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Head and neck cancer (HNC) is the seventh most common cancer globally, resulting in 440 000 deaths per year. While there have been advancements in chemoradiotherapy and surgery, relapse occurs in more than half of HNCs, and these patients have a median survival of 10 months and a 2‐year survival of < 20%. Only a subset of patients displays durable benefits from immunotherapies in metastatic and recurrent HNC, making it critical to understand the tumor microenvironment (TME) underpinning therapy responses in HNC. To recognize biological differences within the TME that may be predictive of immunotherapy response, we applied cutting‐edge geospatial whole‐transcriptome profiling (NanoString GeoMx Digital Spatial Profiler) and spatial proteomics profiling (Akoya PhenoCycler‐Fusion) on a tumor microarray consisting of 25 cores from 12 patients that included 4 immunotherapy‐unresponsive (8 cores) and 2 immunotherapy‐responsive patients (5 cores), as well as 6 immunotherapy naïve patients (12 cores). Through high‐plex, regional‐based transcriptomic mapping of the tumor and TME, pathways involved with the complement system and hypoxia were identified to be differentially expressed in patients who went on to experience a poor immunotherapy response. Single‐cell, targeted proteomic analysis found that immune cell infiltration of the cancer cell mass and interactions of CD8 T cells with tumor and other immune cells were associated with positive immunotherapy response. The relative abundance of specific tumor phenotypes and their interactions with various immune cells was identified to be different between response groups. This study demonstrates how spatial transcriptomics and proteomics can resolve novel alterations in the TME of HNC that may contribute to therapy sensitivity and resistance. [ABSTRACT FROM AUTHOR]

Background: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system characterized by increased inflammation and immune responses, oxidative injury, mitochondrial dysfunction, and iron dyshomeostasis leading to demyelination and axonal damage. In MS, incomplete remyelination results in chronically demyelinated axons and degeneration coinciding with disability. This suggests a failure in the ability to remyelinate in MS, however, the precise underlying mechanisms remain unclear. We aimed to identify proteins whose expression was altered in chronic inactive white matter lesions and periplaque white matter in MS tissue to reveal potential pathophysiological mechanisms. Methods: Laser capture microdissection coupled to proteomics was used to interrogate spatially altered changes in formalin-fixed paraffin-embedded brain tissue from three chronic MS individuals and three controls with no apparent neurological complications. Histopathological maps guided the capture of inactive lesions, periplaque white matter, and cortex from chronic MS individuals along with corresponding white matter and cortex from control tissue. Label free quantitation by liquid chromatography tandem mass spectrometry was used to discover differentially expressed proteins between the various brain regions. Results: In addition to confirming loss of several myelin-associated proteins known to be affected in MS, proteomics analysis of chronic inactive MS lesions revealed alterations in myelin assembly, metabolism, and cytoskeletal organization. The top altered proteins in MS inactive lesions compared to control white matter consisted of PPP1R14A, ERMN, SIRT2, CARNS1, and MBLAC2. Conclusion: Our findings highlight proteome changes in chronic inactive MS white matter lesions and periplaque white matter, which may be crucial for proper myelinogenesis, bioenergetics, focal adhesions, and cellular function. This study highlights the importance and feasibility of spatial approaches such as laser capture microdissection-based proteomics analysis of pathologically distinct regions of MS brain tissue. Identification of spatially resolved changes in the proteome of MS brain tissue should aid in the understanding of pathophysiological mechanisms and the development of novel therapies. [ABSTRACT FROM AUTHOR]

MET inhibitors have demonstrated efficacy in treating patients with non-small cell lung cancer (NSCLC) harboring METex14 skipping alterations. Advancements in spatial profiling technologies have unveiled the complex dynamics of the tumor microenvironment (TME), a crucial factor in cancer progression and therapeutic response.This study uses spatial profiling to investigate the effects of the MET inhibitor tepotinib on the TME in a case of locally advanced NSCLC with a METex14 skipping alteration. A patient with resectable stage IIIB NSCLC, unresponsive to neoadjuvant platinum-based chemotherapy, received tepotinib following the detection of a METex14 skipping alteration. Paired pre- and post-treatment biopsies were subjected to GeoMx Digital Spatial Profiling using the Cancer Transcriptome Atlas and immune-related protein panels to evaluate shifts in the immune TME.Tepotinib administration allowed for a successful lobectomy and a pathological downstaging to stage IA1. The TME was transformed from an immunosuppressive to a more permissive state, with upregulation of antigen-presenting and pro-inflammatory immune cells. Moreover, a marked decrease in immune checkpoint molecules, including PD-L1, was noted. Spatial profiling identified discrete immune-enriched clusters, indicating the role of tepotinib in modulating immune cell trafficking and function. Tepotinib appears to remodel the immune TME in a patient with METex14 skipping NSCLC, possibly increasing responsiveness to immunotherapy.Our study supports the integration of genetic profiling into the management of early and locally advanced NSCLC to guide personalized, targeted interventions. These findings underscore the need to further evaluate combinations of MET inhibitors and immunotherapies.

