Orthotopic Brain Tumor Model Research Articles

Optimization, Characterization, and Comparison of Two Luciferase-Expressing Mouse Glioblastoma Models.

Glioblastoma (GBM) is the most aggressive brain cancer. To model GBM in research, orthotopic brain tumor models, including syngeneic models like GL261 and genetically engineered mouse models like TRP, are used. In longitudinal studies, tumor growth and the treatment response are typically tracked with in vivo imaging, including bioluminescence imaging (BLI), which is quick, cost-effective, and easily quantifiable. However, BLI requires luciferase-tagged cells, and recent studies indicate that the luciferase gene can elicit an immune response, leading to tumor rejection and experimental variation. We sought to optimize the engraftment of two luciferase-expressing GBM models, GL261 Red-FLuc and TRP-mCherry-FLuc, showing differences in tumor take, with GL261 Red-FLuc cells requiring immunocompromised mice for 100% engraftment. Immunohistochemistry and MRI revealed distinct tumor characteristics: GL261 Red-FLuc tumors were well-demarcated with densely packed cells, high mitotic activity, and vascularization. In contrast, TRP-mCherry-FLuc tumors were large, invasive, and necrotic, with perivascular invasion. Quantifying the tumor volume using the HALO® AI analysis platform yielded results comparable to manual measurements, providing a standardized and efficient approach for the reliable, high-throughput analysis of luciferase-expressing tumors. Our study highlights the importance of considering tumor engraftment when using luciferase-expressing GBM models, providing insights for preclinical research design.

Abstract CT056: Development of a novel next-generation RET inhibitor

Abstract Oncogenic RET alterations confer constitutive activation in multiple types of human cancers. Two first-generation selective RET inhibitors selpercatinib and pralsetinib have been approved for patients with RET-driven solid tumors including non-small cell lung cancer (NSCLC) and thyroid cancer. However, critical unmet medical needs remain due to drug resistance caused by RET mutations in patients treated with the first-generation RET inhibitors. To address this emerging clinical issue, we discovered a highly potent and selective RET inhibitor with novel chemical scaffold through structure-based drug design, APS03118. Preclinical studies showed APS03118 had stronger in vitro and in vivo biological activity than the first-generation RET inhibitors targeting a broad range of RET fusions and mutations. Importantly, APS03118 was highly potent against a series of resistant RET mutations including the solvent front mutations RET G810C/R/S (IC50 0.04-5.72 nM) and gatekeeper mutations RET V804E/L/M (IC50 0.04-1.36 nM). Off-target screen analysis using the 468-kinase panel demonstrated APS03118 was highly selective for RET. APS03118 was orally bioavailable with good ADME and PK properties and markedly suppressed tumor growth in various RET-driven mouse xenograft tumor models (tumor growth inhibition 87-108% at 10-30 mg/kg BID). In addition, APS03118 exhibited excellent brain penetrating properties with strong efficacy in the in vivo orthotopic brain tumor model. GLP toxicity studies showed acceptable safety profiles. APS03118 received IND approval from both US FDA and China NMPA, and was granted the fast-track designation by FDA. We recently achieved First-in-Human milestone for APS03118 and the Ph 1 clinical trial is ongoing. As of January 5, 2024, three dose levels have been tested. Plasma exposure of APS03118 increased proportionally following single and repeated administration of APS03118 at different doses. APS03118 was well tolerated in NSCLC and thyroid cancer patients harboring RET fusions or mutations relapsed on the chemotherapy, multi-kinase inhibitor and first-generation RET inhibitor therapy. In addition to the favorable safety profiles, we observed sign of efficacy in the RET altered cancer patients in response to APS03118 treatment even in the early clinical trial testing. Partial response and stable disease were observed in patients 2-4 months after administration of APS03118 and dose limiting toxicity (DLT) dose has not been reached. Dose escalation for APS03118 is continuing. Taken together, APS03118 is a novel highly potent and selective next-generation RET inhibitor overcoming resistance following treatment with the first-generation RET inhibitors. APS03118 warrants further clinical development. Citation Format: Jun Zhong, Ruxue Xiao, Wenya Xie, Minchun Chen, Xiaohu Chen, Hao Wang, Yongbo Liu, Qin Gao, Xiupeng Wang, Zongbao Wang, Qian Chen, Jing Yang, Yizhe Ji, Yuxun Wang. Development of a novel next-generation RET inhibitor [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 CT056.

