Mouse Model Of Glioma Research Articles

Targeted blood-brain barrier penetration and precise imaging of infiltrative glioblastoma margins using hybrid cell membrane-coated ICG liposomes

Surgical resection remains the primary treatment modality for glioblastoma (GBM); however, the infiltrative nature of GBM margins complicates achieving complete tumor removal. Additionally, the blood-brain barrier (BBB) poses a formidable challenge to effective probe delivery, thereby hindering precise imaging-guided surgery. Here, we introduce hybrid cell membrane-coated indocyanine green (ICG) liposomes (HM-Lipo-ICG) as biomimetic near-infrared (NIR) fluorescent probes for targeted BBB penetration and accurate delineation of infiltrative GBM margins. HM-Lipo-ICG encapsulates clinically approved ICG within its core and utilizes a hybrid cell membrane exterior, enabling specific targeting and enhanced BBB permeation. Quantitative assessments demonstrate that HM-Lipo-ICG achieves BBB penetration efficiency 2.8 times higher than conventional ICG liposomes. Mechanistically, CD44 receptor-mediated endocytosis facilitates BBB translocation of HM-Lipo-ICG. Furthermore, HM-Lipo-ICG enables high-contrast NIR imaging, achieving a signal-to-background ratio of 6.5 in GBM regions of an orthotopic glioma mouse model, thereby improving tumor margin detection accuracy fourfold (84.4% vs. 22.7%) compared to conventional ICG liposomes. Application of HM-Lipo-ICG facilitates fluorescence-guided precision surgery, resulting in complete resection of GBM cells. This study underscores the potential of hybrid cell membrane-coated liposomal probes in precisely visualizing and treating infiltrative GBM margins.Graphical abstract

AP-2α decreases TMZ resistance of recurrent GBM by downregulating MGMT expression and improving DNA damage

AimsThe incidence of recurrent gliomas is high, exerting low survival rates and poor prognoses. Transcription factor AP-2α has been reported to regulate the progression of primary glioblastoma (GBM). However, the function of AP-2α in recurrent gliomas is largely unclear. MethodsThe expression of AP-2α and O6-methylguanine DNA-methyltransferase (MGMT) was detected in recurrent glioma tissues and cell lines by Western blots, the regulation mechanisms between AP-2α/MGMT promoter and RA/AP-2α promoter were studied by luciferase reporter assays, EMSA, and chIP assays. The effects of AP-2α and TMZ/RA treatment on cell viability in vitro and in vivo were investigated by MTT assays, γH2AX staining, comet assays and intracranial injection. Key findingsAP-2α expression negatively correlates with the expression of MGMT in glioma samples. AP-2α could directly bind with the promoter of the MGMT gene, suppresses transcriptional levels of MGMT and downregulate MGMT expression in TMZ-resistant U87MG-R and T98G cells, but TMZ treatment decreases AP-2α expression and increases MGMT expression. The extended TMZ treatment and increased TMZ concentrations reversed these effects. Moreover, AP-2α overexpression combines with TMZ to decrease cell viability, concurrently with improved DNA damage marker γH2AX. Furthermore, retinoic acid (RA) activates RAR/RXR heterodimers, which bind to RA-responsive elements (RAREs) of the AP-2α promoter, and activates AP-2α expression in recurrent glioma cells. Finally, in intracranial relapsed glioma mouse model, both RA and TMZ could retard tumor development and prolong the mouse survival. SignificanceAP-2α activation by gene overexpression or RA treatment reveals the suppressive effects on glioma relapse, providing a novel therapeutic strategy against malignant refractory gliomas.

ERK signaling promotes resistance to TRK kinase inhibition in NTRK fusion-driven glioma mouse models

Pediatric-type high-grade gliomas frequently harbor gene fusions involving receptor tyrosine kinase genes, including neurotrophic tyrosine kinase receptor (NTRK) fusions. Clinically, these tumors show high initial response rates to tyrosine kinase inhibition but ultimately recur due to the accumulation of additional resistance-conferring mutations. Here, we develop a series of genetically engineered mouse models of treatment-naive and -experienced NTRK1/2/3 fusion-driven gliomas. All tested NTRK fusions are oncogenic invivo. The NTRK variant, N-terminal fusion partners, and resistance-associated point mutations all influence tumor histology and aggressiveness. Additional tumor suppressor losses greatly enhance tumor aggressiveness. Treatment with TRK kinase inhibitors significantly extends the survival of NTRK fusion-driven glioma mice, but fails to fully eradicate tumors, leading to recurrence upon treatment discontinuation. Finally, we show that ERK activation promotes resistance to TRK kinase inhibition and identify MEK inhibition as a potential combination therapy. These models will be invaluable tools to study therapy resistance of NTRK fusion tumors.

