Invasive Behavior Research Articles

Exploring natural anthraquinones as potential MMP2 inhibitors: A computational study

ObjectiveMatrix metalloproteinase-2 (MMP2) plays a significant role in cleaving extracellular matrix components, leading to many cancer cells' progression and invasion behavior. Therefore, MMP2 inhibition may hold promise for cancer treatment. Anthraquinones have shown antineoplastic effects, some of which have been used in clinical practice as anticancer drugs. This study used a computational drug discovery approach to assess the possible inhibitory effects of selected anthraquinones on MMP2. The results were then compared with that of Captopril, which was considered a standard drug. MethodsThis study used the AutoDock 4.0 tool to evaluate the binding affinity of 21 anthraquinones to the MMP2 catalytic domain. The most favorable scores based on the Gibbs free binding energy scores were given to the highest-ranked ligands. The Discovery Studio Visualizer tool illustrated interactions between MMP2 residues and top-ranked anthraquinones. ResultsA total of 12 anthraquinones were identified with ΔGbinding scores less than − 10 kcal/mol. Pulmatin (Chrysophanol-8-glucoside) was the most potent MMP2 inhibitor, with a ΔGbinding score of − 12.91 kcal/mol. This anthraquinone was able to restrict MMP2 activity within a picomolar range. ConclusionMMP2 inhibition by anthraquinones, notably Pulmatin, may be a useful therapeutic approach for cancer treatment.

WHO histological classification and tumor size are predictors of the locally aggressive behavior of thymic epithelial tumors

ObjectiveThe aim of this study was to explore the influencing factors that affect the local invasive behavior of thymic epithelial tumors (TETs). MethodWe retrospectively analyzed 524 patients with TETs who underwent surgical treatment at our center from January 2010 to January 2022. Cox regression analysis was applied to identify predictors associated with the prognosis of TET. Logistic regression analysis was used to analyze the factors associated with the locally invasive behavior of TETs. Receiver operating characteristic analysis and the Youden index were applied to determine the predictive efficiency and cutoff value. ResultsThere were 275 males and 249 females with a median age of 56 years. Seventy-seven patients had locally invasive behavior. The prognosis of local invasive TETs was significantly worse that of noninvasive TETs (P < 0.001). WHO classification and tumor size were two hazard factors for tumor invasive behavior. The risk of local invasion increased by 2.196 (OR (95 % CI): 1.813–2.659) times for each grade in WHO classification with a change from type A to thymic carcinoma. The tumor size cutoff of 6 cm represented a distinct boundary in predicting the hazard of local invasion (AUC: 0.784, specificity: 0.711, sensitivity: 0.726). ConclusionWHO classification and tumor size are important factors in predicting the locally aggressive behavior of TETs. The invasion capability of TETs is constantly increasing with an escalation in WHO classification. Tumors greater than 6 cm in size have a higher risk for local invasion.

HPV E6/E7: insights into their regulatory role and mechanism in signaling pathways in HPV-associated tumor.

Human papillomavirus (HPV) is a class of envelope-free double-stranded DNA virus. HPV infection has been strongly associated with the development of many malignancies, such as cervical, anal and oral cancers. The viral oncoproteins E6 and E7 perform central roles on HPV-induced carcinogenic processes. During tumor development, it usually goes along with the activation of abnormal signaling pathways. E6 and E7 induces changes in cell cycle, proliferation, invasion, metastasis and other biological behaviors by affecting downstream tumor-related signaling pathways, thus promoting malignant transformation of cells and ultimately leading to tumorigenesis and progression. Here, we summarized that E6 and E7 proteins promote HPV-associated tumorigenesis and development by regulating the activation of various tumor-related signaling pathways, for example, the Wnt/β-catenin, PI3K/Akt, and NF-kB signaling pathway. We also discussed the importance of HPV-encoded E6 and E7 and their regulated tumor-related signaling pathways for the diagnosis and effective treatment of HPV-associated tumors.

