Tumor Cell Migration Research Articles

Hsa_circ_0009092/miR-665/NLK signaling axis suppresses colorectal cancer progression via recruiting TAMs in the tumor microenvironment

BackgroundIt has been demonstrated that circularRNA (circRNAs) plays a critical role in various cancers. While the potential molecular mechanism of circRNAs in the progression of colorectal cancer (CRC) remains uncertain.MethodsDifferentially expressed circRNAs were identified by RNA sequencing. RT-qPCR detected the expression of circ_0009092, miR-665, and NLK in CRC tissues and cells. Functions of circ_0009092 on tumor cell proliferation, migration, and invasion were investigated by a series of in vitro assays. The underlying mechanism of circ_0009092 was explored by bioinformatics analysis, RNA immunoprecipitation (RIP) and luciferase assays. A co-culture assay in vitro was performed to detect the affection of circ_0009092 on macrophage recruitment in the tumor microenvironment (TME). A xenograft mouse model was used to explore the effect of circ_0009092 on tumor growth.ResultsCirc_0009092 was downregulated in CRCand predicted a good prognosis. Overexpression of circ_0009092 reduced tumor cell EMT, proliferation, migration, and invasion in vitro and in vivo. Mechanistically, circ_0009092 elevated the NLK expression via sponging miR-665 and suppressed the Wnt/β-catenin signaling pathway. EIF4EA3 induced circ_0009092 expression in CRC cells. In addition, NLK regulates phosphorylation and O-GlcNAcylation of STAT3 by binding to STAT3, thereby inhibiting CCL2 expression, in which it inhibits macrophage recruitment in the tumor microenvironment (TME).ConclusionEIF4A3 suppressed circ_0009092 biogenesis, whichinhibits CRC progression by sponging miR-665 to downregulate NLK. Circ_0009092/miR-665/NLK suppressed tumor EMT, proliferation, migration, and invasion by acting on the Wnt/β-catenin signaling pathway. NLK directly interacted with STAT3 and decreased the CCL2 expression, inhibiting the recruitment of tumor-associated macrophages (TAMs) in the TME. Our study provided novel insights into the roles of circ_0009092 as a novel promising prognostic and therapeutic target in CRC.

Lipid metabolism-related gene expression in the immune microenvironment predicts prognostic outcomes in renal cell carcinoma.

Rates of renal cell carcinoma (RCC) occurrence and mortality are steadily rising. In an effort to address this issue, the present bioinformatics study was developed with the goal of identifying major lipid metabolism biomarkers and immune infiltration characteristics associated with RCC cases. The Cancer Genome Atlas (TCGA) and E-MTAB-1980 were used to obtain matched clinical and RNA expression data from patients diagnosed with RCC. A LASSO algorithm and multivariate Cox regression analyses were employed to design a prognostic risk model for these patients. The tumor immune microenvironment (TIME) in RCC patients was further interrogated through ESTIMATE, TIMER, and single-cell gene set enrichment analysis (ssGSEA) analyses. Gene Ontology (GO), KEGG, and GSEA enrichment approaches were further employed to gauge the mechanistic basis for the observed results. Differences in gene expression and associated functional changes were then validated through appropriate molecular biology assays. Through the approach detailed above, a risk model based on 8 genes associated with RCC patient overall survival and lipid metabolism was ultimately identified that was capable of aiding in the diagnosis of this cancer type. Poorer prognostic outcomes in the analyzed RCC patients were associated with higher immune scores, lower levels of tumor purity, greater immune cell infiltration, and higher relative immune status. In GO and KEGG enrichment analyses, genes that were differentially expressed between risk groups were primarily related to the immune response and substance metabolism. GSEA analyses additionally revealed that the most enriched factors in the high-risk group included the stable internal environment, peroxisomes, and fatty acid metabolism. Subsequent experimental validation in vitro and in vivo revealed that the most significantly differentially expressed gene identified herein, ALOX5, was capable of suppressing RCC tumor cell proliferation, invasivity, and migration. In summary, a risk model was successfully established that was significantly related to RCC patient prognosis and TIME composition, offering a robust foundation for the development of novel targeted therapeutic agents and individualized treatment regimens. In both immunoassays and functional analyses, dysregulated lipid metabolism was associated with aberrant immunological activity and the reprogramming of fatty acid metabolic activity, contributing to poorer outcomes.