Gambogic acid impairs the maintenance and therapeutic resistance of glioma stem cells by targeting B-cell-specific Moloney leukemia virus insert site 1

BackgroundGlioblastoma (GBM) is the most common and lethal primary brain tumor with low effectiveness of available treatments. The tumor heterogeneity and therapeutic resistance are largely due to the presence of glioma stem cells (GSCs). Therefore, eliminating GSCs can overcome the progression, relapse, and resistance of GBM. Previous studies have shown that gambogic acid (GA), a natural active ingredient, has anti-glioma properties. Nonetheless, it is still unclear whether it has an inhibitory effect on GSCs and what its target might be. This study aimed to investigate the anti-tumor effects of GA on GSCs. In addition, this study found the target of GA in GSCs and elucidated the potential specific mechanisms by conducting both in vitro and in vivo experiments.B-cell-specific Moloney leukemia virus insert site 1 (BMI1) is a key stem cell factor of the polycomb group (PcG) family with important effects on the development, recurrence, and chemoresistance of several cancers. In both normal and cancer stem cells, BMI1 maintains stem cell self-renewal by regulating the cell cycle, cellular immortalization, and senescence. Its high expression in a variety of cancers correlates with poor clinical prognosis and chemoresistance. These mechanisms of BMI1 make it a potential therapeutic target for cancer therapy, and future studies may further reveal the specific roles of BMI1 mechanism and provide a basis for the development of new cancer therapeutic strategies. PurposeThis study investigated the in vitro and in vivo effects of GA in inducing apoptosis in GSCs and inhibiting GSCs self-renewal, as well as its underlying mechanisms. MethodsThis study synthesized biotinylated gambogic acid for the first time and angled for the target of gambogic acid using LC-MS/MS analysis, which has not been reported previously. Human-derived glioma stem cells GSC123 and GSC111 were used for in vitro studies, analyzing functions and mechanisms via microscale thermophoresis (MST), Annexin V/PI staining, Western blotting, immunofluorescence, and co-immunoprecipitation. The orthotopic glioma mouse model was used to assess the anti-tumor effects of GA in vivo. ResultsThis study demonstrated that GA is a specific inhibitor of BMI1, a key regulator controlling stem cell growth and self-renewal. GA binds to BMI1′s RING domain, accelerating K51-dependent degradation and suppressing H2A ubiquitination. Importantly, GA induces apoptosis, and inhibits GSC self-renewal, but minimally impacts neural progenitor cells (NPCs). GA can also be combined effectively with temozolomide and radiotherapy to increase their sensitivities in resistant cells. Furthermore, exogenous induction of BMI1 expression significantly hinders the disruption of GSCs by GA. In vivo, GA inhibits tumorigenicity, enhances the effect of temozolomide, and reduces BMI1 expression. ConclusionThese findings suggest that GA is a potential candidate for targeting GSCs and therefore be used to treat GBM.

Peptide-functionalized gold nanoparticles for boron neutron capture therapy with the potential to use in Glioblastoma treatment

Glioblastoma is a highly aggressive glioma with limited treatment options. Boron neutron capture therapy (BNCT) offers a promising approach for refractory cancers, utilizing boron-10 (10B) and thermal neutrons to generate cytotoxic particles. Effective BNCT depends on selective targeting and retention of 10B in tumors. Current BNCT drugs face issues with rapid clearance and poor tumor accumulation. To address this, we developed gold nanoparticles (AuNPs) functionalized with cyclic arginine-glycine-aspartic acid (cRGD) peptides as a nanocarrier for Sodium Mercaptododecaborate (BSH), resulting in AuNPs-BSH&PEG-cRGD. In vitro, AuNPs-BSH&PEG-cRGD increased 10B content in GL261 glioma cells by approximately 2.5-fold compared to unmodified AuNPs-BSH&PEG, indicating enhanced targeting due to cRGD's affinity for integrin receptor αvβ3. In a subcutaneous glioma mouse model, 6 h post-intratumoral administration, the 10B concentration in tumors was 17.98 μg/g for AuNPs-BSH&PEG-cRGD, significantly higher than 0.45 μg/g for BSH. The tumor-to-blood (T/B) and tumor-to-normal tissue (T/N) ratios were also higher for AuNPs-BSH&PEG-cRGD, suggesting improved targeting and retention. This indicates that AuNPs-BSH&PEG-cRGD may enhance BNCT efficacy and minimize normal tissue toxicity. In summary, this study provides a novel strategy for BSH delivery and may broaden the design vision of BNCT nano-boron capture agents.

