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Background Mucosal head and neck squamous cell carcinoma (HNSCC) is often diagnosed at an advanced stage, where the prognosis is poor due to the high rates of recurrence and metastasis. With approximately one million new cases projected in 2024, worldwide mortality of HNSCC is estimated to reach 50% of detected cases the same year. Patients with early-stage tumours showed a 50–60% five-year survival rate in the US. Immune checkpoint inhibitors (ICIs) have shown promising results in prolonging survival in a subset of patients with recurrent or metastatic disease. However, challenges remain, particularly the limited efficacy of PD-1/PD-L1 blockade therapies. PD-L1 protein expression has been shown to be limited in its predictive power for ICI therapies. Emerging evidence shows that intricate characterisation of the tumour microenvironment (TME) is fundamental to understand interacting cells. This study aims to bridge the gap in understanding the tumor microenvironment by identifying distinct spatial patterns of PD-1/PD-L1 interactions and their association with immunotherapy responses in head and neck squamous cell carcinoma (HNSCC). Methods In this study, we sought to apply a more nuanced approach to understanding cellular interactions by mapping PD-1/PD-L1 interactions across whole-slide HNSCC tissue samples collected prior to ICI therapy. We used a combination of spatial proteomics (Akoya Biosciences) and an in situ proximity ligation assay (isPLA, Navinci Diagnostics) to visualise PD-1/PD-L1 interactions across cell types and cellular neighbourhoods within the tumour TME. Results Our findings indicate the existence of isPLA + PD-1/PD-L1 interactions between macrophages/CD3 T cell-enriched neighbourhoods and tumour cells at the tumour-stroma boundaries in ICI-resistant tumours. The presence of these dense macrophage-tumour layers, which are either absent or dispersed in responders, indicates a barrier that may restrict immune cell infiltration and promote immune escape mechanisms. In contrast, responders had abundant B and T cell aggregates, predominantly around the tumour edges linked to enhanced immune responses to ICI therapy and better clinical outcomes. Conclusion This study highlights the utility of isPLA in detecting distinct tumour-immune interactions within the TME, offering new cellular interaction metrics for stratifying and optimising immunotherapy strategies.

Mammalian systems respond to environmental stress by either adapting or undergoing programmed cell death. While there is general agreement that the caspase family of proteases serve as the effectors of the apoptotic death response, the signaling apparatus involved in the decision to activate the caspase system is less clear. In the past few years, the sphingomyelin and c-Jun Kinase (JNK)/Stress-activated Protein Kinase (SAPK) pathways have been linked to the death response in many cellular systems. These signaling systems are found throughout the animal kingdom, and ceramide signaling is conserved through yeast. Since yeast do not undergo apoptosis, the sphingomyelin pathway appears evolutionarily older than the caspase-mediated death programs. While recent reviews by several groups have broadly surveyed ceramide signaling in apoptosis, this paper examines the role of sphingomyelinases and the JNK/SAPK pathway in coordinate signaling of apoptosis.

BackgroundL-asparaginase is a key component of treatment for patients with acute lymphoblastic leukaemia (ALL) in the UK. Commonly used forms of asparaginase are native E. coli-derived asparaginase (native asparaginase) and pegaspargase in first-line combination therapy, and native Erwinia chrysanthemi-derived asparaginase (Erwinia asparaginase) as second-line treatment. The objective of this study was to evaluate the cost-effectiveness of pegaspargase versus native asparaginase in first-line combination therapy for patients with newly diagnosed ALL. A combined decision tree and health-state transition Markov cost-effectiveness model was developed to assess the relative costs and health outcomes of pegaspargase versus native asparaginase in the UK setting.ResultsIn base case analyses, first-line pegaspargase (followed by Erwinia asparaginase in cases of hypersensitivity) dominated first-line native asparaginase followed by Erwinia asparaginase; i.e. resulted in lower costs and more quality-adjusted life year gain. The favourable hypersensitivity rates and administration profile of pegaspargase led to lifetime cost savings of £4741 versus native asparaginase. Pegaspargase remained cost-effective versus all treatment strategies in all scenario analyses, including use of the 2500 IU/m2 dose, recommended for patients ≤21 years of age.ConclusionsPegaspargase, as part of multi-drug chemotherapy, is a cost-effective option for the treatment of newly diagnosed ALL. Based on this study, The National Institute for Health and Care Excellence Technology Appraisal Committee concluded that it could recommend pegaspargase as a cost-effective use of National Health Service resources in England & Wales for treating ALL in children, young people and adults with untreated, newly diagnosed disease.Trial registrationUKALL 2011, EudraCT number 2010-020924-22; UKALL 2003, EudraCT number 2007-004013-34; UKALL14, EudraCT number 2009-012717-22.