Abstract LB021: BAU-243: A novel BCL-2 inhibitor inducing autophagic cell death in glioblastoma, unveiled through multistep virtual screening, comprehensive in vitro and in vivo animal model evaluation

Abstract The excessive expression of antiapoptotic members in the B-cell leukemia/lymphoma-2 (BCL-2) family proteins emerges as a noteworthy oncogenic target in cancer cells. Employing a multistep virtual screening and filtering approach that integrates structure and ligand-based drug design, we aimed to identify novel and potent BCL-2 inhibitors from a sizable small molecule database, comprising over 210,000 compounds. Subsequently, seven selected molecules underwent rigorous biological activity tests in human cancer cell lines and Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assays. Four of these compounds exhibited notable inhibitory effects on cancer cell proliferation and led to an increase in apoptotic cell fractions. Among the identified compounds, BAU-243 displayed a substantial affinity for Bcl-2, demonstrating efficacy in reducing glioblastoma (GBM) cell proliferation, including cancer-initiating cells. Notably, in contrast to the selective Bcl-2 inhibitor ABT-199, BAU-243 induced autophagic cell death in GBM cells. In vivo investigations using orthotopic brain tumor models revealed a significant reduction in tumor growth with BAU-243 compared to both the vehicle group and animals treated with ABT-199. Computational modeling simulations, in concordance with in vitro findings, proposed that BAU-243 activates autophagic cell death by disrupting the Beclin 1:Bcl-2 complex. This positions BAU-243 as a promising small molecule for the treatment of GBM. Citation Format: Seyma Calis, Berna Dogan, Asuman Celebi, Ozlem Yapicier, Turker Kilic, Eda Tahir Turanli, Timucin Avsar, Serdar Durdagi. BAU-243: A novel BCL-2 inhibitor inducing autophagic cell death in glioblastoma, unveiled through multistep virtual screening, comprehensive in vitro and in vivo animal model evaluation [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 LB021.

492 Novel BH3 Mimetic Bcl-2 Inhibitor Promotes Autophagic Cell Death and Reduces in Vivo Glioblastoma Tumor Growth

INTRODUCTION: Anti-apoptotic members of the Bcl-2 family proteins play central roles in the regulation of cell death in glioblastoma, the most malignant type of brain tumor. Despite the advances in GBM treatment, there is still an urgent need for new therapeutic approaches. METHODS: In silico studies resulted in 500,000 molecules being screened with computer-aided drug design methods and the molecule with the highest binding affinity was selected and named BAU-243. Cell viability, IC50 value, tumor formation potential, tumor spheroid formation potential, tumor invasion studies were investigated following BAU-243 application. The molecular changes caused by Bcl-2 inhibition were demonstrated by quantitative PCR, western blot and immunofluorescence methods. In in vivo studies, glioma tumors were transplanted intracranially into Nod Scid Gamma(NSG) mice using the mouse xenograft model. RESULTS: Here, we report a novel 4-thiazolidinone derivative BH3 mimetic, BAU-243 that binds to Bcl-2 with a high affinity. BAU-243 effectively reduced overall glioblastoma cell proliferation including a subpopulation of cancer-initiating cells in contrast to the selective Bcl-2 inhibitor ABT-199. While ABT-199 successfully induces apoptosis in high BCL2-expressing neuroblastoma SHSY-5Y cells, BAU-243 triggered autophagic cell death rather than apoptosis in A172 cells, indicated by the upregulation of BECN1, ATG5, and MAP1LC3B expression. Lc3b-II, a potent autophagy marker, was significantly upregulated following BAU-243 treatment. Moreover, BAU-243 significantly reduced tumor growth in vivo in orthotopic brain tumor models when compared to the vehicle group, and ABT-199 treated animals. To elucidate the molecular mechanisms of action of BAU-243, we performed computational modeling simulations that were consistent with in vitro results. CONCLUSIONS: Results indicated that BAU-243 activates autophagic cell death by disrupting Beclin 1:Bcl-2 complex, and may serve as a potential small molecule for the treatment of glioblastoma.

