The inhibition of Beclin1-dependent autophagy sensitizes PTC cells to ABT737-induced death.

ABT737 is used as a specific BCL2 inhibitor, which can treat papillary thyroid carcinoma (PTC). However, the effect of ABT737 on PTC cell apoptosis is limited. Moreover, BCL2 inhibition causes the activation of Beclin1-dependent autophagy. Our study aimed to explore the effects of autophagy and Beclin1 on ABT737 efficacy in PTC. The experimental data showed that ABT737 synchronously enhanced autophagic activity and apoptosis level in PTC cells. ABT737 also promoted the dissociation of BCL2-Beclin1 and BCL2-Bax complexes. Autophagy inhibitors, Bafilomycin A1 and 3-MA, enhanced the inhibitory effect of ABT737 on the survival and function in PTC cells. Consistently, autophagy inhibition with Beclin1 pharmacological inhibitor (spautin-1) also enhanced the efficacy of ABT737. Additionally, ABT737 at low-dose promoted LC3 conversion in PTC cells, and did not affect PTC cell apoptosis and survival. However, The efficacy of low-dose of ABT737 in PTC cell apoptosis and survival was displayed with the addition of Bafilomycin A1, 3-MA or spautin-1. In conclusion, the limited role of ABT737 in PTC cell apoptosis is attributed to its promoting effect on Beclin1-dependent autophagy. Therefore, autophagy inhibition based on Beclin1 downregulation can enhance the sensitivity of PTC cells to ABT737-induced death.

TAT-Beclin 1 represses the carcinogenesis of DUSP4-positive PTC by enhancing autophagy.

BACKGROUND: DUSP4 is a pro-tumorigenic molecule of papillary thyroid carcinoma (PTC). DUSP4 also exists as an autophagic regulator. Moreover, DUSP4, as a negative regulator of MAPK, can prevent Beclin 1 from participating in autophagic response. This study aimed to explore whether TAT-Beclin 1, a recombinant protein of Beclin 1, could inhibit the tumorigenesis of DUSP4-positive PTC by regulating autophagy. METHODS: First, we divided PTC tissues into three groups according to DUSP4 expression levels by immunohistochemical analyses, and evaluated the relationship between autophagic molecules (Beclin 1 and LC3II) and DUSP4 using Western blotting assays. After overexpression of DUSP4 by lentiviral transduction, the in vitro and in vivo roles of TAT-Beclin 1 on DUSP4-overexpressed PTC cells were assessed (including autophagic activity, cell survival and function, and tumor growth). The roles of TAT-Beclin 1 in the survival of DUSP4-silenced PTC cells were also evaluated. RESULTS: Our results showed that the expression levels of autophagic proteins decreased with the increase of DUSP4 expression in PTC tissues. In PTC cells, DUSP4 overexpression-inhibited autophagic activity (including Beclin 1 expression, LC3 conversion rate and LC3-puncta formation) and -promoted cell proliferation and migration were reversed by TAT-Beclin 1 administration. In vivo assays also showed that DUSP4-overexpressed PTC cells had stronger tumorigenic ability and weaker autophagic activity, which was blocked by TAT-Beclin 1 administration. CONCLUSION: TAT-Beclin 1, as an autophagic promoter, could repress the carcinogenesis of DUSP4-positive PTC, which implies that the use of TAT-Beclin 1 for the PTC patients' treatment might be determined according to the DUSP4 level in their tumors.

PSC subtyping based on TTF-1 and p40 expression reveals distinct molecular characteristics and therapeutic strategies.

Pulmonary sarcomatoid carcinoma (PSC) is a unique form of poorly differentiated nonsmall cell lung cancer (NSCLC) and is notorious for its highly malignant nature and dismal prognosis. To introduce effective treatment for PSC patients, precise subtyping of PSC is demanding. In our study, TTF-1 and P40 immunohistochemistry (IHC) staining were applied to 56 PSC patients with multiomics data. According to IHC results, we categorized these patients into three subgroups and profiled their molecular contexture using bioinformatic skills. IHC results classified these patients into three subgroups: TTF-1 positive subgroup (n = 27), P40 positive subgroup (n = 15) and double-negative subgroup (n = 14). Spindle cell samples accounted for 35.71% (5/14) of double-negative patients, higher than others (P = .034). The three subgroups were heterogeneous in the genomic alteration spectrum, showing significant differences in the RTK/RAS pathway (P = .004) and the cell cycle pathway (P = .030). The methylation profile of the double-negative subgroup was between the other two subgroups. In similarity analysis, the TTF-1 and p40 subgroups were closely related to LUAD and LUSC, respectively. The TTF-1 positive subgroup had the highest leukocyte fraction (LF) among several cancer types, and the tumor mutation burden (TMB) of the p40 positive subgroup ranked third in the TMB list, suggesting the applicability of immunotherapy for PSC. The study established a new subtyping method of PSC based on IHC results and reveals three subgroups with distinct molecular features, providing evidence for refined stratification in the treatment of PSC.

