Heterogeneous Cell Populations Research Articles

Visualizing scRNA-Seq Data at Population Scale with GloScope.

Increasingly, scRNA-Seq studies explore cell populations across different samples and the effect of sample heterogeneity on organism's phenotype. However, relatively few bioinformatic methods have been developed which adequately address the variation between samples for such population-level analyses. We propose a framework for representing the entire single-cell profile of a sample, which we call a GloScope representation. We implement GloScope on scRNA-Seq datasets from study designs ranging from 12 to over 300 samples and demonstrate how GloScope allows researchers to perform essential bioinformatic tasks at the sample-level, in particular visualization and quality control assessment.

Case report: segmental form of Hailey-Hailey disease

The article presents a clinical case of a rare segmental form of familial benign chronic pemphigus with a positive family history. Patient S., born in 2014, complained of unilateral eruptions in the area of the left retroauricular fold, left axilla, inguinal fold and labia majora on the left, plantar surface of the left foot. Eruptions were represented by erythematous foci, single blisters with serous contents, erosions, crusts. For the entire period, the patient was observed with the following diagnoses: microbial eczema, Sneddon-Wilkinson Disease, Inflammatory Linear Verrucose Epidermal Nevus. Due to the long-term relapsing course and ineffective therapy, additional examination methods were carried out – histological and genetic studies. Only after genetic testing, in which a heterozygous mutation of the ATP2C1 gene was revealed, and geneticist’s consultation, the final diagnosis was established – Hailey-Hailey Disease.It is known, that the segmental form is formed as a result of mosaicism, which can be of two types. The term "mosaicism" refers to the presence in an organism of two or more genetically heterogeneous cell populations formed from a genetically homogeneous zygote. The formation of type 1 mosaicism is based on a de novo postzygotic mutation at an early stage of embryogenesis. In turn, type 2 mosaicism develops as a result of a postzygotic mutation against the background of an already existing prezygotic mutation. Determining the type of mosaicism by genetic testing will make it possible to assess the prognosis of the disease and the chance of transmitting this disease to offspring.

Single-nucleus and spatial transcriptome reveal adrenal homeostasis in normal and tumoural adrenal glands.

The human adrenal gland is a complex endocrine tissue. Studies on adrenal renewal have been limited to animal models or human foetuses. Enhancing our understanding of adult human adrenal homeostasis is crucial for gaining insights into the pathogenesis of adrenal diseases, such as adrenocortical tumours. Here, we present a comprehensive cellular genomics analysis of the adult human normal adrenal gland, combining single-nuclei RNA sequencing and spatial transcriptome data to reconstruct adrenal gland homeostasis. As expected, we identified primary cells of the various zones of the adrenal cortex and medulla, but we also uncovered additional cell types. They constitute the adrenal microenvironment, including immune cells, mostly composed of a large population of M2 macrophages, and new cell populations, including different subpopulations of vascular-endothelial cells and cortical-neuroendocrine cells. Utilizing spatial transcriptome and pseudotime trajectory analysis, we support evidence of the centripetal dynamics of adrenocortical cell maintenance and the essential role played by Wnt/β-catenin, sonic hedgehog, and fibroblast growth factor pathways in the adult adrenocortical homeostasis. Furthermore, we compared single-nuclei transcriptional profiles obtained from six healthy adrenal glands and twelve adrenocortical adenomas. This analysis unveiled a notable heterogeneity in cell populations within the adenoma samples. In addition, we identified six distinct adenoma-specific clusters, each with varying distributions based on steroid profiles and tumour mutational status. Overall, our results provide novel insights into adrenal homeostasis and molecular mechanisms potentially underlying early adrenocortical tumorigenesis and/or autonomous steroid secretion. Our cell atlas represents a powerful resource to investigate other adrenal-related pathologies.

Timing and location dictate monocyte fate and their transition to tumor-associated macrophages.

