Pediatric Tumor Cell Research Articles

A head-to-head comparison of computed tomography- and magnetic resonance imaging-based radiomics in assessing pediatric peripheral neuroblastic tumor cell behavior.

To compare the performance of radiomics from contrast-enhanced computed tomography (CECT) and non-contrast magnetic resonance imaging (MRI) in assessing cellular behavior in pediatric peripheral neuroblastic tumors (PNTs). A retrospective analysis of 81 PNT patients who underwent venous phase CECT, T1-weighted imaging (T1WI), and T2-weighted imaging (T2WI) scans was conducted. The patients were classified into neuroblastoma and ganglioneuroblastoma/ganglioneuroma based on their pathological subtypes. Additionally, they were categorized into favorable histology and unfavorable histology according to the International Neuroblastoma Pathology Classification (INPC). Tumor regions of interest were segmented on CECT, axial T1WI, and axial T2WI images, and radiomics models were developed based on the selected radiomics features. Following five-fold cross-validation, the performance of the radiomics models derived from CECT and MRI was compared using the area under the receiver operating characteristic curve (AUC) and accuracy. For discriminating pathological subtypes, the AUC for CECT radiomics models ranged from 0.765 to 0.870, with an accuracy range of 0.728 to 0.815. In contrast, the AUC for MRI radiomics models ranged from 0.549 to 0.748, with an accuracy range of 0.531 to 0.778. Regarding the discrimination of INPC subgroups, the AUC for CECT radiomics models ranged from 0.503 to 0.759, with an accuracy range of 0.432 to 0.741. Meanwhile, the AUC for MRI radiomics models ranged from 0.512 to 0.739, with an accuracy range of 0.605 to 0.815. CECT radiomics outperforms non-contrast MRI radiomics in evaluating pathological subtypes. When assessing INPC subgroups, CECT radiomics demonstrates comparability with non-contrast MRI radiomics.

Evaluating antitumor activity and response determinants to trastuzumab deruxtecan in pediatric solid tumors.

10049 Background: Trastuzumab deruxtecan (T-DXd) is an anti-HER2 antibody-drug conjugate (ADC) linked to a topoisomerase inhibitor FDA-approved for several indications, including the treatment of HER2+ breast and gastric cancer. Although HER2 amplification is uncommon in pediatric cancers, recent demonstration of T-DXd efficacy in HER2-low breast cancer patients prompted us to examine the potential clinical relevance of T-DXd in pediatrics by evaluating HER2 expression and activity of T-DXd in preclinical pediatric solid tumor models. Methods: Cell viability was assessed in a panel of histologically diverse pediatric solid tumor cell lines and 2 HER2-amplified adult cancer cell lines treated with T-DXd, payload (DXd), and a control IgG-ADC. HER2 protein expression in patient-derived xenograft (PDX) tumors was evaluated by immunohistochemistry (IHC) using 5 clinically validated anti-HER2 antibodies. RNAseq was performed on clinical (n=290) and PDX (n=184) tumors to determine relative expression of ERBB2. In vivo activity of T-DXd was evaluated in osteosarcoma (OS, n=10), Wilms tumor (WT, n=1) and malignant rhabdoid tumor (MRT, n=1) PDX models and a desmoplastic small round cell tumor (DSRCT) cell line xenograft model. Differences in tumor volume and time to disease progression was assessed and compared across treatments and models. Responses were correlated with HER2 expression by IHC. Results: In vitro, HER2-amplified control cell lines demonstrated a >30-fold reduction of IC50 when comparing T-DXd to ADC control. In contrast, the ADC control IC50 was nearly identical to T-DXd across all pediatric cancer cell lines, consistent with the absence of HER2 amplification in these models. We observed focal and membranous staining of HER2 by IHC in PDXs but was quite variable across antibodies tested: WT 0-17% HER2+ cases (n=6), MRT 0-40% (n=5), DSRCT 0-33% (n=24), OS 0-7% (n=30). ERBB2 gene expression was highest in DSRCT followed by WT, OS and MRT. In vivo efficacy studies demonstrated complete and partial responses in OS, WT, and DSRCT and improved disease control rates (OS: 67%, p=0.006, Mann-Whitney; WT: 100%; DSRCT: 100%). However, T-DXd and ADC control demonstrated similar activity in all tumor types with no consistent correlation between in vivo response and HER2 expression. Consistent with these preclinical studies, 4 patients with progressive DSRCT were treated with T-DXd via compassionate/off-label access with signs of clinical and radiographic responses. Conclusions: T-DXd shows significant preclinical antitumor activity across multiple pediatric solid tumors but little correlation with HER2 expression suggesting a mechanism of action similar to the clinical activity observed in HER2-low breast cancer. Xenograft efficacy studies and preliminary clinical experience with T-DXd in DSRCT patients warrant formal clinical trial investigation in this largely incurable disease.

