Gene features of tumor-specific T cells relevant to immunotherapy, targeted therapy and chemotherapy in lung cancer.

1 Background: In lung cancer, the use of small-molecule inhibitors, chemotherapy and immunotherapy has led to unprecedented survival benefits in selected patients. Considering most patients will experience a relapse within a short period of time due to single drug resistance, combination therapy is also particularly important to improve patient prognosis. Therefore, more robust biomarkers to predict responses to immunotherapy, targeted therapy, chemotherapy and rationally drug combination therapies may be helpful in clinical treatment choices. 2 Methods: We defined tumor-specific T cells (TSTs) and their features (TSTGs) by single-cell RNA sequencing. We applied LASSO regression to filter out the most survival-relevant TSTGs to form the Tumor-specific T cell score (TSTS). Immunological characteristics, enriched pathways, and mutation were evaluated in high- and low TSTS groups. 3 Results: We identified six clusters of T cells as TSTs in lung cancer, and four most robust genes from 9 feature genes expressed only on tumor-specific T cells were screened to construct a tumor-specific T cells score (TSTS). TSTS was positively correlated with immune infiltration and angiogenesis and negatively correlated with malignant cell proliferation. Moreover, potential vascular-immune crosstalk in lung cancer provides the theoretical basis for combined anti-angiogenic and immunotherapy. Noticeable, patients in high TSTS had better response to ICB and targeted therapy and patients in the low TSTS group often benefit from chemotherapy. 4 Conclusion: The proposed TSTS is a promising indicator to predict immunotherapy, targeted therapy and chemotherapy responses in lung cancer patients for helping clinical treatment choices.

In vitro determination of osteo-adipogenic lineage choice of bone marrow stromal/stem cells (BMSCs).

Bone marrow stromal/stem cells (BMSCs) are primitive and heterogeneous cells that can be differentiated into osteoblasts, adipocytes and other subsets. Their bone-fat lineage commitment is responsible for the homeostasis of bone marrow microenvironment. However, there are little effective methods and evidence to simultaneously visualise the lineage commitment of BMSCs. Here we provide a bivalent differentiation medium that can enable BMSCs differentiation into osteoblasts and adipocytes in vitro, and establish a method to simultaneously distinguish osteoblasts or adipocytes from the heterogeneous BMSCs based on Alizarin red S and Oil red O staining, which have been used for detection of specific mineralized nodules and lipid droplets, respectively. This assay provides a specifically simple but effective and low-cost method to evaluate the efficiency of osteo-adipogenic (OA) allocation of BMSCs.►Researchers can utilize the bivalent differentiation medium to evaluate the efficiency of osteogenic and adipogenic differentiation of BMSCs in vitro.

Novel-miR-81 Promotes the Chondrocytes Differentiation of Bone Marrow Mesenchymal Stem Cells Through Inhibiting Rac2 Expression.

OBJECTIVE: MicroRNAs (miRNAs) play a key role in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes. Our previous study found that novel-miR-81 can relieve osteoarthritis, but its role in chondrogenic differentiation of BMSCs remains unclear. The purpose of this study was to explore the role of novel-miR-81 in chondrogenic differentiation of BMSCs. METHODS: We used a model in which transforming growth factor (TGF)-ß3-induced BMSCs differentiation into chondrocytes. We detected the expression Sox9, Collagen Ⅱ, Aggrecan, novel-miR-81, and Rac2 by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blot was performed to detect the expression of Sox9, Collagen Ⅱ, and Rac2. Dual-luciferase reporter gene assay confirmed that the association between novel-miR-81 and Rac2. In addition, the ectopic chondrocyte differentiation of BMSCs was performed subcutaneously in nude mice. The effect of novel-miR-81 and Rac2 on ectopic chondrogenic differentiation of BMSCs was determined by immunohistochemical staining. RESULTS: Novel-miR-81 upregulated in chondrogenic differentiation of BMSCs. Rac2 was a key target of novel-miR-81. Mimic novel-miR-81 and siRac2 upregulated the expression of Sox9, Collagen Ⅱ, and Aggrecan. CONCLUSION: Novel-miR-81 promotes the chondrocytes differentiation of BMSCs by inhibiting the expression of target gene Rac2, which provides potential targets for BMSCs transplantation to repair cartilage defects.

Prediction of microvascular invasion in hepatocellular carcinoma based on preoperative Gd-EOB-DTPA-enhanced MRI: Comparison of predictive performance among 2D, 2D-expansion and 3D deep learning models.