Enhancing Photothermal/Photodynamic Therapy for Glioblastoma by Tumor Hypoxia Alleviation and Heat Shock Protein Inhibition Using IR820-Conjugated Reduced Graphene Oxide Quantum Dots.

We use low-molecular-weight branched polyethylenimine (PEI) to produce cytocompatible reduced graphene oxide quantum dots (rGOQD) as a photothermal agent and covalently bind it with the photosensitizer IR-820. The rGOQD/IR820 shows high photothermal conversion efficiency and produces reactive oxygen species (ROS) after irradiation with near-infrared (NIR) light for photothermal/photodynamic therapy (PTT/PDT). To improve suspension stability, rGOQD/IR820 was PEGylated by anchoring with the DSPE hydrophobic tails in DSPE-PEG-Mal, leaving the maleimide (Mal) end group for covalent binding with manganese dioxide/bovine serum albumin (MnO2/BSA) and targeting ligand cell-penetrating peptide (CPP) to synthesize rGOQD/IR820/MnO2/CPP. As MnO2 can react with intracellular hydrogen peroxide to produce oxygen for alleviating the hypoxia condition in the acidic tumor microenvironment, the efficacy of PDT could be enhanced by generating more cytotoxic ROS with NIR light. Furthermore, quercetin (Q) was loaded to rGOQD through π-π interaction, which can be released in the endosomes and act as an inhibitor of heat shock protein 70 (HSP70). This sensitizes tumor cells to thermal stress and increases the efficacy of mild-temperature PTT with NIR irradiation. By simultaneously incorporating the HSP70 inhibitor (Q) and the in situ hypoxia alleviating agent (MnO2), the rGOQD/IR820/MnO2/Q/CPP can overcome the limitation of PTT/PDT and enhance the efficacy of targeted phototherapy in vitro. From in vivo study with an orthotopic brain tumor model, rGOQD/IR820/MnO2/Q/CPP administered through tail vein injection can cross the blood-brain barrier and accumulate in the intracranial tumor, after which NIR laser light irradiation can shrink the tumor and prolong the survival times of animals by simultaneously enhancing the efficacy of PTT/PDT to treat glioblastoma.

Chitosan-coated magnetic graphene oxide for targeted delivery of doxorubicin as a nanomedicine approach to treat glioblastoma

In this study, magnetic graphene oxide (mGO) was first prepared and modified with chitosan to prepare chitosan-coated mGO (mGOC). Gastrin-releasing peptide (GRP)-conjugated mGOC (mGOCG) was then prepared from mGOC. The chemo drug doxorubicin (DOX) was adsorbed to mGOCG surface for dual active/magnetic targeted drug delivery. The DOX loading to mGOCG is 1.71 mg/mg, and drug release is pH-sensitive to facilitate drug delivery in endosomes. In vitro studies confirmed enhanced mGOCG endocytosis by U87 glioblastoma cells, with which enhanced cytotoxicity towards cancer cells could be achieved. This could be revealed from the drastically reduced half-maximal inhibitory concentration of mGOCG/DOX compared with DOX and mGOC/DOX. Furthermore, mGOCG/DOX can be localized under the influence of a magnetic field (MF) to exert this cytotoxic effect. An orthotopic brain tumor model by implanting U87 cells in the intracranial area of BALB/c nude mice was used to study the in vivo anti-tumor efficacy by intravenous injection of different samples and followed with bioluminescence imaging. The tumor size in the mGOCG/DOX + MF group demonstrated the best potency to suppress tumor growth and prolong animal survival time compared with mGOCG/DOX, mGOC/DOX, or DOX groups, indicating this new dual-targeting delivery system for DOX can effectively treat glioblastoma.

