Malignant Glioma Model Research Articles

The Immunomodulatory Effects of Fluorescein-Mediated Sonodynamic Treatment Lead to Systemic and Intratumoral Depletion of Myeloid-Derived Suppressor Cells in a Preclinical Malignant Glioma Model.

Fluorescein-mediated sonodynamic therapy (FL-SDT) is an extremely promising approach for glioma treatment, resulting from the combination of low-intensity focused ultrasound (FUS) with a sonosensitizer. In the present study, we evaluated the efficacy and immunomodulation of SDT with fluorescein as the sonosensitizer in immunocompetent GL261 glioma mice for the first time. In vitro studies demonstrated that the exposure of GL261 cells to FL-SDT induced immunogenic cell death and relevant upregulation of MHC class I, CD80 and CD86 expression. In vivo studies were then performed to treat GL261 glioma-bearing mice with FL-SDT, fluorescein alone, or FUS alone. Perturbation of the glioma-associated macrophage subset within the immune microenvironment was induced by all the treatments. Notably, a relevant depletion of myeloid-derived suppressor cells (MDSCs) and concomitant robust infiltration of CD8+ T cells were observed in the SDT-FL-treated mice, resulting in a significant radiological delay in glioma progression and a consequent improvement in survival. Tumor control and improved survival were also observed in mice treated with FL alone (median survival 41.5 days, p > 0.0001 compared to untreated mice), reflecting considerable modulation of the immune microenvironment. Interestingly, a high circulating lymphocyte-to-monocyte ratio and a very low proportion of MDSCs were predictive of better survival in FL- and FL-SDT-treated mice than in untreated and FUS-treated mice, in which elevated monocyte and MDSC frequencies correlated with worse survival. The immunostimulatory potential of FL-SDT treatment and the profound modulation of most immunosuppressive components within the microenvironment encouraged the exploration of the combination of FL-SDT with immunotherapeutic strategies.

IMMU-52. LOW-INTENSITY PULSED ULTRASOUND CONDUCTS ANTIGEN-INDEPENDENT T CELL HOMING INTO THE BRAIN PARENCHYMA WITH THE INTACT BLOOD-BRAIN BARRIER

Abstract The blood-brain barrier (BBB) protects the brain parenchyma from excessive inflammation but also leads to poor immune cell homing, hampering the success of immunotherapies, such as ones for low-grade glioma, where the BBB is intact. Low-intensity pulsed ultrasound (LIPU) combined with the application of microbubbles allows the non-invasive, transient opening of the BBB and recent studies have observed immune cell infiltration into malignant gliomas following LIPU. While these data warrant mechanistic investigations on the effects of LIPU in preclinical settings, most mouse models of malignant glioma already display BBB disruption. Therefore, to study whether LIPU affects antigen-specific immune cell trafficking through the BBB, we created a novel immunocompetent mouse model of non-tumor bearing mice that display an intact BBB and express a minigene cassette encoding for four immunogenic peptides in glia cells under the glial fibrillary acidic protein (GFAP) promotor. These peptides are either presented by major histocompatibility complex (MHC) class I: human melanoma antigen glycoprotein (gp) 100 and lymphocytic choriomeningitis virus (LCMV)gp31) or class II: LCMV gp66 and tyrosinase-related protein 1. Minigene expression in astrocytes induced antigen-specific T cell proliferation and Interferon-γ release in vitro. Intravenous adoptive T cell transfer of naïve or respectively CD3/CD28-coactivated antigen-specific CD 8 T cells targeting gp100 peptide followed by immediate LIPU treatment induced T cell homing into the CNS parenchyma, and astrocyte-T cell interaction was confirmed by ex-vivo two-photon imaging. Furthermore, some animals that underwent immediate LIPU treatment after transfer of preactivated antigen-specific T cells developed neurological deficits such as ataxia, while no deficits were observed in those animals that underwent LIPU but did not receive T cells or received activated T cells but did not undergo LIFU treatment. In conclusion, LIPU might be a novel effective tool to support antigen-independent immune cell homing with the intact BBB.

ET-5 POTENT BYSTANDER EFFECT IN SUICIDE GENE THERAPY USING TK-EXPRESSING STEM CELLS FROM HUMAN EXFOLIATED DECIDUOUS TEETH