Abstract LB403: Suppression of antigen presentation in pediatric rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma that is diagnosed based on the tumor cell's resemblance to embryonic or poorly differentiated skeletal muscle progenitors. Multiple immunotherapy clinical trials have found no evidence of immune reactivity against RMS, which has been attributed to the relatively low tumor mutation burden in these cancers. However, we made an unexpected observation that many patient RMS tumors had a near absence of MHC-I expression, a key requisite for anti-tumor CD8+ T lymphocytes and many immunotherapies. In this study, we explore the etiology of low antigen presentation in RMS and systematically evaluate therapeutic interventions that sensitize RMS to immunotherapy. Methods: Surface MHC-I expression was evaluated on pediatric tumor cell lines (n=65) from five major pediatric tumor types. RNA-seq, whole exome and genome sequencing data were analyzed from RMS tumors (total of n=247) and iPSC models of skeletal muscle development (PMID: 32011235). Epigentic drugs targeting DNA methylation (decitabine), EZH2 (tazemetostat), Class I/IV HDACs (mocetinostat), and LSD1 (GSK-LSD1) were used at sub-cytotoxic doses on RMS cell lines (n=10) and analyzed for surface MHC-I expression with flow cytometry and gene expression. T cell cytotoxicity assays were performed using engineered human T cells expressing an optimized TCR which recognizes an HLA*A2:01 restricted peptide from PRAME. Results: RMS cell line models exhibited a range of MHC-I expression with most having low or undetectable MHC-I expression, similar to that of a known MHC-I negative tumor type MYCN-amplified neuroblastoma. Exome sequencing of RMS tumors showed no evidence of loss-of-function alterations in essential antigen processing and presentation (APP) pathway genes. Gene expression profiling by RNAseq and western blotting, however, revealed an absence of core APP genes in many RMS tumors and cell lines. We uncovered that during a specific window of embryonic myogenesis, APP is suppressed and that RMS tumors mimicking this state of development show the deepest loss of APP. Multiple classes of epigenetic drugs were evaluated to rescue APP in RMS. Treatment of RMS using a DNA demethylating agent or HDAC inhibitor exhibited the strongest MHC-I restoration across RMS models with no changes observed using LSD1 or EZH2 inhibitors. As a proof-of-concept, we demonstrate that targeting the RMS antigen PRAME using TCR therapy is enhanced by the inclusion of epigenetic therapy. Conclusions and Significance: We revealed that epigenetic suppression of APP is a new hallmark of RMS and normal embryonic skeletal muscle development. Pharmacological reversal of APP pathway downregulation could be achieved using drugs targeting DNA methylation or class I/IV HDACs, and enhanced T cell cytotoxicity against RMS when used in combination. Overall, these findings may explain the historical lack of RMS responsiveness to immunotherapy and unlocks new immunotherapy strategies against RMS that rely on targeting MHC-I antigens. Citation Format: David Milewski, Hsien-Chao Chou, Vineela Gangalapudi, Meijie Tian, Yong Kim, Young Song, Jun Wei, Javed Khan. Suppression of antigen presentation in pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB403.

Cytokine Signaling in Pediatric Kidney Tumor Cell Lines WT-CLS1, WT-3ab and G-401.

Renal tumors comprise ~7% of all malignant pediatric tumors. Approximately 90% of pediatric kidney tumors comprise Wilms tumors, and the remaining 10% include clear cell sarcoma of the kidney, malignant rhabdoid tumor of the kidney, renal cell carcinoma and other rare renal tumors. Over the last 30 years, the role of cytokines and their receptors has been considerably investigated in both cancer progression and anti-cancer therapy. However, more effective immunotherapies require the cytokine profiling of each tumor type and comprehensive understanding of tumor biology. In this study, we aimed to investigate the activation of signaling pathways in response to cytokines in three pediatric kidney tumor cell lines, in WT-CLS1 and WT-3ab cells (both are Wilms tumors), and in G-401 cells (a rhabdoid kidney tumor, formerly classified as Wilms tumor). We observed that interferon-alpha (IFN-α) and interferon-gamma (IFN-γ) very strongly induced the activation of the STAT1 protein, whereas IL-6 and IFN-α activated STAT3 and IL-4 activated STAT6 in all examined tumor cell lines. STAT protein activation was examined by flow cytometry and Western blot using phospho-specific anti-STAT antibodies which recognize only activated (phosphorylated) STAT proteins. Nuclear translocation of phospho-STAT proteins upon activation with specific cytokines was furthermore confirmed by immunofluorescence. Our results also showed that both IFN-α and IFN-γ caused upregulation of major histocompatibility complex (MHC) class I proteins, however, these cytokines did not have any effect on the expression of MHC class II proteins. We also observed that pediatric kidney tumor cell lines exhibit the functional expression of an additional cytokine signaling pathway, the tumor necrosis factor (TNF)-α-mediated activation of nuclear factor kappa B (NF-κB). In summary, our data show that human pediatric renal tumor cell lines are responsive to stimulation with various human cytokines and could be used as in vitro models for profiling cytokine signaling pathways.

