Spheroid Invasion Assay Research Articles

Effect of Sex Steroid Hormones on Tongue Cancer Cells In Vitro.

Tongue cancer is more common in men than in women. Yet the effects of sex steroid hormones on the behaviour of oral tongue squamous cell carcinoma (OTSCC) are not well known. Matrix metalloproteinase 8 (MMP8) is expressed in OTSCC and can degrade estrogen receptors (ERs). Western blot was used to examine the levels of ERβ in OTSCC cell lines (HSC-3 and SCC-25). We evaluated the effects of estradiol and dihydrotestosterone (DHT) on HSC-3 and SCC-25 cell migration, invasion and viability. The effect of estradiol on the invasion of MMP8-overexpressing (MMP8+) and empty vector HSC-3 cells was examined using 3D spheroid invasion assay. Both HSC-3 and SCC-25 cells expressed ERβ. In scratch assay, estradiol, but not DHT, reduced the migration and invasion of HSC-3 and SCC-25 cells. MMP8+ HSC-3 cells showed weaker invasion than empty vector cells, in line with previous reports. However, MMP8 overexpression did not alter the effect of estradiol on HSC-3 cell invasion in spheroid assay. Estradiol inhibited the migration and invasion of OTSCC cells, whereas DHT had no effect. Our data suggest that MMP8 does not modulate the effect of estradiol in OTSCC cells. However, the sex difference in OTSCC incidence might partly be due to protective actions of estradiol in epithelial cell carcinogenesis.

CAFs and Cancer Cells Co-Migration in 3D Spheroid Invasion Assay.

In many solid tumors, collective cell invasion prevails over single-cell dissemination strategies. Collective modes of invasion often display specific front/rear cellular organization, where invasive leader cells arise from cancer cell populations or the tumor stroma. Collective invasion involves coordinated cellular movements which require tight mechanical crosstalk through specific combinations of cell-cell interactions and cell-matrix adhesions.Cancer Associated Fibroblasts (CAFs) have been recently reported to drive the dissemination of epithelial cancer cells through ECM remodeling and direct intercellular contact. However, the cooperation between tumor and stromal cells remains poorly understood. Here we present a simple spheroid invasion assay to assess the role of CAFs in the collective migration of epithelial tumor cells. This method enables the characterization of 3D spheroid invasion patterns through live cell fluorescent labeling combined with spinning disc microscopy. When embedded in extracellular matrix, the invasive strands of spheroids can be tracked and leader/follower organization of CAFs and cancer cells can be quantified.

2D and 3D in vitro assays to quantify the invasive behavior of glioblastoma stem cells in response to SDF-1α.

Invasion is a hallmark of cancer and therefore in vitro invasion assays are important tools in cancer research. We aimed to describe in vitro 2D transwell assays and 3D spheroid assays to quantitatively determine the invasive behavior of glioblastoma stem cells in response to the chemoattractant SDF-1α. Matrigel was used as a matrix in both assays. We demonstrated quantitatively that SDF-1α increased invasive behavior of glioblastoma stem cells in both assays. We conclude that the 2D transwell invasion assay is easy to perform, fast and less complex whereas the more time-consuming 3D spheroid invasion assay is physiologically closer to the in vivo situation.

Abstract A21: TRIM29 promotes invasion and migration of bladder cancer cells by regulating myosin-intermediate filament network and focal adhesion