Abstract Introduction We investigated HSVTK/GCV suicide gene therapy for malignant glioma, and demonstrated the migration ability and antitumor effect of various tissue-derived pluripotent stem cells. In recent years, stem cells from human exfoliated deciduous teeth (SHED), which have excellent ethical and self-renewal ability, have attracted attention, especially in regenerative medicine. In this study, using SHEDTK transfected with TK, we examined the migration ability and antitumor effect against malignant glioma and metastasis models. Methods In vitro assay: Using Matrigel chamber, the migration ability of SHEDTK to conditioned medium (CM) of glioma cells, lung carcinoma cells, and various tumor growth factors (TGF) was examined. The antitumor effect was examined for cell viability by co-culturing SHEDTK and each tumor cell under the addition of GCV. in vivo assay: Using glioma model mice, SHEDTK migrating around the tumor was confirmed in brain sections. In addition, we co-implanted SHEDTK and administered GCV to glioma and metastasis model as therapeutic models, evaluated tumor reduction effect by bioluminescence, and confirmed survival curves. Results In vitro assay: SHEDTK significantly migrated to CM and TGF compared to control. The antitumor effect was observed even when the ratio of each tumor cell to SHEDTK was 128-256 times. in vivo assay: In glioma model mice, SHEDTK migrated around contralateral tumor. In the treatment experiment, the control group died of tumors, whereas the tumor in the treatment group disappeared within the period of GCV administration, and they survived without neurological deficits until 100 days later, and the overall survival time was improved with a statistically significant difference. Conclusions We confirmed the migration ability and antitumor effect of SHEDTK on malignant glioma and metastasis models. Suicide gene therapy using SHEDTK was suggested as novel gene therapy. In the future, we plan to conduct translational research such as preclinical studies and physician -initiated clinical trials.

ApoE-mediated systemic nanodelivery of granzyme B and CpG for enhanced glioma immunotherapy.

The response of malignant glioma to immunotherapy remains gloomy due to its discrete immunological environment and poor brain penetration of immunotherapeutic agents. Here, we disclose that ApoE peptide-mediated systemic nanodelivery of granzyme B (GrB) and CpG ODN co-stimulates enhanced immunotherapy of murine malignant LCPN glioma model. ApoE peptide-functionalized polymersomes encapsulating GrB (ApoE-PS-GrB) could effectively penetrate the blood-brain barrier-mimicking endothelial cell monolayer in vitro and further be taken up by LCPN cells, inducing strong immunogenic cell death (ICD). The co-administration of ApoE-PS-GrB and ApoE-PS-CpG in orthotopic LCPN glioma-bearing mice co-stimulated cytokine production, maturation of dendritic cells (DCs), infiltration of cytotoxic T lymphocytes (CTLs) while reduction of regulatory T lymphocytes (Treg) and M2 phenotype macrophages in the tumor microenvironment, leading to greatly delayed tumor progression and significantly prolonged survival time compared with all controls. The ApoE-mediated systemic nanodelivery of GrB and CpG ODN opens a new pathway for potent immunotherapy of malignant glioma.

Phase 1/2 study of elraglusib (9-ING-41), a small molecule selective glycogen synthase kinase-3 beta (GSK-3β) inhibitor, alone or with irinotecan, temozolomide/irinotecan or cyclophosphamide/topotecan in pediatric patients with refractory malignancies: Interim results.

e22015 Background: GSK-3β overexpression is associated with aggressive malignancies, treatment resistance and poor prognosis. The GSK-3β inhibitor Elraglusib induces apoptosis via NFΚB and p53 pathways and has potent anti-fibrotic and immunomodulatory activity. Adult studies of elraglusib demonstrate clinical activity in pancreatic cancer, melanoma, lymphoma and sarcoma as a single agent or in combination with cytotoxic chemotherapy. Elraglusib is active in in vivo models of neuroblastoma (NBL) and malignant glioma. This first-in-pediatrics study (NCT04239092) is evaluating the safety, pharmacokinetics (PK), and efficacy of elraglusib monotherapy and in combination with chemotherapy in patients with refractory malignancies. Methods: Elraglusib is given intravenously (IV) twice-weekly at 3 dose levels (DL) (9.3, 12.4 and 15 mg/kg) as a single agent or in combination with irinotecan, cyclophosphamide/topotecan or temozolomide/irinotecan in 21-day cycles. A cohort of pts with refractory NBL will be treated at the recommended phase 2 dose (RP2D) of elraglusib with temozolomide/irinotecan. Results: As of January 2022, 23 pts (n = 7 female, median age 14.2 years) have received at least one dose of elraglusib. Tumor types: 5 NBL, 3 diffuse midline glioma (DMG), 3 osteosarcoma (OS), 3 ependymoma (EP), 2 alveolar rhabdomyosarcoma (aRMS), 1 angiosarcoma (AS), 1 Ewing sarcoma (ES), 1 glioblastoma (GBM), 1 hepatoblastoma (HB), 1 embryonal CNS tumor NOS, 1 NUT midline carcinoma, 1 pineoblastoma (PB). Median time from diagnosis is 26 months (range: 6.7 – 156.3) and median number of lines of prior systemic therapy is 2 (range 0-14). Two DLs of single agent (6 pts) have been completed (9.3 and 12.4 mg/kg) without elraglusib-attributable severe adverse events (SAEs). Of the 15 patients on the combination arm with irinotecan or cyclophosphamide/topotecan, a single adverse event (Grade 4 hypotension/infusion reaction) was reported. Grade 1/2 elraglusib attributable-AEs include: transient visual change (n = 10), nausea (n = 7), vomiting (n = 6), fatigue (n = 2), hypotension (n = 2) and infusion reaction (n = 1). One pt with recurrent ES had a radiographic and pathologic CR after 3 cycles of elraglusib/cyclophosphamide/topotecan. 6 pts (26.1%) had SD (2 NBL, 1 aRMS, 1 EP, 1 OS, 1 GBM). 8 pts (35%) remained on study treatment ≥ 3 months (2 NBL, 2 EP, 1 OS, 1 aRMS, 1 ES, 1 PB). Median treatment duration was 40 days (range 1 - 126). 4 pts remain on therapy. Conclusions: Elraglusib is well tolerated as a single agent and with several chemotherapy regimens in this heavily pretreated pediatric population with refractory cancers. It has encouraging antitumor activity, with 1 CR in a patient with recurrent ES. Enrollment is ongoing; a RPD2 has not been reached. Clinical trial information: NCT04239092.