Novel protein products encoded by upstream open reading frames of the MYCN gene in pediatric embryonal tumors.

The MYCC and MYCN loci are each associated with two upstream open reading frames (uORFs) potentially encoding small proteins (9-21 kDa). We previously demonstrated that uORFs mrtl and MYCHEX1 of MYCC are translated, and their protein products may function to regulate the expression of the "parent" oncogene. We hypothesized that a similar relationship might exist between MYCN and its two uORFs: MYCNOT and MNOP, and investigated the uORF-encoded proteins associated with MYCN to confirm their expression and intracellular location in neuroblastoma and medulloblastoma cells and tissues. MNOP, MYCNOT, mrtl, and MYCHEX1 were readily detected via reverse transcription polymerase chain reaction and Western blot analysis in tumor cell lines. In tumor tissue, MNOP protein expression was confirmed; however, MCYNOT generated from alternative splicing MYCNΔ1b mRNA was not detected. Immunofluorescence staining of MYCNOT displayed multiple bright foci in the nucleus and diffuse staining in the cytoplasm, suggesting that this small protein may function in both the nucleus and cytoplasm. Upon JQ1 treatment, MYCN, MYCNOT, and mrtl decreased substantially or disappeared completely in three different tumor cell lines. Significant levels of apoptosis were observed in each pediatric embryonal tumor cell line but not T47D breast carcinoma cells, suggesting that response to JQ1 transcriptional inhibition is greatest in tumor cells, which depend on MYC to maintain an undifferentiated phenotype. In conclusion, both MYCN uORF-encoded proteins MNOP and MYCNOT, together with the two MYCC uORF-encoded proteins mrtl and MYCHEX1 were detected simultaneously in tumor cell lines and tumor tissues. These four distinct proteins are translated from the "5'-untranslated region" of MYCN or MYCC mRNAand display consistent distribution patterns within the cell. Additional studies to further elucidate the physiological and pathological roles of these uORF-encoded proteins are warranted, as insights gained could inform new strategies for modulating MYC-family oncogenes by targeting their uORFs.

SYST-22 INTEGRATING GENE DEPENDENCY INTO A PEDIATRIC-FOCUSED CANCER DATA PORTAL FOR TARGETED THERAPY AND BIOMARKER DISCOVERY

Abstract Pediatric solid cancers, particularly those affecting the central nervous system (CNS), are the leading cause of disease-related deaths in children. Despite significant efforts to sequence pediatric solid tumors, they exhibit a "quiescent genome" with a considerably lower mutational burden than adult cancers. This leads to fewer actionable drug targets and poor response to cancer immunotherapy agents, including checkpoint inhibitors. As a result, it is imperative to move beyond genetic alterations and adopt a combination of functional and integrative approaches to develop novel, targeted treatments that are specifically designed for pediatric cancers. Over the past five years, the Next-Gen Precision Medicine Program team has devoted its efforts to establishing the Childhood Cancer Model Atlas (CCMA) (Sun, Daniel et al. Cancer Cell 2023). The CCMA is the largest collection of high-risk pediatric solid tumor cell lines globally, presenting a tremendous opportunity to integrate molecular features with functional dependencies using state-of-the-art computational approaches. However, the analysis of -omics level data is often limited to laboratories with specialized bioinformatics capabilities. Therefore, the establishment of the CCMA data portal with user-friendly analytical and visualization capabilities is essential to accelerate data-driven discovery and translational research in pediatric cancer on a global scale. The data portal is designed to simplify reporting, automate manual processes, and enhance the quality, usability, and longevity of the CCMA dataset. Its highlighted features concentrate on customized analysis, informative results, and user-friendly data visualization. Furthermore, by connecting the established models and their original tumor with clinical and molecular endpoints, the data portal enables clinical integration within the Australian molecular tumor board framework. This analytical data portal will enable the generation of new research hypotheses, prioritizes therapeutic opportunities, and supports international collaborative efforts in pediatric cancer.

