Tissue Organization Research Articles

Early Radiation-Induced Changes in Lung Tissue and Intercellular Junctions: Implications for Tissue Repair and Fibrosis.

Early changes in lung tissue following ionizing radiation (IR) initiate processes that may lead to either regeneration or fibrosis. Intercellular junction proteins play a crucial role in the organization and function of epithelial tissues, both under normal conditions and after injuries. Alterations in the expression and localization of these proteins can influence the fate of epithelial cells. This study aims to investigate the effects of IR on lung tissue structure, as well as on the levels and distribution of intercellular junction proteins. Wistar rats were subjected to total X-ray irradiation at doses of 2 and 10 Gy. Lung tissue samples were collected for Western blot and histological analysis 72 h post-IR. IR at doses of 2 and 10 Gy led to structural changes in lung tissue and elevated levels of E-cadherin. The 10 Gy IR resulted in increased claudin-4 and occludin in lung parenchyma, decreased claudin-8 and claudin-12 in bronchial epithelium and endothelium, and suppression of apoptosis. Data evaluation indicated that alterations in the protein composition of intercellular junctions are essential processes in lung tissue at early stages after IR, and at least some of these alterations are associated with adaptation.

Poly (A) binding protein 2 is critical for stem cell differentiation during regeneration in the planarian Schmidtea mediterranea.

Post-transcriptional regulation has emerged as a key mechanism for regulating stem cell renewal and differentiation, which is essential for understanding tissue regeneration and homeostasis. Poly(A)-binding proteins are a family of RNA-binding proteins that play a vital role in post-transcriptional regulation by controlling mRNA stability and protein synthesis. The involvement of poly(A) binding proteins in a wide range of cellular functions is increasingly being investigated. In this study, we used the regenerative model planarian organism Schmidtea mediterranea to demonstrate the critical role of poly(A)-binding protein 2 (PABP2) in regulating neoblast maintenance and differentiation. A deficit in PABP2 blocks the transition of neoblasts toward immediate early progenitors, leading to an enhanced pool of non-committed neoblasts and a decreased progenitor population. This is reflected in variations in the transcriptome profile, providing evidence of downregulation in multiple lineages. Thus, an insufficiency of PABP2 resulted in defective formation and organization of tissue, leading to abnormal regeneration. Our study reveals the essential role of PABP2 in regulating genes that mediate stem cell commitment to early progenitors during tissue regeneration.

Mosna reveals different types of cellular interactions predictive of response to immunotherapies and survival in cancer.

Spatially resolved omics enable the discovery of tissue organization of biological or clinical importance. Despite the existence of several methods, performing a rational analysis including multiple algorithms while integrating different conditions such as clinical data is still not trivial. To make such investigations more accessible, we developed mosna, a Python package to analyze spatial omics data with respect to clinical or biological data and to gain insight on cell interaction patterns or tissue architecture of biological relevance. mosna is compatible with all spatial omics methods, it leverages tysserand to build accurate spatial networks, and is compatible with Squidpy. It proposes an analysis pipeline, in which increasingly complex features computed at each step can be explored in integration with clinical data, either with easy-to-use descriptive statistics and data visualization, or by seamlessly training machine learning models and identifying variables with the most predictive power. mosna can take as input any dataset produced by spatial omics methods, including sub-cellular resolved transcriptomics (MERFISH, seqFISH) and proteomics (CODEX, MIBI-TOF, low-plex immuno-fluorescence), as well as spot-based spatial transcriptomics (10x Visium). Integration with experimental metadata or clinical data is adapted to binary conditions, such as biological treatments or response status of patients, and to survival data. We demonstrate the proposed analysis pipeline on two spatially resolved proteomic datasets containing either binary response to immunotherapy or survival data. mosna identifies features describing cellular composition and spatial distribution that can provide biological insight regarding factors that affect response to immunotherapies or survival.