The dual role of POSTN in maintaining glioblastoma stem cells and the immunosuppressive phenotype of microglia in glioblastoma

BackgroundGlioblastoma (GBM) is an immunosuppressive, universally lethal cancer driven by glioblastoma stem cells (GSCs). The interplay between GSCs and immunosuppressive microglia plays crucial roles in promoting the malignant growth of GBM; however, the molecular mechanisms underlying this crosstalk are unclear. This study aimed to investigate the role of POSTN in maintaining GSCs and the immunosuppressive phenotype of microglia.MethodsThe expression of POSTN in GBM was identified via immunohistochemistry, quantitative real-time PCR, and immunoblotting. Tumorsphere formation assay, Cell Counting Kit-8 assay and immunofluorescence were used to determine the key role of POSTN in GSC maintenance. ChIP-seq and ChIP-PCR were conducted to confirm the binding sequences of β-catenin in the promoter region of FOSL1. Transwell migration assays, developmental and functional analyses of CD4+ T cells, CFSE staining and analysis, enzyme-linked immunosorbent assays and apoptosis detection tests were used to determine the key role of POSTN in maintaining the immunosuppressive phenotype of microglia and thereby promoting the immunosuppressive tumor microenvironment. Furthermore, the effects of POSTN on GSC maintenance and the immunosuppressive phenotype of microglia were investigated in a patient-derived xenograft model and orthotopic glioma mouse model, respectively.ResultsOur findings revealed that POSTN secreted from GSCs promotes GSC self-renewal and tumor growth via activation of the αVβ3/PI3K/AKT/β-catenin/FOSL1 pathway. In addition to its intrinsic effects on GSCs, POSTN can recruit microglia and upregulate CD70 expression in microglia through the αVβ3/PI3K/AKT/NFκB pathway, which in turn promotes Treg development and functionality and supports the formation of an immunosuppressive tumor microenvironment. In both in vitro models and orthotopic mouse models of GBM, POSTN depletion disrupted GSC maintenance, decreased the recruitment of immunosuppressive microglia and suppressed GBM growth.ConclusionOur findings reveal that POSTN plays critical roles in maintaining GSCs and the immunosuppressive phenotype of microglia and provide a new therapeutic target for treating GBM.

Galectin-9 Mediates the Functions of Microglia in the Hypoxic Brain Tumor Microenvironment.

Galectin-9 is a multifaceted regulator of various pathophysiological processes that exerts positive or negative effects in a context-dependent manner. Here, we elucidated the distinctive functional properties of galectin-9 on myeloid cells within the brain tumor microenvironment. Galectin-9-expressing cells were abundant at the hypoxic tumor edge in the tumor-bearing ipsilateral hemisphere compared to the contralateral hemisphere in an intracranial mouse brain tumor model. Galectin-9 was highly expressed in microglia and macrophages in tumor-infiltrating cells. In primary glia, both the expression and secretion of galectin-9 were influenced by tumors. Analysis of a human glioblastoma bulk RNA-sequencing dataset and a single-cell RNA-sequencing dataset from a murine glioma model revealed a correlation between galectin-9 expression and glial cell activation. Notably, the galectin-9high microglial subset was functionally distinct from the galectin-9neg/low subset in the brain tumor microenvironment. Galectin-9high microglia exhibited properties of inflammatory activation and higher rates of cell death, whereas galectin-9neg/low microglia displayed a superior phagocytic ability against brain tumor cells. Blockade of galectin-9 suppressed tumor growth and altered the activity of glial and T cells in a mouse glioma model. Additionally, glial galectin-9 expression was regulated by Hif-2α in the hypoxic brain tumor microenvironment. Myeloid-specific Hif-2α deficiency led to attenuated tumor progression. Together, these findings reveal that galectin-9 on myeloid cells is an immunoregulator and putative therapeutic target in brain tumors.

KCa channel targeting impairs DNA repair and invasiveness of patient‐derived glioblastoma stem cells in culture and orthotopic mouse xenografts which only in part is predictable by KCa expression levels

AbstractPrognosis of glioblastoma patients is still poor despite multimodal therapy. The highly brain‐infiltrating growth in concert with a pronounced therapy resistance particularly of mesenchymal glioblastoma stem‐like cells (GSCs) has been proposed to contribute to therapy failure. Recently, we have shown that a mesenchymal‐to‐proneural mRNA signature of patient derived GSC‐enriched (pGSC) cultures associates with in vitro radioresistance and gel invasion. Importantly, this pGSC mRNA signature is prognostic for patients' tumor recurrence pattern and overall survival. Two mesenchymal markers of the mRNA signature encode for IKCa and BKCa Ca2+‐activated K+ channels. Therefore, we analyzed here the effect of IKCa‐ and BKCa‐targeting concomitant to (fractionated) irradiation on radioresistance and glioblastoma spreading in pGSC cultures and in pGSC‐derived orthotopic xenograft glioma mouse models. To this end, in vitro gel invasion, clonogenic survival, in vitro and in vivo residual DNA double strand breaks (DSBs), tumor growth, and brain invasion were assessed in the dependence on tumor irradiation and K+ channel targeting. As a result, the IKCa‐ and BKCa‐blocker TRAM‐34 and paxilline, respectively, increased number of residual DSBs and (numerically) decreased clonogenic survival in some but not in all IKCa‐ and BKCa‐expressing pGSC cultures, respectively. In addition, BKCa‐ but not IKCa‐blockade slowed‐down gel invasion in vitro. Moreover, systemic administration of TRAM‐34 or paxilline concomitant to fractionated tumor irradiation increased in the xenograft model(s) residual number of DSBs and attenuated glioblastoma brain invasion and (numerically) tumor growth. We conclude, that KCa‐blockade concomitant to fractionated radiotherapy might be a promising new strategy in glioblastoma therapy.