MEOX2 homeobox gene promotes growth of malignant gliomas.

Glioblastoma (GBM) is an aggressive tumor that frequently exhibits gain of chromosome 7, loss of chromosome 10, and aberrantly activated receptor tyrosine kinase signaling pathways. Previously, we identified Mesenchyme Homeobox 2 (MEOX2), a gene located on chromosome 7, as an upregulated transcription factor in GBM. Overexpressed transcription factors can be involved in driving GBM. Here, we aimed to address the role of MEOX2 in GBM. Patient-derived GBM tumorspheres were used to constitutively knockdown or overexpress MEOX2 and subjected to in vitro assays including western blot to assess ERK phosphorylation. Cerebral organoid models were used to investigate the role of MEOX2 in growth initiation. Intracranial mouse implantation models were used to assess the tumorigenic potential of MEOX2. RNA-sequencing, ACT-seq, and CUT&Tag were used to identify MEOX2 target genes. MEOX2 enhanced ERK signaling through a feed-forward mechanism. We identified Ser155 as a putative ERK-dependent phosphorylation site upstream of the homeobox-domain of MEOX2. S155A substitution had a major effect on MEOX2 protein levels and altered its subnuclear localization. MEOX2 overexpression cooperated with p53 and PTEN loss in cerebral organoid models of human malignant gliomas to induce cell proliferation. Using high-throughput genomics, we identified putative transcriptional target genes of MEOX2 in patient-derived GBM tumorsphere models and a fresh frozen GBM tumor. We identified MEOX2 as an oncogenic transcription regulator in GBM. MEOX2 increases proliferation in cerebral organoid models of GBM and feeds into ERK signaling that represents a core signaling pathway in GBM.

Lactate-Loaded Nanoparticles Induce Glioma Cytotoxicity and Increase the Survival of Rats Bearing Malignant Glioma Brain Tumor.

A glioblastoma is an aggressive form of a malignant glial-derived tumor with a poor prognosis despite multimodal therapy approaches. Lactate has a preponderant role in the tumor microenvironment, playing an immunoregulatory role as well as being a carbon source for tumor growth. Lactate homeostasis depends on the proper functioning of intracellular lactate regulation systems, such as transporters and enzymes involved in its synthesis and degradation, with evidence that an intracellular lactate overload generates metabolic stress on tumor cells and tumor cell death. We propose that the delivery of a lactate overload carried in nanoparticles, allowing the intracellular release of lactate, would compromise the survival of tumor cells. We synthesized and characterized silica and titania nanoparticles loaded with lactate to evaluate the cellular uptake, metabolic activity, pH modification, and cytotoxicity on C6 cells under normoxia and chemical hypoxia, and, finally, determined the survival of an orthotopic malignant glioma model after in situ administration. A dose-dependent reduction in metabolic activity of treated cells under normoxia was found, but not under hypoxia, independent of glucose concentration. Lactated-loaded silica nanoparticles were highly cytotoxic (58.1% of dead cells) and generated significant supernatant acidification. In vivo, lactate-loaded silica nanoparticles significantly increased the median survival time of malignant glioma-bearing rats (p = 0.005) when administered in situ. These findings indicate that lactate-loaded silica nanoparticles are cytotoxic on glioma cells in vitro and in vivo.