Acid–base and electrolyte changes in dogs after packed red blood cell transfusion

AbstractBackgroundPacked RBC (pRBC) transfusions are often necessary to enhance organ perfusion and tissue oxygenation in cases of severe anemia.ObjectivesWe aimed to describe changes in acid–base and biochemical parameters in dogs after transfusion of pRBC and potential effects on the outcome.MethodsThe prospective observational study included anemic dogs requiring pRBC transfusions. Venous blood gas and electrolytes were measured pre‐transfusion and repeated within 1 h post‐transfusion. Signalment, pre‐existing conditions and underlying disease(s), number of days spent of hospitalization, the quantity of transfused pRBC units, and the outcome were also recorded. Associations between analytical values and all other parameters before and after transfusion were assessed using the Wilcoxon test. A generalized linear model was generated for each of the blood gas parameters post‐transfusion, including the pre‐transfusion values and the characteristics of the animal as fixed effects. The effect of the blood gas parameters on the mortality and hospitalization length was assessed using logistic regression and generalized linear models, respectively.ResultsTwenty‐six dogs with different causes of anemia were included in the study, with dogs remaining hospitalized for a median of 3 days (range: 0–11). Pre‐transfusion heart rate, respiratory rate, Angap, base excess, and lactate were higher than post‐transfusion values. Post‐transfusion results showed an increase in hematocrit, hemoglobin, HCO3‐, PCO2, tCO2, and chloride. Pre‐transfusion hypophosphatemia and receiving an increased volume of transfused pRBC were associated with a longer hospital stay, while higher pre‐transfusion lactate values were associated with higher mortality.ConclusionspRBC transfusion in anemic dogs was associated with an improvement in perfusion and acid–base parameters. Pre‐transfusion lactate may be an indicator of mortality in dogs.

Identification of Reference Gene for Quantitative Gene Expression in Early-Term and Late-Term Cultured Canine Fibroblasts Derived from Ear Skin.

Fibroblasts are cells that reside within the fibrous or loose connective tissues of most mammalian organs. For research purposes, fibroblasts are often subjected to long-term culture under defined conditions, during which their properties can significantly change. It is essential to understand and document these changes to obtain reliable outcomes. For the quantification of specific gene expressions, the most reliable and widely used technique is quantitative real-time polymerase chain reaction (qRT-PCR). Here, we assessed the impact of a reference gene's stability on a qRT-PCR analysis of long-term cultured canine skin fibroblasts. After successfully isolating the fibroblasts from canine skin tissues, they were cultured and evaluated for proliferation and β-galactosidase activity at different passage numbers. With extended culture, the fibroblasts showed a long doubling time and elevated β-galactosidase activity. Using three widely used algorithms, geNorm, Normfinder, and Bestkeeper, we identified HPRT1, YWHAZ, and GUSB as the most stable reference genes for both early- and late-passage fibroblasts. Conventional reference genes such as GAPDH were found to be less stable than those genes. The normalization of Vimentin by the stable genes showed statistical differences, whereas normalization by an unstable gene did not. Collectively, this study indicates that using stable reference genes is essential for accurately and reliably measuring gene expression in both early- and late-passage fibroblasts. These findings provide valuable insights into internal controls for gene expression studies and are expected to be utilized for analyzing gene expression patterns in molecular biology research.

Multiplexed spatial mapping of chromatin features, transcriptome, and proteins in tissues.

The phenotypic and functional states of a cell are modulated by a complex interactive molecular hierarchy of multiple omics layers, involving the genome, epigenome, transcriptome, proteome, and metabolome. Spatial omics approaches have enabled the capture of information from different molecular layers directly in the tissue context. However, current technologies are limited to map one to two modalities at the same time, providing an incomplete representation of cellular identity. Such data is inadequate to fully understand complex biological systems and their underlying regulatory mechanisms. Here we present spatial-Mux-seq, a multi-modal spatial technology that allows simultaneous profiling of five different modalities, including genome-wide profiles of two histone modifications and open chromatin, whole transcriptome, and a panel of proteins at tissue scale and cellular level in a spatially resolved manner. We applied this technology to generate multi-modal tissue maps in mouse embryos and mouse brains, which discriminated more cell types and states than unimodal data. We investigated the spatiotemporal relationship between histone modifications, chromatin accessibility, gene and protein expression in neuron differentiation revealing the relationship between tissue organization, function, and gene regulatory networks. We were able to identify a radial glia spatial niche and revealed spatially changing gradient of epigenetic signals in this region. Moreover, we revealed previously unappreciated involvement of repressive histone marks in the mouse hippocampus. Collectively, the spatial multi-omics approach heralds a new era for characterizing tissue and cellular heterogeneity that single modality studies alone could not reveal.

