Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

A liquid-like coat mediates chromosome clustering during mitotic exit.

The individualization of chromosomes during early mitosis and their clustering upon exit from cell division are two key transitions that ensure efficient segregation of eukaryotic chromosomes. Both processes are regulated by the surfactant-like protein Ki-67, but how Ki-67 achieves these diametric functions has remained unknown. Here, we report that Ki-67 radically switches from a chromosome repellent to a chromosome attractant during anaphase in human cells. We show that Ki-67 dephosphorylation during mitotic exit and the simultaneous exposure of a conserved basic patch induce the RNA-dependent formation of a liquid-like condensed phase on the chromosome surface. Experiments and coarse-grained simulations support a model in which the coalescence of chromosome surfaces, driven by co-condensation of Ki-67 and RNA, promotes clustering of chromosomes. Our study reveals how the switch of Ki-67 from a surfactant to a liquid-like condensed phase can generate mechanical forces during genome segregation that are required for re-establishing nuclear-cytoplasmic compartmentalization after mitosis.

Unbiased spatial proteomics with single-cell resolution in tissues.

Mass spectrometry (MS)-based proteomics has become a powerful technology to quantify the entire complement of proteins in cells or tissues. Here, we review challenges and recent advances in the LC-MS-based analysis of minute protein amounts, down to the level of single cells. Application of this technology revealed that single-cell transcriptomes are dominated by stochastic noise due to the very low number of transcripts per cell, whereas the single-cell proteome appears to be complete. The spatial organization of cells in tissues can be studied by emerging technologies, including multiplexed imaging and spatial transcriptomics, which can now be combined with ultra-sensitive proteomics. Combined with high-content imaging, artificial intelligence and single-cell laser microdissection, MS-based proteomics provides an unbiased molecular readout close to the functional level. Potential applications range from basic biological questions to precision medicine.

The social and structural architecture of the yeast protein interactome.

Cellular functions are mediated by protein-protein interactions, and mapping the interactome provides fundamental insights into biological systems. Affinity purification coupled to mass spectrometry is an ideal tool for such mapping, but it has been difficult to identify low copy number complexes, membrane complexes and complexes that are disrupted by protein tagging. As a result, our current knowledge of the interactome is far from complete, and assessing the reliability of reported interactions is challenging. Here we develop a sensitive high-throughput method using highly reproducible affinity enrichment coupled to mass spectrometry combined with a quantitative two-dimensional analysis strategy to comprehensively map the interactome of Saccharomyces cerevisiae. Thousand-fold reduced volumes in 96-well format enabled replicate analysis of the endogenous GFP-tagged library covering the entire expressed yeast proteome1. The 4,159 pull-downs generated a highly structured network of 3,927 proteins connected by 31,004 interactions, doubling the number of proteins and tripling the number of reliable interactions compared with existing interactome maps2. This includes very-low-abundance epigenetic complexes, organellar membrane complexes and non-taggable complexes inferred by abundance correlation. This nearly saturated interactome reveals that the vast majority of yeast proteins are highly connected, with an average of 16 interactors. Similar to social networks between humans, the average shortest distance between proteins is 4.2 interactions. AlphaFold-Multimer provided novel insights into the functional roles of previously uncharacterized proteins in complexes. Our web portal ( www.yeast-interactome.org ) enables extensive exploration of the interactome dataset.

Genetically encoded multimeric tags for subcellular protein localization in cryo-EM.

Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.

Robust dimethyl-based multiplex-DIA doubles single-cell proteome depth via a reference channel.

Single-cell proteomics aims to characterize biological function and heterogeneity at the level of proteins in an unbiased manner. It is currently limited in proteomic depth, throughput, and robustness, which we address here by a streamlined multiplexed workflow using data-independent acquisition (mDIA). We demonstrate automated and complete dimethyl labeling of bulk or single-cell samples, without losing proteomic depth. Lys-N digestion enables five-plex quantification at MS1 and MS2 level. Because the multiplexed channels are quantitatively isolated from each other, mDIA accommodates a reference channel that does not interfere with the target channels. Our algorithm RefQuant takes advantage of this and confidently quantifies twice as many proteins per single cell compared to our previous work (Brunner et al, PMID 35226415), while our workflow currently allows routine analysis of 80 single cells per day. Finally, we combined mDIA with spatial proteomics to increase the throughput of Deep Visual Proteomics seven-fold for microdissection and four-fold for MS analysis. Applying this to primary cutaneous melanoma, we discovered proteomic signatures of cells within distinct tumor microenvironments, showcasing its potential for precision oncology.

