Preclinical Models Of Glioblastoma Research Articles

Development and Validation of a HPLC-MS/MS Method to Measure Nifuroxazide and Its Application in Healthy and Glioblastoma-Bearing Mice.

Nifuroxazide (NAZ), a nitrofuran derivative used to treat diarrhea, has been recently shown to possess anticancer activity. However, its pharmacokinetic profile is poorly known. The pharmacokinetic profile of NAZ was thus investigated in mice using a newly developed method based on high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). We determined the concentrations of NAZ in the plasma and brain tissue of mice treated with the drug. The method proved to be specific, reproducible, precise, and accurate. It also demonstrated high sensitivity, reaching an LOQ in the order of ppb for both matrices, using samples of 100 µL or 0.2 g. The new HPLC–MS/MS assay was successfully applied to study the pharmacokinetics of NAZ after chronic intraperitoneal administration in mice at a dose of 30 mg/kg. One hour after treatment, plasma concentrations of NAZ were in the range of 336–2640 ng/mL. Moreover, unlike the brains of healthy mice or those with healed mechanical injuries, we found that NAZ was able to cross the injured blood–brain barrier of tumor-infiltrated brains. Thus, following i.p. administration, NAZ reaches systemic levels suitable for testing its efficacy in preclinical models of glioblastoma. Overall, these pharmacokinetic data provide robust evidence supporting the repositioning of NAZ as an antitumor drug.

Impact of gallic acid on tumor suppression: Modulation of redox homeostasis and purinergic response in in vitro and a preclinical glioblastoma model

Glioblastoma (GBM) is the deadliest primary brain tumor in adults due to the high rate of relapse with current treatment. Therefore, the search for therapeutic alternatives is urgent. Gallic acid (GA), a potent natural antioxidant, has antitumor and modulatory actions on purinergic signaling. In this study, we investigated the cytotoxic effects of GA on the rat GBM (C6) cell line and on astrocyte culture and analyzed its role in regulating oxidative stress and purinergic enzymes involved in GBM proliferation. Cells were exposed to GA from 50 to 400 µM for 24 and/or 48 h. Next, the effect of GA was evaluated in the preclinical model of GBM. Wistar rats were treated with 50 or 100 mg/kg of GA for 15 days, and cerebral and systemic redox status and degradation of adenine nucleotides and nucleosides in circulating platelets, lymphocytes, and serum were evaluated. Our results demonstrated that GA has selective anti-glioma activity in vitro, without inducing cytotoxicity in astrocyte. Furthermore, GA prevented oxidative stress and changes in the hydrolysis of nucleotides in GBM cells. The anti-glioma effect was also observed in vivo, as GA reduced tumor volume by 90%. Interestingly, GA decreased the oxidative damage induced by a tumor in the brain, serum, and platelets, and, also prevented changes in the degradation of nucleotides and nucleosides in lymphocytes, platelets, and serum. These results indicate, for the first time, the therapeutic potential of GA in a preclinical model of GBM, whose effects may be related to its role in redox and purinergic modulation.

Orthotopic Glioblastoma Models for Evaluation of the Clinical Target Volume Concept.

Simple SummaryThe accurate and precise definition of the target volume is of enormous importance for the treatment success of radiotherapy. In glioblastoma, the microscopic tumor extension is unclear, which limits the specificity of irradiation leading to either increased risk of local failure or enhanced toxicity rates. In this study, we investigated the microscopic tumor extensions of two different untreated and irradiated orthotopic brain tumor models and correlated this with histologically stained cancer stem cell markers as well as invasion markers and analyses using Matrix-Assisted Laser Desorption/Ionization (MALDI). We found specific MALDI peaks as potential markers for normal brain tissue but also others for demarcation of tumor areas. Furthermore, MMP14 staining revealed mainly positive cells in the tumor border, which could reflect the invasive front in both models. Altogether, the results of this study indicate that an individualized target volume definition for radiotherapy based on biological tumor characteristics in glioblastoma models seems possible.In times of high-precision radiotherapy, the accurate and precise definition of the primary tumor localization and its microscopic spread is of enormous importance. In glioblastoma, the microscopic tumor extension is uncertain and, therefore, population-based margins for Clinical Target Volume (CTV) definition are clinically used, which could either be too small—leading to increased risk of loco-regional recurrences—or too large, thus, enhancing the probability of normal tissue toxicity. Therefore, the aim of this project is to investigate an individualized definition of the CTV in preclinical glioblastoma models based on specific biological tumor characteristics. The microscopic tumor extensions of two different orthotopic brain tumor models (U87MG_mCherry; G7_mCherry) were evaluated before and during fractionated radiotherapy and correlated with corresponding histological data. Representative tumor slices were analyzed using Matrix-Assisted Laser Desorption/Ionization (MALDI) and stained for putative stem-like cell markers as well as invasion markers. The edges of the tumor are clearly shown by the MALDI segmentation via unsupervised clustering of mass spectra and are consistent with the histologically defined border in H&E staining in both models. MALDI component analysis identified specific peaks as potential markers for normal brain tissue (e.g., 1339 m/z), whereas other peaks demarcated the tumors very well (e.g., 1562 m/z for U87MG_mCherry) irrespective of treatment. MMP14 staining revealed only a few positive cells, mainly in the tumor border, which could reflect the invasive front in both models. The results of this study indicate that MALDI information correlates with microscopic tumor spread in glioblastoma models. Therefore, an individualized CTV definition based on biological tumor characteristics seems possible, whereby the visualization of tumor volume and protein heterogeneity can be potentially used to define radiotherapy-sensitive and resistant areas.