Smear Layer Removing and Pulp Dissolving Ability of Sodium Hypochlorite Mixed with Two Etidronate-Containing Irrigants in Continuous Chelation Technique

The study aimed to assess the effect of two etidronate-containing irrigants and EDTA on the ability of sodium hypochlorite (NaOCl) to remove the smear layer and dissolve organic tissues. This study evaluated the following solutions: distilled water, 3% NaOCl, 3% NaOCl + EDTA, and 3% NaOCl mixed with etidronate powder from two manufacturers [Dual Rinse, (DR); IsraDent, (ID)] to obtain 9%, 15%, and 18% solutions. To assess the proteolytic activity, bovine muscle tissue samples (56.1 ± 5.4 mg; n = 10 per group) were immersed in the tested solutions for 15 min. Absolute weight changes and percentages of weight changes (wt%) were calculated. To assess smear layer removal, the root canals of human wisdom teeth were instrumented, irrigated with the tested solutions (n = 10 per group), and evaluated using SEM. Statistical analysis employed an ANOVA with a post-hoc Tukey test and paired t-test, the Kruskal–Wallis test with a pairwise Wilcoxon rank sum test, and the Friedman test with a pairwise Wilcoxon signed-rank test. The mean weight loss in the NaOCl group comprised 17.3 mg (32 wt%). Sequential use of NaOCl and EDTA resulted in a significant increase in proteolytic activity of the former (57 wt%), while mixing these solutions led to a reduction of proteolytic activity (17 wt%). All NaOCl/DR groups exhibited a significantly greater dissolution activity than that of NaOCl alone, with the mean weight loss ranging from 23.3 mg (40 wt%) to 24 mg (41 wt%). ID9% and ID15% significantly decreased the proteolytic activity of NaOCl. In most groups, the apical thirds of the root canals demonstrated a significantly heavier smear layer compared to the middle and coronal thirds. The most effective smear layer removal was observed after irrigation with NaOCl combined with EDTA or DR (all concentrations); however, in the NaOCl + EDTA and DR18% groups, it was accompanied by moderate to severe erosion. Irrigation with ID did not result in smear layer removal or dentin erosion. In conclusion, the smear layer removal and pulp dissolving effects of continuous chelation using etidronate were manufacturer/composition-dependent. DR9% combined with NaOCl demonstrated the most promising results.

Molecular pathology, developmental changes and synaptic dysfunction in (pre-) symptomatic human C9ORF72-ALS/FTD cerebral organoids

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD, even before symptom onset. Because these early disease phenotypes remain incompletely understood, we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients, presymptomatic C9ORF72-HRE (C9-HRE) carriers, and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition, single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids, including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further, molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly, organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology, whereas the extent to which more downstream cellular defects, as found in C9-ALS/FTD models, were detected varied for the different presymptomatic C9-HRE cases. Together, these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies.

Different Biomechanical Cell Behaviors in an Epithelium Drive Collective Epithelial Cell Extrusion.

In vertebrates, many organs, such as the kidney and the mammary gland form ductal structures based on the folding of epithelial sheets. The development of these organs relies on coordinated sorting of different cell lineages in both time and space, through mechanisms that remain largely unclear. Tissues are composed of several cell types with distinct biomechanical properties, particularly at cell-cell and cell-substrate boundaries. One hypothesis is that adjacent epithelial layers work in a coordinated manner to shape the tissue. Using in vitro experiments on model epithelial cells, differential expression of atypical Protein Kinase C iota (aPKCi), a key junctional polarity protein, is shown to reinforce cell epithelialization and trigger sorting by tuning cell mechanical properties at the tissue level. In a broader perspective, it is shown that in a heterogeneous epithelial monolayer, in which cell sorting occurs, forces arising from epithelial cell growth under confinement by surrounding cells with different biomechanical properties are sufficient to promote collective cell extrusion and generate emerging 3D organization related to spheroids and buds. Overall, this research sheds light on the role of aPKCi and the biomechanical interplay between distinct epithelial cell lineages in shaping tissue organization, providing insights into the understanding of tissue and organ development.

3D bioprinting technology and equipment based on microvalve control.

3D bioprinting technology is widely used in biomedical fields such as tissue regeneration and constructing pathological model. The prevailing printing technique is extrusion-based bioprinting. In this printing method, the bioink needs to meet both printability and functionality, which are often conflicting requirements. Therefore, this study has developed an innovative microvalve-based equipment, incorporating components such as pressure control, a three-dimensional motion platform, and microvalve. Here, we present a droplet-based method for constructing complex three-dimensional structures. By leveraging the rapid switching characteristics of the microvalve, this equipment can achieve precise printing of bio-materials with viscosities as low as 10mPa·s, significantly expanding the biofabrication window for bioinks. This technology is of great significance for 3D bioprinting in tissue engineering and lays a solid foundation for the construction of complex artificial organ tissues.