Abstract Development of invasive progression in human bladder cancer is a key determinant of patient outcomes. Therefore, understanding the drivers of bladder cancer invasive progression is critical to improving patient care. We have previously shown that TRIM29 (also known as ataxia-telangiectasia group D complementing gene, ATDC) is highly expressed in muscle-invasive bladder cancer and plays an important role in driving tumor formation and invasion in vitro and in vivo. TRIM29 is upregulated by TP63 in basal subtype tumors, but the mechanism by which TRIM29 promotes tumor invasion was unknown. To elucidate the mechanism by which TRIM29 drives bladder cancer invasion, we developed a 3-D bladder cancer spheroid invasion assay that allows observation of the invasive process of cancer spheroids in a real-time fashion and observation of protein expression using confocal microscopy. Using this system, we confirmed that TRIM29 is required for bladder cancer stromal invasion. To determine if TRIM29 is specifically required for cell migration, which is an important ability contributing to invasive behavior, scratch assays were performed and we found TRIM29 is required for migration. Cancer cell migration requires modulation of cell adhesion and actin/myosin activation. To examine the role of TRIM29 in cell adhesion and motility, gene knockout or knockdowns of TRIM29 were generated in human bladder cancer cell lines (UM-UC5, UC9, UC13, UC14). Interestingly, TRIM29 KO/KD downregulates the expression of keratin 14 (KRT14), a member of type I keratin family that constitutes intermediate filament (IF) network, as well as myosin IIA (MYH9), a non-muscle motor protein that is essential for cell adhesion and migration. Using immunofluorescent confocal microscopy, we found that TRIM29 also modulates formation of focal adhesion plaques in bladder cancer during cell migration. Taken together, these findings suggest a novel mechanism by which TRIM29 regulates bladder cancer cell motility and invasion. Citation Format: Yin Wang, Erica R. Gumkowski, Phillip L. Palmbos. TRIM29 promotes invasion and migration of bladder cancer cells by regulating myosin-intermediate filament network and focal adhesion [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A21.

Omeprazole Inhibits Glioblastoma Cell Invasion and Tumor Growth.

Background: The aryl hydrocarbon receptor (AhR) is expressed in gliomas and the highest staining is observed in glioblastomas. A recent study showed that the AhR exhibited tumor suppressor-like activity in established and patient-derived glioblastoma cells and genomic analysis showed that this was due, in part, to suppression of CXCL12, CXCR4 and MMP9. Methods: Selective AhR modulators (SAhRMs) including AhR-active pharmaceuticals were screened for their inhibition of invasion using a spheroid invasion assay in patient-derived AhR-expressing 15-037 glioblastoma cells and in AhR-silenced 15-037 cells. Invasion, migration and cell proliferation were determined using spheroid invasion, Boyden chambers and scratch assay, and XTT metabolic assays for cell growth. Changes in gene and gene product expression were determined by real-time PCR and Western blot assays, respectively. In vivo antitumorigenic activity of omeprazole was determined in SCID mice bearing subcutaneous patient-derived 15-037 cells. Results: Results of a screening assay using patient-derived 15-037 cells (wild-type and AhR knockout) identified the AhR-active proton pump inhibitor omeprazole as an inhibitor of glioblastoma cell invasion and migration only AhR-expressing cells but not in cells where the AhR was downregulated. Omeprazole also enhanced AhR-dependent repression of the pro-invasion CXCL12, CXCR4 and MMP9 genes, and interactions and effectiveness of omeprazole plus temozolomide were response-dependent. Omeprazole (100 mg/kg/injection) inhibited and delayed tumors in SCID mice bearing patient-derived 15-037 cells injected subcutaneously. Conclusion: Our results demonstrate that omeprazole enhances AhR-dependent inhibition of glioblastoma invasion and highlights a potential new avenue for development of a novel therapeutic mechanism-based approach for treating glioblastoma.

The architecture of co-culture spheroids regulates tumor invasion within a 3D extracellular matrix.

Tumor invasion, the process by which tumor cells break away from their primary tumor and gain access to vascular systems, is an important step in cancer metastasis. Most current 3D tumor invasion assays consisted of single tumor cells embedded within an extracellular matrix (ECM). These assays taught us much of what we know today on how key biophysical (e.g. ECM stiffness) and biochemical (e.g. cytokine gradients) parameters within the tumor microenvironment guided and regulated tumor invasion. One limitation of the single tumor cell invasion assay was that it did not account for cell-cell adhesion within the tumor. In this article, we developed a micrometer scale 3D co-culture spheroid invasion assay that was compatible with microscopic imaging. Micrometer scale co-culture spheroids (1:1 ratio of metastatic breast cancer MDA-MB-231 and non-tumorigenic epithelial MCF-10A cells) were made using an array of microwells, and then were embedded within a collagen matrix in a microfluidic platform. Real time imaging of tumor spheroid invasion revealed that the spatial distribution of the two cell types within the tumor spheroid critically regulated tumor invasion. This work linked tumor architecture with tumor invasion and highlighted the importance of the biophysical cues within the bulk of the tumor in tumor invasion.

