Non-diseased Tissue Research Articles

A point-of-research decision in synovial tissue engineering: Mesenchymal stromal cells, tissue derived fibroblast or CTGF-mediated mesenchymal-to-fibroblast transition

Rheumatoid arthritis (RA) and osteoarthritis (OA) are prevalent inflammatory joint diseases characterized by synovitis, cartilage, and bone destruction. Fibroblast-like synoviocytes (FLSs) of the synovial membrane are a decisive factor in arthritis, making them a target for future therapies. Developing novel strategies targeting FLSs requires advanced in vitro joint models that accurately replicate non-diseased joint tissue. This study aims to identify a cell source reflecting physiological synovial fibroblasts. Therefore, we newly compared the phenotype and metabolism of “healthy” knee-derived FLSs from patients with ligament injuries (trauma-FLSs) to mesenchymal stromal cells (MSCs), their native precursors. We differentiated MSCs into fibroblasts using connective tissue growth factor (CTGF) and compared selected protein and gene expression patterns to those obtained from trauma-FLSs and OA-FLSs. Based on these findings, we explored the potential of an MSC-derived synovial tissue model to simulate a chronic inflammatory response akin to that seen in arthritis. We have identified MSCs as a suitable cell source for synovial tissue engineering because, despite metabolic differences, they closely resemble human trauma-derived FLSs. CTGF-mediated differentiation of MSCs increased HAS2 expression, essential for hyaluronan synthesis. It showed protein expression patterns akin to OA-FLSs, including markers of ECM components and fibrosis, and enzymes leading to a shift in metabolism towards increased fatty acid oxidation. In general, cytokine stimulation of MSCs in a synovial tissue model induced pro-inflammatory and pro-angiogenic gene expression, hyperproliferation, and increased glucose consumption, reflecting cellular response in human arthritis. We conclude that MSCs can serve as a proxy to study physiological synovial processes and inflammatory responses. In addition, CTGF-mediated mesenchymal-to-fibroblast transition resembles OA-FLSs. Thus, we emphasize MSCs as a valuable cell source for tools in preclinical drug screening and their application in tissue engineering.

Smooth muscle-specific deletion of Ccn2 causes severe aorta malformation and atherosclerosis.

Cellular Communication Network Factor 2 (CCN2) is a matricellular protein implicated in fibrotic diseases, with ongoing clinical trials evaluating anti-CCN2-based therapies. By uncovering CCN2 as abundantly expressed in non-diseased artery tissue, this study aimed to investigate the hypothesis that CCN2 plays a pivotal role in maintaining smooth muscle cell (SMC) phenotype and protection against atherosclerosis. Global- and SMC-specific Ccn2 knockout mouse models were employed to demonstrate that Ccn2 deficiency leads to SMC de-differentiation, medial thickening, and aorta elongation under normolipidemic conditions. Inducing hyperlipidemia in both models resulted in severe aorta malformation and a 17-fold increase in atherosclerosis formation. Lipid-rich lesions developed at sites of the vasculature typically protected from atherosclerosis-development by laminar blood flow, covering 90% of aortas, and extending to other vessels, including coronary arteries. Evaluation at earlier time points revealed medial lipid accumulation as a lesion-initiating event. Fluorescently labelled LDL injection followed by confocal microscopy showed increased LDL retention in the medial layer of Ccn2 knockout aortas, likely attributed to marked proteoglycan enrichment of the medial extracellular matrix. Analyses leveraging data from the Athero-Express study cohort indicated relevance of CCN2 in established human lesions, as CCN2 correlated with SMC marker transcripts across 654 transcriptomically profiled carotid plaques. These findings were substantiated through in situ hybridization showing CCN2 expression predominantly in the fibrous cap. This study identifies CCN2 as a major constituent of the normal artery wall, critical in regulating SMC differentiation and aorta integrity, and possessing a protective role against atherosclerosis development. These findings underscore the need for further investigation into the potential effects of anti-CCN2-based therapies on the vasculature.