Introduction: Drug development is systemically inefficient. Research and development costs for novel therapeutics average hundreds of millions to billions of dollars, with the overall likelihood of approval estimated to be as low as 6.7% for oncology drugs. Over half of these failures are due to a lack of drug efficacy. This pervasive and repeated low rate of success exemplifies how preclinical models fail to adequately replicate the complexity and heterogeneity of human cancer. Therefore, new methods of evaluation, early in the development trajectory, are essential both to rule-in and rule-out novel agents with more rigor and speed, but also to spare clinical trial patients from the potentially toxic sequelae (high risk) of testing investigational agents that have a low likelihood of producing a response (low benefit). Methods: The clinical in vivo oncology (CIVO®) platform was designed to change this drug development paradigm. CIVO precisely delivers microdose quantities of up to 8 drugs or combinations directly into patient tumors 4-96 h prior to planned surgical resection. Resected tissue is then analyzed for responses at each site of intratumoral drug exposure. Results: To date, CIVO has been used safely in 6 clinical trials, including 68 subjects, with 5 investigational and 17 approved agents. Resected tissues were analyzed initially using immunohistochemistry and in situ hybridization assays (115 biomarkers). As technology advanced, the platform was paired with spatial biology analysis platforms, to successfully track anti-neoplastic and immunemodulating activity of the injected agents in the intact tumor microenvironment. Discussion: Herein we provide a report of the use of CIVO technology in patients, a depiction of the robust analysis methods enabled by this platform, and a description of the operational and regulatory mechanisms used to deploy this approach in synergistic partnership with pharmaceutical partners. We further detail how use of the CIVO platform is a clinically safe and scientifically precise alternative or complement to preclinical efficacy modeling, with outputs that inform, streamline, and de-risk drug development. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy has been rejuvenated by the growing understanding of the immune system's role in tumor activity over the past two decades. During cancer initiation and progression, tumor cells employ various mechanisms that resemble peripheral immune tolerance to evade the antitumor responses of the immune system. Immune checkpoint molecules are the major mechanism of immune resistance that are exploited by tumor cells to inhibit T‐cell activation and suppress immune responses. The targeting of immune checkpoint pathways has led to substantial improvements in survival rates in a number of solid cancers. However, a lack of understanding of the heterogeneity of the tumor microenvironment (TME) has resulted in inefficient therapy responses. A greater understanding of the TME is needed to identify patients likely to respond, and those that will have resistance to immune checkpoint inhibitors (ICIs). Advancement in spatial single‐cell technologies has allowed deeper insight into the phenotypic and functional diversities of cells in the TME. In this review, we provide an overview of ICI biomarkers and highlight how high‐dimensional spatially resolved, single‐cell approaches provide deep molecular insights into the TME and allow for the discovery of biomarkers of clinical benefit. [ABSTRACT FROM AUTHOR]

BackgroundMultiple sclerosis (MS) is a demyelinating disease of the central nervous system characterized by increased inflammation and immune responses, oxidative injury, mitochondrial dysfunction, and iron dyshomeostasis leading to demyelination and axonal damage. In MS, incomplete remyelination results in chronically demyelinated axons and degeneration coinciding with disability. This suggests a failure in the ability to remyelinate in MS, however, the precise underlying mechanisms remain unclear. We aimed to identify proteins whose expression was altered in chronic inactive white matter lesions and periplaque white matter in MS tissue to reveal potential pathophysiological mechanisms.MethodsLaser capture microdissection coupled to proteomics was used to interrogate spatially altered changes in formalin-fixed paraffin-embedded brain tissue from three chronic MS individuals and three controls with no apparent neurological complications. Histopathological maps guided the capture of inactive lesions, periplaque white matter, and cortex from chronic MS individuals along with corresponding white matter and cortex from control tissue. Label free quantitation by liquid chromatography tandem mass spectrometry was used to discover differentially expressed proteins between the various brain regions.ResultsIn addition to confirming loss of several myelin-associated proteins known to be affected in MS, proteomics analysis of chronic inactive MS lesions revealed alterations in myelin assembly, metabolism, and cytoskeletal organization. The top altered proteins in MS inactive lesions compared to control white matter consisted of PPP1R14A, ERMN, SIRT2, CARNS1, and MBLAC2.ConclusionOur findings highlight proteome changes in chronic inactive MS white matter lesions and periplaque white matter, which may be crucial for proper myelinogenesis, bioenergetics, focal adhesions, and cellular function. This study highlights the importance and feasibility of spatial approaches such as laser capture microdissection-based proteomics analysis of pathologically distinct regions of MS brain tissue. Identification of spatially resolved changes in the proteome of MS brain tissue should aid in the understanding of pathophysiological mechanisms and the development of novel therapies.

The role of the tumor microenvironment (TME) in immuno‐oncology has driven demand for technologies that deliver in situ, or spatial, molecular information. Compartmentalized heterogeneity that traditional methods miss is becoming key to predicting both acquired drug resistance to targeted therapies and patient response to immunotherapy. Here, we describe a novel method for assay‐agnostic spatial profiling and demonstrate its ability to detect immune microenvironment signatures in breast cancer patients that are unresolved by the immunohistochemical (IHC) assessment of programmed cell death ligand‐1 (PD‐L1) on immune cells, which represents the only FDA microenvironment‐based companion diagnostic test that has been approved for triple‐negative breast cancer (TNBC). Two distinct physiological states were found that are uncorrelated to tumor mutational burden (TMB), microsatellite instability (MSI), PD‐L1 expression, and intrinsic cancer subtypes.

Introduction: Drug development is systemically inefficient. Research and development costs for novel therapeutics average hundreds of millions to billions of dollars, with the overall likelihood of approval estimated to be as low as 6.7% for oncology drugs. Over half of these failures are due to a lack of drug efficacy. This pervasive and repeated low rate of success exemplifies how preclinical models fail to adequately replicate the complexity and heterogeneity of human cancer. Therefore, new methods of evaluation, early in the development trajectory, are essential both to rule-in and rule-out novel agents with more rigor and speed, but also to spare clinical trial patients from the potentially toxic sequelae (high risk) of testing investigational agents that have a low likelihood of producing a response (low benefit).Methods: The clinical in vivo oncology (CIVO®) platform was designed to change this drug development paradigm. CIVO precisely delivers microdose quantities of up to 8 drugs or combinations directly into patient tumors 4–96 h prior to planned surgical resection. Resected tissue is then analyzed for responses at each site of intratumoral drug exposure.Results: To date, CIVO has been used safely in 6 clinical trials, including 68 subjects, with 5 investigational and 17 approved agents. Resected tissues were analyzed initially using immunohistochemistry and in situ hybridization assays (115 biomarkers). As technology advanced, the platform was paired with spatial biology analysis platforms, to successfully track anti-neoplastic and immune-modulating activity of the injected agents in the intact tumor microenvironment.Discussion: Herein we provide a report of the use of CIVO technology in patients, a depiction of the robust analysis methods enabled by this platform, and a description of the operational and regulatory mechanisms used to deploy this approach in synergistic partnership with pharmaceutical partners. We further detail how use of the CIVO platform is a clinically safe and scientifically precise alternative or complement to preclinical efficacy modeling, with outputs that inform, streamline, and de-risk drug development.

