CD24 is a novel target of chimeric antigen receptor T cells for the treatment of triple negative breast cancer.

Triple negative breast cancer (TNBC) is a subtype of breast cancer with the highest degree of malignancy and the worst prognosis. The application of immunotherapy for TNBC is limited. This study was to verify the potential application of chimeric antigen receptor-T cells (CAR-T cells) targeting CD24 named as 24BBz in treatment of TNBC. 24BBz was constructed by lentivirus infection and then was co-culture with breast cancer cell lines to evaluate the activation, proliferation and cytotoxicity of engineered T cells. The anti-tumor activity of 24BBz was verified in the subcutaneous xenograft model of nude mice. We found that CD24 gene was significantly up-regulated in breast cancer (BRCA), especially in TNBC. 24BBz showed antigen-specific activation and dose-dependent cytotoxicity against CD24-positive BRCA tumor cells in vitro. Furthermore, 24BBz showed significant anti-tumor effect in CD24-positive TNBC xenografts and T cells infiltration in tumor tissues, while some T cells exhibited exhaustion. No pathological damage of major organs was found during the treatment. This study proved that CD24-specific CAR-T cells have potent anti-tumor activity and potential application value in treatment of TNBC.

AV-casNet: Fully Automatic Arteriole-Venule Segmentation and Differentiation in OCT Angiography.

Automatic segmentation and differentiation of retinal arteriole and venule (AV), defined as small blood vessels directly before and after the capillary plexus, are of great importance for the diagnosis of various eye diseases and systemic diseases, such as diabetic retinopathy, hypertension, and cardiovascular diseases. Optical coherence tomography angiography (OCTA) is a recent imaging modality that provides capillary-level blood flow information. However, OCTA does not have the colorimetric and geometric differences between AV as the fundus photography does. Various methods have been proposed to differentiate AV in OCTA, which typically needs the guidance of other imaging modalities. In this study, we propose a cascaded neural network to automatically segment and differentiate AV solely based on OCTA. A convolutional neural network (CNN) module is first applied to generate an initial segmentation, followed by a graph neural network (GNN) to improve the connectivity of the initial segmentation. Various CNN and GNN architectures are employed and compared. The proposed method is evaluated on multi-center clinical datasets, including 3 ×3 mm2 and 6 ×6 mm2 OCTA. The proposed method holds the potential to enrich OCTA image information for the diagnosis of various diseases.

Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice.

Aging inducing the development of senescent cells (SNCs) in various tissues is considered as the main cause of the age-related diseases. Senotherapy has become a promising anti-aging therapy. However, the effectivity and side-effect of senolytic agents are still concern. Here, we observed the downregulation of senescence-related genes by adoptive infusion of natural killer (NK) cells in 26 cases in peripheral blood CD3+ T cells. NK cell treatment also significantly decreased levels of senescence markers and senescence-associated secretory phenotypes (SASPs) in three senescent adipose tissues when culturing them together. Interestingly, cytotoxic activity of mouse NK cells against SNCs was significantly enhanced by dopamine in vitro through D1-like receptors. Acein, dopamine-releasing peptide, promoted the adoptive infusion of NK cells in effectively eliminating SNCs in a variety of tissues and reduced local and systemic SASPs in aging mice but Acein alone did not have the senolytic effect. These data demonstrated that adoptive infusion of NK cells is an effective means in removing SNCs, and peptide Acein combined with NK cells further enhances this effect in aging mice.

Natural killer cell-derived exosomal miR-1249-3p attenuates insulin resistance and inflammation in mouse models of type 2 diabetes.

Natural killer (NK) cells have been suggested to be associated with type 2 diabetes by regulating systemic inflammation. However, the mechanism by which NK cells regulate insulin sensitivity remains unknown. This study shows that NK-derived exosomes from lean mice attenuate obesity-induced insulin resistance and inflammation in mice of type 2 diabetes. Moreover, lean NK-derived exosomes enhance insulin sensitivity and relieve inflammation in adipocytes and hepatocytes. MiR-1249-3p, which is significantly upregulated in lean NK-derived exosomes, can be transferred from NK cells to adipocytes and hepatocytes via exosomes. NK-derived exosomal miR-1249-3p dramatically induces cellular insulin sensitivity and relieves inflammation. Mechanistically, exosomal miR-1249-3p directly targets SKOR1 to regulate the formation of ternary complex SMAD6/MYD88/SMURF1, which mediates glucose homeostasis by suppressing the TLR4/NF-κB signaling pathway. This study reveals an emerging role for NK-derived exosomal miR-1249-3p in remission of insulin resistance, and provides a series of potential therapeutic targets in type 2 diabetes.