Evaluation the Effect of Sonodynamic Therapy with 5-Aminolevulinic Acid and Sodium Fluorescein by Preclinical Animal Study.

Sonodynamic therapy (SDT) is a novel tumor treatment that combines biosafe sonosensitizers and noninvasive focused ultrasound to eradicate solid tumors. Sonosensitizers such as 5-aminolevulinic acid and fluorescein have great potential in tumor treatment. Here, rodent subcutaneous and brain tumor models were used to evaluate the treatment effect of both 5-ALA- and fluorescein-mediated SDT. The subcutaneous tumor growth rates of both SDT groups were significantly inhibited compared with that of the control groups. For intracranial tumors, 5-ALA-SDT treatment significantly inhibited brain tumor growth, while fluorescein-SDT exerted no therapeutic effect in animals. The distribution of fluorescein in the brain tumor region underwent further assessment. Seven days post tumor implantation, experimental animals received fluorescein and were sacrificed for brain specimen collection. Analysis of the dissected brains revealed no fluorescence signals, indicating an absence of fluorescein accumulation in the early-stage glioma tissue. These data suggest that the fluorescein-SDT treatment response is closely related to the amount of accumulated fluorescein. This study reports the equivalent effects of 5-ALA and fluorescein on the treatment of somatic tumors. For orthotopic brain tumor models, tumor vascular permeability should be considered when choosing fluorescein as a sonosensitizer. In conclusion, both fluorescein and 5-ALA are safe and effective SDT sonosensitizers, and the tumor microenvironment and pathologic type should be considered in the selection of adequate sonosensitizers.

Development of Orthotopic Patient-Derived Xenograft Models of Pediatric Intracranial Tumors.

The development of clinically relevant and reliable models of central nervous system tumors has been instrumental in advancing the field of Neuro-Oncology. The orthotopic intracranial injection is widely used to study the growth, invasion, and spread of tumors in a controlled environment. Orthotopic models are performed to examine tumor cells isolated from a specific region in a patient in the same site or location in an animal model. Orthotopic brain tumor models are also utilized for preclinical testing of therapeutics as they closely recapitulate the behavior of such cancer and the brain environment of patients. Below, we describe our experiences in the development of murine models of pediatric brain tumors including diffuse midline glioma (DMG), glioblastoma (GBM), and medulloblastoma. The method provides an overview of intracranial stereotactic injections in mice.

Synergistically Enhancing Immunotherapy Efficacy in Glioblastoma with Gold-Core Silica-Shell Nanoparticles and Radiation.

Glioblastoma is a highly aggressive brain tumor with universally poor outcomes. Recent progress in immune checkpoint inhibitors has led to increased interest in their application in glioblastoma. Nonetheless, the unique immune milieu in the brain has posed remarkable challenges to the efficacy of immunotherapy. We aimed to leverage the radiation-induced immunogenic cell death to overcome the immunosuppressive network in glioblastoma. We developed a novel approach using the gold-core silica-shell nanoparticles (Au@SiO2 NPs) in combination with low-dose radiation to enhance the therapeutic efficacy of the immune checkpoint inhibitor (atezolizumab) in brain tumors. The biocompatibility, immune cell recruitment, and antitumor ability of the combinatorial strategy were determined using in vitro assays and in vivo models. Our approach successfully induced the migration of macrophages towards brain tumors and promoted cancer cell apoptosis. Subcutaneous tumor models demonstrated favorable safety profiles and significantly enhanced anticancer effects. In orthotopic brain tumor models, the multimodal therapy yielded substantial prognostic benefits over any individual modalities, achieving an impressive 40% survival rate. In summary, the combination of Au@SiO2 NPs and low-dose radiation holds the potential to improve the clinical efficacy of immune checkpoint inhibitors. The synergetic strategy modulates tumor microenvironments and enhances systemic antitumor immunity, paving a novel way for glioblastoma treatment.