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells whose phenotypes and functions are shaped by factors that are incompletely understood. Herein, we asked when and where TAMs arise from blood monocytes and how they evolve during tumor development. We initiated pancreatic ductal adenocarcinoma (PDAC) in inducible monocyte fate-mapping mice and combined single-cell transcriptomics and high-dimensional flow cytometry to profile the monocyte-to-TAM transition. We revealed that monocytes differentiate first into a transient intermediate population of TAMs that generates two longer-lived lineages of terminally differentiated TAMs with distinct gene expression profiles, phenotypes, and intratumoral localization. Transcriptome datasets and tumor samples from patients with PDAC evidenced parallel TAM populations in humans and their prognostic associations. These insights will support the design of new therapeutic strategies targeting TAMs in PDAC.

Deciphering cellular heterogeneity in Spodoptera frugiperda midgut cell line through single cell RNA sequencing

Using the 10x Genomics Chromium single-cell RNA sequencing (scRNA-seq) platform, we discovered unexpected heterogeneity in an established cell line developed from the midgut of the Fall armyworm, Spodoptera frugiperda, a major global pest. We analyzed the sequences of 18,794 cells and identified ten unique cellular clusters, including stem cells, enteroblasts, enterocytes and enteroendocrine cells, characterized by the expression of specific marker genes. Additionally, these studies addressed an important knowledge gap by investigating the expression of genes coding for respiratory and midgut membrane insecticide targets classified by the Insecticide Resistance Action Committee. Dual-fluorescence tagging method, fluorescence microscopy and fluorescence-activated cell sorting confirmed the expression of midgut cell type-specific genes. Stem cells were isolated from the heterogeneous population of SfMG-0617 cells. Our results, validated by KEGG and Gene Ontology analyses and supported by Monocle 3.0, advance the fields of midgut cellular biology and establish standards for scRNA-seq studies in non-model organisms.

Creation of a spatially complex mucus bilayer on an in vitro colon model

The colonic epithelium is comprised of three-dimensional crypts (3D) lined with mucus secreted by a heterogeneous population of goblet cells. In this study, we report the formation of a long-lived, and self-renewing replica of human 3D crypts with a mucus layer patterned in the X–Y–Z dimensions. Primary colon cells were cultured on a shaped scaffold under an air–liquid interface to yield architecturally accurate crypts with a mucus bilayer (605 ± 180 μm thick) possessing an inner (149 ± 50 μm) and outer (435 ± 111 μm) region. Lectins with distinct carbohydrate-binding preferences demonstrated that the mucus in the intercrypt regions was chemically distinct from that above and within the crypts replicating in vivo chemical patterning. Constitutive mucus secretion ejected beads from crypt lumens in 8–10 days, while agonist-stimulated secretion increased mucus thickness by 17-fold in 8 h. The tissue was long-lived, > 50 days, the longest time assessed. In conclusion, the in vitro mucus replicated key physiology of the human mucus, including the bilayer (Z) structure and intercrypt-crypt (X–Y) zones, constitutive mucus flow, spatially complex chemical attributes, and mucus secretion response to stimulation, with the potential to reveal local and global determinants of mucus function and its breakdown in disease.

The Role of Gamma Delta T Lymphocytes in Physiological and Pathological Condition-Focus on Psoriasis, Atopic Dermatitis, Autoimmune Disorders, Cancer and Lymphomas.

Gamma delta (γδ) T cells are a heterogeneous population of cells that play roles in inflammation, host tissue repair, clearance of viral and bacterial pathogens, regulation of immune processes, and tumor surveillance. Recent research suggests that these are the main skin cells that produce interleukin-17 (I-17). Furthermore, γδ T cells exhibit memory-cell-like characteristics that mediate repeated episodes of psoriatic inflammation. γδ T cells are found in epithelial tissues, where many cancers develop. There, they participate in antitumor immunity as cytotoxic cells or as immune coordinators. γδ T cells also participate in host defense, immune surveillance, and immune homeostasis. The aim of this review is to present the importance of γδ T cells in physiological and pathological diseases, such as psoriasis, atopic dermatitis, autoimmune diseases, cancer, and lymphoma.