Preliminary investigation of nitric oxide release from upconverted nanoparticles excited at 808 nm near-infrared for brain tumors

Upconverted UCNPs@mSiO2-NH2 nanoparticles were synthesized via thermal decomposition while employing the energy resonance transfer principle and the excellent near-infrared (NIR) light conversion property of up-conversion. The 808 nm NIR-excited photocontrolled nitric oxide (NO) release platform was successfully developed by electrostatically loading photosensitive NO donor Roussin's black salt (RBS) onto UCNPs@mSiO2-NH2, enabling the temporal, spatial, and dosimetric regulation of NO release for biological applications of NO. The release of NO ranged from 0.015⁓0.099 mM under the conditions of 2.0 W NIR excitation power, 20 min of irradiation time, and UCNPs@mSiO2-NH2&RBS concentration of 0.25⁓1.25 mg/mL. Therefore, this NO release platform has an anti-tumor effect. In vitro experiments showed that under the NIR light, at concentrations of 0.3 mg/mL and 0.8 mg/mL of UCNPs@mSiO2-NH2&RBS, the activity of glioma (U87) and chordoma (U–CH1) cells, as measured by CCK8 assay, was reduced to 50 %. Cell flow cytometry and Western Blot experiments showed that NO released from UCNPs@mSiO2-NH2&RBS under NIR light induced apoptosis in brain tumor cells. In vivo experiments employing glioma and chordoma xenograft mouse models revealed significant inhibition of tumor growth in the NIR and UCNPs@mSiO2-NH2&RBS group, with no observed significant side effects in the mice. Therefore, NO released by UCNPs@mSiO2-NH2&RBS under NIR irradiation can be used as a highly effective and safe strategy for brain tumor therapy.

TRIM37 interacts with EZH2 to epigenetically suppress PTCH1 and regulate stemness in glioma stem cells through sonic hedgehog pathway.

Glioma stem cells (GSCs), which are known for their therapy resistance, play a substantial role in treatment inefficacy for glioblastoma multiforme (GBM). TRIM37, a member of the tripartite motif (TRIM) protein family initially linked to a rare growth disorder, has been recognized for its oncogenic role. However, the mechanism by which TRIM37 regulates tumor growth in glioma and GSCs is unclear. For the in vitro experiments, gene expression was measured by western blotting, RT-qPCR, and immunofluorescence. Cell viability was detected by CCK-8, and cell apoptosis was detected by flow cytometry. The interaction between Enhancer of Zeste Homolog 2 (EZH2) and TRIM37 was verified by co-immunoprecipitation (Co-IP). The interaction between EZH2 and the PTCH1 promoter was verified using dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP). For the in vivo experiments, an orthotopically implanted glioma mouse model was used to validate tumor growth. The expression of TRIM37 is higher in GSCs compared with matched non-GSCs. TRIM37 knockdown promotes apoptosis, decreased stemness in GSCs, and reduces tumor growth in GSCs xenografts of nude mice. TRIM37 and EZH2 co-localize in the nucleus and interact with each other. TRIM37 knockdown or EZH2 inhibition downregulates the protein expressions associated with the Sonic Hedgehog (SHH) pathway. EZH2 epigenetically downregulates PTCH1 to activate SHH pathway in GSCs. TRIM37 maintains the cell growth and stemness in GSCs through the interaction with EZH2. EZH2 activates SHH stem cell signaling pathway by downregulating the expression of SHH pathway suppressor PTCH1. Our findings suggest that TRIM37 may be a potential therapeutic target for GBM.

Efficacy of interstitial photodynamic therapy using talaporfin sodium and a semiconductor laser for a mouse allograft glioma model

To investigate the therapeutic potential of photodynamic therapy (PDT) for malignant gliomas arising in unresectable sites, we investigated the effect of tumor tissue damage by interstitial PDT (i-PDT) using talaporfin sodium (TPS) in a mouse glioma model in which C6 glioma cells were implanted subcutaneously. A kinetic study of TPS demonstrated that a dose of 10 mg/kg and 90 min after administration was appropriate dose and timing for i-PDT. Performing i-PDT using a small-diameter plastic optical fiber demonstrated that an irradiation energy density of 100 J/cm2 or higher was required to achieve therapeutic effects over the entire tumor tissue. The tissue damage induced apoptosis in the area close to the light source, whereas vascular effects, such as fibrin thrombus formation occurred in the area slightly distant from the light source. Furthermore, when irradiating at the same energy density, irradiation at a lower power density for a longer period of time was more effective than irradiation at a higher power density for a shorter time. When performing i-PDT, it is important to consider the rate of delivery of the irradiation light into the tumor tissue and to set irradiation conditions that achieve an optimal balance between cytotoxic and vascular effects.

