Mouse Model Of Glioma Research Articles

Comparing Tumor Cell Invasion and Myeloid Cell Composition in Compatible Primary and Relapsing Glioblastoma.

Simple SummaryWe established a new minimally invasive mouse model for GBM relapse. For this, we utilized orthotopical implantation of HSVTK-transduced GBM cells and pharmacological treatment with GCV. In addition, we implanted patient-derived GBM cells of primary or recurrent tumors. We found that recurrent GBM were more aggressively invasive than primary GBM. Moreover, the recurring tumors had a higher ratio of monocyte-derived macrophages among the entire population of tumor associated myeloid cells. This shift in the composition of tumor-associated immune cells appeared to be independent from cell-death signaling or surgical intervention. This model provides the means to investigate the entire process of tumor relapse and test standard as well as experimental therapeutic strategies for relapsing GBM under defined conditions.Glioblastoma (GBM) recurrence after treatment is almost inevitable but addressing this issue with adequate preclinical models has remained challenging. Here, we introduce a GBM mouse model allowing non-invasive and scalable de-bulking of a tumor mass located deeply in the brain, which can be combined with conventional therapeutic approaches. Strong reduction of the GBM volume is achieved after pharmacologically inducing a tumor-specific cell death mechanism. This is followed by GBM re-growth over a predictable timeframe. Pharmacological de-bulking followed by tumor relapse was accomplished with an orthotopic mouse glioma model. Relapsing experimental tumors recapitulated pathological features often observed in recurrent human GBM, like increased invasiveness or altered immune cell composition. Orthotopic implantation of GBM cells originating from biopsies of one patient at initial or follow-up treatment reproduced these findings. Interestingly, relapsing GBM of both models contained a much higher ratio of monocyte-derived macrophages (MDM) versus microglia than primary GBM. This was not altered when combining pharmacological de-bulking with invasive surgery. We interpret that factors released from viable primary GBM cells preferentially attract microglia whereas relapsing tumors preponderantly release chemoattractants for MDM. All in all, this relapse model has the capacity to provide novel insights into clinically highly relevant aspects of GBM treatment.

OTME-16. Polio virotherapy of murine brain tumors causes microglia/macrophage proliferation and inflammation that is potentiated by immune checkpoint blockade

PVSRIPO is a novel viral immunotherapy that has shown evidence of efficacy in a phase I clinical trial for recurrent GBM, resulting in 21% survival rate at 36 months following treatment. To improve clinical response rate, it is critical to resolve the mechanisms of action and therapy resistance in vivo, thereby designing effective combination therapy strategies.We used immunocompetent mouse models of glioma (CT2A) and metastatic melanoma (B16) to dissect early and late events following virotherapy with PVSRIPO. A blinded systematic review of the pathology from 62 intracranial tumors, collected on different days following PVSRIPO (or control) treatment, was performed. An overall treatment effect, measured by tumor shrinkage, dis-cohesive growth pattern, microglia enrichment, was present in 88% of tumors on day 8, but the tissue response rate fell to 42% on days 10 & 12, and 14% on day 15. The control group showed no treatment effect throughout. RNAseq from the same set of samples showed acute induction of type-I interferon-related inflammation that faded with time in Gene Set Enrichment Analysis. This suggests that sustaining adaptive antitumor immunity elicited by immediate intratumor type-I IFN-dominant inflammation is critical to long term remission. Careful review of the post treatment pathology revealed an early enrichment of both T cells and microglia in the tumor microenvironment with a high Ki-67 proliferation index. We propose that the PVSRIPO therapy effect is dependent on macrophage/microglia mediated cellular immune response, likely in response to direct viral infection. This suggests potential therapeutic interventions, including blockade of the PD1:PD-L1 immune checkpoint, to potentiate antitumor CD8+T cells in response to PVSRIPO therapy. Indeed, combination therapy with αPD-L1 antibody in the CT2A model showed higher long term remission (37%, n=11), compared to either monotherapy; this effect is CD8+T cell- and macrophage-dependent, demonstrated by depletion studies in vivo.

