Mesenchymal-like Cells Research Articles

CNSC-55. UNCOVERING THE ROLE OF COLLAGEN EXTRACELLULAR MATRIX IN GLIOBLASTOMA BRAIN MICROENVIRONMENT TRANSFORMATION

Abstract Glioblastoma (GBM) is a highly malignant brain tumor characterized by diffuse infiltration and significant heterogeneity, without effective treatment. Mesenchymal-like (MES) glioma cells drive tumor aggression by promoting brain invasion and tumor microenvironment remodeling. Previous spatial transcriptomic analyses identified Collagen, particularly COL1A, as a key extracellular matrix (ECM) component, enriched in MES areas termed oncostreams. Depletion of COL1A1 disrupted oncostreams, influencing the tumor microenvironment to support cell survival, and migration. Reactive astrocytes and macrophages, essential components of the brain microenvironment, impact glioma progression. However, the interplay between glioma-secreted collagen and the brain microenvironment remains poorly understood. To elucidate this, we generated orthotopic mouse glioma models with high-collagen (NPD) and low-collagen (NPDshCol1A1). Our data revealed that downregulation of COL1A1 in glioma cells alters the abundance and spatial distribution of reactive astrocytes and macrophages/microglia. Immunofluorescence and flow cytometry analysis showed that tumors with low-collagen had decreased GFAP+ reactive astrocyte cells at the invasive edge and perivascular niche compared to high-collagen gliomas. Moreover, our results shows that high collagen levels may activate astrocytes through distant communication, as evidenced by the higher presence of reactive astrocytes in the contralateral cortex of NPD tumors. However, low-collagen tumors exhibited an increase in astrocytes (GFAP+) in the tumor core. Furthermore, high-collagen gliomas demonstrated increased macrophage infiltration, promoting an immunosuppressive environment. Using multiplex immunohistochemistry assays, we observed that reactive astrocytes along the invasive tumor edge were associated with glioma cells expressing elevated levels of COL1A1 and TGF-β ligand. In summary, our results show that glioma cell-expressed COL1A1 promotes the activation of peritumoral and perivascular brain-resident astrocytes and macrophages. The interactions of reactive astrocyte with glioma cells enriched in collagen form a feedback loop that enhances tumor invasion and vascular proliferation, suggesting that targeting the ECM-astrocyte interactions could disrupt these processes and offer a therapeutic avenue for GBM.

TMIC-22. MYELOID CELLS INDUCE IMMUNOSUPPRESSION AND HYPORESPONSIVENESS OF CHIMERIC ANTIGEN RECEPTOR T CELLS IN THE NEURONAL MICROENVIRONMENT OF GLIOBLASTOMA

Abstract Chimeric antigen receptor T (CART)-cell therapy has shown little success in glioblastoma and other solid tumors, unlike in hematologic malignancies. The immunosuppressive milieu and intratumoral heterogeneity are some obstacles limiting the effectiveness of CART-cell therapy in glioblastoma. Our goal in this study is to investigate how CART-cell transcriptional adaptations and cellular interactions in the glioblastoma microenvironment drive T-cell hyporesponsiveness. Using a human neocortical slice model, we performed physically interacting cell sequencing (PIC-seq) to investigate the transcriptional diversity of NKG2D CAR-T cells and control T-cells in the glioblastoma ecosystem. Through the integration of spatially resolved T-cell receptors sequencing and PIC-seq, we investigated the cellular and receptor-ligand interactions in CART-treated glioblastoma cells. Gene regulatory networks were reconstructed to identify key transcriptional regulators of CD8 CART-cell dysfunction. We used in-silico perturbation to understand transcriptional drivers that could potentially enhance the cytotoxic response of CAR-Ts. Primary patient-derived glioma cells were inoculated into human neocortical slices from surgical access tissue specimens. After 3 days of tumor growth, slices were treated with NKG2D CARTs or control T-cells. We observed that after 2 days of anti-tumor T-cell response in the CART group, tumor growth did not differ between conditions. After 7 days of treatment, PIC-seq was used to profile CARTs (tomato+) and tumor cells (GFP+) as well as physically connected cells (+/+). We identified a transcriptional shift towards T-cell exhaustion in CART cells compared to T-cells lacking the NKG2D construct. This transcriptional phenotype was driven by myeloid-CART and myeloid-tumor interaction. We identified that tumor-associated macrophages with enhanced phagocytic function in proximity to mesenchymal-like tumor cells located within hypoxic niches are the main drivers of T-cell dysfunction. Gene regulatory network analysis demonstrated that MAF and BACH2 are key transcriptional regulators of CART-cell function. Myeloid-tumor and myeloid-T-cell interactions promote an immunosuppressive microenvironment and hyporesponsive T-cells in glioblastoma. Our data has the potential to catalyze the re-engineering and optimization of CART-cell therapies with sustained cytotoxic effect against glioblastoma.