Exploratory Analysis of Serial 18F-fluciclovine PET-CT and Multiparametric MRI during Chemoradiation for Glioblastoma.

Simple SummaryGlioblastoma (GBM), the most common malignant adult primary brain tumour, has a prognosis of ~12–15 months. Poor prognosis is partly due to the inability to accurately define the extent of tumour infiltration; currently demarcated using magnetic resonance imaging (MRI) sequences (e.g., post-contrast T1-weighted (Gd-T1) and dynamic contrast-enhanced (DCE-MRI)). Anti-1-amino-3-18fluorine-fluorocyclobutane-1-carboxylic acid (18F-fluciclovine) positron emission tomography (PET) may depict GBM better than MRI. This prospective pilot study aimed to explore the relationship of 18F-fluciclovine PET, DCE-MRI and Gd-T1 in patients with GBM undergoing standard-of-care adjuvant chemoradiotherapy. A parallel mouse glioma model was used to investigate the relationship between 18F-fluciclovine PET, MRI and tumour biology. Clinical results showed that GBM volume on 18F-fluciclovine PET tended to be larger than Gd-T1 and DCE-MRI in patients with shorter overall survival (OS) but smaller in patients with longer OS. The preclinical study confirmed that 18F-fluciclovine uptake reflected biologically active tumour. Results suggest that 18F-fluciclovine PET may better define GBM infiltration than MRI.Anti-1-amino-3-18fluorine-fluorocyclobutane-1-carboxylic acid (18F-fluciclovine) positron emission tomography (PET) shows preferential glioma uptake but there is little data on how uptake correlates with post-contrast T1-weighted (Gd-T1) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) activity during adjuvant treatment. This pilot study aimed to compare 18F-fluciclovine PET, DCE-MRI and Gd-T1 in patients undergoing chemoradiotherapy for glioblastoma (GBM), and in a parallel pre-clinical GBM model, to investigate correlation between 18F-fluciclovine uptake, MRI findings, and tumour biology. 18F-fluciclovine-PET-computed tomography (PET-CT) and MRI including DCE-MRI were acquired before, during and after adjuvant chemoradiotherapy (60 Gy in 30 fractions with temozolomide) in GBM patients. MRI volumes were manually contoured; PET volumes were defined using semi-automatic thresholding. The similarity of the PET and DCE-MRI volumes outside the Gd-T1 volume boundary was measured using the Dice similarity coefficient (DSC). CT-2A tumour-bearing mice underwent MRI and 18F-fluciclovine PET-CT. Post-mortem mice brains underwent immunohistochemistry staining for ASCT2 (amino acid transporter), nestin (stemness) and Ki-67 (proliferation) to assess for biologically active tumour. 6 patients were recruited (GBM 1–6) and grouped according to overall survival (OS)—short survival (GBM-SS, median OS 249 days) and long survival (GBM-LS, median 903 days). For GBM-SS, PET tumour volumes were greater than DCE-MRI, in turn greater than Gd-T1. For GBM-LS, Gd-T1 and DCE-MRI were greater than PET. Tumour-specific 18F-fluciclovine uptake on pre-clinical PET-CT corresponded to immunostaining for Ki-67, nestin and ASCT2. Results suggest volumes of 18F-fluciclovine-PET activity beyond that depicted by DCE-MRI and Gd-T1 are associated with poorer prognosis in patients undergoing chemoradiotherapy for GBM. The pre-clinical model confirmed 18F-fluciclovine uptake reflected biologically active tumour.

Quinacrine is active in preclinical models of glioblastoma through suppressing angiogenesis, inducing oxidative stress and activating AMPK

The poor prognosis of glioblastoma requires new innovative treatment strategies. We and others have shown that targeting tumor as well as angiogenesis in glioblastoma are effective therapeutic strategies. In line with these efforts, this work reveals that Quinacrine, an antimalarial drug, is a dual inhibitor of angiogenesis and glioblastoma. Using multiple glioblastoma cell lines, we found that Quinacrine inhibited proliferation and induced apoptosis in these cells, and acted in synergy with Temozolomide. Quinacrine potently inhibited tubular structure formations of glioblastoma microvascular endothelial cell (GMVEC) isolated from glioblastoma patients, especially for early stage tubular structure formation. Although Quinacrine induces apoptosis in GMVEC, the anti-angiogenic activity of Quinacrine is independent of its pro-apoptotic activity in GMVECs. Quinacrine inhibits glioblastoma angiogenesis and growth in vivo, and acts synergistically with Temozolomide in inhibiting glioblastoma growth in mice. Mechanistically, we found that Quinacrine acts on glioblastoma through inducing oxidative stress, impairing mitochondrial function and activating AMP-activated protein kinase (AMPK). Our work is the first to demonstrate the anti-angiogenic activity of Quinacrine. Our findings highlight Quinacrine as an attractive candidate to support treatment of glioblastoma.