Apigenin Inhibits the Growth of Hepatocellular Carcinoma Cells by Affecting the Expression of microRNA Transcriptome.

BACKGROUND: Apigenin, as a natural flavonoid, has low intrinsic toxicity and has potential pharmacological effects against hepatocellular carcinoma (HCC). However, the molecular mechanisms involving microRNAs (miRNAs) and their target genes regulated by apigenin in the treatment of HCC have not been addressed. OBJECTIVE: In this study, the molecular mechanisms of apigenin involved in the prevention and treatment of HCC were explored in vivo and in vitro using miRNA transcriptomic sequencing to determine the basis for the clinical applications of apigenin in the treatment of HCC. METHODS: The effects of apigenin on the proliferation, cell cycle progression, apoptosis, and invasion of human hepatoma cell line Huh7 and Hep3B were studied in vitro, and the effects on the tumorigenicity of Huh7 cells were assessed in vivo. Then, a differential expression analysis of miRNAs regulated by apigenin in Huh7 cells was performed using next-generation RNA sequencing and further validated by qRT-PCR. The potential genes targeted by the differentially expressed miRNAs were identified using a curated miRTarBase miRNA database and their molecular functions were predicted using Gene Ontology and KEGG signaling pathway analysis. RESULTS: Compared with the control treatment group, apigenin significantly inhibited Huh7 cell proliferation, cell cycle, colony formation, and cell invasion in a concentration-dependent manner. Moreover, apigenin reduced tumor growth, promoted tumor cell necrosis, reduced the expression of Ki67, and increased the expression of Bax and Bcl-2 in the xenograft tumors of Huh7 cells. Bioinformatics analysis of the miRNA transcriptome showed that hsa-miR-24, hsa-miR-6769b-3p, hsa-miR-6836-3p, hsa-miR-199a-3p, hsa-miR-663a, hsa-miR-4739, hsa-miR-6892-3p, hsa-miR-7107-5p, hsa-miR-1273g-3p, hsa-miR-1343, and hsa-miR-6089 were the most significantly up-regulated miRNAs, and their key gene targets were MAPK1, PIK3CD, HRAS, CCND1, CDKN1A, E2F2, etc. The core regulatory pathways of the up-regulated miRNAs were associated with the hepatocellular carcinoma pathway. The down-regulated miRNAs were hsa-miR-181a-5p and hsa-miR-148a-3p, and the key target genes were MAPK1, HRAS, STAT3, FOS, BCL2, SMAD2, PPP3CA, IFNG, MET, and VAV2, with the core regulatory pathways identified as proteoglycans in cancer pathway. CONCLUSION: Apigenin can inhibit the growth of HCC cells, which may be mediated by up-regulation or down-regulation of miRNA molecules and their related target genes.

Scutellaria barbata D.Don and Oldenlandia diffusa (Willd.) Roxb crude extracts inhibit hepatitis-B-virus-associated hepatocellular carcinoma growth through regulating circRNA expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria barbata D.Don (SB) and Oldenlandia diffusa (Willd.) Roxb are commonly known as Ban Zhi Lian and Bai Hua She Cao in Chinese herbal medicines, respectively. As a pair of herbs, they have traditionally been used as ethnomedicines for clearing away heat and toxins, removing blood stasis, and promoting blood circulation, diuresis, and detumescence. AIM OF THE STUDY: The aim of the present study was to determine the active ingredients in SB and OD extracts and to investigate whether these extracts can inhibit the growth of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) cell lines (HepG2.2.15 and Hep3B) in vitro and in vivo, as well as to explore their mechanisms of action. MATERIALS AND METHODS: We determined the levels of total flavonoids, luteolin, and apigenin in SB and OD extracts via ultraviolet-visible spectrophotometry and high-performance liquid chromatography. The effects of SB and OD extracts on HBV-associated HCC cell growth were assessed by HepG2.2.15 and Hep3B cells phenotype and RNA sequencing of Hep3B cells in vitro, and xenograft models in vivo. RESULTS: The extracts of SB and OD contained total flavonoids. There were active ingredients of luteolin and apigenin in SB, but not in OD. The extracts of SB and OD significantly inhibited HCC growth, migration, invasion, and HBV activity in vitro and in vivo, as well as altered circRNA expression in Hep3B cells. Moreover, we constructed a circRNA-miRNA-mRNA co-expression network. CONCLUSIONS: The extracts of SB and OD may inhibit HCC cell growth and HBV activity in vitro and in vivo through altering circRNA-miRNA-gene expression and that the efficacies of these extracts may be related to the presence of luteolin and apigenin.