Abstract Introduction Triple‐negative breast cancer (TNBC) patients have the poorest clinical outcomes compared to other molecular subtypes of breast cancer. IL6/JAK/STAT3 signalling is upregulated in breast cancer; however, there is limited evidence for its role in TNBC. This study aimed to assess the expression of IL6/JAK/STAT3 in TNBC as a prognostic biomarker. Methods Tissue microarrays consisting of breast cancer specimens from a retrospective cohort (n = 850) were stained for IL6R, JAK1, JAK2 and STAT3 via immunohistochemistry. Staining intensity was assessed by weighted histoscore and analysed for association with survival/clinical characteristics. In a subset of patients (n = 14) bulk transcriptional profiling was performed using TempO‐Seq. Nanostring GeoMx® digital spatial profiling was utilised to establish the differential spatial gene expression in high STAT3 tumours. Results In TNBC patients, high expression of stromal STAT3 was associated with reduced cancer‐specific survival (HR = 2.202, 95% CI: 1.148–4.224, log rank p = 0.018). TNBC patients with high stromal STAT3 had reduced CD4+ T‐cell infiltrates within the tumour (p = 0.001) and higher tumour budding (p = 0.003). Gene set enrichment analysis (GSEA) of bulk RNA sequencing showed high stromal STAT3 tumours were characterised by enrichment of IFNγ, upregulation of KRAS signalling and inflammatory signalling Hallmark pathways. GeoMx™ spatial profiling showed high stromal STAT3 samples. Pan cytokeratin (panCK)‐negative regions were enriched for CD27 (p

The role of the tumor microenvironment (TME) in immuno‐oncology has driven demand for technologies that deliver in situ, or spatial, molecular information. Compartmentalized heterogeneity that traditional methods miss is becoming key to predicting both acquired drug resistance to targeted therapies and patient response to immunotherapy. Here, we describe a novel method for assay‐agnostic spatial profiling and demonstrate its ability to detect immune microenvironment signatures in breast cancer patients that are unresolved by the immunohistochemical (IHC) assessment of programmed cell death ligand‐1 (PD‐L1) on immune cells, which represents the only FDA microenvironment‐based companion diagnostic test that has been approved for triple‐negative breast cancer (TNBC). Two distinct physiological states were found that are uncorrelated to tumor mutational burden (TMB), microsatellite instability (MSI), PD‐L1 expression, and intrinsic cancer subtypes. [ABSTRACT FROM AUTHOR]

ABSTRACT: The noninvasive, longitudinal study of products and food processing is of interest for the dairy industry. Here, we demonstrated that single-sided nuclear magnetic resonance (NMR) can be used for noninvasive monitoring of the cheese ripening process. The maturation of soft-ripened Camembert-like molded cheese samples was monitored for 20 d measuring 1-dimensional and 2-dimensional NMR relaxation and diffusion data at various depths, ranging from the hard surface layer to the soft center. Gelation and gel shrinkage were observed throughout ripening, and a complete loss of free water signal was observed at the cheese rind. Transversal (T2) relaxation distributions include 3 components that evolve with ripening time and position, corresponding to water inside the casein gel network, water trapped in casein, and fat. Two-dimensional T1-T2 relaxation experiments provided enhanced resolution of the 3 components, allowing quantification of the relative proportions of each phase. Furthermore, diffusion (D)-T2 relaxation correlation experiments revealed the bimodal size distribution of fat globules. The study demonstrated that single-sided NMR can provide spatially resolved signal intensity, relaxation, and diffusion parameters that reflect structural changes during the ripening process and can be exploited to understand and monitor the ripening of cheeses.

Tumour heterogeneity is pervasive amongst many cancers and leads to disease progression, and therapy resistance. In this review, using breast cancer as an exemplar, we focus on the recent advances in understanding the interplay between tumour cells and their microenvironment using single cell sequencing and digital spatial profiling technologies. Further, we discuss the utility of lineage tracing methodologies in pre‐clinical models of breast cancer, and how these are being used to unravel new therapeutic vulnerabilities and reveal biomarkers of breast cancer progression. © 2023 The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Simple Summary: Glioblastoma (GBM) are the main primary brain tumors in adults. The role of the immune system in the progression of GBM is still largely under-evaluated. Here, we assessed the distribution of multiple immune biomarkers in a handful of coupled primary and recurrent GBM using both classical immunochemistry and Nanostring Geomix Digital spatial profiling. We observed an absence of correlation between the immune landscape during the evolution of GBM, both in the center and the periphery of the tumor. However, global analyses indicate differential localization of HLA-DR and B7-H3 between primary and recurrent GBM. Only a minority of patients with glioblastoma (GBM) respond to immunotherapy, and always only partially. There is a lack of knowledge on immune distribution in GBM and in its tumor microenvironment (TME). To address the question, we used paired primary and recurrent tumors from GBM patients to study the composition and the evolution of the immune landscape upon treatment. We studied the expression of a handful of immune markers (CD3, CD8, CD68, PD-L1 and PD-1) in GBM tissues in 15 paired primary and recurrent GBM. In five selected patients, we used Nanostring Digital Spatial Profiling (DSP) to obtain simultaneous assessments of multiple biomarkers both within the tumor and the microenvironment in paired primary and recurrent GBM. Our results suggest that the evolution of the immune landscape between paired primary and recurrent GBM tumors is highly heterogeneous. However, our study identifies B3-H7 and HLA-DR as potential targets in primary and recurrent GBM. Spatial profiling of immune markers from matched primary and recurrent GBM shows a nonlinear complex evolution during the progression of cancer. Nonetheless, our study demonstrated a global increase in macrophages, and revealed differential localization of some markers, such as B7-H3 and HLA-DR, between GBM and its TME. [ABSTRACT FROM AUTHOR]