TMIC-70. CORRELATION OF LLT-1 AND NLRC4 INFLAMMASOME AND ITS EFFECT ON GLIOBLASTOMA PROGNOSIS

Abstract PURPOSE LLT-1 is a well-known ligand for the natural killer (NK) cell inhibitory receptor NKRP1A. Here, we examined NLRC4 inflammasome components and LLT-1 expression in glioblastoma (GBM) tissues to elucidate potential associations and interactions between these factors. METHODS Nine GBM tissues were collected for experiments and RNA sequencing, while fifty-two tumor core tissues were collected for RNA sequencing. Expression of LLT-1 and other proteins was assessed by immunofluorescence. Computational analyses utilized RNA-seq data from 296 and 52 patients from the Chinese Glioma Genome Atlas and CHA medical records, respectively, using survival, non-negative matrix factorization clustering, Gene Ontology enrichment, and protein-protein interaction analyses. Receptor-ligand interactions between tumor and immune cells were confirmed by single cell RNA sequencing analysis. RESULTS In GBM tissues, LLT-1 was predominantly colocalized with Glial fibrillary acidic protein (GFAP) -expressing astrocytes, but not with microglial markers like Iba-1. Additionally, LLT-1 and activated NLRC4 inflammasomes were co-expressed mainly in intratumoral astrocytes, suggesting an association between LLT-1, NLRC4, and glioma malignancy. High LLT-1 expression correlates with poor prognosis, particularly in the mesenchymal subtype, and is associated with TNF and NOD-like receptor signaling pathway enrichment, indicating a potential role in tumor inflammation and progression. At the single-cell level, mesenchymal-like malignant cells were highly enriched in the TNF, NLR, and IL-1 signaling pathways compared to other malignant cell types. CONCLUSION We revealed an association between NLRC4 inflammasome activity and LLT-1 expression, suggesting a novel regulatory pathway involving TNF, inflammasomes, and IL-1, potentially offering new anti-glioma approaches by enhancing NK cell-based treatments.

EPCO-34. DECIPHERING THE LONGITUDINAL TRAJECTORIES OF GLIOBLASTOMA BY INTEGRATIVE SINGLE-CELL GENOMICS

Abstract The evolution of cellular heterogeneity in IDH-wildtype glioblastoma (GBM) after standard-of-care therapy remains poorly understood. To address it, we assembled a longitudinal cohort of 121 primary and recurrent GBM specimens from 59 patients, with extensive clinical annotations, and profiled it by single-nucleus RNA-sequencing and bulk tumor DNA sequencing. In most cases, longitudinal samples diverged in their composition of cell types and cell states. However, almost all theoretical trajectories were observed in our cohort such that the overall distribution of cell types and cell states was comparable between primary and recurrent samples. The most consistent longitudinal effect (66% of patients) was a lower malignant cell fraction at recurrence and a reciprocal increase in proportions of glio-neuronal TME cell types; in some cases, this was further accompanied by a coordinated shift of malignant cells towards neuronal-like states. MGMT methylation and radiation-related small deletion phenotypes were linked to particular trajectories, with depletion of mesenchymal-like cells and enrichment of hypoxia-related malignant cells, respectively. Importantly, changes in malignant states were also associated with specific changes in TME composition. In summary, our analysis highlights diverse longitudinal GBM trajectories that are shaped by treatment response and TME interactions.