Purpose: To evaluate and compare the predictive performance of different deep learning models using gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced MRI in predicting microvascular invasion (MVI) in hepatocellular carcinoma. Methods: The data of 233 patients with pathologically confirmed hepatocellular carcinoma (HCC) treated at our hospital from June 2016 to June 2021 were retrospectively analyzed. Three deep learning models were constructed based on three different delineate methods of the region of interest (ROI) using the Darwin Scientific Research Platform (Beijing Yizhun Intelligent Technology Co., Ltd., China). Manual segmentation of ROI was performed on the T1-weighted axial Hepatobiliary phase images. According to the ratio of 7:3, the samples were divided into a training set (N=163) and a validation set (N=70). The receiver operating characteristic (ROC) curve was used to evaluate the predictive performance of three models, and their sensitivity, specificity and accuracy were assessed. Results: Among 233 HCC patients, 109 were pathologically MVI positive, including 91 men and 18 women, with an average age of 58.20 ± 10.17 years; 124 patients were MVI negative, including 93 men and 31 women, with an average age of 58.26 ± 10.20 years. Among three deep learning models, 2D-expansion-DL model and 3D-DL model showed relatively good performance, the AUC value were 0.70 (P=0.003) (95% CI 0.57-0.82) and 0.72 (P<0.001) (95% CI 0.60-0.84), respectively. In the 2D-expansion-DL model, the accuracy, sensitivity and specificity were 0.7143, 0.739 and 0.688. In the 3D-DL model, the accuracy, sensitivity and specificity were 0.6714, 0.800 and 0.575, respectively. Compared with the 3D-DL model (based on 3D-ResNet), the 2D-DL model is smaller in scale and runs faster. The frames per second (FPS) for the 2D-DL model is 244.7566, which is much larger than that of the 3D-DL model (73.3374). Conclusion: The deep learning model based on Gd-EOB-DTPA-enhanced MRI could preoperatively evaluate MVI in HCC. Considering that the predictive performance of 2D-expansion-DL model was almost the same as the 3D-DL model and the former was relatively easy to implement, we prefer the 2D-expansion-DL model in practical research.

Atractylodes lancea volatile oils target ADAR2-miR-181a-5p signaling to mesenchymal stem cell chondrogenic differentiation.

Atractylodeslancea Rhizoma (Rhizoma atractylodis [RA]) has long been recommended for the treatment of arthritis in traditional Chinese medicine, but its mechanism of action is still unclear. RA contains a large amount of Atractylodes lancea volatile oils (Atr). In this study, we investigated whether Atr can promote mesenchymal stem cells (MSCs) chondrogenic differentiation. The Atr were extracted from RA by steam distillation method, and the effect of Atr on MSCs was detected by the CCK8 assay. The optimal concentration of Atr for MSCs cultivation was 3 µg/ml. The differentially expressed miR-181a-5p was screened by miRNA microarray assay, and its mimics and inhibitors were transfected into MSCs. It was found that the inhibitor of miR-181a-5p could upregulate cartilage-specific genes such as SOX9, COL2A1, and ACAN. Meanwhile, we also found that the expression of gene editing enzyme ADAR2 was significantly increased in the chondrogenic differentiation of MSCs induced by Atr, and the bases of precursor sequence of miR-181a-5p were changed from A to G. After ADAR2 deletion, the expression of cartilage-specific genes was significantly down-regulated and the precursor sequence bases of miR-181a-5p were not changed. Bioinformatics analysis revealed that the predicted target gene of miR-181a-5p was yingyang1 (YY1), and the targeting relationship was verified by dual-luciferase reporter assay. After deleting YY1, the expression of cartilage-specific genes was significantly down-regulated. In conclusion, our study demonstrated that Atr can promote chondrogenic differentiation of MSC through regulation of the ADAR2-miR-181a-5p signaling pathway. This may provide a new insight into the possible mechanism of traditional Chinese medicine (Atr) in treating inflammatory joint diseases.

TIGER: A Web Portal of Tumor Immunotherapy Gene Expression Resource.

Immunotherapy is a promising cancer treatment method; however, only a few patients benefit from it. The development of new immunotherapy strategies and effective biomarkers of response and resistance is urgently needed. Recently, high-throughput bulk and single-cell gene expression profiling technologies have generated valuable resources. However, these resources are not well organized and systematic analysis is difficult. Here, we present TIGER, a tumor immunotherapy gene expression resource, which contains bulk transcriptome data of 1508 tumor samples with clinical immunotherapy outcomes and 11,057 tumor/normal samples without clinical immunotherapy outcomes, as well as single-cell transcriptome data of 2,116,945 immune cells from 655 samples. TIGER provides many useful modules for analyzing collected and user-provided data. Using the resource in TIGER, we identified a tumor-enriched subset of CD4+ T cells. Patients with melanoma with a higher signature score of this subset have a significantly better response and survival under immunotherapy. We believe that TIGER will be helpful in understanding anti-tumor immunity mechanisms and discovering effective biomarkers. TIGER is freely accessible at http://tiger.canceromics.org/.

