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In this Resource study, the authors used Direct RNA Sequencing (DRS) to quantitatively examine the transcriptional profile of microglia, focusing specifically on the proteins important for binding endogenous ligands and potential pathogens—a collection they term the 'sensome'. They also compare this profile to that of peripheral macrophages. Microglia, the principal neuroimmune sentinels of the brain, continuously sense changes in their environment and respond to invading pathogens, toxins and cellular debris. Microglia exhibit plasticity and can assume neurotoxic or neuroprotective priming states that determine their responses to danger. We used direct RNA sequencing, without amplification or cDNA synthesis, to determine the quantitative transcriptomes of microglia of healthy adult and aged mice. We validated our findings using fluorescence dual in situ hybridization, unbiased proteomic analysis and quantitative PCR. We found that microglia have a distinct transcriptomic signature and express a unique cluster of transcripts encoding proteins for sensing endogenous ligands and microbes that we refer to as the sensome. With aging, sensome transcripts for endogenous ligand recognition were downregulated, whereas those involved in microbe recognition and host defense were upregulated. In addition, aging was associated with an overall increase in the expression of microglial genes involved in neuroprotection.

Gliomas are the most fatal malignant cerebral tumors. Temozolomide (TMZ), as the primary chemotherapy drug, has been widely used in clinics. However, resistance of TMZ still remains to poor defined. LncRNAs have been reported to play crucial roles in progression of various cancers and resistance of multiple drugs. However, the biological function and underlying mechanisms of most lncRNAs in glioma still remains unclear. Based on the TCGA database, a total of 94 differentially expressed lncRNAs, including 16 up-regulated genes and 78 downregulated genes were identified between gliomas and normal brain tissues. Subsequently, lncRNA DLEU1, HOTAIR, and LOC00132111 were tested to be significantly related to overall survival (OS) between high- and low-expression groups. Additionally, we verified that lncRNA DLEU1 was high expressed in 108 gliomas, compared with 19 normal brain tissues. And high expression of lncRNA DLEU1 predicted a poor prognosis (HR = 1.703, 95%CI: 1.133–2.917, p -value = 0.0159). Moreover, functional assays revealed that knockdown of lncRNA DLEU1 could suppress the proliferation by inducing cell cycle arrest at G1 phase and reducing the S phase by down-regulating the CyclinD1 and p -AKT, as the well as migration and invasion by inhibiting the epithelial–mesenchymal transition (EMT) markers, such as ZEB1, N-cadherin, β-catenin and snail in glioma cells. Furthermore, silencing lncRNA DLEU1 suppressed TMZ-activated autophagy via regulating the expression of P62 and LC3, and promoted sensitivity of glioma cells to TMZ by triggering apoptosis. Conclusively, our study indicated that lncRNA DLEU1 might perform as a prognostic potential target and underlying therapeutic target for sensitivity of glioma to TMZ.

BackgroundTumor cells employ multiple tactics to evade immune cell activities. Programmed cell death ligand 1 (PD-L1) is one of the key antigens presented on tumor cells that bind to programmed cell death protein 1 (PD-1) and block immune response. Multiple immunotherapies targeting PD-1/PD-L1 blockade have been approved by FDA. Currently, patients are tested for PD-L1 protein expression to be considered for PD-1/PD-L1 checkpoint inhibitor immunotherapies. Clinical outcomes of these immunotherapies, however, do not always correlate to the expression level of PD-L1. There is a need for better biomarkers that are more predictive of clinical outcomes. Direct detection of PD-1/PD-L1 interactions in patient tissues is likely to have better correlation to the therapeutic effect of checkpoint inhibitors than PD-L1 test alone.We have developed an assay that enables visualization of protein-protein interaction with multiomic context of tumor immune microenvironment (TIME). We observed PD-1/PD-L1 interaction, individual proteins, and mRNA at high spatial resolution in various tumor tissues using new workflow enabled by high sensitivity and specificity of RNAscopeTM technology.MethodsTo visualize protein-protein interactions in situ, we developed a novel technique enabling multiplexed detection of protein and mRNA targets on a single slide. Oligonucleotide-conjugated antibodies were prepared to integrate the detection of protein-protein interaction and proteins into semi-automated co-detection workflow based on RNAscopeTM technology, performed on Leica BOND instrument. The protocol allows imaging of up to 4 targets per cycle, and three cycles of detection. Protein and highly expressed mRNA targets were imaged first, followed by the detection of protein-protein interaction and mRNAs with lower expression level, with tyramide signal amplification.ResultsNew assay for co-detection of protein-protein interaction, protein, and mRNA (up to 12-plex detection) was developed. First, we verified the PD-1/PD-L1 interaction signals generated with this assay, are detected where PD-1 and PD-L1 signals overlap using sequential immunofluorescence. Next, to characterize TIME surrounding the interaction-positive region, the panel was expanded to include cell phenotyping protein markers for both immune and tumor cells such as CD3, CD4, CD8 and PanCK, as well as mRNA markers for chemokines and cytokines such as CXCL10, IFNG and TNFA. The interaction signals appear as a group of punctate dots implying possibility for semi-quantitative analysis.ConclusionsNew RNAscopeTM multiomics workflow is a powerful technique to resolve PD-1/PD-L1 interaction in the context of TIME. Spatial multiomic analyses of ligand-receptor interaction will expand our knowledge of tumor immune evasion strategies and potentially offer new patient stratification strategy for checkpoint inhibitor immunotherapies.