Label-Free Single-Cell SERS Detection and Fluorescence Imaging of Molecular Responses to Endoplasmic Reticulum Stress under Electrical Stimulation.

The endoplasmic reticulum (ER) is one of the most important organelles in eukaryotic cells, in which most proteins and lipids are synthesized to regulate complex cellular processes. Generally, the excessive accumulation of unfolded or misfolded proteins can disturb ER homeostasis and induce endoplasmic reticulum stress (ERS). Howbeit, the molecular stress responses within ERS and metastatic behaviors of tumor cells during electrical stimulation (ES) are still poorly investigated and remain a challenge. In this study, by the combined use of fluorescence imaging, ER-targeting plasmonic nanoprobes were developed to trace molecular stress response profiling within the ER during a constant-voltage ES process at ∼1 V based on label-free surface-enhanced Raman spectroscopy (SERS). The excess accumulation of β-misfolded proteins was found after the ES, leading to breaking of the ER homeostasis and further inducing mitochondrial dysfunction. Notably, the excessive stress of ER under ES can destroy the calcium ion balance and induce significant upregulation of calreticulin expression. Importantly, the content ratio of two kinds of cadherin between E-cadherin and N-cadherin was gradually improved with the voltages boosted. Meanwhile, the epithelial adhesion factor expression was ascended with voltages amplified, leading to inhibiting tumor cell migration at low voltages or death under higher voltages (∼1 V). This study provides cellular insights into the ES approach for tumor therapy and also provides a simple and effective method for detecting molecular stress responses in endoplasmic reticulum stress.

Anticancer effect of superoxide dismutase on canine mammary gland tumour in vitro.

Reactive oxygen species (ROS) have been shown to promote tumour growth and metastasis in human cell lines. The superoxide anion (•O2 - ) is produced during ROS formation and is involved in tumour cell signalling. Superoxide dismutase (SOD) has been applied to canine mammary gland tumours to investigate its antitumour effects in vitro. Cell proliferation, cell cycle cell migration assays, reverse transcription-quantitative polymerase chain reaction, and western blot analysis were performed to determine the effects of SOD on canine mammary tumour cell line. SOD treatment resulted in anti-proliferative effects and mediated cell cycle arrest in the canine mammary gland tumour cell lines (CIPp and CIPm). It also downregulated the expression of N-cadherin and Vimentin. The results confirmed that SOD inhibits tumour cell proliferation and migration, thus supporting the potential applications of SOD as a chemotherapeutic agent for canine mammary gland tumours.

Multi-Enzyme Co-Expressed Nanomedicine for Anti-Metastasis Tumor Therapy by Up-Regulating Cellular Oxidative Stress and Depleting Cholesterol.

Tumor cells movement and migration are inseparable from the integrity of lipid rafts and the formation of lamellipodia, and lipid rafts are also a prerequisite for the formation of lamellipodia. Therefore, destroying the lipid rafts is an effective strategy to inhibit tumor metastasis. Herein, a multi-enzyme co-expressed nanomedicine: cholesterol oxidase (CHO) loaded Co─PN3 single-atom nanozyme (Co─PN3 SA/CHO) that can up-regulate cellular oxidative stress, disrupt the integrity of lipid rafts, and inhibit lamellipodia formation to induce anti-metastasis tumor therapy, is developed. In this process, Co─PN3 SA can catalyze oxygen (O2 )and hydrogen peroxide (H2 O2 ) to generate reactive oxygen species (ROS) via oxidase-like and Fenton-like properties. The doping of P atoms optimizes the adsorption process of the intermediate at the active site and enhances the ROS generation properties of nanomedicine. Meantime, O2 produced by catalase-like catalysis can combine with excess cholesterol to generate more H2 O2 under CHO catalysis, achieving enhanced oxidative damage to tumor cells. Most importantly, cholesterol depletion in tumor cells also disrupts the integrity of lipid rafts and inhibits the formation of lamellipodia, greatly inhibiting the proliferation and metastasis of tumor cells. This strategy by up-regulating cellular oxidative stress and depleting cellular cholesterol constructs a new idea for anti-metastasis-oriented cancer therapy strategies.

Dynamics of Fibril Collagen Remodeling by Tumor Cells: A Model of Tumor-Associated Collagen Signatures.

