HCC. R848@M2pep-MPsAFP target and reprogram immunosuppressive M2-like tumor-associated macrophages (TAMs) into M1-like phenotype. Meanwhile, R848@M2pep-MPsAFP-reprogrammed TAMs act as antigen-presenting cells, not only presenting AFP antigen to activate CD8+ T cell-mediated antitumor immunity, but also providing an intra-tumoral niche to maintain and differentiate stem-like CD8+ T cells. Combination immunotherapy with anti-PD-1 antibody generates strong antitumor immune memory and induces abundant stem-like CD8+ T cell proliferation and differentiation to terminally exhausted CD8+ T cells for long-term immune surveillance in orthotopic and autochthonous HCC preclinical models in male mice. We also show that the R848-loaded engineered MPs derived from macrophages overexpressing a model antigen ovalbumin (OVA) can improve anti-PD-1 therapy in melanoma B16-OVA tumor-bearing mice. Our work presents a facile and generic strategy for personalized cancer immunotherapy to boost anti-PD-1 therapy.

Hydrophobicity-Adaptive Polymers Trigger Fission of Tumor-Cell-Derived Microparticles for Enhanced Anticancer Drug Delivery.

Tumor-cell-derived microparticles (MPs) can function as anticancer drug-delivery carriers. However, short blood circulation time, large-size-induced insufficient tumor accumulation and penetration into tumor parenchyma, as well as limited cellular internalization by tumor cells and cancer stem cells (CSCs), and difficult intracellular drug release restrict the anticancer activity of tumor-cell-derived MP-based drug-delivery systems. In this work, hydrophobicity-adaptive polymers based on poly(N-isopropylacrylamide) are anchored to tumor-cell-derived MPs for enhanced delivery of the anticancer drug doxorubicin (DOX). The polymers are hydrophilic in blood to prolong the circulation time of DOX-loaded MPs (DOX@MPs), while rapidly switching to hydrophobic at the tumor acidic microenvironment. The hydrophobicity of polymers drives the fission of tumor-cell-derived MPs to form small vesicles, facilitating tumor accumulation, deep tumor penetration, and efficient internalization of DOX@MPs into tumor cells and CSCs. Subsequently, the hydrophobicity of polymers in acidic lysosomes further promotes DOX release to nuclei for strong cytotoxicity against tumor cells and CSCs. The work provides a facile and simple strategy for improved anticancer drug delivery of tumor-cell-derived MPs.

Compact optical fiber photoacoustic gas sensor with integrated multi-pass cell.

Optical fiber acoustic sensors with miniature size and high sensitivity are attractive to develop compact photoacoustic spectroscopy. Here, a compact photoacoustic gas sensor was demonstrated by utilizing a diaphragm-based fiber-optic Fabry-Perot cavity as both the acoustic sensor and the multipass cell. A nanoscale graphite film was used as the flexible diaphragm to increase the acoustic sensitivity of the Fabry-Perot cavity and the cavity inner surface was coated with highly-reflective Au film to form a multipass cell for amplification of the photoacoustic signal. With a laser power of 20 mW at 1532.8 nm, the sensor demonstrated a low detection limit of ∼ 50 ppb for C2H2 gas with an integration time of ∼ 100 s. The optical fiber photoacoustic gas sensor with a millimeter-scale diameter and ppb-level detection limit is promising for trace gas sensing in various areas including industrial process and environmental monitoring.

Septin11 promotes hepatocellular carcinoma cell motility by activating RhoA to regulate cytoskeleton and cell adhesion.

