Tissue Slices Research Articles

Generation of human fetal brain organoids and their CRISPR engineering for brain tumor modeling.

The developing human brain displays unique features that are difficult to study in animal models. Current in vitro models based on human brain tissue face several challenges, including the limited cellular heterogeneity in two- or three-dimensional neural stem cell cultures, while tissue slice cultures suffer from short survival. We recently established culture conditions to derive organoid cultures directly from human fetal brain tissue by preserving tissue integrity, which can be long-term expanded and display cellular heterogeneity and complex organization. In this Protocol, we describe detailed procedures to establish human fetal brain organoids (FeBOs) that broadly retain regional characteristics, along with procedures for their passaging and characterization. In addition, we describe genome engineering approaches applied to FeBOs to generate mutant FeBO lines that serve as versatile bottom-up brain cancer models. Lastly, we exemplify various downstream applications applicable to both healthy and mutant FeBOs. Scientists with experience in tissue culture can expect the establishment of human FeBO cultures to take 2-3 weeks, while genome engineering of FeBOs takes 2-4 months.

Organoids, tissue slices and organotypic cultures: advancing our understanding of pancreatic ductal adenocarcinoma through in vitro and ex vivo models.

Pancreatic ductal adenocarcinoma (PDAC) has one of the worst prognoses of all common solid cancers. For the large majority of PDAC patients, only systemic therapies with very limited efficacy are indicated. In addition, immunotherapies have not brought the advances seen in other cancer types. Several key characteristics of PDAC contribute to poor treatment outcomes, and in this review, we will discuss how these characteristics are best captured in currently available ex vivo or in vitro model systems. For instance, PDAC is hallmarked by a highly desmoplastic and immune-suppressed tumor microenvironment that impacts disease progression and therapy resistance. Also, large differences in tumor biology exist between and within tumors, complicating treatment decisions. Furthermore, PDAC has a very high propensity for locally invasive and metastatic growth. The use of animal models is often not desirable or feasible and several in vitro and ex vivo model systems have been developed, such as organotypic cocultures and tissue slices, among others. However, the absence of a full host organism impacts the ability of these models to accurately capture the characteristics that contribute to poor outcomes in PDAC. We will discuss the caveats and advantages of these model systems in the context of PDAC's key characteristics and provide recommendations on model choice and the possibilities for optimization. These considerations should be of use to researchers aiming to study PDAC in the in vitro setting.

Achieving theranostic probes targeting BRD3/BRD4 for imaging and therapy of tumor.

The BET family proteins play pleiotropic roles in the tumorigenesis and growth of various human malignancies that have aroused great interests as the cancer therapeutic targets. Therefore, it's significant to develop labeling toolkits for the dynamic monitoring of BET family proteins in living tumor cells and tissue slices. In particular, there are few small-molecule fluorescent probes based on BET family proteins developed for real-time imaging of these proteins in tumor cells and tissues and treatment of breast cancer. In general, antibodies of BET family proteins are chosen for imaging of these proteins. However, the cost of these antibodies is more expensive than small molecules and the operation is relatively complicated. Moreover, the antibodies for imaging are not capable of the therapy for breast cancer. Thus, it's essential to exploit a novel imaging system for BET family proteins which is easy-operating and economical, with achieving therapeutic effects simultaneously. Therefore, a series of fluorescent probes (17-21) targeting BET family proteins were developed. Through the evaluation studies, probe 17 showed advantages in docking studies, analysis of cell viability, and imaging studies. Importantly, probe 17 was capable of distinguishing tumor cells and tissue slices and made a distinction between them by labeling BRD3 and BRD4 proteins. Importantly, probe 17 showed higher resolution in mouse tumor and human tumor slice imaging than BRD3 and BRD4 antibodies. Moreover, compared with these antibodies, probe 17 was more stable, more economical and easier to operate in the imaging assays. In addition, it also played an anti-tumor role by inducing cell apoptosis, proliferation inhibition, and cycle arrest in tumor cells. All these features render probe 17 to perform as an effective labeling toolkit compatible for imaging studies of BRD3/BRD4, as well as an approach to diagnosis and treatment of breast cancer.