MiR-1 Activates NF-κB to Promote the Differentiation of Bone Marrow Mesenchymal Stem Cells in Mouse Models of Glioma

NF-κB activation and its abnormal expression are involved in the progression of glioma. miRNA plays a crucial role in bone diseases. The role of NF-κB is becoming more and more important. The purpose of this study is to explore the mechanism by how miR-1 regulates NF-κB signaling. C57 glioma mouse models were divided into osteoporosis (OP) group and control group. qPCR was used to measure miR-1 levels in OP and control mice. Bone marrow mesenchymal stem cells (BMSCs) were cultured and transfected with miR-1 specific siRNA to establish miR-1 knockout cell model followed by analysis of cell apoptosis, expression of NF-κB signaling molecules by western blot. qPCR results showed that miR-1 levels in OP mice were significantly reduced compared to control mice. A large number of siRNA particles were observed in transfected BMSCs under a fluorescence microscope. qPCR results showed that siRNA transfection significantly suppressed miR-1, indicating successful transfection. Flow cytometry revealed significant differences in cell apoptosis between miR-1 siRNA group and the NC group. Western blot indicated miR-1 promoted BMSCs differentiation via NF-κB mediated up-regulation of ALP activity. The expression of miR-1 is low in BMSCs of mice with glioma. In addition, BMSCs differentiation is enhanced by NF-κB activation via up-regulating miR-1.

Abstract CT240: LUMINOS-101: Phase 2 study of PVSRIPO with pembrolizumab in recurrent glioblastoma

Abstract Background: The prognosis for patients (pts) with recurrent (r) glioblastoma (GBM) is poor, with no highly effective approved therapies. Treatment failure may result from poor penetration of drugs through the blood-brain barrier and the immunosuppressive nature of the tumor microenvironment (TME). PVSRIPO, a recombinant poliovirus (PV):rhinovirus chimera, is a novel, non-neurovirulent, intratumoral immunotherapy. Trial results in rGBM pts showed greater long-term survival with PVSRIPO monotherapy (21%, 36-60 months [mos]) vs. criteria-matched external controls (4%, 36 mos; 2%, 60 mos; Desjardins 2018 NEJM). PVSRIPO targets CD155 (PV receptor), expressed on solid tumors and APC. PVSRIPO IT infection results in inflammatory-mediated destruction of tumor cells but non-lethal lingering infection in TME APC. This leads to a type-I/III interferon-dominant inflammation and ultimately, tumor antigen-specific T cell activation and recruitment (Brown 2017 Sci Transl Med), which is potentiated by immunologic recall to intratumoral replicating virus via prior vaccination. Induction of type-1 IFN dominant inflammation and compensatory activation of the PD-1:PD-L1 immune checkpoint (IC) pathway support investigation of PVSRIPO in combination with PD-1/L1 IC inhibitors. Immunologically cold mouse glioma models showed PVSRIPO+anti-PD-1 therapy resulted in greater anti-tumor response than either agent. Trial design/objectives: LUMINOS-101 (NCT04479241) is a phase 2, multicenter, open-label, single-arm study of intratumoral infusion of PVSRIPO (day 1: 5×107 TCID50) followed by the anti-PD-1 monoclonal antibody pembrolizumab (200mg IV q3w) in adult pts with rGBM. The trial objective is to evaluate the anti-tumor activity, safety, and tolerability of the combination. Eligibility criteria: Pts ≥18 years who had prior PV and boost IPOL® immunizations, histologically confirmed supratentorial rGBM, infusible 1 to ≤5.5cm enhancing disease, confirmed disease progression following prior therapies, and KPS ≥70 will be enrolled. Key exclusion criteria include multifocal disease; discontinuation of prior anti-PD-1/L1 agent for toxicity; prior intratumoral therapy, immunotherapy, or radiotherapy within 12 weeks; high-dose systemic corticosteroids; chemotherapy, anti-VEGF, or TTF therapy ≤1-6 weeks depending on the therapy; serious cerebral herniation syndrome; extensive leptomeningeal, subependymal, or ≥1cm enhancing disease crossing the midline; and severe active comorbidities. Endpoints: Primary endpoints are objective response rate, duration of response, and safety. Secondary endpoints include overall and progression-free survival and disease control rate and duration. Exploratory endpoints include assessment of tumor and blood for biomarkers of response. The study is currently enrolling in the US, and results will inform the design of a randomized phase 3 trial. Citation Format: Andrea True Kelly, Prakash Ambady, Michael Brown, Nicholas Butowski, Robert Cavaliere, William Curry, Annick Desjardins, Lisa Franklin, Henry Friedman, Matthias Gromeier, LeAnn Jackson, Lori Mixson, Shirley Ong, Dina Randazzo, Andrew Sloan, Patrick Wen, Garrett Nichols. LUMINOS-101: Phase 2 study of PVSRIPO with pembrolizumab in recurrent glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT240.