MiR-423 Promotes Breast Cancer Invasion by Activating NF-κB Signaling.

ObjectiveBreast cancer has become the most common malignancy among women worldwide; therefore, novel diagnostic and prognostic markers and therapeutic targets are urgently required. NF-κB signaling plays a pivotal role in enhancing breast cancer malignant phenotypes, especially cancer invasion and metastasis, which is the main cause of death in cancer patients. TNIP2, an important inhibitor of the NF-κB pathway, is known to involve a negative feedback loop of the NF-κB signaling cascade and to regulate tumor aggressiveness in various cancer types. However, the mRNA level of TNIP2 is barely altered in breast cancer; thus, the mechanism that regulates TNIP2 in breast cancer still needs to be elucidated.MethodsWe analyzed the expression and prognosis of miR-423 in a TCGA BRCA miRNA cohort and in clinical specimens. We detected the invasive capacity through a Matrigel-coated Transwell penetration assay, a three-dimensional (3D) spheroid invasion assay and a wound healing assay. Then, we applied luciferase assays, real-time PCR assays and Western blotting to further study the mechanism.ResultsIn this study, analysis of the TCGA BRCA miRNA cohort and clinical specimens demonstrated that miR-423 was upregulated in human breast cancers and was positively correlated with clinical stage, poor overall survival and metastasis classification. Moreover, the invasiveness of breast cancer cells was enhanced by ectopic expression of miR-423 and inhibited by miR-423 downregulation. Mechanistically, upregulation of miR-423 led to activation of the NF-κB signaling pathway and elevated expression of snail and twist, while repression of miR-423 inhibited this pathway. Furthermore, the results indicated that TNIP2 is a target gene of miR-423, and suppression of TNIP2 resulted in increased invasiveness in miR-423-silenced cells.ConclusionOur results suggest that miR-423 is a crucial factor that enhances breast cancer cell invasion through the NF-κB signaling pathway and shed light on miR-423 as a promising prognostic and therapeutic marker for metastatic breast cancer.

A rapid quantification of invasive phenotype in head and neck squamous cell carcinoma: A novel 3D pillar array system.

The widely used in vitro invasion assays for head and neck squamous cell carcinoma (HNSCC) are wound healing, transwell, and organotypic assays. However, these are still lab-intensive and time-consuming tasks. For the rapid detection and high throughput screening of invasiveness in 3D condition, we propose a novel spheroid invasion assay using commercially available pillar platform system. Using the pillar-based spheroid invasion assay, migration and invasion was evaluated in three patient-derived cells (PDCs) of HNSCC. Immunofluorescence of live cells was used for the quantitative measurement of migratory and invaded cells attached to the pillar. Expression of epithelial-mesenchymal transition (EMT)-related gene (snai1/2) was measured by qRT-PCR. We also tested the impact of drug treatments (cisplatin, docetaxel) on the changes in the invasive phenotype. All PDCs successfully formed spheroid at 4days and can be measured invasiveness within 7days. Intriguingly, one PDC (#1) obtained from the advanced stage showed robust migration, invasion and higher transcription of snai1/2, compared with the other two PDCs. Furthermore, the invasion ratio of the control spheroids was about 70% while the invasion ratios of drug-treated spheroids were lower than 50%, and the difference showed statistical significance (p<0.01). The presented spheroid invasion assay using pillar array could be useful for the evaluation of cancer cell behavior and physiology in response to diverse therapeutic drugs.