Enhanced Safety and Antitumor Efficacy of Switchable Dual Chimeric Antigen Receptor-Engineered T Cells against Solid Tumors through a Synthetic Bifunctional PD-L1-Blocking Peptide.

Chimeric antigen receptor (CAR)-engineered T cells (CAR-Ts) therapy has an excellent efficacy in cancer treatment, especially its impressive results in hematological malignancies. Unfortunately, its application on solid tumors is challenged by the off-target effects caused by lacking of tumor specific antigens and the immunosuppression caused by the tumor microenvironment. We constructed a switchable dual receptor CAR-T cell (sdCAR-T) whose activity relied upon double antigens (mesothelin and fluorescein isothiocyanate) and was strictly controlled by a "switch" (FPBM) consisting of a PD-L1 blocking peptide conjugated to fluorescein isothiocyanate. SdCAR-T cells were activated only when FPBM and cognate tumor cells expressing both PD-L1 and mesothelin coexist. Importantly, long-term proliferation experiments in vitro and the pharmacodynamic study in vivo showed a stronger antitumor activity of this system compared to the second generation mesothelin CAR-T cells. In view of this novel treatment paradigm being safer and more effective than traditional CAR-T cells, it may become a new strategy for the treatment of solid tumors.

Antisense oligonucleotides targeting lncRNA AC104041.1 induces antitumor activity through Wnt2B/ß-catenin pathway in head and neck squamous cell carcinomas.

Long non-coding RNAs (lncRNAs) contribute to the initiation and progression of various tumors, including head and neck squamous carcinoma (HNSCC), which is a common malignancy with high morbidity and low survival rate. However, the mechanism of lncRNAs in HNSCC tumorigenesis remains largely unexplored. In this work, we identified a novel lncRNA AC104041.1 which is highly upregulated and correlated with poor survival in HNSCC patients. Moreover, AC104041.1 overexpression significantly promoted tumor growth and metastasis of HNSCC in vitro and in vivo. Mechanistically, AC104041.1 mainly located in the cytoplasm and could function as ceRNA (competing endogenous RNA) for miR-6817-3p, thereby stabilized Wnt2B, and consequently inducing ß-catenin nuclear translocation and activation. Moreover, we demonstrate that salinomycin, which as a highly effective antibiotic in the elimination of cancer stem cells through the Wnt/ß-catenin signaling, could enhance the inhibition of tumor growth by antisense oligonucleotides (ASO) targeting AC104041.1 in HNSCC cells and PDXs (patient-derived xenograft) model. Thus, our data provide preclinical evidence to support a novel strategy of ASOs targeting AC104041.1 in combination with salinomycin and may as a beneficial treatment approach for HNSCC.

Micropeptide MIAC Inhibits HNSCC Progression by Interacting with Aquaporin 2.

Several important micropeptides encoded by noncoding RNAs have been identified in recent years; however, there have never been any reports of micropeptides in head and neck squamous cell carcinoma (HNSCC). Here we report the discovery and characterization of a human endogenous peptide named micropeptide inhibiting actin cytoskeleton (MIAC). Comprehensive analysis of the TCGA (The Cancer Genome Atlas) database (n = 500), clinical fresh samples (n = 94), and tissue microarrays (n = 60) revealed that lower MIAC expression is correlated with poor overall survival of HNSCC patients. Meanwhile, RNA-sequencing analysis of 9657 human tissues across 32 cancer types from TCGA cohorts found that MIAC is significantly associated with the progression of 5 other different tumors. Mechanistically, MIAC directly interacts with AQP2 (Aquaporin 2) to inhibit the actin cytoskeleton by regulating SEPT2 (Septin 2)/ITGB4 (Integrin Beta 4) and ultimately suppressing the tumor growth and metastasis of HNSCC. Collectively, the mechanism investigation and evaluation of MIAC activity in vivo and in vitro highlights that MIAC plays an important role in HNSCC tumorigenesis.