Argyrin F Treatment‐Induced Vulnerabilities Lead to a Novel Combination Therapy in Experimental Glioma

AbstractGlioblastoma are incurable primary tumors of the central nervous system that frequently harbor molecular alterations in the retinoblastoma pathway with subsequent cell cycle abnormalities. It is aimed to investigate the anti‐glioma activity of the novel cell cycle‐stabilizing compound Argyrin F and its potential treatment‐induced vulnerabilities to exploit possibilities for novel rational combination therapies. Human and murine glioma cells are used, cytotoxicity and clonogenic survival assays, cell cycle analyses, and immunoblots are performed. Residual freshly resected tissue is used for a preclinical glioma model ex vivo, i.e., patient‐derived microtumors (PDMs) for co‐culture experiments with autologous tumor‐infiltrating lymphocytes (TILs). Mass spectrometry‐based immunopeptidomics are performed and the orthotopic syngeneic SMA560/VM/Dk glioma mouse model is used. Argyrin F‐induced cell cycle arrest and reduced clonogenic survival is observed. Argyrin F‐treated experimental glioma in vivo displays 4.6‐fold more glioma‐infiltrating CD8+ T cells. A distinctive treatment‐induced immunopeptidome is discovered. The combination of Argyrin F plus PD‐1 antibody increases cellular toxicity in PDM/TILs co‐cultures ex vivo and prolonged overall survival compared with monotherapies in vivo. Thus, Argyrin F leads to anti‐glioma effects and increases the immunogenicity, paving the way for a novel combination therapy of Argyrin F plus PD‐1 blockade.

The lipid-lowering drug fenofibrate combined with si-HOTAIR can effectively inhibit the proliferation of gliomas

BackgroundFenofibrate is a fibric acid derivative known to have a lipid-lowering effect. Although fenofibrate-induced peroxisome proliferator-activated receptor alpha (PPARα) transcription activation has been shown to play an important role in the malignant progression of gliomas, the underlying mechanisms are poorly understood.MethodsIn this study, we analyzed TCGA database and found that there was a significant negative correlation between the long noncoding RNA (lncRNA) HOTAIR and PPARα. Then, we explored the molecular mechanism by which lncRNA HOTAIR regulates PPARα in cell lines in vitro and in a nude mouse glioma model in vivo and explored the effect of the combined application of HOTAIR knockdown and fenofibrate treatment on glioma invasion.ResultsFor the first time, it was shown that after knockdown of the expression of HOTAIR in gliomas, the expression of PPARα was significantly upregulated, and the invasion and proliferation ability of gliomas were obviously inhibited. Then, glioma cells were treated with both the PPARα agonist fenofibrate and si-HOTAIR, and the results showed that the proliferation and invasion of glioma cells were significantly inhibited.ConclusionsOur results suggest that HOTAIR can negatively regulate the expression of PPARα and that the combination of fenofibrate and si-HOTAIR treatment can significantly inhibit the progression of gliomas. This introduces new ideas for the treatment of gliomas.

HGG-41. CHARACTERIZATION OF THE IMMUNE RESPONSE FOLLOWING VARIOUS RADIOTHERAPY TREATMENTS IN GLIOBLASTOMA

Malignant gliomas represent 6.5% of all childhood brain neoplasms with a 5-years survival rate of less than 20%. Current standard of care for these tumors include radiotherapy; recent data in solid tumors indicate that adequate radiation protocols may synergize with immunotherapy strategies for better outcomes. Nonetheless, a great discrepancy between preclinical studies and clinical trials outcomes persists, the basis of which is not fully understood. One hypothesis may be due to different radiation protocols used. We used the GL261 syngeneic mouse model of glioma to test this hypothesis and characterize the immune response to radiotherapy, with either a single dose of 10Gy, a dose often used in preclinical models, or a fractionated treatment of 2Gy for five consecutive days (2Gyx5), as fractioned radiotherapy is most often used in patients. The immune content of the brain and the blood was assessed by flow cytometry in un-irradiated (control), 10Gyx1 and 2Gyx5 treated mice for three weeks after radiation. In the brain, both radiation regimens drastically reduced the number of CD45+ cells for the first two weeks after treatment. When compared to controls, 10Gyx1 but not 2Gyx5 treated mice showed a significant increase in tumor infiltrating lymphocytes (CD3+) starting from the second week following treatment. This effect persisted until three weeks post treatment. The 10Gyx1 dose was better tolerated by the resident microglia (CD45lowCD11b+) when compared to the 2Gyx5 treatment. Our data describe the dynamics through which the immune microenvironment responds to two radiation regimens over time. Our results show that 10Gyx1 is the most effective regimen to impede tumor growth and to induce lymphocyte infiltration once the system recovers from the treatment. Our work suggests that, in the GL261 model, the fractionated radiation treatment we tested may be less optimal in priming glioma cells to the immune system.