Introduction: Triple‐negative breast cancer (TNBC) patients have the poorest clinical outcomes compared to other molecular subtypes of breast cancer. IL6/JAK/STAT3 signalling is upregulated in breast cancer; however, there is limited evidence for its role in TNBC. This study aimed to assess the expression of IL6/JAK/STAT3 in TNBC as a prognostic biomarker. Methods: Tissue microarrays consisting of breast cancer specimens from a retrospective cohort (n = 850) were stained for IL6R, JAK1, JAK2 and STAT3 via immunohistochemistry. Staining intensity was assessed by weighted histoscore and analysed for association with survival/clinical characteristics. In a subset of patients (n = 14) bulk transcriptional profiling was performed using TempO‐Seq. Nanostring GeoMx® digital spatial profiling was utilised to establish the differential spatial gene expression in high STAT3 tumours. Results: In TNBC patients, high expression of stromal STAT3 was associated with reduced cancer‐specific survival (HR = 2.202, 95% CI: 1.148–4.224, log rank p = 0.018). TNBC patients with high stromal STAT3 had reduced CD4+ T‐cell infiltrates within the tumour (p = 0.001) and higher tumour budding (p = 0.003). Gene set enrichment analysis (GSEA) of bulk RNA sequencing showed high stromal STAT3 tumours were characterised by enrichment of IFNγ, upregulation of KRAS signalling and inflammatory signalling Hallmark pathways. GeoMx™ spatial profiling showed high stromal STAT3 samples. Pan cytokeratin (panCK)‐negative regions were enriched for CD27 (p < 0.001), CD3 (p < 0.05) and CD8 (p < 0.001). In panCK‐positive regions, high stromal STAT3 regions had higher expression of VEGFA (p < 0.05). Conclusion: High expression of IL6/JAK/STAT3 proteins was associated with poor prognosis and characterised by distinct underlying biology in TNBC. [ABSTRACT FROM AUTHOR]

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Single-cell and single-population RNA sequencing (RNA-seq) is a rapidly evolving new field of intense investigation. Recent studies indicate unique transcriptomic profiles are derived based on the spatial localization of neurons within circuits and regions. Individual neuronal subtypes can have vastly different transcriptomic fingerprints, well beyond the basic excitatory neuron and inhibitory neuron designations. To study single-population gene expression profiles of spatially characterized neurons, we have developed a methodology combining laser capture microdissection (LCM), RNA purification of single populations of neurons, and subsequent library preparation for downstream applications, including RNA-seq. LCM provides the benefit of isolating single neurons characterized by morphology or via transmitter-identified and/or receptor immunoreactivity and enables spatial localization within the sample. We utilize unfixed human postmortem and mouse brain tissue that is frozen to preserve RNA quality in order to isolate the desired neurons of interest. Microisolated neurons are then pooled for RNA purification utilizing as few as 250 individual neurons from a tissue section, precluding extraneous nonspecific tissue contaminants. Library preparation is performed from picogram RNA quantities extracted from LCM-captured neurons. Single-population RNA-seq analysis demonstrates that microisolated neurons from both postmortem human and mouse brain tissues are viable for transcriptomic profiling, including differential gene expression assessment and bioinformatic pathway inquiry. [ABSTRACT FROM AUTHOR]

The tumor microenvironment (TME) is composed of many different cellular and acellular components that together drive tumor growth, invasion, metastasis, and response to therapies. Increasing realization of the significance of the TME in cancer biology has shifted cancer research from a cancer-centric model to one that considers the TME as a whole. Recent technological advancements in spatial profiling methodologies provide a systematic view and illuminate the physical localization of the components of the TME. In this review, we provide an overview of major spatial profiling technologies. We present the types of information that can be extracted from these data and describe their applications, findings and challenges in cancer research. Finally, we provide a future perspective of how spatial profiling could be integrated into cancer research to improve patient diagnosis, prognosis, stratification to treatment and development of novel therapeutics. Elhanani et al. review major spatial profiling technologies and their application, findings, and challenges in cancer research. They propose how spatial profiling could be integrated into cancer research to improve patient diagnosis, prognosis, stratification to treatment, and development of novel therapeutics. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is known to present with pulmonary and extra‐pulmonary organ complications. In comparison with the 2009 pandemic (pH1N1), SARS‐CoV‐2 infection is likely to lead to more severe disease, with multi‐organ effects, including cardiovascular disease. SARS‐CoV‐2 has been associated with acute and long‐term cardiovascular disease, but the molecular changes that govern this remain unknown. In this study, we investigated the host transcriptome landscape of cardiac tissues collected at rapid autopsy from seven SARS‐CoV‐2, two pH1N1, and six control patients using targeted spatial transcriptomics approaches. Although SARS‐CoV‐2 was not detected in cardiac tissue, host transcriptomics showed upregulation of genes associated with DNA damage and repair, heat shock, and M1‐like macrophage infiltration in the cardiac tissues of COVID‐19 patients. The DNA damage present in the SARS‐CoV‐2 patient samples, were further confirmed by γ‐H2Ax immunohistochemistry. In comparison, pH1N1 showed upregulation of interferon‐stimulated genes, in particular interferon and complement pathways, when compared with COVID‐19 patients. These data demonstrate the emergence of distinct transcriptomic profiles in cardiac tissues of SARS‐CoV‐2 and pH1N1 influenza infection supporting the need for a greater understanding of the effects on extra‐pulmonary organs, including the cardiovascular system of COVID‐19 patients, to delineate the immunopathobiology of SARS‐CoV‐2 infection, and long term impact on health. [ABSTRACT FROM AUTHOR]

