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E-cadherin and p120 catenin (p120) are essential for epithelial homeostasis, but can also exert pro-tumorigenic activities. Here, we resolve this apparent paradox by identifying two spatially and functionally distinct junctional complexes in non-transformed polarized epithelial cells: one growth suppressing at the apical zonula adherens (ZA), defined by the p120 partner PLEKHA7 and a non-nuclear subset of the core microprocessor components DROSHA and DGCR8, and one growth promoting at basolateral areas of cell–cell contact containing tyrosine-phosphorylated p120 and active Src. Recruitment of DROSHA and DGCR8 to the ZA is PLEKHA7 dependent. The PLEKHA7–microprocessor complex co-precipitates with primary microRNAs (pri-miRNAs) and possesses pri-miRNA processing activity. PLEKHA7 regulates the levels of select miRNAs, in particular processing of miR-30b, to suppress expression of cell transforming markers promoted by the basolateral complex, including SNAI1, MYC and CCND1. Our work identifies a mechanism through which adhesion complexes regulate cellular behaviour and reveals their surprising association with the microprocessor. Anastasiadis and colleagues report the presence of two spatially and functionally distinct E-cadherin-based junctional complexes, which influence cell behaviour either by regulating miRNA processing or by promoting p120 catenin and Src signalling.

We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+) and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A), and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER- cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5' end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

The sequencing by the PolyA selection is the most common approach for library preparation. With limited amount or degraded RNA, alternative protocols such as the NuGEN have been developed. However, it is not yet clear how the different library preparations affect the downstream analyses of the broad applications of RNA sequencing. Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina's mRNA-Seq PolyA selection and NuGEN ENCORE library preparation. The following analyses and comparisons were conducted: 1) the numbers of genes captured by each protocol; 2) the impact of protocols on differentially expressed gene detection between biological replicates; 3) expressed single nucleotide variant (SNV) detection; 4) non-coding RNAs, particularly lincRNA detection; and 5) intragenic gene expression. Sequences from the NuGEN protocol had lower (75%) alignment rate than the PolyA (over 90%). The NuGEN protocol detected fewer genes (12-20% less) with a significant portion of reads mapped to non-coding regions. A large number of genes were differentially detected between the two protocols. About 17-20% of the differentially expressed genes between biological replicates were commonly detected between the two protocols. Significantly higher numbers of SNVs (5-6 times) were detected in the NuGEN samples, which were largely from intragenic and intergenic regions. The NuGEN captured fewer exons (25% less) and had higher base level coverage variance. While 6.3% of reads were mapped to intragenic regions in the PolyA samples, the percentages were much higher (20-25%) for the NuGEN samples. The NuGEN protocol did not detect more known non-coding RNAs such as lincRNAs, but targeted small and \"novel\" lincRNAs. Different library preparations can have significant impacts on downstream analysis and interpretation of RNA-seq data. The NuGEN provides an alternative for limited or degraded RNA but it has limitations for some RNA-seq applications.

Our goal in these analyses was to use genomic features from a test set of primary breast tumors to build an integrated transcriptome landscape model that makes relevant hypothetical predictions about the biological and/or clinical behavior of HER2-positive breast cancer. We interrogated RNA-Seq data from benign breast lesions, ER+, triple negative, and HER2-positive tumors to identify 685 differentially expressed genes, 102 alternatively spliced genes, and 303 genes that expressed single nucleotide sequence variants (eSNVs) that were associated with the HER2-positive tumors in our survey panel. These features were integrated into a transcriptome landscape model that identified 12 highly interconnected genomic modules, each of which represents a cellular processes pathway that appears to define the genomic architecture of the HER2-positive tumors in our test set. The generality of the model was confirmed by the observation that several key pathways were enriched in HER2-positive TCGA breast tumors. The ability of this model to make relevant predictions about the biology of breast cancer cells was established by the observation that integrin signaling was linked to lapatinib sensitivity in vitro and strongly associated with risk of relapse in the NCCTG N9831 adjuvant trastuzumab clinical trial dataset. Additional modules from the HER2 transcriptome model, including ubiquitin-mediated proteolysis, TGF-beta signaling, RHO-family GTPase signaling, and M-phase progression, were linked to response to lapatinib and paclitaxel in vitro and/or risk of relapse in the N9831 dataset. These data indicate that an integrated transcriptome landscape model derived from a test set of HER2-positive breast tumors has potential for predicting outcome and for identifying novel potential therapeutic strategies for this breast cancer subtype.