STAIG: Spatial transcriptomics analysis via image-aided graph contrastive learning for domain exploration and alignment-free integration

Spatial transcriptomics is an essential application for investigating cellular structures and interactions and requires multimodal information to precisely study spatial domains. Here, we propose STAIG, a deep-learning model that integrates gene expression, spatial coordinates, and histological images using graph-contrastive learning coupled with high-performance feature extraction. STAIG can integrate tissue slices without prealignment and remove batch effects. Moreover, it is designed to accept data acquired from various platforms, with or without histological images. By performing extensive benchmarks, we demonstrate the capability of STAIG to recognize spatial regions with high precision and uncover new insights into tumor microenvironments, highlighting its promising potential in deciphering spatial biological intricates.

Mapping the topography of spatial gene expression with interpretable deep learning.

Spatially resolved transcriptomics technologies provide high-throughput measurements of gene expression in a tissue slice, but the sparsity of these data complicates analysis of spatial gene expression patterns. We address this issue by deriving a topographic map of a tissue slice-analogous to a map of elevation in a landscape-using a quantity called the isodepth. Contours of constant isodepths enclose domains with distinct cell type composition, while gradients of the isodepth indicate spatial directions of maximum change in expression. We develop GASTON (gradient analysis of spatial transcriptomics organization with neural networks), an unsupervised and interpretable deep learning algorithm that simultaneously learns the isodepth, spatial gradients and piecewise linear expression functions that model both continuous gradients and discontinuous variation in gene expression. We show that GASTON accurately identifies spatial domains and marker genes across several tissues, gradients of neuronal differentiation and firing in the brain, and gradients of metabolism and immune activity in the tumor microenvironment.

Intracerebral Hemorrhage-Associated Iron Release Leads to Pericyte-Dependent Cerebral Capillary Function Disruption

Intracerebral hemorrhage leads to immediate brain injury due to local mechanical damage, on which current treatment approaches are focused, but it also induces secondary brain injury. The purpose of this study is to characterize blood components, degradation products and their effects in secondary brain injury. Immunocyto- and immunohistochemistry, Fluorescence-Activated Cell Sorting, WST-1 assays and RNA sequencing were applied using human cell cultures and human ex vivo brain tissue slices. Brain tissue was immediately collected, cooled and sliced during neurosurgical operations to perform experiments on living tissue slices of the human brain. Among the blood degradation products, free iron (Fe2+ and Fe3+), but not hemoglobin, induced detrimental effects on pericyte function and survival (78.5% vs. 94.3%; p-value < 0.001). RNA sequencing revealed ferroptosis as the underlining cellular mechanism, mediated via GPX-4 (log2 fold change > 1.0, p-value < 1.08 × 10−30) in pathway analysis and eventually resulting in oxidative cell death. Pericytes located at cerebral capillary branching sites were specifically affected by ferroptosis, leading to capillary disruption and vasoconstriction, which were partially prevented by ferrostatin-1. Free iron induces the pericyte-dependent disruption of cerebral capillary function and represents a therapeutic target to attenuate secondary injury after intracerebral hemorrhage.

DOP064 Evaluation of pharmacological interventions in human Precision-Cut Intestinal Slices: insights into tissue-resident immunity in Inflammatory Bowel Diseases