Binary fabrication of decellularized lung extracellular matrix hybridgels for in vitro chronic obstructive pulmonary disease modeling

Limited treatments and a lack of appropriate animal models have spurred the study of scaffolds to mimic lung disease in vitro. Decellularized human lung and its application in extracellular matrix (ECM) hydrogels has advanced the development of these lung ECM models. Controlling the biochemical and mechanical properties of decellularized ECM hydrogels continues to be of interest due to inherent discrepancies of hydrogels when compared to their source tissue. To optimize the physiologic relevance of ECM hydrogel lung models without sacrificing the native composition we engineered a binary fabrication system to produce a Hybridgel composed of an ECM hydrogel reinforced with an ECM cryogel. Further, we compared the effect of ECM-altering disease on the properties of the gels using elastin poor Chronic Obstructive Pulmonary Disease (COPD) vs non-diseased (ND) human lung source tissue. Nanoindentation confirmed the significant loss of elasticity in hydrogels compared to that of ND human lung and further demonstrated the recovery of elastic moduli in ECM cryogels and Hybridgels. These findings were supported by similar observations in diseased tissue and gels. Successful cell encapsulation, distribution, cytotoxicity, and infiltration were observed and characterized via confocal microscopy. Cells were uniformly distributed throughout the Hybridgel and capable of survival for 7 days. Cell-laden ECM hybridgels were found to have elasticity similar to that of ND human lung. Compositional investigation into diseased and ND gels indicated the conservation of disease-specific elastin to collagen ratios. In brief, we have engineered a composited ECM hybridgel for the 3D study of cell-matrix interactions of varying lung disease states that optimizes the application of decellularized lung ECM materials to more closely mimic the human lung while conserving the compositional bioactivity of the native ECM. Statement of significanceThe lack of an appropriate disease model for the study of chronic lung diseases continues to severely inhibit the advancement of treatments and preventions of these otherwise fatal illnesses due to the inability to recapture the biocomplexity of pathologic cell-ECM interactions. Engineering biomaterials that utilize decellularized lungs offers an opportunity to deconstruct, understand, and rebuild models that highlight and investigate how disease specific characteristics of the extracellular environment are involved in driving disease progression. We have advanced this space by designing a binary fabrication system for a ECM Hybridgel that retains properties from its source material required to observe native matrix interactions. This design simulates a 3D lung environment that is both mechanically elastic and compositionally relevant when derived from non-diseased tissue and pathologically diminished both mechanically and compositionally when derived from COPD tissue. Here we describe the ECM hybridgel as a model for the study of cell-ECM interactions involved in COPD.

In vivo movement of retinoblastoma-related protein (RBR) towards cytoplasm during mitosis in Arabidopsisthaliana

Retinoblastoma protein is central in signaling networks of fundamental cell decisions such as proliferation and differentiation in all metazoans and cancer development. Immunostaining and biochemical evidence demonstrated that during interphase retinoblastoma protein is in the nucleus and is hypophosphorylated, and during mitosis is in the cytoplasm and is hyperphosphorylated. The purpose of this study was to visualize in vivo in a non-diseased tissue, the dynamic spatial and temporal nuclear exit toward the cytoplasm of this protein during mitosis and its return to the nucleus to obtain insights into its potential cytosolic functions. Using high-resolution time-lapse images from confocal microscopy, we tracked in vivo the ortholog in plants the RETINOBLASTOMA RELATED (RBR) protein tagged with Green Fluorescent Protein (GFP) in Arabidopsis thaliana's root. RBR protein exits from dense aggregates in the nucleus before chromosomes are in prophase in less than 2 min, spreading outwards as smaller particles projected throughout the cytosol during mitosis like a diffusive yet controlled event until telophase, when the daughter's nuclei form; RBR returns to the nuclei in coordination with decondensing chromosomal DNA forming new aggregates again in punctuated larger structures in each corresponding nuclei. We propose RBR diffused particles in the cytoplasm may function as a cytosolic sensor of incoming signals, thus coordinating re-aggregation with DNA is a mechanism by which any new incoming signals encountered by RBR may lead to a reconfiguration of the nuclear transcriptomic context. The small RBR diffused particles in the cytoplasm may preserve topologic-like properties allowing them to aggregate and restore their nuclear location, they may also be part of transient cytoplasmic storage of the cellular pre-mitotic transcriptional context, that once inside the nuclei may execute both the pre mitosis transcriptional context as well as new transcriptional instructions.

The spatially resolved transcriptome signatures of glomeruli in chronic kidney disease.