Septins as GTPases in the cytoskeleton, are linked to a broad spectrum of cellular functions, including cell migration and the progression of hepatocellular carcinoma (HCC). However, roles of SEPT11, the new member of septin, have been hardly understood in HCC. In the study, the clinical significance and biological function of SEPT11 in HCC was explored. SEPT11 was screened out by combining ATAC-seq with mRNA-seq. Role of SEPT11 in HCC was further investigated by using overexpression, shRNA and CRISPR/Cas9-mediated SEPT11-knockout cells or in vivo models. We found RNA-seq and ATAC-seq highlights LncRNA AY927503 (AY) induced SEPT11 transcription, resulting in Rho GTPase activation and cytoskeleton actin aggregation. The GTP-binding protein SEPT11 is thus considered, as a downstream factor of AY, highly expressed in various tumors, including HCC, and associated with poor prognosis of the patients. In vitro, SEPT11 overexpression promotes the migration and invasion of HCC cells, while SEPT11-knockout inhibits migration and invasion. In vivo, SEPT11-overexpressed HCC cells show high metastasis incidents but don't significantly affect proliferation. Meanwhile, we found SEPT11 targets RhoA, thereby regulating cytoskeleton rearrangement and abnormal cell adhesion through ROCK1/cofilin and FAK/paxillin signaling pathways, promoting invasion and migration of HCC. Further, we found SEPT11 facilitates the binding of GEF-H1 to RhoA, which enhances the activity of RhoA. Overall, our study confirmed function of SEPT11 in promoting metastasis in HCC, and preliminarily explored its related molecular mechanism. SEPT11 acts as an oncogene in HCC, also draws further interest regarding its clinical application as a potential therapeutic target.

Long Non-coding RNA CBR3 Antisense RNA 1 is Downregulated in Colorectal Cancer and Inhibits miR-29a-Mediated Cell Migration and Invasion.

Although CBR3 Antisense RNA 1 (CBR3-AS1) has been characterized as an oncogenic long non-coding RNA (lncRNA) in several cancers, a recent study reported the downregulation of CBR3-AS1 in colorectal cancer (CRC). Therefore, we analyzed its role in CRC. CBR3-AS1 and microRNA-29a (miR-29a) expression in tissue samples from CRC patients were analyzed by RT-qPCR. The interaction between CBR3-AS1 and miR-29a was predicted by IntaRNA and validated by RNA pull-down assay. The location of CBR3-AS1 was analyzed by nuclear fractionation assay. CBR3-AS1 overexpression was performed to analyze its role in miR-29a expression. The roles of CBR3-AS1 and miR-29a in CRC cell migration and invasion were analyzed by Transwell assay. CBR3-AS1 was downregulated, and miR-29a was upregulated in CRC. CBR3-AS1 and miR-29a directly interacted with each other. CBR3-AS1 was localized in both nucleus and cytoplasm fractions. CBR3-AS1 overexpression failed to alter miR-29a expression but reduced its enhancing effects on cell invasion and migration. CBR3-AS1 is downregulated in CRC and inhibits miR-29a-mediated cell migration and invasion by sponging miR-29a.

MicroRNA-124 ameliorates autophagic dysregulation in glaucoma via regulation of P2X7-mediated Akt/mTOR signaling.

Glaucoma is a neurodegenerative disease that leads to irrevocable blindness. In glaucoma, even though axonal damage and function deficit culminates in retinal ganglion cell (RGC) degeneration, our knowledge on the autophagic mechanisms and the role of specific microRNAs is still limited. In this study, we investigated the role of microRNA-124 (MiR-124) in surgically induced glaucomatous neurodegeneration using a mouse model. Animals were segregated into four cohorts of 10 each: (i) sham-operated (n = 10); (ii) surgically induced glaucoma (SIG; n = 10); (iii) SIG + miR-124 mimic; (iv) SIG + miR-NC. Chronic elevation of intraocular pressure (IOP) is a critical risk factor for glaucoma. In our study, chronically elevated IOP caused anterograde axonal transport (AAT) defect, increased the autophagic activity (manifested by significantly (p < 0.05) increased LC3-II/LC3-I ratio, beclin-1 and Atg7 protein expressions) and also downmodulated the protein expression of p-Akt and p-mTOR, mediated by the purinergic P2 receptor subtype 7 (P2X7) upmodulation-leading to retinal degeneration. However, administration of miR-124 mimic improved the retinal integrity and function, as indicated by the improved AAT function, normalized the autophagic dysfunction, modulated the protein expression of P2X7-mediated p-Akt and p-mTOR. Hence, we propose that development of miR-124-based advanced therapies might be a potential avenue in the treatment of glaucomatous neurodegeneration.

Study of the G Protein Nucleolar 2 Value in Liver Hepatocellular Carcinoma Treatment and Prognosis.