Objectives The sequencing by the PolyA selection is the most common approach for library preparation. With limited amount or degraded RNA, alternative protocols such as the NuGEN have been developed. However, it is not yet clear how the different library preparations affect the downstream analyses of the broad applications of RNA sequencing. Methods and Materials Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina's mRNA-Seq PolyA selection and NuGEN ENCORE library preparation. The following analyses and comparisons were conducted: 1) the numbers of genes captured by each protocol; 2) the impact of protocols on differentially expressed gene detection between biological replicates; 3) expressed single nucleotide variant (SNV) detection; 4) non-coding RNAs, particularly lincRNA detection; and 5) intragenic gene expression. Results Sequences from the NuGEN protocol had lower (75%) alignment rate than the PolyA (over 90%). The NuGEN protocol detected fewer genes (12-20% less) with a significant portion of reads mapped to non-coding regions. A large number of genes were differentially detected between the two protocols. About 17-20% of the differentially expressed genes between biological replicates were commonly detected between the two protocols. Significantly higher numbers of SNVs (5-6 times) were detected in the NuGEN samples, which were largely from intragenic and intergenic regions. The NuGEN captured fewer exons (25% less) and had higher base level coverage variance. While 6.3% of reads were mapped to intragenic regions in the PolyA samples, the percentages were much higher (20-25%) for the NuGEN samples. The NuGEN protocol did not detect more known non-coding RNAs such as lincRNAs, but targeted small and \"novel\" lincRNAs. Conclusion Different library preparations can have significant impacts on downstream analysis and interpretation of RNA-seq data. The NuGEN provides an alternative for limited or degraded RNA but it has limitations for some RNA-seq applications.

Evan Eichler and colleagues analyze copy number variation in 15,767 children with intellectual disability, developmental delay, congenital birth defects and/or other related phenotypes. They identify 59 likely pathogenic CNV regions, including 14 new candidate regions, and estimate that ~14% of disorders in this sample collection are caused by large CNVs. To understand the genetic heterogeneity underlying developmental delay, we compared copy number variants (CNVs) in 15,767 children with intellectual disability and various congenital defects (cases) to CNVs in 8,329 unaffected adult controls. We estimate that ∼14.2% of disease in these children is caused by CNVs >400 kb. We observed a greater enrichment of CNVs in individuals with craniofacial anomalies and cardiovascular defects compared to those with epilepsy or autism. We identified 59 pathogenic CNVs, including 14 new or previously weakly supported candidates, refined the critical interval for several genomic disorders, such as the 17q21.31 microdeletion syndrome, and identified 940 candidate dosage-sensitive genes. We also developed methods to opportunistically discover small, disruptive CNVs within the large and growing diagnostic array datasets. This evolving CNV morbidity map, combined with exome and genome sequencing, will be critical for deciphering the genetic basis of developmental delay, intellectual disability and autism spectrum disorders.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/β sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/β cDNAs from CD8EXP into Jurkat 76 cells (TCR-/-) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.