Expression pattern of osteopontin isoforms in malignant melanoma cell lines.

Osteopontin (OPN) is a secreted integrin-binding protein that plays a role in inflammation, cellular viability, cell adhesion and migration, cancer development, and diabetes through different mechanisms. The splice variants of OPN can play essential roles in cancer development, progression, and metastasis formation; however, limited data are available about the role of OPN isoforms in human malignant melanoma. Our goal was to define the gene expression patterns of five OPN variants (OPN4, OPN5, OPNa, OPNb, and OPNc), integrin, and CD44 receptor genes in primary and metastatic melanoma-originated cell lines (n = 19), and to explore the association of the expression patterns with clinicopathological parameters. We evaluated the invasive property of the cell lines and investigated the potential association between the invasion and gene expression of OPN isoforms. We found a significant rise in the expression of OPNc in the invasive cell lines compared to the noninvasive cells and detected significantly higher expression of the OPN splice variants in melanoma cell lines originating from more advanced stages tumors than cell lines originating from early-stage melanomas. The correlation analysis revealed that all five OPN variants positively correlated with ITGB3 and ITGA9, whereas OPN5 positively correlated with ITGB1, ITGAV, ITGA6, and CD44. OPN can activate extracellular signal-regulated kinase signaling through binding to α9β1 integrin, promoting melanoma tumor cell migration. It is possible that such associations between OPN splice variants and integrin receptors may play a role in melanoma progression. In conclusion, our findings suggest that high expression of OPNc correlates with the invasive behavior of melanoma cells.

In Vitro Organoid-Based Assays Reveal SMAD4 Tumor-Suppressive Mechanisms for Serrated Colorectal Cancer Invasion.

Colon cancer is the third most prominent cancer and second leading cause of cancer-related deaths in the United States. Up to 20% of colon cancers follow the serrated tumor pathway driven by mutations in the MAPK pathway. Loss of SMAD4 function occurs in the majority of late-stage colon cancers and is associated with aggressive cancer progression. Therefore, it is important to develop technology to accurately model and better understand the genetic mechanisms behind cancer invasion. Organoids derived from tumors found in the Smad4KO BRAFV600E/+ mouse model present multiple phenotypes characteristic of invasion both in ex vivo and in vivo systems. Smad4KO BRAFV600E/+ tumor organoids can migrate through 3D culture and infiltrate through transwell membranes. This invasive behavior can be suppressed when SMAD4 is re-expressed in the tumor organoids. RNA-Seq analysis reveals that SMAD4 expression in organoids rapidly regulates transcripts associated with extracellular matrix and secreted proteins, suggesting that the mechanisms employed by SMAD4 to inhibit invasion are associated with regulation of extracellular matrix and secretory pathways. These findings indicate new models to study SMAD4 regulation of tumor invasion and an additional layer of complexity in the tumor-suppressive function of the SMAD4/Tgfβ pathway.