Rapid and In-Depth Coverage of the (Phospho-)Proteome With Deep Libraries and Optimal Window Design for dia-PASEF.

Data-independent acquisition (DIA) methods have become increasingly attractive in mass spectrometry-based proteomics because they enable high data completeness and a wide dynamic range. Recently, we combined DIA with parallel accumulation-serial fragmentation (dia-PASEF) on a Bruker trapped ion mobility (IM) separated quadrupole time-of-flight mass spectrometer. This requires alignment of the IM separation with the downstream mass selective quadrupole, leading to a more complex scheme for dia-PASEF window placement compared with DIA. To achieve high data completeness and deep proteome coverage, here we employ variable isolation windows that are placed optimally depending on precursor density in the m/z and IM plane. This is implemented in the freely available py_diAID (Python package for DIA with an automated isolation design) package. In combination with in-depth project-specific proteomics libraries and the Evosep liquid chromatography system, we reproducibly identified over 7700 proteins in a human cancer cell line in 44 min with quadruplicate single-shot injections at high sensitivity. Even at a throughput of 100 samples per day (11 min liquid chromatography gradients), we consistently quantified more than 6000 proteins in mammalian cell lysates by injecting four replicates. We found that optimal dia-PASEF window placement facilitates in-depth phosphoproteomics with very high sensitivity, quantifying more than 35,000 phosphosites in a human cancer cell line stimulated with an epidermal growth factor in triplicate 21 min runs. This covers a substantial part of the regulated phosphoproteome with high sensitivity, opening up for extensive systems-biological studies.

diaPASEF: parallel accumulation-serial fragmentation combined with data-independent acquisition.

Data-independent acquisition modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection windows collectively cover the entire m/z range, overall, only a few per cent of all incoming ions are isolated for mass analysis. Here, we make use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro) to devise a scan mode that samples up to 100% of the peptide precursor ion current in m/z and mobility windows. We extend an established targeted data extraction workflow by inclusion of the ion mobility dimension for both signal extraction and scoring and thereby increase the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.

AlphaMap: an open-source Python package for the visual annotation of proteomics data with sequence-specific knowledge.

SUMMARY: Integrating experimental information across proteomic datasets with the wealth of publicly available sequence annotations is a crucial part in many proteomic studies that currently lacks an automated analysis platform. Here, we present AlphaMap, a Python package that facilitates the visual exploration of peptide-level proteomics data. Identified peptides and post-translational modifications in proteomic datasets are mapped to their corresponding protein sequence and visualized together with prior knowledge from UniProt and with expected proteolytic cleavage sites. The functionality of AlphaMap can be accessed via an intuitive graphical user interface or-more flexibly-as a Python package that allows its integration into common analysis workflows for data visualization. AlphaMap produces publication-quality illustrations and can easily be customized to address a given research question. AVAILABILITY AND IMPLEMENTATION: AlphaMap is implemented in Python and released under an Apache license. The source code and one-click installers are freely available at https://github.com/MannLabs/alphamap. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation.

Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.

A Review of Approaches for Mitigating Effects from Variable Operational Environments on Piezoelectric Transducers for Long-Term Structural Health Monitoring.

Extending the service life of ageing infrastructure, transportation structures, and processing and manufacturing plants in an era of limited resources has spurred extensive research and development in structural health monitoring systems and their integration. Even though piezoelectric transducers are not the only sensor technology for SHM, they are widely used for data acquisition from, e.g., wave-based or vibrational non-destructive test methods such as ultrasonic guided waves, acoustic emission, electromechanical impedance, vibration monitoring or modal analysis, but also provide electric power via local energy harvesting for equipment operation. Operational environments include mechanical loads, e.g., stress induced deformations and vibrations, but also stochastic events, such as impact of foreign objects, temperature and humidity changes (e.g., daily and seasonal or process-dependent), and electromagnetic interference. All operator actions, correct or erroneous, as well as unintentional interference by unauthorized people, vandalism, or even cyber-attacks, may affect the performance of the transducers. In nuclear power plants, as well as in aerospace, structures and health monitoring systems are exposed to high-energy electromagnetic or particle radiation or (micro-)meteorite impact. Even if environmental effects are not detrimental for the transducers, they may induce large amounts of non-relevant signals, i.e., coming from sources not related to changes in structural integrity. Selected issues discussed comprise the durability of piezoelectric transducers, and of their coupling and mounting, but also detection and elimination of non-relevant signals and signal de-noising. For long-term service, developing concepts for maintenance and repair, or designing robust or redundant SHM systems, are of importance for the reliable long-term operation of transducers for structural health monitoring.

Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase.

Post-translational modification of proteins with ubiquitin and ubiquitin-like proteins (Ubls) is central to the regulation of eukaryotic cellular processes. Our ability to study the effects of ubiquitylation, however, is limited by the difficulty to prepare homogenously modified proteins in vitro and by the impossibility to selectively trigger specific ubiquitylation events in living cells. Here we combine genetic-code expansion, bioorthogonal Staudinger reduction and sortase-mediated transpeptidation to develop a general tool to ubiquitylate proteins in an inducible fashion. The generated ubiquitin conjugates display a native isopeptide bond and bear two point mutations in the ubiquitin C terminus that confer resistance toward deubiquitinases. Nevertheless, physiological integrity of sortase-generated diubiquitins in decoding cellular functions via recognition by ubiquitin-binding domains is retained. Our approach allows the site-specific attachment of Ubls to nonrefoldable, multidomain proteins and enables inducible and ubiquitin-ligase-independent ubiquitylation of proteins in mammalian cells, providing a powerful tool to dissect the biological functions of ubiquitylation with temporal control.

The Swiss Primary Hypersomnolence and Narcolepsy Cohort study (SPHYNCS): Study protocol for a prospective, multicentre cohort observational study.

Narcolepsy type 1 (NT1) is a disorder with well-established markers and a suspected autoimmune aetiology. Conversely, the narcoleptic borderland (NBL) disorders, including narcolepsy type 2, idiopathic hypersomnia, insufficient sleep syndrome and hypersomnia associated with a psychiatric disorder, lack well-defined markers and remain controversial in terms of aetiology, diagnosis and management. The Swiss Primary Hypersomnolence and Narcolepsy Cohort Study (SPHYNCS) is a comprehensive multicentre cohort study, which will investigate the clinical picture, pathophysiology and long-term course of NT1 and the NBL. The primary aim is to validate new and reappraise well-known markers for the characterization of the NBL, facilitating the diagnostic process. Seven Swiss sleep centres, belonging to the Swiss Narcolepsy Network (SNaNe), joined the study and will prospectively enrol over 500 patients with recent onset of excessive daytime sleepiness (EDS), hypersomnia or a suspected central disorder of hypersomnolence (CDH) during a 3-year recruitment phase. Healthy controls and patients with EDS due to severe sleep-disordered breathing, improving after therapy, will represent two control groups of over 50 patients each. Clinical and electrophysiological (polysomnography, multiple sleep latency test, maintenance of wakefulness test) information, and information on psychomotor vigilance and a sustained attention to response task, actigraphy and wearable devices (long-term monitoring), and responses to questionnaires will be collected at baseline and after 6, 12, 24 and 36 months. Potential disease markers will be searched for in blood, cerebrospinal fluid and stool. Analyses will include quantitative hypocretin measurements, proteomics/peptidomics, and immunological, genetic and microbiota studies. SPHYNCS will increase our understanding of CDH and the relationship between NT1 and the NBL. The identification of new disease markers is expected to lead to better and earlier diagnosis, better prognosis and personalized management of CDH.

MaxQuant.Live Enables Global Targeting of More Than 25,000 Peptides.