Circ_0027599 elevates RUNX1 expression via sponging miR-21-5p on gastric cancer progression.

BACKGROUND: Increasing evidence has shown that circular RNAs (circRNAs) serve as vital regulators in tumour progression. In this study, we focused on the functions of circ_0027599 in gastric cancer (GC) progression. METHODS: The levels of circ_0027599, runt-related transcription factor 1 (RUNX1) mRNA and microRNA-21-5p (miR-21-5p) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) assay. The protein levels of RUNX1, E-Cadherin, vimentin and N-Cadherin were measured by Western blot assay. Cell viability, colony formation, metastasis and cell cycle process were evaluated by Cell Counting Kit-8 (CCK-8) assay, colony formation assay, transwell assay and flow cytometry analysis, respectively. The interaction between circ_0027599 and miR-21-5p and the interaction between miR-21-5p and RUNX1 were verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The role of circ_0027599 in tumour growth in vivo was investigated by murine xenograft model assay. RESULTS: Circ_0027599 and RUNX1 were downregulated in GC tissues and cells. Circ_0027599 level was associated with the overall survival of GC patients. Circ_0027599 or RUNX1 overexpression inhibited GC cell viability, colony formation, migration, invasion and cell cycle process in vitro. For mechanism analysis, circ_0027599 positively regulated RUNX1 expression via functioning as the sponge for miR-21-5p. RUNX1 inhibition reversed circ_0027599 overexpression mediated malignant behaviours of GC cells. Moreover, circ_0027599 overexpression repressed tumour growth in vivo. CONCLUSION: Circ_0027599 overexpression repressed GC progression via modulation of miR-21-5p/RUNX1 axis, which might illumine a novel therapeutic target for GC.

A novel tumor mutational burden estimation model as a predictive and prognostic biomarker in NSCLC patients.

BACKGROUND: Tumor mutational burden (TMB) has both prognostic value in resected non-small cell lung cancer (NSCLC) patients and predictive value for immunotherapy response. However, TMB evaluation by whole-exome sequencing (WES) is expensive and time-consuming, hampering its application in clinical practice. In our study, we aimed to construct a mutational burden estimation model, with a small set of genes, that could precisely estimate WES-TMB and, at the same time, has prognostic and predictive value for NSCLC patients. METHODS: TMB estimation model was trained based on genomic data from 1056 NSCLC samples from The Cancer Genome Atlas (TCGA). Validation was performed using three independent cohorts, including Rizvi cohort and our own Asian cohorts, including 89 early-stage and n late-stage Asian NSCLC patients, respectively. TCGA data were obtained on September 3, 2018. The two Asian cohort studies were performed from September 1, 2018, to March 5, 2019. Pearson's correlation coefficient was used to assess the performance of estimated TMB with WES-TMB. The Kaplan-Meier survival analysis was applied to evaluate the association of estimated TMB with disease-free survival (DFS), overall survival (OS), and response to anti-programmed death-1 (PD-1) and anti-programmed death-ligand 1 (PD-L1) therapy. RESULTS: The estimation model, consisted of only 23 genes, correlated well with WES-TMB both in the training set of TCGA cohort and validation set of Rizvi cohort and our own Asian cohort. Estimated TMB by the 23-gene panel was significantly associated with DFS and OS in patients with early-stage NSCLC and could serve as a predictive biomarker for anti-PD-1 and anti-PD-L1 treatment response. CONCLUSIONS: The 23-gene panel, instead of WES or the currently used panel-based methods, could be used to assess the WES-TMB with a high relevance. This customized targeted sequencing panel could be easily applied into clinical practice to predict the immunotherapy response and prognosis of NSCLC.

Efficient and tumor-specific knockdown of MTDH gene attenuates paclitaxel resistance of breast cancer cells both in vivo and in vitro.

