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Spatial transcriptomics of histological sections have revolutionized research in life sciences and enabled unprecedented insights into genetic processes involved in tissue reorganization. However, in contrast to genomic analysis, the actual biomolecular composition of the sample has fallen behind, leaving a gap of potentially highly valuable information. Raman microspectroscopy provides untargeted spatiomolecular information at high resolution, capable of filling this gap. In this study we demonstrate spatially resolved Raman “spectromics” to reveal homogeneity, heterogeneity and dynamics of cell matrix on molecular levels by repurposing state-of-the-art bioinformatic analysis tools commonly used for transcriptomic analyses. By exploring sections of murine myocardial infarction and cardiac hypertrophy, we identify myocardial subclusters when spatially approaching the pathology, and define the surrounding metabolic and cellular (immune-) landscape. Our innovative, label-free, non-invasive “spectromics” approach could therefore open perspectives for a profound characterization of histological samples, while additionally allowing the combination with consecutive downstream analyses of the very same specimen. Spatial transcriptomics of histological sections have revolutionized basic research, while the actual biomolecular composition of the sample has fallen behind. Here, the authors propose a novel approach to analyze untargeted spatiomolecular Raman spectroscopy data through bioinformatic tools developed for transcriptomic analyses, and integrate them with additional Omics techniques.

Immune-checkpoint inhibitors (ICI) have transformed oncological therapy. Up to 20% of all non-small cell lung cancers (NSCLCs) show durable responses upon treatment with ICI, however, robust markers to predict therapy response are missing. Here we show that blood platelets interact with lung cancer cells and that PD-L1 protein is transferred from tumor cells to platelets in a fibronectin 1, integrin α5β1 and GPIbα-dependent manner. Platelets from NSCLC patients are found to express PD-L1 and platelet PD-L1 possess the ability to inhibit CD4 and CD8 T-cells. An algorithm is developed to calculate the activation independent adjusted PD-L1 payload of platelets (pPD-L1 Adj. ), which is found to be superior in predicting the response towards ICI as compared to standard histological PD-L1 quantification on tumor biopsies. Our data suggest that platelet PD-L1 reflects the collective tumor PD-L1 expression, plays important roles in tumor immune evasion and overcomes limitations of histological quantification of often heterogeneous intratumoral PD-L1 expression. The definition of biomarkers to predict therapy responses to immune-checkpoint inhibitors represent an unmet clinical need. Here the authors provide evidences that platelet-derived PD-L1 could serve as a prognostic and predictive biomarker in patients with non-small cell lung cancer.

Deciphering cellular components and the spatial interaction network of the tumor immune microenvironment (TIME) of solid tumors is pivotal for understanding biologically relevant cross-talks and, ultimately, advancing therapies. Multiplexed tissue imaging provides a powerful tool to elucidate spatial complexity in a holistic manner. We established and cross-validated a comprehensive immunophenotyping panel comprising over 121 markers for multiplexed tissue imaging using MACSima™ imaging cyclic staining (MICS) alongside an end-to-end analysis workflow. Applying this panel and workflow to primary cancer tissues, we characterized tumor heterogeneity, investigated potential therapeutical targets, conducted in-depth profiling of cell types and states, sub-phenotyped T cells within the TIME, and scrutinized cellular neighborhoods of diverse T cell subsets. Our findings highlight the advantage of spatial profiling, revealing immunosuppressive molecular signatures of tumor-associated myeloid cells interacting with neighboring exhausted, PD1 high T cells in the TIME of hepatocellular carcinoma (HCC). This study establishes a robust framework for spatial exploration of TIMEs in solid tumors and underscores the potency of multiplexed tissue imaging and ultra-deep cell phenotyping in unraveling clinically relevant tumor components.

Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction–modification and CRISPR–Cas systems 1 . In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors 2 – 4 . However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation. The nucleus-like compartment formed in bacteria during infection by jumbo phage 201phi2-1 is composed of the bacteriophage protein chimallin, which can self-assemble into closed compartments in vitro.

Background Pictorial blood loss assessment charts (PBACs) represent the most widely used method to assess menstrual blood loss (MBL) in clinical trials. The aims of this review were to: (1) determine the diagnostic accuracy of PBACs that have been validated against the reference alkaline hematin technique; (2) categorize the pitfalls of using obsolete and nonvalidated charts; (3) provide guidelines for development of a new PBAC or use of an existing chart to measure MBL in clinical trials; and (4) consider the feasibility of using pictorial charts in primary care. Methods A literature review was conducted using Embase and MEDLINE databases. The review identified reports of women with self-perceived or actual heavy menstrual bleeding (HMB), bleeding disorders, abnormal uterine bleeding, leiomyomata (uterine fibroids) or endometriosis, and women undergoing treatment for HMB, as well as those with normal menstrual periods. Data were reviewed from studies that focused on the development and validation of PBACs and from those that used derivative noncertified charts to assess HMB. Results Nine studies reported validation of PBAC scoring systems against the alkaline hematin technique. Across these studies, the sensitivity was 58–97%, the specificity was 7.5–95.5%, the positive and negative likelihood ratios were 1.1–13.8 and 0.14–0.56, respectively, and the diagnostic odds ratio was 2.6–52.4. The cut-off score above which the diagnosis of HMB was made ranged from 50 to 185. Several modifications of these PBACs were used in other studies; however, objective confirmation of their validity was not reported. Overall, there was widespread inconsistency of chart design, scoring systems, diagnostic cut-off limits and post-treatment outcome measures. Conclusions PBACs are best suited to the controlled and specific environment of clinical studies, where clinical outcome parameters are defined. The current lack of standardization precludes widespread use of the PBAC in primary care. Review registration number PROSPERO international prospective register of systematic reviews: CRD42016030083.