Focused Ultrasound-Enhanced Liquid Biopsy: A Promising Diagnostic Tool for Brain Tumor Patients.

The performance of minimally invasive molecular diagnostic tools in brain tumors, such as liquid biopsy, has so far been limited by the blood-brain barrier (BBB). The BBB hinders the release of brain tumor biomarkers into the bloodstream. The use of focused ultrasound in conjunction with microbubbles has been shown to temporarily open the BBB (FUS-BBBO). This may enhance blood-based tumor biomarker levels. This systematic review provides an overview of the data regarding FUS-BBBO-enhanced liquid biopsy for primary brain tumors. A systematic search was conducted in PubMed and Embase databases with key terms "brain tumors", "liquid biopsy", "FUS" and their synonyms, in accordance with PRISMA statement guidelines. Five preclinical and two clinical studies were included. Preclinical studies utilized mouse, rat and porcine glioma models. Biomarker levels were found to be higher in sonicated groups compared to control groups. Both stable and inertial microbubble cavitation increased biomarker levels, whereas only inertial cavitation induced microhemorrhages. In clinical studies involving 14 patients with high-grade brain tumors, biomarker levels were increased after FUS-BBBO with stable cavitation. In conclusion, FUS-BBBO-enhanced liquid biopsy using stable cavitation shows diagnostic potential for primary brain tumors. Further research is imperative before integrating FUS-BBBO for liquid biopsy enhancement into clinical practice.

Multi-omics-based investigation of Bifidobacterium's inhibitory effect on glioma: regulation of tumor and gut microbiota, and MEK/ERK cascade.

Glioma, the most prevalent primary tumor of the central nervous system, is characterized by a poor prognosis and a high recurrence rate. The interplay between microbes, such as gut and tumor microbiota, and the host has underscored the significant impact of microorganisms on disease progression. Bifidobacterium, a beneficial bacterial strain found in the human and animal intestines, exhibits inhibitory effects against various diseases. However, the existing body of evidence pertaining to the influence of Bifidobacterium on glioma remains insufficient. Here, we found that Bifidobacterium reduces tumor volume and prolongs survival time in an orthotopic mouse model of glioma. Experiments elucidated that Bifidobacterium suppresses the MEK/ERK cascade. Additionally, we noted an increase in the α-diversity of the tumor microbiota, along with an augmented relative abundance of Bifidobacterium in the gut microbiota. This rise in Bifidobacterium levels within the intestine may be attributed to a concurrent increase in Bifidobacterium within the glioma. Additionally, Bifidobacterium induced alterations in serum metabolites, particularly those comprised of organonitrogen compounds. Thus, our findings showed that Bifidobacterium can suppress glioma growth by inhibiting the MEK/ERK cascade and regulating tumor, and gut microbiota, and serum metabolites in mice, indicating the promising therapeutic prospects of Bifidobacterium against glioma.

Overcoming the Blood-Brain Tumor Barrier with Docetaxel-Loaded Mesoporous Silica Nanoparticles for Treatment of Temozolomide-Resistant Glioblastoma.

While temozolomide (TMZ) has been a cornerstone in the treatment of newly diagnosed glioblastoma (GBM), a significant challenge has been the emergence of resistance to TMZ, which compromises its clinical benefits. Additionally, the nonspecificity of TMZ can lead to detrimental side effects. Although TMZ is capable of penetrating the blood-brain barrier (BBB), our research addresses the need for targeted therapy to circumvent resistance mechanisms and reduce off-target effects. This study introduces the use of PEGylated mesoporous silica nanoparticles (MSN) with octyl group modifications (C8-MSN) as a nanocarrier system for the delivery of docetaxel (DTX), providing a novel approach for treating TMZ-resistant GBM. Our findings reveal that C8-MSN is biocompatible in vitro, and DTX@C8-MSN shows no hemolytic activity at therapeutic concentrations, maintaining efficacy against GBM cells. Crucially, in vivo imaging demonstrates preferential accumulation of C8-MSN within the tumor region, suggesting enhanced permeability across the blood-brain tumor barrier (BBTB). When administered to orthotopic glioma mouse models, DTX@C8-MSN notably prolongs survival by over 50%, significantly reduces tumor volume, and decreases side effects compared to free DTX, indicating a targeted and effective approach to treatment. The apoptotic pathways activated by DTX@C8-MSN, evidenced by the increased levels of cleaved caspase-3 and PARP, point to a potent therapeutic mechanism. Collectively, the results advocate DTX@C8-MSN as a promising candidate for targeted therapy in TMZ-resistant GBM, optimizing drug delivery and bioavailability to overcome current therapeutic limitations.