Effect of dual properties of regorafenib in osteosarcoma on tumor progression and bone parameters in mouse preclinical models.

e23501 Background: Osteosarcoma (OS) constitutes the most frequent malignant primary bone tumor in children and young adults. Actual treatments in OS are based in a multimodal therapy that combines pre- and post-surgery polychemotherapies including cisplatin, doxorubicin, ifosfamide and/or methotrexate. Regardless the use of this polychemotherapeutic approach, the five years overall survival for patients has not improved during the last four decades and there is a clinical need to identify new active drugs in OS. An interesting strategy implies the use of compounds that showed therapeutic benefits in other solid tumors. Regorafenib, a small molecule with multi-target tyrosine kinase inhibitory activity against FGFR-1 and -2, KIT, PDGFR, RAF, RET, TIE2 and VEGFR-1, -2 and -3, showed an anti-angiogenic/oncogenic properties in various solid tumors that includes metastatic colorectal cancer, hepatocarcinoma and breast cancer. In addition, regorafenib also inhibits in vitro the proliferation of several pediatric solid tumor cell lines such as rhabdomyosarcoma, Ewing sarcoma and OS. Methods: The present work aims to decipher the in vitro and in vivo effects of regorafenib in OS preclinical models. Results: The treatment of MNNG-HOS, KHOS and 143B osteosarcoma cell lines with Regorafenib resulted in the inhibition of cell proliferation in a dose dependent manner. The gene expression profiles of MNNG-HOS and 143B cells treated or not with regorafenib and analyzed by NanoString technology revealed a specific regulation of molecular pathways by regorafenib in both cell lines. The in vivo effect of regorafenib was then analyzed in immunocompetent (mouse MOS-J cells inoculated in C57Bl/6 mice) and immunodeficient (human MNNG-HOS and 143B OS cells injected in Nude mice) murine osteosarcoma models. In all the cases, a significant reduction of primary tumor growth was observed as well as a significant decrease of lung metastases compared to untreated mice. Interestingly, the cortical and trabecular bone mineral density were significantly increased in regorafenib treated mice compared to untreated groups as demonstrated by X ray microtomodensitometry in all models. Conclusions: Our results support the use of regorafenib as an alternative treatment in metastatic OS and demonstrated for the first time the its bone protective effect.

CSIG-29. EMPLOYING THE ZIKA VIRUS AS ONCOLYTIC VIROTHERAPY AGAINST PAEDIATRIC NERVOUS SYSTEM CANCER CELLS

Abstract BACKGROUND Malignant paediatric nervous system tumours, such as medulloblastoma, ATRT and high-risk neuroblastoma commonly harbour tumour cells with stem-like features which are highly tumorigenic and resistant to conventional therapies. These tumours can exhibit high lethality and may result in severe sequelae that significantly affect paediatric patients' quality of life. Oncolytic virotherapy exploits viruses that preferentially infect and destroy tumour cells. These viruses present a unique advantage in targeting highly heterogeneous cancers as they possess a secondary mechanism of action, through which they induce an anti-tumoral immune response. The Zika virus (ZIKV) is capable of infecting and destroying aggressive human paediatric brain tumour and neuroblastoma cells in vitro. ZIKV effectively reduces brain tumour size in mice (xenograft model) and canines (naturally occurring) and can induce an immune response against canine brain tumours. METHODS Employing global expression omics profiling of ZIKV infection and mapping of viral protein-host protein interactions, we aim to elucidate the mechanisms which underpin ZIKVs therapeutic properties, both at the molecular and cellular pathway levels. RESULTS Through extensive transcriptome profiling of ZIKV-infected paediatric brain tumour, neuroblastoma and NPCs, we have identified a variety of pathways which are involved in the ZIKV oncolytic response in the tumour cells and its neuro-dysregulation of NPCs. Despite both brain tumour and neuroblastoma cells undergoing ZIKV-induced oncolysis, we observed there to be a heterogeneous response within these different tumour cells at the molecular level to lead to oncolysis. Additionally, the infected tumour cells demonstrate elevated immune system profiles which alludes to the immune response that ZIKV may raise within the patient’s body against the paediatric tumour. Analysing our findings alongside the neuro-dysregulation we observe in our ZIKV-infected NPCs is allowing us to build a safety profile for employing a ZIKV-based therapy, whilst contributing to the growing knowledge of Congenital ZIKV Syndrome.

Evaluation of Antitumor and On-Target Activity of HDAC Inhibitors with the Zebrafish Embryo Xenograft Model.

Reliable preclinical drug testing models for cancer research are urgently needed with zebrafish embryo models emerging as a powerful vertebrate model for xenotransplantation studies. Here, we describe the evaluation of toxicity, efficacy, and on-target activity of histone deacetylase (HDAC) inhibitors in a zebrafish embryo yolk sac xenotransplantation model of medulloblastoma and neuroblastoma cells. For this, we performed toxicity assays with our zebrafish drug library consisting of 28 clinically relevant targeted as well as chemotherapeutic drugs with zebrafish embryos. We further engrafted zebrafish embryos with fluorescently labeled pediatric tumor cells (SK-N-BE(2)-C, HD-MB03, or MED8A) and monitored the progression after HDAC inhibitor treatment of xenotransplanted tumors through tumor volume measurements with high-content confocal microscopy in a multi-well format. The on-target activity of HDAC inhibitors was verified through immunohistochemistry staining on paraffin-embedded early larvae. Overall, the zebrafish embryo xenotransplantation model allows for fast and cost-efficient in vivo evaluation of targeted drug toxicity, efficacy, and on-target activity in the field of precision oncology.

Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors

BackgroundPediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Glypican 2 (GPC2) is a cell surface oncoprotein expressed in neuroblastoma...

TBIO-07. Pediatric tumor classification through genome-wide methylation profiling of extracellular vesicle DNA

BACKGROUND: Genome-wide methylation profiling reliably classifies pediatric central nervous system (CNS) tumors. Extracellular vesicles (EVs) are released by pediatric CNS tumor cells (pCC) and contain high molecular weight tumor DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated, whether the DNA in pCC-derived EVs reflects genome-wide tumor methylation profiles and allows tumor subtype classification. Currently, the tests are being expanded to include blood samples (n=80 patients). METHODS: DNA was isolated from EVs secreted by pediatric CNS tumor cells (pCC) as well as from the shortly cultured tumor cells and from the original tumor samples (n=4 patients). Pediatric Fibroblasts and EVs derived thereof were used as a non-tumorous control. EVs were classified by nanoparticle analysis (NTA), immunoblotting, imaging flow cytometry (IFCM and electron microscopy. Genome-wide DNA methylation profiling was performed using an 850k Illumina EPIC array and results were classified according to the DKFZ brain tumor classifier and further analysed by t-SNE and Copy number alteration analysis (CNA). RESULTS: The size range of pCC-derived EVs was 120-150 nm, as measured by NTA. The majority of secreted EVs exhibited high expression of common EV markers (i.e. CD9, CD63 and CD81), as characterized by IFCM. Genome-wide DNA methylation profiling of pCC-derived EVs correctly identified the methylation class of the original tumor (i.e. pilocytic astrocytoma, medulloblastoma). In addition, t-SNE analysis and copy number alterations matched the pattern of the parental pCC and original tumor samples. CONCLUSION: EV DNA faithfully reflects the tumor methylation class and copy number alterations present in the parental cells and the original tumor. Methylation profiling of circulating tumor EV DNA could become a useful tool to detect and classify pediatric CNS tumors.

MODL-28. Patient-derived, three-dimensional organoid platform for pediatric brain tumor modeling

Brain tumors have become the leading cause of cancer-related death in children. An important hurdle to scientific and clinical progress in the field has been the limited availability of preclinical tumor models. Historically, few pediatric brain tumor cell lines have been established and these often poorly recapitulate the phenotypes of the original tumors. In recent years, the Children’s Brain Tumor Network (CBTN) has accelerated the development of patient-derived cell lines and xenografts, offering these resources to the community through open-source access. While these models are extremely valuable, their development process can be lengthy and result in clonally selected lines which presents a challenge for studying complex tumor biology. To address the need for three-dimensional tissue culture, our group in conjunction with CBTN, utilized organoid culture from fresh tissue specimens obtained directly from surgical resection of various pediatric brain tumor histologies. This resulted in the development and banking of over 30 organoid models, which included ependymoma, high-grade glioma, medulloblastoma, atypical teratoid-rhabdoid tumor, diffuse midline glioma, and low-grade glioma diagnoses. Tissue was processed within an hour post extraction and cultured with universal media composition for each diagnosis. Organoid growth was observed within 2-3 weeks of initiation and continued for up to three months before banking. Banked organoids established growth upon return to culture. Phenotypic analysis revealed organoid cell composition that represented clinical histology. Importantly, organoids returned to culture post-banking demonstrated similar cell composition to those in the original culture, indicating their utility for subsequent preclinical testing. Here we provide a simple and efficient workflow for the generation and characterization of three-dimensional tumor organoids generated from fresh surgical pediatric brain tumor tissue. The platform has the potential to accelerate investigations into tumor biology and empower a diverse array of translational studies for the pediatric brain tumor field.