Sequential fate-switches in stem-like cells drive the tumorigenic trajectory from human neural stem cells to malignant glioma

Glioblastoma (GBM) is an incurable and highly heterogeneous brain tumor, originating from human neural stem/progenitor cells (hNSCs/hNPCs) years ahead of diagnosis. Despite extensive efforts to characterize hNSCs and end-stage GBM at bulk and single-cell levels, the de novo gliomagenic path from hNSCs is largely unknown due to technical difficulties in early-stage sampling and preclinical modeling. Here, we established two highly penetrant hNSC-derived malignant glioma models, which resemble the histopathology and transcriptional heterogeneity of human GBM. Integrating time-series analyses of whole-exome sequencing, bulk and single-cell RNA-seq, we reconstructed gliomagenic trajectories, and identified a persistent NSC-like population at all stages of tumorigenesis. Through trajectory analyses and lineage tracing, we showed that tumor progression is primarily driven by multi-step transcriptional reprogramming and fate-switches in the NSC-like cells, which sequentially generate malignant heterogeneity and induce tumor phenotype transitions. We further uncovered stage-specific oncogenic cascades, and among the candidate genes we functionally validated C1QL1 as a new glioma-promoting factor. Importantly, the neurogenic-to-gliogenic switch in NSC-like cells marks an early stage characterized by a burst of oncogenic alterations, during which transient AP-1 inhibition is sufficient to inhibit gliomagenesis. Together, our results reveal previously undercharacterized molecular dynamics and fate choices driving de novo gliomagenesis from hNSCs, and provide a blueprint for potential early-stage treatment/diagnosis for GBM.

EXTH-41. THE microRNA miR-138 MAY BE RESPONSIBLE FOR SOME OF THE EFFECTS OF METABOLIC KETOSIS ON MALIGNANT GLIOMA

Abstract We and others have demonstrated that altering metabolism through the use of a ketogenic diet (KD) has numerous effects in mouse models of malignant glioma including slowed growth, reduced hypoxia and angiogenesis, enhanced survival and potentiation of the effects of radiation and chemotherapy. These effects are recapitulated in vitro when the ketone β-hydroxybutyrate (BHB) is added to mouse and human malignant glioma cells grown under high glucose conditions. BHB has been demonstrated to be an epigenetic modifier, providing a potential mechanism for the pluripotent effects of ketones even in the presence of high glucose. Therefore, we have analyzed alterations in microRNA (miRNA) expression in tumors and cells treated with a KD or BHB, respectively. MicroRNAs are short, single-stranded, noncoding RNAs that posttranscriptionally control gene expression. Each miRNA can affect the expression of many genes and they are implicated in all hallmarks of cancer. We found a number of miRNAs deregulated in tumor tissue from mice fed a KD, particularly mmu-miR-138 which was increased over 30-fold relative to tumors from mice fed a standard diet. MiR-138 is involved in a variety of anti-tumor effects that correlate with our data from KD treated gliomas. We have shown that ketones radiosensitize glioma cells, potentially through a reduction in DNA damage repair. MiR-138 has been shown to reduce the expression of H2AX which plays a key role in sensing and repairing DNA damage. Hypoxia inducible factor-1α (HIF-1α) is also a target of miR-138, and we have demonstrated a reduction in hypoxia, angiogenesis and the expression of HIF-1α in tumors from mice fed a KD. Furthermore, when we genetically engineered tumor cells to express miR-138 and injected them into mice, only necrotic tumor foci were found. Thus, we suggest that miR-138 is an important regulatory mechanism for the anti-tumor actions of metabolic ketosis in glioma.

Downregulation of the CCL2/CCR2 and CXCL10/CXCR3 axes contributes to antitumor effects in a mouse model of malignant glioma

Glioblastoma multiforme involves glioma stem cells (GSCs) that are resistant to various therapeutic approaches. Here, we studied the importance of paracrine signaling in the glioma microenvironment by focusing on the celecoxib-mediated role of chemokines C–C motif ligand 2 (CCL2), C-X-C ligand 10 (CXCL10), and their receptors, CCR2 and CXCR3, in GSCs and a GSC-bearing malignant glioma model. C57BL/6 mice were injected with orthotopic GSCs intracranially and divided into groups administered either 10 or 30 mg/kg celecoxib, or saline to examine the antitumor effects associated with chemokine expression. In GSCs, we analyzed cell viability and expression of chemokines and their receptors in the presence/absence of celecoxib. In the malignant glioma model, celecoxib exhibited antitumor effects in a dose dependent manner and decreased protein and mRNA levels of Ccl2 and CxcL10 and Cxcr3 but not of Ccr2. CCL2 and CXCL10 co-localized with Nestin+ stem cells, CD16+ or CD163+ macrophages and Iba-1+ microglia. In GSCs, celecoxib inhibited Ccl2 and Cxcr3 expression in a nuclear factor-kappa B-dependent manner but not Ccr2 and CxcL10. Moreover, Ccl2 silencing resulted in decreased GSC viability. These results suggest that celecoxib-mediated regulation of the CCL2/CCR2 and CXCL10/ CXCR3 axes may partially contribute to glioma-specific antitumor effects.

A Chimeric Signal Peptide-Galectin-3 Conjugate Induces Glycosylation-Dependent Cancer Cell-Specific Apoptosis.