Abstract P45: REDUCED IMMUNOGENICITY DRIVES SUPERIORITY OF THE NANOLUCIFERASE (NLUC) REPORTER IN AN IMMUNOCOMPETENT GL261 MOUSE GLIOMA MODEL

Abstract Bioluminescence imaging (BLI) with luciferase-based reporters is a robust approach for monitoring tumour growth and treatment response in preclinical in vivo cancer models. Luciferases are considered foreign molecules that could elicit immune responses when introduced in immunocompetent live animal models [1, 2]. The immunogenicity of luciferases and other transgenic imaging reporters, though often overlooked, was recently discussed in relation to mouse cancer models [3-5]. Herein, we investigated the immunogenicity of two luciferase reporters: (1) red-shifted firefly luciferase (RFLuc, 61 kDa) and (2) deep-sea shrimp nanoluciferase (nLuc, 19 kDa) in an orthotopic brain tumour model using GL261 mouse glioma cells in a syngeneic C57BL/6J mouse. We evaluated their suitability for monitoring of brain tumour growth kinetics by BLI. Mice were implanted with 2 × 104 either GL261, GL261-NLuc or GL261-RFLuc cells in the right cerebrum using a stereotaxic device. GL261-RFLuc tumours could only be detected by BLI for 2 weeks, and the mice did not die of disease (median overall survival; mOS > 35 days). In contrast, GL261-nLuc cells formed lethal brain tumours that grew in volume as determined by BLI. GL261-NLuc implanted mice exhibited mOS comparable to mice implanted with GL261 cells (24.5 days vs. 23 days). These observations indicated that nLuc is not significantly immunogenic in mouse brains and did not cause rejection of the tumour cells as observed with RFluc. Cytokine expression profiles and immune cell profiles of the tumour-bearing brain hemispheres were comparable between tumours from GL261 and GL261-NLuc. In contrast, GL261-RFLuc tumours exhibited elevated CD8+ T cells, particularly effector (CD62L¯CD44+) and granzyme B-expressing (GZB+) CD8+ T cells. GL261-nLuc formed stable glioma tumors comparable to the original GL261 mouse model and is a suitable in vivo preclinical model for evaluating experimental therapeutic interventions against gliomas, when paired with Promega’s proprietary fluoro-furimazine substrate. Funding: This research was supported by Singapore’s Health and Biomedical Sciences (HBMS) Industry Alignment Fund Pre-Positioning (IAF-PP) grant H18/01/a0/018 (to AMC) administered by A*STAR and Singapore-MIT Alliance for Research and Technology (SMART) Innovation Grant (ING-000913 BIO) (to AMC). Citation Format: Carla Bianca Luena Victorio, Wisna Novera, Arun Ganasarajah, Joanne Ong, Peter Li, Sven Hans Petersen, Rasha Msallam, Ann-Marie Chacko. REDUCED IMMUNOGENICITY DRIVES SUPERIORITY OF THE NANOLUCIFERASE (NLUC) REPORTER IN AN IMMUNOCOMPETENT GL261 MOUSE GLIOMA MODEL [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P45.

Single-cell analysis of 5-aminolevulinic acid intraoperative labeling specificity for glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor, and resection is a key part of the standard of care. In fluorescence-guided surgery (FGS), fluorophores differentiate tumor tissue from surrounding normal brain. The heme synthesis pathway converts 5-aminolevulinic acid (5-ALA), a fluorogenic substrate used for FGS, to fluorescent protoporphyrin IX (PpIX). The resulting fluorescence is believed to be specific to neoplastic glioma cells, but this specificity has not been examined at a single-cell level. The objective of this study was to determine the specificity with which 5-ALA labels the diversity of cell types in GBM. The authors performed single-cell optical phenotyping and expression sequencing-version 2 (SCOPE-seq2), a paired single-cell imaging and RNA sequencing method, of individual cells on human GBM surgical specimens with macroscopically visible PpIX fluorescence from patients who received 5-ALA prior to surgery. SCOPE-seq2 allowed the authors to simultaneously image PpIX fluorescence and unambiguously identify neoplastic cells from single-cell RNA sequencing. Experiments were also conducted in cell culture and co-culture models of glioma and in acute slice cultures from a mouse glioma model to investigate cell- and tissue-specific uptake and secretion of 5-ALA and PpIX. SCOPE-seq2 analysis of human GBM surgical specimens revealed that 5-ALA treatment resulted in labeling that was not specific to neoplastic glioma cells. The cell culture further demonstrated that nonneoplastic cells could be labeled by 5-ALA directly or by PpIX secreted from surrounding neoplastic cells. Acute slice cultures from mouse glioma models showed that 5-ALA preferentially labeled GBM tumor tissue over nonneoplastic brain tissue with significant labeling in the tumor margins, and that this contrast was not due to blood-brain barrier disruption. Together, these findings support the use of 5-ALA as an indicator of GBM tissue but question the main advantage of 5-ALA for specific intracellular labeling of neoplastic glioma cells in FGS. Further studies are needed to systematically compare the performance of 5-ALA to that of potential alternatives for FGS.