MODL-05 Metronomic Intrathecal Delivery of CDK4/6 Inhibitors in Preclinical Models of Pediatric Brain Tumors

INTRODUCTION: CDK4/6 inhibitors have shown promise against central nervous system (CNS) tumors in vitro. This class of drugs relies on long-term exposure. Their use in early phase clinical studies in children with CNS tumors has defined dose limitations due to systemic toxicity. We have sought to circumvent these limitations in using CDK4/6 inhibitors for pediatric CNS tumors by first demonstrating enhanced efficiency with long-term administration and exploiting prolonged intrathecal delivery (IT). METHODS: Pediatric CNS tumor cell lines were used for cell viability assays: ATRT (BT-12, BT-16), CPC (CCHE-45), diffuse midline glioma (DIPG-XIII, HSJD-007), and medulloblastoma (DAOY); the assays were conducted at 24h, 72h, and 7d post-administration of CDK4/6 inhibitors (abemaciclib, palbociclib, ribociclib). Half maximal growth inhibitory concentrations (GI50) and areas under the curve (AUC) were compared for short-term (24h, 72h) and long-term (7d) dose-response curves. Toxicity with chronic IT administration was assessed using a neurobehavioral safety profile of 7-day continuous infusion of 2.5mM palbociclib (n = 5) into the mouse lateral ventricle compared with vehicle (n = 4). RESULTS: Our results demonstrate increased CDK4/6 inhibitor potency with longer administration. The greatest reductions in short-term to long-term GI50 were observed in ATRT, CPC, and DIPG across all inhibitors. The most pronounced time-dependent efficacy was observed with palbociclib for ATRT and abemaciclib for CPC and DIPG. AUCs significantly decreased (P < 0.05) with increasing drug exposure time across all inhibitors. 7-day intraventricular palbociclib infusion was equivalent in safety to PBS at doses ranging from 1,000 to 10,000-fold the in vitro GI50. CONCLUSIONS: The efficiency of CDK4/6 inhibitors in pediatric CNS tumors is enhanced with prolonged exposure. Long-term IT administration can achieve high CNS doses without associated systemic toxicities. Translational efforts using a metronomic IT strategy are logical to explore for pediatric CNS tumors which have potential for a leptomeningeal disease pattern.

Synthetic extracellular matrices and astrocytes provide a supportive microenvironment for the cultivation and investigation of primary pediatric gliomas.

Pediatric gliomas comprise a diverse set of brain tumor entities that have substantial long-term ramifications for patient survival and quality of life. However, the study of these tumors is currently limited due to a lack of authentic models. Additionally, many aspects of pediatric brain tumor biology, such as tumor cell invasiveness, have been difficult to study with currently available tools. To address these issues, we developed a synthetic extracellular matrix (sECM)-based culture system to grow and study primary pediatric brain tumor cells. We developed a brain-like sECM material as a supportive scaffold for the culture of primary, patient-derived pediatric glioma cells and established patient-derived cell lines. Primary juvenile brainstem-derived murine astrocytes were used as a feeder layer to support the growth of primary human tumor cells. We found that our culture system facilitated the proliferation of various primary pediatric brain tumors, including low-grade gliomas, and enabled ex vivo testing of investigational therapeutics. Additionally, we found that tuning this sECM material allowed us to assess high-grade pediatric glioma cell invasion and evaluate therapeutic interventions targeting invasive behavior. Our sECM culture platform provides a multipurpose tool for pediatric brain tumor researchers that enables both a wide breadth of biological assays and the cultivation of diverse tumor types.

Pediatric brain tumor cell lines exhibit miRNA-depleted, Y RNA-enriched extracellular vesicles.

Medulloblastoma (MB) and diffuse infiltrative pontine glioma (DIPG) are malignant pediatric tumors. Extracellular vesicles (EVs) and their bioactive cargoes have been implicated in tumorigenesis. Most studies have focused on adult tumors, therefore the role of EVs and the noncoding RNA (ncRNA) landscape in pediatric brain tumors is not fully characterized. The overall aim of this pilot study was to isolate EVs from MB and DIPG patient-derived cell lines and to explore the small ncRNA transcriptome. EVs from 3 DIPG and 4MB patient-derived cell lines were analyzed. High-throughput next generation sequencing interrogated the short non-coding RNA (ncRNA) transcriptome. Known and novel miRNAs were quantified. Differential expression analysis, in silico target prediction, and functional gene enrichment were performed. EV secretomes from MB and DIPG patient-derived cell lines demonstrated discrete ncRNA biotypes. Notably, miRNAs were depleted and Y RNAs were enriched in EV samples. Hierarchical cluster analysis revealed high discrimination in miRNA expression between DIPG and MB cell lines and RNA-Seq identified novel miRNAs not previously implicated in MB or DIPG pathogenesis. Known and putative target genes of dysregulated miRNAs were identified. Functional annotation analysis of the target genes for differentially expressed EV-and parental-derived miRNAs revealed significant cancer-related pathway involvement. This hypothesis-generating study demonstrated that pediatric brain tumor-derived cell lines secrete EVs comprised of various ncRNA cargoes. Validation of these findings in patient samples may provide new insights into the pediatric brain tumor microenvironment and identification of novel therapeutic candidates.