Integrative analysis of bulk and single-cell RNA sequencing reveals the gene expression profile and the critical signaling pathways of type II CPAM

BackgroudType II congenital pulmonary airway malformation (CPAM) is a rare pulmonary microcystic developmental malformation. Surgical excision is the primary treatment for CPAM, although maternal steroids and betamethasone have proven effective in reducing microcystic CPAM. Disturbed intercellular communication may contribute to the development of CPAM. This study aims to investigate the expression profile and analyze intercellular communication networks to identify genes potentially associated with type II CPAM pathogenesis and therapeutic targets.MethodsRNA sequencing (RNA-seq) was performed on samples extracted from both the cystic area and the adjacent normal tissue post-surgery in CPAM patients. Iterative weighted gene correlation network analysis (iWGCNA) was used to identify genes specifically expressed in type II CPAM. Single-cell RNA-seq (scRNA-seq) was integrated to unveil the heterogeneity in cell populations and analyze the communication and interaction within epithelial cell sub-populations.ResultsA total of 2,618 differentially expressed genes were identified, primarily enriched in cilium-related biological process and inflammatory response process. Key genes such as EDN1, GPR17, FPR2, and CHRM1, involved in the G protein-coupled receptor (GPCR) signaling pathway and playing roles in cell differentiation, apoptosis, calcium homeostasis, and the immune response, were highlighted based on the protein-protein interaction network. Type II CPAM-associated modules, including ciliary function-related genes, were identified using iWGCNA. By integrating scRNA-seq data, AGR3 (related to calcium homeostasis) and SLC11A1 (immune related) were identified as the only two differently expressed genes in epithelial cells of CPAM. Cell communication analysis revealed that alveolar type 1 (AT1) and alveolar type 2 (AT2) cells were the predominant communication cells for outgoing and incoming signals in epithelial cells. The ligands and receptors between epithelial cell subtypes included COLLAGEN genes enriched in PI3K-AKT singaling and involved in epithelial to mesenchymal transition.ConclusionsIn summary, by integrating bulk RNA-seq data of type II CPAM with scRNA-seq data, the gene expression profile and critical signaling pathways such as GPCR signaling and PI3K-AKT signaling pathways were revealed. Abnormally expressed genes in these pathways may disrupt epithelial-mesenchymal transition and contribute to the development of CPAM. Given the effectiveness of prenatal treatments of microcystic CPAM using maternal steroids and maternal betamethasone administration, targeting the genes and signaling pathways involved in the development of CPAM presents a promising therapeutic strategy.

Stimulation-induced cytokine polyfunctionality as a dynamic concept

Cytokine polyfunctionality is a well-established concept in immune cells, especially T cells, and their ability to concurrently produce multiple cytokines has been associated with better immunological disease control and subsequent effectiveness during infection and disease. To date, only little is known about the secretion dynamics of those cells, masked by the widespread deployment of mainly time-integrated endpoint measurement techniques that do not easily differentiate between concurrent and sequential secretion. Here, we employed a single-cell microfluidic platform capable of resolving the secretion dynamics of individual PBMCs. To study the dynamics of poly-cytokine secretion, as well as the dynamics of concurrent and sequential polyfunctionality, we analyzed the response at different time points after ex vivo activation. First, we observed the simultaneous secretion of cytokines over the measurement time for most stimulants in a subpopulation of cells only. Second, polyfunctionality generally decreased with prolonged stimulation times and revealed no correlation with the concentration of secreted cytokines in response to stimulation. However, we observed a general trend towards higher cytokine secretion in polyfunctional cells, with their secretion dynamics being distinctly different from mono-cytokine-secreting cells. This study provided insights into the distinct secretion behavior of heterogenous cell populations after stimulation with well-described agents and such a system could provide a better understanding of various immune dynamics in therapy and disease.

Stimulation-induced cytokine polyfunctionality as a dynamic concept.