Abstract 5455: Nuclear export inhibitor selinexor synergizes with proteasome inhibitor marizomib in preclinical models of glioblastoma

Abstract Background: Glioblastoma (GBM) is an aggressive brain cancer with poor prognosis and limited treatment options. Selinexor is a first-in-class oral inhibitor of Exportin 1 (XPO1/CRM1) with broad anti-cancer activities and is clinically approved to treat multiple myeloma and diffuse large B cell lymphoma. In a phase 2 study of recurrent GBM, monotherapy of selinexor demonstrated clinically relevant disease control (Lassman 2021 Clin Cancer Res). Our previous preclinical studies identified synergy between selinexor and proteasome inhibitors in GBM in vitro. Among the proteasome inhibitors tested, we chose to focus on marizomib due to its ability to cross the blood-brain-barrier, supporting its use in GBM. We tested GBM cell lines and mouse xenograft models to evaluate the anti-GBM effects of selinexor and marizomib in combination. Methods: Cytotoxic effects of selinexor and marizomib were evaluated with two GBM cell lines LN18 and U87MG. The anti-GBM effects of selinexor and marizomib were further investigated in vivo with U87MG mouse xenograft models of GBM. Female nude mice carrying U87MG subcutaneous implants were treated with vehicle, selinexor, marizomib or the combination of the two agents (six mice per treatment group). Tumor volume and body weight were monitored throughout the study. Tumor samples were collected at the end of the study for transcriptomic analysis and immunohistochemistry tests. Results: Synergistic anti-GBM effects of selinexor and marizomib were observed in both LN18 and U87MG cells in vitro. In mouse xenograft models of U87MG, tumor growth was significantly inhibited by selinexor and marizomib single agents relative to controls, at Day 15 of the study, tumor growth inhibition (TGI) was 60.0% (p=0.0009) for selinexor (10 mg/kg, once per week), 49.2% (p=0.0069) for marizomib once weekly at 75 µg/kg, and 33.1% (p=0.0991) for marizomib once every 2 weeks at 75 µg/kg. Synergistic anti-GBM effect was observed in the combination groups, with a TGI of 99.6% (p<0.0001) for selinexor with once weekly marizomib, and a TGI of 90.0% (p<0.0001) for selinexor with once every other week marizomib. Conclusions: Synergistic anti-GBM effects of selinexor and marizomib in preclinical models warrants further investigation. The synergism is likely due to enhanced inhibition of the NFκB signaling pathways as previously demonstrated for selinexor and proteasome inhibitor combination. Citation Format: Hua Chang, Leah Henegar, Christopher J. Walker, Trinayan Kashyap, Marie Maloof, Feng Wang, Kathleen Martyn, Shira Orr, Sharon Tamir, Michael G. Kauffman, Sharon Shacham, Yosef Landesman. Nuclear export inhibitor selinexor synergizes with proteasome inhibitor marizomib in preclinical models of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5455.

Abstract 349: Pharmacokinetics- and pharmacodynamics-based evaluation of quisinostat as a radiosensitizer in preclinical models of human glioblastoma

Abstract Glioblastoma (GBM), the most common and aggressive primary brain tumor affecting adults, is characterized by an aberrant yet druggable epigenetic landscape. Class I histone deacetylases (HDACs) mediate chromatin compaction and are frequently overexpressed in GBM. Hence, over the last decade, there has been considerable interest in HDAC inhibitors (HDACi) for the treatment of malignant brain tumors. However, to date almost all HDACi tested clinically have failed to provide significant therapeutic benefit to primary and recurrent GBM patients. These HDACi are broad-spectrum with poor or unknown pharmacokinetic profiles and have narrow therapeutic window. Isoform specificity for HDACi is important given that not all HDAC enzymes are equally expressed in GBM. Recently, we uncovered the functional importance of HDAC1 in therapy-resistant glioma stem cells, where we found that its expression increases with brain tumor grade and is correlated with decreased survival. While no class I HDAC isoform-specific inhibitors are currently available, the second-generation HDACi, quisinostat harbors high specificity for HDAC1. In this study, we assessed the pharmacokinetic, pharmacodynamic and radiosensitizing properties of quisinostat in preclinical models of human GBM. In vitro analyses conducted in patient-derived glioma stem cell (GSC) lines revealed that quisinostat exhibited potent growth inhibition in multiple GSC lines (IC50 ~ 50-86 nM), and induced global histone hyperacetylation, elevated DNA damage, cell death, cell cycle arrest and decreased expression of multiple key stem cell proteins. To determine the efficacy and drug distribution profile of quisinostat in vivo, athymic mice with orthotopic or flank tumors were treated with an optimized treatment schedule. At various timepoints during treatment, plasma and brain/tumor tissue were collected to measure total and unbound drug levels by liquid chromatography tandem mass spectrometry (LC-MS/MS) and assess pharmacodynamic changes within tumor tissues. We found that quisinostat reduces tumor burden in flank and orthotopic models of GBM and extends survival when administered in combination with radiation therapy in vivo. Together, these results provide a rationale for developing quisinostat as a potential combination therapy with radiation in the treatment of GBM. Citation Format: Costanza Lo Cascio, Tigran Margaryan, Ernesto Luna Melendez, James McNamara, William Knight, Sarah Himes, Connor White, Alexis Giff, Jesus Peralta, Nader Sanai, Artak Tovmasyan, Shwetal Mehta. Pharmacokinetics- and pharmacodynamics-based evaluation of quisinostat as a radiosensitizer in preclinical models of human glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 349.

