bluuebunny/crossref_metadata_2025_split huggingface dataset:
EXTH-28. NLGN4X TCR TRANSGENIC T CELLS TARGETING EXPERIMENTAL GLIOMAS
Authors: Krämer Christopher , Kilian Michael , Will Rainer , Sanghvi Khwab , Green Edward , Friedrich Mirco , Wick Wolfgang , Platten Michael , Bunse Lukas 
Published: November 2021

Abstract

BACKGROUND
The application of personalized vaccines has shown to be effective in patients with newly diagnosed glioblastoma in phase 1 clinical trials. Responses of CD8 T cells directed against the glioma-associated antigen Neuroligin-4, X-linked (NLGN4X) were reported in multipeptide vaccine trials in patients with glioblastoma. Here, we characterized the functional status of NLGN4X TCR transgenic T cells in vitro and assessed their therapeutic capacity in vivo.

METHODS
TCR encoding sequences were delivered by lentiviral transduction to activated T cells from healthy donors. After confirmation of TCR surface expression T cells were used for a functional in vitro characterization. For in vivo assessment of NLGN4X-specific TCR transgenic T cells, NLGN4X-expressing U87 glioma cells were injected into the flank of NSG MHCI/MHC II knockout mice, which do not develop graft versus host disease. TCR transgenic T cells were injected intravenously on day 11 and day 18 and tumor size was monitored.

RESULTS
TCR transgenic T cells depicted stable surface expression for at least 11 days in vitro after transduction. Thereby, murine TCR beta constant region positive T cells featured a polyfunctional phenotype demonstrated by a significant increase of Interferon-γ and TNF-α and remained reactive to the NLGN4X epitope for at least 7 days. Additionally, NLGN4X TCR transgenic T cells showed significantly increased antigen-specific production of the cytolytic protein granzyme B and elevated levels of perforin. In a novel xenograft mouse model NLGN4X TCR transgenic T cells slowed the tumor growth compared to the initial tumor size until day 25 after tumor inoculation.

DISCUSSION
We demonstrate that NLGN4X TCR transgenic T cells specifically and consistently recognize their corresponding immunogenic sequence and target antigen-overexpressing glioma cells. We present first evidence of in vivo reactivity, while further experiments are required to assess the full therapeutic potential of NLGN4X-TCR-transgenic T cell therapy for glioma patients.
EXTH-32. NOVEL THIORIDAZINE DERIVATES: ANTIPROLIFERATIVE AND APOPTOSIS-INDUCING ACTIVITY ON GLIOBLASTOMA CELLS IN VITRO
Authors: Schwab Stephanie , Sarnow Katharina , Alme Eirin , Goldbrunner Roland , Bjørsvik Hans-René , Bjerkvig Rolf 
Published: November 2021

Abstract

BACKGROUND
Although withdrawn from the market due to cardiotoxicity, we showed that the antipsychotic drug Thioridazine shows chemosensitizing effects in combination with Temozolomide (TMZ) for the treatment of glioblastoma multiforme (GBM). Based on our prior observations, the aim of this study was through medicinal chemistry, to design and synthesize new compounds based on Thioridazines tricyclic structure, and to determine their therapeutic potential.

METHODS
Fourteen compounds were synthesized where variations were made within the tricyclic side chains. The newly synthesized compounds were screened for therapeutic efficacy with or without TMZ using a WST-1 cell viability assay and real-time imaging system (IncuCyte). Tests were performed on both monolayer cell cultures, as well as on glioma stem cell spheroids (GSC). The therapeutic effects were also studied on human astrocytes (NHA) as well as on rat brain organoids (BO). Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis was performed after 48 hours of treatment.

RESULTS
Following an extensive screening, we identified two novel compounds (EA01 and EA02) that at concentrations of 4 and 9.5 µM showed a strong cytotoxicity on GBM cell lines (U-87 MG p&lt; 0,0001, U251 p&lt; 0,0001, LN18 p= 0,0004) as well as on glioma stem cells (GSC) (P3 p&lt; 0,0001) compared to NHA and BOs respectively. Also, when BOs were confronted with GSC spheres in an invasion assay, a selective cytotoxicity was observed in the GSCs. Mechanistically, we show that both compounds induce apoptosis in GBM cells. Moreover, intravenous delivery of increasing concentrations of EA01 and EA02 revealed no toxicity in animals at concentrations up to 21 mg/kg.