Many solid tumors are characterized by a dense extracellular matrix (ECM) composed of various ECM fibril proteins. These proteins provide structural support and a biological context for the residing cells. The reciprocal interactions between growing and migrating tumor cells and the surrounding stroma result in dynamic changes in the ECM architecture and its properties. With the use of advanced imaging techniques, several specific patterns in the collagen surrounding the breast tumor have been identified in both tumor murine models and clinical histology images. These tumor-associated collagen signatures (TACS) include loosely organized fibrils far from the tumor and fibrils aligned either parallel or perpendicular to tumor colonies. They are correlated with tumor behavior, such as benign growth or invasive migration. However, it is not fully understood how one specific fibril pattern can be dynamically remodeled to form another alignment. Here, we present a novel multi-cellular lattice-free (MultiCell-LF) agent-based model of ECM that, in contrast to static histology images, can simulate dynamic changes between TACSs. This model allowed us to identify the rules of cell-ECM physical interplay and feedback that guided the emergence and transition among various TACSs.

ASIC1a contributes to the epithelial-mesenchymal transformation of breast cancer by activating the Ca2+ /β-catenin pathway.

Breast cancer is the most common cancer in the world, with metastasis being one of the leading causes of death among patients. The acidic environment of breast cancer tissue promotes tumor cell invasion and migration by inducing epithelial-mesenchymal transformation (EMT) in tumor cells, but the exact mechanisms are not yet fully understood. This study investigated the expression of acid-sensitive ion channel 1a (ASIC1a) in breast cancer tissue samples and explored the mechanisms by which ASIC1a mediates the promotion of EMT in breast cancer cells in an acidic microenvironment through in vivo and in vitro experiments. The results showed that first, the expression of ASIC1a was significantly upregulated in breast cancer tissue and was correlated with the TNM (tumor node metastasis) staging of breast cancer. Furthermore, ASIC1a expression was higher in tumors with lymph node metastasis than in those without. Second, the acidic microenvironment promoted [Ca2+ ]i influx via ASIC1a activation and regulated the expression of β-catenin, Vimentin, and E-cadherin, thus promoting EMT in breast cancer cells. Inhibition of ASIC1a activation with PcTx-1 could suppress EMT in breast cancer cells. Finally, in vivo studies also showed that inhibition of ASIC1a could reduce breast cancer metastasis, invasion, and EMT. This study suggests that ASIC1a expression is associated with breast cancer staging and metastasis. Therefore, ASIC1a may become a new breast cancer biomarker, and the elucidation of the mechanism by which ASIC1a promotes EMT in breast cancer under acidic microenvironments provides evidence for the use of ASIC1a as a molecular target for breast cancer treatment.

Sulfated Triterpene Glycosides from the Far Eastern Sea Cucumber Cucumaria djakonovi: Djakonoviosides C1, D1, E1, and F1; Cytotoxicity against Human Breast Cancer Cell Lines; Quantitative Structure-Activity Relationships.

Four new mono- and trisulfated triterpene penta- and tetraosides, djakonoviosides C1 (1), D1 (2), E1 (3), and F1 (4) were isolated from the Far Eastern sea cucumber Cucumaria djakonovi (Cucumariidae, Dendrochirotida), along with six known glycosides found earlier in other Cucumaria species. The structures of unreported compounds were established on the basis of extensive analysis of 1D and 2D NMR spectra as well as by HR-ESI-MS data. The set of compounds contains six different types of carbohydrate chains including two new ones. Thus, djakonovioside C1 (1) is characterized by xylose as the second residue, that was a branchpoint in the pentasaccharide chain. Meanwhile, only quinovose and rarely glucose have been found earlier in pentasaccharide chains branched at C-2 of the second sugar unit. Djakonovioside E1 (3) is characterized by a tetrasaccharide trisulfated chain, with glucose as the second residue. So, in the series of isolated glycosides, three types of sugars in the second position were presented: the most common, quinovose-in six compounds; glucose-in three substances; and the rare xylose-in one glycoside. The set of aglycones was composed of holostane- and non-holostane-type polycyclic systems; the latter comprised normal and reduced side chains. Noticeably, isokoreoside A (9), isolated from C. djakonovi, was a single glycoside having a 9(11)-double bond, indicating two oxidosqualenecyclases are operating in the process of the biosynthesis of aglycones. Some of the glycosides from C. djakonovi, which were characterized by pentasaccharide branched chains containing one to three sulfate groups, are chemotaxonomic features of the representatives of the genus Cucumaria. The assortment of sugar parts of Cucumaria's glycosides was broadened with previously undescribed penta- and tetrasaccharide moieties. The metabolic network of sugar parts and aglycones is constructed based on biogenetic relationships. The cytotoxic action of compounds 1-10, isolated from C. djakonovi, against human breast cancer cell lines was investigated along with the hemolytic activity. Erythrocytes were, as usual, more sensitive to the membranolytic action of the glycosides than cancer cells. The triple-negative breast cancer MDA-MB-231 cell line was more vulnerable to the action of glycosides in comparison with the other tested cancer cells, while the MCF-7 cell line was less susceptible to cytotoxic action. Djakonovioside E1 (3) demonstrated selective action against ER-positive MCF-7 and triple-negative MDA-MB-231 cell lines, while the toxic effect in relation to normal mammary epithelial cells (MCF-10A) was absent. Cucumarioside A2-5 (6) inhibited the formation and growth of colonies of cancer cells to 44% and tumor cell migration to 85% of the control. Quantitative structure-activity relationships (QSAR) were calculated on the basis of the correlational analysis of the physicochemical properties and structural features of the glycosidic molecules and their membranolytic activity. QSAR revealed the extremely complex nature of such relationships, but these calculations correlated well with the observed SAR.