Abstract 6388: The effect of oncolytic virus therapy on neoantigen specific immune responses

Abstract Background: Patients with malignant brain tumor, glioblastoma (GBM), do not benefit from promising immunotherapies that use checkpoint inhibitors, such as PD-1 and CTLA-4 blockade antibodies. Several reasons may underlie this clinical result, particularly because of immunogenically cold nature of the GBM microenvironment, which is characterized by the low level of infiltrating cytotoxic CD8+ T cells and by an enriched myeloid suppressor cell population. We and other groups hypothesize that oncolytic virus (OVs)-based treatments can overcome this problem. Our preclinical GBM models and an ongoing clinical trial with oncolytic herpes simplex virus-1 (oHSV-1) at our institution (NCT03152318) demonstrated that OV treatment leads to an increase in the influx of CD8+ and CD4+ T cells into TME. However, it remained unknown whether these T cells are specific to the tumor. Here, we evaluated T-cell responses against newly identified neoantigens from murine syngeneic glioma line in response to the intratumoral OV treatments. Results: To evaluate the effect of oHSV-1 treatment on neoantigen-specific responses, we first performed whole-exome and RNA sequencing of murine GBM for the identification of tumor-specific mutations. Putative MHC I antigens were predicted with netMHCpan V4 and immunogenicity of candidates, that were selected based on affinity to MHC I molecules, were checked by IFN-gamma enzyme-linked immunospot (ELISpot) assay. With our workflow, 91 H2-Db-restricted and 77 H2-Kb-restricted peptides were identified. From this group, 8 Db- and 9 Kb-restricted peptides were selected based on their highest MHC affinity. We then asked which of these neoantigens would show enhanced presentation upon oHSV infection in vivo. To determine that we first implanted GBM tumors in the brains of syngeneic mice. At Day7 post-tumor implantation, the oHSV-1 (or vehicle control), was in situ stereotactically administered. Seven days later, splenocytes, PBMCs, and TILs were harvested to be screened. TILs from the oHSV-1 treated mice, but not those from the control group reacted to the putative neoantigens, and this activation was similar in magnitude to the response to the immunodominant HSV peptide. We are currently performing immunopeptidome analyses to confirm the binding of these neoepitopes to MHC upon oHSV treatment and in vivo studies to determine how T cells specific to these MHC-I restricted neoepitopes contribute to the anti-tumor effect. Conclusion: Our data with the preclinical model of GBM show for the first time that infection with an oHSV-1 does indeed lead to novel TIL activation against tumor neoantigens that were not presented without OV infection. Thus, oncolytic viruses may offer an in-situ vaccination approach to stimulate neoantigen-specific immune responses. Citation Format: Raziye Piranlioglu, Junfeng Liu, Anan Islam, Alex Ling, Naoyuki Shono, William F. Goins, Hiroshi Nakashima, E. Antonio Chiocca. The effect of oncolytic virus therapy on neoantigen specific immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6388.

Abstract 6023: Selinexor synergizes with temozolomide in preclinical models of glioblastoma independent of MGMT promoter methylation