TMOD-06. LOSS OF DICER COOPERATES WITH TUMOR SUPPRESSORS TO INITIATE METASTATIC MEDULLOBLASTOMA

To determine the role of microRNA regulation in brain tumor development, we incorporated a conditional allele of the microRNA processing enzyme Dicer to a previously characterized glioma mouse model based on inactivation of the tumor suppressors Nf1, Trp53, and Pten using the Nestin-creERT2 transgene. Loss of Dicer and tumor suppressors at adult ages led to glioma development; however, mutant mice tamoxifen induced at early postnatal ages developed medulloblastoma instead of glioma. The switch in tumor spectrum occurred with 100% penetrance and tumors were histologically indistinguishable from human medulloblastoma (MB). The minimum genetic mutations required for MB formation were Dicer and Trp53. Nf1 was dispensable, while additional loss of Pten produced more invasive tumors and leptomeningeal metastases. The time window for initiation of tumorigenesis was until the 2nd postnatal week, coinciding with the disappearance of the external granule layer (EGL), where cerebellar granule neuron precursors (CGNPs) undergo proliferation. Analysis of pre-symptomatic mutant mice showed proliferative defects and retained cells in the EGL, suggesting that the tumors may arise from CGNPs. However, targeting a subset of CGNPs using Math1-creERT2 did not lead to MB development, suggesting that an earlier EGL precursor may be required for tumorigenesis. Analysis of tumor transcriptome and MB subtype-specific genes and markers show that Dicer tumors most resemble extremely high risk p53-mutated SHH MB. Small RNA and mRNA sequencing analyses showed downregulation of microRNAs and dysregulation of its targets such as N-Myc. These studies demonstrate a role for microRNAs in MB development and show a fully penetrant genetic mouse model of highly metastatic MB.

LUMINOS-101: Phase 2 study of PVSRIPO with pembrolizumab in recurrent glioblastoma.

TPS2065 Background: The prognosis for patients (pts) with recurrent (r) glioblastoma (GBM) is poor, with no highly effective approved therapies. Treatment failure may result from poor penetration of drugs through the blood-brain barrier and the immunosuppressive nature of the tumor microenvironment (TME). PVSRIPO, a recombinant poliovirus (PV):rhinovirus chimera, is a novel, non-neurovirulent, intratumoral immunotherapy. Trial results in rGBM pts show greater long-term survival with PVSRIPO monotherapy (21%, 36-60 months [mos]) vs criteria-matched external controls (4%, 36 mos; 2%, 60 mos; Desjardins 2018 NEJM). PVSRIPO targets CD155 (PV receptor), expressed on solid tumors and on APC. PVSRIPO infection results in inflammatory-mediated destruction of tumor cells but non-lethal lingering infection in TME APC. This leads to type I/III interferon-dominant inflammation and, ultimately, tumor antigen-specific T cell activation and recruitment (Brown 2017 Sci Transl Med), which is potentiated by immunologic recall to intratumoral replicating virus via prior vaccination. Induction of type 1 IFN dominant inflammation and compensatory activation of the PD-1:PD-L1 immune checkpoint (IC) pathway support investigation of PVSRIPO in combination with PD-1/L1 IC inhibitors. Immunologically cold mouse glioma models show PVSRIPO+anti-PD-1 therapy resulted in greater anti-tumor response than either agent. Methods: LUMINOS-101 is a phase 2, multicenter, open-label, single-arm study of intratumoral infusion of PVSRIPO (Day 1: 5x107 TCID50) followed by the anti-PD-1 monoclonal antibody pembrolizumab (200mg IV q3w) in adult pts with rGBM. The trial objective is to evaluate anti-tumor activity and safety and tolerability of the combination. Eligibility criteria include pts ≥18 years who had prior PV and boost IPOL® immunizations, histologically confirmed supratentorial rGBM, infusible 1 to ≤5.5cm enhancing disease, confirmed disease progression following prior therapies, and KPS ≥70. Key exclusion criteria include multifocal disease; discontinuation of prior anti-PD-1/L1 agent for toxicity; prior intratumoral therapy, immunotherapy, or radiotherapy within 12 weeks; high-dose systemic corticosteroids; chemotherapy, anti-VEGF, or TTF therapy ≤1-6 weeks depending on the therapy; serious cerebral herniation syndrome; extensive leptomeningeal, subependymal, or ≥1cm enhancing disease crossing the midline; and severe active comorbidities. Primary endpoints are objective response rate, duration of response, and safety. Secondary endpoints include overall and progression-free survival and disease control rate and duration. Exploratory endpoints include assessment of tumor and blood for biomarkers of response. The initially planned safety lead-in period is now fully enrolled. Recruitment is ongoing in the US, and results will inform the design of a randomized phase 3 trial. Clinical trial information: NCT04479241.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Targeting CSF1R Alone or in Combination with PD1 in Experimental Glioma.