Cullin-7 (CUL7) is overexpressed in glioma cells and promotes tumorigenesis via NF-\u03baB activation

BackgroundCullin-7 (CUL7) is a member of the DOC domain-containing cullin family and is involved in the regulation of cell transformation. However, the clinical significance, potential mechanism and upstream regulators of CUL7 in malignant gliomas remain to be determined.MethodsExpression level data and clinical information were obtained via the Cancer Genome Atlas (TCGA) database, the Chinese Glioma Genome Atlas (CGGA) database, immunohistochemistry (IHC) and western blot analysis. Gene set enrichment analysis (GSEA) was used to explore the potential molecular mechanisms of CUL7. RNA silencing was performed using siRNA or lentiviral constructs in U87MG and U251 glioma cell lines and GSC267 glioma stem cells. CUL7 overexpression was performed using the GV141-CUL7 plasmid construct. In addition, overexpression of miR-3940-5p was performed and validated by quantitative real-time PCR (qRT-PCR). Cells were characterized in vitro or in vivo to evaluate their molecular status, cell proliferation, invasion, and migration by Cell Counting Kit (CCK)-8, EdU, flow cytometry, colony formation, Transwell and 3D tumour spheroid invasion assays. Coimmunoprecipitation (co-IP) and western blotting were performed to test the mechanisms of activation of the NF-κB signalling pathway.ResultsHigh CUL7 expression was associated with a high tumour grade, a mesenchymal molecular glioma subtype and a poor prognosis in patients. Gene silencing of CUL7 in U87MG and U251 cells significantly inhibited tumour growth, invasion and migration in vitro and in vivo. Western blot analysis revealed that cyclin-dependent kinase inhibitors and epithelial-mesenchymal transition (EMT) molecular markers changed under CUL7 silencing conditions. In contrast, CUL7 overexpression promoted tumour growth, invasion and migration. Gene set enrichment analysis (GSEA) and western blot analysis revealed that CUL7 was positively associated with the NF-κB pathway. Moreover, with coimmunoprecipitation assays, we discovered that CUL7 physically associated with MST1, which further led to ubiquitin-mediated MST1 protein degradation, which promoted activation of the NF-κB signalling pathway. Finally, CUL7 was found to be downregulated by miR-3940-5p, which suppressed the development of gliomas.ConclusionsOur findings indicate that CUL7 plays a significant role in promoting tumorigenesis via NF-κB activation and that it can be negatively regulated by miR-3940-5p in human gliomas. Furthermore, CUL7 might be a candidate molecular target for the treatment of glioma.

The critical effects of matrices on cultured carcinoma cells: Human tumor-derived matrix promotes cell invasive properties

The interaction between squamous cell carcinoma (SCC) cells and the tumor microenvironment (TME) plays a major role in cancer progression. Therefore, understanding the TME is essential for the development of cancer therapies.We used four (primary and metastatic) head and neck (HN) SCC cell lines and cultured them on top of or within 5 matrices (mouse sarcoma-derived Matrigel®, rat collagen, human leiomyoma-derived Myogel, human fibronectin and human fibrin). We performed several assays to study the effects of these matrices on the HNSCC behavior, such as proliferation, migration, and invasion, as well as cell morphology, and molecular gene profile.Carcinoma cells exhibited different growth patterns depending on the matrix. While fibrin enhanced the proliferation of all the cell lines, collagen did not. The effects of the matrices on cancer cell migration were cell line dependent. Carcinoma cells in Myogel-collagen invaded faster in scratch wound invasion assay. On the other hand, in the spheroid invasion assay, three out of four cell lines invaded faster in Myogel-fibrin. These matrices significantly affected hundreds of genes and a number of pathways, but the effects were cell line dependent.The matrix type played a major role in HNSCC cell phenotype. The effects of the ECMs were either constant, or cell line dependent. Based on these results, we suggest to select the most suitable matrix, which provides the closest condition to the in vivo TME, in order to get reliable results in in vitro experiments.

Novel invasion indices quantify the feed-forward facilitation of tumor invasion by macrophages