Here, we used digital spatial profiling (DSP) to describe the glomerular transcriptomic signatures that may characterize the complex molecular mechanisms underlying progressive kidney disease in Alport syndrome, focal segmental glomerulosclerosis, and membranous nephropathy. Our results revealed significant transcriptional heterogeneity among diseased glomeruli, and this analysis showed that histologically similar glomeruli manifested different transcriptional profiles. Using glomerular pathology scores to establish an axis of progression, we identified molecular pathways with progressively decreased expression in response to increasing pathology scores, including signal recognition particle-dependent cotranslational protein targeting to membrane and selenocysteine synthesis pathways. We also identified a distinct signature of upregulated and downregulated genes common to all the diseases investigated when compared with nondiseased tissue from nephrectomies. These analyses using DSP at the single-glomerulus level could help to increase insight into the pathophysiology of kidney disease and possibly the identification of biomarkers of disease progression in glomerulopathies.

Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity

BackgroundCancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms.ResultsIn sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms.ConclusionThis work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

Decreased inflammatory profile in oral leukoplakia tissue exposed to cold physical plasma ex vivo.

Oral leukoplakia (OL) is an unfavorable oral disease often resistant to therapy. To this end, cold physical plasma technology was explored as a novel therapeutic agent in an experimental setup. Biopsies with a diameter of 3 mm were obtained from non-diseased and OL tissues. Subsequently, cold atmospheric pressure plasma (CAP) exposure was performed ex vivo in the laboratory. After 20 h of incubation, biopsies were cryo-conserved, and tissue sections were quantified for lymphocyte infiltrates, discriminating between naïve and memory cytotoxic and T-helper cells. In addition, the secretion pattern related to inflammation was investigated in the tissue culture supernatants by quantifying 10 chemokines and cytokines. In CAP-treated OL tissue, significantly decreased overall lymphocyte numbers were observed. In addition, reduced levels were observed when discriminating for the T-cell subpopulations but did not reach statistical significance. Moreover, CAP treatment significantly reduced levels of C-X-C motif chemokine 10 (CXCL10) and granulocyte-macrophage colony-stimulating factor in the OL biopsies' supernatants. In idiopathically inflamed tissues, ex vivo CAP exposure reduced T-cells and CXCL10 as well but also led to markedly increased interleukin-1β secretion. Our findings suggest CAP to have immuno-modulatory properties, which could be of therapeutic significance in the therapy of OL. Future studies should investigate the efficacy of CAP therapy in vivo in a larger cohort.

Functional Profiling of Soft Tissue Sarcoma Using Mechanistic Models.

Soft tissue sarcoma is an umbrella term for a group of rare cancers that are difficult to treat. In addition to surgery, neoadjuvant chemotherapy has shown the potential to downstage tumors and prevent micrometastases. However, finding effective therapeutic targets remains a research challenge. Here, a previously developed computational approach called mechanistic models of signaling pathways has been employed to unravel the impact of observed changes at the gene expression level on the ultimate functional behavior of cells. In the context of such a mechanistic model, RNA-Seq counts sourced from the Recount3 resource, from The Cancer Genome Atlas (TCGA) Sarcoma project, and non-diseased sarcomagenic tissues from the Genotype-Tissue Expression (GTEx) project were utilized to investigate signal transduction activity through signaling pathways. This approach provides a precise view of the relationship between sarcoma patient survival and the signaling landscape in tumors and their environment. Despite the distinct regulatory alterations observed in each sarcoma subtype, this study identified 13 signaling circuits, or elementary sub-pathways triggering specific cell functions, present across all subtypes, belonging to eight signaling pathways, which served as predictors for patient survival. Additionally, nine signaling circuits from five signaling pathways that highlighted the modifications tumor samples underwent in comparison to normal tissues were found. These results describe the protective role of the immune system, suggesting an anti-tumorigenic effect in the tumor microenvironment, in the process of tumor cell detachment and migration, or the dysregulation of ion homeostasis. Also, the analysis of signaling circuit intermediary proteins suggests multiple strategies for therapy.