Abstract Background Inflammatory Bowel Diseases (IBD), including Crohn's Disease (CD) and Ulcerative Colitis (UC), are marked by dysregulated immune responses and significant T cell infiltration, leading to chronic intestinal inflammation. The global burden of IBD is increasing, yet its pathogenesis remains only partly understood. Our study aims to gain deeper insights into the immunological responsiveness of diseased tissue using ex vivo primary human intestinal tissue slices (Precision Cut Intestinal Slices; PCIS). PCIS are ultrathin tissue slices of the intestine of human donors that remain viable in culture, preserve intestinal architecture, and maintain all relevant intestinal cells including tissue-resident immune cells. Here, we specifically investigated the effects of the JAK1 inhibitor filgotinib and the calcineurin inhibitor pimecrolimus on tissue-resident immune cell responses in PCIS ex vivo. Methods PCIS from ileum and colon resections of Crohn’s disease (CD) and non-CD patients were stimulated ex vivo with Concanavalin A, anti-CD3/CD28 antibodies and IL-1β. Induced responses were modulated with anti-inflammatory treatments such as pimecrolimus and filgotinib for 24h. We evaluated tissue viability (LDH-assay), cytokine release (multiplex ELISA), and PCIS morphology (H&E-staining and Immune fluorescence). Results CD and non-CD intestinal tissue slices maintained viability and intestinal morphology, including disease-specific features in ex vivo culture such as immune cell infiltrates and higher abundance of CD4+ and CD8+ T cells. Ex vivo treatment with various immune stimulations revealed distinct disease-specific cytokine release patterns, with a primary focus on Th1 and Th17 responses, but also including other Th cell, innate and epithelial responses. Filgotinib (1-100 μM) dose-dependently inhibited the secretion of general proinflammatory and T cell-specific cytokines (maximal decrease e.g. IL-8: 8-fold, IL-6: 50-fold, IL-17A: 27-fold). Pimecrolimus (25 µM) specifically decreased T cell-specific cytokines (maximal decrease e.g. IL-2: 13-fold, IL-17A: 22-fold, IL-17F: 27-fold or IFNγ: 7-fold) while the release of general proinflammatory mediators like IL-8 was less affected. Conclusion Our data show the anti-inflammatory effects of filgotinib and pimecrolimus in primary intestinal tissue slices ex vivo. These effects include modulation of various T cell-associated cytokines and other molecules involved in the calcineurin and JAK-STAT pathways. Notably, the different treatments showed distinct effects on cytokine release profiles. This study underscores the potential of PCIS to elucidate the mechanisms of JAK1 inhibitors, advancing IBD treatment strategies and providing a test platform for new drugs in a complex human tissue system.

Spatiotemporal network dynamics and structural correlates in the human cerebral cortex in vitro.

Elucidating human cerebral cortex function is essential for understanding the physiological basis of both healthy and pathological brain states. We obtained extracellular local field potential recordings from cortical slices of neocortical tissue from refractory epilepsy patients. Multi-electrode recordings were combined with histological information, providing a two-dimensional spatiotemporal characterization of human cortical dynamics in control conditions and following modulation of the excitation/inhibition balance. Slices expressed spontaneous rhythmic activity consistent with slow wave activity, comprising alternating active (Up) and silent (Down) states (Up-duration: 0.08±0.03s, Down-duration: 2.62±2.12s, frequency: 0.75±0.39Hz). Up states propagated from deep to superficial layers, with faster propagation speeds than in other species (vertical: 64.6mm/s; horizontal: 65.9mm/s). GABAA blockade progressively transformed the emergent activity into epileptiform discharges, marked by higher firing rates, faster network recruitment and propagation, and infraslow rhythmicity (0.01Hz). This dynamical characterization broadens our understanding of the mechanistic organization of the human cortical network at the micro- and mesoscale.

Tissue optical clearing and expanding solution for improved nuclear segmentation

AbstractOver the recent years, fluorescence microscopy has seen substantial progress, transitioning from primarily qualitative analysis to a profoundly quantitative methodology, yet limitations in imaging depth and data quality persist due to tissue opacity. Tissue optical clearing (TOC) methodologies were implemented to overcome this hurdle, enabling the imaging and quantitative analysis of large tissue samples such as entire murine organs. Further improvements, including expansion microscopy merge TOC benefits with improved imaging resolution, converting cells and tissues into tissue‒gel hybrids. Yet, its intricate protocols lead to a remarkable, ∼10‒20× tissue volume expansion, making the acquisition of even thin sections exceedingly challenging in terms of time, labor, and data handling. Here, we present a novel solution, Improved Segmentation by gEntle Expansion (ISEE), for both TOC and gentle (1.6‒1.7×) tissue expansion for significantly improved precision of fluorescent signal segmentation, while maintaining tolerable increase during data acquisition in everyday laboratory practice. When applied to thick ∼40‒200 µm tissue slices, ISEE renders them transparent and ultimately expanded within minutes, in a simple, one‐step process compatible with immunohistochemistry as well as with all major murine organs.

Protocol for generating protein profiles and distance-based network analysis of murine tissue slices.

We introduce a protocol for spatial proteomics using thin cryotome sections of mouse skeletal muscle tissue. We describe steps for preparing muscle sections and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses to generate spatial protein profiles along the longitudinal skeletal muscle axis. We detail procedures for scanning longitudinal protein profiles and replacing missing data using a sliding window approach. This protocol has potential for applications in spatial proteomics to other tissues with asymmetric patterns such as the brain and heart tissue. For complete details on the use and execution of this protocol, please refer to Schmidt etal.1.