Quantitative and reliable measurement of cellular invasion is important to understand a range of biological processes such as cancer metastasis and angiogenesis. Spheroid invasion assays are an attractive in vitro platform because they effectively mimic the tumor cell invasion of solid tissues. Here, we developed an image analysis–based method to quantify the invasiveness of HT1080 human fibrosarcoma tumor cell spheroids. We segmented a cell-covered area into three subareas using objectively set threshold pixel intensities and calculated invasion indices using these subareas. Comparison with conventional parameters for spheroid invasion assays, such as area, length, and detached cells, showed that our indices present the invasion event at an early time and without being convoluted by proliferation. As an application, we then examined paracrine interactions between LLC1 mouse lung carcinoma cells and Raw264.7 mouse macrophage cells with our developed analysis method. We found that the invasion of tumor spheroids was increased by a macrophage-conditioned medium, concomitantly with a decrease in tumor cell proliferation. Importantly, invasion was further enhanced by a conditioned medium from activated macrophages by co-culture with tumor cells. Thus, our indices reveal that tumor cell invasion is facilitated in a feed-forward manner by communication between tumor cells and macrophages in the tumor microenvironment.

Suppression of LIM Kinase 1 and LIM Kinase 2 Limits Glioblastoma Invasion.

The aggressive brain tumor glioblastoma (GBM) is characterized by rapid cellular infiltration of brain tissue, raising the possibility that disease progression could potentially be slowed by disrupting the machinery of cell migration. The LIM kinase isoforms LIMK1 and LIMK2 (LIMK1/2) play important roles in cell polarization, migration, and invasion and are markedly upregulated in GBM and many other infiltrative cancers. Yet, it remains unclear whether LIMK suppression could serve as a viable basis for combating GBM infiltration. In this study, we investigated effects of LIMK1/2 suppression on GBM invasion by combining GBM culture models, engineered invasion paradigms, and mouse xenograft models. While knockdown of either LIMK1 or LIMK2 only minimally influenced invasion in culture, simultaneous knockdown of both isoforms strongly reduced the invasive motility of continuous culture models and human GBM tumor-initiating cells (TIC) in both Boyden chamber and 3D hyaluronic acid spheroid invasion assays. Furthermore, LIMK1/2 functionally regulated cell invasiveness, in part, by disrupting polarized cell motility under confinement and cell chemotaxis. In an orthotopic xenograft model, TICs stably transduced with LIMK1/2 shRNA were implanted intracranially in immunocompromised mice. Tumors derived from LIMK1/2 knockdown TICs were substantially smaller and showed delayed growth kinetics and more distinct margins than tumors derived from control TICs. Overall, LIMK1/2 suppression increased mean survival time by 30%. These findings indicate that LIMK1/2 strongly regulate GBM invasive motility and tumor progression and support further exploration of LIMK1/2 as druggable targets. SIGNIFICANCE: Targeting the actin-binding proteins LIMK1 and LIMK2 significantly diminishes glioblastoma invasion and spread, suggesting the potential value of these proteins as therapeutic targets.

Nitidine Chloride Is a Potential Alternative Therapy for Glioma Through Inducing Endoplasmic Reticulum Stress and Alleviating Epithelial-Mesenchymal Transition

Background: Malignant glioma is a lethal brain tumor that is highly resistant to standard therapy. Our research aims to explore the suppressive effects of nitidine chloride (NC) on gliomas and the mechanisms involved, showing that it is a potential agent for integrative therapy of gliomas. Methods: After glioma cells were treated with NC, several experiments were performed to evaluate NC’s antitumor effects. CCK-8 assay was used to detect viability. Transwell and 3-dimensional spheroid invasion assays were used to evaluate motility of glioma in vitro, and the sphere-formation assay showed NC’s influence on glioma stem cells. Apoptosis and intracellular reactive oxygen species were measured by means of flow cytometry. Subcellular structures were observed through transmission electron microscopy. Western blot analysis reflected expression of endoplasmic reticulum (ER) stress and epithelial-mesenchymal transition (EMT) marker proteins. An orthotopic xenograft model was established to investigate the tumor suppressive effects in vivo. Results: Nitidine chloride inhibited glioma cell migration and invasion in vitro, downregulated the EMT proteins, and suppressed sphere formation of glioma stem cells. Furthermore, NC induced persistent ER stress that contributed to apoptosis and reactive oxygen species production. The xenograft model showed that NC effectively restricted glioma growth and invasion in vivo. Furthermore, we confirmed the signaling pathways that ER stress downregulates C/EBPβ and slug, as well as inhibition of the AKT/GSK3β/β-catenin axis caused by NC, in U-87 MG. Conclusion: We demonstrated that NC inhibits gliomas in vitro and in vivo by activating ER stress and downregulating EMT, which provides a basis for glioma therapy.