[Attach importance to the training of critical care physicians in China].

At the beginning of 2020, China is experiencing an unprecedented coronavirus disease 2019 (COVID-19) epidemic. Under the wise leadership of the CPC Central Committee, we have made brilliant achievements in fighting against the 2019 novel coronavirus with the strength of the whole country. Among them, the majority of medical workers in China played a leading role in the fight against the epidemic. In particular, critical care physicians actively signed up and took the initiative to carry out the treatment for COVID-19 patients in Wuhan region, where the epidemic situation was the most serious. It highlights the technological advantages and importance of critical care specialists facing major epidemic and critical patients in China. Therefore, we should attach importance to the development of critical care medicine and the training of physicians in China.

Automated quantification of superficial retinal capillaries and large vessels for diabetic retinopathy on optical coherence tomographic angiography.

Optical coherence tomography angiography (OCTA) is a relatively new technique with capillary-level resolution, which has shown great potential for the diagnosis of diabetic retinopathy (DR). A fully automatic algorithm for the quantitative measurement of microcirculatory changes in sight-threatening DR is presented. The foveal avascular zone (FAZ) segmentation was improved with a graph-theoretic method and the large vessels and capillaries were separately identified and analyzed. The method was evaluated in healthy and diabetic eyes with various stages of retinopathy. Results showed that, compared with the healthy group, the diabetic group showed a significantly larger large vessel density, but a significantly smaller capillary density (P < .001). Circularity of FAZ was significantly smaller while nonperfusion area was significantly larger in the diabetic group. The combined variable of all image metrics reached an area under the ROC of 0.853 (95% CI, 0.784-0.923) for mild to moderate nonproliferative DR and 0.950 (95% CI, 0.922-0.979) for proliferative DR. Microvascular and FAZ changes with various DR stages can be accurately delineated using the developed automatic program. Quantitative metrics on OCTA serve as potential biomarkers for the staging of DR.

Significantly increased anti-tumor activity of carcinoembryonic antigen-specific chimeric antigen receptor T cells in combination with recombinant human IL-12.

BACKGROUND AIMS: Chimeric antigen receptor T cells (CAR-T cells) have been successfully used in treatments of hematological tumors, however, their anti-tumor activity in solid tumor treatments was limited. As IL-12 increases T-cell immune functions, we designed carcinoembryonic antigen (CEA) specific CAR-T (CEA-CAR-T) cells and, for the first time, used them in combination with recombinant human IL-12 (rhIL-12) to treat several types of solid tumors. METHODS: In vitro anti-tumor activity of CEA-CAR-T cells in combination with rhIL-12 was confirmed by evaluation of CEA-CAR-T cell activation, proliferation, and cytotoxicity after co-incubation with CEA-positive or CEA-negative human tumor cells. In vivo anti-tumor activity of CEA-CAR-T cells in combination with rhIL-12 was confirmed in a xenograft model in nude mice for treatments of several types of solid tumors. RESULTS: In vitro experiments confirmed that rhIL-12 significantly increased the activation, proliferation, and cytotoxicity of CEA-CAR-T cells. Similarly, in vivo experiments found that CEA-CAR-T cells in combination with rhIL-12 had significantly enhanced anti-tumor activity than CEA-CAR-T cells in growth inhibition of newly colonized colorectal cancer cell HT-29, pancreatic cancer cell AsPC-1, and gastric cancer cell MGC803. CONCLUSIONS: These works confirmed that simultaneous use of cytokines, for example, rhIL-12, can increase the anti-tumor activity of CAR-T cells, especially for treatments of several types of solid tumors.

MicroRNA-6809-5p mediates luteolin-induced anticancer effects against hepatoma by targeting flotillin 1.

