Posttranscriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among posttranscriptional processes, alternative polyadenylation (APA) is ...globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in patients with acute myeloid leukemia (AML) by performing 3‘ region extraction and deep sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3‘ untranslated region (UTR) shortening and coding sequence lengthening due to differences in poly(A) site (PAS) usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS data set. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mechanistic target of rapamycin complex 1 (mTORC1) signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block in patients with AML.
•FIP1L1 knockdown reverses global APA dysregulation in t(8;21) AML and promotes differentiation by 3'UTR lengthening of AML1-ETO.•Targeting APA induces differentiation across AML subtypes by downregulation of MYC and attenuation of oncogenic growth signaling pathways.
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Epithelial-mesenchymal transition (EMT) is a pivotal mechanism for cancer dissemination. However, EMT-regulated individual cancer cell invasion is difficult to detect in clinical samples. Emerging ...evidence implies that EMT is correlated to collective cell migration and invasion with unknown mechanisms. We show that the EMT transcription factor Snail elicits collective migration in squamous cell carcinoma by inducing the expression of a tight junctional protein, claudin-11. Mechanistically, tyrosine-phosphorylated claudin-11 activates Src, which suppresses RhoA activity at intercellular junctions through p190RhoGAP, maintaining stable cell-cell contacts. In head and neck cancer patients, the Snail-claudin-11 axis prompts the formation of circulating tumour cell clusters, which correlate with tumour progression. Overexpression of snail correlates with increased claudin-11, and both are associated with a worse outcome. This finding extends the current understanding of EMT-mediated cellular migration via a non-individual type of movement to prompt cancer progression.
Cell cultured meat (CCM) production is an innovative technology that does not depend on livestock farming practices to produce meat. The construction of structured CCM requires a three-dimensional ...(3D) scaffold to mimic the extracellular matrix to provide mechanical support for the cells. Furthermore, the 3D scaffolds should be edible and have good biocompatibility and tissue-like texture. Here, we demonstrated a 3D edible chitosan‑sodium alginate-collagen/gelatin (CS-SA-Col/Gel) scaffold that can support the adhesion and proliferation of porcine skeletal muscle satellite cells, culminating in the construction of a structured CCM model. The 3D edible scaffolds were prepared by freeze-drying using electrostatic interactions between chitosan and sodium alginate. Initially, the physicochemical properties and structural characteristics of different scaffolds were explored, and the biocompatibility of the scaffolds was evaluated using the C2C12 cell model. The results showed that the 2-CS-SA-Col1-Gel scaffold provided stable mechanical support and abundant adhesion sites for the cells. Subsequently, we inoculated porcine skeletal muscle satellite cells on the 2-CS-SA-Col1-Gel scaffold and induced differentiation for a total of 14 days. Immunofluorescence staining results showed cytoskeleton formation, and Western blotting (WB) and qPCR results showed upregulation of skeletal proteins and myogenic genes. Ultimately, the structured CCM model has similar textural properties (chewiness, springiness and resilience) and appearance to those of fresh pork. In conclusion, the method of constructing 3D edible scaffolds to prepare structured CCM models exhibits the potential to produce cell cultured meat.
•Construction of edible scaffolds with good biocompatibility through electrostatic interactions.•Constructing cell culture meat models based on 3D scaffolds.•Scaffolds provide mechanical support for structured culture meat models•Obtain porcine-derived CCM with similar texture to real meat.
Abstract
Cancer immunotherapy targeting co-inhibitory pathways by checkpoint blockade shows remarkable efficacy in a variety of cancer types. However, only a minority of patients respond to treatment ...due to the stochastic heterogeneity of tumor microenvironment (TME). Recent advances in single-cell RNA-seq technologies enabled comprehensive characterization of the immune system heterogeneity in tumors but posed computational challenges on integrating and utilizing the massive published datasets to inform immunotherapy. Here, we present Tumor Immune Single Cell Hub (TISCH, http://tisch.comp-genomics.org), a large-scale curated database that integrates single-cell transcriptomic profiles of nearly 2 million cells from 76 high-quality tumor datasets across 27 cancer types. All the data were uniformly processed with a standardized workflow, including quality control, batch effect removal, clustering, cell-type annotation, malignant cell classification, differential expression analysis and functional enrichment analysis. TISCH provides interactive gene expression visualization across multiple datasets at the single-cell level or cluster level, allowing systematic comparison between different cell-types, patients, tissue origins, treatment and response groups, and even different cancer-types. In summary, TISCH provides a user-friendly interface for systematically visualizing, searching and downloading gene expression atlas in the TME from multiple cancer types, enabling fast, flexible and comprehensive exploration of the TME.
Neutrophils are the most abundant circulating leukocyte population with critical roles in immune defense, regulation of innate and adaptive immune systems, and disease pathogenesis. Our progress in ...understanding precise mechanisms of neutrophil activation, recruitment, and function has been hampered by the lack of optimized and standardized methods for the characterization and phenotyping of this readily activated population. By comparing eight methods of neutrophil characterization, we demonstrate that the level of neutrophil activation and degranulation is associated with specific experimental conditions and the number and type of manipulation steps employed. Staining whole blood at 4 °C and removal of remaining unbound antibodies prior to one-step fixation and red blood cell lysis minimizes neutrophil activation, decreases phenotypic alterations during processing, and prevents nonspecific antibody binding. The effects of anticoagulants used for collection, processing delays, and time and temperature during sample analysis on neutrophil phenotype are addressed. The presented data provide a foundation for higher quality standards of neutrophil characterization improving consistency and reproducibility among studies.