Background Glioblastoma is the most common primary malignant brain tumor. Because of the limited understanding of its pathogenesis, the prognosis of glioblastoma remains poor. This study was conducted to explore potential competing endogenous RNA (ceRNA) network chains and biomarkers in glioblastoma by performing integrated bioinformatics analysis. Methods Transcriptome expression data from The Cancer Genome Atlas database and Gene Expression Omnibus were analyzed to identify differentially expressed genes between glioblastoma and normal tissues. Biological pathways potentially associated with the differentially expressed genes were explored by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, and a protein-protein interaction network was established using the STRING database and Cytoscape. Survival analysis using Gene Expression Profiling Interactive Analysis was based on the Kaplan–Meier curve method. A ceRNA network chain was established using the intersection method to align data from four databases (miRTarBase, miRcode, TargetScan, and lncBace2.0), and expression differences and correlations were verified by quantitative reverse-transcription polymerase chain reaction analysis and by determining the Pearson correlation coefficient. Additionally, an MTS assay and the wound-healing and transwell assays were performed to evaluate the effects of complement C1s (C1S) on the viability and migration and invasion abilities of glioblastoma cells, respectively. Results We detected 2842 differentially expressed (DE) mRNAs, 2577 DE long non-coding RNAs (lncRNAs), and 309 DE microRNAs (miRNAs) that were dysregulated in glioblastoma. The final ceRNA network consisted of six specific lncRNAs, four miRNAs, and four mRNAs. Among them, four DE mRNAs and one DE lncRNA were correlated with overall survival ( p  < 0.05). C1S was significantly correlated with overall survival ( p = 0.015). In functional assays, knockdown of C1S inhibited the proliferation and invasion of glioblastoma cell lines. Conclusions We established four ceRNA networks that may influence the occurrence and development of glioblastoma. Among them, the MIR155HG/has-miR-129-5p/C1S axis is a potential marker and therapeutic target for glioblastoma. Knockdown of C1S inhibited the proliferation, migration, and invasion of glioblastoma cells. These findings clarify the role of the ceRNA regulatory network in glioblastoma and provide a foundation for further research.

BackgroundStudying cell-cell interactions can have important implications in immuno-oncology, inflammation, and neuroscience. Tissue heterogeneity poses immense challenges to understanding underlying molecular mechanisms using techniques such as qRT-PCR or bulk sequencing. While single-cell RNA sequencing can provide information about precise cellular composition of tissues, spatial context is lost. With platforms such as RNAscope, target gene and protein expression can be visualized to characterize cell types and tissue neighborhoods. Here, we demonstrate a novel method for the simultaneous detection of RNA and protein using a modified co-detection assay.MethodsThis novel co-detection assay enables visualization of a combination of up to 12 RNA and/or protein targets on the same sample. We used a set of antibodies targeting key immune and tumor cell markers- PD1, CD3, CD4, CD8, CD68, FOXP3 and KRT17, along with RNA biomarkers to interrogate the tumor microenvironment (TME) in human FFPE tumor samples.ResultsUsing a combination of RNA and protein targets, we characterized different cell types such as T cells, macrophages, and tumor cells in the tumor immune microenvironment (TIME). Co-expression of RNA and protein targets resulted in detection of specific tumor infiltrating immune cell populations and analysis of their activation states.ConclusionsThe assay offers a powerful technique for visualizing target RNA biomarkers in specific cell-types identified by cell-marker protein expression. This is a valuable tool for multiomic analysis and accurate interrogation of complex tissues to obtain insights into novel biomarkers and therapeutic targets.