Therapeutic Anti-KIR Antibody of 1-7F9 Attenuates the Antitumor Effects of Expanded and Activated Human Primary Natural Killer Cells on In Vitro Glioblastoma-like Cells and Orthotopic Tumors Derived Therefrom.

Glioblastoma (GBM) is the leading malignant intracranial tumor, where prognosis for which has remained extremely poor for two decades. Immunotherapy has recently drawn attention as a cancer treatment, including for GBM. Natural killer (NK) cells are immune cells that attack cancer cells directly and produce antitumor immunity-related cytokines. The adoptive transfer of expanded and activated NK cells is expected to be a promising GBM immunotherapy. We previously established an efficient expansion method that produced highly purified, activated primary human NK cells, which we designated genuine induced NK cells (GiNKs). The GiNKs demonstrated antitumor effects in vitro and in vivo, which were less affected by blockade of the inhibitory checkpoint receptor programmed death 1 (PD-1). In the present study, we assessed the antitumor effects of GiNKs, both alone and combined with an antibody targeting killer Ig-like receptor 2DLs (KIR2DL1 and DL2/3, both inhibitory checkpoint receptors of NK cells) in vitro and in vivo with U87MG GBM-like cells and the T98G GBM cell line. Impedance-based real-time cell growth assays and apoptosis detection assays revealed that the GiNKs exhibited growth inhibitory effects on U87MG and T98G cells by inducing apoptosis. KIR2DL1 blockade attenuated the growth inhibition of the cell lines in vitro. The intracranial administration of GiNKs prolonged the overall survival of the U87MG-derived orthotopic xenograft brain tumor model. The KIR2DL1 blockade did not enhance the antitumor effects; rather, it attenuated it in the same manner as in the in vitro experiment. GiNK immunotherapy directly administered to the brain could be a promising immunotherapeutic alternative for patients with GBM. Furthermore, KIR2DL1 blockade appeared to require caution when used concomitantly with GiNKs.

P03.11.B MITOTIC ENRICHMENT AS AN EFFICIENT RADIOSENSITIZATION STRATEGY FOR INTRACRANIAL TUMORS

Abstract BACKGROUND Radiotherapy remains one of the most effective modalities for anticancer treatment and part of standard of care for many primary and metastatic brain tumors. Boosting the efficacy of radiotherapy is therefore a logical avenue to improve patient survival. We have developed a radiosensitization strategy called ‘induction of mitotic enrichment’. It has long been known that the radiosensitivity of a cell depends on the phase of the cell cycle and that mitotic cells are especially vulnerable. Enriching the tumor for mitotic cells by arresting them during division prior to each radiotherapy fraction should therefore render the tumor population more sensitive to irradiation. Ideally, induction of mitotic enrichment should be reversible and non-cytotoxic to prevent healthy tissue toxicity and be compatible with clinically applied fractionated radiotherapy regimens. MATERIAL AND METHODS Mitotic enrichment in vitro is established using flow cytometry and radiosensitization is assessed using colony formation assays. In vivo, several independent luciferase-tagged orthotopic brain tumor models are used to investigate mitotic enrichment (phospho-histone 3 immunohistochemistry), tumor growth, survival and healthy tissue toxicity (body weight, blood liver enzymes, γH2AX and cleaved-caspase 3 immunohistochemistry). Pharmacokinetic studies are quantified using LC-MS/MS. All experiments on primary brain tumors included serum-free neurosphere culture models. RESULTS We have now identified an orally available targeted tubulin polymerization inhibitor that can achieve repeated and reversible mitotic enrichment for up to 10 hours prior to radiotherapy, without causing cytotoxicity in vitro or healthy tissue toxicity in vivo. This tubulin inhibitor (ABT-751) efficiently radiosensitizes a range of preclinical tumor models representing adult and pediatric, primary and metastatic brain tumors. Importantly, ABT-751 also improves survival in mouse models of adult glioblastoma (GBM), but only in a mitotic enrichment setup when given several hours prior to radiotherapy to allow accumulation in mitosis. Of note, induction of mitotic enrichment does not exacerbate healthy tissue toxicity. CONCLUSION Induction of mitotic enrichment by ABT-751 radiosensitizes adult GBM to fractionated radiotherapy. We are currently expanding our preclinical development of mitotic enrichment as a radiosensitization strategy to other targets and cancers and have designed a phase 0/I trial for adult GBM to demonstrate induction of mitotic enrichment and tolerability when combined with radiotherapy. We expect to start enrollment in this trial soon.