OSM May Serve as a Biomarker of Poor Prognosis in Clear Cell Renal Cell Carcinoma and Promote Tumor Cell Invasion and Migration.

Currently, the role of oncostatin M (OSM) in clear cell renal cell carcinoma (ccRCC) has not been investigated. This study will explore the impact of OSM on ccRCC expression, prognosis, and cell function. In this study, we used The Cancer Genome Atlas (TCGA) database to evaluate OSM expression characteristics, pathogenic factor distribution, and prognostic aspects in ccRCC. We also combined this analysis with qRT-PCR to verify OSM mRNA expression levels at the tissue level. Then, the effects of OSM on the proliferation, invasion, and migration abilities of ccRCC cells were explored through CCK8, Transwell, Western blotting, and immunofluorescence experiments. Finally, the oncogenic mechanisms associated with OSM in ccRCC were explored through signaling pathway enrichment and single-cell analysis. The results demonstrated that OSM was significantly more expressed in ccRCC than in normal tissues. According to the survival analysis, OSM in ccRCC was considerably worse in the group with high expression than in the group with low expression. Also, the univariate and multivariate Cox analyses of clinical characteristics show that OSM in ccRCC may be able to predict a poor prognosis on its own as a biomarker. In vitro cellular experiments demonstrated that high OSM expression had no discernible impact on ccRCC cell proliferation compared to the control group, but it did promote tumor cell invasion and migration. Signaling pathways and single-cell analysis revealed that OSM might promote ccRCC invasion and migration through M2 macrophages. In conclusion, OSM may serve as an independent poor prognostic biomarker in ccRCC and promote tumor cell invasion and migration. This discovery is expected to provide a new therapeutic target for patients with recurrent and metastatic ccRCC.

Remodeling of intestinal epithelium derived extracellular vesicles by nanoparticles and its bioeffect on tumor cell migration

Extracellular vesicles (EVs) are an effective tool to elucidate the bioeffect of nanomedicines. To clarify the interaction between oral nanomedicines and intestinal epithelial cells, and their bioeffects on downstream cells, polystyrene nanoparticles (PS-NPs) with different sizes were used as the model nanomedicines for EVs induction. Caco-2 monolayers were selected as the model of the intestinal epithelium and DLD-1 cells as the colorectal cancer model proximal to the gastrointestinal tract. It is found that compared with small-sized (25, 50, 100 nm) PS-NPs, the large-sized (200 and 500 nm) exhibited higher co-localization with multivesicular bodies and lysosomes, and more significant reduction of lysosomal acidification in Caco-2 cells. Proteomic and western-blotting analysis showed that the EVs remodeled by large-sized PS-NPs exhibited a higher extent of protein expression changes. The in vitro and in vivo signaling pathway detection in DLD-1 cells and DLD-1 cell xenograft nude mice showed that the remodeled EVs by large-sized PS-NPs inhibited the activation of multiple signaling pathways including Notch3, EGF/EGFR, and PI3K/Akt pathways, which resulted in the inhibition of tumor cell migration. These results primarily clarify the regulation mechanisms of nanomedicines-EVs-receptor cells chain. It provides a new perspective for the rational design and bioeffect evaluation of oral drug nanomaterials and sets up the fundamental knowledge for novel tumor therapeutics in the future.