Abstract Background: The alkylating agent temozolomide (TMZ) is widely applied as an effective first line treatment to glioblastoma (GBM), but its effectiveness is substantially reduced by the MGMT O6-methylguanine-DNA methyltransferase that repairs the main cytotoxic methylguanine generated by TMZ. Thus, TMZ is more effective in patients who have GBM with MGMT gene silencing promoter hypermethylation. Selinexor is an oral selective inhibitor of Exportin-1 (XPO1/CRM1) with efficacy in various solid and hematological cancers and clinically approved to treat multiple myeloma and diffuse large B cell lymphoma. Selinexor also crosses the blood brain barrier and demonstrates clinically relevant disease control with manageable and reversible side effects in a phase 2 clinical trial for patients with recurrent GBM as a monotherapy (Lassman 2021 Clin Cancer Res). To evaluate the effects of selinexor and TMZ combination, preclinical studies were performed with GBM cell lines in vitro and in mouse xenograft models in vivo. Methods: Cytotoxic effects of selinexor and TMZ were evaluated with two GBM cell lines LN18 and U87MG. The MGMT promoter is hypermethylated in U87MG cells while unmethylated in LN18 cells. The anti-cancer effects of selinexor (20 mg/kg, PO, once weekly) and TMZ (50 mg/kg, PO, QDX5, week 1 only) were also tested in a mouse xenograft model of U87MG using different dosing schedules (8 mice per treatment group). Results: Synergistic cytotoxic effects of selinexor and TMZ were observed in vitro in both LN18 and U87MG cells, regardless of the methylation status of MGMT promoter. In mouse xenograft models of U87MG, at Day 29 of study, compared with the control group, tumor growth inhibition (TGI) is 64.5% (p=0.0002) for selinexor and 67.2% (p=0.0001) for TMZ. The combination of the two agents completely inhibited tumor growth, with a TGI of 101.9% (p<0.0001) for a concomitant schedule and a TGI of 104.1% (p<0.0001) for a sequential schedule. Conclusion: The selinexor and TMZ combination demonstrated synergistic anti-cancer effects in preclinical GBM models. Previous studies have shown that selinexor treatment reduces DNA damage repair proteins involved in multiple DNA repair pathways, which would provide a mechanism to enhance the cytotoxic effects of TMZ in GBM cells independent of MGMT promoter methylation. This combination is now being investigated in an ongoing GBM clinical trial (XPORT-GBM-029, NCT04421378). Citation Format: Leah Henegar, Hua Chang, Christopher J. Walker, Trinayan Kashyap, Marie Maloof, Feng Wang, Kathleen Martyn, Shira Orr, Sharon Tamir, Michael G. Kauffman, Sharon Shacham, Yosef Landesman. Selinexor synergizes with temozolomide in preclinical models of glioblastoma independent of MGMT promoter methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6023.

A randomized phase II study of anlotinib combined with STUPP versus STUPP alone in patients with newly diagnosed glioblastoma (GBM).

TPS2079 Background: Postoperative with radiotherapy, and concomitant and up to 6 cycles of maintenance temozolomide (TMZ) chemotherapy (STUPP regimen) is now the standard treatment for newly diagnosed GBM, while the effectiveness is limited. Anlotinib is a novel, multi-kinase inhibitor against both tumor angiogenesis and tumor cell proliferation with encouraging findings in preclinical GBM models. Our previous single-arm study (NCT04119674) showed that the addition of anlotinb to STUPP exhibited promising progression-free survival (PFS) in patients with GBM. Therefore, a phase II trial was designed to assessed the efficacy and safety of anlotinib with the STUPP regimen. Methods: This is a multicenter, randomized, controlled, phase II superiority trial. Approximately 150 patients will be randomly assigned 1:1 to receive TMZ-based radiochemotherapy with anlotinib or TMZ-based radiochemotherapy with placebo. All patients will receive radiotherapy (fractionated focal irradiation in daily fractions of 1.8–2.0 Gy given 5 days per week for 6 weeks, for a total of 54–60 Gy) concurrently with TMZ (75 mg/m2 QD) and anlotinib (10 mg QD, d1–14/3wks) or placebo. Anlotinib or placebo is administrated during the 28-day treatment break. Adjuvant therapy start four weeks after radiotherapy completion, including six cycles of TMZ (150–200mg/m2, d1–5/4wks) and eight cycles of anlotinib (10 mg QD, d1–14/3wks) or placebo. Patients who complete adjuvant therapy are administrated anlotinib or placebo continuously until the disease progressed or unacceptable toxic effects developed. Eligibility criteria include histologically confirmed newly diagnosed GBM and a World Health Organization (WHO) performance status ≤ 2. Additional inclusion criteria include patients between 18 and 75 years old with adequate wound healing of craniotomy or biopsy; adequate hematologic, hepatic, and renal function; acceptable blood coagulation levels; and the stable or decreasing usage of glucocorticoid doses within 5 days before randomization. Exclusion criteria include potentially fatal intracranial hemorrhage on magnetic resonance imaging (MRI); tumors located only in the brainstem; prior chemotherapy or radiotherapy administered for GBM; and IDH1/2 mutation. The primary objective of this study is to compare PFS accessed by Independent Review Committee in patients receiving anlotinib in addition to standard treatment with patients receiving standard treatment only. Secondary endpoints include overall survival (OS), PFS accessed by investigators, overall response rate (ORR), severity and frequency of AEs (CTCAE V5.0), and quality of life (QOL). The disease control rate (DCR) and duration of response (DOR) will also be explored. Clinical trial information: NCT04959500. Research Sponsor: Chia Tai Tianqing Pharmaceutical Group Co., Ltd .

Thiazolidine-2,4-dione derivative exhibits antitumoral effect and reverts behavioral and metabolic changes in a model of glioblastoma.