Disease Severity Impairs Generation of Intestinal Organoid Cultures From Inflammatory Bowel Disease Patients

IntroductionChronic inflammation of the intestinal epithelium is an underlying cause of the two main types of inflammatory bowel disease (IBD), ulcerative colitis (UC), and Crohn's disease (CD). Ex vivo organoids derived from the intestinal epithelium are a useful model to study IBD. Whether such cultures can be established from surgically resected diseased IBD intestinal tissues has not been fully explored. In this study, we tested our ability to establish organoids from nondiseased and diseased IBD intestinal tissues. Materials and methodsFrom 12 UC patients (n = 54 tissues) and 20 CD patients (n = 49 tissues), tissues were collected from multiple colonic regions, and for CD, the terminal ileum was also surveyed. Organoids were cultured in Matrigel domes using defined media. In primary tissues, we conducted immunohistochemical analysis for mucin 2 (MUC2) and Alcian blue staining for goblet cells. Organoids were stained for Ki67, E-cadherin, and MUC2. ResultsFor UC, we were highly successful establishing organoids from nondiseased tissue (n = 12 of 13, 92%). This success rates dropped from tissues with mild (n = 6 of 9, 67%), moderate (n = 2 of 9, 22%), or severe disease (n = 1 of 23, 4%). The rates from nondiseased CD tissues were reduced (n = 11 of 23, 48%) in comparison to such tissues from UC patients. In UC, goblet cells and MUC2 were reduced in diseased tissues and these phenotypes were retained in organoids. ConclusionsOrganoids can be readily derived from nondiseased surgically resected IBD tissues. While more work is needed to improve their derivation from diseased tissue, our study supports the use of organoids to study IBD pathophysiology.

Unraveling the Mechanism of Epichaperome Modulation by Zelavespib: Biochemical Insights on Target Occupancy and Extended Residence Time at the Site of Action.

Drugs with a long residence time at their target sites are often more efficacious in disease treatment. The mechanism, however, behind prolonged retention at the site of action is often difficult to understand for non-covalent agents. In this context, we focus on epichaperome agents, such as zelavespib and icapamespib, which maintain target binding for days despite rapid plasma clearance, minimal retention in non-diseased tissues, and rapid metabolism. They have shown significant therapeutic value in cancer and neurodegenerative diseases by disassembling epichaperomes, which are assemblies of tightly bound chaperones and other factors that serve as scaffolding platforms to pathologically rewire protein-protein interactions. To investigate their impact on epichaperomes in vivo, we conducted pharmacokinetic and target occupancy measurements for zelavespib and monitored epichaperome assemblies biochemically in a mouse model. Our findings provide evidence of the intricate mechanism through which zelavespib modulates epichaperomes in vivo. Initially, zelavespib becomes trapped when epichaperomes bound, a mechanism that results in epichaperome disassembly, with no change in the expression level of epichaperome constituents. We propose that the initial trapping stage of epichaperomes is a main contributing factor to the extended on-target residence time observed for this agent in clinical settings. Zelavespib's residence time in tumors seems to be dictated by target disassembly kinetics rather than by frank drug-target unbinding kinetics. The off-rate of zelavespib from epichaperomes is, therefore, much slower than anticipated from the recorded tumor pharmacokinetic profile or as determined in vitro using diluted systems. This research sheds light on the underlying processes that make epichaperome agents effective in the treatment of certain diseases.

Pyrroloquinoline quinone drives ATP synthesis in vitro and in vivo and provides retinal ganglion cell neuroprotection

Retinal ganglion cells are highly metabolically active requiring strictly regulated metabolism and functional mitochondria to keep ATP levels in physiological range. Imbalances in metabolism and mitochondrial mechanisms can be sufficient to induce a depletion of ATP, thus altering retinal ganglion cell viability and increasing cell susceptibility to death under stress. Altered metabolism and mitochondrial abnormalities have been demonstrated early in many optic neuropathies, including glaucoma, autosomal dominant optic atrophy, and Leber hereditary optic neuropathy. Pyrroloquinoline quinone (PQQ) is a quinone cofactor and is reported to have numerous effects on cellular and mitochondrial metabolism. However, the reported effects are highly context-dependent, indicating the need to study the mechanism of PQQ in specific systems. We investigated whether PQQ had a neuroprotective effect under different retinal ganglion cell stresses and assessed the effect of PQQ on metabolic and mitochondrial processes in cortical neuron and retinal ganglion cell specific contexts. We demonstrated that PQQ is neuroprotective in two models of retinal ganglion cell degeneration. We identified an increased ATP content in healthy retinal ganglion cell-related contexts both in in vitro and in vivo models. Although PQQ administration resulted in a moderate effect on mitochondrial biogenesis and content, a metabolic variation in non-diseased retinal ganglion cell-related tissues was identified after PQQ treatment. These results suggest the potential of PQQ as a novel neuroprotectant against retinal ganglion cell death.