Hypoxia-induced circRTN4IP1 promotes progression and glycolysis of hepatocellular carcinoma cells.

Hypoxia is one of the hallmarks of solid tumors, especially in hepatocellular carcinoma (HCC). CircRNAs are reported to be tightly connected to hypoxia and also have essential roles in cancer progression. However, many circRNAs implicated in hypoxia-mediated HCC progression are still unclear and require further exploration. In this study, a hypoxia cell model was structured by exposing cells to hypoxia conditions (1% O2) and normoxia conditions (21% O2) as a control. The effects of hypoxia and normoxia on cell viability, migration, invasion, and glycolysis were examined. The expressions of circRNARTN4IP1 under hypoxia were identified. Finally, molecular mechanisms and biological function of circRTN4IP1 were explored. We confirmed that hypoxia treatment facilitated capacities of proliferation, migration, invasion, and glycolysis in tumor cells. Hypoxia induced a significant increase expression of circRTN4IP1 in cells. Functionally, knockdown of circRTN4IP1 inhibited cell malignant progression and glycolysis under hypoxia HCC cells. Mechanistically, HIF1A targeted the promoter region of circRTN4IP1 and positively regulated the expression of circRTN4IP1. In addition, circRTN4IP1 targeted miR-532-5p/G6PC3 axis. In short, hypoxia induced activation of the HIF1A/circRTN4IP1/miR-532-5p/G6PC3 signaling axis, which promoted proliferation, migration, invasion, and glycolysis of HCC cells. This study may reveal a possible mechanism driving the progression of hypoxia HCC, so as to find potential effective candidates for targeting hypoxia microenvironment therapy.

Crucial role of hsa-mir-503, hsa-mir-1247, and their validation in prostate cancer.

Prostate cancer (PC) is a common urinary system malignancy, and advanced PC patients had a poor prognosis due to recurrence or distant metastasis. Therefore, it's imperative to reveal more details in tumorigenesis and prognosis of PC patients. The miRNA and mRNA expression profile data of 485 PC patients were obtained from The Cancer Genome Atlas database. The univariate Cox regression was applied to screen miRNAs relating to prognosis of PC. Then miRTarBase was used to predict target mRNAs of miRNAs. The hsa-mir-503/hsa-mir-1247 knockdown in 22RV1 cells was established to evaluate the effect of these two miRNAs on tumor cell migration and invasion ability. Flow cytometry was used to detect the effect of hsa-mir-503/hsa-mir-1247 knockdown on 22RV1 apoptosis rate. Univariate Cox regression analysis identified hsa-mir-503 as a poor and hsa-mir-1247 as a favorable prognostic marker. Totally 649 target mRNAs were screened, among which DUSP19, FGF2, and SLC2A5 had a negative correlation with hsa-mir-503, while FGF2 and VSTM4 had a positive correlation with hsa-mir-1247. In 22RV1 cells, hsa-mir-503 was up-regulated, and hsa-mir-1247 was down-regulated. hsa-mir-503 knockdown attenuated the migration and invasion of 22RV1 cells, while hsa-mir-1247 knockdown exhibited the opposite effect. In addition, hsa-mir-503 knockdown promoted 22RV1 cell apoptosis. hsa-mir-1247 overexpression significantly inhibited the tumor growth of PC in vivo. Herein, we demonstrated that hsa-mir-503 and hsa-mir-1247 could serve as new prognostic markers of PC, and hsa-mir-1247 had great potential to inhibit PC progression by suppressing the migration and invasion ability in vitro and in vivo.

Prediction and related genes of cancer distant metastasis based on deep learning

Cancer metastasis is one of the main causes of cancer progression and difficulty in treatment. Genes play a key role in the process of cancer metastasis, as they can influence tumor cell invasiveness, migration ability and fitness. At the same time, there is heterogeneity in the organs of cancer metastasis. Breast cancer, prostate cancer, etc. tend to metastasize in the bone. Previous studies have pointed out that the occurrence of metastasis is closely related to which tissue is transferred to and genes. In this paper, we identified genes associated with cancer metastasis to different tissues based on LASSO and Pearson correlation coefficients. In total, we identified 45 genes associated with bone metastases, 89 genes associated with lung metastases, and 86 genes associated with liver metastases. Through the expression of these genes, we propose a CNN-based model to predict the occurrence of metastasis. We call this method MDCNN, which introduces a modulation mechanism that allows the weights of convolution kernels to be adjusted at different positions and feature maps, thereby adaptively changing the convolution operation at different positions. Experiments have proved that MDCNN has achieved satisfactory prediction accuracy in bone metastasis, lung metastasis and liver metastasis, and is better than other 4 methods of the same kind. We performed enrichment analysis and immune infiltration analysis on bone metastasis-related genes, and found multiple pathways and GO terms related to bone metastasis, and found that the abundance of macrophages and monocytes was the highest in patients with bone metastasis.