RNA binding protein ZCCHC24 promotes tumorigenicity in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) lacks the expression of hormone and HER2 receptors and is highly malignant with no effective therapeutic targets. In TNBC, the cancer stem-like cell (CSC) population is considered to be the main cause of resistance to treatment. Thus, the therapeutic targeting of this population could substantially improve patient survival. Here, we identify the RNA-binding protein ZCCHC24 as enriched in the mesenchymal-like TNBC population. ZCCHC24 promotes the expression of a set of genes related to tumorigenicity and treatment resistance by directly binding to the cis-element "UGUWHWWA" in their mRNAs, thereby stabilizing them. One of the ZCCHC24 targets, ZEB1, is a transcription factor that promotes the expression of cancer stemness genes and reciprocally induces ZCCHC24 expression. ZCCHC24 knockdown by siRNAs shows a therapeutic effect and reduces the mesenchymal-like cell population in TNBC patient-derived xenografts. ZCCHC24 knockdown also has additive effects with the BET inhibitor JQ1 in suppressing tumor growth in TNBC patient-derived xenografts.

GRHL2 regulates keratinocyte EMT-MET dynamics and scar formation during cutaneous wound healing

After cutaneous wounds successfully heal, keratinocytes that underwent the epithelial-mesenchymal transition (EMT) regain their epithelial characteristics, while in scar tissue, epidermal cells persist in a mesenchymal state. However, the regulatory mechanisms governing this reversion are poorly understood, and the impact of persistent mesenchymal-like epidermal cells in scar tissue remains unclear. In the present study, we found that during wound healing, the regulatory factor GRHL2 is highly expressed in normal epidermal cells, downregulated in EMT epidermal cells, and upregulated again during the process of mesenchymal-epithelial transition (MET). We further demonstrated that interfering with GRHL2 expression in epidermal cells can effectively induce the EMT. Conversely, the overexpression of GRHL2 in EMT epidermal cells resulted in partial reversion of the EMT to an epithelial state. To investigate the effects of failed MET in epidermal cells on skin wound healing, we interfered with GRHL2 expression in epidermal cells surrounding the cutaneous wound. The results demonstrated that the persistence of epidermal cells in the mesenchymal state promoted fibrosis in scar tissue, manifested by increased thickness of scar tissue, deposition of collagen and fibronectin, as well as the activation of myofibroblasts. Furthermore, the miR-200s/Zeb1 axis was perturbed in GRHL2 knockdown keratinocytes, and transfection with miR-200s analogs promoted the reversion of EMT in epidermal cells, which indicates that they mediate the EMT process in keratinocytes. These results suggest that restoration of the epithelial state in epidermal cells following the EMT is essential to wound healing, providing potential therapeutic targets for preventing scar formation.

Molecular Subtypes Are Associated With Clinical Benefit in Cisplatin-Treated Metastatic Urothelial Cancer Patients.

Cisplatin-based combination chemotherapy (CHT) is standard of care in metastatic urothelial cancer (mUC); however, no predictive molecular biomarkers are available for clinical use. The aim of this study was to investigate the impact of molecular subtypes in relation to treatment response and survival in patients with mUC treated with first-line CHT. Molecular subtype classification according to the Lund Taxonomy (LundTax) was performed by tumor transcriptomic profiling and immunostaining in a retrospective cohort. Molecular subtypes were investigated in relation to the primary end point overall response rate (ORR) and secondary end points progression-free survival (PFS) and overall survival (OS). Differential gene expression and association to treatment response were explored. Ninety-five patients with mUC were classified into urothelial-like (Uro, 43%), genomically unstable (GU, 26%), basal squamous-like (Ba/Sq, 20%), mesenchymal-like (Mes-like, 8%), and small cell neuroendocrine-like (Sc/NE, 3%) subtypes. Patients with Mes-like tumors had lower ORR (14%) compared with Uro (70%), GU (77%), Ba/Sq (75%), and Sc/NE (67%; odds ratio, 0.06 [95% CI, 0.01 to 0.54], P = .012). Furthermore, patients with Mes-like tumors had significantly shorter PFS (hazard ratio [HR], 5.18 [95% CI, 2.28 to 11.76], P < .001) and OS (HR, 3.19 [95% CI, 1.45 to 7.03], P = .004). Patients with Uro and GU showed the longest survival. In responders, an enrichment of downregulated stromal- and immune-related genes was seen. Downregulation of interferon-induced transmembrane protein 2 was associated with increased ORR and improved OS. This study identifies different CHT responses by LundTax molecular subtypes in patients with mUC, where the Mes-like subtype was associated with lower response rate and shorter survival.