BackgroundSpatial biology methods are increasingly used for the characterization of complex tissue microenvironments, the understanding of which can shed light on fundamental biological mechanisms and better inform development of targeted therapeutics.RNAscope™ in situ hybridization (ISH) technology, capable of highly sensitive single-molecule RNA detection, can be combined with immunohistochemistry (IHC) or immunofluorescence (IF) for the co-detection of clinically relevant biomarkers on the same slide with morphological context. This application is especially important in immuno-oncology research to profile immune cell populations using protein markers and characterize their activation states by detecting cytokine and chemokine expression with RNA. However, RNAscope necessitates the use of proteases to digest RNA-associated proteins and facilitate probe access to RNA targets, which can negatively impact epitopes targeted by some antibodies.Previously, we developed the Integrated Co-detection Workflow (ICW) to partially solve this problem, with the fixation of the primary antibody-target complex prior to the protease application. While ICW rescues signal for many previously incompatible antibodies, proteases can still adversely impact some of the primary antibody-target complex. To address this deficiency, we have developed a novel RNA-protein co-detection workflow that eliminates the need for protease, resulting in high detection sensitivities for both protein and RNA markers.MethodsTo maintain the same RNA detection sensitivity without the use of proteases, we formulated a new protease-free pretreatment buffer to replace the existing protease step within the current workflow which allows adequate accessibility of RNAscope™ probes to the target RNAs. Following this protease-free pretreatment, tissue specimens were assayed with RNAscope Multiplex to detect RNA species in the tissue, followed by standard IF staining to co-detect protein biomarkers. Antibodies which previously exhibited degraded protein signal in both sequential ISH-IF and ICW were tested in the new protocol.ResultsHere, we present results from FFPE human tissues to co-detect several protease-sensitive antibodies, including degranulating cytotoxic lymphocyte marker CD107a along with human house-keeping genes TBP, POLR2A and PPIB. The protease-free RNAscope co-detection workflow restored the protein staining pattern at nominal antibody concentrations used for IHC while maintaining mRNA dot counts for TBP, POLR2A and PPIB, indicating minimal impact of protease-free pretreatment buffer on both RNA and protein signal.ConclusionsThe protease-free RNAscope co-detection workflow will serve as a powerful multi-omics staining technique for a wider range of antibodies by enabling visualization of RNA-protein co-detection for the comprehensive profiling of tissue microenvironments, facilitating faster breakthroughs in the discovery of therapeutics.