Selective Gamma-Secretase Inhibition by CHF5074 Attenuates Inflammation and Neovascularization in a Murine Model of Choroidal Neovascularization

Purpose Chronic inflammation plays an important role in the pathogenesis of choroidal neovascularization (CNV). This study aimed to investigate the effect of the CHF5074, a γ-secretase inhibitor, on angiogenesis in a laser-induced CNV model and elucidate its possible molecular mechanism. Methods Male C57/BL6J mice aged between 6 to 8 weeks were employed to set up a laser-induced model of CNV. Then, CHF5074 was injected intraperitoneally on the day after laser modeling, as well as on the second, third, and fourth days. Immunofluorescence staining was used to evaluate the retinal and choroidal complex. The markers used were CD31 for neovascularization and IBA1 for microglia staining in ocular tissue slices. Fundus fluorescein angiography on days 3d, 7d, and 14d analyzed neovascularization and leakage areas. Inflammatory indicators were examined by Western blot (WB) and enzyme-linked immunosorbent assay (ELISA). High-throughput whole-tissue sequencing of retinal choroids identified relevant cell pathways. Key regulatory factors modulated by CHF5074 were identified via WB. Co-culture of BV2 cells and human umbilical vein endothelial cells (HUVEC) were used to explore the function of CHF5074 on the inhibition of tube formation. Results CHF5074 significantly decreased CD31 expression in the choroid on 3d, 7d, and 14d post-laser modeling (p < 0.05) and decreased both neovascularization and leakage areas (p < 0.05). Additionally, CHF5074 significantly lowered TNF-α, IL-10, and IL-1β expression levels in the choroid (p < 0.05), as demonstrated by WB analysis and ELISA. High-throughput whole-tissue sequencing identified P38-MAPK, JNK, and Wnt signaling pathways associated with neovascularization. CHF5074 decreased P38 protein phosphorylation (p < 0.05) as confirmed by WB analysis. CHF5074 inhibited the tube formation of HUVECs co-cultured with LPS and ATP-treated BV2 cells. Conclusion CHF5074 significantly suppresses angiogenesis in laser-induced choroidal neovascularization models, suggesting its potential as a novel agent for preventing and treating CNV.

Ultrafast multicellular calcium imaging of calcium spikes in mouse beta cells in tissue slices.

The crucial steps in beta cell stimulus-secretion coupling upon stimulation with glucose are oscillatory changes in metabolism, membrane potential, intracellular calcium concentration, and exocytosis. The changes in membrane potential consist of bursts of spikes, with silent phases between them being dominated by membrane repolarization and absence of spikes. Assessing intra- and intercellular coupling at the multicellular level is possible with ever-increasing detail, but our current ability to simultaneously resolve spikes from many beta cells remains limited to double-impalement electrophysiological recordings. Since multicellular calcium imaging of spikes would enable a better understanding of coupling between changes in membrane potential and calcium concentration in beta cell collectives, we set out to design an appropriate methodological approach. Combining the acute tissue slice method with ultrafast calcium imaging, we were able to resolve and quantify individual spikes within bursts at a temporal resolution of >150 Hz over prolonged periods, as well as describe their glucose-dependent properties. In addition, by simultaneous patch-clamp recordings we were able to show that calcium spikes closely follow membrane potential changes. Both bursts and spikes coordinate across islets in the form of intercellular waves, with bursts typically displaying global and spikes more local patterns. This method and the associated findings provide additional insight into the complex signaling within beta cell networks. Once extended to tissue from diabetic animals and human donors, this approach could help us better understand the mechanistic basis of diabetes and find new molecular targets.

Minimal differences observed when comparing the morphological profiling of microglia obtained by confocal laser scanning and optical sectioning microscopy.