Defining heritability, plasticity, and transition dynamics of cellular phenotypes in somatic evolution.

Single-cell sequencing has characterized cell state heterogeneity across diverse healthy and malignant tissues. However, the plasticity or heritability of these cell states remains largely unknown. To address this, we introduce PATH (phylogenetic analysis of trait heritability), a framework to quantify cell state heritability versus plasticity and infer cell state transition and proliferation dynamics from single-cell lineage tracing data. Applying PATH to a mouse model of pancreatic cancer, we observed heritability at the ends of the epithelial-to-mesenchymal transition spectrum, with higher plasticity at more intermediate states. In primary glioblastoma, we identified bidirectional transitions between stem- and mesenchymal-like cells, which use the astrocyte-like state as an intermediary. Finally, we reconstructed a phylogeny from single-cell whole-genome sequencing in B cell acute lymphoblastic leukemia and delineated the heritability of B cell differentiation states linked with genetic drivers. Altogether, PATH replaces qualitative conceptions of plasticity with quantitative measures, offering a framework to study somatic evolution.

Lipid-Laden Macrophages Recycle Myelin to Feed Glioblastoma.

Tumor-associated microglia and macrophages (TAM) make up the largest immune cell population in the glioblastoma (GBM) tumor microenvironment. Given the heterogeneity and plasticity of TAMs in the GBM tumor microenvironment, understanding the context-dependent cancer cell-TAM symbiotic interaction is crucial for understanding GBM biology and developing effective therapies. In a recent issue of Cell, Kloosterman and colleagues identified a subpopulation of glycoprotein nonmetastatic melanoma protein Bhigh lipid-laden microglia and macrophages (LLM) in GBM. Mesenchymal-like GBM cells help generate the LLM phenotype. Reciprocally, LLMs are epigenetically rewired to recycle myelin and transfer the lipid from myelin to cancer cells, fueling mesenchymal-like GBM progression in a liver X receptor/ABCA1-dependent manner. Together, leveraging LLMs opens new therapeutic possibilities for rewiring the metabolism-mediated tumor-TAM interaction during GBM progression.

Gene expression-phenotype association study reveals the dual role of TNF-α/TNFR1 signaling axis in confined breast cancer cell migration

AimsWhile enhanced tumor cell migration is a key process in the tumor dissemination, mechanistic insights into causal relationships between tumor cells and mechanical confinement are still limited. Here we combine the use of microfluidic platforms to characterize confined cell migration with genomic tools to systematically unravel the global signaling landscape associated with the migratory phenotype of breast cancer (BC) cells. Meterials and methodsThe spontaneous migration capacity of seven BC cell lines was evaluated in 3D microfluidic devices and their migration capacity was correlated with publicly available molecular signatures. The role of identified signaling pathways on regulating BC migration capacity was determined by receptor stimulation through ligand binding or inhibition through siRNA silencing. Downstream effects on cell migration were evaluated in microfluidic devices, while the molecular changes were monitored by RT-qPCR. Key findingsExpression of 715 genes was correlated with BC cells migratory phenotype, revealing TNF-α as one of the top upstream regulators. Signal transduction experiments revealed that TNF-α stimulates the confined migration of triple negative, mesenchymal-like BC cells that are also characterized by high TNFR1 expression, but inhibits the migration of epithelial-like cells with low TNFR1 expression. TNFR1 was strongly associated with the migration capacity and triple-negative, mesenchymal phenotype. Downstream of TNF/TNFR1 signaling, transcriptional regulation of NFKB seems to be important in driving cell migration in confined spaces. SignificanceTNF-α/TNFR1 signaling axis reveals as a key player in driving BC cells confined migration, emerging as a promising therapeutic strategy in targeting dissemination and metastasis of triple negative, mesenchymal BC cells.