The aim of the present study was to evaluate the anti-glioma activity of 3-(4-fluorobenzyl)-5-(4-methoxybenzylidene)thiazolidine-2,4-dione (AV23) in a preclinical model of glioblastoma, as well as behavioral parameters and toxicological profile. The implantation of C6 cells in the left striatum of male Wistar rats was performed by stereotaxic surgery. After recovery, animals were treated with vehicle (canola oil) or AV23 (10mg/kg/day) intragastrically for 15 days. It was found that AV23 reduced tumor volume by 90%. Serum biochemical parameters such as triglycerides, cholesterol, HDL-cholesterol, LDL-cholesterol, albumin, aspartate aminotransferase, urea, creatinine and total proteins were not changed; however, there was a slight increase in alanine aminotransferase. The compound AV23 reverted the hypoglycemia and the reduction in body weight caused by glioblastoma. Additionally, AV23 was able to revert the reduction of locomotion caused by the tumor implantation. Therefore, the compound AV23 can be considered a promising candidate in the treatment of glioblastoma.

Non-metabolic functions of phosphofructokinase-1 orchestrate tumor cellular invasion and genome maintenance under bevacizumab therapy.

Glioblastoma (GBM) is a highly lethal malignancy for which neoangiogenesis serves as a defining hallmark. The anti-VEGF antibody, bevacizumab, has been approved for the treatment of recurrent GBM, but resistance is universal. We analyzed expression data of GBM patients treated with bevacizumab to discover potential resistance mechanisms. Patient-derived xenografts (PDXs) and cultures were interrogated for effects of phosphofructokinase-1, muscle isoform (PFKM) loss on tumor cell motility, migration, and invasion through genetic and pharmacologic targeting. We identified PFKM as a driver of bevacizumab resistance. PFKM functions dichotomize based on subcellular location: cytosolic PFKM interacted with KIF11, a tubular motor protein, to promote tumor invasion, whereas nuclear PFKM safeguarded genomic stability of tumor cells through interaction with NBS1. Leveraging differential transcriptional profiling, bupivacaine phenocopied genetic targeting of PFKM, and enhanced efficacy of bevacizumab in preclinical GBM models in vivo. PFKM drives novel molecular pathways in GBM, offering a translational path to a novel therapeutic paradigm.

Analytic approaches to clinical validation of results from preclinical models of glioblastoma: A systematic review.

Analytic approaches to clinical validation of results from preclinical models are important in assessment of their relevance to human disease. This systematic review examined consistency in reporting of glioblastoma cohorts from The Cancer Genome Atlas (TCGA) or Chinese Glioma Genome Atlas (CGGA) and assessed whether studies included patient characteristics in their survival analyses. We searched Embase and Medline on 02Feb21 for studies using preclinical models of glioblastoma published after Jan2008 that used data from TCGA or CGGA to validate the association between at least one molecular marker and overall survival in adult patients with glioblastoma. Main data items included cohort characteristics, statistical significance of the survival analysis, and model covariates. There were 58 eligible studies from 1,751 non-duplicate records investigating 126 individual molecular markers. In 14 studies published between 2017 and 2020 using TCGA RNA microarray data that should have the same cohort, the median number of patients was 464.5 (interquartile range 220.5-525). Of the 15 molecular markers that underwent more than one univariable or multivariable survival analyses, five had discrepancies between studies. Covariates used in the 17 studies that used multivariable survival analyses were age (76.5%), pre-operative functional status (35.3%), sex (29.4%) MGMT promoter methylation (29.4%), radiotherapy (23.5%), chemotherapy (17.6%), IDH mutation (17.6%) and extent of resection (5.9%). Preclinical glioblastoma studies that used TCGA for validation did not provide sufficient information about their cohort selection and there were inconsistent results. Transparency in reporting and the use of analytic approaches that adjust for clinical variables can improve the reproducibility between studies.

Tannic Acid Attenuates Peripheral and Brain Changes in a Preclinical Rat Model of Glioblastoma by Modulating Oxidative Stress and Purinergic Signaling.

Glioblastoma (GB) is a highly aggressive and invasive brain tumor; its treatment remains palliative. Tannic acid (TA) is a polyphenol widely found in foods and possesses antitumor and neuroprotective activities. This study aimed to investigate the effect of TA on oxidative stress parameters and the activity of ectonucleotidases in the serum, platelets, and lymphocytes and/or in the brain of rats with preclinical GB. Rats with GB were treated intragastrically with TA (50mg/kg/day) for 15days or with a vehicle. In the platelets of the animals with glioma, the adenosine triphosphate (ATP) and adenosine monophosphate (AMP) hydrolysis and the catalase (CAT) activity decreased. Besides, the adenosine diphosphate (ADP) hydrolysis, adenosine (Ado) deamination, and the reactive oxygen species (ROS) and nitrite levels were increased in glioma animals; however, TA reversed ROS and nitrite levels and AMP hydrolysis alterations. In lymphocytes from animals with glioma, the ATP and ADP hydrolysis, as well as Ado deamination were increased; TA treatment countered this increase. In the brain of the animals with glioma, the ROS, nitrite, and thiobarbituric acid reactive substance (TBARS) levels increased and the thiol (SH) levels and CAT and superoxide dismutase (SOD) activities were decreased; TA treatment decreased the ROS and TBARS levels and restored the SOD activity. In the serum of the animals with glioma, the ATP hydrolysis decreased; TA treatment restored this parameter. Additionally, the ROS levels increased and the SH and SOD activity decreasedby glioma implant; TA treatment enhanced nitrite levels andreversed SOD activity. Altogether, our results suggest that TA is an important target in the treatment of GB, as it modulates purinergic and redox systems.