LIHC (liver hepatocellular carcinoma) mostly occurs in patients with chronic liver disease. It is primarily induced by a vicious cycle of liver injury, inflammation, and regeneration that usually last for decades. The G protein nucleolar 2 (GNL2), as a protein-encoding gene, is also known as NGP1, Nog2, Nug2, Ngp-1, and HUMAUANTIG. Few reports are shown towards the specific biological function of GNL2. Meanwhile, it is still unclear whether it is related to the pathogenesis of carcinoma up to date. Here, our study attempts to validate the role and function of GNL2 in LIHC via multiple databases and functional assays. After analysis of gene expression profile from The Cancer Genome Atlas (TCGA) database, GNL2 was largely heightened in LIHC, and its overexpression displayed a close relationship with different stages and poor prognosis of carcinoma. After enrichment analysis, the data revealed that the genes coexpressed with GNL2 probably participated in ribosome biosynthesis which was essential for unrestricted growth of carcinoma. Cell functional assays presented that GNL2 knockdown by siRNA in LIHC cells MHCC97-H and SMCC-7721 greatly reduced cell proliferation, migration, and invasion ability. All in all, these findings capitulated that GNL2 could be a promising treatment target and prognosis biomarker for LIHC.

GATA4-driven miR-206-3p signatures control orofacial bone development by regulating osteogenic and osteoclastic activity.

Growth disorders in the orofacial bone development process may lead to orofacial deformities. The balance between bone matrix formation by mesenchymal lineage osteoblasts and bone resorption by osteoclasts is vital for orofacial bone development. Although the mechanisms of orofacial mesenchymal stem cells (OMSCs) in orofacial bone development have been studied intensively, the communication between OMSCs and osteoclasts remains largely unclear. Methods: We used a neural crest cell-specific knockout mouse model to investigate orofacial bone development in GATA-binding protein 4 (GATA4) morphants. We investigated the underlying mechanisms of OMSCs-derived exosomes (OMExos) on osteoclastogenesis and bone resorption activity in vitro. miRNAs were extracted from OMExos, and differences in miRNA abundances were determined using an Affymetrix miRNA array. Luciferase reporter assays were used to validate the binding between GATA4 and miR-206-3p in OMSCs and to confirm the putative binding of miR-206-3p and its target genes in OMSCs and osteoclasts. The regulatory mechanism of the GATA4-miR-206-3p axis in OMSC osteogenic differentiation and osteoclastogenesis was examined in vitro and in vivo. Results: Wnt1-Cre;Gata4fl/fl mice (cKO) not only presented inhibited bone formation but also showed active bone resorption. Osteoclasts cocultured in vitro with cKO OMSCs presented an increased capacity for osteoclastogenesis, which was exosome-dependent. Affymetrix miRNA array analysis showed that miR-206-3p was downregulated in exosomes from shGATA4 OMSCs. Moreover, the transcriptional activity of miR-206-3p was directly regulated by GATA4 in OMSCs. We further demonstrated that miR-206-3p played a key role in the regulation of orofacial bone development by directly targeting bone morphogenetic protein-3 (Bmp3) and nuclear factor of activated T -cells, cytoplasmic 1 (NFATc1). OMExos and agomiR-206-3p enhanced bone mass in Wnt1-cre;Gata4fl/fl mice by augmenting trabecular bone structure and decreasing osteoclast numbers. Conclusion: Our findings confirm that miR-206-3p is an important downstream factor of GATA4 that regulates the functions of OMSCs and osteoclasts. These results demonstrate the efficiency of OMExos and microRNA agomirs in promoting bone regeneration, which provide an ideal therapeutic tool for orofacial bone deformities in the future.

CHAF1A overexpression in human retinoblastoma promotes cell proliferation and suppresses apoptosis.