Abstract 1437: Generating primary mouse models of diffuse midline glioma through a combination of the RCAS/tv-a retrovirus system and CRISPR/Cas9 gene editing

Abstract Diffuse midline gliomas (DMGs) are deadly brain tumors that arise in the brainstem of children and young adults, resulting in a median survival of less than two years. Genetically engineered mouse models (GEMMs) are critical to studying tumorigenesis and tumor-immune interactions in DMG, which may inform the design of new urgently-needed treatment approaches. One approach to generate DMG GEMMs uses RCAS/tv-a, an avian retrovirus gene delivery system to express transgenes in specific brain cell lineages in the mouse brain. However, this approach often requires resource-intensive breeding to generate mice with the desired genetic alterations in their germline. As a result, we aimed to establish a new approach that combines this system with CRISPR/Cas9 gene editing technology. This methodology has been described for model adult supratentorial gliomas in mice but has not been described for pediatric or brainstem gliomas. To express Cas9 and the avian retrovirus receptor TVA in mouse neural stem cells, we bred mice of genotype Nestin-TVA; Nestin-Cre; LoxP-STOP-LoxP-Cas9-EGFP. RCAS retrovirus plasmids were generated containing various guide RNAs (gRNAs) targeting DMG-relevant genes for disruption by Cas9, including Trp53, Cdkn2a, Atm, Ppm1d, Pten, and a non-targeting control (Cntl). For each gRNA, two plasmids were generated, one with a platelet-derived growth factor beta ligand (PDGFB) oncogene driver and one with a blue fluorescent protein (BFP) marker. In a pilot experiment, avian fibroblast cells transduced with RCAS-Trp53_gRNA_BFP and RCAS-Cntl_gRNA-PDGFB were injected into postnatal day 4 mouse brainstems of the aforementioned genotype and observed for tumor formation. High grade tumor formation was observed. We also observed high grade tumors in mice of genotype Nestin-TVA; LoxP-STOP-LoxP-Cas9-EGFP when injected with an additional RCAS-Cre construct, indicating that Cre can be expressed from the mouse germline or delivered via a retrovirus payload to drive tumorigenesis. Immunohistochemistry confirmed tumor features that model human DMG, expression of Cas9 and PDGFB, and loss of p53. Thus, RCAS/tv-a and CRISPR/Cas9 can be combined to model pediatric glioma tumorigenesis in the mouse brainstem. This approach to mouse modeling will streamline future GEMM DMG research by allowing new genes of interest to be rapidly perturbed by simply generating RCAS retrovirus constructs. The workflow may also enable sophisticated experiments such as pooled in vivo genetic experiments that screen gRNAs targeting various tumor suppressor genes or therapeutic targets. Citation Format: Sophie R. Wu, Harrison Q. Liu, Joshua Tolliver, Spencer M. Maingi, Vennesa Valentine, Nerissa T. Williams, Zachary J. Reitman. Generating primary mouse models of diffuse midline glioma through a combination of the RCAS/tv-a retrovirus system and CRISPR/Cas9 gene editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1437.

Abstract 487: Peptide amphiphiles hitchhike on endogenous biomolecules for enhanced drug delivery

Abstract Peptide amphiphiles (PAs) have been applied as building blocks for self-assembled nanostructures with various morphologies. These self-assembled nanostructures are promising drug delivery platforms as they are naturally biodegradable and biocompatible with low immunogenicity. One primary focus in drug delivery using the PA nanostructures has been to develop strongly assembled structures to avoid disassembly in the blood and allow them to carry payloads to their intended targets. In this work, rather than developing stable self-assembled PA structures, we focused on weakly assembled PA nanostructures to investigate their interactions with blood components and to understand how these interactions affect their biodistribution. We found that weakly assembled PA nanostructures (SA-E) quickly disassembled in plasma and reassembled with lipoproteins (LPs), which significantly prolonged the blood circulation of SA-E in mice. We showed that SA-E crossed the endothelial barriers through transcytosis only when they were assembled with LPs. Cellular uptake studies showed that SA-E accumulated in cancer cells through lipid-raft-mediated uptake. Interestingly, their cellular internalization was independent of LP receptors, suggesting that SA-E assembles with different endogenous biomolecules in vivo. By exploiting these endogenous interactions, SA-E demonstrated high tumor accumulation in a myriad of small animal tumor models of breast, colon, pancreatic, brain, skin, prostate, or lung cancers. SA-E demonstrated large tumor-to-background signal ratios in live animals (>5), extended retention (>2 weeks), and excellent tissue distribution (tumor to liver signal >3) in these tumor models. Encouraged by the excellent tumor accumulation of SA-E, we prepared SA-E drug conjugate using a highly potent and toxic chemotherapeutic agent, Monomethyl auristatin E (MMAE). SA-E-MMAE conjugate demonstrated strong antitumor efficacy in aggressive breast and glioma models in mice with reduced side effects. With its simple and modular design and universal tumor accumulation mechanism, SA-E represents a promising platform for broad applications in cancer therapy. Citation Format: Jared M. Fischer, Morgan Stewart, Mingchong Dai, Samuel Drennan, Sinan Sabuncu, Benjamin Kingston, Isabel Dengos, Li Xiang, Karla Bonic, Xin Yi, Srivathsan Ranganathan, Bruce Branchaud, Adem Yildirim. Peptide amphiphiles hitchhike on endogenous biomolecules for enhanced drug delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 487.