10000-SPE-1 STEM CELL-BASED GENE THERAPY FOR MALIGNANT GLIOMA USING GENOME-EDITED HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract Glioblastoma is the most aggressive primary brain tumor, and is characterized by diffuse infiltration into the normal brain parenchyma. New therapeutic approaches targeting invasive biological behaviour are warranted. In the present study, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited induced pluripotent stem cells (iPSCs) have high tumor-trophic migratory capacity and stable constitutive therapeutic transgene expression, which leads to strong anti-tumor effects against glioma stem cell (GSC) models. The present study provides answers to some important research questions associated with stem cell-based gene therapy. First, the tumor-trophic migratory capacities of human iPSC-derived NSCs (iPSC-NSCs), fetal NSCs, and mesenchymal stem cells (MSCs) were quantitatively evaluated by spatiotemporal methodologies. We demonstrated that iPSC-NSCs have a higher tumor-trophic migratory capacity than MSCs in the brain. Self-repulsive action and pathotropism were important for the migration of iPSC-NSCs: ephrin ligand/receptor mediated repulsion of iPSC-NSCs and CXCL12-CXCR4 interactions between GSCs and iPSC-NSCs. Second, a prodrug converting enzyme fusion gene was selected as a therapeutic gene in human iPSCs. In general, stable constitutive transgene expression by viral vectors was difficult in human iPSCs. Furthermore, viral vectors integrate randomly into the host genome, which raises concerns about transgene silencing, insertional mutagenesis, and oncogene activation. In the present study, several common insertion sites including GAPDH, ACTB, and AAVS1, were compared. The most appropriate gene locus that achieved stable constitutive transgene expression was determined via CRISPR/Cas9-mediated genome editing. Third, we revealed the novel mechanism of action using iPSC-NSCs expressing CD-UPRT, in which ferroptosis was associated with enhanced anti-tumor immune responses. We demonstrated that the established iPSC-NSCs had strong therapeutic efficacy in GSC animal models. Finally, predictive biomarkers for the efficacy of the present treatment strategy were established. We will conduct a clinical trial of this treatment strategy. This research concept can disseminate biological, medical and engineering advances.

Impaired dNKAP function drives genome instability and tumorigenic growth in Drosophila epithelia.

Mutations or dysregulated expression of NF-kappaB-activating protein (NKAP) family genes have been found in human cancers. How NKAP family gene mutations promote tumor initiation and progression remains to be determined. Here, we characterized dNKAP, the Drosophila homolog of NKAP, and showed that impaired dNKAP function causes genome instability and tumorigenic growth in a Drosophila epithelial tumor model. dNKAP-knockdown wing imaginal discs exhibit tumorigenic characteristics, including tissue overgrowth, cell-invasive behavior, abnormal cell polarity, and cell adhesion defects. dNKAP knockdown causes both R-loop accumulation and DNA damage, indicating the disruption of genome integrity. Further analysis showed that dNKAP knockdown induces c-Jun N-terminal kinase (JNK)-dependent apoptosis and causes aberrant cell proliferation in distinct cell populations. Activation of the Notch and JAK/STAT signaling pathways contributes to the tumorigenic growth of dNKAP-knockdown tissues. Furthermore, JNK signaling is essential for dNKAP depletion-mediated cell invasion. Transcriptome analysis of dNKAP-knockdown tissues confirmed the misregulation of signaling pathways involved in promoting tumorigenesis and revealed abnormal regulation of metabolic pathways. dNKAP knockdown and oncogenic Ras, Notch, or Yki mutations show synergies in driving tumorigenesis, further supporting the tumor-suppressive role of dNKAP. In summary, this study demonstrates that dNKAP plays a tumor-suppressive role by preventing genome instability in Drosophila epithelia and thus provides novel insights into the roles of human NKAP family genes in tumor initiation and progression.

Machine Learning Methods Based on CT Features Differentiate G1/G2 From G3 Pancreatic Neuroendocrine Tumors

To identify CT features for distinguishing grade 1 (G1)/grade 2 (G2) from grade 3 (G3) pancreatic neuroendocrine tumors (PNETs) using different machine learning (ML) methods. A total of 147 patients with 155 lesions confirmed by pathology were retrospectively included. Clinical-demographic and radiological CT features was collected. The entire cohort was separated into training and validation groups at a 7:3 ratio. Least absolute shrinkage and selection operator (LASSO) algorithm and principal component analysis (PCA) were used to select features. Three ML methods, namely logistic regression (LR), support vector machine (SVM), and K-nearest neighbor (KNN) were used to build a differential model. Receiver operating characteristic (ROC) curves and precision-recall curves for each ML method were generated. The area under the curve (AUC), accuracy rate, sensitivity, and specificity were calculated. G3 PNETs were more likely to present with invasive behaviors and lower enhancement than G1/G2 PNETs. The LR classifier yielded the highest AUC of 0.964 (95% confidence interval [CI]: 0.930, 0.972), with 95.4% accuracy rate, 95.7% sensitivity, and 92.9% specificity, followed by SVM (AUC: 0.957) and KNN (AUC: 0.893) in the training group. In the validation group, the SVM classier reached the highest AUC of 0.952 (95% CI: 0.860, 0.981), with 91.5% accuracy rate, 97.3% sensitivity, and 70% specificity, followed by LR (AUC: 0.949) and KNN (AUC: 0.923). The LR and SVM classifiers had the best performance in the training group and validation group, respectively. ML method could be helpful in differentiating between G1/G2 and G3 PNETs.