Simple SummaryGlioblastomas are incurable tumors of the central nervous system. Currently, treatment strategies combine neurosurgical intervention, radiation therapy, and chemotherapy. Yet, clinical experience shows that tumors acquire escape mechanisms. Furthermore, the tumor-associated microenvironment, including macrophages expressing the receptor CSF1R, promote and nourish tumor cells. The so-called PD1/PDL1 axis is a major reason why tumors can grow with a “magic hat”; i.e., unrecognized from the immune system. The aim of our study was to assess treatment strategies that target macrophages in the microenvironment by blocking CSF1R alone or in combination with PD1 blockade. Using an immune competent mouse model and an ex vivo microtumor model using freshly resected glioblastoma material, we observed prolonged survival of treated mice and an improved “attack” of the immune system. We conclude that targeting CSF1R is a promising strategy that should be explored in clinical trials, potentially in combination with PD1 blockade.Glioblastoma is an aggressive primary tumor of the central nervous system. Targeting the immunosuppressive glioblastoma-associated microenvironment is an interesting therapeutic approach. Tumor-associated macrophages represent an abundant population of tumor-infiltrating host cells with tumor-promoting features. The colony stimulating factor-1/ colony stimulating factor-1 receptor (CSF-1/CSF1R) axis plays an important role for macrophage differentiation and survival. We thus aimed at investigating the antiglioma activity of CSF1R inhibition alone or in combination with blockade of programmed death (PD) 1. We investigated combination treatments of anti-CSF1R alone or in combination with anti-PD1 antibodies in an orthotopic syngeneic glioma mouse model, evaluated post-treatment effects and assessed treatment-induced cytotoxicity in a coculture model of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) ex vivo. Anti-CSF1R monotherapy increased the latency until the onset of neurological symptoms. Combinations of anti-CSF1R and anti-PD1 antibodies led to longterm survivors in vivo. Furthermore, we observed treatment-induced cytotoxicity of combined anti-CSF1R and anti-PD1 treatment in the PDM/TILs cocultures ex vivo. Our results identify CSF1R as a promising therapeutic target for glioblastoma, potentially in combination with PD1 inhibition.

TNF\u03b1 secreted by glioma associated macrophages promotes endothelial activation and resistance against anti-angiogenic therapy

One of the most prominent features of glioblastoma (GBM) is hyper-vascularization. Bone marrow-derived macrophages are actively recruited to the tumor and referred to as glioma-associated macrophages (GAMs) which are thought to provide a critical role in tumor neo-vascularization. However, the mechanisms by which GAMs regulate endothelial cells (ECs) in the process of tumor vascularization and response to anti-angiogenic therapy (AATx) is not well-understood. Here we show that GBM cells secrete IL-8 and CCL2 which stimulate GAMs to produce TNFα. Subsequently, TNFα induces a distinct gene expression signature of activated ECs including VCAM-1, ICAM-1, CXCL5, and CXCL10. Inhibition of TNFα blocks GAM-induced EC activation both in vitro and in vivo and improve survival in mouse glioma models. Importantly we show that high TNFα expression predicts worse response to Bevacizumab in GBM patients. We further demonstrated in mouse model that treatment with B20.4.1.1, the mouse analog of Bevacizumab, increased macrophage recruitment to the tumor area and correlated with upregulated TNFα expression in GAMs and increased EC activation, which may be responsible for the failure of AATx in GBMs. These results suggest TNFα is a novel therapeutic that may reverse resistance to AATx. Future clinical studies should be aimed at inhibiting TNFα as a concurrent therapy in GBMs.

Novel LncRNA OXCT1-AS1 indicates poor prognosis and contributes to tumorigenesis by regulating miR-195/CDC25A axis in glioblastoma