G-Protein Subunit Gamma 4 as a Potential Biomarker for Predicting the Response of Chemotherapy and Immunotherapy in Bladder Cancer.

BACKGROUND: GNG4, a member of the G-protein γ family, is a marker of poor overall survival (OS) rates in some malignancies. However, the potential role of GNG4 in bladder cancer (BLCA) is unknown. It is also unclear whether GNG4 may be utilized as a marker to guide chemotherapy or immunotherapy. METHODS: Single-cell RNA sequencing data were used to explore the expression of GNG4 in tumor microenvironment of BLCA. Bulk RNA sequencing data from TCGA were used to evaluate the relationship between GNG4 expression and biological features, such as immune cell infiltrations and gene mutations. The associations between GNG4 expression and survival in BLCA patients under or not under immunotherapy were evaluated using seven BLCA cohorts. RESULTS: GNG4 was specifically expressed in exhausted CD4+ T cells. And the high expression of the GNG4 was associated with high level of immune cell infiltration. The high-GNG4-expression group displayed a better response to immunotherapy, whereas patients in the low-GNG4-expression group often benefited from chemotherapy. Moreover, the high-GNG4 group was more similar to the basal group, whereas the low-GNG4 group was similar to the luminal group. CONCLUSIONS: GNG4 may be a potential biomarker for the prediction of the response to therapy in BLCA. Higher GNG4 expression can be used as a predictor of response to immunotherapy, and lower GNG4 expression can be used as a predictor of response to chemotherapy.

Dual-acting antitumor agents targeting the A2A adenosine receptor and histone deacetylases: Design and synthesis of 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine derivatives.

Based on its inhibition by antagonists, the A2A adenosine receptor (A2AAR) has attracted attention as an anti-tumor drug target; however, in preclinical models and clinical trials, A2AAR antagonists have so far shown only limited efficacy as standalone therapies. The design of dual-acting compounds, targeting the A2AAR and histone deacetylases (HDACs), is used here as an approach to the discovery of novel and more potent antitumor agents. Based on the core structures of the A2AAR antagonists V-2006 and CPI-444, novel 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine derivatives were designed as such dual-acting compounds. The binding affinities for A2AAR of all the new compounds were tested, and their HDAC inhibitory activity was evaluated. Compounds with balanced A2AAR antagonism and HDAC inhibition were tested for their in vitro anti-proliferative activity and pharmacokinetic properties. One of the compounds, 14c (4-(2-(6-Amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-(2-amino-phenyl)benzamide) showed an overall favorable pharmacokinetic profile; in the mouse MC38 xenograft model, it showed potent anti-tumor effects with inhibition rates of 44% (90 mg/kg, po, bid) and 85% (60 mg/kg, ip, bid), respectively.

MeRIPseqPipe: an integrated analysis pipeline for MeRIP-seq data based on Nextflow.

SUMMARY: MeRIPseqPipe is an integrated and automatic pipeline that can provide users a friendly solution to perform in-depth mining of MeRIP-seq data. It integrates many functional analysis modules, range from basic processing to downstream analysis. All the processes are embedded in Nextflow with Docker support, which ensures high reproducibility and scalability of the analysis. MeRIPseqPipe is particularly suitable for analyzing a large number of samples at once with a simple command. The final output directory is structured based on each step and tool. And visualization reports containing various tables and plots are provided as HTML files. AVAILABILITY AND IMPLEMENTATION: MeRIPseqPipe is freely available at https://github.com/canceromics/MeRIPseqPipe. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The Crosstalk Between Malignant Cells and Tumor-Promoting Immune Cells Relevant to Immunotherapy in Pancreatic Ductal Adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) is dominated by an immunosuppressive microenvironment, which makes immune checkpoint blockade (ICB) often non-responsive. Understanding the mechanisms by which PDAC forms an immunosuppressive microenvironment is important for the development of new effective immunotherapy strategies. Methods: This study comprehensively evaluated the cell-cell communications between malignant cells and immune cells by integrative analyses of single-cell RNA sequencing data and bulk RNA sequencing data of PDAC. A Malignant-Immune cell crosstalk (MIT) score was constructed to predict survival and therapy response in PDAC patients. Immunological characteristics, enriched pathways, and mutations were evaluated in high- and low MIT groups. Results: We found that PDAC had high level of immune cell infiltrations, mainly were tumor-promoting immune cells. Frequent communication between malignant cells and tumor-promoting immune cells were observed. 15 ligand-receptor pairs between malignant cells and tumor-promoting immune cells were identified. We selected genes highly expressed on malignant cells to construct a Malignant-Immune Crosstalk (MIT) score. MIT score was positively correlated with tumor-promoting immune infiltrations. PDAC patients with high MIT score usually had a worse response to immune checkpoint blockade (ICB) immunotherapy. Conclusion: The ligand-receptor pairs identified in this study may provide potential targets for the development of new immunotherapy strategy. MIT score was established to measure tumor-promoting immunocyte infiltration. It can serve as a prognostic indicator for long-term survival of PDAC, and a predictor to ICB immunotherapy response.