Mass spectrometry (MS)-based proteomics is often performed in a shotgun format, in which as many peptide precursors as possible are selected from full or MS1 scans so that their fragment spectra can be recorded in MS2 scans. Although achieving great proteome depths, shotgun proteomics cannot guarantee that each precursor will be fragmented in each run. In contrast, targeted proteomics aims to reproducibly and sensitively record a restricted number of precursor/fragment combinations in each run, based on prescheduled mass-to-charge and retention time windows. Here we set out to unify these two concepts by a global targeting approach in which an arbitrary number of precursors of interest are detected in real-time, followed by standard fragmentation or advanced peptide-specific analyses. We made use of a fast application programming interface to a quadrupole Orbitrap instrument and real-time recalibration in mass, retention time and intensity dimensions to predict precursor identity. MaxQuant.Live is freely available (www.maxquant.live) and has a graphical user interface to specify many predefined data acquisition strategies. Acquisition speed is as fast as with the vendor software and the power of our approach is demonstrated with the acquisition of breakdown curves for hundreds of precursors of interest. We also uncover precursors that are not even visible in MS1 scans, using elution time prediction based on the auto-adjusted retention time alone. Finally, we successfully recognized and targeted more than 25,000 peptides in single LC-MS runs. Global targeting combines the advantages of two classical approaches in MS-based proteomics, whereas greatly expanding the analytical toolbox. MaxQuant.Live builds on the fast application programming interface of quadrupole Orbitrap mass analyzers to control data acquisition in real-time (freely available at www.maxquant.live). Its graphical user interface enables advanced data acquisition strategies, such as in-depth characterization of peptides of interest. Online recalibration in mass, retention time, and intensity dimensions extends this concept to more than 25,000 peptides per run. Our "global targeting" strategy combines the best of targeted and shotgun approaches.

MaxQuant.Live Enables Global Targeting of More Than 25,000 Peptides.

Mass spectrometry (MS)-based proteomics is often performed in a shotgun format, in which as many peptide precursors as possible are selected from full or MS1 scans so that their fragment spectra can be recorded in MS2 scans. Although achieving great proteome depths, shotgun proteomics cannot guarantee that each precursor will be fragmented in each run. In contrast, targeted proteomics aims to reproducibly and sensitively record a restricted number of precursor/fragment combinations in each run, based on prescheduled mass-to-charge and retention time windows. Here we set out to unify these two concepts by a global targeting approach in which an arbitrary number of precursors of interest are detected in real-time, followed by standard fragmentation or advanced peptide-specific analyses. We made use of a fast application programming interface to a quadrupole Orbitrap instrument and real-time recalibration in mass, retention time and intensity dimensions to predict precursor identity. MaxQuant.Live is freely available (www.maxquant.live) and has a graphical user interface to specify many predefined data acquisition strategies. Acquisition speed is as fast as with the vendor software and the power of our approach is demonstrated with the acquisition of breakdown curves for hundreds of precursors of interest. We also uncover precursors that are not even visible in MS1 scans, using elution time prediction based on the auto-adjusted retention time alone. Finally, we successfully recognized and targeted more than 25,000 peptides in single LC-MS runs. Global targeting combines the advantages of two classical approaches in MS-based proteomics, whereas greatly expanding the analytical toolbox.

Online Parallel Accumulation-Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer.

In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra are acquired in about 100 microseconds with time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive because of its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 600,000 fragmentation spectra in standard 120 min LC runs are achievable, which can be used for near exhaustive precursor selection in complex mixtures or accumulating the signal of weak precursors. In 120 min single runs of HeLa digest, MaxQuant identified more than 6,000 proteins without matching to a library and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,500 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored.

Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli.

Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Glycoconjugate vaccines consisting of bacterial surface polysaccharides conjugated to carrier proteins are the most effective vaccines for controlling invasive bacterial infections. Nevertheless, the development of a multivalent conjugate vaccine to prevent Shigellosis has been hampered by the complex manufacturing process as the surface polysaccharide for each strain requires extraction, hydrolysis, chemical activation and conjugation to a carrier protein. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of glycoconjugates. Herein, the Shigella dysenteriae type 1 (Sd1) O-polysaccharide is expressed and its functional assembly on an E. coli glycosyl carrier lipid is demonstrated by HPLC analysis and mass spectrometry. The polysaccharide is enzymatically conjugated to specific asparagine residues of the carrier protein by co-expression of the PglB oligosaccharyltransferase and the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa. The extraction and purification of the Shigella glycoconjugate (Sd1-EPA) and its detailed characterization by the use of physicochemical methods including NMR and mass spectrometry is described. The report shows for the first time that bioconjugation provides a newly developed and improved approach to produce an Sd1 glycoconjugate that can be characterized using state-of-the-art techniques. In addition, this generic process together with the analytical methods is ideally suited for the production of additional Shigella serotypes, allowing the development of a multivalent Shigella vaccine.