BackgroundLong noncoding RNAs (lncRNAs) contribute to multiple biological processes in human glioblastoma (GBM). However, identifying a specific lncRNA target remains a challenge. In this study, bioinformatics methods and competing endogenous RNA (ceRNA) network regulatory rules were used to identify GBM-related lncRNAs and revealed that OXCT1 antisense RNA 1 (OXCT1-AS1) is a potential therapeutic target for the treatment of glioma.MethodsBased on the Gene Expression Omnibus (GEO) dataset, we identified differential lncRNAs, microRNAs and mRNAs and constructed an lncRNA-associated ceRNA network.The novel lncRNA OXCT1-AS1 was proposed to function as a ceRNA, and its potential target miRNAs were predicted through the database LncBase Predicted v.2. The expression patterns of OXCT1-AS1 in glioma and normal tissue samples were measured. The effect of OXCT1-AS1 on glioma cells was checked using the Cell Counting Kit 8 assay, cell colony formation assay, Transwell assay and flow cytometry in vitro. The dual-luciferase activity assay was performed to investigate the potential mechanism of the ceRNA network. Finally, orthotopic mouse models of glioma were created to evaluate the influence of OXCT1-AS1 on tumour growth in vivo.ResultsIn this study, it was found that the expression of lncRNA OXCT1-AS1 was upregulated in both The Cancer Genome Atlas (TCGA) GBM patients and GBM tissue samples, and high expression of OXCT1-AS1 predicted a poor prognosis. Suppressing OXCT1-AS1 expression significantly decreased GBM cell proliferation and inhibited cell migration and invasion. We further investigated the potential mechanism and found that OXCT1-AS1 may act as a ceRNA of miR-195 to enhance CDC25A expression and promote glioma cell progression. Finally, knocking down OXCT1-AS1 notably attenuated the severity of glioma in vivo.ConclusionOXCT1-AS1 inhibits glioma progression by regulating the miR-195-5p/CDC25A axis and is a specific tumour marker and a novel potential therapeutic target for glioma treatment.

Temozolomide-Induced Changes in Gut Microbial Composition in a Mouse Model of Brain Glioma.

BackgroundGut microbiota is associated with the progression of brain tumors. However, the alterations in gut microbiota observed during glioma growth and temozolomide (TMZ) therapy remain poorly understood.MethodsC57BL/6 male mice were implanted with GL261 glioma cells. TMZ/sodium carboxymethyl cellulose (SCC) was administered through gavage for five consecutive days (from 8 to 12 days after implantation). Fecal samples were collected before (T0) and on days 7 (T1), 14 (T2), and 28 (T3) after implantation. The gut microbiota was analyzed using 16S ribosomal DNA sequencing followed by absolute and relative quantitation analyses.ResultsNineteen genera were altered during glioma progression with the most dramatic changes in Firmicutes and Bacteroidetes phyla. During glioma growth, Lactobacillus abundance decreased in the early stage (T1) and then gradually increased (T2, T3); Intestinimonas abundance exhibited a persistent increase; Anaerotruncus showed a transient increase (T2) and then a subsequent decrease (T3). Similar longitudinal changes in Intestinimonas and Anaerotruncus abundance were observed in TMZ-treated mice, but the decrease of Anaerotruncus at T3 in the TMZ-treated group was less than that in the vehicle-treated group. No significant change in Lactobacillus was observed after TMZ treatment. Additionally, compared to vehicle control, TMZ treatment led to an enrichment in Akkermansia and Bifidobacterium.ConclusionGlioma development and progression altered the composition of gut microbiota. Induction of Akkermansia and Bifidobacterium as well as the prevention of the reduction in Anaerotruncus may contribute to the anti-tumor effect of TMZ. This study helps to reveal the association between levels of specific microbial species in the gut and the anti-tumor effect of TMZ.

Inflammatory tumor microenvironment responsive neutrophil exosomes-based drug delivery system for targeted glioma therapy

Clinical treatment of malignant glioma remains a major challenge due to high infiltrative growth and chemotherapeutic resistance of tumors and the presence of the blood brain barrier (BBB). Advanced nanoplatforms that can efficiently cross the BBB and target to brain tumor are urgently needed. Encouraged by the intrinsic inflammatory chemotaxis and excellent BBB-crossing capability of neutrophils, a bioinspired neutrophil-exosomes (NEs-Exos) system for delivering loaded doxorubicin (DOX) drug for glioma treatment is proposed and systematically investigated. In vivo zebrafish and C6-Luc glioma-bearing mice models show that NEs-Exos carrying the drug rapidly penetrate the BBB and migrate into the brain. Additionally, a transwell BBB model and mouse brain inflammatory study show that NEs-Exos can respond chemotactically to inflammatory stimuli and target infiltrating tumor cells in inflamed brain tumors. Moreover, intravenous injection of NEs-Exos/DOX efficiently suppress tumor growth and prolong survival time in a glioma mouse model. On the basis of these results, NEs-Exos are confirmed to have neutrophil-like chemotactic function and BBB penetration. This novel NEs-Exos/DOX delivery platform represents a promising chemotherapeutic approach for clinical treatment of glioma and other solid tumor or brain diseases.

POT1 Regulates Proliferation and Confers Sexual Dimorphism in Glioma.