End-to-end multimodal image registration via reinforcement learning.

Multimodal image registration is a vital initial step in several medical image applications for providing complementary information from different data modalities. Since images with different modalities do not exhibit the same characteristics, finding their accurate correspondences remains a challenge. For convolutional multimodal registration methods, two components are quite significant: descriptive image feature as well as the suited similarity metric. However, these two components are often custom-designed and are infeasible to the high diversity of tissue appearance across modalities. In this paper, we translate image registration into a decision-making problem, where registration is achieved via an artificial agent trained by asynchronous reinforcement learning. More specifically, convolutional long-short-term-memory is incorporated after stacked convolutional layers in this method to extract spatial-temporal image features and learn the similarity metric implicitly. A customized reward function driven by landmark error is advocated to guide the agent to the correct registration direction. A Monte Carlo rollout strategy is also leveraged to perform as a look-ahead inference in the testing stage, to increase registration accuracy further. Experiments on paired CT and MR images of patients diagnosed as nasopharyngeal carcinoma demonstrate that our method achieves state-of-the-art performance in medical image registration.

Effect of Flow State of Pure Aluminum and A380 Alloy on Porosity of High Pressure Die Castings.

Air entrapment defects prevent the heat treatment from improving the mechanical properties of die castings, which limits the die casting of high-performance components. The flow pattern of the filling process is complicated and experimental analysis is difficult in thin-walled complex die castings. In this study, we constructed a shock absorption tower to observe in real-time the filling process of pure aluminum and A380 aluminum alloy at different fast injection speeds. The degree of breakup of pure aluminum was larger than that of A380 during the filling process, which caused the porosity of pure aluminum to be greater than that of the A380 at each observation position. Re-Oh diagrams explained the difference in porosity between the two metals. The porosity in different regions was closely related to the flow state of aluminum liquid. In addition to porosity measurements, we specifically analyzed the relationship between the porosity of the flowback zone, the final filling zone, and the near-tail zone of cylinder. At the same injection velocity, the porosity at flowback zone was greater than that at the final filling position, the porosity at final filling position was larger than that at the near-tail zone of cylinder, and the final filling position changed as the injection velocity changed.

TET2-Mediated Spatiotemporal Changes of 5-Hydroxymethylcytosine During Organogenesis in the Late Mouse Fetus.

Genomic DNA demethylation is important for mammalian embryonic development and organ function. 5-Hydroxymethylcytosine (5hmC) is considered a novel epigenetic marker. Ten-eleven translocation (TET) enzymes convert 5-methylcytosine (5mC) to 5hmC. To explore the dynamic changes of epigenetic modifications during organogenesis in the late mouse fetus, the regional distribution and histological localization of 5hmC and TET enzymes was investigated by immunohistochemical method. The liver of mouse fetus gradually matured from embryonic day (E) 12.5 to E18.5.5mC was positive in developing liver at E16.5 and E18.5. 5hmC, TET2 and TET3 were strongly positive in hepatocytes and oval cells at E18.5. The small intestinal villi were formed at E16.5. The striate border and goblet cells appeared at E18.5. 5mC was detectable from E12.5 to E18.5. 5hmC and TET2 were positive in small intestine at E12.5, E14.5, and E18.5. The alveolar was formed at E18.5. 5mC and 5hmC were detectable from E12.5 to E18.5. Only TET2 was positive in the lung of the late Kunming mouse fetus. For vertebra, mesenchymal cells formed hyaline cartilage at E15.5 and then ossify at E16.5 and E18.8. 5mC, 5hmC, and TET2 were detectable in chondrocytes and osteocytes during the late Kunming mouse fetal; TET1 expressed from E14.5 to E16.5 and TET3 expressed in bone matrix at E18.5. In summary, TET2 was strongly expressed in liver, small intestinal, lung, and vertebra in the late Kunming mouse fetus. These findings suggested that TET2 may play a more critical role than TET1 and TET3 during organogenesis in the late stage of Kunming mouse embryo. Anat Rec, 302:954-963, 2019. © 2018 Wiley Periodicals, Inc.