BACKGROUND: Drug resistance of paclitaxel (TAX), the first-line chemotherapy drug for breast cancer, was reported to develop in 90% of patients with breast cancer, especially metastatic breast cancer. Investigating the mechanism of TAX resistance of breast cancer cells and developing the strategy improving its therapeutic efficiency are crucial to breast cancer cure. METHODS AND RESULTS: We here report an elegant nanoparticle (NP)-based technique that realizes efficient breast cancer treatment of TAX. Using lentiviral vector-mediated gene knockdown, we first demonstrated that TAX therapeutic efficiency was closely correlated with metadherin (MTDH) gene expression in breast cancer cell lines. This finding was also supported by efficacy of TAX treatment in breast cancer patients from our clinical studies. Specifically, TAX treatment became more effective when MTDH expression was decreased in MCF-7 cancer cells by the blocking nuclear factor-kappa B (NF-κB) pathway. Based on these findings, we subsequently synthesized a polymeric NP that could co-deliver MTDH-small interfering RNA (MTDH-siRNA) and TAX into the breast cancer tumors in tumor-bearing mice. The NPs were composed of a cationic copolymer, which wrapped TAX in the inside and adsorbed the negatively charged siRNA on their surface with high drug-loading efficiency and good stability. CONCLUSIONS: NP-based co-delivery approach can effectively knock down the MTDH gene both in vitro and in vivo, which dramatically inhibits breast tumor growth, achieving effective TAX chemotherapy treatment without overt side effects. This study provides a potential therapeutic strategy for the treatment of a wide range of solid tumors highly expressing MTDH.

Fine-tuned h-ferritin nanocage with multiple gold clusters as near-infrared kidney specific targeting nanoprobe.

When stabilized and functionalized by biomolecules, noble metal (such as gold and silver) cluster-based hybrid nanocomposites have shown great promise for biomedical applications, due to their unique physiochemical properties originating from the inorganic elements and specific functionality and biocompatibility from their biological components. Although certain promise for bioimaging, biosensing, and biomimetic catalysis has been demonstrated, it is still a great challenge to integrate the defined functionality of the biomolecules with enhanced or novel physiochemical properties of the metal clusters, under control at the molecular level. Herein, based on molecular dynamics simulation of a gold (Au) cluster assembly, we designed near-infrared (NIR) fluorescent hybrid nanocomposites with multiple Au clusters within an apo H-ferritin (HFt) nanocage. The fluorescence quantum yield of near-infrared (NIR) Au-HFt is about 63.4% and the emission peak is 810 nm. The NIR Au-HFt is one of the first native protein-guided Au cluster-based nanomaterials for in vivo biowindow imaging. In vivo fluorescent imaging and quantification of Au element confirmed that Au-HFt not only retained the kidney targeting properties of HFt well (about 10 times higher Au concentration in kidney than in liver and spleen, the most common organs for nanoparticle accumulation), but also gained strong NIR imaging capability for live animals. The NIR Au-HFt showed powerful tissue penetrating ability, strong fluorescent efficiency, and excellent kidney targeting specificity. These results thus open new opportunities for kidney disease imaging and theranostic applications.

In vitro and in vivo antioxidant and antimutagenic activities of polyphenols extracted from hops (Humulus lupulus L.).

BACKGROUND: Hops (Humulus lupulus L.) contain 40-140 mg g(-1) polyphenols. The objective of this study was to determine the phenolic composition of a high-purity (total phenolic content = 887 mg g(-1) ) hop polyphenol extract (HPE) and evaluate its antioxidant activities in vivo and in vitro and its antimutagenic activity. The antioxidant activity of HPE was compared with the activity of green tea polyphenols. RESULTS: The phenolic compositions of HPE were more than 55% proanthocyanidins and more than 28% flavonoid glycosides. In vitro, HPE effectively scavenged α,α-diphenyl-ß-picrylhydrazyl, hydroxyl and superoxide anion radicals, and inhibited DNA oxidative damage. In vivo, oral HPE at a polyphenol dose of 200-800 mg kg(-1) body weight significantly prevented a bromobenzene-induced decrease in liver superoxide dismutase and glutathione peroxidase activity, and decreased levels of liver thiobarbituric acid reactive substances in bromobenzene-treated mice. An oral dose of 20-80 mg kg(-1) body weight HPE significantly reduced the frequency of bone marrow micronuclei induced by cyclophosphamide. The antioxidant activities of hop polyphenols in vitro and in vivo were higher than green tea polyphenols at the same concentration. CONCLUSION: Hop polyphenols had the same or higher antioxidant activity than tea polyphenols. Hop polyphenols might be useful as natural antioxidants and antimutagens.