Multiplexed imaging, which enables spatial localization of proteins and RNA to cells within tissues, complements existing multi-omic technologies and has deepened our understanding of health and disease. CODEX, a multiplexed single-cell imaging technology, utilizes a microfluidics system that incorporates DNA barcoded antibodies to visualize 50 + cellular markers at the single-cell level. Here, we discuss the latest applications of CODEX to studies of cancer, autoimmunity, and infection as well as current bioinformatics approaches for analysis of multiplexed imaging data from preprocessing to cell segmentation and marker quantification to spatial analysis techniques. We conclude with a commentary on the challenges and future developments for multiplexed spatial profiling. [ABSTRACT FROM AUTHOR]

Technological progress over the past 15 years has fueled an explosion in genome‐wide chromatin profiling tools that take advantage of low‐cost short‐read sequencing technologies to map particular chromatin features. Here, we survey the recent development of epigenomic tools that provide precise positions of chromatin proteins genome‐wide in intact cells or nuclei. Some profiling tools are based on tethering Micrococcal Nuclease to chromatin proteins of interest in situ, whereas others similarly tether Tn5 transposase to integrate DNA sequencing adapters (tagmentation) and so eliminate the need for library preparation. These in situ cleavage and tagmentation tools have gained in popularity over the past few years, with many protocol enhancements and adaptations for single‐cell and spatial chromatin profiling. The application of experimental and computational tools to address problems in gene regulation, eukaryotic development, and human disease are helping to define the emerging field of chromatin structural epigenomics. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is an extremely aggressive and incurable primary brain tumor with a 10-year survival of just 0.71%. Cancer stem cells (CSCs) are thought to seed GBM's inevitable recurrence by evading standard of care treatment, which combines surgical resection, radiotherapy, and chemotherapy, contributing to this grim prognosis. Effective targeting of CSCs could result in insights into GBM treatment resistance and development of novel treatment paradigms. There is a major ongoing effort to characterize CSCs, understand their interactions with the tumor microenvironment, and identify ways to eliminate them. This review discusses the diversity of CSC lineages present in GBM and how this glioma stem cell (GSC) mosaicism drives global intratumoral heterogeneity constituted by complex and spatially distinct local microenvironments. We review how a tumor's diverse CSC populations orchestrate and interact with the environment, especially the immune landscape. We also discuss how to map this intricate GBM ecosystem through the lens of metabolism and immunology to find vulnerabilities and new ways to disrupt the equilibrium of the system to achieve improved disease outcome. [ABSTRACT FROM AUTHOR]

Simple Summary: Immune checkpoint inhibitors (ICIs) aim to re-establish cancer immune control by modulating immune-inhibitory signaling pathways. ICIs are currently approved in breast cancer treatment act by blocking cell anti-PD-1/PD-L1 interactions. Nonetheless, as many mechanisms of immune escape can underlie the insurgence of cancer cells, most patients progress to ICIs, even when combined with chemotherapy. Multiplexed single-cell spatially resolved tissue analysis, by combining monoclonal antibodies with different reporters, can obtain precise single-cell epitope colocalization and thus allow to infer cellular functional states, while conserving their spatial coordinates. In this review, we highlight the potential of this technology in the context of breast cancer by selecting relevant prognostic and predictive markers through the lens of the cancer-immunity cycle. Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of many solid tumors. In breast cancer (BC), immunotherapy is currently approved in combination with chemotherapy, albeit only in triple-negative breast cancer. Unfortunately, most patients only derive limited benefit from ICIs, progressing either upfront or after an initial response. Therapeutics must engage with a heterogeneous network of complex stromal–cancer interactions that can fail at imposing cancer immune control in multiple domains, such as in the genomic, epigenomic, transcriptomic, proteomic, and metabolomic domains. To overcome these types of heterogeneous resistance phenotypes, several combinatorial strategies are underway. Still, they can be predicted to be effective only in the subgroups of patients in which those specific resistance mechanisms are effectively in place. As single biomarker predictive performances are necessarily suboptimal at capturing the complexity of this articulate network, precision immune-oncology calls for multi-omics tumor microenvironment profiling in order to identify unique predictive patterns and to proactively tailor combinatorial treatments. Multiplexed single-cell spatially resolved tissue analysis, through precise epitope colocalization, allows one to infer cellular functional states in view of their spatial organization. In this review, we discuss—through the lens of the cancer-immunity cycle—selected, established, and emerging markers that may be evaluated in multiplexed spatial protein panels to help identify prognostic and predictive patterns in BC. [ABSTRACT FROM AUTHOR]

Macrophages are key regulators of inflammation and repair, but their heterogeneity and multiple roles in the liver are not fully understood. We aimed herein to map the intrahepatic macrophage populations and their function(s) during acute liver injury. We used flow cytometry, gene expression analysis, multiplex-immunofluorescence, 3D-reconstruction, and spatial image analysis to characterize the intrahepatic immune landscape in mice post-CCl4-induced acute liver injury during three distinct phases: necroinflammation, and early and late repair. We observed hepatocellular necrosis and a reduction in liver resident lymphocytes during necroinflammation accompanied by the infiltration of circulating myeloid cells and upregulation of inflammatory cytokines. These parameters returned to baseline levels during the repair phase while pro-repair chemokines were upregulated. We identified resident CLEC4F+ Kupffer cells (KCs) and infiltrating IBA1+CLEC4F- monocyte-derived macrophages (MoMFs) as the main hepatic macrophage populations during this response to injury. While occupying most of the necrotic area, KCs and MoMFs exhibited distinctive kinetics, distribution and morphology at the site of injury. The necroinflammation phase was characterized by low levels of KCs and a remarkable invasion of MoMFs suggesting their potential role in phagoctosing necrotic hepatocytes, while opposite kinetics/distribution were observed during repair. During the early repair phase, yolksac - derived KCs were restored, whereas MoMFs diminished gradually then dissipated during late repair. MoMFs interacted with hepatic stellate cells during the necroinflammatory and early repair phases, potentially modulating their activation state and influencing their fibrogenic and pro-repair functions that are critical for wound healing. Altogether, our study reveals novel and distinctspatial and temporal distribution of KCs and MoMFs and provides insights into their complementary roles during acute liver injury. [ABSTRACT FROM AUTHOR]