Cytokine polyfunctionality is a well-established concept in immune cells, especially T cells, and their ability to concurrently produce multiple cytokines has been associated with better immunological disease control and subsequent effectiveness during infection and disease. To date, only little is known about the secretion dynamics of those cells, masked by the widespread deployment of mainly time-integrated endpoint measurement techniques that do not easily differentiate between concurrent and sequential secretion. Here, we employed a single-cell microfluidic platform capable of resolving the secretion dynamics of individual PBMCs. To study the dynamics of poly-cytokine secretion, as well as the dynamics of concurrent and sequential polyfunctionality, we analyzed the response at different time points after ex vivo activation. First, we observed the simultaneous secretion of cytokines over the measurement time for most stimulants in a subpopulation of cells only. Second, polyfunctionality generally decreased with prolonged stimulation times and revealed no correlation with the concentration of secreted cytokines in response to stimulation. However, we observed a general trend towards higher cytokine secretion in polyfunctional cells, with their secretion dynamics being distinctly different from mono-cytokine-secreting cells. This study provided insights into the distinct secretion behavior of heterogenous cell populations after stimulation with well-described agents and such a system could provide a better understanding of various immune dynamics in therapy and disease.

Single-cell mass cytometry in immunological skin diseases.

Immune-related skin diseases represent a collective of dermatological disorders intricately linked to dysfunctional immune system processes. These conditions are primarily characterized by an immoderate activation of the immune system or deviant immune responses, involving diverse immune components including immune cells, antibodies, and inflammatory mediators. However, the precise molecular dysregulation underlying numerous individual cases of these diseases and unique subsets respond under disease conditions remains elusive. Comprehending the mechanisms and determinants governing the homeostasis and functionality of diseases could offer potential therapeutic opportunities for intervention. Mass cytometry enables precise and high-throughput quantitative measurement of proteins within individual cells by utilizing antibodies labeled with rare heavy metal isotopes. Imaging mass cytometry employs mass spectrometry to obtain spatial information on cell-to-cell interactions within tissue sections, simultaneously utilizing more than 40 markers. The application of single-cell mass cytometry presents a unique opportunity to conduct highly multiplexed analysis at the single-cell level, thereby revolutionizing our understanding of cell population heterogeneity and hierarchy, cellular states, multiplexed signaling pathways, proteolysis products, and mRNA transcripts specifically in the context of many autoimmune diseases. This information holds the potential to offer novel approaches for the diagnosis, prognostic assessment, and monitoring responses to treatment, thereby enriching our strategies in managing the respective conditions. This review summarizes the present-day utilization of single-cell mass cytometry in studying immune-related skin diseases, highlighting its advantages and limitations. This technique will become increasingly prevalent in conducting extensive investigations into these disorders, ultimately yielding significant contributions to their accurate diagnosis and efficacious therapeutic interventions.

Breast Cancer Stem Cells and Tumor Heterogeneity: Characteristics and Therapeutic Strategies.

Breast cancer is one of the most frequently detected malignancies worldwide. It is responsible for more than 15% of all death cases caused by cancer in women. Breast cancer is a heterogeneous disease representing various histological types, molecular characteristics, and clinical profiles. However, all breast cancers are organized in a hierarchy of heterogeneous cell populations, with a small proportion of cancer stem cells (breast cancer stem cells (BCSCs)) playing a putative role in cancer progression, and they are responsible for therapeutic failure. In different molecular subtypes of breast cancer, they present different characteristics, with specific marker profiles, prognoses, and treatments. Recent efforts have focused on tackling the Wnt, Notch, Hedgehog, PI3K/Akt/mTOR, and HER2 signaling pathways. Developing diagnostics and therapeutic strategies enables more efficient elimination of the tumor mass together with the stem cell population. Thus, the knowledge about appropriate therapeutic methods targeting both "normal" breast cancer cells and breast cancer stem cell subpopulations is crucial for success in cancer elimination.

GSK3 regulation Wnt/β-catenin signaling affects adipogenesis in bovine skeletal muscle fibro/adipogenic progenitors