A Sequential Homologation of Alkynes and Aldehydes for Chain Elongation with Optional (13)C-Labeling.

Terminal alkynes (RCCH) are homologated by a sequence of ruthenium-catalyzed anti-Markovnikov hydration of alkyne to aldehyde (RCH2CHO), followed by Bestmann-Ohira alkynylation of aldehyde to chain-elongated alkyne (RCH2CCH). Inverting the sequence by starting from aldehyde brings about the reciprocal homologation of aldehydes instead. The use of (13)C-labeled Bestmann-Ohira reagent (dimethyl ((1-(13)C)-1-diazo-2-oxopropyl)phosphonate) for alkynylation provides straightforward access to singly or, through additional homologation, multiply (13) C-labeled alkynes. The labeled alkynes serve as synthetic platform for accessing a multitude of specifically (13)C-labeled products. Terminal alkynes with one or two (13)C-labels in the alkyne unit have been submitted to alkyne-azide click reactions; the copper-catalyzed version (CuAAC) was found to display a regioselectivity of >50,000:1 for the 1,4- over the 1,5-triazine isomer, as shown analytically by (13)C NMR spectroscopy.

Nanoengineered diamond waveguide as a robust bright platform for nanomagnetometry using shallow nitrogen vacancy centers.

Photonic structures in diamond are key to most of its application in quantum technology. Here, we demonstrate tapered nanowaveguides structured directly onto the diamond substrate hosting shallow-implanted nitrogen vacancy (NV) centers. By optimization based on simulations and precise experimental control of the geometry of these pillar-shaped nanowaveguides, we achieve a net photon flux up to ∼ 1.7 × 10(6) s(-1). This presents the brightest monolithic bulk diamond structure based on single NV centers so far. We observe no impact on excited state lifetime and electronic spin dephasing time (T2) due to the nanofabrication process. Possessing such high brightness with low background in addition to preserved spin quality, this geometry can improve the current nanomagnetometry sensitivity ∼ 5 times. In addition, it facilitates a wide range of diamond defects-based magnetometry applications. As a demonstration, we measure the temperature dependency of T1 relaxation time of a single shallow NV center electronic spin. We observe the two-phonon Raman process to be negligible in comparison to the dominant two-phonon Orbach process.

Epithelial and tumor-associated endothelial expression of B7-H3 in cervical carcinoma: relation with CD8+ intraepithelial lymphocytes, FIGO stage, and phosphohistone H3 (PHH3) reactivity.

B7-H3 is a transmembrane protein and a member of the B7 family of immune regulatory ligands. It exerts both inhibitory and stimulatory effects on the activation of T cells. We investigated the expression of B7-H3 in invasive squamous cell carcinoma (ISCC) of the uterine cervix by immunohistochemistry, and aimed to determine whether expression of this factor is involved in the progression of the morphologic spectrum from normal cervical epithelia to cervical intraepithelial neoplasia and cervical ISCC. In addition, we sought to examine the relation of B7-H3 to the abundance of tumor-infiltrating and tumor-associated CD8(+) lymphocytes and to the evidence of phosphohistone H3, which is a core histone protein detected during mitosis. B7-H3 immunostaining was scored with regard to quantity and intensity of positively stained cells, and was noted in membranous and cytoplasmic patterns in epithelial cells and on endothelia of stromal blood vessels. Compared with those in intraepithelial neoplasias, immunoscores were significantly increased in ISCC (P<0.0001 for epithelial and endothelial expression, respectively). High scoring was associated with International Federation of Gynecology and Obstetrics stages IB and higher. Immunoscores of epithelial and endothelial B7-H3 expression were correlated significantly (P=0.0358). Epithelial and endothelial expression of B7-H3 was inversely related with CD8(+) tumor-infiltrating lymphocyte (P<0.0001). Moderate/strong B7-H3 epithelial as well as endothelial expression was mutually increased with intermediate/strong phosphohistone H3 scores (P=0.0396 and P=0.0483, respectively). There was no statistical relation with survival; however, no patient with negative scoring died of her tumor. Our results indicate that B7-H3 expression in cervical ISCC may play an important role in overcoming CD8(+) T-cell immunoregulation to acquire aggressive growth.