CONCLUSION
We developed two new tricyclic therapeutic compounds that show a strong selective cytotoxicity in GBM cells with limited systemic toxicity in animals. Ongoing studies are investigating the therapeutic potential of EA01 and EA02 in orthotopic xenografts in vivo.
EXTH-37. PAK4 INHIBITION REPROGRAMS VASCULAR MICROENVIRONMENT AND IMPROVES CAR T IMMUNOTHERAPY IN GLIOBLASTOMA
Authors: Yang Fan , Binder Zev , O’Rourke Donald M , Fan Yi 
Published: November 2021

Abstract
Malignant solid tumors are characterized by aberrant vascularity that inhibits T cell adhesion to vasculature and impedes T cell delivery into the tumors, contributing to glioblastoma (GBM) resistance to T cell-based immunotherapy such as adoptive chimeric antigen receptor (CAR)–T transfer. Vascular abnormality is driven by pro-angiogenic pathway activation and genetic reprogramming in tumor endothelial cells (ECs). Here, our kinome-wide functional screening of mesenchymal-like transcriptional activation in human GBM-derived ECs identifies p21-activated kinase 4 (PAK4) as a selective regulator of genetic programming and aberrant vascularization in tumor. Genetic ablation of PAK4 reprograms EC transcriptome and inhibits mesenchymal-like transformation. Interestingly, deficiency of PAK4 induces adhesion protein re-expression in tumor ECs, reduces vascular abnormalities in tumors, improves T cell infiltration into the tumors, and inhibits GBM growth in mice. Moreover, pharmacological PAK4 inhibition normalizes the tumor vascular microenvironment and sensitizes GBM to Egfrviii CAR–T cell immunotherapy. Finally, we reveal a MEF2D/ZEB1- and SLUG-mediated mechanism by which PAK4 reprograms the EC transcriptome and downregulates claudin-14 and VCAM-1 expression, enhancing vessel permeability and reducing T cell adhesion to the endothelium. Thus, targeting PAK4-mediated EC plasticity may offer exciting opportunities to recondition the vascular microenvironment and strengthen cancer immunotherapy.
EXTH-40. MULTIDIMENSIONAL INTERROGATION OF GLIOBLASTOMA MICROENVIRONMENT TREATMENT RESPONSE IN EXPLANT CULTURES
Authors: Shekarian Tala , Bartoszek-Kandler Ewelina , Zinner Carl , Schuerch Christian , Hutter Gregor 
Published: November 2021

Abstract
The immune tumor microenvironment (iTME) of glioblastoma (GBM) contains microglial, macrophage, other myeloid cell populations and as adaptive immune cells. Recent therapeutic strategies for GBM aim at targeting iTME components to induce antitumoral immunity. A patient-tailored, ex vivo drug testing and response analysis platform would facilitate personalized therapy planning, provide insights into treatment-induced immune mechanisms in the iTME, and enable the discovery of biomarkers of response and resistance. Here, we generated patient-derived, live 3D GBM bioreactors from different tumor regions to assess iTME treatment responses to microglia modulators and immune checkpoint inhibitors. Intact GBM tissue specimens from the tumor center and periphery were cultured for 7 days in the presence or absence of anti-PD1, anti-CD47 antibodies or their combination. Tissues were analyzed by CODEX highly multiplexed microscopy using an immune-centered 54-marker panel, and changes in cytokine and chemokine levels in culture supernatants were investigated. A computational pipeline for integrative therapy response assessment was implemented. Explant cultures from n=8 IDH wt GBM were subjected to this integrative personalized analysis. Tissue integrity after 3D bioreactor cultures was comparable to tissue taken directly after surgery. FFPE CODEX workflow was feasible with adequate staining quality in bioreactor cultures. 850'000 single cells were segmented and clustered. Cellular composition between tumor center and the peripheral invasion zone differed significantly in immune phenotypes, cytokine profile and response to innate, adaptive or combinatorial local immunotherapies. Multiplexed cytokine analysis revealed IFNγ response signatures in a subset of center samples, whereas the peripheral invasion zone displayed a blunted cytokine response. This cytokine signature corresponded to cellular composition shifts within specific cellular neighborhoods. CD4 and CD8 T cells were invigorated and left their vascular niche. Our study demonstrates that local immunotherapies enable an active antitumoral immune response within the tumor center, and provides a multidimensional personalized framework for immunotherapy response assessment.
EXTH-42. COMBINATION OF MAPK PATHWAY INHIBITORS AND IMMUNE CHECKPOINT BLOCKADE IN BRAF-MUTANT HIGH-GRADE GLIOMA
Authors: Park Jong-Whi , Grossauer Stefan , Wang Wei , Daynac Mathieu , Pitteri Sharon , Monje Michelle , Grant Gerald , Petritsch Claudia 
Published: November 2021

Abstract

BACKGROUND
Despite successes, clinical MAPK pathway inhibitors show limited anti-tumor activity in the majority of patients with BRAF-mutant high-grade glioma. Because of the presence of higher fraction of CD8+ tumor-infiltrating T cells in MAPK pathway-altered glioma, we explored the possibility that combined BRAF and MEK inhibition with immune checkpoint blockade enhances anti-tumor response.