Abstract B119: Inhibition of UCHL1 blocks proliferation and metastasis in HIF-1 dependent tumor malignancy

Abstract Hypoxia-inducible factor 1 (HIF-1) is a key transcription factor for tumor malignancy. Recently, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, has been reported to stabilize HIF-1α by releasing the pVHL complex conjugated Ub moieties. In this study, we investigated whether inhibition of UCHL1 can be an effective strategy for HIF-1-related tumor chemotherapy. First, we found that UCHL1 inhibition by siRNA or LDN57444, a well-known UCHL1 inhibitor, drastically lowered HIF-1α protein levels in UCHL1-expressing MDA-MB-436 breast cancer cells, by Western blotting analysis and immunostaining experiments. In UCHL1 non-expressing MDA-MB-231 cells, the ectopic expression of UCHL1 significantly increased HIF-1α protein expression levels, which was canceled by the treatment of LDN57444. We further analyzed whether the UCHL1 inhibitor could affect the HIF-dependent transactivation using 5HRE-luciferase reporter assay and RT-qPCR. LDN57444 dose-dependently inhibited the HIF activity and decreased the transcription of HIF downstream genes in MDA-MB-436 cells. Next we tested the effects of the UCHL1 inhibitor on tumor cell migration. LDN57444 significantly blocked cell migration in MDA-MB-436 cells in wound healing and transwell migration assays, suggesting a crucial role of the enzyme in tumor migration. We finally investigated potential roles of UCHL1 in tumor malignancy by using a 3D spheroid culture model, which has been more widely applied in the preclinical studies to provide more physiologically relevant information. Image analysis using open source image processing suites, AnaSP and ReViSP revealed that ectopic expression of UCHL1 in MDA-MB-231 cells significantly upregulated malignancy representing factors such as integrity and volume. Whole cell number of UCHL1 transfected MDA-MB-231 spheroids was also much higher than that of the mock treated spheroids when measured by 3D cell viability assay. Depletion of UCHL1 or inhibition of UCHL1 enzyme activity in MDA-MB-436 cells, on the contrary, downregulated these malignant factors. Besides, in spheroid viability staining and spheroid invasion assay, LDN57444 abolished the UCHL1 mediated cell proliferation and invasiveness as well. Moreover, the ectopic expression of UCHL1 in MDA-MB-231 cells stabilized HIF-1α protein level and promoted the expression of HIF-1 target genes in the 3D model as in the case of 2D monolayer culture. In conclusion, we clearly proved the involvement of UCHL1 in the HIF-1 induced tumor malignancy employing the 3D spheroid culture model. Our research indicates that the UHCL1-HIF-1 pathway is a promising therapeutic target for cancer chemotherapy. Citation Format: Xuebing Li, Akira Hattori, Senye Takahashi, Yoko Goto, Hiroshi Harada, Hideaki Kakeya. Inhibition of UCHL1 blocks proliferation and metastasis in HIF-1 dependent tumor malignancy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B119. doi:10.1158/1535-7163.TARG-19-B119

Nuclear receptor 4A2 (NR4A2) is a druggable target for glioblastomas.

The orphan nuclear receptor 4A2 (NR4A2) has been extensively characterized in subcellular regions of the brain and is necessary for the function of dopaminergic neurons. The NR4A2 ligand, 1,1-bis (31-indoly1)-1-(p-chlorophenyl)methane (DIM-C-pPhCl) inhibits markers of neuroinflammation and degeneration in mouse models and in this study we investigated expression and function of NR4A2 in glioblastoma (GBM). Established and patient-derived cell lines were used as models and the expression and functions of NR4A2 were determined by western blots and NR4A2 gene silencing by antisense oligonucleotides respectively. Effects of NR4A2 knockdown and DIM-C-pPhCl on cell growth, induction of apoptosis (Annexin V Staining) and migration/invasion (Boyden chamber and spheroid invasion assay) and transactivation of NR4A2-regulated reporter genes were determined. Tumor growth was investigated in athymic nude mice bearing U87-MG cells as xenografts. NR4A2 knockdown and DIM-C-pPhCl inhibited GBM cell and tumor growth, induced apoptosis and inhibited migration and invasion of GBM cells. DIM-C-pPhCl and related analogs also inhibited NR4A2-regulated transactivation (luciferase activity) confirming that DIM-C-pPhCl acts as an NR4A2 antagonist and blocks NR4A2-dependent pro-oncogenic responses in GBM. We demonstrate for the first time that NR4A2 is pro-oncogenic in GBM and thus a potential druggable target for patients with tumors expressing this receptor. Moreover, our bis-indole-derived NR4A2 antagonists represent a novel class of anti-cancer agents with potential future clinical applications for treating GBM.