Epigallocatechin Gallate Derivative Y6 Reverses Oxaliplatin Resistance in Hepatocellular Carcinoma via Targeting the MiR-338-3p/HIF-1α/TWIST Axis to Inhibit EMT.

The emergence of drug resistance to oxaliplatin (OXA) is one of the critical obstacles in the therapy of advanced Hepatocellular Carcinoma (HCC). As an ethyl derivative of the natural compound epigallocatechin gallate (epigallocatechin-3-gallate, EGCG), Y6 was found to be able to enhance the sensitivity of HCC cells to doxorubicin. This study aimed to investigate the effect of Y6 on oxaliplatin resistance in HCC. MTT was used to determine the reversal effect of Y6 on OXA resistance. To further explore the reversal mechanism, we treated OXA alone or in combination with Y6 or EGCG in drugresistant cells and observed the morphological changes of the cells. At the same time, transwell assay was used to detect the invasion and migration ability of cells. Moreover, Real-time PCR and Western blot analysis were performed to determine the expression levels of the miR-338-3p gene, HIF-1α/Twist proteins, and EMT-related proteins. We found that Y6 could inhibit the proliferation of HCC cells and effectively reverse the drug resistance of oxaliplatin-resistant human liver cancer cells (SMMC-7721/OXA) to OXA, and the reversal effect was more significant than that of its lead drug EGCG. Most of the cells in the control group and OXA group showed typical mesenchymal-like cell morphology, while most of the cells in co-administration groups showed typical epithelioid cell morphology, and the ability of the cells to invade and migrate decreased dramatically, particularly in Y6 plus OXA group. At the same time, Y6 could up-regulate the EMT epithelial marker protein E-cadherin and down-regulate the interstitial marker protein Vimentin. In addition, in co-administration groups, the expression of miR-338-3p was up-regulated, while the expression of HIF-1α and Twist was down-regulated. Y6 significantly enhanced the susceptibility of drug-resistant cells to OXA, and the process may be related to the regulation of miR-338-3p/HIF-1α / TWIST pathway to inhibit EMT. Therefore, Y6 could be considered an effective medication resistance reversal agent, which could improve the therapeutic effect for hepatocellular cancer patients.

Unleashing the pathological role of epithelial-to-mesenchymal transition in diabetic nephropathy: The intricate connection with multifaceted mechanism

Renal epithelial-to-mesenchymal transition (EMT) is a process in which epithelial cells undergo biochemical changes and transform into mesenchymal-like cells, resulting in renal abnormalities, including fibrosis. EMT can cause diabetic nephropathy through triggering kidney fibrosis, inflammation, and functional impairment. The diverse molecular pathways that drive EMT-mediated renal fibrosis are not utterly known. Targeting key signaling pathways involved in EMT may help ameliorate diabetic nephropathy and improve renal function. In such settings, understanding precisely the complicated signaling networks is critical for developing customized therapies to intervene in EMT-mediated diabetic nephropathy.

Correlation of LLT-1 and NLRC4 inflammasome and its effect on glioblastoma prognosis.

LLT-1 is a well-known ligand for the natural killer (NK) cell inhibitory receptor NKRP1A. Here, we examined NLRC4 inflammasome components and LLT-1 expression in glioblastoma (GBM) tissues to elucidate potential associations and interactions between these factors. GBM tissues were collected for RNA sequencing (RNA-seq) and Immunofluorescent experiments. Colocalization of LLT-1 and other proteins was assessed by immunofluorescence. Computational analyses utilized RNA-seq data from 296 to 52 patients from the Chinese Glioma Genome Atlas and CHA medical records, respectively. These data were subjected to survival, non-negative matrix factorization clustering, Gene Ontology enrichment, and protein-protein interaction analyses. Receptor-ligand interactions between tumor and immune cells were confirmed by single-cell RNA-seq analysis. In GBM tissues, LLT-1 was predominantly colocalized with glial fibrillary acidic protein (GFAP)-expressing astrocytes, but not with microglial markers like Iba-1. Additionally, LLT-1 and activated NLRC4 inflammasomes were mainly co-expressed in intratumoral astrocytes, suggesting an association between LLT-1, NLRC4, and glioma malignancy. High LLT-1 expression correlates with poor prognosis, particularly in the mesenchymal subtype, and is associated with TNF and NOD-like receptor signaling pathway enrichment, indicating a potential role in tumor inflammation and progression. At the single-cell level, mesenchymal-like malignant cells showed high NF, NLR, and IL-1 signaling pathway enrichment compared to other malignant cell types. We revealed an association between NLRC4 inflammasome activity and LLT-1 expression, suggesting a novel regulatory pathway involving TNF, inflammasomes, and IL-1, potentially offering new NK-cell-mediated anti-glioma approaches.