BACKGROUND: Luteolin (3,4,5,7-tetrahydroxy flavone) is a natural flavonoid abundant in fruits and vegetables. Although luteolin has shown pro-apoptotic activity in hepatocellular carcinoma (HCC) cells, the underlying molecular mechanism has not yet been clarified. PURPOSE: The aim of this study is to identify novel miRNAs involved in the action of luteolin in HCC cells and to explore the biological roles of these miRNAs. METHODS: The effect of luteolin on HCC cell growth was assessed using CCK-8 colony formation assay, flow cytometric analysis in vitro, and a xenograft model in vivo. miRNA expression profiles were assessed using next-generation sequencing. Differentially expressed miRNAs were validated by quantitative PCR. Bioinformatics analysis and luciferase reporter assay were utilized to confirm the binding of miR-6809-5p to the 3'-untranslated region (3'-UTR) of flotillin 1 (FLOT1). Furthermore, the effects of ectopic FLOT1 and miR-6809-5 expression on cell proliferation, colony formation, and cell apoptosis were also assessed. Western blotting analysis was used to detect activation of multiple signaling molecules including Erk1/2, p38, JNK, and NF-κB/p65 in the MAPK pathway. RESULTS: It was found that luteolin significantly inhibited HCC growth and caused apoptosis and cell cycle arrest at the G0/G1 phase in Huh7 cells, at the G2/M phase in HepG2 cells in vitro. Tumorigenic studies revealed that luteolin treatment significantly suppressed HCC growth in vivo. miR-6809-5p was upregulated by luteolin. Overexpression of miR-6809-5p suppressed HCC cell growth, while knockdown of miR-6809-5p reversed the anticancer effect of luteolin. With regards to its signaling mechanism, miR-6809-5p directly targets FLOT1in HCC cells. Enforced expression of FLOT1 prevented miR-6809-5p-mediated growth suppression. Downregulation of FLOT1 exerted growth-suppressive effects on HCC cells. Multiple signaling pathways including Erk1/2, p38, JNK, and NF-κB/p65 were inactivated by miR-6809-5p overexpression or FLOT1 downregulation. CONCLUSION: These findings indicated that miR-6809-5p mediates the growth-suppressive activity of luteolin in HCC, which is causally linked to FLOT1 downregulation. Induction of miR-6809-5p may provide therapeutic benefits in the treatment of HCC.

Recombination of a dual-CAR-modified T lymphocyte to accurately eliminate pancreatic malignancy.

BACKGROUND: The therapeutic application of T cells endowing with chimeric antigen receptors (CARs) is faced with "on-target, off-tumor" toxicity against solid tumors, particularly in the treatment of the pancreatic cancer. To our best knowledge, the pancreatic cancer cell line AsPC-1 often highly expressed some distinct tumor-associated antigens, such as carcino-embryonic antigen (CEA) and mesothelin (MSLN). Therefore, in this research, we have characterized dual-receptor CAR-modified T cells (dCAR-T) that exert effective and safe cytotoxicity against AsPC-1 cells. METHODS: Based on the dual signaling pathway of wild T cells, we designed a novel dCAR diagram specific for CEA and MSLN, which achieved comparable activity relative to that of conventional CAR-T cells (CEA-CAR T or MSLN-CAR T). In this dCAR, a tandem construct containing two physically separate structures, CEA-CD3ζ and MSLN-4/1BB signaling domains were effectively controlled with tumor antigens CEA and MSLN, respectively. Finally, the activity of dCAR-T cells has been verified via in vitro and in vivo experiments. RESULTS: In the presence of cognate tumor cells (AsPC-1) expressing both CEA and MSLN, dCAR-T cells exerted high anti-tumor activity relative to that of other single-receptor CAR-T cells bearing only one signaling pathway (e.g., Cζ-CAR and MBB-CAR). In a xenograft model, dCAR-T cells significantly inhibited the growth of AsPC-1 cells yet no effect on the growth of non-cognate tumor cells. Furthermore, the released cytokines and T cell persistence in mice were comparable with that of conventional CAR-T cells, obtaining specific and controllable cytotoxicity. CONCLUSIONS: A novel type of CAR-T cells, termed dCAR-T, was designed with specific activities, that is, significant cytotoxicity for two antigen-positive tumor cells yet no cytotoxicity for single antigen-positive tumor cells. Dual-targeted CAR-T cells can be precisely localized at the tumor site and can exert high cytotoxicity against tumor cells, alleviating "on-target, off-tumor" toxicity and enabling accurate application of CAR-T cell therapy.