Mechano-growth factor enhances differentiation of bone marrow-derived mesenchymal stem cells.

OBJECTIVES: To investigate the effect of mechano-growth factor (MGF) on the differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) in vitro. RESULTS: Flow cytometry assay identified the isolated cells were human bone marrow mesenchymal stem cells, which had differentiation ability when cultured with specific induction culture media. Alizarin Red S, Oil Red O and Alcian Blue staining showed osteogenic, adipogenic and chondrogenic differentiation were significantly increased after hBMSCs were treated with MGF E peptide. Collagen II expression was considerably increased after hBMSCs were induced with chondrogenic induction culture medium supplemented with TGF-ß3 and MGF E peptide. Overexpression of MGF by an expression plasmid further confirmed the MGF could enhance tri-lineage differentiation of hBMSCs. Moreover, we found that hBMSCs proliferation rate was decreased and G1 phase of the cell cycle was lengthened after MGF treatment when compared to the control group. CONCLUSIONS: MGF can enhance differentiation of hBMSCs during specific induction culture media induction by lengthening G1 phase of cell cycle.

Mechano growth factor (MGF) and transforming growth factor (TGF)-ß3 functionalized silk scaffolds enhance articular hyaline cartilage regeneration in rabbit model.

Damaged cartilage has poor self-healing ability and usually progresses to scar or fibrocartilaginous tissue, and finally degenerates to osteoarthritis (OA). Here we demonstrated that one of alternative isoforms of IGF-1, mechano growth factor (MGF) acted synergistically with transforming growth factor ß3 (TGF-ß3) embedded in silk fibroin scaffolds to induce chemotactic homing and chondrogenic differentiation of mesenchymal stem cells (MSCs). Combination of MGF and TGF-ß3 significantly increased cell recruitment up to 1.8 times and 2 times higher than TGF-ß3 did in vitro and in vivo. Moreover, MGF increased Collagen II and aggrecan secretion of TGF-ß3 induced hMSCs chondrogenesis, but decreased Collagen I in vitro. Silk fibroin (SF) scaffolds have been widely used for tissue engineering, and we showed that methanol treated pured SF scaffolds were porous, similar to compressive module of native cartilage, slow degradation rate and excellent drug released curves. At 7 days after subcutaneous implantation, TGF-ß3 and MGF functionalized silk fibroin scaffolds (STM) recruited more CD29+/CD44+cells (P<0.05). Similarly, more cartilage-like extracellular matrix and less fibrillar collagen were detected in STM scaffolds than that in TGF-ß3 modified scaffolds (ST) at 2 months after subcutaneous implantation. When implanted into articular joints in a rabbit osteochondral defect model, STM scaffolds showed the best integration into host tissues, similar architecture and collagen organization to native hyaline cartilage, as evidenced by immunostaining of aggrecan, collagen II and collagen I, as well as Safranin O and Masson's trichrome staining, and histological evalution based on the modified O'Driscoll histological scoring system (P<0.05), indicating that MGF and TGF-ß3 might be a better candidate for cartilage regeneration. This study demonstrated that TGF-ß3 and MGF functionalized silk fibroin scaffolds enhanced endogenous stem cell recruitment and facilitated in situ articular cartilage regeneration, thus providing a novel strategy for cartilage repair.

Human iPSC-derived neural crest stem cells promote tendon repair in a rat patellar tendon window defect model.

Induced pluripotent stem cells (iPSCs) hold great potential for cell therapy and tissue engineering. Neural crest stem cells (NCSCs) are multipotent that are capable of differentiating into mesenchymal lineages. In this study, we investigated whether iPSC-derived NCSCs (iPSC-NCSCs) have potential for tendon repair. Human iPSC-NCSCs were suspended in fibrin gel and transplanted into a rat patellar tendon window defect. At 4 weeks post-transplantation, macroscopical observation showed that the repair of iPSC-NCSC-treated tendons was superior to that of non-iPSC-NCSC-treated tendons. Histological and mechanical examinations revealed that iPSC-NCSCs treatment significantly enhanced tendon healing as indicated by the improvement in matrix synthesis and mechanical properties. Furthermore, transplanted iPSC-NCSCs produced fetal tendon-related matrix proteins, stem cell recruitment factors, and tenogenic differentiation factors, and accelerated the host endogenous repair process. This study demonstrates a potential strategy of employing iPSC-derived NCSCs for tendon tissue engineering.