While widefield microscopy has long been constrained by out-of-focus scattering, advancements have generated a solution in the form of confocal laser scanning microscopy (cLSM) and optical sectioning microscopy using structured illumination (OSM). In this study, we aim to investigate, using microglia branching, if cLSM and OSM can produce images with comparable morphological characteristics. By imaging the somatosensory microglia from a tissue slice of a 3-week-old mouse and establishing morphological parameters that characterizes the microglial branching pattern, we were able to show that there is no difference in total length of the branch tree, number of branches, mean branch length and number of primary to terminal branches. We did find that area-based parameters such as mean occupied area and mean surveillance area were bigger in cLSM isolated microglia compared to OSM ones. Additionally, by investigating the difference in acquisition time between techniques and personal costs we were able to establish that the amortization could be made in 6.11 ± 2.93 years in the case of countries with a Human Development Index (HDI) = 7-9 and 7.06 ± 3.13 years, respectably, for countries with HDI < 7. As such, OSM systems seem a valid option if one just wants basic histological evaluation, and cLSM should be considered for groups that demand higher resolution or volumetric images.

DeST-OT: Alignment of spatiotemporal transcriptomics data.

Spatially resolved transcriptomics (SRT) measures mRNA transcripts at thousands of locations within a tissue slice, revealing spatial variations in gene expression and cell types. SRT has been applied to tissue slices from multiple time points during the development of an organism. We introduce developmental spatiotemporal optimal transport (DeST-OT), a method to align spatiotemporal transcriptomics data using optimal transport (OT). DeST-OT uses semi-relaxed OT to model cellular growth, death, and differentiation processes. We also derive a growth distortion metric and a migration metric to quantify the plausibility of spatiotemporal alignments. DeST-OT outperforms existing methods on the alignment of spatiotemporal transcriptomics data from developing mouse kidney and axolotl brain. DeST-OT estimated growth rates also provide insights into the gene expression programs governing the growth and differentiation of cells over space and time.

A 3D-printed multi-compartment organ-on-chip platform with a tubing-free pump models communication with the lymph node.

Multi-organ-on-chip systems (MOOCs) have the potential to mimic communication between organ systems and reveal mechanisms of health and disease. However, many existing MOOCs are challenging for non-experts to implement due to complex tubing, electronics, or pump mechanisms. In addition, few MOOCs have incorporated immune organs such as the lymph node (LN), limiting their applicability to model critical events such as vaccination. Here we developed a 3D-printed, user-friendly device and companion tubing-free impeller pump with the capacity to co-culture two or more tissue samples, including a LN, under a recirculating common media. Native tissue structure and immune function were incorporated by maintaining slices of murine LN tissue ex vivo in 3D-printed mesh supports for at least 24 h. In a two-compartment model of a LN and an upstream injection site in mock tissue, vaccination of the multi-compartment chip was similar to in vivo vaccination in terms of locations of antigen accumulation and acute changes in activation markers and gene expression in the LN. We anticipate that in the future, this flexible platform will enable models of multi-organ immune responses throughout the body.

BETA CELL DYSFUNCTION OCCURS INDEPENDENTLY OF INSULITIS IN TYPE 1 DIABETES PATHOGENESIS.

During type 1 diabetes (T1D) progression, beta cells become dysfunctional and exhibit reduced first-phase insulin release. While this period of beta cell dysfunction is well established, its cause and underlying mechanism remain unknown. To address this knowledge gap, live human pancreas tissue slices were prepared from autoantibody-negative organ donors without diabetes (ND), donors positive for one or more islet autoantibodies (AAb+), and donors with T1D within 0-4 years of diagnosis (T1D+). Dynamic imaging and physiological secretion studies were conducted to assess the extent and impact of T cell infiltration on beta cell function through immunolabeling, Ca2+ imaging, and perifusion assays for insulin secretion. Beta cells were identified in living slices by ENTPD3 cell surface staining. Beta cells from both ND and AAb+ donors exhibited normal cytosolic Ca2+ mobilization in response to high glucose and KCl. Beta cells from donors with T1D had significantly diminished Ca2+ responses to high glucose but preserved responses to KCl. In T1D, glucose responsiveness was impaired in both T cell-infiltrated and non-infiltrated insulin positive islets, supporting the concept that beta cell dysfunction is independent of close spatial association between beta cells and T cells. Fixed tissue staining and gene expression profiling of laser-capture microdissected islets revealed significant decreases in markers of glucose metabolism to ATP in beta cells from donors with T1D but no changes in endoplasmic reticulum (ER) stress markers. From these data, we posit that functional defects occur in beta cells during T1D pathogenesis independent of local T cell infiltration and beta cell destruction. Additionally, these beta cell metabolic defects contribute to the clinical manifestation of dysglycemia at T1D diagnosis despite a remaining mass of beta cells.