Identification of Synthetic Activators of Cancer Cell Migration by Hybrid Deep Learning.

Deep convolutional neural networks (CNNs) are a method of choice for image recognition. Herein a hybrid CNN approach is presented for molecular pattern recognition in drug discovery. Using self-organizing map images of molecular pharmacophores as input, CNN models were trained to identify chemokine receptor CXCR4 modulators with high accuracy. This machine learning classifier identified first-in-class synthetic CXCR4 full agonists. The receptor-activating effects were confirmed by intracellular cAMP response and in a phenotypic spheroid invasion assay of medulloblastoma cell invasion. Additional macromolecular targets of the small molecules were predicted in silico and tested in vitro, revealing modulatory effects on dopamine receptors and CCR1. These results positively advocate the applicability of molecular image recognition by CNNs to ligand-based virtual compound screening, and demonstrate the complementarity of machine intelligence and human expert knowledge.

The Collagen-Prolyl Hydroxylases promote proliferation and invasion in GBM

Abstract Glioblastoma Multiforme (GBM) is the most aggressive form of brain tumour, with a median survival time of just 14 months. Current standard therapy includes surgical excision, followed by chemotherapy and radiotherapy, however complete surgical excision is not usually possible due to the diffuse and infiltrative nature of the disease. To combat this, therapies inhibiting the invasion of leader migratory cells are an attractive area of research. The collagen prolyl-4 hydroxylases (C-P4Hs) are a family of proteins which catalyse the hydroxylation of proline residues on pre-pro-collagen, leading to the formation of the stable triple-helical structure of functional collagen. Collagen is a highly abundant protein, and important in several oncogenic cellular processes including proliferation, invasion and migration. We hypothesis that by inhibiting the C-P4Hs, the pro-tumorigenic effect of collagen will be inhibited in GBM. To explore this, we treated cells with ethyl 3,4-dihydroxybenzoate (EDHB) (a C-P4H inhibitor) and observed the resultant effect in GBM cells lines and primary cells through proliferation assays and 3D tumour spheroid invasion assays. Our results indicate that the addition of EDHB significantly reduces proliferation and invasion in both cell lines and primary GBM samples. Furthermore, we show that the C-P4Hs are upregulated in hypoxic conditions, which are commonly found in the clinical tumour microenvironment. Our study reveals that the C-P4H proteins may be an attractive therapeutic target to reduce invasion and proliferation and may be especially attractive as adjuvants with a cytotoxic agent such as temozolomide.

P1.14-20 Tarloxotinib as a Novel Therapeutic Strategy for Oncogenic Alterations Across the ErbB Family of Receptors