Abstract 3056: Copper chelation modulates YTHDF2 RNA-binding protein localization and global translation, in neuronal cancers

Abstract Cancers are dependent on copper for growth, angiogenesis and metastasis and copper chelators are in clinical trials against breast cancer, however the mechanisms of this emerging cancer treatment are still largely unexplored. We previously demonstrated that copper chelation has efficacy in mouse models of neuroblastoma and glioma. Multiomics analyses in neuronal cancers suggested that RNA-binding proteins were deregulated by copper chelator treatment. We found that the “RNA Binding” gene set was the most significantly ranked molecular function list using differentially expressed RNAs after copper chelation treatment (GO:0003723, p-value 1.02e−32). We subsequently found that the copper chelator drug decreased global protein synthesis, MYC/MYCN expression, as well as the amount a key methylated (m6A) RNA-binding protein called YTHDF2 in glioma cells, by RNA-seq (-log2FC 0.3, p-value 0.005) and western blot. This downregulation was also clear in neuroblastoma and glioblastoma cell lines. Interestingly, copper chelation also shifted YTHDF2 protein location into the nucleus. YTHDF2 knock-out (DepMAP Chronos dependency score, -0.32) and knock-down studies (siRNA pool, 38% decrease in cell confluence) in neuroblastoma cell lines and others, indicated that YTHDF2 depletion is selective against neuronal cancer cell growth. YTHDF2 was found to be a MYCN transcriptional target. Moreover, as YTHDF2 protein also binds MYCN transcripts (the principal oncogene in neuroblastoma), and copper signaling mRNAs, it appears that copper signaling, YTHDF2 and MYCs operates in an oncogenic-axis. Ongoing studies are investigating novel YTHDF2 inhibitors and their neuronal cancer efficacy. Our work represents a significant breakthrough in drugging oncogenic post-transcriptional processes either directly, or indirectly via depleting copper. Citation Format: Jessica L. Bell, Domenico K. Abruzzese, Erin Kasiou, Vu Vu Pham, Jean B. Bertoldo, Filip Michniewicz, Orazio Vittorio. Copper chelation modulates YTHDF2 RNA-binding protein localization and global translation, in neuronal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3056.

Folic acid-decorated astrocytes-derived exosomes enhanced the effect of temozolomide against glioma.

A direct strategy to achieve specific treatments and reduce side effects is through cell type-specific drug delivery. Exosomes (Exos) can be modified with folic acid (FA) to prepare drug delivery systems targeting tumor cells that highly express FA receptors. This study aimed to produce an exo drug delivery system with FA decoration and temozolomide (TMZ) loading to improve the sustained TMZ release and targeting. We used DSPE-PEG2000-FA to modify exos derived from astrocyte U-87 to prepare FA-modified exos (Astro-exo-FA). TMZ was encapsulated into Astro-exo-FA or Astro-exo through electroporation to produce TMZ@Astro-exo and TMZ@Astro-exo-FA. In vitro drug release was examined using the dialysis bag method. Through cell experiments in vitro and mouse glioma models in vivo, the effect of TMZ@Astro-exo-FA on U-87 cells was determined. Exo properties were not affected by FA modification and TMZ loading. The drug release rate of TMZ@Astro-exo-FA was slower. TMZ@Astro-exo-FA uptake by U-87 cells was higher compared to TMZ@Astro-exo, indicating that TMZ@Astro-exo-FA has a stronger targeting toward U-87 cells. TMZ@Astro-exo-FA remarkably reduced U-87 cell proliferation, migration, and invasion compared with TMZ@Astro-exo and free TMZ. Treatment with TMZ@Astro-exo-FA reduced the side effects of TMZ (minimal change in body weight), prolonged survival, and inhibited tumor growth in mouse glioma models, and its efficacy was stronger than that of TMZ@Astro-exo and free TMZ. TMZ@Astro-exo-FA could enhance the effect of TMZ against glioma, providing novel ideas for drug targeting delivery and exploring exos as drug carriers against glioma.