Advanced optical neuromonitoring of cerebral hemodynamics with hybrid diffuse optical spectroscopy (DOS) and diffuse correlation spectroscopy (DCS) methods holds promise for non-invasive characterization of brain health in critically ill patients. However, the methods’ fiber-coupled patient interfaces (probes) are challenging to apply in emergent clinical scenarios that require rapid and reproducible attachment to the head. To address this challenge, we developed a novel chassis-based optical probe design for DOS/DCS measurements and validated its measurement accuracy and reproducibility against conventional, manually held measurements of cerebral hemodynamics in pediatric swine (n = 20). The chassis-based probe design comprises a detachable fiber housing which snaps into a 3D-printed, circumferential chassis piece that is secured to the skin. To validate its reproducibility, eight measurement repetitions of cerebral tissue blood flow index (BFI), oxygen saturation (StO 2 ), and oxy-, deoxy- and total hemoglobin concentration were acquired at the same demarcated measurement location for each pig. The probe was detached after each measurement. Of the eight measurements, four were acquired by placing the probe into a secured chassis, and four were visually aligned and manually held. We compared the absolute value and intra-subject coefficient of variation (CV) of chassis versus manual measurements. No significant differences were observed in either absolute value or CV between chassis and manual measurements (p > 0.05). However, the CV for BFI (mean ± SD: manual, 19.5% ± 9.6; chassis, 19.0% ± 10.8) was significantly higher than StO 2 (manual, 5.8% ± 6.7; chassis, 6.6% ± 7.1) regardless of measurement methodology (p<0.001). The chassis-based DOS/DCS probe design facilitated rapid probe attachment/re-attachment and demonstrated comparable accuracy and reproducibility to conventional, manual alignment. In the future, this design may be adapted for clinical applications to allow for non-invasive monitoring of cerebral health during pediatric critical care.

Neuronal activity-driven mechanisms influence glioblastoma cell proliferation and invasion, while glioblastoma remodels neuronal circuits. Although a subpopulation of malignant cells enhances neuronal connectivity, their impact on the immune system remains unclear. Here, we show that glioblastoma regions with enhanced neuronal connectivity exhibit regional immunosuppression, characterized by distinct immune cell compositions and the enrichment of anti-inflammatory tumor-associated macrophages (TAMs). In preclinical models, knockout of Thrombospondin-1 (TSP1/ Thbs1 ) in glioblastoma cells suppresses synaptogenesis and glutamatergic neuronal hyperexcitability. Furthermore, TSP1 knockout restores antigen presentation-related genes, promotes the infiltration of pro-inflammatory TAMs and CD8 + T-cells in the tumor, and alleviates TAM-mediated T-cell suppression. Pharmacological inhibition of glutamatergic signaling also shifts TAMs toward a less immunosuppressive state, prolongs survival in mice, and shows the potential to enhance the efficacy of immune cell-based therapy. These findings confirm that glioma-neuronal circuit remodeling is strongly linked with regional immunosuppression and suggest that targeting glioma-neuron-immune crosstalk could provide avenues for immunotherapy. Immunotherapy has yet to demonstrate efficacy for patients with glioblastoma. Here, the authors employ human single-cell RNA-seq, spatial transcriptomics, and a preclinical mouse model to show that glioblastoma cell-derived synaptogenic factor Thrombospondin-1 promotes neuronal circuit remodeling and regional immunosuppression, highlighting a potential therapeutic target.

Interleukin-2 (IL-2) is an important cytokine that helps T cells destroy tumors and virus-infected cells. IL-2 has great therapeutic promise but is limited by toxic side effects and its capacity to both activate and repress immune responses. Sockolosky et al. set out to improve IL-2–based immunotherapy by engineering synthetic IL-2–receptor pairs (i.e., IL-2 and its receptor, IL-2R) (see the Perspective by Mackall). Engineered complexes transmitted IL-2 signals but only interacted with each other and not with endogenous IL-2/IL-2R. Treatment of mice with IL-2 improved the ability of engineered T cells to reject tumors with no obvious side effects. This type of approach may provide a way to mitigate toxicities associated with some cytokine-based immunotherapies. Science , this issue p. 1037 ; see also p. 990 Engineered cytokines are able to improve immunotherapy in mouse tumor models. Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal ( ortho ) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of ortho IL-2Rβ into T cells enabled the selective cellular targeting of ortho IL-2 to engineered CD4 + and CD8 + T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. Ortho IL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.