ESCO2 promotes hypopharyngeal carcinoma progression in a STAT1-dependent manner

BackgroundThe establishment of sister chromatid cohesion N-acetyltransferase 2 (ESCO2) is involved in the development of multiple malignancies. However, its role in hypopharyngeal carcinoma (HPC) progression remains uncharacterized.MethodsThis study employed bioinformatics to determine the ESCO2 expression in head and neck squamous cell carcinoma (HNSC) and normal tissues. In vitro cell proliferation, migration, apoptosis, and/or cell cycle distribution assays were used to determine the function of ESCO2 and its relationship with STAT1. Xenograft models were established in nude mice to determine ESCO2 in HPC growth in vivo. Co-immunoprecipitation/mass spectrometry (Co-IP/MS) was conducted to identify the potential ESCO2 binding partners.ResultsWe found that ESCO2 expression was elevated in HNSC tissues, and ESCO2 depletion suppressed tumor cell migration in vitro and inhibited tumor growth in vitro and in vivo. Co-IP/MS and immunoblotting assays revealed the interaction between ESCO2 and STAT1 in HPC cells. STAT1-overexpression compromised ESCO2-mediated suppressive effects on HPC cell proliferation, viability, and migration.ConclusionsThese findings suggest that ESCO2 is crucial in promoting HPC malignant progression through the STAT1 pathway and provides novel therapeutic targets for HPC treatment.

CD15 Is a Risk Predictor and a Novel Target in Clear Cell Renal Cell Carcinoma

Introduction: Tumor cells use adhesion molecules like CD15 or sialylCD15 (sCD15) for metastatic spreading. We analyzed the expression of CD15 and sCD15 in clear cell renal cell carcinoma (ccRCC) regarding prognosis. Methods: A tissue microarray containing tissue specimens of 763 patients with ccRCC was immunohistochemically stained for CD15 and sCD15, their expression quantified using digital image analysis, and the impact on patients’ survival analyzed. The cell lines 769p and 786o were stimulated with CD15 or control antibody in vitro and the effects on pathways activating AP-1 and tumor cell migration were examined. Results: ccRCC showed a broad range of CD15 and sCD15 expression. A high CD15 expression was significantly associated with favorable outcome (p < 0.01) and low-grade tumor differentiation (p < 0.001), whereas sCD15 had no significant prognostic value. Tumors with synchronous distant metastasis had a significantly lower CD15 expression compared to tumors without any (p < 0.001) or with metachronous metastasis (p < 0.01). Tumor cell migration was significantly reduced after CD15 stimulation in vitro, but there were no major effects on the activating pathways of AP-1. Conclusion: CD15, but not sCD15, qualifies as a biomarker for risk stratification and as an interesting novel target in ccRCC. Moreover, the data indicate a contribution of CD15 to metachronous metastasis. Further research is warranted to decipher the intracellular pathways of CD15 signaling in ccRCC in order to characterize the CD15 effects on ccRCC more precisely.

Design, Synthesis, and Antitumor Activity Evaluation of Proteolysis-Targeting Chimeras as Degraders of Extracellular Signal-Regulated Kinases 1/2.

Inhibition of the extracellular signal-regulated kinases 1/2 (ERK1/2) alone or in combination with other targets has emerged as a promising treatment strategy for a variety of human tumors. In addition to the development of inhibitors, the development of ERK1/2 degraders is an alternative approach to decrease its activity. We synthesized proteolysis-targeting chimeras (PROTACs) as effective ERK1/2 degraders, among which B1-10J showed high degradative activity, with DC50 of 102 nM and cytotoxic IC50 of 2.2 μM against HCT116 cells. Moreover, B1-10J dose-dependently inhibited tumor cell migration. Xenograft experiments in nude mice demonstrated that B1-10J inhibited HCT116 tumor cell growth and achieved significant regression of tumors at a daily dose of 25 mg/kg.

