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Boor, P.

Publications and source records attributed to Boor, P..

At least 19 recordsLinked to original sources

Hypothermic and Normothermic Machine Perfusion Differentially Shape the Renal Cellular Landscape

The shortage of donor organs has led to an increased use of extended-criteria donors and donation after circulatory death for kidney transplantation, both of which are associated with graft injury. Although machine perfusion (MP) offers a preservation strategy, the most beneficial approach for kidneys with respect to effective graft preservation remains unclear. We compared oxygenated hypothermic (HMP) and normothermic machine perfusion (NMP) using a murine kidney perfusion model regarding intra-graft immune cell composition, viability, and inflammatory responses. NMP resulted in more severe tubular injury, accompanied by increased immune cell mobilization out of the graft. NMP increased early apoptosis among kidney-resident immune-cell populations, whereas endothelial cells showed increased late apoptosis after both strategies. Proximal tubular-cell apoptosis remained comparable to naive tissue, and neither modality reduced reactive oxygen species levels. Both protocols induced antioxidant and hypoxia-responsive genes compared with static cold storage, while NMP elicited a stronger inflammatory response. Treatment with the senolytic ABT-263 demonstrated the models feasibility for investigating renal injury, cellular viability, and immune activation during MP.

immunology↗

Cyclic uremia without kidney injury drives cardiovascular disease with rapid onset of immunosenescence and heart failure

End-stage kidney disease (ESKD) is a major driver of cardiovascular disease (CVD) and requires dialysis treatment leading to a unique metabolic phenotype with cyclic transitions between accumulation and clearance of uremic toxins. Although ESKD patients display the highest cardiovascular mortality among CKD patients, the effect of cyclic uremia on cardiovascular disease remains subject to discussion. Existing mouse models rely on induction of kidney injury and do not resolve the interconnected pathophysiological effects of metabolic, hypertensive and endocrine renal failure. They are therefore unable to distinguish uremia-driven effects from well-established drivers of cardiovascular disease such as hypertension. Here, we established a simple, reproducible, and sex-inclusive mouse model of uremia in absence of kidney injury or hypertension leveraging a bistable vesico-peritoneal shunt (VPS). Strikingly, we identify cyclic uremia as an independent driver of ESKD-induced CVD, that induces heart failure with preserved ejection fraction (HFpEF), vascular inflammation and immunosenescence. As such, the VPS represents the first preclinical model that enables investigation of cyclic uremia as a driver of cardiovascular disease.

systems biology↗

Chronic exposure to low-concentration urban PM2.5 accelerates maladaptive repair after ischemic injury via mitochondrial dysfunction and lysosomal stress.

BackgroundFine particulate matter (PM2.5), airborne particles with an aerodynamic diameter [&le;]2.5 m that can penetrate deep into the lungs and enter the circulation, is increasingly recognized as a risk factor for chronic kidney disease (CKD) with long-term exposure. We previously demonstrated that high-dose PM2.5 exposure prior to ischemia-reperfusion injury (IRI) aggravates acute kidney injury (AKI). Here, we investigated how prolonged, low-concentration urban PM2.5 exposure (<15 {micro}g/m3) affects kidney repair after AKI. MethodsSix-week-old mice underwent bilateral IRI or sham surgery, followed by six months of exposure to either filtered air or ambient PM2.5 exposure in a unique exposome chamber. Kidneys were analyzed using pathomics, electron and super-resolution microscopy, immunohistochemistry, transcriptomics, and LC-MS lipidomics/metabolomics. Complementary in vitro hypoxia-reoxygenation and PM2.5 exposure experiments were performed in proximal tubular epithelial cells. ResultsLong-term PM2.5 exposure had minimal effects in sham-operated mice, including no significant changes in body weight or kidney function. Despite preserved kidney function, IRI+PM2.5 mice exhibited reduced weight gain, a marked expansion of the interstitial area, attributable to enhanced fibrosis and inflammatory responses, microvascular rarefaction, and endothelial-to-mesenchymal transition, consistent with maladaptive repair features. Proximal tubules displayed mitochondrial injury, glycolytic reprogramming, lipid accumulation, and a senescent phenotype. Energy Dispersive X-ray (EDX) microscopy confirmed PM2.5-derived elements within proximal tubules lysosomes, accompanied by lysosomal stress. Transcriptional signature-based drug screening identified nicotinamide as a compound capable of reversing PM2.5-induced metabolic alterations; in vitro validation confirmed restoration of mitochondrial function. ConclusionsTogether, these findings show that chronic post-AKI exposure to PM2.5 at levels currently considered safe by regulatory bodies drives maladaptive repair and accelerates CKD progression through mitochondrial dysfunction, lysosomal stress senescence in proximal tubules, due to local PM2.5 element accumulation. Translational StatementAcute kidney injury frequently progresses to chronic kidney disease due to maladaptive repair, yet environmental drivers of this transition remain underrecognized. Using a controlled exposome chamber, we demonstrate that chronic exposure to low, real-world concentrations of urban PM2.5 during post-ischemic recovery results in the accumulation of PM2.5-derived elements within proximal tubular lysosomes, leading to organelle dysfunction, metabolic reprogramming, lipid accumulation, and a senescence-like phenotype. Importantly, transcriptomics-based drug repurposing identified nicotinamide as a candidate compound capable of reversing metabolic dysfunction in injured proximal tubular cells subjected to hypoxia-reoxygenation and PM2.5 exposure, an effect validated in vitro.