EXTH-34. ION CHANNELS AS A THERAPEUTIC TARGET IN PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract Pediatric Glioblastoma Multiforme (GBM) is a leading cause of CNS cancer-related death in children. The scarcity of treatments for GBM has opened the field to new pathways in adults, such as tumour treating fields(TTF). Studies have identified the clinical benefit of electrotherapy in combination with chemotherapeutics, however the mechanistic action is unclear. Increasing evidence suggests that ion channels not only regulate electrical signalling of excitable cells, but are also crucial in the development/progression of brain tumours. Ion channels are essential in cell-cycle control, invasion and migration of cancer cells, therefore presenting as valuable therapeutic targets. Candidate ion channel genes(ICG) associated with high-grade glioma (HGG) were identified via analysis of inhouse and publicly available data sets. Expression patterns of selected ICGs were assessed along with clinician correlations.. Protein and RNA expression of target ICGs was assessed in pGBM cell lines/ TMAs. Finally, the Human ClariomTMS array was used for whole transcriptome gene expression analysis of paediatric GBM cell lines treated with tumour treating fields or low frequency electrical fields via deep brain stimulating electrodes. Paediatric brain tumour cells were exposed to genetic and pharmacological manipulation of CLIC1 and CLIC4 ion channels individually or in combination. We have shown that HGG exhibit increased expression of CLIC4 and CLIC1 at protein and RNA levels in both publically available data sets and inhouse cell lines / TMAs. Clinical correlation determined that high CLIC4 and CLIC1 expression was associated with poor overall survival. Further to this, DNA array analysis revealed a downregulation of CLIC1 and CLIC4 ion channels genes in KNS42 cells when treated with electrotherapy compared to untreated cells. Inhibition of CLIC1 and CLIC4 reduces Cl- flux across cell membranes and reduces cell proliferation. These data provide rationale that manipulation of ICGs will reduce the capacity of childhood brain tumours to proliferate and invade.

Steroidal alkaloids isolated from Veratrum grandiflorum Loes. as novel Smoothened inhibitors with anti-proliferation effects on DAOY medulloblastoma cells

Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and other tumors. Therefore, small molecular inhibitors of Hh pathway are urgently needed. In this study, three new steroidal alkaloids, ⊿5 (20R, 24R) 23-oxo-24-methylsolacongetidine, ⊿5 (20S, 24R) 23-oxo-24-methylsolacongetidine and veralinine 3-O-α-l-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside, together with six known alkaloids, 20-epi-verazine, verazine, protoverine 15-(l)-2′-methylbutyrate, jervine, veramarine and β1-chaconine, were isolated and determined from Veratrum grandiflorum Loes. The dual-luciferase bioassay indicated that all compounds exhibited significant inhibitions of Hh pathway with IC50 values of 0.72–14.31 μM against Shh-LIGHT 2 cells. To determine whether these Hh pathway inhibitors act with the Smoothened (Smo) protein, which is an important oncoprotein and target for this pathway, BODIPY-cyclopamine (BC) competitive binding assay was preferentially performed. Compared with BC alone, all compounds obviously reduced the fluorescence intensities of BC binding with Smo in Smo-overexpression HEK293T cells through fluorescence microscope and flow cytometer. By directly interacting with Smo, it revealed that they were actually novel natural Smo inhibitors. Then, their anti-tumor effects were investigated against the human MB cell line DAOY, which is a typical pediatric brain tumor cells line with highly expressed Hh pathway. Interestingly, most of compounds had slight proliferation inhibitions on DAOY cells after treatment for 24 h same as vismodegib, while β1-chaconine showed the strongest inhibitory effect on the growth of DAOY with IC50 value of 5.35 μM. In conclusion, our studies valuably provide several novel natural Smo inhibitors for potential targeting treatment of Hh-dependent tumors.

Design, synthesis and evaluation of novel indole-2-carboxamides for growth inhibition of Mycobacterium tuberculosis and paediatric brain tumour cells.