MiR-4763-3p accelerates lipopolysaccharide-induced cardiomyocyte apoptosis and inflammatory response by targeting IL10RA.

In order to investigate miR-4763-3p and associated genes' roles in myocarditis, AC16 cell line was divided into LPS + miR-4763-3p inhibitor, LPS + NC inhibitor, LPS + miR-4763-3p inhibitor + si-IL10RA and NC groups, and Q-PCR was used to find out whether miR-4763-3p was expressed; Targetscan, Genecards, and MiRDB were used to estimate the miR-4763-3p target; Targetscan was used to display binding sites. Western blot assay was undertaken to detect Bax, Bcl-2, and IL10RA expression. Proliferation and apoptosis were processed using CCK8 and the flow cytometry assay, respectively. Migration and invasion were confirmed utilizing Transwell test. ELISA assay was processed to show the content of IL-6, IL-1ß, IL-10 and TGF-ß in the cell culture supernatant. After being exposed to LPS, cardiomyocyte cells expressed more miR-4763-3p. MiR-4763-3p inhibitor accelerated proliferation, migration and invasion behavior, while it also decreased apoptosis rate in LPS-treated cardiomyocyte cells. MiR-4763-3p inhibitor attenuated the inflammatory response by up-regulating Bax expression and down-regulating Bcl-2 level in LPS-treated cardiomyocyte cells. In cardiomyocyte cells treated with LPS, MiR-4763-3p expression was elevated. si-IL10RA The miR-4763-3p inhibitor restored its effects. MiR-4763-3p accelerates lipopolysaccharide-induced cardiomyocyte apoptosis and inflammatory response by targeting IL10RA, which might be a potential target for myocarditis.

Dynamic compartmentalization of the pro-invasive transcription factor NHR-67 reveals a role for Groucho in regulating a proliferative-invasive cellular switch in C. elegans.

A growing body of evidence suggests that cell division and basement membrane invasion are mutually exclusive cellular behaviors. How cells switch between proliferative and invasive states is not well understood. Here, we investigated this dichotomy in vivo by examining two cell types in the developing Caenorhabditis elegans somatic gonad that derive from equipotent progenitors, but exhibit distinct cell behaviors: the post-mitotic, invasive anchor cell and the neighboring proliferative, non-invasive ventral uterine (VU) cells. We show that the fates of these cells post-specification are more plastic than previously appreciated and that levels of NHR-67 are important for discriminating between invasive and proliferative behavior. Transcription of NHR-67 is downregulated following post-translational degradation of its direct upstream regulator, HLH-2 (E/Daughterless) in VU cells. In the nuclei of VU cells, residual NHR-67 protein is compartmentalized into discrete punctae that are dynamic over the cell cycle and exhibit liquid-like properties. By screening for proteins that colocalize with NHR-67 punctae, we identified new regulators of uterine cell fate maintenance: homologs of the transcriptional co-repressor Groucho (UNC-37 and LSY-22), as well as the TCF/LEF homolog POP-1. We propose a model in which the association of NHR-67 with the Groucho/TCF complex suppresses the default invasive state in non-invasive cells, which complements transcriptional regulation to add robustness to the proliferative-invasive cellular switch in vivo.