Postoperative radiotherapy with concomitant temozolomide plus anlotinib for newly diagnosed glioblastoma: Study protocol for a multicenter, double-blind, randomized phase II trial

Background and Aim: Postoperative radiotherapy with concomitant temozolomide (TMZ) followed by ≤ six cycles of adjuvant TMZ chemotherapy (Stupp regimen) is the standard treatment for newly diagnosed glioblastoma (GBM); however, its effectiveness is limited. Anlotinib inhibits both tumor angiogenesis and tumor cell proliferation by targeting multiple kinases, and showing promising results in preclinical GBM models. We designed a Phase II trial to assess the efficacy and safety of the Stupp regimen plus anlotinib. Methods/Design: This is a multicenter, double-blind, randomized, placebo-controlled trial with an expected 150 patients randomly assigned 1:1 ratio to receive TMZ-based radiochemotherapy with anlotinib (Stupp regimen + anlotinib) or placebo (Stupp regimen + placebo). Postoperative patient recruitment will begin in July 2021, the analysis of primary outcome measures will be completed in July 2023, and the study will finish in July 2024. Major eligibility criteria include histologically confirmed newly diagnosed GBM and an Eastern Cooperative Oncology Group performance score ≤2. Other criteria for inclusion include age ≥18 years and lack of significant comorbidity. In this study, the primary endpoint will be the median progression-free survival (PFS). Secondary endpoints include 1-year overall survival rate, PFS at 6 months, overall response rate, duration of response, disease control rate, quality of life, and toxicity. The protocol has received approval from Sun Yat-sen University Cancer Center Ethics Committee (Approval No. B2021-073-01; approved on April 19, 2021). Discussion: Updated treatment modalities are required for newly diagnosed GBM. Our proposed treatment modality of the Stupp regimen combined with anlotinib therapy are waiting conclusion. Trial Registration: This study was registered with ClinicalTrials.gov under identifier NCT04959500 on July 13, 2021.

Transitioning pre-clinical glioblastoma models to clinical settings with biomarkers identified in 3D cell-based models: A systematic scoping review.

Glioblastoma (GBM) remains incurable despite the overwhelming discovery of 2-dimensional (2D) cell-based potential therapeutics since the majority of them have met unsatisfactory results in animal and clinical settings. Incremental empirical evidence has laid the widespread need of transitioning 2D to 3-dimensional (3D) cultures that better mimic GBM's complex and heterogenic nature to allow better translation of pre-clinical results. This systematic scoping review analyses the transcriptomic data involving 3D models of GBM against 2D models from 22 studies identified from four databases (PubMed, ScienceDirect, Medline, and Embase). From a total of 499 genes reported in these studies, 313 (63%) genes were upregulated across 3D models cultured using different scaffolds. Our analysis showed that 4 of the replicable upregulated genes are associated with GBM stemness, epithelial to mesenchymal transition (EMT), hypoxia, and migration-related genes regardless of the type of scaffolds, displaying close resemblances to primitive undifferentiated tumour phenotypes that are associated with decreased overall survival and increased hazard ratio in GBM patients. The upregulation of drug response and drug efflux genes (e.g. cytochrome P450s and ABC transporters) mirrors the GBM genetic landscape that contributes to in vivo and clinical treatment resistance. These upregulated genes displayed strong protein-protein interactions when analysed using an online bioinformatics software (STRING). These findings reinforce the need for widespread transition to 3D GBM models as a relatively inexpensive humanised pre-clinical tool with suitable genetic biomarkers to bridge clinical gaps in potential therapeutic evaluations.

Functional Characterization of Brain Tumor-Initiating Cells and Establishment of GBM Preclinical Models that Incorporate Heterogeneity, Therapy, and Sex Differences.

Glioblastoma (GBM) is the most common primary brain cancer in adults where tumor cell heterogeneity and sex differences influence clinical outcomes. Here, we functionally characterize three male and three female patient-derived GBM cell lines, identify protumorigenic BTICs, and create novel male and female preclinical models of GBM. Cell lines were evaluated on the following features: proliferation, stemness, migration, tumorigenesis, clinical characteristics, and sensitivity to radiation, TMZ, rhTNFSF10 (rhTRAIL), and rhBMP4 All cell lines were classified as GBM according to epigenetic subtyping, were heterogenous and functionally distinct from one another, and re-capitulated features of the original patient tumor. In establishing male and female preclinical models, it was found that two male-derived GBM cell lines (QNS108 and QNS120) and one female-derived GBM cell line (QNS315) grew at a faster rate in female mice brains. One male-derived GBM cell line (QNS108) decreased survival in female mice in comparison with male mice. However, no survival differences were observed for mice injected with a female-derived cell line (QNS315). In summary, a panel of six GBM patient-derived cell lines were functionally characterized, and it was shown that BTIC lines can be used to construct sex-specific models with differential phenotypes for additional studies.