PURPOSE: Retinoblastoma causes significant human mortality especially in children. Although retinoblastoma may be treated if detected at early stage, however, it becomes destructive at advanced stages. The treatment involves surgery and chemotherapy. However, the chemotherapeutic agents have severe adverse effects. Therefore, development of viable drugs and identification of novel molecular therapeutic targets may enable efficient management of retinoblastoma. This study was designed to examine the expression profile of Chromatin Assembly Factor-1 (CHAF1A) and explore its therapeutic implications in retinoblastoma. METHODS: The expression of CHAF1A was determined by qRT-PCR. MTT assay was used for the determination of the cell viability. Apoptosis was detected by acridine orange (AO)/ethidium bromide (EB) and annexin V/propidium iodide (PI) assay. Cell cycle analysis was determined by flow cytometery. Protein expression was determined by western blot analysis. RESULTS: The results showed that CHAF1A is significantly upregulated in human retinoblastoma, with 7.3 folds upregulation in retinoblastoma cells relative to normal cells. Knockdown of CHAF1A resulted in significant decline in the viability of the RB355 retinoblastoma cells. The flow cytometric analysis showed that knockdown of CHAF1A caused arrest of the RB355 cells at G0/G1 phase of the cell cycle. This was also linked with significant downregulation of cyclin D1 and cyclin E1. The AO/EB staining assay showed that CHAF1A knockdown promotes apoptosis which is associated with downregulation of Bcl-2 and upregulation of Bax. CONCLUSION: Taken together, these results suggest that CHAF1A is upregulated in retinoblastoma and regulates its proliferation and apoptosis. As such CHAF1A may act as biomarker as well as therapeutic target for the management of retinoblastoma.

Rho-GEF trio regulates osteoclast differentiation and function by Rac1/Cdc42.

Many bone diseases result from abnormal bone resorption by osteoclasts (OCs). Studying OC related regulatory genes is necessary for the development of new therapeutic strategies. Rho GTPases have been proven to regulate OC differentiation and function and only mature OCs can carry out bone resorption. Here we demonstrate that Rac1 and Cdc42 exchange factor Triple functional domain (Trio) is critical for bone resorption caused by OCs. In this study, we created LysM-Cre;Triofl/fl conditional knockout mice in which Trio was conditionally ablated in monocytes. LysM-Cre;Triofl/fl mice showed increased bone mass due to impaired bone resorption caused by OCs. Furthermore, our in vitro analysis indicated that Trio conditional deficiency significantly suppressed OC differentiation and function. At the molecular level, Trio deficiency significantly inhibited the expression of genes critical for osteoclastogenesis and OC function. Mechanistically, our researches suggested that perturbed Rac1/Cdc42-PAK1-ERK/p38 signaling could be used to explain the lower ability of bone resorption in CKO mice. Taken together, this study indicates that Trio is a regulator of OCs. Studying the role of Trio in OCs provides a potential new insight for the treatment of OC related bone diseases.

Relieving immunosuppression during long-term anti-angiogenesis therapy using photodynamic therapy and oxygen delivery.

Angiogenesis is an irreplaceable therapeutic cancer target, where anti-angiogenesis are drugs that are limited by their hydrophobicity and low therapeutic effects. What is more, the long-term shutdown of tumor blood vessel density also aggravates hypoxia and causes immunosuppression in the tumor microenvironment (TME). In order to solve these shortcomings, we developed a single therapeutic agent based on a bovine serum albumin nanocarrier that can co-deliver the anti-angiogenic drug Sorafenib ("S") and the photosensitizer Ce6 ("C") along with a molecular oxygen supply based on MnO2 ("M") as a convenient one-pot formulated nanoscale agent (SCM@BSA). Compared with anti-angiogenesis monotherapy, SCM@BSA can not only improve upon the solubility and therapeutic effects of anti-angiogenesis agents, but it also reshapes the immunosuppressive TME during anti-angiogenic therapy. Together, these results point out that SCM@BSA synthesized via a very simple method can solve the shortcomings usually experienced during long-term anti-angiogenic therapy.

Co-delivery of Bee Venom Melittin and a Photosensitizer with an Organic-Inorganic Hybrid Nanocarrier for Photodynamic Therapy and Immunotherapy.