WITHDRAWN: Primary large cell neuroendocrine carcinoma of the bladder: A case report and review of the literature

Large cell neuroendocrine carcinoma (LCNEC) of the bladder is a rare bladder tumor. Due to its invasive clinical behavior, most patients already have muscle layer invasion and/or metastasis at the time of diagnosis. We report a 72-year-old female patient who presented with gross hematuria. A solitary tumor located in the anterior wall of the bladder was found by cystoscopy. Biopsy pathological examination suggested urothelial carcinoma (UC) of the bladder. The patient underwent partial cystectomy and was eventually diagnosed as LCNEC (pT2bN0M0) based on the results of postoperative immunohistochemical examination.

Dynamic compartmentalization of the pro-invasive transcription factor NHR-67 reveals a role for Groucho in regulating a proliferative-invasive cellular switch in C. elegans

A growing body of evidence suggests that cell division and basement membrane invasion are mutually exclusive cellular behaviors. How cells switch between proliferative and invasive states is not well understood. Here, we investigated this dichotomy in vivo by examining two cell types in the developing Caenorhabditis elegans somatic gonad that derive from equipotent progenitors, but exhibit distinct cell behaviors: the post-mitotic, invasive anchor cell and the neighboring proliferative, non-invasive ventral uterine (VU) cells. We show that the fates of these cells post-specification are more plastic than previously appreciated and that levels of NHR-67 are important for discriminating between invasive and proliferative behavior. Transcription of NHR-67 is downregulated following post-translational degradation of its direct upstream regulator, HLH-2 (E/Daughterless) in VU cells. In the nuclei of VU cells, residual NHR-67 protein is compartmentalized into discrete punctae that are dynamic over the cell cycle and exhibit liquid-like properties. By screening for proteins that colocalize with NHR-67 punctae, we identified new regulators of uterine cell fate maintenance: homologs of the transcriptional co-repressor Groucho (UNC-37 and LSY-22), as well as the TCF/LEF homolog POP-1. We propose a model in which the association of NHR-67 with the Groucho/TCF complex suppresses the default invasive state in non-invasive cells, which complements transcriptional regulation to add robustness to the proliferative-invasive cellular switch in vivo.

Myricetin ameliorates the IL-21-induced tumorigenic phenotype of adjuvant-induced arthritis FLS by modulating the choline kinase signaling cascade.

The synovial intimal lining is mainly governed by fibroblast-like synoviocytes (FLS), which portray a transformed tumor-like phenotype in rheumatoid arthritis (RA). Among the diverse cytokines that engender FLS, interleukin-21 (IL-21) was reported to stimulate hyperproliferation and perpetuate inflammation. Recently, choline kinase (ChoKα) has been reported to be an essential enzyme aiding RA-FLS hyperproliferation by altering phosphatidylcholine biosynthesis. The current study aimed to elucidate the therapeutic efficacy of myricetin, a flavonoid, in abating the IL-21-induced tumor-like phenotype of adjuvant-induced arthritis (AIA)-FLS via the ChoKα signaling cascade. Our results showed that myricetin suppressed IL-21 receptor expression and activation of the ChoKα signaling cascade (N-Ras, Ral-GDS, and PI3K) in IL-21-induced AIA-FLS. Consequently, myricetin treatment decreased ChoKα and PLD2 enzymatic activity and inhibited the proliferative, migratory, and invasive properties of AIA-FLSs. Our results demonstrated that myricetin could be a promising anti-arthritic compound by abating IL-21-induced hyperproliferation, migration, and invasive behavior of AIA-FLS by downregulating the ChoKα signaling cascade.

Expression of Gata Binding Protein 3 as a Prognostic Factor in Urogenital Lesions and Its Association With Morphology.