Exploitation of altered glycosylation in cancer is a major goal for the design of new cancer therapy. Here, we designed a novel secreted chimeric signal peptide-Galectin-3 conjugate (sGal-3) and investigated its ability to induce cancer-specific cell death by targeting aberrantly N-glycosylated cell surface receptors on cancer cells. sGal-3 was genetically engineered from Gal-3 by extending its N-terminus with a noncleavable signal peptide from tissue plasminogen activator. sGal-3 killing ability was tested on normal and tumor cells in vitro and its antitumor activity was evaluated in subcutaneous lung cancer and orthotopic malignant glioma models. The mechanism of killing was investigated through assays detecting sGal-3 interaction with specific glycans on the surface of tumor cells and the elicited downstream proapoptotic signaling. We found sGal-3 preferentially binds to β1 integrin on the surface of tumor cells due to aberrant N-glycosylation resulting from cancer-associated upregulation of several glycosyltransferases. This interaction induces potent cancer-specific death by triggering an oncoglycan-β1/calpain/caspase-9 proapoptotic signaling cascade. sGal-3 could reduce the growth of subcutaneous lung cancers and malignant gliomas in brain, leading to increased animal survival. We demonstrate that sGal-3 kills aberrantly glycosylated tumor cells and antagonizes tumor growth through a novel integrin β1-dependent cell-extrinsic apoptotic pathway. These findings provide proof-of-principle that aberrant N-oncoglycans represent valid cancer targets and support further translation of the chimeric sGal-3 peptide conjugate for cancer therapy.

Downregulation of PD-L1 via FKBP5 by celecoxib augments antitumor effects of PD-1 blockade in a malignant glioma model.

BackgroundAntitumor therapies targeting programmed cell death-1 (PD-1) or its ligand-1 (PD-L1) are used in various cancers. However, in glioblastoma (GBM), the expression of PD-L1 varies between patients, and the relationship between this variation and the efficacy of anti-PD-1 antibody therapy remains unclear. High expression levels of PD-L1 affect the proliferation and invasiveness of GBM cells. As COX-2 modulates PD-L1 expression in cancer cells, we tested the hypothesis that the COX-2 inhibitor celecoxib potentiates anti-PD-1 antibody treatment via the downregulation of PD-L1.MethodsSix-week-old male C57BL/6 mice injected with murine glioma stem cells (GSCs) were randomly divided into four groups treated with vehicle, celecoxib, anti-PD-1 antibody, or celecoxib plus anti-PD-1 antibody and the antitumor effects of these treatments were assessed. To verify the mechanisms underlying these effects, murine GSCs and human GBM cells were studied in vitro.ResultsCompared with that with each single treatment, the combination of celecoxib and anti-PD-1 antibody treatment significantly decreased tumor volume and prolonged survival. The high expression of PD-L1 was decreased by celecoxib in the glioma model injected with murine GSCs, cultured murine GSCs, and cultured human GBM cells. This reduction was associated with post-transcriptional regulation of the co-chaperone FK506-binding protein 5 (FKBP5).ConclusionsCombination therapy with anti-PD-1 antibody plus celecoxib might be a promising therapeutic strategy to target PD-L1 in glioblastoma. The downregulation of highly-expressed PD-L1 via FKBP5, induced by celecoxib, could play a role in its antitumor effects.

GCN2 is essential for CD8+ T cell survival and function in murine models of malignant glioma.

Amino acid deprivation is a strategy that malignancies utilize to blunt anti-tumor T-cell immune responses. It has been proposed that amino acid insufficiency in T-cells is detected by GCN2 kinase, which through phosphorylation of EIF2α, shuts down global protein synthesis leading to T-cell arrest. The role of this amino acid stress sensor in the context of malignant brain tumors has not yet been studied, and may elucidate important insights into the mechanisms of T-cell survival in this harsh environment. Using animal models of glioblastoma and animals with deficiency in GCN2, we explored the importance of this pathway in T-cell function within brain tumors. Our results show that GCN2 deficiency limited CD8+ T-cell activation and expression of cytotoxic markers in two separate murine models of glioblastoma in vivo. Importantly, adoptive transfer of antigen-specific T-cells from GCN2 KO mice did not control tumor burden as well as wild-type CD8+ T-cells. Our in vitro and in vivo data demonstrated that reduction in amino acid availability caused GCN2 deficient CD8+ T-cells to become rapidly necrotic. Mechanistically, reduced CD8+ T-cell activation and necrosis was due to a disruption in TCR signaling, as we observed reductions in PKCθ and phoshpo-PKCθ on CD8+ T-cells from GCN2 KO mice in the absence of tryptophan. Validating these observations, treatment of wild-type CD8+ T-cells with a downstream inhibitor of GCN2 activation also triggered necrosis of CD8+ T-cells in the absence of tryptophan. In conclusion, our data demonstrate the vital importance of intact GCN2 signaling on CD8+ T-cell function and survival in glioblastoma.