Clarifying the cellular origin and regulatory mechanisms of intramuscular fat (IMF) deposition is crucial for improving beef quality. Here, we used single-nucleus RNA sequencing to analyze the structure and heterogeneity of skeletal muscle cell populations in different developmental stages of Yanbian cattle and identified eight cell types in two developmental stages of calves and adults. Among them, fibro/adipogenic progenitors (FAPs) expressing CD29 (ITGA7)pos and CD56 (NCAM1)neg surface markers were committed to IMF deposition in beef cattle and expressed major Wnt ligands and receptors. LY2090314/XAV-939 was used to activate/inhibit Wnt/β-catenin signal. The results showed that the blockade of Glycogen Synthase Kinase 3 (GSK3) by LY2090314 promoted the stabilization of β-catenin and reduced the expression of genes related adipogenic differentiation (e.g., PPARγ and C/EBPα) in bovine FAPs, confirming the anti-adipogenic effect of GSK3. XAV-939 inhibition of the Wnt/β-catenin pathway promoted the lipid accumulation capacity of FAPs. Furthermore, we found that blocking GSK3 enhanced the paracrine effects of FAPs-MuSCs and increased myotube formation in muscle satellite cells (MuSCs). Overall, our results outline a single-cell atlas of skeletal muscle development in Yanbian cattle, revealed the role of Wnt/GSK3/β-catenin signaling in FAPs adipogenesis, and provide a theoretical basis for further regulation of bovine IMF deposition.

Dual roles of myeloid-derived suppressor cells in various diseases: a review.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that originate from bone marrow stem cells. In pathological conditions, such as autoimmune disorders, allergies, infections, and cancer, normal myelopoiesis is altered to facilitate the formation of MDSCs. MDSCs were first shown to promote cancer initiation and progression by immunosuppression with the assistance of various chemokines and cytokines. Recently, various studies have demonstrated that MDSCs play two distinct roles depending on the physiological and pathological conditions. MDSCs have protective roles in autoimmune disorders (such as uveoretinitis, multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, type 1 diabetes, autoimmune hepatitis, inflammatory bowel disease, alopecia areata, and systemic lupus erythematosus), allergies, and organ transplantation. However, they play negative roles in infections and various cancers. Several immunosuppressive functions and mechanisms of MDSCs have been determined in different disease conditions. This review comprehensively discusses the associations between MDSCs and various pathological conditions and briefly describes therapeutic approaches.

Fast and efficient isolation of murine circulating tumor cells using screencell technology for pre-clinical analyzes

Circulating tumor cells (CTCs) represent a rare and heterogeneous population of cancer cells that are detached from the tumor site and entered blood or lymphatic circulation. Once disseminated in distant tissues, CTCs could remain dormant or create a tumor mass causing serious danger for patients. Many technologies exist to isolate CTCs from patients’ blood samples, mostly based on microfluidic systems or by sorting them according to their surface antigens, notably EpCAM, and/or cytokeratins for carcinoma. ScreenCell has developed an easy-to-use, antigen-independent, rapid, cost-effective, and efficient technology that isolates CTCs according to their bigger size compared to the blood cells. This study provides the technical information necessary to isolate and characterize CTCs from mouse blood. By using blood samples from transgenic mice with breast cancer or from WT mice in which we spiked cancer cells, we showed that ScreenCell technology is compatible with standard EDTA blood collection tubes. Furthermore, the ScreenCell Cyto kit could treat up to 500 µl and the ScreenCell MB kit up to 200 µl of mouse blood. As the ScreenCell MB kit captures unaltered live CTCs, we have shown that their DNA could be efficiently extracted, and the isolated cells could be grown in culture. In conclusion, ScreenCell provides a rapid, easy, antigen-independent, cost-effective, and efficient technology to isolate and characterize CTCs from the blood samples of cancer patients and murine models. Thanks to this technology CTCs could be captured fixed or alive. Murine cancer models are extensively used in pre-clinical studies. Therefore, this study demonstrates the crucial technical points necessary while manipulating mouse blood samples using ScreenCell technology.

Pilot Study for Isolation of Stromal Vascular Fraction with Collagenase Using an Automated Processing System.