Pseudoginsenoside GQ mitigates chronic intermittent hypoxia-induced cognitive damage by modulating microglia polarization.

Obstructive sleep apnea (OSA), a state of sleep disruption, is characterized by recurrent apnea, chronic intermittent hypoxia (CIH) and hypercapnia. Previous studies have showed that CIH-induced neuroinflammatory plays a crucial role in cognitive deficits. Pseudoginsenoside GQ (PGQ) is a new oxytetracycline-type saponin formed by the oxidation and cyclization of the 20(S) Rg3 side chain. Rg3 has been found to afford anti-inflammatory effects, while whether PGQ plays a role of anti-neuroinflammatory remains unclear. The purpose of this study was to investigate whether PGQ attenuates CIH-induced neuroinflammatory and cognitive impairment and the possible mechanism it involves. We found that PGQ significantly ameliorated CIH-induced spatial learning deficits, and inhibited microglial activation, pro-inflammatory cytokine release, and neuronal apoptosis in the hippocampus of CIH mice. In addition, PGQ pretreatment promoted microglial M1 to M2 phenotypic transition in IH-induced BV-2 microglial, as well as indirectly inhibited IH-induced neuronal injury via modulation of microglia polarization. Furthermore, we noted that activation of HMGB1/TLR4/NF-κB signaling pathway induced by IH was inhibited by PGQ. Molecular docking results revealed that PGQ could bind to the active sites of HMGB1 and TLR4. Taken together, this work supports that PGQ inhibits M1 microglial polarization via the HMGB1/TLR4/NF-κB signaling pathway, and indirectly exerts neuroprotective effects, suggesting that PGQ may be a potential therapeutic strategy for cognitive impairment accompanied OSA.

Tumor fibroblast specific activation of a hybrid ferritin nanocage-based optical probe for tumor microenvironment imaging.

Ferritin-based FAP-α-responsive fluorescence nanoprobes could be activated immediately as they penetrate the tumor blood vessels and come across the FAP-α molecules on the membrane of CAFs in the tumor microenvironment instead of reaching a certain depth into tumor tissue to interact with tumor cells. This probe may become a promising nanomaterial for highly specific and sensitive tumor imaging through responding to the enzymes in the tumor microenvironment.

PHACTR1 promotes the mobility of papillary thyroid carcinoma cells by inducing F-actin formation.

Papillary thyroid carcinoma (PTC) limits effective biomarkers for predicting prognosis and targeted therapy. Phosphatase and actin regulator 1 (PHACTR1) is a mobility-promoting molecule due to its regulation on F-actin formation, which is valuable for the investigation of PTC. Our study aimed to investigate the relationship between PHACTR1 and PTC carcinogenesis, especially mobility. Our results displayed that PHACTR1 expression was elevated in metastatic or larger PTC tissues. In addition, PTC cells K1 with more obvious mobility had higher PHACTR1 expression whereas weakly mobile cells TPC-1 was contrary. Moreover, PHACTR1 silencing inhibited the invasion, migration and tumorigenicity of K1 cells, while PHACTR1 overexpression promoted the invasion, migration and tumorigenicity in TPC-1 cells. Furthermore, PHACTR1 overexpression increased the fluorescent intensity of F-actin in TPC-1 cells. Importantly, the enhanced invasion and migration in TPC-1 cells caused by PHACTR1 overexpression were significantly reversed by the disruption of F-actin assembly with swinholide A. In conclusion, PHACTR1 can promote the mobility of PTC cells, which results in the carcinogenesis of PTC. PHACTR1-regulated F-actin formation determines the mobility of PTC cells. Therefore, PHACTR1 can function as a potential biomarker for predicting prognosis and targeting therapy in PTC.

Abnormal activation of NF-κB and MAPK signaling pathways affect osimertinib resistance and influence the recruitment of myeloid-derived suppressor cells to shape the immunosuppressive tumor immune microenvironment.