molecular biology↗

Polyploid cardiomyocytes define disease-specific transcriptional states in the mammalian heart

The adult mammalian heart has a limited regenerative capacity. Following injury, cardiomyocytes undergo a hypertrophic response accompanied by polyploidization, which has been described as a barrier to proliferation and regeneration of the heart1,2. However, the unique molecular programs of polyploidy, or genome multiplied cardiomyocytes, and their influence on the disease-related myocardial remodelling process remains unclear. Here, we integrate single-nuclei and high-resolution spatial multi-omics across human, rat, and mouse hearts to define novel cardiac cell states and their tissue niches in ischemic and non-ischemic heart disease. Computational analysis across scales allowed us to generate detailed networks of the cardiac tissue remodelling process as well as tissue and sub-cellular environments uniquely enriched in polyploid cardiomyocytes or their diploid origins. We identify a conserved, dichotomous transcriptional program distinguishing diploid from polyploid cardiomyocytes. Polyploid cardiomyocytes demonstrated rewired metabolic and chromatin-remodeling transcriptional programs and recapitulate the gene signature of immature human fetal cardiomyocytes. Notably, we observe that polyploid cardiomyocytes--rather than the general myocyte population--are the primary sites of enrichment for major heart-failure drug targets, including the mineralocorticoid, {beta}1-adrenergic, and glucagon-like peptide-1 receptors. Based on our cross-species dataset we further identified TNIK, a Wnt-pathway regulator expressed in polyploid cardiomyocytes across species, as a potential therapeutic target and demonstrate that pharmacological TNIK inhibition improves cardiac function after myocardial infarction in rats. Together, this species-spanning, disease-resolved study redefines cardiomyocyte heterogeneity in heart disease and suggests a therapeutic path to heart failure treatment by targeting polyploid cardiomyocytes.

genomics↗

The role of Platelet-derived growth factor (PDGF) in uremic vascular calcification.

Vascular calcification is common in chronic kidney disease (CKD), contributing to increased cardiovascular morbidity and mortality. One of the proposed mechanisms of driving vascular calcification is a phenotypic switch of vascular smooth muscle cells (VSMCs). The platelet-derived growth factors (PDGFs) and their receptors (PDGFRs), particularly PDGFR-{beta}, were shown to modulate the VSMC phenotype. However, their role in uremic vascular calcification remained unclear. We adapted an ex vivo calcification model using murine aortas to simulate uremic conditions. Compared to control conditions, incubation with hemodialysate from CKD patients or using aortas from CKD animals both resulted in significantly increased PDGFR-{beta} phosphorylation and vascular calcification. Inhibition of PDGF signaling using soluble PDGFR-{beta} or the small molecule tyrosine kinase inhibitor imatinib significantly reduced uremic calcification and enhanced vascular elasticity. Next, we generated transgenic mice with a VSMC-specific, inducible expression of constitutively active PDGFR-{beta}. The aortas of these mice exhibited significantly increased vascular calcification ex vivo, which was further aggravated by uremic conditions. We established an in vivo model of accelerated vascular calcification and CKD in the transgenic mice, showing significantly aggravated vascular calcification and phenotypic switching of VSMCs compared to non-transgenic littermates. Finally, increased expression of phosphorylated PDGFR-{beta} and a VSMC phenotypic switching were detected in human arteries from patients with CKD compared to those without CKD. In conclusion, PDGFR-{beta} contributes to CKD-associated vascular calcification, representing a potential novel therapeutic target.