The omnipresent threat of tuberculosis (TB) and the scant treatment options thereof necessitate the development of new antitubercular agents, preferably working via a novel mechanism of action distinct from the current drugs. Various studies identified the mycobacterial membrane protein large 3 transporter (MmpL3) as the target of several classes of compounds, including the indole-2-caboxamides. Herein, several indoleamide analogues were rationally designed, synthesised, and evaluated for their antitubercular and antitumour activities. Compound 8g displayed the highest activity (MIC = 0.32 μM) against the drug-sensitive (DS) Mycobacterium tuberculosis (M. tb) H37Rv strain. This compound also exhibited high selective activity towards M. tb over mammalian cells [IC50 (Vero cells) = 40.9 μM, SI = 128], suggesting its minimal cytotoxicity. In addition, when docked into the MmpL3 active site, 8g adopted a binding profile similar to the indoleamide ligand ICA38. A related compound 8f showed dual antitubercular (MIC = 0.62 μM) and cytotoxic activities against paediatric glioblastoma multiforme (GBM) cell line KNS42 [IC50 (viability) = 0.84 μM]. Compound 8f also showed poor cytotoxic activity against healthy Vero cells (IC50 = 39.9 μM). Compounds 9a and 15, which were inactive against M. tb, showed potent cytotoxic (IC50 = 8.25 and 5.04 μM, respectively) and antiproliferative activities (IC50 = 9.85 and 6.62 μM, respectively) against KNS42 cells. Transcriptional analysis of KNS42 cells treated with compound 15 revealed a significant downregulation in the expression of the carbonic anhydrase 9 (CA9) and the spleen tyrosine kinase (SYK) genes. The expression levels of these genes in GBM tumours were previously shown to contribute to tumour progression, suggesting their involvement in our observed antitumour activities. Compounds 9a and 15 were selected for further evaluations against three different paediatric brain tumour cell lines (BT12, BT16 and DAOY) and non-neoplastic human fibroblast cells HFF1. Compound 9a showed remarkable cytotoxic (IC50 = 0.89 and 1.81 μM, respectively) and antiproliferative activities (IC50 = 7.44 and 6.06 μM, respectively) against the two tested atypical teratoid/rhabdoid tumour (AT/RT) cells BT12 and BT16. Interestingly, compound 9a was not cytotoxic when tested against non-neoplastic HFF1 cells [IC50 (viability) = 119 μM]. This suggests that an indoleamide scaffold can be fine-tuned to confer a set of derivatives with selective antitubercular and/or antitumour activities.

THER-03. IN VITRO EVALUATION OF THE EFFECT OF CANNABIDIOL ON PAEDIATRIC BRAIN TUMOUR CELL LINES USING A PULSED TREATMENT REGIME

Paediatric brain tumours are the second most common cancer after haematological malignancies. Intermittent dosing regimens are typical for chemotherapy drugs in order to avoid excessive damage to organs and avoid the onset of late effects. Cannabidiol (CBD) has been shown to have cytotoxic properties on paediatric brain tumour cell lines. Although CBD is far less toxic and damaging than the classical chemotherapy options which are currently available to children suffering with brain tumours, there are some possible side effects. Given that the half-life of the drug is 24 hours, it was important to establish the nature of the effect of cumulative dosing on top of the remaining drug in the system. The pHGG cell line, SF188 was cultured in different concentrations of CBD with either 1, 2 or 3 doses being given on consecutive days. 24 hours after the last dose the cells were analysed using the resazurin assay. It was observed that the amount of drug required for an EC50 to be obtained decreased; 17.6µM (1 dose), 8µM (2 doses), 5µM (3 doses) and that cell survival was reduced to nearly 0% in those cells which received multiple does of CBD at 17.6µM. In order to mimic the intermittent dosing regime, the cells were returned to the incubator for 4 days before the resazurin assay was repeated. The decrease in viability was maintained over the extended culture period meaning that the ability of even the apparent “healthy” cells to proliferate had been permanently affected.

THER-04. IS THERE A ROLE FOR CANNABIDIOL IN THE TREATMENT OF CHILDHOOD BRAIN TUMOURS?

Brain tumours are the leading cause of cancer related death in children with limited treatment options and high recurrence rates. Recent evidence suggests there may be anti-tumoral properties of cannabinoids, and of cannabidiol (CBD) in particular. We evaluated the effect of CBD on paediatric brain tumour cell lines in 2D and 3D spheroids; pHGG (SF188), ependymoma (BxD-1425EPN) and human astrocytes. At the CBD EC50 concentration, astrocytic cell death was insignificant. 3D spheroids decreased in size by approximately 20% when cultured in CBD compared to cells only after 5-day exposure. Cell death increased with time after a single dose of CBD. Western Blot showed an increase in Lc3b expression (autophagy) after 24 hours incubation (early cell death) with CBD in both BxD-1425EPN and SF188 with PARP expression (apoptosis) increased after 5 days incubation (late cell death). Cell cycle analysis showed a decrease of cells in G1 and no change in G2 indicating cell cycle arrest. In hypoxia, SF188 and BxD-1425EPN cells showed decreased cell death after 24 hours and 5 days when compared to normoxia and an EC50 within acceptable limits could not be achieved. SF188 cells pre-treated with receptor antagonists indicate that CBD was not acting through CB1, CB2, GPR18, PPARα or PPARγ receptors but may act as a partial agonist of the TRPV1 and 5-HT1A receptors and a full agonist of the GPR55 receptor (resazurin assay). This provides evidence that CBD is effective at killing paediatric brain tumour cells and does not have a significant effect on normal astrocytes.