Evaluating cancer cell line and patient-derived xenograft recapitulation of tumor and non-diseased tissue gene expression profiles in silico.

Preclinical models like cancer cell lines and patient-derived xenografts (PDXs) are vital for studying disease mechanisms and evaluating treatment options. It is essential that they accurately recapitulate the disease state of interest to generate results that will translate in the clinic. Prior studies have demonstrated that preclinical models do not recapitulate all biological aspects of human tissues, particularly with respect to the tissue of origin gene expression signatures. Therefore, it is critical to assess how well preclinical model gene expression profiles correlate with human cancer tissues to inform preclinical model selection and data analysis decisions. Here we evaluated how well preclinical models recapitulate human cancer and non-diseased tissue gene expression patterns in silico with respect to the full gene expression profile as well as subsetting by the most variable genes, genes significantly correlated with tumor purity, and tissue-specific genes. By using publicly available gene expression profiles across multiple sources, we evaluated cancer cell line and patient-derived xenograft recapitulation of tumor and non-diseased tissue gene expression profiles in silico. We found that using the full gene set improves correlations between preclinical model and tissue global gene expression profiles, confirmed that glioblastoma (GBM) PDX global gene expression correlation to GBM tumor global gene expression outperforms GBM cell line to GBM tumor global gene expression correlations, and demonstrated that preclinical models in our study often failed to reproduce tissue-specific expression. While including additional genes for global gene expression comparison between cell lines and tissues decreases the overall correlation, it improves the relative rank between a cell line and its tissue of origin compared to other tissues. Our findings underscore the importance of using the full gene expression set measured when comparing preclinical models and tissues and confirm that tissue-specific patterns are better preserved in GBM PDX models than in GBM cell lines. Future studies can build on these findings to determine the specific pathways and gene sets recapitulated by particular preclinical models to facilitate model selection for a given study design or goal.

The bacteriology of diabetic foot ulcers and infections and incidence of Staphylococcus aureus Small Colony Variants.

Introduction. Uninfected diabetes-related foot ulcer (DFU) progression to diabetes-related foot infection (DFI) is a prevalent complication for patients with diabetes. DFI often progresses to osteomyelitis (DFI-OM). Active (growing) Staphylococcus aureus is the most common pathogen in these infections. There is relapse in 40-60 % of cases even when the initial treatment at the DFI stage apparently clears infection.Hypothesis. S. aureus adopts the quasi-dormant Small Colony Variant (SCV) state during DFU and consequently infection, and when present in DFI cases also permits survival in non-diseased tissues as a reservoir to cause relapse.Aim. The aim of this study was to investigate the bacterial factors that facilitate persistent infections.Methodology. People with diabetes were recruited from two tertiary hospitals. Clinical and bacterial data was taken from 153 patients with diabetes (51 from a control group with no ulcer or infection) and samples taken from 102 patients with foot complications to identify bacterial species and their variant colony types, and then compare the bacterial composition in those with uninfected DFU, DFI and those with DFI-OM, of whom samples were taken both from wounds (DFI-OM/W) and bone (DFI-OM/B). Intracellular, extracellular and proximal 'healthy' bone were examined.Results. S. aureus was identified as the most prevalent pathogen in diabetes-related foot pathologies (25 % of all samples). For patients where disease progressed from DFU to DFI-OM, S. aureus was isolated as a diversity of colony types, with increasing numbers of SCVs present. Intracellular (bone) SCVs were found, and even within uninfected bone SCVs were present. Wounds of 24 % of patients with uninfected DFU contained active S. aureus. All patients with a DFI with a wound but not bone infection had previously had S. aureus isolated from an infection (including amputation), representing a relapse.Conclusion. The presence of S. aureus SCVs in recalcitrant pathologies highlights their importance in persistent infections through the colonization of reservoirs, such as bone. The survival of these cells in intracellular bone is an important clinical finding supporting in vitro data. Also, there seems to be a link between the genetics of S. aureus found in deeper infections compared to those only found in DFU.