systems biology↗

IFNα Inhibits JAK2V617F-Driven Neoplastic Vasculogenesis and Endothelial-to-Mesenchymal Transition in a 3D iPSC-Based Bone Marrow Niche Model

The vascular niche is a critical regulator of hematopoiesis and disease progression in myeloproliferative neoplasms (MPN). The presence of JAK2V617F+ endothelial cells (EC) in MPN patients and their association with cardiovascular complications highlight the need to understand and therapeutically target this compartment. Using patient-specific induced pluripotent stem cells (iPSC) harboring JAK2WT or the MPN-driver JAK2V617F (heterozygous, JAK2V617FHET, or homozygous, JAK2V617FHOM), we identified zygosity-dependent transcriptional profiles in iPSC-derived EC (iEC) at baseline and following interferon-alpha (IFN) treatment. JAK2V617FHET iEC exhibited an endothelial-to-mesenchymal transition (EndMT) signature, while JAK2V617FHOM iEC showed suppression of translation and ribosome biogenesis. Leveraging iPSC-based 3D assembloids that mimic the bone marrow (BM) niche, we showed that JAK2V617F-driven EndMT is inhibited by tyrosine kinase inhibitors and IFN. In both JAK2V617F-driven polycythemia vera and TPO-driven myelofibrosis murine models, scRNA-seq analysis of the BM vascular niche consistently revealed inflammatory and EndMT-associated signatures in arterial and arteriolar EC. Notably, dysregulation of ribosome- and translation-related pathways emerged in the myelofibrosis model and at advanced disease stages in JAK2V617F-driven polycythemia vera, indicating progressive vascular remodeling with disease evolution. Chronic pegylated IFN treatment in vivo effectively reversed these pathological changes. IFNs anti-EndMT activity was further validated in BM biopsies from MPN patients undergoing IFN therapy. This is the first study to define MPN stage-dependent vascular remodeling and zygosity-specific endothelial effects of JAK2V617F, and to directly link IFN-mediated EndMT inhibition as a novel antifibrotic mechanism. Our 3D assembloids provide a translational platform for mechanistic studies and therapeutic targeting of the BM microenvironment in MPN. Bullet PointsO_LIArterial vascular remodeling emerges as a novel hallmark of MPN, characterized by TNF-inflammation, ribosomal dysregulation and EndMT. C_LIO_LIIFN restores neoplastic endothelial dysfunction, highlighting its role as a vascular niche-modulating and anti-fibrotic agent in MPN. C_LI

cancer biology↗

The proteomic landscape of proteotoxic stress in a fibrogenic liver disease

Protein misfolding diseases, including alpha-1 antitrypsin deficiency (AATD), pose significant health challenges, with their cellular progression still poorly understood1-3. We utilize spatial proteomics by mass spectrometry and machine learning to map AATD in human liver tissue. Combining Deep Visual Proteomics (DVP) with single-cell analysis4,5, we probe intact patient biopsies to resolve molecular events during hepatocyte stress in pseudo-time across fibrosis stages. We achieve unprecedented proteome depth of up to 3,800 proteins from a third of a single cell in formalin-fixed, paraffin-embedded (FFPE) tissue. This dataset revealed a potentially clinically actionable peroxisomal upregulation that precedes the canonical unfolded protein response. Our single-cell proteomics data show alpha-1 antitrypsin accumulation is largely cell-intrinsic, with minimal stress propagation between hepatocytes. We integrated proteomic data with AI-guided image-based phenotyping across multiple disease stages, revealing a terminal hepatocyte state characterized by globular protein aggregates and distinct proteomic signatures, notably including elevated TNFSF10/TRAIL expression. This phenotype may represent a critical disease progression stage. Our study offers novel insights into AATD pathogenesis and introduces a powerful methodology for high-resolution, in situ proteomic analysis of complex tissues. This approach holds potential to unravel molecular mechanisms in various protein misfolding disorders, setting a new standard for understanding disease progression at the single-cell level in human tissue.