BackgroundSpatial biology has transformed our understanding of the tumor microenvironment (TME) by enabling the study of tissue composition and intercellular interactions at a single-cell level while preserving spatial context.1–3 Hyperplex immunofluorescence (IF) techniques allow the simultaneous detection of multiple protein biomarkers, enabling immune cell profiling in the TME.4 Similarly, RNA in situ hybridization (ISH) techniques have enabled the detection of RNA biomarkers, such as soluble factors with high sensitivity and specificity.5 Combining the detection of key RNA and protein targets can provide valuable insights into unique infiltrating immune cell populations and their activation states.In this study, we propose a novel approach that combines RNAscope™ and sequential immunofluorescence (seqIF™) protocols for the simultaneous detection of RNA and protein targets. The integrated same-slide multi-omics protocol is automated on the COMET™ platform, an advanced platform for tissue staining that uses precise temperature control and automation capabilities, ensuring reproducibility and efficiency in the workflow.MethodsThe RNAscope HiPlex assay was automated on COMET™ for RNA detection and combined with the seqIF™ protocol for the detection of protein biomarkers. By integrating the two, we could sequentially detect multiple RNAs and proteins in the same tissue sections, preserving the spatial relationship between different molecular species.ResultsWe developed an integrated protocol for RNA and protein detection with three cycles of RNA detection (four fluorescent channels per cycle), enabling a total of 12-plex RNA panel detection, followed by consecutive cycles of seqIF™, with two protein markers detected per cycle. We included antibodies to detect infiltration of T cells, B cells, macrophages, and other immune cells in combination with RNA probes for key biomarkers such as chemokines and cytokines. The automated process on COMET™ seamlessly synchronized all protocol steps, including imaging, and allowed multi-omics analysis without any user intervention. By combining RNA and protein codetection, we gained extensive insights into the TME molecular landscape, uncovering co-expression patterns and relationships between RNA and proteins within individual cells.ConclusionsOur results demonstrate the successful implementation of the combined RNAscope and seqIF™ protocols on COMET™. Preserving spatial context and intercellular relationships, this approach offers a more holistic understanding of the TME molecular landscape and the complex cellular interactions exhibited by different cell populations.Multi-omics analysis on the same slide will allow a better comprehension of the interplay between transcriptomics and proteomics information, opening new perspectives for personalized medicine and the discovery of novel therapeutic targets.ReferencesBosisio FM, Van Herck Y, Messiaen J, Bolognesi MM, Marcelis L, Van Haele M, Cattoretti G, Antoranz A, De Smet F. Next-Generation Pathology Using Multiplexed Immunohistochemistry: Mapping Tissue Architecture at Single-Cell Level. Front Oncol. 2022 Jul 29;12:918900.Binnewies M, Roberts EW, Kersten K, Chan V, Fearon DF, Merad M, Coussens LM, Gabrilovich DI, Ostrand-Rosenberg S, Hedrick CC, Vonderheide RH, Pittet MJ, Jain RK, Zou W, Howcroft TK, Woodhouse EC, Weinberg RA, Krummel MF. Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat Med. 2018 May;24(5):541–550.Vitale I, Shema E, Loi S, Galluzzi L. Intratumoral heterogeneity in cancer progression and response to immunotherapy. Nat Med. 2021 Feb;27(2):212–224.Hickey JW, Neumann EK, Radtke AJ, Camarillo JM, Beuschel RT, Albanese A, McDonough E, Hatler J, Wiblin AE, Fisher J, Croteau J, Small EC, Sood A, Caprioli RM, Angelo RM, Nolan GP, Chung K, Hewitt SM, Germain RN, Spraggins JM, Lundberg E, Snyder MP, Kelleher NL, Saka SK. Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging. Nat Methods. 2022 Mar;19(3):284–295.Palla G, Fischer DS, Regev A, Theis FJ. Spatial components of molecular tissue biology. Nat Biotechnol. 2022 Mar;40(3):308–318.

Background Folate receptor alpha (FOLR1/FRA) is expressed in a number of epithelial cancers and in particular epithelial ovarian cancer (EOC), especially of the serous histotype. Recent studies have shown that EOC originates from the fallopian tube fimbriae rather than from epithelial cells lining the ovary. We have previously shown by immunohistochemistry a strong correlation between FRA expression in EOC and normal and fallopian adenocarcinoma. Folate receptor beta (FOLR2/FRB) has been described to be expressed by macrophages both in inflammatory disorders and certain epithelial cancers. Given the high sequence identity of these two folate receptor family members we sought to investigate the architectural and cell-specific expression of these two receptors in gynecologic tissues. Methods RNA scope, a novel chromogenic in situ hybridization assay tool, was used to examine expression of the alpha (FOLR1) and beta (FOLR2) isoforms of folate receptor relative to each other as well as to the macrophage markers CD11b and CD68, in samples of normal fallopian tube and fallopian adenocarcinoma as well as normal ovary and EOC. Results We demonstrated expression of both FOLR1 and FOLR2 in EOC, normal fallopian tube and fallopian adenocarcinoma tissue while very little expression of either marker was observed in normal ovary. Furthermore, FOLR2 was shown to be expressed almost exclusively in macrophages, of both the M1 and M2 lineages, as determined by co-expression of CD11b and/or CD68, with little or no expression in epithelial cells. Conclusions These findings further substantiate the hypothesis that the cell of origin of EOC is tubal epithelium and that the beta isoform of folate receptor is primarily restricted to macrophages. Further, macrophages expressing FOLR2 may represent tumor associated or infiltrating macrophages (TAMs) in epithelial cancers.

In order to expand the application of the electrodeposited Ni-Fe alloy foil, their mechanical and magnetic properties were studied after heat treatment. The development of grain growth during annealing was in-situ online investigated using a heating stage microscope, and the texture was analyzed via X-ray diffraction (XRD) and electron back-scattered diffraction (EBSD). The results indicated that abnormal grain growth usually occurred during annealing at 1000–1050 °C. The {111} oriented grains preferentially grew as the annealing temperature and holding time increased. The plasticities of the electrodeposited Ni-Fe alloy foils after heat treatment were better than those of the original samples. The excellent ductility was obtained without a loss in magnetic properties after annealing at 1100 °C for 6 h.