Urogenital malignancies, encompassing urinary bladder cancer, prostate cancer, and renal cell carcinoma, pose significant diagnostic challenges due to overlapping histopathological features. GATA binding protein 3 (GATA3), a transcription factor associated with urothelial tissue, has shown promise as a potential diagnostic marker.This study aimed to investigate the incidence of these malignancies, explore GATA3's involvement in urothelial cancer (UC), and determine its role in distinguishing urogenital malignancies. A cross-sectional, retro-prospective, hospital-based study was conducted from May 2019 to April 2021. The surgical samples of patients who underwent transurethral resection of bladder tumour (TURBT), transurethral resection of the prostate (TURP), radical cystoprostatectomy, total and partial radical nephrectomy specimens during the study period were reviewed. Patients diagnosed with urinary bladder neoplasm and high-grade prostate neoplasm along with chromophobe, oncocytic, sarcomatoid variant and clear cell carcinoma, renalcell carcinoma were included. Immunohistochemical analysis of GATA3 expression was performed, with scoring based on nuclear staining intensity and percentage of tumor cells labeled. The study included 64 patients, predominantly males over 60 years. Personal habits revealed a high prevalence of smoking (85.9%). The most prevalent symptom was hematuria (75.0%), followed by hematuria with urgency (20.3%). The most common site of lesion was posterolateral (31.3%). Urothelial cancer was the most common malignancy, primarily high-grade. Strong positive GATA3 expression was significantly associated with high-grade UC (p=0.01) and invasion (p=0.01). However, low-grade UC and papillary urothelial neoplasm of low malignant potential exhibited moderate GATA3 expression. GATA3 demonstrated potential for distinguishing UC from other histological types. GATA3 expression correlates with high-grade urothelial cancer and invasive behavior, suggesting its utility as a diagnostic marker in challenging cases.

Monoclonal antibody cetuximab impairs matrix metalloproteinases 2 and 9, epithelial-mesenchymal transition and cell migration in feline oral squamous cell carcinoma in vitro.

Feline oral squamous cell carcinoma (FOSCC) is characterised by invasive and metastatic behaviour and is poorly responsive to current treatments, hence the need for new therapeutic strategies. FOSCC shares molecular targets with human head and neck squamous cell carcinoma (HNSCC), among these the epidermal growth factor receptor. Cetuximab is an anti-epidermal growth factor receptor monoclonal antibody employed in the therapy of HNSCC and, interestingly, previous work in vitro suggested that it displays cytostatic and cytotoxic properties also against FOSCC. With the present study, we aimed at further investigating the effects of cetuximab on invasion and metastasis pathways proven to be relevant in human patients. To this purpose, FOSCC cell lines SCCF1, SCCF2 and SCCF3 were treated with cetuximab for 48/72 h and subjected to Western blot for matrix metalloproteinases-2/9 (MMP-2/9) and epithelial-mesenchymal transition markers vimentin, E-, P- and N-cadherin. Treatment with cetuximab resulted in downregulation of MMP-2/-9 in all of the three cell lines in a dose-dependent manner. Moreover, cetuximab downregulated vimentin and P-cadherin in SCCF1, upregulated E-cadherin whilst downregulating P-/N-cadherins in SCCF2, and impaired P-/N-cadherins in SCCF3. An in vitro scratch test also demonstrated that cetuximab delayed cell migration in SCCF3. These data suggest that cetuximab mitigates invasion and metastasis processes by impairing MMPs and epithelial-mesenchymal transition pathways in FOSCC, indicating that this monoclonal antibody may help to counteract malignant progression and improve the management of locally invasive disease.

Alteration of Cadherin 3 Expression and DNA Methylation in Association with Aggressive Renal Cell Carcinoma.

Cadherins (calcium-dependent adhesion proteins) are important in cellular adhesion and may play a role in the development and progression of renal cell carcinoma (RCC). This study investigated changes in cadherin 3 (CDH3; P-cadherin) mRNA expression, DNA methylation, and protein expression in RCC and compared the results with the histopathological and clinical characteristics of patients. The possible contribution of CDH3 to tumor cell invasiveness was tested in a functional assay using siRNA-based suppression of CDH3 expression and subsequent real-time impedance analysis using a Matrigel invasion model. Our analyses revealed a tumor-specific loss of CDH3 mRNA expression, CDH3 DNA hypermethylation, and loss of distal tubular and collecting duct CDH3 protein expression in RCC. A relatively higher methylation level in tumors was associated with a loss of cell differentiation and higher clinical stage. siRNA-induced suppression of CDH3 expression modulated the invasion characteristics of tumor cells in the impedance-based real-time cellular analysis. Our results indicate that loss of CDH3 expression is common in RCC and may contribute to the pathogenesis of a subset of RCC. Further studies to reveal the mechanisms of loss of expression and its effects on the invasive behavior of renal tumor cells are required.