In vivo imaging of adipose-derived stem cell sheets on biodegradable nonwoven fabric using X-ray CT

BackgroundA biodegradable nonwoven fabric that can be used to extract adipose-derived stem cells (ADSCs) from adipose tissue slices was developed, which were cultured rapidly without enzymatic treatment. The extracted and cultured ADSCs remain on the nonwoven fabric and form a thick cell sheet. The aim was to use the thick cell sheet as a treatment by transplanting it into the living body. In addition, the expectation was that it will be possible to observe the cell sheet in the living body using X-ray computed tomography (CT) because the nonwoven fabric used to produce the cell sheet contains 50% (by weight) hydroxyapatite.ResultsThick cell sheets of ADSCs supported by two layers of nonwoven fabric were cut to size and transplanted into the cheeks of rats. No health damage was observed in the rats in which the cell sheets were implanted, except for one in which the surgery appeared to have failed. X-ray CT imaging showed that the fabric of the implanted cell sheet biodegraded over 12 weeks. Changes in the position, shape, and size of the cell sheet within the rat’s body were tracked by X-ray CT. The thick cell sheets, which can be easily produced by simply seeding tissue slices, can be cut into appropriate shapes and transplanted safely, and it was confirmed that they slowly biodegraded when transplanted into the rats’ bodies.ConclusionsWe demonstrated not only that the thick ADSC sheets can be transplanted successfully into animals, but also that the transplanted sheets can be observed in vivo by X-ray CT, which also allows changes in the ADSC sheets to be tracked. The results suggest that the biodegradable nonwoven fabric will be a useful transplantation device to ensure cell engraftment throughout the affected area, and facilitate monitoring of the transplant’s subsequent status. We expect that this transplantation device will promote the development of regenerative therapy.

Deep learning for opportunistic, end-to-end automated assessment of epicardial adipose tissue in pre-interventional, ECG-gated spiral computed tomography

ObjectivesRecently, epicardial adipose tissue (EAT) assessed by CT was identified as an independent mortality predictor in patients with various cardiac diseases. Our goal was to develop a deep learning pipeline for robust automatic EAT assessment in CT.MethodsContrast-enhanced ECG-gated cardiac and thoraco-abdominal spiral CT imaging from 1502 patients undergoing transcatheter aortic valve replacement (TAVR) was included. Slice selection at aortic valve (AV)-level and EAT segmentation were performed manually as ground truth. For slice extraction, two approaches were compared: A regression model with a 2D convolutional neural network (CNN) and a 3D CNN utilizing reinforcement learning (RL). Performance evaluation was based on mean absolute z-deviation to the manually selected AV-level (Δz). For tissue segmentation, a 2D U-Net was trained on single-slice images at AV-level and compared to the open-source body and organ analysis (BOA) framework using Dice score. Superior methods were selected for end-to-end evaluation, where mean absolute difference (MAD) of EAT area and tissue density were compared. 95% confidence intervals (CI) were assessed for all metrics.ResultsSlice extraction using RL was slightly more precise (Δz: RL 1.8 mm (95% CI: [1.6, 2.0]), 2D CNN 2.0 mm (95% CI: [1.8, 2.3])). For EAT segmentation at AV-level, the 2D U-Net outperformed BOA significantly (Dice score: 2D U-Net 91.3% (95% CI: [90.7, 91.8]), BOA 85.6% (95% CI: [84.7, 86.5])). The end-to-end evaluation revealed high agreement between automatic and manual measurements of EAT (MAD area: 1.1 cm2 (95% CI: [1.0, 1.3]), MAD density: 2.2 Hounsfield units (95% CI: [2.0, 2.5])).ConclusionsWe propose a method for robust automatic EAT assessment in spiral CT scans enabling opportunistic evaluation in clinical routine.Critical relevance statementSince inflammatory changes in epicardial adipose tissue (EAT) are associated with an increased risk of cardiac diseases, automated evaluation can serve as a basis for developing automated cardiac risk assessment tools, which are essential for efficient, large-scale assessment in opportunistic settings.Key PointsDeep learning methods for automatic assessment of epicardial adipose tissue (EAT) have great potential.A 2-step approach with slice extraction and tissue segmentation enables robust automated evaluation of EAT.End-to-end automation enables large-scale research on the value of EAT for outcome analysis.Graphical

First-in-human Study of ABY-029, a Novel Fluorescent Peptide that Targets Epidermal Growth Factor Receptor, Applied to Soft-Tissue Sarcomas.