This concise review provides the broad background and selection from the literature for a Keynote lecture at EHSF 2022 on state of the art technologies in inflammation research, with an emphasis on disease of the skin and the nervous system. The value of ex vivo skin explant models is discussed, as well as the innovative use of animal models, wherein the crucial roles of antigen experience and "wild" microbiota are emphasized. Spectral flow cytometry allowing large surface marker panels to be explored is touched upon, as well as multiplex technology for cytokines and other analytes important for inflammation and tissue damage. Single‐cell sequencing and in situ transcriptomics (spatial profiling) now provide exciting granular information on functional cell subsets, interactions and plasticity. A selection of novel research and diagnostic tools for antibodies against linear peptides or gangliosides is presented. Finally, the review discusses a new anti‐inflammatory strategy against skin inflammation with a panel of protease inhibitors derived from the protein fraction of industrial starch potatoes. [ABSTRACT FROM AUTHOR]

An array of phenotypically diverse myeloid cells and macrophages (MC&M) resides in the tumor microenvironment, requiring multiplexed detection systems for visualization. Here we report an automated, multiplexed staining approach, named PLEXODY, that consists of five MC&M-related fluorescently-tagged antibodies (anti - CD68, - CD163, - CD206, - CD11b, and - CD11c), and three chromogenic antibodies, reactive with high- and low-molecular weight cytokeratins and CD3, highlighting tumor regions, benign glands and T cells. The staining prototype and image analysis methods which include a pixel/area-based quantification were developed using tissues from inflamed colon and tonsil and revealed a unique tissue-specific composition of 14 MC&M-associated pixel classes. As a proof-of-principle, PLEXODY was applied to three cases of pancreatic, prostate and renal cancers. Across digital images from these cancer types we observed 10 MC&M-associated pixel classes at frequencies greater than 3%. Cases revealed higher frequencies of single positive compared to multi-color pixels and a high abundance of CD68+/CD163+ and CD68+/CD163+/CD206+ pixels. Significantly more CD68+ and CD163+ vs. CD11b+ and CD11c+ pixels were in direct contact with tumor cells and T cells. While the greatest percentage (~70%) of CD68+ and CD163+ pixels was 0-20 microns away from tumor and T cell borders, CD11b+ and CD11c+ pixels were detected up to 240 microns away from tumor/T cell masks. Together, these data demonstrate significant differences in densities and spatial organization of MC&M-associated pixel classes, but surprising similarities between the three cancer types.

The 2023 Annual Review Issue of The Journal of Pathology, Recent Advances in Pathology, contains 12 invited reviews on topics of current interest in pathology. This year, our subjects include immuno‐oncology and computational pathology approaches for diagnostic and research applications in human disease. Reviews on the tissue microenvironment include the effects of apoptotic cell‐derived exosomes, how understanding the tumour microenvironment predicts prognosis, and the growing appreciation of the diverse functions of fibroblast subtypes in health and disease. We also include up‐to‐date reviews of modern aspects of the molecular basis of malignancies, and our final review covers new knowledge of vascular and lymphatic regeneration in cardiac disease. All of the reviews contained in this issue are written by expert groups of authors selected to discuss the recent progress in their particular fields and all articles are freely available online (https://pathsocjournals.onlinelibrary.wiley.com/journal/10969896). © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Single-cell omics aim at charting the different types and properties of all cells in the human body in health and disease. Over the past years, myriads of cellular phenotypes have been defined by methods that mostly required cells to be dissociated and removed from their original microenvironment, thus destroying valuable information about their location and interactions. Growing insights, however, are showing that such information is crucial to understand complex disease states. For decades, pathologists have interpreted cells in the context of their tissue using low-plex antibody- and morphology-based methods. Novel technologies for multiplexed immunohistochemistry are now rendering it possible to perform extended single-cell expression profiling using dozens of protein markers in the spatial context of a single tissue section. The combination of these novel technologies with extended data analysis tools allows us now to study cell-cell interactions, define cellular sociology, and describe detailed aberrations in tissue architecture, as such gaining much deeper insights in disease states. In this review, we provide a comprehensive overview of the available technologies for multiplexed immunohistochemistry, their advantages and challenges. We also provide the principles on how to interpret high-dimensional data in a spatial context. Similar to the fact that no one can just “read” a genome, pathological assessments are in dire need of extended digital data repositories to bring diagnostics and tissue interpretation to the next level. [ABSTRACT FROM AUTHOR]

Single-cell omics aim at charting the different types and properties of all cells in the human body in health and disease. Over the past years, myriads of cellular phenotypes have been defined by methods that mostly required cells to be dissociated and removed from their original microenvironment, thus destroying valuable information about their location and interactions. Growing insights, however, are showing that such information is crucial to understand complex disease states. For decades, pathologists have interpreted cells in the context of their tissue using low-plex antibody- and morphology-based methods. Novel technologies for multiplexed immunohistochemistry are now rendering it possible to perform extended single-cell expression profiling using dozens of protein markers in the spatial context of a single tissue section. The combination of these novel technologies with extended data analysis tools allows us now to study cell-cell interactions, define cellular sociology, and describe detailed aberrations in tissue architecture, as such gaining much deeper insights in disease states. In this review, we provide a comprehensive overview of the available technologies for multiplexed immunohistochemistry, their advantages and challenges. We also provide the principles on how to interpret high-dimensional data in a spatial context. Similar to the fact that no one can just “read” a genome, pathological assessments are in dire need of extended digital data repositories to bring diagnostics and tissue interpretation to the next level.