There are many potential therapeutic applications for autologous adipose-derived stromal cells. These cells are found in a heterogeneous population isolated from adipose tissue called the stromal vascular fraction (SVF). Closed automated systems are available to release cells from the adherent stroma. Here, we test one system to evaluate the heterogeneous output for yield, purity, cellular characterization, and stemness criteria. The SVF was isolated from three donors using the Automated Cell Station (ACS) from BSL Co., Ltd., Busan, Republic of Korea. The SVF cellular output was characterized for cell yield and viability, immunophenotyping analysis, pluripotent differentiation potential, adhesion to plastic, and colony-forming units. Additionally, the SVF was tested for endotoxin and collagenase residuals. The SVF yield from the ACS system was an average volume of 7.9 ± 0.5 mL containing an average of 19 × 106 nucleated cells with 85 ± 12% viability. Flow cytometry identified a variety of cells, including ASCs (23%), macrophages (24%), endothelial cells (5%), pericytes (4%), and transitional cells (0.5%). The final concentrated product contained cells capable of differentiating into adipogenic, chondrogenic, and osteogenic phenotypes. Furthermore, tests for SVF sterility and purity showed no evidence of endotoxin or collagenase residuals. The ACS system can efficiently process cells from adipose tissue within the timeframe of a single surgical procedure. The cellular characterization indicated that this system can yield a sterile and concentrated SVF output, providing a valuable source of ASCs within the heterogeneous cell population.

Assessment and process optimization of high throughput biofabrication of immunocompetent breast cancer model for drug screening applications

Recent advancements in 3D cancer modeling have significantly enhanced our ability to delve into the intricacies of carcinogenesis. Despite the pharmaceutical industry’s substantial investment of both capital and time in the drug screening and development pipeline, a concerning trend persists: drug candidates screened on conventional cancer models exhibit a dismal success rate in clinical trials. One pivotal factor contributing to this discrepancy is the absence of drug testing on pathophysiologically biomimetic 3D cancer models during pre-clinical stages. Unfortunately, current manual methods of 3D cancer modeling, such as spheroids and organoids, suffer from limitations in reproducibility and scalability. In our study, we have meticulously developed 3D bioprinted breast cancer model utilizing decellularized adipose tissue-based hydrogel obtained via a detergent-free decellularization method. Our innovative printing techniques allows for rapid, high-throughput fabrication of 3D cancer models in a 96-well plate format, demonstrating unmatched scalability and reproducibility. Moreover, we have conducted extensive validation, showcasing the efficacy of our platform through drug screening assays involving two potent anti-cancer drugs, 5-Fluorouracil and PRIMA-1Met. Notably, our platform facilitates effortless imaging and gene expression analysis, streamlining the evaluation process. In a bid to enhance the relevance of our cancer model, we have introduced a heterogeneous cell population into the DAT-based bioink. Through meticulous optimization and characterization, we have successfully developed a biomimetic immunocompetent breast cancer model, complete with microenvironmental cues and diverse cell populations. This breakthrough paves the way for rapid multiplex drug screening and the development of personalized cancer models, marking a paradigm shift in cancer research and pharmaceutical development.

Type VI Collagen Deficiency Causes Enhanced Periodontal Tissue Destruction

The periodontal ligament (PDL) is a fibrillar connective tissue that lies between the alveolar bone and the tooth and is composed of highly specialized extracellular matrix (ECM) molecules and a heterogeneous population of cells that are responsible for collagen formation, immune response, bone formation, and chewing force sensation. Type VI collagen (COL6), a widely distributed ECM molecule, plays a critical role in the structural integrity and mechanical properties of various tissues including muscle, tendon, bone, cartilage, and skin. However, its role in the PDL remains largely unknown. Our study shows that deficiency of COL6 impairs PDL fibrillogenesis and exacerbates tissue destruction in ligature-induced periodontitis (LIP). We found that COL6-deficient mice exhibited increased bone loss and degraded PDL in LIP and that fibroblasts expressing high levels of Col6α2 are pivotal in ECM organization and cell-ECM interactions. Moreover, COL6 deficiency in the PDL led to an increased number of fibroblasts geared toward the inflammatory response. We also observed that cultured COL6-deficient fibroblasts from the PDL exhibited decreased expression of genes related to collagen fiber turnover and ECM organization as well as migration and proliferation. Our findings suggest that COL6 plays a crucial role in the PDL, influencing fibroblast function in fibrillogenesis and affecting the immune response in periodontitis. These insights advance our understanding of the molecular mechanisms underlying PDL maturation and periodontal disease.

Understanding mechanisms of JAK1 inhibition on synovial fibroblasts using combinatorial approaches of bulk and single cell RNAseq analyses.