Conformational States of the CXCR4 Inhibitor Peptide EPI-X4-A Theoretical Analysis.

EPI-X4, an endogenous peptide inhibitor, has exhibited potential as a blocker of CXCR4-a G protein-coupled receptor. This unique inhibitor demonstrates the ability to impede HIV-1 infection and halt CXCR4-dependent processes such as tumor cell migration and invagination. Despite its promising effects, a comprehensive understanding of the interaction between EPI-X4 and CXCR4 under natural conditions remains elusive due to experimental limitations. To bridge this knowledge gap, a simulation approach was undertaken. Approximately 150,000 secondary structures of EPI-X4 were subjected to simulations to identify thermodynamically stable candidates. This simulation process harnessed a self-developed reactive force field operating within the ReaxFF framework. The application of the Two-Phase Thermodynamic methodology to ReaxFF facilitated the derivation of crucial thermodynamic attributes of the EPI-X4 conformers. To deepen insights, an ab initio density functional theory calculation method was employed to assess the electrostatic potentials of the most relevant (i.e., stable) EPI-X4 structures. This analytical endeavor aimed to enhance comprehension of the inhibitor's structural characteristics. As a result of these investigations, predictions were made regarding how EPI-X4 interacts with CXCR4. Two pivotal requirements emerged. Firstly, the spatial conformation of EPI-X4 must align effectively with the CXCR4 receptor protein. Secondly, the functional groups present on the surface of the inhibitor's structure must complement the corresponding features of CXCR4 to induce attraction between the two entities. These predictive outcomes were based on a meticulous analysis of the conformers, conducted in a gaseous environment. Ultimately, this rigorous exploration yielded a suitable EPI-X4 structure that fulfills the spatial and functional prerequisites for interacting with CXCR4, thus potentially shedding light on new avenues for therapeutic development.

Modelling the disease: H2S-sensitivity and drug-resistance of triple negative breast cancer cells can be modulated by embedding in isotropic micro-environment

Three-dimensional (3D) cell culture systems provide more physiologically relevant information, representing more accurately the actual microenvironment where cells reside in tissues. However, the differences between the tissue culture plate (TCP) and 3D culture systems in terms of tumour cell growth, proliferation, migration, differentiation and response to the treatment have not been fully elucidated. Tumoroid microspheres containing the MDA-MB 231 breast cancer cell line were prepared using either tunable PEG-fibrinogen (PFs) or tunable PEG-silk fibroin (PSFs) hydrogels, respectively named MDAPFs and MDAPSFs. The cancer cells in the tumoroids showed changes both in globular morphology and at the protein expression level. A decrease of both Histone H3 acetylation and cyclin D1 expression in all 3D systems, compared to the 2D cell culture, was detected in parallel to changes of the matrix stiffness. The effects of a glutathionylated garlic extract (GSGa), a slow H2S-releasing donor, were investigated on both tumoroid systems. A pro-apoptotic effect of GSGa on tumour cell growth in 2D culture was observed as opposed to a pro-proliferative effect apparent in both MDAPFs and MDAPSFs. A dedicated ad hoc 3D cell migration chip was designed and optimized for studying tumour cell invasion in a gel-in-gel configuration. An anti-cell-invasion effect of the GSGa was observed in the 2D cell culture, whereas a pro-migratory effect in both MDAPFs and MDAPSFs was observed in the 3D cell migration chip assay. An increase of cyclin D1 expression after GSGa treatment was observed in agreement with an increase of the cell invasion index. Our results suggest that the “dimensionality” and the stiffness of the 3D cell culture milieu can change the response to both the gasotransmitter H2S and doxorubicin due to differences in both H2S diffusion and changes in protein expression. Moreover, we uncovered a direct relation between the cyclin D1 expression and the stiffness of the 3D cell culture milieu, suggesting the potential causal involvement of the cyclin D1 as a bio-marker for sensitivity of the tumour cells to their matrix stiffness. Therefore, our hydrogel-based tumoroids represent a valid tunable model for studying the physically induced transdifferentiation (PiT) of cancer cells and as a more reliable and predictive in vitro screening platform to investigate the effects of anti-tumour drugs.