BackgroundThe tumor microenvironment (TME) is composed of highly heterogeneous extracellular structures and cell types such as endothelial cells, immune cells, and fibroblasts that dynamically influence and communicate with each other. The constant interaction between a tumor and its microenvironment plays a critical role in cancer development and progression and can significantly affect a tumor’s response to therapy and capacity for multi-drug resistance. High resolution analyses of gene and protein expression with spatial context can provide deeper insights into the interactions between tumor cells and surrounding cells within the TME, where a better understanding of the underlying biology can improve treatment efficacy and patient outcomes. Here, we demonstrated the ability to perform streamlined multi-omic tumor analyses by utilizing the 10X Genomics Visium Spatial Gene Expression Solution for FFPE with multiplex protein enablement. This technique simultaneously assesses gene and protein expression to elucidate the immunological profile and microenvironment of different breast cancer samples in conjunction with standard pathological methods.MethodsSerial (5 µm) sections of FFPE human breast cancer samples were placed on Visium Gene Expression (GEX) slides. The Visium GEX slides incorporate ~5,000 molecularly barcoded, spatially encoded capture spots onto which tissue sections are placed, stained, and imaged. Following incubation with a human whole transcriptome, probe-based RNA panel and an immuno-oncology oligo-tagged antibody panel, developed with Abcam conjugated antibodies, the tissues are permeabilized and the representative probes are captured. Paired GEX and protein libraries are generated for each section and then sequenced on an Illumina NovaSeq at a depth of ~50,000 reads per spot. Resulting reads from both libraries are aligned and overlaid with H&E-stained tissue images, enabling analysis of both mRNA and protein expression. Additional analyses and data visualizations were performed on the Loupe Browser v4.1 desktop software.ConclusionsSpatial transcriptomics technology complements pathological examination by combining histological assessment with the throughput and deep biological insight of highly-multiplexed protein detection and RNA-seq. Taken together, our work demonstrated that Visium Spatial technology provides a spatially-resolved, multi-analyte view of the tumor microenvironment, where a greater understanding of cellular behavior in and around tumors can help drive discovery of new biomarkers and therapeutic targets.

THE sphingomyelin pathway, initiated by hydrolysis of sphingo-myelin to ceramide and stimulation of a Ser/Thr ceramide-activated protein (CAP) kinase, mediates tumour necrosis factor-α (TNF-α) and interleukin-1β action 1–4 . CAP kinase is membrane-bound and proline-directed, recognizing the minimal substrate motif Thr-Leu-Pro (ref. 5). TNF may use the sphingomyelin pathway to signal Raf1 to activate the MAP kinase cascade 6–8 . Evidence shows that cytoplasmic Rafl binds to GTP-ras upon cellular stimulation, is recruited to the plasma membrane, and activated 9,11 . How membrane-bound Rafl is activated is uncertain, but regulation of its kinase activity may involve its phosphorylation 12–19 . Specific Raf kinases, however, have not hitherto been identified. Here we report that CAP kinase phosphorylates Rafl on Thr 269, increasing its activity towards MEK (MAP kinase or ERK kinase). Moreover, in intact HL-60 cells, CAP kinase complexes with Rafl and, in response to TNF and ceramide analogues, phosphorylates and activates Rafl, implicating CAP kinase as a link between the TNF receptor and Rafl.

Biofilm-forming mercury-resistant marine bacterium Bacillus cereus BW-201B has been explored to evident that the bacterial biofilm-EPS (exopolymers) trap inorganic mercury but subsequently release EPS-bound mercury for induction of mer operon-mediated volatilization of inorganic mercury. The isolate was able to tolerate 50 ppm of mercury and forms biofilm in presence of mercury. mer operon-mediated volatilization was confirmed, and –SH was found to be the key functional group of bacterial EPS responsible for mercury binding. Biofilm-EPS-bound mercury was found to be internalized to the bacterial system as confirmed by reversible conformational change of –SH group and increased expression level of merA gene in a timescale experiment. Biofilm-EPS trapped Hg after 24 h of incubation, and by 96 h, the volatilization process reaches to its optimum confirming the internalization of EPS-bound mercury to the bacterial cells. Biofilm disintegration at the same time corroborates the results.