Radiomics characterization of tissues in an animal brain tumor model imaged using dynamic contrast enhanced (DCE) MRI

Here, we investigate radiomics-based characterization of tumor vascular and microenvironmental properties in an orthotopic rat brain tumor model measured using dynamic-contrast-enhanced (DCE) MRI. Thirty-two immune compromised-RNU rats implanted with human U-251N cancer cells were imaged using DCE-MRI (7Tesla, Dual-Gradient-Echo). The aim was to perform pharmacokinetic analysis using a nested model (NM) selection technique to classify brain regions according to vasculature properties considered as the source of truth. A two-dimensional convolutional-based radiomics analysis was performed on the raw-DCE-MRI of the rat brains to generate dynamic radiomics maps. The raw-DCE-MRI and respective radiomics maps were used to build 28 unsupervised Kohonen self-organizing-maps (K-SOMs). A Silhouette-Coefficient (SC), k-fold Nested-Cross-Validation (k-fold-NCV), and feature engineering analyses were performed on the K-SOMs’ feature spaces to quantify the distinction power of radiomics features compared to raw-DCE-MRI for classification of different Nested Models. Results showed that eight radiomics features outperformed respective raw-DCE-MRI in prediction of the three nested models. The average percent difference in SCs between radiomics features and raw-DCE-MRI was: 29.875% ± 12.922%, p < 0.001. This work establishes an important first step toward spatiotemporal characterization of brain regions using radiomics signatures, which is fundamental toward staging of tumors and evaluation of tumor response to different treatments.

466 Robust Model of Microglia Replacement With Circulation-Derived Microglia-Like Cells (CDMCs) in CNS Malignancies

INTRODUCTION: Tumor-associated microglia and macrophages (TAMs) represent a main cell type of brain malignancies and demonstrate complex interactions with cancerous cells and the tumor microenvironment. These interactions have important implications for the progression and treatment of malignant central nervous system (CNS) tumors. TAMs have therefore emerged as therapeutic targets, though their potential to date has been unfulfilled. METHODS: C57BL/6 mice were preconditioned for BMT with retroorbital busulfan injections (25 mg/kg), and native microglia were depleted via PLX5622 (1200 mg/kg, chow). Following transplantation, animals underwent intracranial stereotactic injection of syngeneic cell lines of either high-grade glioma (GL261) or breast cancer metastasis (E0771). Following animal sacrifice, we used single cell RNA sequencing of CD45-positive cells to profile the transcriptomic identity of glioma tumor-associated CDMCs (TA-CDMCs). RESULTS: Lineage tracing proved high intra- and peri-tumoral chimerism of BMT-graft derived cells among CNS myeloid cells in both models two weeks after tumor cell injection, demonstrating a robust ability to modify the tumor-immune microenvironment in animal models. TA-CDMCs showed a similarly activated morphology as naÏve TAMs under immunohistochemistry. Pathway enrichment analysis revealed an upregulation of immune response pathways in TA-CDMCs compared to regular TAMs, including the overexpression of genes relevant for the regulation of T cell activation, suggesting possible modification of the glioma immune-environment following CDMC repopulation. CONCLUSIONS: The present study offered a robust proof-of-concept that highly efficient TAM replacement could be achieved in orthotopic murine brain tumor models. Additionally, it appears that replaced CDMCs retain unique transcriptional patterns in the tumor-immune microenvironment, with the upregulation of several key immune response pathways. These efforts may represent a novel route of tumor-directed immunotherapy and offer exciting opportunities for ex-vivo CDMC manipulation to specifically target neoplastic cells.