The ErbB family of receptors tyrosine kinases (EGFR, HER2, HER3, and HER4) have been implicated in multiple different tumor types. The implementation of comprehensive next generation sequencing has allowed the identification of diverse gene alterations that function as oncogene drivers in these receptors. Some of the non-common gene alterations identified are resistant to marketed EGFR/HER2 inhibitors. Tarloxotinib is a prodrug that generates a potent and irreversible pan-HER inhibitor (tarloxotinib-E) under hypoxic conditions associated with tumors. In this study we evaluated the effect of tarloxotinib on several types of oncogenic mutations and fusions that involve the ErbB family of receptors. cDNAs encoding EGFR kinase domain duplications (EGFR-EGFR), EGFR-RAD51 gene fusion, EGFR-ERBB4, ERBB2-GRB7 and EZR-ERBB4 gene fusion were expressed in Ba/F3 cells. Using spheroid assays we evaluated the proliferation of A172 glioblastoma cell line treated with tarloxotinib, tarloxotinib-E or 1st, 2ndor 3rdgeneration EGFR/HER2 TKIs. We analyze the on target and signaling effects elicited by tarloxotinib-E via immunoblots. Using a nude mice xenograft model of the human derived cell line CUTO17 wi the EGFR exon 20 insertion p.N771_H773dupNPH, we evaluated tumor, tissue and blood drug levels by mass spectrometry and the effect of tarloxotinib on tumor growth. Our results demonstrate that tarloxotinib-E inhibits phosphorylation of EGFR with a kinase duplication and inhibits proliferation in a spheroid invasion assay in A172 cells. In the CUTO17 EGFR exon 20 model, treatment with tarloxotinib inhibited tumor growth. Intratumor levels of tarloxotinib-E were ∼20 times higher than skin and ∼50 times higher than plasma demonstrating selective tumor conversion of tarloxotinib. Cell growth inhibition (EC50) of novel HER family fusions (EGFR-EGFR, EGFR-RAD51, EGFR-ERBB4, ERRB2-GRB7 and EZR-ERBB4) will be presented. Tarloxotinib is a potent irreversible inhibitor in vitro for cells that harbor oncogenic alterations across the ERBB gene family, including EGFR kinase domain duplications, ErbB fusions and exon-20 insertions. Tarloxotinib is selectively activated in hypoxic tumor regions demonstrating a novel mechanism to generate a therapeutic window and avoid on-target EGFR-related toxicities.

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy.

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

P14.52 Differential methylation of a single CpG site in the CD95 ligand promoter affects gene activity and correlates with invasiveness of glioma cells

Abstract BACKGROUND CD95 ligand (TNFSF6/APO-1L/FASLG) is a member of the Tumour Necrosis Factor Super Family (TNF-SF). Binding of the CD95 ligand (CD95L) to CD95 expressed on intrinsically apoptosis-resistant cancer cells like glioblastoma, triggers intracellular signal transduction resulting in increased tumour growth and invasiveness. A phase II clinical trial in patients with recurrent glioblastoma (NCT01071837) treated by Asunercept (a recombinant glycosylated fusion protein, which selectively binds and inhibits CD95L activity) was followed by a genome-wide assessment of DNA methylation. Here, a CpG-site (designated CpG2) within the CD95L-promotor was identified, exhibiting differential methylation between Asunercept responders (PFS&gt;5 months) and non-responders (PFS &lt; 2 months) with a significant survival benefit achieved in Asunercept treated patients with low CpG2 methylation (HR 0.34, p = 0.025). METHODS AND RESULTS We have performed in vitro studies to establish a link between the CpG2 methylation status and cellular characteristics of glioblastoma cell lines. A 3-dimensional spheroid invasion assay showed that highly methylated glioma cells like T98G did not grow and invade the surrounding matrix as aggressively compared to spheroids formed from low methylated glioma cells, e.g. U87-MG. Invasive growth of U87-MG spheroids in these assays was suppressed in the presence of Asunercept. A 1 kb fragment of the T98G CD95L promoter was subsequently cloned into a CpG-free reporter gene plasmid. Luciferase-based reporter gene assays of in vitro methylated and unmethylated plasmids in transfected HEK cells indicate that the CD95L promoter, despite the observed sparseness of CpG sites, is at least partially regulated by its methylation level. Furthermore, mutational disruption of the CpG2 site completely silenced reporter gene activity in vitro, which insinuates that both methylation level and gene promoter sequence are involved in regulation of the CD95L gene. CONCLUSION In essence, the level of CpG2 methylation correlates to aggressiveness of glioma derived cancer spheroids in vitro, and methylation of the CD95L promoter in glioblastoma tissue from patients might warrant use as a potential biomarker predicting response to therapy with Asunercept. We are currently developing a specific and sensitive assay to quantify CpG2 methylation considered as a companion diagnostic for further clinical studies.