Integrins Can Act as Suppressors of Ras-Mediated Oncogenesis in the Drosophila Wing Disc Epithelium.

Cancer is the second leading cause of death worldwide. Key to cancer initiation and progression is the crosstalk between cancer cells and their microenvironment. The extracellular matrix (ECM) is a major component of the tumour microenvironment and integrins, main cell-ECM adhesion receptors, are involved in every step of cancer progression. However, accumulating evidence has shown that integrins can act as tumour promoters but also as tumour suppressor factors, revealing that the biological roles of integrins in cancer are complex. This incites a better understating of integrin function in cancer progression. To achieve this goal, simple model organisms, such as Drosophila, offer great potential to unravel underlying conceptual principles. Here, we find that in the Drosophila wing disc epithelium the βPS integrins act as suppressors of tumours induced by a gain of function of the oncogenic form of Ras, RasV12. We show that βPS integrin depletion enhances the growth, delamination and invasive behaviour of RasV12 tumour cells, as well as their ability to affect the tumour microenvironment. These results strongly suggest that integrin function as tumour suppressors might be evolutionarily conserved. Drosophila can be used to understand the complex tumour modulating activities conferred by integrins, thus facilitating drug development.

TMIC-77. UNVEILING THE DEPTHS: EXPLORING DIFFUSE MIDLINE GLIOMA, TUMORIGENESIS, AND RADIATION RESISTANCE

Abstract Diffuse Midline Glioma (DMG) is a highly aggressive form of brainstem cancer that primarily affects children, leading to a grim prognosis and limited treatment options. Despite extensive research efforts, many crucial aspects of DMG development and progression remain poorly understood. One significant knowledge gap lies in comprehending the invasive nature of DMG cells, which enables their escape from the primary tumor site and dissemination to other regions of the brain. Additionally, understanding the molecular basis that drives this invasive behavior is essential but remains largely unexplored, necessitating further investigation. To address these critical knowledge gaps, we have generated one of the largest single-cell multi-omic datasets ( &amp;gt;300,000 cells) specifically focusing on pons development in humans and its relation to DMG. This dataset provides a valuable resource for investigating the intricacies of tumorigenesis and the development of resistance to radiation therapy. Comprehensive analyses of this dataset shed light on the molecular events driving DMG progression and radiation resistance, ultimately paving the way for the discovery of novel therapeutic targets and the development of innovative strategies to combat this devastating pediatric cancer. In summary, our study aims to deepen our understanding of DMG by elucidating the mechanisms associated with histone mutations, unraveling the invasive behavior of DMG cells, and exploring the factors contributing to radiation resistance. Through these endeavors, we hope to make significant advancements in the field and improve the outcomes for patients affected by DMG.