Germline POT1 mutations are found in a spectrum of cancers and confer increased risk. Recently, we identified a series of novel germline POT1 mutations that predispose carrier families to the development of glioma. Despite these strong associations, how these glioma-associated POT1 mutations contribute to glioma tumorigenesis remains undefined. Here we show that POT1-G95C increases proliferation in glioma-initiating cells in vitro and in progenitor populations in the developing brain. In a native mouse model of glioma, loss of Pot1a/b resulted in decreased survival in females compared with males. These findings were corroborated in human glioma, where low POT1 expression correlated with decreased survival in females. Transcriptomic and IHC profiling of Pot1a/b-deficient glioma revealed that tumors in females exhibited decreased expression of immune markers and increased expression of cell-cycle signatures. Similar sex-dependent trends were observed in human gliomas that had low expression of POT1. Together, our studies demonstrate context-dependent functions for POT1 mutation or loss in driving progenitor proliferation in the developing brain and sexual dimorphism in glioma. SIGNIFICANCE: This study shows that manipulation of POT1 expression in glioma has sex-specific effects on tumorigenesis and associated immune signatures.

A tumor-promoting role for soluble T\u03b2RIII in glioblastoma

PurposeMembers of the transforming growth factor (TGF)-β superfamily play a key role in the regulation of the malignant phenotype of glioblastoma by promoting invasiveness, angiogenesis, immunosuppression, and maintaining stem cell-like properties. Betaglycan, a TGF-β coreceptor also known as TGF-β receptor III (TβRIII), interacts with members of the TGF-β superfamily and acts as membrane-associated or shed molecule. Shed, soluble TβRIII (sTβRIII) is produced upon ectodomain cleavage of the membrane-bound form. Elucidating the role of TβRIII may improve our understanding of TGF-β pathway activity in glioblastomaMethodsProtein levels of TβRIII were determined by immunohistochemical analyses and ex vivo single-cell gene expression profiling of glioblastoma tissue respectively. In vitro, TβRIII levels were assessed investigating long-term glioma cell lines (LTCs), cultured human brain-derived microvascular endothelial cells (hCMECs), glioblastoma-derived microvascular endothelial cells, and glioma-initiating cell lines (GICs). The impact of TβRIII on TGF-β signaling was investigated, and results were validated in a xenograft mouse glioma modelResultsImmunohistochemistry and ex vivo single-cell gene expression profiling of glioblastoma tissue showed that TβRIII was expressed in the tumor tissue, predominantly in the vascular compartment. We confirmed this pattern of TβRIII expression in vitro. Specifically, we detected sTβRIII in glioblastoma-derived microvascular endothelial cells. STβRIII facilitated TGF-β-induced Smad2 phosphorylation in vitro and overexpression of sTβRIII in a xenograft mouse glioma model led to increased levels of Smad2 phosphorylation, increased tumor volume, and decreased survivalConclusionsThese data shed light on the potential tumor-promoting role of extracellular shed TβRIII which may be released by glioblastoma endothelium with high sTβRIII levels.

BIMG-03. MOLECULAR IMAGING OF GLUCOSE METABOLISM FOR INTRAOPERATIVE FLUORESCENCE GUIDANCE DURING GLIOMA SURGERY

PURPOSEThis study evaluated the utility of using molecular imaging of fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) as a discriminatory marker for intraoperative tumor border identification in a mouse glioma model.PROCEDURES2-NBDG and were assessed in GL261 and U251 orthotopic tumor bearing mice. Intraoperative fluorescence of 2-NBDG administered topical and intravenous in normal and tumor regions was assessed with operating microscope, handheld confocal laser scanning endomicroscope (CLE) and benchtop confocal laser scanning microscope (LSM). Additionally, 2-NBDG fluorescence in tumors was compared to 5-aminolevulinic acid-induced protoporphyrin IX fluorescence.RESULTSIntravenously administered 2-NBDG was detectable in brain tumor and absent in contralateral normal brain parenchyma on wide field operating microscopy imaging. Intraoperative and benchtop CLE showed preferential 2-NBDG accumulation in the cytoplasm of glioma cells (tumor-background ratio of 2.76±0.43). Topically administered 2-NBDG did not create a sufficient tumor-background contrast for white field operating microscopy imaging, or under benchtop LSM (tumor-background ratio 1.42 ± 0.72). However, topical 2-NBDG did create sufficient contrast to evaluate cellular tissue architecture and differentiate tumor cells from normal brain parenchyma. PpIX imaging resulted in a more specific delineation of gross tumor margins than IV or topical 2-NBDG, and a significantly higher tumor-normal brain fluorescence intensity ratio.CONCLUSIONAfter intravenous administration, 2-NBDG selectively accumulated in the experimental brain tumors and provided bright contrast under wide field fluorescence imaging with a clinical grade operating microscope. Topical 2-NBDG was able to create a sufficient contrast to differentiate tumor from normal brain cells based on visualization of cellular architecture with CLE. 5-ALA demonstrated superior specificity in outlining tumor margins and significantly higher tumor-background contrast. Given its non-toxicity, using 2-NBDG as a topical molecular marker for noninvasive in vivo intraoperative microscopy is encouraging, and warrants further clinical evaluation.