METHODS
We engineered mice to carry BRAF V600E expression and CDKN2A deletion in various hemispheric areas. We treated syngeneic tumor-bearing mice with dabrafenib, trametinib, anti-PD-L1 and anti-CTLA-4 antibodies, and analyzed the tumor immune infiltrate by high-dimensional single-cell mass cytometry (CyTOF). RNA sequencing and Gene Set Enrichment Analysis were conducted using patient-derived BRAF-mutant glioma lines upon the inhibitor treatment.

RESULTS
The transcriptome analysis demonstrated that antigen processing and presentation feature is strongly enriched upon dual MAPK pathway inhibition. Consistent with these molecular changes, dabrafenib and trametinib treatment led to dynamic changes in tumor-infiltrating immune cells, including CD8+ and CD4+ T cells. In line with this, combination of MAPK pathway and immune checkpoint inhibitors elicit a significant survival benefit over MAPK pathway inhibition alone in mice with orthotopic BRAF-mutant glioma.

CONCLUSIONS
Clinically relevant molecular targeted therapy by dabrafenib and trametinib and immune checkpoint blockade synergize in pre-clinical models.
EXTH-47. SAFETY AND EFFECTS OF TRANSCRANIAL MAGNETIC STIMULATION ON GLIOBLASTOMA MEASURED IN VITRO
Authors: O'Neal Christen , Scott Sydney , Stephens Tressie , McKernan Patrick , Chakraborty Arpan , Babu Anish , Evans Alexander , Glenn Chad , Battiste James 
Published: November 2021

Abstract

BACKGROUND
Although gross total resection (GTR) with chemoradiation is the standard of care for treating glioblastoma (GBM), tumor infiltration and treatment sequelae can impair activity of eloquent regions. Transcranial magnetic stimulation (TMS) has been explored as an adjunct therapy to rehabilitation for post-stroke motor deficits. TMS could be effective for postoperative rehabilitation in GBM, but its effect on GBM cells has not been evaluated. While TMS utilizes magnetic fields to induce electrical currents at low frequencies to cause neuronal excitation or inhibition, tumor-treating fields (TTF) utilize electrical currents with intermediate frequency to exert anti-mitotic effects, demonstrating promise as an adjunctive therapy in recurrent GBM. Although similarities exist between electrical and magnetic fields, the effects of magnetically induced electrical currents at low frequencies via TMS must be studied systematically in vitro on GBM cell lines.

METHODS
We studied the effect of theta burst stimulation (TBS), a form of patterned TMS, on in vitro G55 cell viability using colony forming assays. We compared TMS-treated cells to controls using a combination of parameters: continuous versus intermittent TBS (cTBS and iTBS), 300 versus 600 pulses, stimulation intensity of 32% versus 60%, and no pre-TMS chemotherapy versus 100 nM or 100 µM temozolomide (TMZ). Viability measurements between controls and TMS were analyzed using analysis of variance (ANOVA). Independent t-tests were used to analyze effects of stimulation parameters on viability percent difference within each TMZ condition.

RESULTS
There was no statistically significant increase in viability between control and TMS conditions for any of the stimulation parameters (+/- TMZ) while some showed decreased viability of GBM cells.

CONCLUSIONS
TMS did not significantly increase GBM viability compared to controls. Future studies include validation in other cell lines and characterization of the effects of stimulation parameters in conjunction with TMZ and dexamethasone, (often administered concurrently with GBM treatment).
EXTH-61. MODULATION OF THE IL-27 RECEPTOR SIGNALING PATHWAY IN GLIOBLASTOMA AND ONCOLYTIC VIROTHERAPY
Authors: Panagioti Eleni , Nowicki Michal , Goins William , Macias Jorge Jimenez , Escobar Julia , Zdioruk Mykola , Saini Shikha , Ligon Keith , Anderson Ana Carrizosa , Kuchroo Vijay Kumar , Lawler Sean , Chiocca E Antonio 
Published: November 2021