Abstract 4075: Overcoming glioma immunoediting and MHC class I loss during adoptive cellular therapy

Abstract Introduction: Adoptive T cell immunotherapy (ACT) leads to 30% long-term cures of intractable models of malignant glioma. While promising, glioma-bearing hosts succumb to disease for unclear reasons. Therefore, we studied glioma escape variants after immunoediting by ACT. Since the escaped tumors displayed marked gene loss making them distinct from original tumor, we anticipated that successful future strategies would need to be tailored to the new antigenic signature while using combinatorial approaches to overcome other mechanisms of escape. We therefore studied the hypothesis that a polyclonal population of ex vivo-expanded T cells specific for the total tumor RNA of glioma escape variants could successfully retarget glioma escape variants, despite immunoediting pressure. Methods: RNAseq revealed that tumor escape variants from KR158B (TOGA1.1/1.2) displayed a loss of ~80% of the genes that were present in KR158B and GL261 glioma. We regenerated polyclonal T cells against TOGA using total tumor RNA from TOGA tumors. We used restimulation co-cultures of T cells with tumor and IFN-γ ELISAs for T cell activation. In vivo therapeutic models required re-implantation of TOGA tumors intracranially after gross dissection and passaging in vitro. Flow cytometry determined marker expression in tumors. Results: TOGA1.1 or TOGA1.2-specific T cells recognized cognate TOGA1.1 or TOGA1.2 cells but not KR158B or the opposite glioma escape variant. When we utilized TOGA-specific T cells in vivo for TOGA-bearing mice, median survival was prolonged from 24 to 33 days (p=.0003). However, all animals succumbed to disease. We then evaluated additional mechanisms of escape. At endpoint, MHC class I on tumors was ~50% of its original expression after antigen-matched ACT (KR158B-bearing: 70% to 40% post-ACT; TOGA-bearing: 22% to 10%). Additionally, KR158B and TOGA-specific T cells express ~50% PD-1 both post-expansion ex vivo and in the tumor-infiltrating lymphocytes post-treatment while tumor-infiltrating NK cells express 30% PD-1. Given the role of NK cells in targeting MHC Ilo tumors, and the recent discoveries that PD-1 checkpoint blockade works on T cells and NK cells, we investigated the impact of PD-1 and antigen-matched ACT in KR158B or TOGA-bearing animals. This revealed that the combination generates 71% long-term cures in KR158B primary glioma while promoting an increase in activated NK and T cells. Treatment of TOGA-bearing animals is underway and will include the depletion of either CD8 T cells or NK cells to determine the relative contributions of each cell type. Conclusions: Glioma escape variants retain expression of immunogenic antigens. Generation of ACT with specificity for escape variants provides exquisite antigen-specific recognition of the immunoedited tumors. Combinatorial use of antigen-specific ACT with PD-1 blockade provides a flexible and specific platform for treatment of primary and escaped tumors. Citation Format: Tyler J. Wildes, Catherine T. Flores, Kyle Dyson, Connor Francis, Adam Grippin, Bayli Divita Dean, Duane A. Mitchell. Overcoming glioma immunoediting and MHC class I loss during adoptive cellular therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4075.

Differential Effects of Ang-2/VEGF-A Inhibiting Antibodies in Combination with Radio- or Chemotherapy in Glioma.

Antiangiogenic strategies have not shown striking antitumor activities in the majority of glioma patients so far. It is unclear which antiangiogenic combination regimen with standard therapy is most effective. Therefore, we compared anti-VEGF-A, anti-Ang2, and bispecific anti-Ang-2/VEGF-A antibody treatments, alone and in combination with radio- or temozolomide (TMZ) chemotherapy, in a malignant glioma model using multiparameter two-photon in vivo microscopy in mice. We demonstrate that anti-Ang-2/VEGF-A lead to the strongest vascular changes, including vascular normalization, both as monotherapy and when combined with chemotherapy. The latter was accompanied by the most effective chemotherapy-induced death of cancer cells and diminished tumor growth. This was most probably due to a better tumor distribution of the drug, decreased tumor cell motility, and decreased formation of resistance-associated tumor microtubes. Remarkably, all these parameters where reverted when radiotherapy was chosen as combination partner for anti-Ang-2/VEGF-A. In contrast, the best combination partner for radiotherapy was anti-VEGF-A. In conclusion, while TMZ chemotherapy benefits most from combination with anti-Ang-2/VEGF-A, radiotherapy does from anti-VEGF-A. The findings imply that uninformed combination regimens of antiangiogenic and cytotoxic therapies should be avoided.