DDRE-16. CYSTEINE IS AN ESSENTIAL AMINO ACID IN GLIOMAS

BACKGROUNDCysteine is a non-essential amino acid, since it can be synthetized from methionine through the transsulfuration pathway; moreover, cysteine is also uptake from the diet as cystine. We have investigated the metabolism of cysteine in glioma cell lines, and how cysteine/cystine-deprivation alters their antioxidant response in addition to the effect of this nutrient restriction to viability and proliferation in vitro and in vivo.METHODSCysteine metabolism was investigated through LCMS-based 13C-tracing experiments involving different probes such as 13C-methyl-Methionine, 13C-C3-Cysteine, 13C-C3,3’-Cystine, 13C-C3-Serine and 13C-U-Glutamine and the expression levels of key enzymes in the transsulfuration pathway were also explored. Finally, a mouse model of IDH1 mutant glioma was subjected to a cysteine/cystine-free diet and tumor metabolism was analyzed by LCMS.RESULTSWe demonstrated that exogenous cysteine/cystine are crucial for glutathione synthesis, and impact growth and viability. We also found that methionine cycle is disconnected from the transsulfuration pathway based on 13C-tracing data and protein expression levels of cystathionine synthase and cystathioninase. Accordingly, cysteine-related metabolites such as GSH, involved in REDOX hemostasis, are downregulated, revealing a hypersensitive phenotype to ROS. Animal models upon a cysteine/cystine-free diet experienced an increase in survival and elevated levels of oxidative stress in tumor tissue.CONCLUSIONThis results presented herein reveal an alternative therapeutic approach combining cysteine/cysteine-deprivation diets and treatments involving ROS production by limiting the ability of glioma cells to quench oxidative stress through dietary interventions.

Abstract S05-02: Plant viral nanoparticle-based adjuvants for cancer immunotherapy and COVID-19 vaccines

Abstract Plant virus nanoparticles are recognized as a platform technology for potential applications in nanomedicine. While non-infectious toward mammals, many plant virus nanoparticles are recognized by the immune system and act as potent adjuvants, both in the classical setting of vaccination and cancer immunotherapy. In the context of cancer immunotherapy, we have demonstrated potent efficacy of plant virus, particularly cowpea mosaic virus (CPMV) when applied intratumorally; CPMV signals through and activates multiple toll-like receptors, which primes a potent innate immune activation leading to reprogramming of the tumor microenvironment from an immunosuppressed to immune-activated state. Data in mouse models of melanoma, colon cancer, ovarian cancer, glioma, and breast cancer demonstrate potent efficacy through priming of the innate immune system leading to adaptive and systemic anti-tumor immunity and most importantly immune memory. Efficacy was also replicated in canine patients with melanoma. Given the promise of the plant virus nanotechnology as an adjuvant for cancer immunotherapy, we also have begun to utilize the technology as a vaccination platform for the generation of COVID-19 vaccine candidates. We formulated multivalent COVID-19 vaccine candidates using plant virus nanoparticles displaying SARS-CoV-2 S protein peptide B cell epitopes. The plant virus nanoparticle confers efficient lymphatic trafficking and targeting of antigen-presenting cells; the adjuvant properties of the nanotechnology lead to robust antibody levels against the target, S protein. While other vaccine candidates have advanced through clinical testing, our approach has unique advantages: The high thermal and pH stability of the plant virus nanotechnology alleviates cold chain requirements; further, the stability of the platform technology enables its integration into vaccine delivery devices such as microneedle patches that can be shipped globally and be self-administered. In this presentation, we will discuss our data on the cancer immunotherapy and highlight the potential to pivot the technology for infectious disease vaccines with unique attributes. This work was funded in part through grants from the National Cancer Institute (U01CA218292) and National Science Foundation (RAPID CMMI-2027668). Citation Format: Nicole F. Steinmetz, Jonathan K. Pokorski, Steven N. Fiering, P. Jack Hoopes, Sourabh Shukla, Oscar A. Ortega-Rivera, Steven King, Cyril J. Empig. Plant viral nanoparticle-based adjuvants for cancer immunotherapy and COVID-19 vaccines [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr S05-02.