Abstract-Cyclic mechanical strain produced by pulsatile blood flow regulates the orientation of endothelial cells lining blood vessels and influences critical processes such as angiogenesis. Mechanical stimulation of stretch-activated calcium channels is known to mediate this reorientation response; however, the molecular basis remains unknown. Here, we show that cyclically stretching capillary endothelial cells adherent to flexible extracellular matrix substrates activates mechanosensitive TRPV4 (transient receptor potential vanilloid 4) ion channels that, in turn, stimulate phosphatidylinositol 3-kinase-dependent activation and binding of additional ␤1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking antibodies and knock down of TRPV4 channels using specific small interfering RNA suppress strain-induced capillary cell reorientation. Thus, mechanical forces that physically deform extracellular matrix may guide capillary cell reorientation through a strain-dependent "integrin-to-integrin" signaling mechanism mediated by force-induced activation of mechanically gated TRPV4 ion channels on the cell surface. Key Words: mechanical strain Ⅲ integrin Ⅲ TRPV4 Ⅲ endothelial cell Ⅲ reorientation Ⅲ cytoskeleton M echanical forces regulate vascular growth and development by influencing endothelial cell growth, survival, differentiation and migration. 1,2 Local mechanical cues conveyed by extracellular matrix (ECM) attributable to cyclic deformation of blood vessels, hemodynamic forces, or cellgenerated traction forces are also potent inducers of directional capillary blood vessel growth and vascular remodeling in vitro and in vivo. [3][4][5][6][7][8][9][10] For example, the initial step in neovascularization involves reorientation of a subset of capillary endothelial (CE) cells that spread and migrate perpendicular to the main axis of the preexisting vessel toward the angiogenic stimulus 11 ; however, the molecular mechanism responsible for this CE cell reorientation response is unknown. Many cell types, including large vessel endothelial cells, realign perpendicular to the direction of the applied force when they experience cyclic stretching (mechanical strain). [12][13][14][15] In the case of macrovascular endothelium, this reorientation response can be prevented by treatment with chemical inhibitors of stretch-activated (SA) ion channels. 15 But neither the identity of these channels nor the mechanism by which they elicit cell reorientation is known.Endothelial cells express most members of the transient receptor potential (TRP) family of ion channels 16 -18 and TRP vanilloid (TRPV)4 has been reported to mediate flowinduced vasodilation in large vessel endothelium. 19 -22 Here, we show that calcium influx through TRPV4 channels stimulated by mechanically stretching CE cells through their integrin-extracellular matrix (ECM) adhesions promotes cell reorientation by activating phosphatidylinositol 3-kinase (PI3K), thereby stimulating activ...

Key Points Question Is tumor mutation burden (TMB) associated with improved outcomes of programmed cell death–1 (PD-1)/programmed death ligand–1 (PD-L1) inhibition across PD-L1 expression levels in non–small cell lung cancer (NSCLC)? Findings In this cohort study of 1552 patients with NSCLC, the group with high TMB had improved response rates and survival after receiving PD-1/PD-L1 inhibition therapy across PD-L1 expression subgroups compared with the group with low TMB. High TMB levels were associated with increased CD8-positive T-cell infiltration and distinct immune response gene expression signatures. Meaning These findings suggest that in NSCLC, a high number of nonsynonymous tumor mutations is associated with immune cell infiltration and inflammatory T-cell expression signatures, leading to increased sensitivity to PD-1/PD-L1 inhibition across PD-L1 expression subgroups.

MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.

Summary MicroRNAs are a class of short ~22 nucleotide RNAs predicted to regulate nearly half of all protein coding genes, including many involved in basal cellular processes and organismal development. Although a global reduction in miRNAs is commonly observed in various human tumors, complete loss has not been documented, suggesting an essential function for miRNAs in tumorigenesis. Here we present the finding that transformed or immortalized Dicer1-null somatic cells can be isolated readily in vitro, maintain the characteristics of DICER1-expressing controls and remain stably proliferative. Furthermore, Dicer1-null cells from a sarcoma cell line, though depleted of miRNAs, are competent for tumor formation. Hence, miRNA levels in cancer may be maintained in vivo by a complex stabilizing selection in the intratumoral environment.