ATRT-09. MULTI-OMICS SEQUENCING OF ATYPICAL TERATOID RHABDOID TUMORS UNVEILS A SHARED GROUND-STATE POPULATION PROMOTING SUBGROUP-SPECIFIC DIFFERENTIATION TRAJECTORIES

Abstract BACKGROUND Atypical teratoid rhabdoid tumors (ATRTs) remain a significant clinical challenge in pediatric neurooncology. Despite the simplicity of the genomic profile, mostly characterized by the functional loss of SMARCB1 and in rare cases SMARCA4, ATRTs are molecularly and epigenetically diverse, with three main molecular subgroups identified: ATRT-TYR, ATRT-SHH and ATRT-MYC. METHODS To address the lack of effective mechanism-of-action based treatments and comprehend the biology of these subgroups, we conducted 10X single-nucleus transcriptome (n = 12) and multiome (transcriptome + ATAC) (n = 7) sequencing on tumor samples (ATRT-TYR = 6, ATRT-SHH = 9, ATRT-MYC = 4). Validation datasets included 10X single-cell transcriptomics (n = 1), 10X single-nucleus transcriptomics of cell lines (n = 5), and single-cell (n = 3) and single-nucleus (n = 9) SMART-seq transcriptomics. Separation of malignant cells from tumor microenvironment (TME) cells was achieved by comparison of expression profiles against literature-based marker genesets of normal cell types. Tumor cell populations were characterized based on enrichment in fetal cell types and recurrent tumor programs across cancer types, coupled with non-negative matrix factorization (NMF) and differential expression analyses. RESULTS ATRT-TYR exhibited choroid plexus-like and cilia-like cell populations, ATRT-SHH displayed radial glia-like, OPC-like and NPC-like cell populations and ATRT-MYC mainly demonstrated a mesenchymal-like cell population. Each subgroup represented unique differentiation trajectories. However, we identified a population of intermediate neuronal precursor-like cells (IPC-like) shared across all subgroups, expressing markers of pluripotent stem cells and cell cycle. RNA + ATAC transcription factor (TF) enrichment analysis for IPC-like cells revealed TFs linked to each subgroup’s differentiation trajectory, suggesting IPC-like cells as a putative ground-state tumor cell population driving differentiation of each subgroup. Functional validation of these findings in ATRT organoid models is currently ongoing. CONCLUSIONS This study elucidates defined ATRT subgroup-specific differentiation trajectories that may provide guidance for the development of subgroup-based therapies.