Functional specialization of short-lived and long-lived macrophage subsets in human tonsils.

Macrophages play a central role in tissue homeostasis and host defense. However, the properties of human macrophages in non-diseased tissues remain poorly understood. Here, we characterized human tonsil macrophages and identified three subsets with distinct phenotype, transcriptome, life cycle, and function. CD36hi macrophages were related to monocytes, while CD36lo macrophages showed features of embryonic origin and CD36int macrophages had a mixed profile. scRNA-seq on non-human primate tonsils showed that monocyte recruitment did not pre-exist an immune challenge. Functionally, CD36hi macrophages were specialized for stimulating T follicular helper cells, by producing Activin A. Combining reconstruction of ligand-receptor interactions and functional assays, we identified stromal cell-derived TNF-α as an inducer of Activin A secretion. However, only CD36hi macrophages were primed for Activin A expression, via the activity of IRF1. Our results provide insight into the heterogeneity of human lymphoid organ macrophages and show that tonsil CD36hi macrophage specialization is the result of both intrinsic features and interaction with stromal cells.

M6 A mRNA methylation in human brain is disrupted in Lewy body disorders.

N6-methyladenosine modification of RNA (m6 A) regulates translational control which may influence neuronal dysfunction underlying neurodegenerative diseases. Using microscopy and a machine learning approach, we performed cellular profiling of m6 A-RNA abundance and YTHDF1/YTHDF3 m6 A reader expression within four regions of the human brain from non-affected individuals and individuals with Parkinson's disease, Dementia with Lewy bodies (DLB) or Mild Cognitive Impairment (MCI). In non-diseased tissue, we found m6 A-modified RNAs showed cell-type and sub-compartment-specific variation. YTHDF1 and YTHDF3 showed opposing expression patterns in the cerebellum, and frontal and cingulate cortices. Machine learning quantitative image analysis revealed that m6 A-modified transcripts were significantly altered in localisation and abundance in disease tissue with significant decreases in m6 A-RNAs in Parkinson's disease, and significant increases in m6 A-RNA abundance in Dementia with Lewy bodies. MCI tissue showed variability across regions but similar to DLB, in brain areas with an overall significant increase in m6 A-RNAs, modified RNAs within dendritic processes were reduced. Using mass spectrometry proteomic datasets to corroborate our findings, we found significant changes in YTHDF3 and m6 A anti-reader protein abundance in Alzheimer's disease (AD) and asymptomatic AD/MCI tissue and correlation with cognitive resilience. These results provide evidence for disrupted m6 A regulation in Lewy body diseases and a plausible mechanism through which RNA processing could contribute to the formation of Lewy bodies and other dementia-associated pathological aggregates. The findings suggest that manipulation of epitranscriptomic processes influencing translational control may lead to new therapeutic approaches for neurodegenerative diseases.

A Comprehensive Landscape of Imaging Feature-Associated RNA Expression Profiles in Human Breast Tissue.

The expression abundance of transcripts in nondiseased breast tissue varies among individuals. The association study of genotypes and imaging phenotypes may help us to understand this individual variation. Since existing reports mainly focus on tumors or lesion areas, the heterogeneity of pathological image features and their correlations with RNA expression profiles for nondiseased tissue are not clear. The aim of this study is to discover the association between the nucleus features and the transcriptome-wide RNAs. We analyzed both microscopic histology images and RNA-sequencing data of 456 breast tissues from the Genotype-Tissue Expression (GTEx) project and constructed an automatic computational framework. We classified all samples into four clusters based on their nucleus morphological features and discovered feature-specific gene sets. The biological pathway analysis was performed on each gene set. The proposed framework evaluates the morphological characteristics of the cell nucleus quantitatively and identifies the associated genes. We found image features that capture population variation in breast tissue associated with RNA expressions, suggesting that the variation in expression pattern affects population variation in the morphological traits of breast tissue. This study provides a comprehensive transcriptome-wide view of imaging-feature-specific RNA expression for healthy breast tissue. Such a framework could also be used for understanding the connection between RNA expression and morphology in other tissues and organs. Pathway analysis indicated that the gene sets we identified were involved in specific biological processes, such as immune processes.