Versatile and Tunable Performance of PVA/PAM Tridimensional Hydrogel Models for Tissues and Organs: Augmenting Realism in Advanced Surgical Training.

The increasing complexity and difficulty of surgical procedures have led to a rise in medical errors within clinical settings in recent years. Gastrointestinal diseases, in particular, present significant medical challenges and impose substantial economic burdens, underscoring the urgent need for experiential, high-fidelity gastrointestinal surgical training tools. This study leverages patient-specific computed tomography (CT) and magnetic resonance imaging (MRI) data, combined with 3D printed manufacturing, to develop hydrogel organ models with tunable performance and tissue-mimicking softness. These properties are achieved by regulating the freeze-thaw cycles, cross-linking agents, and the concentration of incorporated antibacterial nanoparticles in DN hydrogels. Through the application of indirect 3D printing and the "sacrificial material method", we successfully fabricate organ tissues such as the stomach, intestines, and blood vessels with high precision. In ex vivo surgical training demonstrations, these tissue-like soft hydrogels provide an effective platform for preoperative simulation and surgical training in digestive surgery, accommodating various surgical procedures and accurately simulating intraoperative bleeding. The development of advanced bionic organ models with specific and tunable characteristics based on DN hydrogels is poised to significantly advance surgical training, medical device testing, and the reform of medical education.

Scalable imaging-free spatial genomics through computational reconstruction.

Tissue organization arises from the coordinated molecular programs of cells. Spatial genomics maps cells and their molecular programs within the spatial context of tissues. However, current methods measure spatial information through imaging or direct registration, which often require specialized equipment and are limited in scale. Here, we developed an imaging-free spatial transcriptomics method that uses molecular diffusion patterns to computationally reconstruct spatial data. To do so, we utilize a simple experimental protocol on two dimensional barcode arrays to establish an interaction network between barcodes via molecular diffusion. Sequencing these interactions generates a high dimensional matrix of interactions between different spatial barcodes. Then, we perform dimensionality reduction to regenerate a two-dimensional manifold, which represents the spatial locations of the barcode arrays. Surprisingly, we found that the UMAP algorithm, with minimal modifications can faithfully successfully reconstruct the arrays. We demonstrated that this method is compatible with capture array based spatial transcriptomics/genomics methods, Slide-seq and Slide-tags, with high fidelity. We systematically explore the fidelity of the reconstruction through comparisons with experimentally derived ground truth data, and demonstrate that reconstruction generates high quality spatial genomics data. We also scaled this technique to reconstruct high-resolution spatial information over areas up to 1.2 centimeters. This computational reconstruction method effectively converts spatial genomics measurements to molecular biology, enabling spatial transcriptomics with high accessibility, and scalability.

Biofabrication of engineered tissues by 3D bioprinting of tissue specific high cell-density bioinks.

Bioprinting of high cell-density bioinks is a promising technique for cellular condensation-based tissue engineering and regeneration medicine. However, it remains difficult to create precisely controlled complex structures and organization of tissues with high cell-density bioink-based bioprinting for tissue specific condensation. In this study, we present newly biofabricated tissues from directly assembled, tissue specific, high cell-density bioinks which have been three-dimensionally printed into a photocrosslinkable and biodegradable hydrogel microparticle supporting bath. Three types of tissue specific high cell-density bioinks have been prepared with individual stem cells or stem cell aggregates by incorporation of growth factor-loaded gelatin microparticles. The bioprinted tissue specific high cell-density bioinks in the photocrosslinked microgel supporting bath condense together and differentiate down tissue-specific lineages to form multi-phase tissues (e.g., osteochondral tissues). By changing the growth factors and cell types, these tissue specific high cell-density bioinks enable engineering of various functional tissues with controlled architecture and organization of cells.

Dynamics of Acute Infection with Mammarenavirus Wenzhouense in Rattus norvegicus.

While Mammarenavirus Wenzhouense (WENV) is broadly distributed across Asia, the dynamics of WENV infection remain unclear. In this study, a field-derived strain of WENV was used to inoculate Sprague Dawley (SD) rats by intramuscular injection, and the process of viral infection was observed over the course of 28 d. Viral RNA became detectable in the blood at 3 dpi and remained detectable for about 12 d. In most organ tissues, viral RNA peaked at 7 dpi, and then began to decline by 14 d, but remained detectable in intestine and brain tissues at 21 and 28 dpi. Viral shedding was detected from fecal samples for 5 d, from 6 to 11 dpi using qRT-PCR, and was recovered from feces collected at 8 dpi. Horizontal contact infection occurred among cage-mates at 14 and 21 dpi. Antibodies against the nucleocapsid were detected at 5 dpi, and then increased and persisted until the end of the experiment. These results enabled us to determine the kinetics of viremic response, viral shedding in feces, and horizontal transmission dynamics, as well as the potential sites for WENV replication and viral maintenance in nature.