Abstract 2739: The ketone body β-hydroxybutyrate alters expression of PD-L1 on malignant glioma cells but does not directly affect T cells in vitro

Abstract Patients with newly diagnosed glioblastoma multiforme (GBM), the most aggressive grade of glioma, have an average life expectancy of ~15 months, and less than 6% survive five years, despite the current standard of care, which consists of aggressive surgery followed by radiation and chemotherapy. Advances in the survival of GBM patients require the design of new therapeutic approaches. One novel strategy being explored in our laboratory and others involves using ketogenic therapy (KT). This approach involves shifting the body's metabolic physiology by increasing the production of ketone bodies such as β-hydroxybutyrate (BHB) and decreasing blood glucose. We have demonstrated that nutritionally-induced KT enhances survival and potentiates standard therapy in a mouse model of malignant glioma. Our previous study shows that KT works in part as an immune adjuvant, boosting tumor-reactive immune responses in the microenvironment by alleviating immune suppression in vivo. In particular we found that KT reduced expression of programmed cell death-1 (PD-1) on tumor-infiltrating T cells and its ligand PD-L1 on tumor cells. The PD-1/PD-L1 axis represents a crucial mechanism of glioma-mediated immunosuppression and is a major focus of immunotherapy in GBM and other cancers. The current study aims to determine the role of BHB in the anti-tumor and immune-enhancing effects of KT. We show that in vitro treatment with BHB induces a variety of molecular changes in glioma cells including reduced protein expression of PD-L1. Given that our in vivo data suggests that KT enhances T cell function against tumors we also sought to determine the impact of BHB on T cell function in vitro. Activated CD8+ and CD4+ T cells were cultured with varying doses of BHB prior to flow cytometric analysis. There were no significant changes in expression of immune checkpoint receptors (PD-1, CTLA-4 and LAG-3), production of anti-tumor promoting cytokines (IFN-y, TNF, IL-2) or viability in either T cell population following treatment with any dose of BHB. This suggests that BHB and KT may indirectly impact T cell function by influencing the way tumor cells manipulate the microenvironment. Taken together this data helps us better understand the anti-tumor mechanisms underlying KT and warrants further exploration of combinational treatment strategies using this approach and immunotherapies for GBM. Citation Format: Eric C. Woolf, Kavita R. Manhas, Deborah R. Healey, Joseph N. Blattman, Adrienne C. Scheck. The ketone body β-hydroxybutyrate alters expression of PD-L1 on malignant glioma cells but does not directly affect T cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2739.

PO-227 Sensitising glioma cells to fatty acid oxidation inhibitor by modulation of SLC22A5 transporter activity

IntroductionGliomas are the most common primary malignant brain tumour in adults, but current treatment for glioblastoma multiforme (GBM) is insufficient. It was shown that primary-cultured human GBM cells depend on fatty acid oxidation (FAO) for proliferation, and that FAO inhibition in vivo by etomoxir prolongs survival in the mouse model of malignant glioma. Etomoxir, an inhibitor of FAO rate-limiting enzyme – carnitine palmitoylotransferase 1 (CPT1), was tested in clinical trials, which were terminated due to high toxicity. Carnitine is a CPT1 substrate, and SLC22A5 is the only high-affinity carnitine transporter in the plasma membrane expressed in the central nervous system. Moreover, HEK293 cells overexpressing SLC22A5 show increased transport of several chemotherapeutics, the process that may inhibit carnitine transport into the cell. This study is aimed at establishing the role of SLC22A5 in glioma cells and at assessing its potential for sensitising them to lower, less toxic doses of etomoxir.Material and methodsData from REMBRANDT and a Western Blot analysis of non-transformed astrocytes and several glioma cell lines were used to assess SLC22A5 expression both on the mRNA level and the protein level, respectively. Transport of radiolabeled carnitine was measured to verify whether anti-cancer drugs transported by SLC22A5 are able to inhibit carnitine transport in U87-MG, LN229 and T89G glioma cells. MTT assay and BrdU staining were used to test the viability and proliferation of glioma cells after administering SLC22A5-transported drugs alone and combined with etomoxir.Results and discussionsStatistical analysis of data from REMBRANDT showed significantly higher SLC22A5 expression in glioma patient-derived tissues when compared to normal tissues, an observation confirmed by a Western Blot analysis of astrocytes and several glioma cell lines. Out of several tested drugs, vinorelbine and vincristine are most efficient in carnitine transport inhibition in all of the tested lines. The viability and proliferation are significantly reduced when the foregoing are used in combination with etomoxir.ConclusionProper carnitine delivery by SLC22A5 transporter is important for glioblastoma metabolism and SLC22A5-transported chemotherapeutics may induce cell death both by their regular mechanism of action and by inhibition of carnitine delivery to glioma cells, thus sensitising them to lower, less toxic doses of CPT1 inhibitors.This project is financed by grant 2016/23/N/NZ3/02430 from National Science Centre in Poland.