Understanding morphological rib abnormalities in Atlantic salmon

Poor rib health in Atlantic salmon (Salmo salar L.) can lead to negative effects on welfare and fillet quality. The present research aimed to enhance the comprehension of long-term rib morphology within a broader musculoskeletal framework of harvest-sized salmon (3 kg), examining a population fed different smoltification diets. Generally, X-ray examinations revealed the highest concentration of abnormalities in the anterior-central area of the rib cage, and the mid-distal rib parts. The different diets, based on either marine (M-group) or vegetal (V-group) ingredients, modulated the morphology of the ribs in the central area of the rib cage. Compared to the V-group, the M-group had a lower prevalence of generalized radiolucency (GR) (19 vs. 26% of mid-distal rib parts), and axis deformations/malformations (12 vs. 17% of mid rib parts). Histological examination of GR, unrelated to near inflammatory processes, revealed potential degradation/transformation of central core chondrocytes to mesenchymal-like cells. The newly formed cavity progressed/expanded through osteolysis until it reached the periosteum. In parallel, there were progressive alterations of the concentric appositional growth pattern of the compact bone, with an increase of the external collagen layer under the cellular periosteum. Disorganized collagen-rich islands were also seen in the compact bone, as were signs of new osteoid deposition in the cavity. The degenerative process in GR was accompanied by an 18–20% increase in the cross-sectional diameter of the rib. The M-group presented numerically higher mechanical rib strength (p = 0.06), and phosphorus content (p = 0.07). The sensitivity of rib morphology to smoltification diet was higher than vertebrae in the long-term. This study enhances understanding of the etiology of the primary morphological rib abnormalities in Atlantic salmon, particularly in GR, by providing detailed histomorphology of pathological changes. The study associates these changes with altered rib development, degeneration, and possibly osteomalacia.

Expression of the apelin receptor, a novel potential therapeutic target, and its endogenous ligands in diverse stem cell populations in human glioblastoma.

Glioblastoma multiforme (GBM) is one of the most common and lethal forms of brain cancer, carrying a very poor prognosis (median survival of ~15 months post-diagnosis). Treatment typically involves invasive surgical resection of the tumour mass, followed by radiotherapy and adjuvant chemotherapy using the alkylating agent temozolomide, but over half of patients do not respond to this drug and considerable resistance is observed. Tumour heterogeneity is the main cause of therapeutic failure, where diverse progenitor glioblastoma stem cell (GSC) lineages in the microenvironment drive tumour recurrence and therapeutic resistance. The apelin receptor is a class A GPCR that binds two endogenous peptide ligands, apelin and ELA, and plays a role in the proliferation and survival of cancer cells. Here, we used quantitative whole slide immunofluorescent imaging of human GBM samples to characterise expression of the apelin receptor and both its ligands in the distinct GSC lineages, namely neural-progenitor-like cells (NPCs), oligodendrocyte-progenitor-like cells (OPCs), and mesenchymal-like cells (MES), as well as reactive astrocytic cells. The data confirm the presence of the apelin receptor as a tractable drug target that is common across the key cell populations driving tumour growth and maintenance, offering a potential novel therapeutic approach for patients with GBM.

A Genuinely Hybrid, Multiscale 3D Cancer Invasion and Metastasis Modelling Framework

We introduce in this paper substantial enhancements to a previously proposed hybrid multiscale cancer invasion modelling framework to better reflect the biological reality and dynamics of cancer. These model updates contribute to a more accurate representation of cancer dynamics, they provide deeper insights and enhance our predictive capabilities. Key updates include the integration of porous medium-like diffusion for the evolution of Epithelial-like Cancer Cells and other essential cellular constituents of the system, more realistic modelling of Epithelial–Mesenchymal Transition and Mesenchymal–Epithelial Transition models with the inclusion of Transforming Growth Factor beta within the tumour microenvironment, and the introduction of Compound Poisson Process in the Stochastic Differential Equations that describe the migration behaviour of the Mesenchymal-like Cancer Cells. Another innovative feature of the model is its extension into a multi-organ metastatic framework. This framework connects various organs through a circulatory network, enabling the study of how cancer cells spread to secondary sites.

Abstract 274: Cancer-associated fibroblasts exhibit increased motility under hypoxic conditions via interaction with cancer cells

Abstract Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment, playing various roles through interactions with cancer cells and other stromal cell types. In a 3D co-culture system of CAFs and lung cancer cells, a specific subgroup of CAFs exhibited increased motility, while another subgroup of CAFs remained stationary. Using the photoconvertible fluorescent protein Dendra2, we isolated these distinct CAF subgroups and conducted RNA sequencing. Gene set enrichment and gene ontology analysis revealed that upregulated genes in motile CAFs are associated with hypoxia and glycolysis. Conditioned medium from mesenchymal-like cancer cells enhanced the expression of genes related to hypoxia and glycolysis in CAFs. Among these genes, Aldoa and Ldha were chosen for knockdown in CAFs, resulting in a reduction in CAF motility. We confirmed that mesenchymal-like cancer cells promoted CAF motility and intensified hypoxic conditions within the co-culture system, which was monitored using a hypoxia reporter. Ceruloplasmin (CP) was identified as a secretory protein from mesenchymal-like cancer cells through mass spectrometry. Knockdown of CP led to the inhibition of CAF motility. In conclusion, the motility of CAFs is enhanced under hypoxic conditions facilitated by the secretion of CP from mesenchymal-like cancer cells in the lung tumor microenvironment. Citation Format: Jihye Park, Sieun Lee, Jonathan M. Kurie, Young-Ho Ahn. Cancer-associated fibroblasts exhibit increased motility under hypoxic conditions via interaction with cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 274.