Among all of the Super Elongation Complex (SEC) components, ELL1 (also known as ELL) is the only bona fide elongation factor that directly stimulates transcription elongation by RNA polymerase II. However, the mechanism(s) of functional regulation of ELL1 (referred to as ELL hereafter), through its stabilization, is completely unknown. Here, we report a function of human DBC1 in regulating ELL stability involving HDAC3, p300, and Siah1. Mechanistically, we show that p300-mediated site-specific acetylation increases, whereas HDAC3-mediated deacetylation decreases, ELL stability through polyubiquitylation by the E3 ubiquitin ligase Siah1. DBC1 competes with HDAC3 for the same binding sites on ELL and thus increases its acetylation and stability. Knockdown of DBC1 reduces ELL levels and expression of a significant number of genes, including those involved in glucose metabolism. Consistently, Type 2 diabetes patient-derived peripheral blood mononuclear cells show reduced expression of DBC1 and ELL and associated key target genes required for glucose homeostasis. Thus, we describe a pathway of regulating stability and functions of key elongation factor ELL for expression of diverse sets of genes, including ones that are linked to Type 2 diabetes pathogenesis.

Multiplex immunofluorescence and immunohistochemistry (mIF/IHC) are increasingly employed antibody-based technologies that use tissue sparingly and facilitate the detection of co-localized or neighboring biomarkers. Specifically, these platforms enable spatial analyses of the tumor microenvironment as well as extended applications, for example, describing normal tissue anatomy, autoimmunity, infectious diseases, etc. mIF/IHC has greatly enhanced biomarker discovery efforts, and a growing number of studies suggest superiority to traditional IHC. Standardization of staining approaches, reporting of image analysis strategies and resultant data is critical for facilitating cross-study comparisons, validation, deployment, and generalization of findings. To address this challenge, The Society for Immunotherapy of Cancer (SITC) previously published two articles providing best practice guidelines for mIF/IHC staining, image analysis, and data sharing. Here, SITC convened stakeholders to develop the third article in the series, a consensus checklist for scientific reporting of mIF/IHC data to support and complement the best practice guidelines. The checklist includes critical components of mIF/IHC applications to be defined within publications such as detailed descriptions of analytical validation; image acquisition, selection, and registration methods; and cell clustering and spatial analysis strategies, amongst others. Such information will help with data reproducibility and comparison across studies towards future drug and assay development.

BackgroundThe spatial interactions between the immune system and tumor cells greatly influence antitumoral immunity, patient prognosis, and therapeutic efficacy. However, few methods exist to query large numbers of immune biomarkers at subcellular spatial resolution. Launched earlier this year, the CosMx™ Spatial Molecular Imager (SMI) platform captures high-plex single cell and subcellular detection of proteins from FFPE tissues. To detect these key drivers of cancer progression and immune cell activation states and functions, we designed and validated a high-plex protein panel for the CosMx SMI platform. This panel contains 4 markers for cell segmentation and pre-experimental imaging and 64 barcoded antibodies emphasizing immuno-oncology focused targets.MethodsThe CosMx protein assay uses antibodies conjugated with oligonucleotides, which are detected using universal, multi-analyte CosMx readout reagents. The fully automated CosMx instrument carries a widefield water immersion objective with 1.1 NA. The CosMx Human Immuno-oncology panel was optimized to comprehensively phenotype lymphoid and stromal lineages within the tumor microenvironment. The CosMx protein assay reagents were validated on multi-organ FFPE tissue microarrays covering prevalent solid tumor types and matched controls, and 52 human FFPE cell lines, including overexpression lines for key targets such as GITR, CD278, PD-L1, and PD-1.ResultsWe achieved 86% sensitivity and 90% specificity across well-characterized human cell lines compared to GeoMx spatial profiling, and further benchmarked to multiple orthogonal datasets (e.g., the Human Protein Atlas, low-plex IHC). Within the tissue sample profiled, we captured immune cell localization across and within the tumor, key signaling markers related to lymphoid and myeloid activation such as checkpoint engagement, and myeloid cell polarization and antigen cross-presentation markers. We made the data from the study profiling cancer tissue freely available online. This includes the protein images, AI-based cell segmentation, and the per-cell protein abundance profiles.ConclusionsCosMx SMI is a high-plex spatial multiomics platform that enables detection of more than 64 proteins at subcellular resolution in real-world FFPE tissues. Our 64-plex human immuno-oncology protein panel enables in-depth study of the tumor microenvironment, including markers covering cell typing and lineage, immune activation, and checkpoints. This new platform will enable researchers to collect high-plex protein immunophenotyping data and understand immune molecular mechanisms with full spatial context.FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.