LILRB2-containing small extracellular vesicles from glioblastoma promote tumor progression by promoting the formation and expansion of myeloid-derived suppressor cells

BackgroundLeukocyte immunoglobulin-like receptor subfamily B2 (LILRB2) was reported to be an inhibitory molecule with suppressive functions. sEVs mediate communication between cancer cells and other cells. However, the existence of LILRB2 on sEVs in circulation and the function of sEVs-LILRB2 are still unknown. This study aims to investigate the role of LILRB2 in GBM and determine how LILRB2 in sEVs regulates tumor immunity.MethodsLILRB2 expression in normal brain and GBM tissues was detected by immunohistochemistry, and the effect of LILRB2 on prognosis was evaluated in an orthotopic brain tumor model. Next, a subcutaneous tumor model was constructed to evaluate the function of pirb in vivo. The immune cells in the tumor sites and spleen were detected by immunofluorescence staining and flow cytometry. Then, the presence of pirb in sEVs was confirmed by WB. The percentage of immune cells after incubation with sEVs from GL261 (GL261-sEVs) or sEVs from GL261-pirb+ (GL261-sEVs-pirb) was detected by flow cytometry. Then, the effect of pirb on sEVs was evaluated by a tumor-killing assay and proliferation assay. Finally, subcutaneous tumor models were constructed to evaluate the function of pirb on sEVs.ResultsLILRB2 was overexpressed in human GBM tissue and was closely related to an immunosuppressive TME in GBM. Then, a protumor ability of LILRB2 was observed in subcutaneous tumor models, which was related to lower CD8 + T cells and higher MDSCs (myeloid-derived suppressor cells) in the tumor and spleen compared to those of the control group. Next, we found that pirb on sEVs (sEVs-pirb) inhibits the function of CD8 + T cells by promoting the formation and expansion of MDSCs. Furthermore, the protumor function of sEVs-pirb was demonstrated in subcutaneous tumor models.ConclusionWe discovered that LILRB2/pirb can be transmitted between GBM cells via sEVs and that pirb on sEVs induces the formation and expansion of MDSCs. The induced MDSCs facilitate the formation of an immunosuppressive TME.Graphical

Translational Orthotopic Models of Glioblastoma Multiforme.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumorsdisplaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Characterization of the Response of 9L and U-251N Orthotopic Brain Tumors to 3D Conformal Radiation Therapy.

In a study employing MRI-guided stereotactic radiotherapy (SRS) in two orthotopic rodent brain tumor models, the radiation dose yielding 50% survival (the TCD50) was sought. Syngeneic 9L cells, or human U-251N cells, were implanted stereotactically in 136 Fischer 344 rats or 98 RNU athymic rats, respectively. At approximately 7 days after implantation for 9L, and 18 days for U-251N, rats were imaged with contrast-enhanced MRI (CE-MRI) and then irradiated using a Small Animal Radiation Research Platform (SARRP) operating at 220 kV and 13 mA with an effective energy of ∼70 keV and dose rate of ∼2.5 Gy per min. Radiation doses were delivered as single fractions. Cone-beam CT images were acquired before irradiation, and tumor volumes were defined using co-registered CE-MRI images. Treatment planning using MuriPlan software defined four non-coplanar arcs with an identical isocenter, subsequently accomplished by the SARRP. Thus, the treatment workflow emulated that of current clinical practice. The study endpoint was animal survival to 200 days. The TCD50 inferred from Kaplan-Meier survival estimation was approximately 25 Gy for 9L tumors and below 20 Gy, but within the 95% confidence interval in U-251N tumors. Cox proportional-hazards modeling did not suggest an effect of sex, with the caveat of wide confidence intervals. Having identified the radiation dose at which approximately half of a group of animals was cured, the biological parameters that accompany radiation response can be examined.