In order to understand deposit formation during urea selective catalytic reduction (SCR) resulting from isocyanic acid (HNCO) formation, the present study investigates the potential of HNCO hydrolysis by spatially resolved gas phase concentration profiles along a single catalyst channel of commercial Cu‐zeolite and V‐based SCR catalysts. The spatially resolved profiles, obtained in a special hot gas test rig via capillary technique, provide information on reaction rates of HNCO hydrolysis, NH3 adsorption and NO conversion, hereby revealing a better performance of the standard V‐based catalyst regarding the HNCO hydrolysis, which is attributed to the TiO2 support. [ABSTRACT FROM AUTHOR]

Recent advances in single-cell technologies, particularly single-cell RNA-sequencing (scRNA-seq), have permitted high throughput transcriptional profiling of a wide variety of biological systems. As scRNA-seq supports inference of cell-cell communication, this technology has and continues to anchor groundbreaking studies into the efficacy and mechanism of novel immunotherapies for cancer treatment. In this review, we will highlight methods developed to infer inter- and intracellular signaling from scRNA-seq and discuss how they have contributed to studies of immunotherapeutic intervention in the tumor microenvironment (TME). However, a central challenge remains in validating the hypothesized cell-cell interactions. Therefore, this review will also cover strategies for integration of these scRNA-seq-derived interaction networks with existing experimental and computational approaches. Integration of these networks with imaging, protein secretion measurements, and network analysis and mathematical modeling tools addresses challenges that remain with scRNA-seq to enhance studies of immunosuppressive and immunotherapy-altered signaling in the TME. [ABSTRACT FROM AUTHOR]

A high-resolution vector-sum phase shifter fabricated using a 28 nm CMOS was developed for a 79 GHz imaging radar. The phase shifter incorporates a process and temperature (P/T) stable in-phase (I)/quadrature-phase (Q) signal generator with a robust matching network and highly linear variable gain amplifiers (VGAs) with a crossing metal-line layout. These techniques contribute to 0.5° phase-setting resolution and a 0.9° root-mean-square (rms) phase error with no calibration process. The developed phase shifters were applied to a 79 GHz imaging radar prototype that consists of eight-channel transmitters and eight-channel receivers. The measurement results show that our prototype can recognize human posture, recognize multiple people, and monitor vital signs. [ABSTRACT FROM AUTHOR]

The paper presents an option of the steam condensing turbine K-325-23.5 (K-300 series) low pressure cylinder flow part improvement due to the last stage modernization. The K-325-23.5 turbine is designed to replace the outdated K-300 series turbines, which together with the K-200 series turbines form the basis of Ukraine's thermal energy. In the modernized flow part, new last stage guide apparatus blades with a complex circular lean near the hub are used. The purpose of the modernization was to increase the low-pressure cylinder efficiency in the "bad" condenser vacuum to ensure that it did not "decrease" its efficiency at rated operating modes. The modernized low-pressure cylinder flow part is developed with the usage of modern methods of the viscous three-dimensional flow calculation based on the numerical integration of the Reynolds-averaged Navier-Stoks equations. For the turbulent effects, a two-parameter differential SST Menter turbulence model is applied, and for the hydraulic fluid real properties, the IAPWS-95 state equation is used. To construct the axial blades three-dimensional geometry, the original method, the initial data for which was the limited number of parameterized quantities, was used. The applied methods of gas-dynamic calculations and design of flow turbomachines are implemented in the IPMFlow software package, which is the development of the FlowER and FlowER-U software packages. The researched low-pressure cylinder flow part is limited by the last two stages (4th and 5th). A difference grid with a total element volume of more than 3 million is used to construct the calculation area. The research examined more than 20 options of the last stage stator blades. In the modernized flow part of the low-pressure cylinder last stage at rated operating mode, the gain of the efficiency coefficient (efficiency) is 0.9% and power – 0.61 MW. In the mode of "bad" condenser vacuum (with high pressure) a significant increase is achieved: efficiency – by 11.5%, power increased by almost 2 MW.

Recent advances in single-cell technologies, particularly single-cell RNA-sequencing (scRNA-seq), have permitted high throughput transcriptional profiling of a wide variety of biological systems. As scRNA-seq supports inference of cell-cell communication, this technology has and continues to anchor groundbreaking studies into the efficacy and mechanism of novel immunotherapies for cancer treatment. In this review, we will highlight methods developed to infer inter- and intracellular signaling from scRNA-seq and discuss how they have contributed to studies of immunotherapeutic intervention in the tumor microenvironment (TME). However, a central challenge remains in validating the hypothesized cell-cell interactions. Therefore, this review will also cover strategies for integration of these scRNA-seq-derived interaction networks with existing experimental and computational approaches. Integration of these networks with imaging, protein secretion measurements, and network analysis and mathematical modeling tools addresses challenges that remain with scRNA-seq to enhance studies of immunosuppressive and immunotherapy-altered signaling in the TME.

Immune responses require complex, spatially coordinated interactions between immune cells and their tissue environment. For decades, we have imaged tissue sections to visualize a limited number of immune-related macromolecules in situ, functioning as surrogates for cell types or processes of interest. However, this inevitably provides a limited snapshot of the tissue's immune landscape. Recent developments in high-throughput spatial -omics technologies, particularly spatial transcriptomics, and its application to human samples has facilitated a more comprehensive understanding of tissue immunity by mapping fine-grained immune cell states to their precise tissue location while providing contextual information about their immediate cellular and tissue environment. These data provide opportunities to investigate mechanisms underlying the spatial distribution of immune cells and its functional implications, including the identification of immune niches, although the criteria used to define this term have been inconsistent. Here, we review recent technological and analytic advances in multiparameter spatial profiling, focusing on how these methods have generated new insights in translational immunology. We propose a 3-step framework for the definition and characterization of immune niches, which is powerfully facilitated by new spatial profiling methodologies. Finally, we summarize current approaches to analyze adaptive immune repertoires and lymphocyte clonal expansion in a spatially resolved manner., Competing Interests: Disclosure statement C.Y.C.L. is funded by the Gates Cambridge scholarship trust and University of Cambridge School of Clinical Medicine Elmore fund. D.M. is funded by the UK Medical Research Council. J.M. was supported by a Jean Shanks/Pathological Society Clinical Lecturer Grant (JSPS CLG 2019 02). M.R.C. is supported by a Wellcome Trust Investigator Award (220268/Z/20/Z), by a WellcomeDiscovery Award (227890/ Z/23/Z), and by the National Institute for Health Research Cambridge Biomedical Research Centre. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Upon transmission to the human host, Plasmodium sporozoites exit the skin, are taken up by the blood stream, and then travel to the liver where they infect and significantly modify a single hepatocyte. Low infection rates within the liver have made proteomic studies of infected hepatocytes challenging, particularly in vivo, and existing studies have been largely unable to consider how protein and phosphoprotein differences are altered at different spatial locations within the heterogeneous liver. Using digital spatial profiling, we characterized changes in host signaling during Plasmodium yoelii infection in vivo without disrupting the liver tissue. Moreover, we measured alterations in protein expression around infected hepatocytes and identified a subset of CD163+ Kupffer cells that migrate towards infected cells during infection. These data offer the first insight into the heterogeneous microenvironment that surrounds the infected hepatocyte and provide insights into how the parasite may alter its milieu to influence its survival and modulate immunity.