MODL-42. BLOOD-BRAIN TUMOR BARRIER ORGANOID MODEL FOR THERAPEUTIC DELIVERY TO GLIOBLASTOMA

Abstract The integrity of the blood-brain barrier (BBB) is compromised in brain tumors, leading to the formation of the blood-brain tumor barrier (BTB). While the BTB is generally more permeable than the BBB, it is nevertheless heterogeneously restrictive to the entry of therapeutic agents to brain tumors. Modeling the BTB accurately is crucial for understanding its physiological properties and developing effective treatments. Our lab has previously introduced a 3D in vitro human BBB organoid platform that reproduces key BBB markers and predicts compound permeability across the BBB in vivo. Here, we describe incorporating human GBM stem cells (GSC) into the organoid to form BTB organoids, presenting a novel scalable multicellular human brain tumor organoid model that simulates the BTB to facilitate therapeutic development in neuro-oncology. This model involves the self-assembly of essential BTB components, including human brain microvascular endothelial cells, astrocytes, brain pericytes and patient-derived GSCs, co-cultured under ultralow attachment conditions. Immunofluorescence microscopy results show reduced ZO-1 and Mfsd2a expression in the BTB compared to the control BBB organoids, indicating tight junction disruption and BBB breakdown by the GSCs. Correspondingly, we observe increased dextran permeability in the BTB organoids compared to the control BBB organoids, which is dependent on the GSC count per organoid. Electron and confocal microscopy reveal disorganized neovascular structures and invasive GSC behavior in the BTB organoids. Lastly, using a known GBM-targeting peptide (BTP-7), as well as a known BBB-penetrating adeno-associated virus (AAV.CPP.16) as proofs-of-concept, we demonstrate the efficacy of using the BTB organoids for modeling therapeutic delivery to brain tumors. In summary, the BTB organoid platform provides a tractable in vitro system reflecting the brain tumor microenvironment, enabling the study of GBM cell properties, investigation of barrier behavior, and modeling therapeutic delivery to brain tumors.

CRIP1: Role in the pathogenesis of hypertension by regulating interactions with cytoskeletal proteins

Abstract Background Hypertension is a complex polygenic disease and the underlying molecular mechanisms remain unknown. In an interdisciplinary approach, we identified cysteine-rich protein 1 (CRIP1) in human monocytes as being strongly associated with hypertension, suggesting a link between CRIP1 and the pathophysiology of hypertension via the immune system. The identification of CRIP1 protein interaction partners and associated signaling pathways would provide insight into the pathophysiological significance of CRIP1 under hypertensive conditions. Objective The overall aim of our project is to evaluate the functional role of CRIP1 in hypertension and cardiovascular disease (CVD). Here, using systems medicine approaches including experimental settings and human cohort data, we focused on the identification of novel CRIP1 protein interaction partners that may be associated with the pathogenesis of hypertension and cardiovascular disease. Methods Monocytic CRIP1 was immunoprecipitated and co-immunoprecipitated proteins in the eluate were analyzed by LC-MS/MS and Pathway and Process Enrichment Analysis and validated by immunoblot techniques. Silencing of CRIP1 was established in the monocyte-like cell line THP-1 using a vector-based shRNA system. The association of CRIP1 with genes encoding cytoskeletal proteins was accessed in a human cohort by transcriptome-based analysis of human monocytes in the Guttenberg-Gesundheitsstudie (GHS; n = 1527). Results First, a Yeast Two-Hybrid Assay was performed and two potential protein interaction partners were predicted for CRIP1: KTN1 and TPM2. To obtain comprehensive information on interacting proteins, a co-immunoprecipitation assay with a specific antibody against monocytic CRIP1 confirmed these as interaction partners and revealed further protein interactions with proteins that are crucial for monocyte migration and invasion behavior of monocytes by LC-MS/MS and Pathway and Process Enrichment Analysis, and immunoblot analysis. In an in vitro experiment with the human monocytic cell line THP-1, downregulation of CRIP1 by &amp;gt;70% significantly reduce the abundance of these proteins. In addition, in a transcriptome-based analysis of human monocytes, most of the genes encoding proteins of microtubule cytoskeleton organization (GO:0000226), actin filament and cytoskeleton organization (GO:0007015 and GO:0030036), Arp2/3 complex-mediated actin nucleation (GO:0034314), and cytoskeleton-dependent intracellular transport (GO:0030705) were associated with CRIP1 expression. Conclusion As CRIP1 expression in human monocytes has already been linked to hypertension, our results suggest that CRIP1 may play a role in the pathogenesis of this disease by regulating interactions with cytoskeletal proteins that lead to impaired monocyte migration and invasion behaviour. Further experiments are underway to translate our findings into clinical application.