Abstract C010: Genes with African ancestry-specific expression in TNBC tumors also show population-specific baseline differences in normal mammary tissue

Abstract Our recent efforts to define ancestry-specific drivers in triple negative breast tumors (TNBC) has been sharply focused on identifying African (AFR) ancestry-associated gene signatures, which we have reported to be distinct from traditional approaches using SRR to categorize patients. In our AFR ancestry enriched TNBC cohort, we have identified a 613 gene signature associated with AFR ancestry, which is enriched in previously implicated canonical pathways, such as RNA post-transcriptional modification and chronic inflammation. We also reported a significant enrichment in disease/function terms associated with immune cell trafficking, where increasing African ancestry was associated with predicted activation of immune cell migration, and inhibition of immune cell activation. While these associations were determined in the TNBC tumor environment, involvement of this 613 AFR ancestry-associated gene signature has yet to be explored in the context of non-diseased breast/mammary tissue. At present, we obtained gene expression (TPM) and ancestry data from the GTEx cohort. Of 459 patients with gene expression data available for breast/mammary tissue, 47 of those were found to be of African ancestry, and 20 identified as female, and were used in subsequent analyses. We found that 17 of the 613 AFR ancestry-associated genes from TNBC tumors were also associated with AFR ancestry in non-diseased breast/mammary tissue. Interestingly we observed that some genes had differencing directionality/correlation between non-diseased and tumor tissue. Of the 17 genes, 7 were positively associated with AFR ancestry in both cohorts, including: AGMAT, CYBA, HEATR3, KCNAB2, PTGES, RAB42, and SHPK. Eight genes were found to be positively associated with AFR in normal, but negatively associated in TNBC (AC068279.3, AP001434.2, C12orf60, FNDC3B, HNRNPA1P50, RP11-214O14.1, SNORA53 and SPATA20P1). The remaining 2 were negatively associated with AFR in normal, and positively associated in tumor (AVPR2 and FAM26D). Tumor agnostic survival analysis using TCGA BRCA cohort data shows some race-specific differences in overall survival of these genes. Specifically, higher expression of AVPR2 and CYBA, and lower expression of AGMAT and SNORA53 show benefit among AA patients, but not EA patients (ns). Pathway analysis of the 17 genes shows significant enrichment with growth of mammary tumor (p = 7.3 E-07) and inflammatory response (p = 2.5 E-07) disease and function terms, specifically related to predicted activation of IFNG, AKT1, and FOS. Ancestry-specific differences in gene expression that are present in non-malignant tissue may implicate baseline differences that could play a significant role in later tumor etiology. Interestingly, 10 of 17 genes showed differing association with African ancestry, potentially revealing malignancy-induced changes to these pathways. Further analyses and curation of robust and diverse datasets of non-diseased tissue is needed to fully contextualize these findings. Citation Format: Rachel Martini, Nicolas Robine, Ishmael Kyei, Ernest Adjei, Mahteme Bekele, Olivier Elemento, Lisa Newman, Melissa Davis. Genes with African ancestry-specific expression in TNBC tumors also show population-specific baseline differences in normal mammary tissue [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C010.

Early tissue growth and cell fate determination following segmental esophageal repair using a tissue engineered esophageal implant composed of a polyurethane scaffold seeded with autologous adipose-derived mesenchymal stromal cells