CD8(+) CTL play important roles against malignancy in both active and passive immunotherapy. Nonetheless, the success of antitumor CTL responses may be improved by additional therapeutic modalities. ...Radiotherapy, which has a long-standing use in treating neoplastic disease, has been found to induce unique biologic alterations in cancer cells affecting Fas gene expression, which, consequently, may influence the overall lytic efficiency of CTL. Here, in a mouse adenocarcinoma cell model, we examined whether exposure of these tumor cells to sublethal doses of irradiation 1) enhances Fas expression, leading to more efficient CTL killing via Fas-dependent mechanisms in vitro; and 2) improves antitumor activity in vivo by adoptive transfer of these Ag-specific CTL. Treatment of carcinoembryonic Ag-expressing MC38 adenocarcinoma cells with irradiation (20 Gy) in vitro enhanced Fas expression at molecular, phenotypic, and functional levels. Furthermore, irradiation sensitized these targets to Ag-specific CTL killing via the Fas/Fas ligand pathway. We examined the effect of localized irradiation of s.c. growing tumors on the efficiency of CTL adoptive immunotherapy. Irradiation caused up-regulation of Fas by these tumor cells in situ, based on immunohistochemistry. Moreover, localized irradiation of the tumor significantly potentiated tumor rejection by these carcinoembryonic Ag-specific CTL. Overall, these results showed for the first time that 1) regulation of the Fas pathway in tumor cells by irradiation plays an important role in their sensitization to Ag-specific CTL; and 2) a combination regimen of tumor-targeted irradiation and CTL promotes more effective antitumor responses in vivo, which may have implications for the combination of immunotherapy and radiation therapy.
Increasing evidence suggests that asthma is a heterogeneous disorder regulated by distinct molecular mechanisms. In a cross-sectional study of asthmatics of varying severity (n = 51), endobronchial ...tissue gene expression analysis revealed three major patient clusters: TH2-high, TH17-high, and TH2/17-low. TH2-high and TH17-high patterns were mutually exclusive in individual patient samples, and their gene signatures were inversely correlated and differentially regulated by interleukin-13 (IL-13) and IL-17A. To understand this dichotomous pattern of T helper 2 (TH2) and TH17 signatures, we investigated the potential of type 2 cytokine suppression in promoting TH17 responses in a preclinical model of allergen-induced asthma. Neutralization of IL-4 and/or IL-13 resulted in increased TH17 cells and neutrophilic inflammation in the lung. However, neutralization of IL-13 and IL-17 protected mice from eosinophilia, mucus hyperplasia, and airway hyperreactivity and abolished the neutrophilic inflammation, suggesting that combination therapies targeting both pathways may maximize therapeutic efficacy across a patient population comprising both TH2 and TH17 endotypes.
The intestinal mucosal barrier (IMB), which consists of mechanical barrier, chemical barrier, biological barrier and immune barrier, plays an important role in the maintenance of intestinal ...epithelium integrity and defense against invasion of bacteria, endotoxins and foreign antigens. Impaired IMB, characterized by increased intestinal mucosal permeability (IMP) and decreased transmembrane resistance (TR), has been implicated in the pathogenesis of various digestive, urinary, circulatory, neurological and metabolic dysfunctions. Electrophysiological recording of TR in the ex vivo intestinal tissues or cultured epithelial cell monolayers, or biochemical quantification of transepithelial movement of orally-administered molecular probes or specific endogenous protein molecules has frequently been used in the evaluation of IMB. In this paper, the composition and function of IMB will be summarized, with emphasis on the evaluation methods of IMP.
In CLL data from chromosome banding analysis (CBA) have been scarce due to the low proliferative activity of CLL cells in vitro. We improved the cultivation technique using an immunostimulatory ...CpG-oligonucleotide DSP30 and IL-2. A total of 506 CLL samples were analysed with CBA and interphase FISH using probes for the detection of trisomy 12, IgH rearrangements and deletions of 6q21, 11q22.3 (ATM), 13q14 (D13S25 and D13S319) and 17p13 (TP53). A total of 500 of 506 (98.8%) cases were successfully stimulated for metaphase generation and are subject to this study. Aberrations were detected in 415 of 500 (83.0%) cases by CBA and in 392 of 500 (78.4%) cases by FISH. CBA detected 832 abnormalities and FISH only 502. Therefore, CBA offers important information in addition to FISH. (1) CLL is characterized mainly by genomic imbalances and reciprocal translocations are rare. (2) A subgroup with complex aberrant karyotype (16.4%) is identified which is associated with an unmutated IgV(H) status and CD38 expression (P=0.034 and 0.02, respectively). (3) Additional abnormalities are detectable providing new biological insights into different CLL subclasses revealing a much more heterogeneous pattern of cytogenetic abnormalities as assumed so far based on FISH data only. Therefore, prospective clinical trials should evaluate the prognostic impact of newly available CBA data.
Quantitative measurements of cell-generated forces have heretofore required that cells be cultured on two-dimensional substrates. We describe a technique to quantitatively measure three-dimensional ...traction forces exerted by cells fully encapsulated in well-defined elastic hydrogel matrices. Using this approach we measured traction forces for several cell types in various contexts and revealed patterns of force generation attributable to morphologically distinct regions of cells as they extend into the surrounding matrix.
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