Transcriptional and phenotypical alterations associated with a gradual benzo[a]pyrene-induced transition of human bronchial epithelial cells into mesenchymal-like cells

The role of benzo[a]pyrene (BaP), a prominent genotoxic carcinogen and aryl hydrocarbon receptor (AhR) ligand, in tumor progression remains poorly characterized. We investigated the impact of BaP on the process of epithelial-mesenchymal transition (EMT) in normal human bronchial epithelial HBEC-12KT cells. Early morphological changes after 2-week exposure were accompanied with induction of SERPINB2, IL1, CDKN1A/p21 (linked with cell cycle delay) and chemokine CXCL5. After 8-week exposure, induction of cell migration and EMT-related pattern of markers/regulators led to induction of further pro-inflammatory cytokines or non-canonical Wnt pathway ligand WNT5A. This trend of up-regulation of pro-inflammatory genes and non-canonical Wnt pathway constituents was observed also in the BaP-transformed HBEC-12KT-B1 cells. In general, transcriptional effects of BaP differed from those of TGFβ1, a prototypical EMT inducer, or a model non-genotoxic AhR ligand, TCDD. Carcinogenic polycyclic aromatic hydrocarbons could thus induce a unique set of molecular changes linked with EMT and cancer progression.

Abstract 5466: In vivo characterization of neuroblastoma intratumoral heterogeneity

Abstract Background: Neuroblastoma is a pediatric solid tumor that arises from neural crest-derived progenitor cells of the peripheral sympathetic nervous system (PSNS). Neuroblastoma patients display a significant level of genetic and phenotypic heterogeneity, with amplification of the MYCN oncogene associated with poor clinical outcomes. To better understand underlying genetic mechanisms associated with aggressive neuroblastoma, our group incorporated patient-relevant loss-of-function mutations into the established MYCN-driven zebrafish model of neuroblastoma (Tg(dbh:EGFP-MYCN)), which resulted in increased tumor penetrance and a highly metastatic phenotype. Aims &amp; Methods: We aimed to investigate metastases-associated intratumoral heterogeneity in vivo using our patient-relevant metastatic zebrafish models, with functional validations performed using human neuroblastoma cell lines. Results: Single-cell RNA sequencing of metastatic zebrafish neuroblastoma tumors revealed intra-tumor cell heterogeneity, with a subset of cells highly expressing markers of more differentiated adrenergic-like cell fates (th, dbh, phox2a) while others enriched for markers of less differentiated mesenchymal-like progenitor cells (prrx1a, vim, fn1a). Differential cell labeling in zebrafish neuroblastoma suggests distinct tumor cell populations for follow-up validation and characterization throughout metastatic progression. Conclusion: Single-cell RNA sequencing of zebrafish metastatic neuroblastoma showed evidence of intratumoral heterogeneity that warrants further investigation. We are characterizing neuroblastoma intratumoral heterogeneity in our in vivo metastatic zebrafish models to examine the association between neuroblastoma tumor composition and metastatic potential. Altogether, this work aims to improve our understanding of the evolution of tumor cell heterogeneity associated with neuroblastoma metastasis and uncover novel therapeutic opportunities to improve patient outcomes. Citation Format: Willow R. Squires, Alex Weiss, Sarah Cohen-Gogo, Adam Shlien, David Kaplan, Meredith S. Irwin, Madeline N. Hayes. In vivo characterization of neuroblastoma intratumoral heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5466.