Modelling the microenvironment of the most aggressive brain tumours for preclinical studies

Preclinical and clinical cancer studies use unrepresentative tumour models that do not properly simulate the intricate pathobiology of the human tumour and its complex microenvironment. This is of critical importance for the brain tumour glioblastoma (GBM), one of the most malignant cancers, which almost always relapses despite treatment. Due to the intertumoral and intratumoral heterogeneity of GBM, it is highly resistant to conventional treatment with radiotherapy and chemotherapy. In this review, we describe the heterogeneity of GBM and the unique microenvironment of brain tumours as well as their importance for GBM progression and to set up clinically relevant GBM models for successful preclinical research and drug screening. A combination of cancer cells, cancer stem cells, and cancer-associated cells create an intricate and diverse tumour microenvironment. GBM is very adept at recruiting healthy cells to support itself. The function of these cancer-associated cells varies in different niches, but they all share the common function of promoting tumour growth and spread. Glioblastoma stem cells are one of the central components of GBM treatment resistance, invasiveness, and cancer stem cell niche relationships. All of these aspects are critical when selecting and designing preclinical GBM models.

IMMU-21. DIFFERENTIAL EXPRESSION OF ADHESION MOLECULES DEFINES MYELOID CELL INFILTRATION IN GLIOBLASTOMA AND COMPRISES A THERAPEUTIC TARGET

Abstract A potently immunosuppressive tumor microenvironment facilitates progression of glioblastoma (GBM). Immunotherapies have had variable success in improving the outcome of GBM patients, suggesting that there is a need to gain insight into the mechanisms of immunosuppression. Myeloid-derived suppressor cells (MDSCs) associate with poor prognosis and treatment resistance of GBM patients, but the distinct role of individual populations is not well-defined. We previous showed that monocytic MDSCs (mMDSCs) accumulated in tumors of mice and patients, while granulocytic MDSCs (gMDSCs) mainly remained in the circulation. Furthermore, nonspecific targeting of mMDSCs with chemotherapies provided therapeutic benefit in preclinical models of GBM, suggesting that mMDSCs drive disease progression. To investigate the differential function of mMDSCs versus gMDSCs in GBM, we adoptively transferred bone marrow-derived MDSC subsets into tumor-bearing mice. Mice that received mMDSCs succumbed to disease sooner compared to control mice, which was not observed with gMDSC transfer. To determine the basis of this pro-tumorigenic activity of mMDSCs, we performed ATAC-sequencing and comparison of differentially accessible regions indicated that cell adhesion pathways were significantly upregulated in mMDSCs. Aligned with this epigenetic profile, mMDSCs from bone marrow and blood had significantly higher surface integrin β1 and integrin β7 expression compared to gMDSCs. To evaluate the role of integrins in MDSC behavior, we pre-treated mMDSCs with anti-integrin β1 prior to adoptive transfer. Blockade of integrin β1 interfered with the pro-tumorigenic role of mMDSCs compared to isotype controls. Similarly, blockade of integrin β1 and integrin β7 systemically extended the survival duration of tumor-bearing mice. Finally, high expression of integrin β1 and integrin β7 served as a poor prognostic indicator in GBM patients. Our findings indicate that modulation of immunosuppressive myeloid cells by leveraging differences in adhesion mechanisms represents a potential immunotherapeutic option for GBM.

ITVT-02. Elucidating the pleiotropic effects of verteporfin photodynamic therapy in preclinical glioblastoma models

Abstract Glioblastoma (GBM) ranks the highest in mortality rate, incidence, and aggressiveness of primary brain tumor types. The highly malignant nature of GBM makes it difficult for mainstay treatments to have an effect beyond stabilizing the disease. Deregulation of receptor tyrosine kinase (RTKs), such as EGFR and PI3K, have been implicated in GBM tumorigenesis. The expression of EGFR has been linked to hippo pathway transcriptional co-activators YAP and TAZ that bind to TEAD co-factors to drive the transcription of target genes. The convergence of EGFR signaling and the hippo pathway regulates stem cell programs, including proliferation, survival, and self-renewal. Verteporfin (VP), an FDA-approved macular degeneration therapy, has antitumor effects in in vitro and in vivo GBM preclinical models and phase I/II clinical trials for patients with EGFR mutated/amplified GBM by abrogating YAP/TAZ-TEAD interactions. As a porphyrin derivative, VP can exert therapeutic and photodynamic effects in the presence of 689 nm light; however, the efficacy of VP-PDT has not been explored in GBM. Our results indicate that VP-PDT reduces cell viability to a greater extent than solitary VP treatment (viability — 0.7 uM VP: 97%, 0.7 uM VP-PDT: 46%). The antitumor effects of VP-PDT are two pronged involving 1) inhibition of live cell dynamics, including migration and intravasation, by downregulating hippo pathway constituents YAP, TAZ and TEAD and transcriptional target EGFR and 2) induction of programmed cell death by reactive oxygen species. Our results suggest that VP-PDT can be a potential avenue for treating these incurable tumors.