Abstract
Glioblastoma (GBM) is the most common primary malignant brain tumor in adults with median overall survival of 14-16 months. Glioblastoma progression involves multiple immunosuppressive pathways hindering the success of cancer immunotherapy. Oncolytic virotherapy is a promising approach to reprogram the glioblastoma microenvironment and restore anti-tumor immunity. Interleukin (IL)-27 receptor signaling pathway regulates development of effector T cells, IL-10 producing T regulatory type 1 (Tr1) cells and induction of co-inhibitory receptors associated with T cell exhaustion in cancer. In this study, we sought to tackle local and systemic GBM-induced immunosuppression using the novel rQNestin34.5v2 oncolytic herpes simplex virus type-1 (HSV-1), engineered to selectively replicate in glioma cells together with genetic disruptions of the IL-27 receptor signaling pathway. The antitumor activity of HSV-1 rQNestin34.5v2 was evaluated in IL-27ra-/- and wild-type C57BL/6 mice harboring orthotopic CT-2A and GL261 glioblastoma and in athymic nude mice harboring orthotopic patient derived glioblastoma xenografts (PDXs) selected from patients that had been treated in a clinical trial of intratumoral administration of HSV-1 rQNestin34.5v2 (ClinicalTrials.gov: NCT03152318). HSV-1 rQNestin34.5v2 exhibited superior capacity to infect glioma cells in vitro triggering release of proinflammatory cytokines and inducing immunogenic cell death in infected cells. Immune phenotyping of GL261 and CT-2A gliomas revealed astrocytes and microglia express the highest levels of IL-27R. Lack of IL-27R signaling decreased expression of the co-inhibitory receptors PD-1 and Tim-3 on circulating CD4+ T cells suggesting altered CD4+ T cell responses. In the tumor microenvironment, lack of IL-27R signaling decreased immune suppression as evidenced by reduced interleukin (IL)-10 secretion by glioma cells. Despite positive functional modifications on CD4+ T cells, global lack of IL-27R signaling promoted tumor growth accompanied by increased circulating myeloid derived suppressor cells (MDSCs) in the GL261 model. Experiments to address the immunoregulatory role of IL-27R signaling during HSV-1 rQNestin34.5v2 virotherapy-induced glioma tissue destruction are ongoing.
EXTH-67. THERAPEUTIC EFFICACY OF GC1118, A NOVEL ANTI-EGFR ANTIBODY, AGAINST GLIOBLASTOMA WITH HIGH EGFR AMPLIFICATION IN PATIENT-DERIVED XENOGRAFTS
Authors: Choi Seung Won 
Published: November 2021

Abstract
We aimed to evaluate the preclinical efficacy of GC1118, a novel anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), against glioblastoma (GBM) tumors using patient-derived xenograft (PDX) models. A total of 15 distinct GBM PDX models were used to evaluate the therapeutic efficacy of GC1118. Genomic data derived from PDX models were analyzed to identify potential biomarkers associated with the anti-tumor efficacy of GC1118. A patient-derived cell-based high-throughput drug screening assay was performed to further validate the efficacy of GC1118. Compared to cetuximab, GC1118 exerted comparable growth inhibitory effects on the GBM tumors in the PDX models. We confirmed that GC1118 accumulated within the tumor by crossing the blood–brain barrier in in vivo specimens and observed the survival benefit in GC1118-treated intracranial models. Genomic analysis revealed high EGFR amplification as a potent biomarker for predicting the therapeutic efficacy of GC1118 in GBM tumors. In summary, GC1118 exerted a potent anti-tumor effect on GBM tumors in PDX models, and its therapeutic efficacy was especially pronounced in the tumors with high EGFR amplification. Our study supports the importance of patient stratification based on EGFR copy number variation in clinical trials for GBM. The superiority of GC1118 over other EGFR mAbs in GBM tumors should be assessed in future studies.
EXTH-70. ESTABLISHMENT OF INTRAVENTRICULAR SHH INHIBITION AS A THERAPEUTIC OPTION IN YOUNG PATIENTS WITH MEDULLOBLASTOMA
Authors: Kresbach Catena , Schoof Melanie , Leven Tara , Holst Lea , Yorgan Timur , Wrzeszcz Antonina , Rutkowski Stefan , Schüller Ulrich 
Published: November 2021