ABY-029, an anti-epidermal growth factor receptor (EGFR) Affibody® molecule conjugated to IRDye 800CW, recently underwent first-in-human testing in soft-tissue sarcoma (STS). FDA Exploratory Investigational New Drug status was obtained for the Phase 0 clinical trial in which study objectives were to determine whether biological variance ratio (BVR) of 10 was achievable, fluorescence intensity correlated with EGFR expression, and doses were well tolerated. Patients (N=12) with STS were recruited based on positive EGFR immunohistochemical staining of diagnostic biopsies. ABY-029 was administered at micro- (30 nanomole, n=3), medium (90 nanomole, n=3), or high dose (171 nanomole, n=6), 1-3 hours prior to surgery. Following tumor resection, ex vivo tissue was imaged to determine mean fluorescence intensity (MFI), BVR, and other contrast measures. EGFR expression was correlated with immunohistochemistry. For micro-, medium, and high doses, mean BVR (SD) in cross-sectional slices were 4 (4), 10 (6), and 7 (8), respectively, for the whole tumor region and 6 (5), 13 (11), and 8 (6), respectively, for pathology-confirmed regions-of-interest. Strong linear correlations were found between all ABY-029 contrast metrics and total EGFR (r≥0.86, p<0.029) in cross-sectional tissue slices, and MFI and EGFR percent area (r=0.63, p<0.0001) in excised region-of-interest tissue sections. No ABY-029 related adverse events were observed. When administered above the microdose, ABY-029 demonstrated high correlation to EGFR expression and contrast values that were encouraging for translation to clinical practice. Contrast was similar to those observed with antibody agents, but with substantially reduced imaging-to-resection time, and no drug-related adverse events.

Diagnosis of fibrotic interstitial lung diseases based on the combination of label-free quantitative multiphoton fiber histology and machine learning.

Interstitial lung disease (ILD), characterized by inflammation and fibrosis, often suffers from low diagnostic accuracy and consistency. Traditional H&E staining primarily reveals cellular inflammation with limited detail on fibrosis. To address these issues, we introduce a pioneering label-free quantitative multiphoton fiber histology (MPFH) technique that delineates the intricate characteristics of collagen and elastin fibers for ILDs diagnosis. We acquired co-located multiphoton and H&E-stained images from a single tissue slice. Multiphoton imaging was performed on the deparaffinized section to obtain fibrotic tissue information, followed by H&E staining to capture cellular information. This approach was tested in a blinded diagnostic trial among 7 pathologists involving 14 relatively normal lung patients and 31 ILD patients (11 idiopathic pulmonary fibrosis (IPF) / usual interstitial pneumonia (UIP), 14 nonspecific interstitial pneumonia (NSIP), and 6 pleuroparenchymal fibroelastosis (PPFE)). A customized algorithm extracted quantitative fiber indicators from multiphoton images. These indicators, combined with clinical and radiological features, were used to develop an automatic multi-class ILDs classifier. Using MPFH, we can acquire high-quality, co-localized images of collagen fibers, elastin fibers, and cells. We found that the type, distribution, and degree of fibrotic proliferation can effectively distinguish between different subtypes. The blind study showed MPFH enhanced diagnostic consistency (kappa values from 0.56 to 0.72) and accuracy (from 73.0% to 82.5%, p=0.0090). The combination of quantitative fiber indicators effectively distinguished between different tissues, with areas under the receiver operating characteristic curves exceeding 0.92. The automatic classifier achieved 93.8% accuracy, closely paralleling the 92.2% accuracy of expert pathologists. The outcomes of our research underscore the transformative potential of MPFH in the field of f-ILD diagnostics. By integrating quantitative analysis of fiber characteristics with advanced machine learning algorithms, MPFH facilitates the automatic and accurate identification of various fibrotic disease subtypes, showcasing a significant leap forward in precision diagnostics.