Localization and Tissue Tropism of Ostreid Herpesvirus 1 in Blood Clam Anadara broughtonii.

OsHV-1 caused detrimental infections in a variety of bivalve species of major importance to aquaculture worldwide. Since 2012, there has been a notable increase in the frequency of mass mortality events of the blood clam associated with OsHV-1 infection. The pathological characteristics, tissue and cellular tropisms of OsHV-1 in A. broughtonii remain unknown. In this study, we sought to investigate the distribution of OsHV-1 in five different organs (mantle, hepatopancreas, gill, foot, and adductor muscle) of A. broughtonii by quantitative PCR, histopathology and in situ hybridization (ISH), to obtain insight into the progression of the viral infection. Our results indicated a continuous increase in viral loads with the progression of OsHV-1 infection, reaching a peak at 48 h or 72 h post-infection according to different tissues. Tissue damage and necrosis, as well as colocalized OsHV-1 ISH signals, were observed primarily in the connective tissues of various organs and gills. Additionally, minor tissue damage accompanied by relatively weak ISH signals was detected in the foot and adductor muscle, which were filled with muscle tissue. The predominant cell types labeled by ISH signals were infiltrated hemocytes, fibroblastic-like cells, and flat cells in the gill filaments. These results collectively illustrated the progressive alterations in pathological confusion and OsHV-1 distribution in A. broughtonii, which represent most of the possible responses of cells and tissues to the virus.

Thrombospondins: Conserved mediators and modulators of metazoan extracellular matrix.

This review provides a personal overview of significant scientific developments in the thrombospondin field during the course of my career. Thrombospondins are multidomain, multimeric, calcium-binding extracellular glycoproteins with context-specific roles in tissue organisation. They act at cell surfaces and within ECM to regulate cell phenotype and signalling, differentiation and assembly of collagenous ECM, along with tissue-specific roles in cartilage, angiogenesis and synaptic function. More recently, intracellular, homeostatic roles have also been identified. Resolution of structures for the major domains of mammalian thrombospondins has facilitated major advances in understanding thrombospondin biology from molecule to tissue; for example, in illuminating molecular consequences of disease-causing coding mutations in human pseudoachrondroplasia. Although principally studied in vertebrates, thrombospondins are amongst the most ancient of animal ECM proteins, with many invertebrates encoding a single thrombospondin and the thrombospondin gene family of vertebrates originating through gene duplications. Moreover, thrombospondins form one branch of a thrombospondin superfamily that debuted at the origin of metazoans. The super-family includes additional sub-groups, present only in invertebrates, that differ in N-terminal domain organisation, share the distinctive TSP C-terminal region domain architecture and, to the limited extent studied to date, apparently contribute to tissue development and organisation. Finally, major lines of translational research are discussed, related to fibrosis; TSP1, TSP2 and inhibition of angiogenesis; and the alleviation of chronic cartilage tissue pathologies in pseudoachrondroplasia.

Custom microfluidic chip design enables cost-effective three-dimensional spatiotemporal transcriptomics with a wide field of view.

Spatial transcriptomic techniques offer unprecedented insights into the molecular organization of complex tissues. However, integrating cost-effectiveness, high throughput, a wide field of view and compatibility with three-dimensional (3D) volumes has been challenging. Here we introduce microfluidics-assisted grid chips for spatial transcriptome sequencing (MAGIC-seq), a new method that combines carbodiimide chemistry, spatial combinatorial indexing and innovative microfluidics design. This technique allows sensitive and reproducible profiling of diverse tissue types, achieving an eightfold increase in throughput, minimal cost and reduced batch effects. MAGIC-seq breaks conventional microfluidics limits by enhancing barcoding efficiency and enables analysis of whole postnatal mouse sections, providing comprehensive cellular structure elucidation at near single-cell resolution, uncovering transcriptional variations and dynamic trajectories of mouse organogenesis. Our 3D transcriptomic atlas of the developing mouse brain, consisting of 93 sections, reveals the molecular and cellular landscape, serving as a valuable resource for neuroscience and developmental biology. Overall, MAGIC-seq is a high-throughput, cost-effective, large field of view and versatile method for spatial transcriptomic studies.