BackgroundTumor infiltrating lymphocytes (TILs) reflect adaptive antitumor immune responses in cancer and are generally associated with favorable prognosis. However, the relationships between TILs subsets and their spatial arrangement with clinical benefit from immune checkpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC) remains less explored.MethodsWe used multiplexed quantitative immunofluorescence panels to determine the association of major TILs subpopulations, CD8+ cytotoxic T cells, CD4+ helper T cells and CD20+ B cells, and T cell exhaustion markers, programmed cell death protein-1 (PD-1),lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin mucin-3 (TIM-3) with outcomes in a multi-institutional cohort of baseline tumor samples from 179 patients with NSCLC treated with ICI. The analysis of full-face tumor biopsies including numerous fields of view allowed a detailed spatial analysis and assessment of tumor immune heterogeneity using a multiparametric quadratic entropy metric (Rao’s Q Index (RQI)).ResultsTILs were preferentially located in the stromal tissue areas surrounding tumor-cell nests and CD8+ T cells were the most abundant subset. Higher density of stromal CD8+ cytotoxic T cells was significantly associated with longer survival, and this effect was more prominent in programmed death ligand-1 (PD-L1) positive cases. The role of baseline T cell infiltration to stratify PD-L1 expressing cases was confirmed measuring the T cell receptor-burden in an independent NSCLC cohort studied with whole-exome DNA sequencing. High levels of LAG-3 on T cells or elevated RQI heterogeneity index were associated with worse survival in the cohort.ConclusionBaseline T cell density and T cell exhaustion marker expression can stratify outcomes in PD-L1 positive patients with NSCLC treated with ICI. Spatial immune heterogeneity can be measured using the RQI and is associated with survival in NSCLC.

MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function.

Variations in microRNA (miRNA) gene and/or target repertoire are likely to be key drivers of phenotypic differences between species. To better understand these changes, we developed a computational method that identifies signatures of species-specific target site gain and loss associated with miRNA acquisition. Interestingly, several of the miRNAs implicated in mouse 3′ UTR evolution derive from a single rapidly expanded rodent-specific miRNA cluster. Located in the intron of Sfmbt2, a maternally imprinted polycomb gene, these miRNAs (referred to as the Sfmbt2 cluster) are expressed in both embryonic stem cells and the placenta. One abundant miRNA from the cluster, miR-467a, functionally overlaps with the mir-290-295 cluster in promoting growth and survival of mouse embryonic stem cells. Predicted novel targets of the remaining cluster members are enriched in pathways regulating cell survival. Two relevant species-specific target candidates, Lats2 and Dedd2, were validated in cultured cells. We suggest that the rapid evolution of the Sfmbt2 cluster may be a result of intersex conflict for growth regulation in early mammalian development and could provide a general model for the genomic response to acquisition of miRNAs and similar regulatory factors.

Abstract-Looping is a vital event during early cardiac morphogenesis, as the initially straight heart tube bends and twists into a curved tube, laying out the basic pattern of the future fourchambered heart. Despite intensive study for almost a century, the biophysical mechanisms that drive this process are not well understood. To explore a recently proposed hypothesis for looping, we constructed a finite element model for the embryonic chick heart during the first phase of looping, called c-looping. The model includes the main structures of the early heart (heart tube, omphalomesenteric veins, and dorsal mesocardium), and the analysis features realistic three-dimensional geometry, nonlinear passive and active material properties, and anisotropic growth. As per our earlier hypothesis for c-looping, actin-based morphogenetic processes (active cell shape change, cytoskeletal contraction, and cell migration) are simulated in specific regions of the model. The model correctly predicts the initial gross morphological shape changes of the heart, as well as distributions of morphogenetic stresses and strains measured in embryonic chick hearts. The model was tested further in studies that perturbed normal cardiac morphogenesis. The model, taken together with the new experimental data, supports our hypothesis for the mechanisms that drive early looping.