cell biology↗

Rewired type I IFN signaling is linked to age-dependent differences in COVID-19

Advanced age is the most important risk factor for severe disease or death from COVID-19, but a thorough mechanistic understanding of the molecular and cellular underpinnings is lacking. Multi-omics analysis of samples from SARS-CoV-2 infected persons aged 1 to 84 years, revealed a rewiring of type I interferon (IFN) signaling with a gradual shift from signal transducer and activator of transcription 1 (STAT1) to STAT3 activation in monocytes, CD4+ T cells and B cells with increasing age. Diversion of interferon IFN signaling was associated with increased expression of inflammatory markers, enhanced release of inflammatory cytokines, and delayed contraction of infection-induced CD4+ T cells. A shift from IFN-responsive germinal center B (GCB) cells towards CD69high GCB and atypical B cells corresponded to the formation of IgA in children while complement fixing IgG was dominant in adults. Our data provide a mechanistic basis for inflammation-prone responses to infections and associated pathology during aging. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/619479v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@723896org.highwire.dtl.DTLVardef@e12b2aorg.highwire.dtl.DTLVardef@d8de22org.highwire.dtl.DTLVardef@1df96ec_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

Pathophysiological response in experimental trauma-related acute kidney injury

BackgroundTrauma and shock often severely affect the kidneys. This can lead to trauma-related acute kidney injury (TRAKI), which significantly increases the risk of adverse outcomes. MethodsTo study the pathophysiology of TRAKI, we developed a murine model of combined blunt thoracic trauma and pressure-controlled hemorrhage that induces mild transient TRAKI. ResultsThe mice showed early and transient increased plasma creatinine, urea, NGAL, and urine albumin, resolving 5 days after TRAKI induction. Despite normal kidney morphology, significant damage to proximal tubular cells and a loss of the brush border was observed. This included kidney stress responses, e.g., with induced heme oxygenase-1 expression in tubules. The upregulation of inflammatory mediators and kidney injury markers was followed by elevated leukocyte numbers, mainly consisting of monocytes/macrophages. Proteomic analyses revealed a distinct time course of intrarenal processes after trauma. 3D x-ray-based whole-organ histology by contrast-enhanced microcomputed tomography showed significant impairment of capillary blood flow, especially during the first day post THS, which was partly resolved by day 5. ConclusionsOur novel model of murine TRAKI has revealed previously unknown aspects of the complex temporal pathophysiological response of the kidney along the nephron after trauma and hemorrhage, which may provide mechanistic starting points for future therapeutic approaches.

pathology↗

CREB determines the expression of ST2 in Tregs and mediates the balance between type 1 and type 2 immune responses

Regulatory T cells (Tregs) are gatekeepers of immune homeostasis and characterized by expression of Foxp3, which maintains Treg identity. Here we demonstrate that in mice with a Foxp3-specific knockout of CREB, enhanced numbers of Tregs are found in vivo in spleen, lung and colon. These Tregs display a reduced Foxp3 expression, but enhanced expression of the IL-33 receptor (ST-2), IL-10, IL-13, and CREM. CREB deficient Tregs were highly suppressive in vitro and prevented disease activity in CD4 T cell mediated transfer colitis in an IL-10 dependent way. Mechanistically CREB fulfils dual roles in Tregs. First it downregulates Foxp3 expression, however in cooperation with CREM, CREB expression in Tregs alters chromatin accessibility to the ST-2 region and thereby influences T cell specific immune responses mediated by IL-10. Brief summary: Mice with a Foxp3-specific knockout of CREB display enhanced expression of IL-13, IL-10, ST-2 and CREM, which prevents gut inflammation GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/601312v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@a2d341org.highwire.dtl.DTLVardef@1db6852org.highwire.dtl.DTLVardef@19e024borg.highwire.dtl.DTLVardef@a8c84d_HPS_FORMAT_FIGEXP M_FIG C_FIG Created by Biorender

immunology↗

Detection of PatIent-Level distances from single cell genomics and pathomics data with Optimal Transport (PILOT)