BACKGROUND: Osimertinib is the first-line treatment for patients with epidermal growth factor receptor (EGFR) mutations, but the treatment options after drug resistance are limited. Previous studies have suggested that EGFR is in an immunosuppressive tumor immune microenvironment (TIME). However, the evolution of TIME after osimertinib resistance and whether this resistance can be overcome by targeting TIME needs to be further investigated. METHODS: The remodeling process and mechanism of TIME during the treatment with osimertinib were studied. RESULTS: The proportion of EGFRL858R+T790M mutant tumor immune infiltrating cells was extremely low. Osimertinib treatment transiently triggered inflammatory cells, but several immunosuppressive cells infiltrated after drug resistance and formed a myeloid-derived suppressor cell (MDSC)-enriched TIME. The programmed cell death protein-1 monoclonal antibody was not able to reverse the MDSC-enriched TIME. Further analysis revealed that the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways recruited a large number of MDSCs via cytokines. Finally, MDSC secreted high levels of interleukin-10 and arginase-1 and created an immunosuppressive TIME. CONCLUSIONS: Thus, our findings lay the foundation for the evolution of TIME in osimertinib treatment, establish the mechanism of immunosuppressive TIME after osimertinib resistance, and propose potential solutions.

ZEB1 Is Regulated by K811 Acetylation to Promote Stability, NuRD Complex Interactions, EMT, and NSCLC Metastasis.

Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to posttranslational modifications (PTM). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non-small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (∼225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, whereas the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition. IMPLICATIONS: The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in patients with NSCLC.

ZEB1 is regulated by K811 acetylation to promote stability, NuRD complex interactions, EMT, and NSCLC metastasis.

Epithelial-to-mesenchymal transition results in loss of specialized epithelial cell contacts and acquisition of mesenchymal invasive capacity. The transcription repressor zinc finger E-box-binding homeobox 1 (ZEB1) binds to E-boxes of gene promoter regions to suppress the expression of epithelial genes. ZEB1 has inconsistent molecular weights, which have been attributed to post-translational modifications (PTMs). We performed mass spectrometry and identified K811 acetylation as a novel PTM in ZEB1. To define the role of ZEB1 acetylation in regulating function, we generated ZEB1 acetyl-mimetic (K811Q) and acetyl-deficient (K811R) mutant-expressing non-small cell lung cancer cell lines (NSCLC). We demonstrate that the K811R ZEB1 (125 kDa) has a shorter protein half-life than wild-type (WT) ZEB1 and K811Q ZEB1 (&tilde225 kDa), suggesting that lack of ZEB1 acetylation in the lower molecular weight form affects protein stability. Further, the acetylated form of ZEB1 recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that WT ZEB1 and K811Q ZEB1 downregulate the expression of epithelial genes to promote lung adenocarcinoma invasion and metastasis, while the K811R ZEB1 does not. Our findings establish that the K811 acetylation promotes ZEB1 protein stability, interaction with other protein complexes, and subsequent invasion/metastasis of lung adenocarcinoma via epithelial-to-mesenchymal transition. Implications: The molecular mechanisms by which ZEB1 is regulated by K811 acetylation to promote protein stability, NuRD complex and promoter interactions, and function are relevant to the development of treatment strategies to prevent and treat metastasis in NSCLC patients.

Genetic trajectory and clonal evolution of multiple primary lung cancer with lymph node metastasis.

Multiple primary lung cancer (MPLC) with lymph node metastasis (LNM) is a rare phenomenon of multifocal lung cancer. The genomic landscapes of MPLC and the clonal evolution pattern between primary lung lesions and lymph node metastasis haven't been fully illustrated. We performed whole-exome sequencing (WES) on 52 FFPE (Formalin-fixed Paraffin-Embedded) samples from 11 patients diagnosed with MPLC with LNM. Genomic profiling and phylogenetic analysis were conducted to infer the evolutional trajectory within each patient. The top 5 most frequently mutated genes in our study were TTN (76.74%), MUC16 (62.79%), MUC19 (55.81%), FRG1 (46.51%), and NBPF20 (46.51%). For most patients in our study, a substantial of genetic alterations were mutually exclusive among the multiple pulmonary tumors of the same patient, suggesting their heterogenous origins. Individually, the genetic profile of lymph node metastatic lesions overlapped with that of multiple lung cancers in different degrees but are more genetically related to specific pulmonary lesions. SETD2 was a potential metastasis biomarker of MPLC. The mean putative neo-antigen number of the primary tumor (646.5) is higher than that of lymph node metastases (300, p = 0.2416). Primary lung tumors and lymph node metastases are highly heterogenous in immune repertoires. Our findings portrayed the comprehensive genomic landscape of MPLC with LNM. We characterized the genomic heterogeneity among different tumors. We offered novel clues to the clonal evolution between MPLC and their lymphatic metastases, thus advancing the treatment strategies and preventions of MPLC with LNM.

pan-MHC and cross-Species Prediction of T Cell Receptor-Antigen Binding.