Abstract
Medulloblastoma is the most common malignant brain tumor in children. The embryonal tumor arises in the posterior fossa and disseminates via the cerebrospinal fluid. Medulloblastoma divides in four molecular subgroups, one of which is characterized by mutations in the sonic-hedgehog (SHH) -pathway. SHH inhibition provides an elegant way of targeted therapy. The small molecule Vismodegib allosterically inhibits Smoothened (SMO), an upstream activator of SHH. Unfortunately, Vismodegib has shown to cause irreversible premature epiphyseal growth plate fusions in preclinical studies and clinical trials, preventing its systemic application in children (Kimura et al. 2008; Robinson et al. 2017). We established an intraventricular therapy with Vismodegib, combining the benefits of targeted drug delivery and minimizing systemic side effects. In a mouse model for SHH medulloblastoma, we compare intraventricular, oral and placebo treatment regarding effects on survival, tumor biology, and bone morphology. Math1-cre::Ptch1 Fl/Fl mice show a homozygous loss of PTCH1 in Math1-positive cells, resulting in SHH-pathway overactivation and development of SHH medulloblastomas. At postnatal day 11-13, Math1-cre::Ptch1 Fl/Fl mice were randomized in four treatment arms: group A (n= 9) received placebo intrathecally, B (n= 9) received Vismodegib 200 mg/kd/d orally, C (n= 19) received Vismodegib 0.2 mg/kg/d intrathecally, and D (n= 8) received Vismodegib 1.6 mg/kg/d intrathecally. Kaplan-Meier survival analysis showed a significant survival benefit for 1.6 mg/kg/d intraventricular Vismodegib compared to placebo (p= 0.012). Bone histology and X-ray analysis of intraventricular treated mice showed intact femoral and tibial growth plates, in contrast to orally treated mice that developed skeletal malformations. Further analyses such as DNA sequencing, gene expression analysis, and histological evaluation are ongoing and will add to the picture of the anti-tumor effects of intraventricular SHH-inhibition. Based on the preliminary experimental results, we conclude that intrathecal application of a SMO-inhibitor might evolve as a promising new way of targeted treatment of SHH medulloblastomas.
EXTH-74. INCREASING CANCER CELL MEMBRANE PERMEABILITY THROUGH APPLICATION OF TUMOR TREATING FIELDS (TTFIELDS)
Authors: Voloshin Tali , Koltun Bella , Koren Lilach , Porat Yaara , Volodin Alexandra , Kaynan Noa , Klein-Goldberg Anat , Paz Rom , Brant Boris , Zemer-Tov Efrat , Haber Adi , Giladi Moshe , Weinberg Uri , Palti Yoram 
Published: November 2021

Abstract

INTRODUCTION
Tumor Treating Fields (TTFields) are intermediate frequency, alternating electric fields with anti-mitotic effects on cancerous cells. TTFields are delivered non-invasively through arrays placed on the skin at the tumor region. TTFields therapy is approved in several territories for treatment of glioblastoma (GBM) and mesothelioma. Recently, TTFields have been shown to increase GBM cell membrane permeability. The current study aimed to explore this effect in multiple cell lines and examine the potential benefits of combining TTFields with other anticancer agents.

METHODS
TTFields were delivered to GBM (U-87 MG), uterine sarcoma (MES-SA), and breast adenocarcinoma (MCF-7) cell lines for 72hr across a range of frequencies (50-500kHz). Cytotoxicity of TTFields was examined by cell counts, and intracellular accumulation of 7-aminoactinomycin D (7-AAD) was measured by flow cytometry. Exposing the cells to 7-AAD at different time points relative to TTFields application cessation was used to determine the kinetics of cell membrane permeability. The potential of TTFields to facilitate intracellular accumulation of anthracycline chemotherapeutics was tested in chemotherapy-sensitive and chemotherapy-resistant cells.

RESULTS
Elevated intracellular accumulation of 7-AAD was observed in all examined cell lines treated with TTFields, at an optimal frequency that differed from that for maximal TTFields-induced cytotoxicity. No intracellular accumulation of 7-AAD was seen for measurements performed after termination of TTFields application, indicating that increased cell membrane permeability by TTFields was temporary and reversible. Lastly, the accumulation of chemotherapeutic agents in chemotherapy-resistant cancer cells was elevated to the same extent as in matched chemotherapy-sensitive cells when TTFields were delivered concomitant with chemotherapy.

CONCLUSIONS
TTFields increased cancer cell permeability in a transient and reversible manner across multiple cancer cell types. The increased permeability enhanced intracellular accumulation of chemotherapeutics, even within chemotherapy-resistant cells.