Although clinical applications represent the next challenge in single-cell genomics and digital pathology, we still lack computational methods to analyze single-cell and pathomics data to find sample level trajectories or clusters associated with diseases. This remains challenging as single-cell/pathomics data are multi-scale, i.e., a sample is represented by clusters of cells/structures and samples cannot be easily compared with each other. Here we propose PatIent Level analysis with Optimal Transport (PILOT). PILOT uses optimal transport to compute the Wasserstein distance between two individual single-cell samples. This allows us to perform unsupervised analysis at the sample level and uncover trajectories or cellular clusters associated with disease progression. We evaluate PILOT and competing approaches in single-cell genomics and pathomics studies involving various human diseases with up to 600 samples/patients and millions of cells or tissue structures. Our results demonstrate that PILOT detects disease-associated samples from large and complex single-cell and pathomics data. Moreover, PILOT provides a statistical approach to delineate non-linear changes in cell populations, gene expression, and tissue structures related to the disease trajectories supporting interpretation of predictions.

bioinformatics↗

PI 3-kinase isoform p110alpha controls smooth muscle cell functionality and protects against aortic aneurysm formation

BackgroundCatalytic class IA PI 3-kinase isoform p110 is a crucial regulator of cellular proliferation and survival in numerous cell types. While p110 is critically involved in pathogenic vascular remodeling, its physiological role for vascular integrity under stress conditions has not been studied. We report a protective function of smooth muscle p110 against abdominal aortic aneurysm (AAA) formation. Methods & ResultsIn mice lacking p110 in smooth muscle cells (sm-p110-/-), perfusion of the infrarenal aorta with porcine pancreatic elastase (PPE) yielded substantially enhanced AAA formation compared to wild type controls. This disease phenotype is partly attributable to a subtle preexisting vascular phenotype under basal conditions, as sm-p110-/- mice displayed a smaller media area, deranged aortic wall structure (detached smooth muscle cells, increased apoptotic cell death), and a diminished functional responsiveness of aortic rings to vasodilators. Furthermore, p110 is also implicated in regenerative processes during AAA development: Whereas wild type mice showed increased media hypertrophy, neointima formation and proliferation upon PPE intervention, these vascular remodeling processes were diminished in sm-p110-/- mice. Concomitantly, increased numbers of elastic fiber breaks and ECM degradation were detected in sm-p110-/- aorta. Mechanistically, we found that lack of p110 expression impaired smooth muscle cell proliferation, expression of contractile marker genes and production of elastin fibers. This phenotype largely depended on reduced phosphorylation and inactivation of FOXO1, as specific FOXO1 inhibition fully rescued proliferation of p110-/- smooth muscle cells, and knockdown of FOXO1 increased expression of calponin and elastin. ConclusionsSmooth muscle p110 protects against AAA disease by maintaining aortic wall homoeostasis and promoting SMC proliferation to compensate for cell loss during AAA development. Our findings have potential implications for current approaches aimed at p110 inhibition for cancer therapy and suggest new pharmacological strategies to activate p110 signaling in AAA disease.