Profiling the binding of T cell receptors (TCRs) of T cells to antigenic peptides presented by MHC proteins is one of the most important unsolved problems in modern immunology. Experimental methods to probe TCR-antigen interactions are slow, labor-intensive, costly, and yield moderate throughput. To address this problem, we developed pMTnet-omni, an Artificial Intelligence (AI) system based on hybrid protein sequence and structure information, to predict the pairing of TCRs of αß T cells with peptide-MHC complexes (pMHCs). pMTnet-omni is capable of handling peptides presented by both class I and II pMHCs, and capable of handling both human and mouse TCR-pMHC pairs, through information sharing enabled this hybrid design. pMTnet-omni achieves a high overall Area Under the Curve of Receiver Operator Characteristics (AUROC) of 0.888, which surpasses competing tools by a large margin. We showed that pMTnet-omni can distinguish binding affinity of TCRs with similar sequences. Across a range of datasets from various biological contexts, pMTnet-omni characterized the longitudinal evolution and spatial heterogeneity of TCR-pMHC interactions and their functional impact. We successfully developed a biomarker based on pMTnet-omni for predicting immune-related adverse events of immune checkpoint inhibitor (ICI) treatment in a cohort of 57 ICI-treated patients. pMTnet-omni represents a major advance towards developing a clinically usable AI system for TCR-pMHC pairing prediction that can aid the design and implementation of TCR-based immunotherapeutics.

Nanoparticle-induced exosomes target antigen-presenting cells to initiate Th1-type immune activation.

The mechanisms associated with the induction of systemic immune responses by nanoparticles are not fully understood, but their elucidation is critical to address safety issues associated with the broader medical application of nanotechnology. In this study, a key role of nanoparticle-induced exosomes (extracellularly secreted membrane vesicles) as signaling mediators in the induction of T helper cell type 1 (Th1) immune activation is demonstrated. In vivo exposure to magnetic iron oxide nanoparticles (MIONs) results in significant exosome generation in the alveolar region of Balb/c mice. These act as a source of nanoparticle-induced, membrane-bound antigen/signaling cargo, which transfer their components to antigen-presenting cells (APCs) in the reticuloendothelial system. Through exosome-initiated signals, immature dendritic cells (iDCs) undergo maturation and differentiation to the DC1 subtype, while macrophages go through classical activation and differentiation to the M1 subtype. Simultaneously, iDCs and macrophages release various Th1 cytokines (including interleukin-12 and tumor necrosis factor α) driving T-cell activation and differentiation. Activated APCs (especially DC1 and M1 subtypes) consequently prime T-cell differentiation towards a Th1 subtype, thereby resulting in an orchestrated Th1-type immune response. Th1-polarized immune activation is associated with delayed-type hypersensitivity, which might underlie the long-term inflammatory effects frequently associated with nanoparticle exposure. These studies suggest that nanoparticle-induced exosomes provoke the immune activation and inflammatory responses that can accompany nanoparticle exposure.

N6-methylandenosine-related immune genes correlate with prognosis and immune landscapes in gastric cancer.

Objectives: This study aimed to probe into the significance of N6-methyladenosine (m6A)-related immune genes (m6AIGs) in predicting prognoses and immune landscapes of patients with gastric cancer (GC). Methods: The clinical data and transcriptomic matrix of GC patients were acquired from The Cancer Genome Atlas database. The clinically meaningful m6AIGs were acquired by univariate Cox regression analysis. GC patients were stratified into different clusters via consensus clustering analysis and different risk subgroups via m6AIGs prognostic signature. The clinicopathological features and tumor microenvironment (TME) in the different clusters and different risk subgroups were explored. The predictive performance was evaluated using the KM method, ROC curves, and univariate and multivariate regression analyses. Moreover, we fabricated a nomogram based on risk scores and clinical risk characteristics. Biological functional analysis was performed based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. The connectivity map was used to screen out potential small molecule drugs for GC patients. Results: A total of 14 prognostic m6AIGs and two clusters based on 14 prognostic m6AIGs were identified. A prognostic signature based on 4 m6AIGs and a nomogram based on independent prognostic factors was constructed and validated. Different clusters and different risk subgroups were significantly correlated with TME scores, the distribution of immune cells, and the expression of immune checkpoint genes. Some malignant and immune biological processes and pathways were correlated with the patients with poor prognosis. Ten small molecular drugs with potential therapeutic effect were screened out. Conclusions: This study revealed the prognostic role and significant values of m6AIGs in GC, which enhanced the understanding of m6AIGs and paved the way for developing predictive biomarkers and therapeutic targets for GC.