Photodynamic therapy (PDT) is a clinical cancer treatment modality based on the induction of therapeutic reactive oxygen species (ROS), which can trigger immunogenic cell death (ICD). With the aim of simultaneously improving both PDT-mediated intracellular ROS production and ICD levels, we designed a serum albumin (SA)-coated boehmite ("B"; aluminum hydroxide oxide) organic-inorganic scaffold that could be loaded with chlorin e6 (Ce6), a photosensitizer, and a honey bee venom melittin (MLT) peptide, denoted Ce6/MLT@SAB. Ce6/MLT@SAB was anchored by a boehmite nanorod structure and exhibited particle size of approximately 180 nm. Ce6/MLT@SAB could significantly reduce hemolysis relative to that of free MLT, while providing MLT-enhanced PDT antitumor effects in vitro. Compared with Ce6@SAB, Ce6/MLT@SAB improved Ce6 penetration of cancer cells both in vitro and in vivo, thereby providing enhanced intracellular ROS generation with 660 nm light treatment. Following phototreatment, Ce6/MLT@SAB-treated cells displayed significantly improved levels of ICD and abilities to activate dendritic cells. In the absence of laser irradiation, multidose injection of Ce6/MLT@SAB could delay the growth of subcutaneous murine tumors by more than 60%, compared to controls. When combined with laser irradiation, a single injection and phototreatment with Ce6/MLT@SAB eradicated one-third of subcutaneous tumors in treated mice. The addition of an immune checkpoint blockade to Ce6/MLT@SAB phototreatment further augmented antitumor effects, generating increased numbers of CD4+ and CD8+ T cells in tumors with concomitant reduction of myeloid-derived suppressor cells.

Antitumor effect of Quercetin on Y79 retinoblastoma cells via activation of JNK and p38 MAPK pathways.

BACKGROUND: Quercetin (QCT) is a flavonol present in many vegetables, it is proved to show chemo preventive effect against lung, cervical, prostate, breast and colon cancer due to its anti-inflammatory, anti-tumor and anti-oxidant property. Looking into the reported chemo-preventive effect we speculated antitumor activity in retinoblastoma (RB) Y79 cells, we also studied the molecular mechanism for antitumor activity. METHODS: The effect of QCT on Y79 cell viability count was done by cell counting kit, cell cycle distribution, apoptosis studies and mitochondrial membrane potential was evaluated by flow cytometry. Protein expression was done by western blot analysis. RESULTS: The outcomes of study showed that QCT reduced Y79 cell viability and caused arrest of G1 phase in cell cycle via decreasing the expression levels of cyclin-dependent kinase (CDK)2/6 and cyclin D3 and by increasing the levels of both CDK inhibitor proteins p21 and p27. Apoptosis of Y79 cells mediated by QCT occurred via activation of both caspases-3/-9. Flow cytometry studies showed that QCT caused collapse in mitochondrial membrane potential (ΔΨm) in Y79 cells. Western blot studies confirmed that QCT brought about phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). We also established that inhibitors of JNK and p38 MAPK suppressed QCT mediated activation of both caspases-3/-9 and subdued the apoptosis of cancerous Y79 cells. CONCLUSION: All the results of the study suggest that QCT induced the apoptosis of Y79 cells via activation of JNK and p38 MAPK pathways, providing a novel treatment approach for human RB.

Evaluation of p53 gene expression and prognosis characteristics in uveal melanoma cases.

OBJECTIVE: The objective of this study was to evaluate the correlation between the expression of p53 gene and the prognosis after local excision in uveal melanoma. MATERIALS AND METHODS: Real-time polymerase chain reaction (RT-PCR) test and Western blot were used to detect the expression of p53 in C918, MUM-2B, and D78 cell lines at the levels of messenger RNA (mRNA) and protein. Immunohistochemistry staining was done in the tissues of 68 patients, which were diagnosed with uveal melanoma. Furthermore, the effects of p53 protein on the invasion abilities of both the cell lines were studied by transinfection of p53 small interfering RNA. The clinical and prognostic data regarding the effect of p53 protein on the patient's prognosis were calculated and further analyzed by Kaplan-Meier univariate analysis method. RESULTS: The results of RT-PCR and Western blot revealed that p53 mRNAs were highly expressed in C918 and MUM-2B cells. The high expression rate of p53 among the 88 uveal melanoma tissues was 77.27%. Transinfection of p53 serine could inhibit the expression of p53 in uveal melanoma and the invasion ability of the cells. This study found that the high expression of p53 and the prognosis of uveal melanoma patients were statistically correlated. CONCLUSION: The expression of p53 protein in uveal melanoma was unusual and was associated with the invasion ability of uveal melanoma. This indicates that the highest expression of p53 protein indicates worse prognosis of uveal melanoma patients.