cell biology↗

Plasmacytoid dendritic cells regulate megakaryocyte and platelet homeostasis

Platelet homeostasis is essential for vascular integrity and immune defense. While the process of platelet formation by fragmenting megakaryocytes (thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of megakaryocytes by their progenitor cells (megakaryopoiesis) remains unclear. Here we use intravital 2 photon microscopy to track individual megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as crucial bone marrow niche cells that regulate megakaryopoiesis. pDCs monitor the bone marrow for platelet-producing megakaryocytes and deliver IFN- to the megakaryocytic niche to trigger local on-demand proliferation of megakaryocyte progenitors. This fine-tuned coordination between thrombopoiesis and megakaryopoiesis is crucial for megakaryocyte and platelet homeostasis in steady state and stress. However, uncontrolled pDC function within the megakaryocytic niche is detrimental. Accordingly, we show that pDCs activated by SARS-CoV2 drive inappropriate megakaryopoiesis associated with thrombotic complications. Together, we uncover a hitherto unknown megakaryocytic bone marrow niche maintained by the constitutive delivery of pDC-derived IFN-.

immunology↗

Monocyte-derived macrophages aggravate pulmonary vasculitis via cGAS/STING/IFN-mediated nucleic acid sensing

Autoimmune vasculitis is a group of life-threatening diseases, whose underlying pathogenic mechanisms are incompletely understood, hampering development of targeted therapies. Here, we demonstrate that patients suffering from anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) showed increased activity of the DNA sensor cGAS and enhanced IFN-I signature. To identify potential therapeutic targets, we developed a mouse model for pulmonary AAV that mimics severe disease in patients. Immunogenic DNA accumulated during disease onset, triggering cGAS/STING/IRF3-dependent IFN-I release that promoted endothelial damage, pulmonary hemorrhages, and lung dysfunction. Macrophage subsets played dichotomic roles in disease. While recruited monocyte-derived macrophages were major disease drivers by producing most IFN-{beta}, resident alveolar macrophages contributed to tissue homeostasis by clearing red blood cells and limiting infiltration of IFN-{beta}-producing macrophages. Moreover, pharmacological inhibition of STING, IFNAR-I or its downstream JAK/STAT signaling reduced disease severity and accelerated recovery. Our study unveils the importance of STING/IFN-I axis in promoting pulmonary AAV progression and identifies cellular and molecular targets to ameliorate disease outcome. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/493983v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@521964org.highwire.dtl.DTLVardef@13506org.highwire.dtl.DTLVardef@1403dbeorg.highwire.dtl.DTLVardef@958cc4_HPS_FORMAT_FIGEXP M_FIG C_FIG SummaryKessler et al. identify aberrant DNA recognition by cGAS/STING axis and IFN-I production by inflammatory macrophages as a major driver of severe ANCA-associated vasculitis (AAV). Pharmacological interventions blocking this pathway ameliorate disease and accelerate recovery, identifying potential targets for therapeutic intervention in patients.

immunology↗

Adversarial attacks and adversarial robustness in computational pathology

Artificial Intelligence (AI) can support diagnostic workflows in oncology by aiding diagnosis and providing biomarkers. AI applications are therefore expected to evolve from academic prototypes to commercial products in the coming years. However, AI applications are vulnerable to adversarial attacks, such as malicious interference with test data aiming to cause misclassifications. Therefore, it is essential for the use of AI-based diagnostic devices to secure them against such attacks before widespread use. Unfortunately, no resistant systems exist in computational pathology so far. To address this problem, we investigate the susceptibility of convolutional neural networks (CNNs) to multiple types of white- and black-box attacks. We demonstrate that both attacks can easily confuse CNNs in clinically relevant pathology tasks and impair classification performance. Classical adversarially robust training and dual batch normalization (DBN) are possible mitigation strategies but require precise knowledge of the type of attack used in the inference. We demonstrate that vision transformers (ViTs) perform equally well compared to CNNs at baseline and are orders of magnitude more robust to different types of white-box and black-box attacks. At a mechanistic level, we show that this is associated with a more robust latent representation of clinically relevant categories in ViTs compared to CNNs. Our results are in line with previous theoretical studies. We show that ViTs are robust learners in computational pathology. This implies that large-scale rollout of AI models in computational pathology should rely on ViTs rather than CNN-based classifiers to provide inherent protection against adversaries.

bioinformatics↗

PD1+ T-cells correlate with Nerve Fiber Density as a prognostic biomarker in patients with resected perihilar cholangiocarcinoma.