Molecular profiling of human non-small cell lung cancer by single-cell RNA-seq.

BACKGROUND: Lung cancer, one of the most common malignant tumors, exhibits high inter- and intra-tumor heterogeneity which contributes significantly to treatment resistance and failure. Single-cell RNA sequencing (scRNA-seq) has been widely used to dissect the cellular composition and characterize the molecular properties of cancer cells and their tumor microenvironment in lung cancer. However, the transcriptomic heterogeneity among various cancer cells in non-small cell lung cancer (NSCLC) warrants further illustration. METHODS: To comprehensively analyze the molecular heterogeneity of NSCLC, we performed high-precision single-cell RNA-seq analyses on 7364 individual cells from tumor tissues and matched normal tissues from 19 primary lung cancer patients and 1 pulmonary chondroid hamartoma patient. RESULTS: In 6 of 16 patients sequenced, we identified a significant proportion of cancer cells simultaneously expressing classical marker genes for two or even three histologic subtypes of NSCLC-adenocarcinoma (ADC), squamous cell carcinoma (SCC), and neuroendocrine tumor (NET) in the same individual cell, which we defined as mixed-lineage tumor cells; this was verified by both co-immunostaining and RNA in situ hybridization. These data suggest that mixed-lineage tumor cells are highly plastic with mixed features of different types of NSCLC. Both copy number variation (CNV) patterns and mitochondrial mutations clearly showed that the mixed-lineage and single-lineage tumor cells from the same patient had common tumor ancestors rather than different origins. Moreover, we revealed that patients with high mixed-lineage features of different cancer subtypes had worse survival than patients with low mixed-lineage features, indicating that mixed-lineage tumor features were associated with poorer prognosis. In addition, gene signatures specific to mixed-lineage tumor cells were identified, including AKR1B1. Gene knockdown and small molecule inhibition of AKR1B1 can significantly decrease cell proliferation and promote cell apoptosis, suggesting that AKR1B1 plays an important role in tumorigenesis and can serve as a candidate target for tumor therapy of NSCLC patients with mixed-lineage tumor features. CONCLUSIONS: In summary, our work provides novel insights into the tumor heterogeneity of NSCLC in terms of the identification of prevalent mixed-lineage subpopulations of cancer cells with combined signatures of SCC, ADC, and NET and offers clues for potential treatment strategies in these patients.

Single-cell transcriptomic profiling reveals the tumor heterogeneity of small-cell lung cancer.

Small-cell lung cancer (SCLC) is the most aggressive and lethal subtype of lung cancer, for which, better understandings of its biology are urgently needed. Single-cell sequencing technologies provide an opportunity to profile individual cells within the tumor microenvironment (TME) and investigate their roles in tumorigenic processes. Here, we performed high-precision single-cell transcriptomic analysis of ~5000 individual cells from primary tumors (PTs) and matched normal adjacent tissues (NATs) from 11 SCLC patients, including one patient with both PT and relapsed tumor (RT). The comparison revealed an immunosuppressive landscape of human SCLC. Malignant cells in SCLC tumors exhibited diverse states mainly related to the cell cycle, immune, and hypoxic properties. Our data also revealed the intratumor heterogeneity (ITH) of key transcription factors (TFs) in SCLC and related gene expression patterns and functions. The non-neuroendocrine (non-NE) tumors were correlated with increased inflammatory gene signatures and immune cell infiltrates in SCLC, which contributed to better responses to immune checkpoint inhibitors. These findings indicate a significant heterogeneity of human SCLC, and intensive crosstalk between cancer cells and the TME at single-cell resolution, and thus, set the stage for a better understanding of the biology of SCLC as well as for developing new therapeutics for SCLC.