Exposure routes of microplastics (MPs) to humans and possible risks of MPs to human health from food and the environment: a short review

This review aims to document the exposure routes of microplastics (MPs) to humans from food and the environment and assess the possible effects of MPs and associated chemical pollutants on human health. MPs are small plastic particles that are less than five millimeters (<5 mm) in size. Humans can be exposed to MPs by ingesting contaminated water and food or inhaling contaminated air. MPs have been detected in human lungs, blood, placenta, and faeces. MPs may cause various effects on human health, including DNA damage, cellular damage, inflammation, oxidative stress, cancer, fetal growth, and inflammatory bowel disease. Both plastic additive chemicals and pollutants adsorbed onto MPs are harmful to humans. They are persistent (persists long in the environment), toxic (poisonous), and bioaccumulative (accumulate in tissues of food and human organs). These chemicals are also endocrine-disrupting (can alter functions of the endocrine system) and carcinogenic (can cause cancer). Exposure to these chemicals (additives and adsorbents) can have long-term effects on human health.

Characterization of mesothelin gene expression in dogs and overexpression in canine mesotheliomas.

Canine mesotheliomas are uncommon malignant tumors typically detected late. Minimally invasive diagnostic biomarkers would facilitate diagnosis at earlier stages, thereby improving clinical outcomes. We hypothesized that mesothelin could be used as a reliable diagnostic biomarker for canine mesotheliomas since it has been used as a cancer biomarker for human mesothelioma. We aimed to explore and characterize mesothelin gene expression in dogs and assess its use as a diagnostic biomarker for canine mesotheliomas. We quantified expressed canine mesothelin transcripts via reverse transcription polymerase chain reaction (RT-PCR) and sequenced them using ribonucleic acid (RNA) extracted from a canine mesothelioma cell line. After confirming mesothelin expression, we assessed its levels in major organ tissues and compared them with those in the mesothelioma tissues using quantitative PCR (qPCR). Mesothelin overexpression in mesotheliomas was detected, and we further compared its levels using qPCR between mesotheliomas and non-mesotheliomas using tumor tissues and clinical sample effusions, confirming its significance as a diagnostic biomarker for canine mesothelioma. Mesothelin complementary deoxyribonucleic acid (cDNA) was amplified via RT-PCR, yielding a single band of expected upon DNA electrophoresis. Sequence analyses confirmed it as a predicted canine mesothelin transcript from the genome sequence database. Comparative sequence analysis of the deduced amino acid sequence of the expressed canine mesothelin demonstrated molecular signature similarities with the human mesothelin. However, the pre-sequence of canine mesothelin lacks the mature megakaryocyte potentiating factor (MPF) portion, which is typically cleaved post-translationally with furin. Mesothelin expression was quantified via qPCR revealing low levels in the mesothelial and lung tissues, with negligible expression in the other major organs. Canine mesothelin exhibited significantly higher expression in the canine mesotheliomas than in the noncancerous tissues. Moreover, analysis of clinical samples using qPCR demonstrated markedly elevated mesothelin expression in canine mesotheliomas compared to non-mesothelioma cases. Canine mesothelin exhibits molecular and biological characteristics akin to human mesothelin. It could serve as a vital biomarker for diagnosing canine mesotheliomas, applicable to both tissue- and effusion-based samples.

Characterization of tumor heterogeneity through segmentation-free representation learning.

The interaction between tumors and their microenvironment is complex and heterogeneous. Recent developments in high-dimensional multiplexed imaging have revealed the spatial organization of tumor tissues at the molecular level. However, the discovery and thorough characterization of the tumor microenvironment (TME) remains challenging due to the scale and complexity of the images. Here, we propose a self-supervised representation learning framework, CANVAS, that enables discovery of novel types of TMEs. CANVAS is a vision transformer that directly takes high-dimensional multiplexed images and is trained using self-supervised masked image modeling. In contrast to traditional spatial analysis approaches which rely on cell segmentations, CANVAS is segmentation-free, utilizes pixel-level information, and retains local morphology and biomarker distribution information. This approach allows the model to distinguish subtle morphological differences, leading to precise separation and characterization of distinct TME signatures. We applied CANVAS to a lung tumor dataset and identified and validated a monocytic signature that is associated with poor prognosis.