Background & AimsPerihilar cholangiocarcinoma (pCCA) is a rare hepatobiliary malignancy. Nerve fiber invasion (NFI) shows cancer invading the nerve and is considered an aggressive feature. Nerve fiber density (NFD) consists of small nerve fibers without cancer invasion and is divided into high NFD (high numbers of small nerve fibers) or low NFD (low numbers of small nerve fibers). We aim to explore differences in immune cell populations and survival. MethodsWe applied multiplex immunofluorescence (mIF) on 47 pCCA surgically resected patients and investigated the immune cell composition in the tumor microenvironment (TME) of different nerve fiber phenotypes (NFI, high and low NFD). Extensive group comparison was carried out and the association with overall survival (OS) was assessed. ResultsThe NFI ROI was measured with highest CD68+ macrophage levels among 3 ROIs (NFI compared to tumor free p= 0.016 and to tumor p=0.034). Further, for NFI patients the density of co-inhibitory markers CD8+PD1+ and CD68+PD1+ were more abundant in the tumor rather than NFI ROI (p= 0.004 and p= 0.0029 respectively). Comparison between patients with NFD and NFI groups, the signals of co-expression of CD8+PD1+ as well as CD68+PD1+ were significantly higher in the high NFD group (p= 0.027 and p= 0.044, respectively). The OS for high NFD patients was 92 months median OS (95% CI:41-142), for low NFD patients 20 months ((95% CI: 4-36) and for NFI group of patients 19 months (95% CI 7-33). The OS for high NFD patients was significantly better compared to low NFD (p= 0.046) and NFI (p= 0.032). ConclusionsPD1+ T-cells correlate with high NFD as a prognostic biomarker, the biological pathway behind this needs to be investigated. Lay summaryNerve fibers play a dual role in the tumor microenvironment in pCCA. In our previous study, we showed that the presence of high numbers of small nerve fibers is associated with a better overall survival. In addition, we found that in high NFD patients PD1+ T-cells are significantly overexpressed. Therefore, we present high NFD as a promising prognostic biomarker. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/475344v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@e30fadorg.highwire.dtl.DTLVardef@11a4e4aorg.highwire.dtl.DTLVardef@a04d8org.highwire.dtl.DTLVardef@1c425db_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

Benchmarking artificial intelligence methods for end-to-end computational pathology

Artificial intelligence (AI) can extract subtle visual information from digitized histopathology slides and yield scientific insight on genotype-phenotype interactions as well as clinically actionable recommendations. Classical weakly supervised pipelines use an end-to-end approach with residual neural networks (ResNets), modern convolutional neural networks such as EfficientNet, or non-convolutional architectures such as vision transformers (ViT). In addition, multiple-instance learning (MIL) and clustering-constrained attention MIL (CLAM) are being used for pathology image analysis. However, it is unclear how these different approaches perform relative to each other. Here, we implement and systematically compare all five methods in six clinically relevant end-to-end prediction tasks using data from N=4848 patients with rigorous external validation. We show that histological tumor subtyping of renal cell carcinoma is an easy task which approaches successfully solved with an area under the receiver operating curve (AUROC) of above 0.9 without any significant differences between approaches. In contrast, we report significant performance differences for mutation prediction in colorectal, gastric and bladder cancer. Weakly supervised ResNet-and ViT-based workflows significantly outperformed other methods, in particular MIL and CLAM for mutation prediction. As a reason for this higher performance we identify the ability of ResNet and ViT to assign high prediction scores to highly informative image regions with plausible histopathological image features. We make all source codes publicly available at https://github.com/KatherLab/HIA, allowing easy application of all methods on any end-to-end problem in computational pathology.

bioinformatics↗

CALR frameshift mutations in MPN patient-derived iPS cells accelerate maturation of megakaryocytes

Calreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor, and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem (iPS) cells to establish a MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in CD15+ granulocytic cells derived from homozygous CALR-mutant iPS cells, rescued by repairing the mutation using CRISPR/Cas9. iPS cell-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelets protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated vs. unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis, dependent on its zygosity and found known and novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs.

cancer biology↗