Targeting AML by chimeric antigen receptor T-cells (CAR-T) is challenging due to the promiscuous expression of AML-associated antigens in healthy hematopoiesis and high degree of inter- and intratumoral heterogeneity. Here, we present single-cell expression data of AML-associated antigens in 30 primary pediatric AML samples. We identified CD33, CD38, CD371, IL1RAP and CD123 as the most frequently expressed. Notably, high variability was observed not only across the different patient samples but also among leukemic cells of the same patient suggesting the necessity of multiplexed targeting approaches. To address this need, we utilized our modular Adapter CAR (AdCAR) platform, enabling precise qualitative and quantitative control over CAR-T-cell function. We show highly efficient and target-specific activity for newly generated adapter molecules (AMs) against CD33, CD38, CD123, CD135 and CD371, both in vitro and in vivo. We reveal that inherent intratumoral heterogeneity in antigen expression translates into antigen escape and therapy failure to monotargeted CAR-T therapy. Further, we demonstrate in PDX models that rational combinatorial targeting by AdCAR-T-cells can cure heterogenic disease. In conclusion, we elucidate the clinical relevance of heterogeneity in antigen expression in pediatric AML and present a novel concept for precision immunotherapy by combinatorial targeting utilizing the AdCAR platform.

Background Since the publication over 50 years ago of the alkaline hematin method for quantifying menstrual blood loss (MBL) many new approaches have been developed to assess MBL. The aim of this systematic review is to determine for methods of measuring MBL: ability to distinguish between normal and heavy menstrual bleeding (HMB); practicalities and limitations in the research setting; and suitability for diagnosing HMB in routine clinical practice. Methods Embase®™, MEDLINE®, and ClinicalTrials.gov were screened for studies on the development/validation of MBL assessment methods in women with self-perceived HMB, actual HMB or uterine fibroids, or patients undergoing treatment for HMB. Studies using simulated menstrual fluid and those that included women with normal MBL as controls were also eligible for inclusion. Extracted data included study population, results of validation, and advantages/disadvantages of the technique. Results Seventy-one studies fulfilled the inclusion criteria. The sensitivity and/or specificity of diagnosing HMB were calculated in 16 studies of methods involving self-perception of MBL (11 pictorial), and in one analysis of the menstrual-fluid-loss (MFL) method; in 13 of these studies the comparator was the gold standard alkaline hematin technique. Sensitivity and specificity values by method were, respectively: MFL model, 89, 98%; pictorial blood loss assessment chart (PBAC), 58–99%, 7.5–89%; menstrual pictogram, 82–96%, 88–94%; models/questionnaires, 59–87%, 62–86%, and complaint of HMB, 74, 74%. The power of methods to identify HMB was also assessed using other analyses such as comparison of average measurements: statistical significance was reported for the PBAC, MFL, subjective complaint, and six questionnaires. In addition, PBAC scores, menstrual pictogram volumes, MFL, pad/tampon count, iron loss, and output from three questionnaires correlated significantly with values from a reference method in at least one study. In general, pictorial methods have been more comprehensively validated than questionnaires and models. Conclusions Every method to assess MBL has limitations. Pictorial methods strike a good balance between ease of use and validated accuracy of MBL determination, and could complement assessment of HMB using quality of life (QoL) in the clinical and research setting. Trial registration PRISMA registration number: CRD42016032956 .