IntroductionEnd-stage or chronic esophageal disease may eventually lead to surgical intervention and could potentially result in an esophagectomy, followed by a gastric pull-up or colon interposition procedure. Biostage's Cellspan™ Esophageal Implant (CEI) is designed to repair and replace full-circumferential esophageal surgical resections (≤6 cm) using autologous adipose derived mesenchymal stromal cells (Ad-MSCs) seeded on a retrievable polyurethane scaffold. The use of a segmental implant has the advantage of preserving the native non-diseased esophageal tissue as well as the stomach or intestinal tissue following esophagectomy. The mechanism of action, the fate of the Ad-MSC component (biodistribution) and the process of early tissue regrowth/wound repair following implantation remains to be elucidated. MethodsCEIs seeded with Ad-MSCs transduced with green fluorescent protein (GFP) were implanted into a pig model of esophageal segmental resection. A 5 cm full-circumferential esophageal resection was performed followed by CEI implantation using an end-to-end anastomoses to bridge the gap between the 2 native esophageal ends. Cell fate and tissue development were assessed at 14 (N = 3), 21 (N = 3), and 28 days (N = 3), post-CEI implantation. ResultsAll animals in all groups exhibited a contiguous biologic esophageal conduit with a denuded patent lumen at necropsy. Epithelial cell proliferation/regrowth was evident from both anastomotic margins toward the implant center. Morphometric analysis indicated an increase in epithelial regrowth and a concomitant reduction in denuded tissue from day 14 to day 28. Histological evaluation revealed fibrovascular tissue and neovascularization on the adventitial side, with no discernible differences in tissue organization between 14 and 28 day implants. The majority of GFP + cells were on the abluminal esophageal surface and localized around vascular structures. No GFP + cells were detected in lymph nodes or on retrieved scaffolds. ConclusionThese findings support luminal continuity by day 14 post-implantation with Ad-MSC derived pericytes contributing to the neo-fibrovascular tissue. Morphometric analysis of the lumenal surface indicates that the process of lumenal re-epithelialization results from epithelial cell proliferation from the implant margins towards the center of the implant.

Substrate stiffness engineered to replicate disease conditions influence senescence and fibrotic responses in primary lung fibroblasts.

In fibrosis remodelling of ECM leads to changes in composition and stiffness. Such changes can have a major impact on cell functions including proliferation, secretory profile and differentiation. Several studies have reported that fibrosis is characterised by increased senescence and accumulating evidence suggests that changes to the ECM including altered composition and increased stiffness may contribute to premature cellular senescence. This study investigated if increased stiffness could modulate markers of senescence and/or fibrosis in primary human lung fibroblasts. Using hydrogels representing stiffnesses that fall within healthy and fibrotic ranges, we cultured primary fibroblasts from non-diseased lung tissue on top of these hydrogels for up to 7days before assessing senescence and fibrosis markers. Fibroblasts cultured on stiffer (±15kPa) hydrogels showed higher Yes-associated protein-1 (YAP) nuclear translocation compared to soft hydrogels. When looking at senescence-associated proteins we also found higher secretion of receptor activator of nuclear factor kappa-B ligand (RANKL) but no change in transforming growth factor-β1 (TGF-β1) or connective tissue growth factor (CTGF) expression and higher decorin protein deposition on stiffer matrices. With respect to genes associated with fibrosis, fibroblasts on stiffer hydrogels compared to soft had higher expression of smooth muscle alpha (α)-2 actin (ACTA2), collagen (COL) 1A1 and fibulin-1 (Fbln1) and higher Fbln1 protein deposition after 7days. Our results show that exposure of lung fibroblasts to fibrotic stiffness activates genes and secreted factors that are part of fibrotic responses and part of the Senescence-associated secretory phenotype (SASP). This overlap may contribute to the creation of a feedback loop whereby fibroblasts create a perpetuating cycle reinforcing progression of a fibrotic response.

Widespread expression of the ancient HERV-K (HML-2) provirus group in normal human tissues.

Human endogenous retrovirus (HERV) transcripts are known to be highly expressed in cancers, yet their activity in nondiseased tissue is largely unknown. Using the GTEx RNA-seq dataset from normal tissue sampled at autopsy, we characterized individual expression of the recent HERV-K (HML-2) provirus group across 13,000 different samples of 54 different tissues from 948 individuals. HML-2 transcripts could be identified in every tissue sampled and were elevated in the cerebellum, pituitary, testis, and thyroid. A total of 37 different individual proviruses were expressed in 1 or more tissues, representing all 3 LTR5 subgroups. Nine proviruses were identified as having long terminal repeat (LTR)-driven transcription, 7 of which belonged to the most recent LTR5HS subgroup. Proviruses of different subgroups displayed a bias in tissue expression, which may be associated with differences in transcription factor binding sites in their LTRs. Provirus expression was greater in evolutionarily older proviruses with an earliest shared ancestor of gorilla or older. HML-2 expression was significantly affected by biological sex in 1 tissue, while age and timing of death (Hardy score) had little effect. Proviruses containing intact gag, pro, and env open reading frames (ORFs) were expressed in the dataset, with almost every tissue measured potentially expressing at least 1 intact ORF (gag).