A novel BH3 mimetic Bcl-2 inhibitor promotes autophagic cell death and reduces in vivo Glioblastoma tumor growth

Anti-apoptotic members of the Bcl-2 family proteins play central roles in the regulation of cell death in glioblastoma (GBM), the most malignant type of brain tumor. Despite the advances in GBM treatment, there is still an urgent need for new therapeutic approaches. Here, we report a novel 4-thiazolidinone derivative BH3 mimetic, BAU-243 that binds to Bcl-2 with a high affinity. BAU-243 effectively reduced overall GBM cell proliferation including a subpopulation of cancer-initiating cells in contrast to the selective Bcl-2 inhibitor ABT-199. While ABT-199 successfully induces apoptosis in high BCL2-expressing neuroblastoma SHSY-5Y cells, BAU-243 triggered autophagic cell death rather than apoptosis in GBM A172 cells, indicated by the upregulation of BECN1, ATG5, and MAP1LC3B expression. Lc3b-II, a potent autophagy marker, was significantly upregulated following BAU-243 treatment. Moreover, BAU-243 significantly reduced tumor growth in vivo in orthotopic brain tumor models when compared to the vehicle group, and ABT-199 treated animals. To elucidate the molecular mechanisms of action of BAU-243, we performed computational modeling simulations that were consistent with in vitro results. Our results indicate that BAU-243 activates autophagic cell death by disrupting the Beclin 1:Bcl-2 complex and may serve as a potential small molecule for treating GBM.

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.

Homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors.

SUMMARYGlioblastoma multiforme (GBM) is the most aggressive and fatal disease of all brain tumor types. Most therapies rarely provide clinically meaningful outcomes in the treatment of GBM. Although antibody-drug conjugates (ADCs) are promising anticancer drugs, no ADCs have been clinically successful for GBM, primarily because of poor blood-brain barrier (BBB) penetration. Here, we report that ADC homogeneity and payload loading rate are critical parameters contributing to this discrepancy. Although both homogeneous and heterogeneous conjugates exhibit comparable in vitro potency and pharmacokinetic profiles, the former shows enhanced payload delivery to brain tumors. Our homogeneous ADCs provide improved antitumor effects and survival benefits in orthotopic brain tumor models. We also demonstrate that overly drug-loaded species in heterogeneous conjugates are particularly poor at crossing the BBB, leading to deteriorated overall brain tumor targeting. Our findings indicate the importance of homogeneous conjugation with optimal payload loading in generating effective ADCs for intractable brain tumors.

Recycling of SLC38A1 to the plasma membrane by DSCR3 promotes acquired temozolomide resistance in glioblastoma.

Glioblastoma multiforme (GBM) is a primary brain tumor with devastating prognosis. Although the O6-methylguanine-DNA methyltransferase (MGMT) leads to inherent temozolomide (TMZ) resistance, approximately half of GBMs were sufficient to confer acquired TMZ resistance, which express low levels of MGMT. The purpose of this study was to investigate the underlying mechanisms of the acquired TMZ resistance in MGMT-deficient GBM. The function of Down syndrome critical region protein 3 (DSCR3) on MGMT-deficient GBM was investigated in vitro and in an orthotopic brain tumor model in mice. Purification of plasma membrane proteins by membrane-cytoplasmic separation and subsequent label free-based quantitative proteomics were used to identified potential protein partners for DSCR3. Immunofluorescence was performed to show the reverse transport of solute carrier family 38 member 1 (SLC38A1) mediated by DSCR3. DSCR3 is upregulated in MGMT-deficient GBM cells during TMZ treatment. Both DSCR3 and SLC38A1 were highly expressed in recurrent GBM patients. Silencing DSCR3 or SLC38A1 expression can increase TMZ sensitivity in MGMT-deficient GBM cells. Combination of proteomics and in vitro experiments show that DSCR3 directly binds internalized SLC38A1 to mediate its sorting into recycling pathway, which maintains the abundance on plasma membrane and enhances uptake of glutamine in MGMT-deficient GBM cells. DSCR3 is a crucial regulator of acquired TMZ resistance in MGMT-deficient GBM. The DSCR3-dependent recycling of SLC38A1 maintains its abundance on plasma membrane, leading to tumor progression and acquired TMZ resistance in MGMT-deficient GBM.