A major challenge hampering therapeutic advancements for high-risk sarcoma patients is the broad spectrum of molecularly distinct sarcoma types and the corresponding lack of suitable model systems. Here we describe the development of a genetically-controlled, yet versatile mouse modeling platform allowing delivery of different genetic lesions by muscle electroporation (EPO) in wildtype mice. This EPO-GEMM (EPO-based genetically engineered mouse model) platform allows the generation of ten genetically distinct sarcomas on an isogenic background, including the first model of ETV6::NTRK3 -driven sarcoma. Comprehensive histological and molecular profiling reveals that this mouse sarcoma cohort recapitulates a spectrum of molecularly diverse sarcomas with gene fusions acting as major determinants of sarcoma biology. Integrative cross-species analyses show faithful recapitulation of human sarcoma subtypes, including expression of relevant immunotherapy targets. Comparison of syngeneic allografting methods enables reliable preservation and scalability of sarcoma-EPO-GEMMs for preclinical treatment trials, such as NTRK inhibitor therapy in an immunocompetent background. Sarcomas are a group of mesenchymal malignancies which are molecularly heterogeneous. Here, the authors develop an in vivo muscle electroporation system for gene delivery to generate distinct subtypes of orthotopic genetically engineered mouse models of sarcoma, as well as syngeneic allograft models with scalability for preclinical assessment of therapeutics.

Urolithiasis is one of the most common diseases worldwide, characterized by high morbidity and significant treatment-related costs, with a rising prevalence of up to 20%. The relapse rate within the first 10 years after initial treatment is estimated to be about 60%. Given the increasing prevalence, healthcare-related costs associated with urinary tract stones in the USA are expected to reach up to US $1.24 billion annually by 2030. Current prophylactic therapy for urolithiasis recurrence includes lifestyle modifications, citrate supplementation, and pharmaceuticals. However, a high number of cases remain unresponsive to available pharmacological therapies. Though initially developed for the treatment of Diabetes mellitus, SGLT-2 inhibitors have shown promise in decreasing cardiac and renal endpoints across multiple indications. Recent registry studies have indicated that patients receiving SGLT-2 inhibitors exhibit lower rates of urolithiasis incidence, suggesting a potential reduction in recurrence rates and associated mortality. We hypothesize that SGLT-2 inhibitors (Dapagliflozin), owing to their multiple pleiotropic effects, may offer a viable treatment option for the prophylaxis of high-risk calcium oxalate kidney stones and reduce urinary calcium oxalate output. This study will proceed in two phases: an exploratory phase and a randomized controlled phase. In the exploratory phase, 22 participants with indications for dapagliflozin treatment will be evaluated before and after treatment initiation to ascertain the concrete effect size regarding oxalate and calcium-sparing effects. This data will inform the calculation of the study sample size (ranging from 17 to 104 participants) to include high-risk calcium oxalate kidney stone formers in a randomized controlled crossover study design. Treatment phases-one with dapagliflozin and one with placebo-will alternate with wash-out phases involving placebo. The primary outcome is the reduction of oxalate excretion in 24-hour urine samples compared to baseline values after 8 weeks of therapy. Secondary objectives include analysing effects on kidney function, the frequency of urolithiasis, and treatment tolerance. Additionally, in-depth metabolomics analyses will explore pathophysiological pathways during treatment. Investigators, patients, and research staff will be blinded to the randomization list. This study was initially registered under EudraCT (Nr:2022-000994-13) and has been transitioned to CTIS (Nr: 2024-519371-25-00) to comply with EU Regulation 536/2014, ensuring streamlined management and transparency. Dapagliflozin's pleiotropic effects may provide a novel prophylactic treatment option for urolithiasis. This study aims to evaluate potential treatment effects in a prospective RCT and elucidate potential pathophysiological pathways through in-depth metabolomics analyses. SGLT-2 inhibitors have the potential to transform the landscape of urolithiasis treatment, reduce the healthcare burden on individuals and the system, and significantly improve patient quality of life.

ObjectiveThe substitution of an in-study control population with a historical control (HC) population is considered a viable option for reducing the necessary recruitment of control patients. However, it is necessary to evaluate whether this method is applicable to studies on indications targeting endometriosis-associated pelvic pain (EAPP). This study aims to evaluate the potential bias in the results of an EAPP study with an HC arm.MethodsFor this case study, we re-evaluated data from a randomised, placebo-controlled trial using dienogest daily to treat EAPP with an HC arm based on data from a second randomised, placebo-controlled trial in the same indication. Propensity Score (PS) matching was used to match between the treatment and HC arm on all baseline variables. To evaluate the effect of matching on the introduced bias, we evaluated efficacy parameters with the full treatment and control group, as well as the matched group.ResultsThe difference between means (placebo minus treatment) in change in pain, as measured on the Visual Analogue Scale from baseline to end of treatment, deviates in the comparison treatment/pool of HC (7.15 (0.22 to 14.08)) from the overall in-study group (reference: 11.89 (6.06 to 17.73)). After PS matching on the baseline variables, the difference between means (11.79 (4.09 to 19.5)) is close to the reference.ConclusionsUsing HC with PS matching has proven to be useful in the setting of treating EAPP, while emphasis must be given to the selection mechanism and the underlying assumptions. This case study has shown that even for studies which are very similar in design, heterogeneity and between-study variations are present. With the use of an HC arm, it was possible to reproduce similar results than in the original study, while the PS matching improved the comparability considerably. For the main endpoint, PS matching could reproduce the original study results.Trial registration numberNCT00225199, NCT00185341