Upon transmission to the human host, Plasmodium sporozoites exit the skin, are taken up by the blood stream, and then travel to the liver where they infect and significantly modify a single hepatocyte. Low infection rates within the liver have made proteomic studies of infected hepatocytes challenging, particularly in vivo , and existing studies have been largely unable to consider how protein and phosphoprotein differences are altered at different spatial locations within the heterogeneous liver. Using digital spatial profiling, we characterized changes in host signaling during Plasmodium yoelii infection in vivo without disrupting the liver tissue. Moreover, we measured alterations in protein expression around infected hepatocytes and identified a subset of CD163+ Kupffer cells that migrate towards infected cells during infection. These data offer the first insight into the heterogeneous microenvironment that surrounds the infected hepatocyte and provide insights into how the parasite may alter its milieu to influence its survival and modulate immunity. [ABSTRACT FROM AUTHOR]

Systemic inflammation measured by the acute-phase protein CRP associates with poor outcome across cancer types. In contrast, local tumor-associated inflammation, primarily evaluated by T-lymphocytes, correlates with favorable prognosis. Yet, little is known whether these two responses are related or opposing processes and why elevated CRP in relation to cancer is detrimental for clinical outcome. As proof of concept, we developed a platform combining multiplexed IHC and digital imaging, enabling a virtual readout of both lymphoid and myeloid immune markers and their spatial patterns in the primary tumors of resected stage II and III colon cancer (CC) patients with and without accompanying systemic inflammation. Twenty-one patients with elevated CRP (>30 mg/l) and 15 patients with low CRP (<10 mg/l) were included in the analyses. Whole slides from the primary tumors were stained for markers of adaptive (CD8+, CD4+, foxp3 regulatory T cells, CD20+ B cells) and innate (CD68+ macrophages, CD66b+ neutrophils) immunity and the immune checkpoint molecule PD-L1. Associations between individual immune markers, preoperative CRP values, mismatch repair status (MMR), and risk of recurrence or death were assessed. Unsupervised hierarchical clustering was used to explore whether distinct immune phenotypes were present. Tumors from systemically inflamed patients (CRP >30 mg/l) displayed significantly more myeloid features in terms of higher densities of CD66b+neutrophils (p = 0.001) and CD68+macrophages (p = 0.04) and less lymphoid features (lower CD8 T cell, p = 0.03, and foxp3 regulatory T cell densities, p = 0.03) regardless of MMR status. Additionally, systemically inflamed patients harbored lower mean distances between neutrophils and tumor cells within the TME. Intriguingly, microsatellite instable (MSI) tumor status correlated with systemic inflammation. However, using a combinatorial approach, we found that regardless of an adaptive composite score (compounded CD4+ and CD8+ T cells), a high innate score (CD66b+ neutrophils and CD68+ macrophages) associated significantly with elevated CRP. In conclusion, tumor-associated systemic inflammation correlated with a myeloid-dominated TME in a small cohort of resectable CC patients. Our data highlight the importance of a comprehensive immune classification of tumors including players of innate immunity and support a role for CRP as an informative biomarker of the immune response taking place at the tumor site. [ABSTRACT FROM AUTHOR]

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Malignant primary and secondary brain tumors pose a major health challenge, and the incidence of these tumors is rising. The brain tumor microenvironment (TME) is highly complex and thought to impact treatment resistance and failure. To enable a greater understanding of the milieu of cells in the brain TME, advances in imaging and sequential profiling of proteins/mRNA have given rise to the field of spatial transcriptomics. These technologies provide a greater depth of understanding of the tissue architecture, cellular and spatial profiles, including cellular activation status, which may provide insights into effective therapies for brain cancers. In this review, we provide an overview of spatial profiling technologies at the forefront in the field and describe the applications for brain cancer. Brain tumors are often resistant to treatment, and display both an immunosuppressive and heterogeneous tumor microenvironment. Next-generation imaging and multi-omics technologies are providing a tool for intricately characterizing their tissue biology. This information will aid in the design of effective therapies and begin to provide an understanding of therapy resistance. [ABSTRACT FROM AUTHOR]

Immunotherapies have shown long‐lasting and unparalleled responses for cancer patients compared to conventional therapy. However, they seem to only be effective in a subset of patients. Therefore, it has become evident that a greater understanding of the tumor microenvironment (TME) is required to understand the nuances which may be at play for a favorable outcome to therapy. The immune contexture of the TME is an important factor in dictating how well a tumor may respond to immune checkpoint inhibitors. While traditional immunohistochemistry techniques allow for the profiling of cells in the tumor, this is often lost when tumors are analysed using bulk tissue genomic approaches. Moreover, the actual cellular proportions, cellular heterogeneity and deeper spatial distribution are lacking in characterisation. Advances in tissue interrogation technologies have given rise to spatially resolved characterisation of the TME. This review aims to provide an overview of the current methodologies that are used to profile the TME, which may provide insights into the immunopathology associated with a favorable outcome to immunotherapy. [ABSTRACT FROM AUTHOR]