Combination Therapy of Intravenously Injected Microglia and Radiation Therapy Prolongs Survival in a Rat Model of Spontaneous Malignant Glioma

The aim of this study was to investigate the efficacy of combination therapy with intravenously injected microglia (MI) and radiation therapy (RT) for malignant glioma in rats. Transgenic rats expressing v-erbB and spontaneously developing malignant glioma were used. The rats were divided into 4 groups: control (n=19), RT alone (n=10), MI alone (n=9), and combination MI and RT (MI+RT) (n=10). Cranial x-ray irradiation (8Gy per fraction; once per week) was performed at 50 and 51weeks of age. Cultured rat microglial cells (5×106cells/rat) were intravenously injected via the tail vein within 30minutes after RT. No evidence of side effects, including thrombosis or graft-versus-host disease, was noted. Rats treated with RT alone, MI alone, MI+RT, and control survived 60.9, 56.3, 66.0, and 56.1weeks, respectively. The survival period of MI+RT was significantly longer than that of control (P=.014), MI alone (P=.027), and RT alone (P=.049). Immunohistochemical analysis showed a significantly higher number of tumor-infiltrated MI in the RT alone (P=.041) and MI+RT groups (P=.014) compared with the control. Significantly more CD8-positive lymphocytes were observed in the MI+RT group (P=.049) compared with the control. A positive correlation was found between the number of MI and CD8-positive lymphocytes (R2=0.556). A positive correlation was also found between CD8-positive lymphocytes and survival periods (R2=0.460). MI+RT increased infiltrated MI and CD8-positive T cells and prolonged survival in transgenic rats that spontaneously developed malignant glioma. Combined immunocellular therapy and RT may provide a novel treatment strategy for malignant glioma.

In vivo tracking of the tropism of mesenchymal stem cells to malignant gliomas using reporter gene-based MR imaging.

Mesenchymal stem cells (MSCs) have emerged as a promising cellular vehicle for gene therapy of malignant gliomas due to their property of tumor tropism. However, MSCs may show bidirectional and divergent effects on tumor growth. Therefore, a robust surveillance system with a capacity for noninvasive monitoring of the homing, distribution and fate of stem cells in vivo is highly desired for developing stem cell-based gene therapies for tumors. In this study, we used ferritin gene-based magnetic resonance imaging (MRI) to track the tumor tropism of MSCs in a rat orthotopic xenograft model of malignant glioma. MSCs were transduced with lentiviral vectors expressing ferritin heavy chain (FTH) and enhanced green fluorescent protein (eGFP). Intra-arterial, intravenous and intertumoral injections of these FTH transgenic MSCs (FTH-MSCs) were performed in rats bearing intracranial orthotopic C6 gliomas. The FTH-MSCs were detected as hypointense signals on T2- and T2*-weighted images on a 3.0 T clinical MRI. After intra-arterial injection, 17% of FTH-MSCs migrated toward the tumor and gradually diffused throughout the orthotopic glioma. This dynamic process could be tracked in vivo by MRI up to 10 days of follow-up, as confirmed by histology. Moreover, the tumor tropism of MSCs showed no appreciable impact on the progression of the tumor. These results suggest that FTH reporter gene-based MRI can be used to reliably track the tropism and fate of MSCs after their systemic transplantation in orthotopic gliomas. This real-time in vivo tracking system will facilitate the future development of stem cell-based therapies for malignant gliomas.

Organotypic brain explant culture as a drug evaluation system for malignant brain tumors.

Therapeutic options for malignant brain tumors are limited, with new drugs being continuously evaluated. Organotypic brain slice culture has been adopted for neuroscience studies as a system that preserves brain architecture, cellular function, and the vascular network. However, the suitability of brain explants for anticancer drug evaluation has been unclear. We here adopted a mouse model of malignant glioma based on expression of H‐RasV12 in Ink4a/Arf −/− neural stem/progenitor cells to establish tumor‐bearing brain explants from adult mice. We treated the slices with cisplatin, temozolomide, paclitaxel, or tranilast and investigated the minimal assays required to assess drug effects. Serial fluorescence‐based tumor imaging was sufficient for evaluation of cisplatin, a drug with a pronounced cytotoxic action, whereas immunostaining of cleaved caspase 3 (a marker of apoptosis) and of Ki67 (a marker of cell proliferation) was necessary for the assessment of temozolomide action and immunostaining for phosphorylated histone H3 (a marker of mitosis) allowed visualization of paclitaxel‐specific effects. Staining for cleaved caspase 3 was also informative in the assessment of drug toxicity for normal brain tissue. Incubation of explants with fluorescently labeled antibodies to CD31 allowed real‐time imaging of the microvascular network and complemented time‐lapse imaging of tumor cell invasion into surrounding tissue. Our results suggest that a combination of fluorescence imaging and immunohistological staining allows a unified assessment of the effects of various classes of drug on the survival, proliferation, and invasion of glioma cells, and that organotypic brain slice culture is therefore a useful tool for evaluation of antiglioma drugs.