MODL-11. EXPLORING THE THERAPEUTIC POTENTIAL OF YAP-TEAD INHIBITORS FOR TARGETING GBM CELL MIGRATION AT THE INFILTRATIVE MARGIN

Abstract Glioblastoma (GBM) is an aggressive brain tumor with a median overall survival of only 12-15 months. Despite remarkable cellular and molecular heterogeneity, all GBM subtypes exhibit an infiltrating growth pattern which leads to incomplete surgical resection and inevitable recurrence, representing a major obstacle for effective treatment and an unmet need for therapy. Our research project aims to investigate the efficacy of YAP-TEAD inhibitors, including Verteporfin (VP), on migratory properties of GBM cells at the interface between the tumor core and the infiltrative tumor margin. Recently, several groups have implicated Hippo pathway YAP-TEAD activation in GBM growth and mesenchymal transition, and our laboratory demonstrated anti-migration and anti-invasion efficacy of the YAP-TEAD inhibitor VP across GBM subtypes, in patient-derived cell and xenograft (PDX) mouse models. Here we set to develop an ex vivo model for high-throughput testing of various YAP-TEAD inhibitors using organotypic slice cultures obtained from PDX and primary glioblastoma resection specimens, focusing on tumor areas around the infiltrative margin. Tumor cells within 250µ-thick organotypic slices were infected with GFP labeled retrovirus and subjected to confocal time-lapse imaging 72-96 hours later. Slices were treated first with vehicle (DMSO) for 4 hours, and subsequently with the YAP-TEAD inhibitor VP for 4 hours. To assess drug impact on tumor cell migration, we gathered imaging data on migration properties such as speed and directionality. Analysis of a PDX model generated from a de-novo GBM surgical resection (G-17969) revealed significantly decreased distance traveled in GFP+ cells treated with VP, compared to DMSO (p-value = 0.0002, n=50 cells). These pilot results corroborate other studies demonstrating anti-invasion properties of VP, contributing towards our goal of establishing a preclinical pipeline for rapid, high-throughput screening of anti-invasion drug inhibitors ex vivo. Key words: Glioblastoma, cell migration, YAP-TEAD inhibitors, time-lapse imaging

CNSC-29. NKCC1 SIGNALING IN GLIOBLASTOMA REGULATES NEURONAL HYPEREXCITABILITY THROUGH GABAERGIC TONE

Abstract Over 90% of patients with glioma experience tumor-associated epilepsy, however first-line treatment fails to control seizures in most patients. Thus, there is a critical need to identify novel drivers of glioblastoma-induced neuronal hyperexcitability. We therefore used high-density electrode arrays to record local field potentials in vivo from gliomas. We then developed cerebral organoid and mouse models using primary glioblastoma cells from patients and neurons derived from induced pluripotent stem cells. Single-cell RNA sequencing (13,730 cells analyzed) in this system identified genes elevated in glioma-intrinsic neurons, including the sodium-potassium-chloride co-transporter 1 (NKCC1), which mediates chloride (Cl-) influx into cells. This elevated NKCC1 expression and subsequent increase of intracellular Cl- sets the reversal potential of GABAAR-mediated currents promoting depolarizing excitatory GABAergic responses. Electrophysiological analysis of glioma-neuron co-cultures using multi-electrode array and live-cell calcium imaging demonstrated increased network burst synchrony in the presence of NKCC1-high expressing tumor cells. Single-nucleus sequencing (54,000 cells analyzed) of embryonic neurons in co-culture with GBM cells further confirmed the elevated expression of circuit assembly genes including neuronal NKCC1. Live-cell imaging of iPSC-induced neuron organoids co-cultured with NKCC1-high-and low-GBM cells revealed increased migration and integration of NKCC1-elevated tumor cells. Mechanistic and functional studies validating therapeutic vulnerabilities to NKCC1 silencing by shRNA knockdown and by the FDA-approved diuretic drug, bumetanide was performed in vitro. The reduction in neuronal synchrony induced by NKCC1 downregulation correlated with a decrease in extracellular GABA and glutamate, suggesting a strong association between excitatory GABAergic and glutamate signaling with neuronal hypersynchrony in glioblastoma. Patient-derived xenograft in vivo experiments evaluated the effect of NKCC1 on tumor growth and progression and NKCC1-high GBM cells exhibited greater tumor burden compared to control mice. Collectively, these findings reveal a novel mechanism driving neuronal hyperactivity in glioblastoma and identifies potential therapeutic vulnerabilities for the treatment of glioma-associated epilepsy.