The aim of these studies was to characterise the molecular effects of a tool JAK1 inhibitor on cultured primary fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) through both total and individual cell analysis. RA-FLS cultures from 6 (Bulk RNA-seq) or 4 (ScRNA-seq) donors were pre-treated with various concentrations (100 nM and 1μM) of ABT-317 with/without exposure to 25% SEB-conditioned PBMC medium to mimic the RA inflammatory milieu. Cells were subjected to both bulk RNA-seq (36 libraries) and single cell RNA-seq (scRNA-seq; 24 libraries) to identify biological processes impacted by CM and ABT-317 treatments. In our bulk RNA-seq analysis, a total of 2,605 differentially expressed genes (DEGs) were identified between CM-stimulation and unstimulated groups, while 1,122 DEGs were found between ABT-317 1μM and DMSO in CM-stimulated groups using thresholds of log2 (fold change) ≥ |0.58| and FDR ≤ 10%. Both bulk and single cell mRNA analysis of RA-FLS treated with a combination of CM and ABT-317 demonstrated the expected changes in inflammatory pathways such as interferon and IL-6 signalling. However, other non-inflammation associated pathways were also altered by ABT-317. In addition, the single cell analysis highlighted that FLS segregate into distinctive clusters upon combination CM and ABT-317 treatment, suggesting JAK inhibition can drive RA-FLS into multiple heterogenous cell populations. Interestingly, one of the unique RA-FLS clusters that emerged from the CM and ABT-317 treatment showed matrix metalloproteinase-3 (MMP3)high expression as well as several gene signatures that are not found in any other ABT-317 derived clusters. JAK inhibition with ABT-317 is effective at globally inhibiting CM-induced pro- and non-inflammatory pathways in FLS cultures, but also results in several distinct fibroblast populations with unique gene-associated pathways. This study advances the molecular understanding of JAK1 inhibitor effects on fibroblasts that may contribute to clinical efficacy.

NFS-22. INVESTIGATING MALIGNANT TRANSFORMATION IN NF1 PEDIATRIC GLIOMAS USING AN IPSCS-DERIVED CEREBRAL ORGANOID MODEL

Abstract BACKGROUND Neurofibromatosis Type 1 (NF1) is an autosomal dominant tumor predisposition syndrome affecting 1 in 3000 children around the world. Although NF1 inactivation is recognized as the cause of low-grade glioma (LGG) development, little is known about subsequent genetic alterations that lead to malignant transformation. One of the limitations in understanding the molecular events underlying the malignant transformation of NF1 gliomas is the lack of appropriate NF1 preclinical models for its comprehensive profiling. OBJECTIVE Brain organoids have emerged as powerful in vitro models for studying the interaction between tumor cells and the tumor microenvironment. In this study, we present a novel cerebral organoid model, developed to investigate the molecular events leading to malignant transformation in NF1 pediatric gliomas. METHODS Induced pluripotent stem cells (iPSCs)-derived cerebral organoids were generated using NF1wt and NF1mut iPSCs, the latter reprogrammed from patients with plexiform neurofibromas (Carlos III Health Institute, Spain). Immunofluorescence analysis was performed at four different developmental stages to confirm their correct cytoarchitectural development and differentiation into heterogeneous cell populations. Furthermore, four mCherry-labelled isogenic glioma cells lines, RES 186 wild type, NF1-/-, CDKN2a-/-,NF1-/-/CDKN2a-/- were co-cultured with the developed cerebral organoids and their migration potential and invasiveness were compared using two-photon microscopy. RESULTS/DISCUSSION NF1mut cerebral organoids display ventricular-like zone at early stages of development (d15-d30), characterized by luminal Sox2+ neural stem cells surrounded by Tuj1+ immature neuron. These organized structures disappear at later stages, while progenitor cells start to differentiate into fully mature neurons and glial cells (d45-d60). Additionally, after 10 days of co-culture, all four RES186 isogenic cell lines infiltrated the organoid structure with different invasion patterns. The established organoid model will provide a platform for evaluating the impact of sequential mutational events on LGG transformation, taking into consideration the role exerted by the tumor microenvironment in supporting tumor cell invasion and migration.