GL261 Tumor Cells Research Articles

IMMU-43. GUT MICROBIOTA SHAPE IMMUNE PATTERNS IN NAÏVE AND GLIOMA-BEARING HUMANIZED MICROBIOME MICE

Abstract Immunotherapy increases survival in mouse models of glioblastoma, but this is not observed in clinical trials. To address this issue, we employed a humanized microbiome (HuM) mouse model where fecal matter is transplanted from healthy human donors into gnotobiotic mice. Our previous research found that in the GL261 model of glioma, HuM mouse lines could be classified as either responders or nonresponders to anti-PD-1 therapy based solely on the gut microbiota unique to each HuM line. Here, we sought to determine the immune differences between responder and nonresponder HuM lines in both naïve (tumor-free) and glioma conditions. Single-cell RNA-sequencing (scRNA-seq) analysis of naïve colonic CD45+ cells showed that responder colons had higher frequencies of neutrophils and naïve B cells and lower frequencies of plasmablast and plasma cells. Notably, colonic neutrophils in naïve responders upregulated H2-Eb1 and Il1a compared to nonresponders. To validate the scRNA-seq data, flow cytometry was also performed on the naïve lymph nodes (LNs) and brains. The gut-draining LNs of responders had a higher frequency of CD45+ immune cells and CD4+CD8+ T cells and a lower frequency of Tregs than nonresponders. In naïve brains, responders had a higher frequency of CD45+ cells and macrophages than nonresponders and trended toward having fewer CD206+ macrophages. When mice were injected with GL261 tumor cells and treated with one dose of anti-PD-1, there was a trend for an increase in Nos2+ CD11b+ cells in both frequency and count compared to nonresponders, indicating an early and robust anti-tumor myeloid response in the responders. In summary, these results show how variations in the microbiota of HuM mice impact the immune environment across multiple organs in the baseline and tumor states, which potentially accounts for the positive response to anti-PD-1 therapy in the glioma model.

Abstract 1105: Carbon ion radiotherapy eradicates glioblastoma via tumor immune environment reprograming

Abstract Background and purpose: Glioblastoma multiforme (GBM) is a stroma rich immune cold tumor with still dismal prognosis of despite multimodal therapy consisting of postoperative surgery and radiochemotherapy. Consequently, all randomized Phase III clinical GBM trials attempting to integrate immune checkpoint blockade (ICB) have failed to demonstrate efficacy. We have recently shown that tumor immune microenvironment (TIME) reprograming via Bintrafusp alpha (Anti-PD-L1/TGF-beta bispecific molecule) combined with conventional photon radiotherapy elicits beneficial effects and prolong survival in experimental models (Lan, Knoll, Moustafa et al. Cancer Cell 2021). Moreover, eradication of radioresistant i.e., stem cell like, invasive and hypoxic tumor subpopulations by high linear energy transfer (LET) carbon ion radiotherapy (CIRT) was shown to generate an immune permissive niche in experimental GBM models (Chiblak et al. JCI Insight 2019, Tang et al. IJRBP 2022). Therefore, we aimed to systematically study the effect of CIRT vs. conventional photon radiotherapy (RT) in two (GL261 and SB28) orhotopic syngeneic mouse GBM models. Methods: Tumor growth monitoring was conducted longitudinally by means of bioluminescence and small animal MR-imaging. Mice were irradiated in five fractions (Fx) with a total dose of 15Gy RT vs. 5, 10 and 15Gy CIRT. Results: Intriguingly, all GL261 harbouring mice were cured after 5 × 3Gy Fx CIRT with no sign of tumor recurrence up to 80 days post irradiation. In contrast, no tumor control was found after 5 × 3Gy Fx RT led despite a significant prolongation of OS. The tumor control probability 50% (TCP50) for CIRT in GL261 was estimated to be 10Gy in 5 Fx. To asses if the curative effect was related to tumor eradication or reconstitution of immune surveillance. GL261 tumor cells were implanted in the contralateral hemisphere of cured mice. Single cell and Bulk RNAseq as well as immunostaining were conducted to deconvolute TIME effects post CIRT. Conclusions: In line with mechanistic studies, lack of tumor growth indicating persistent immunity against GL261 was found in CIRT cured mice. Similar experiments are conducted in SB28 model and will be presented. Together, these encouraging data indicate potential benefits for high-LET CIRT in immune modulation of GBM in experimental models that warrants for study and optimization towards clinical translation. Citation Format: Federica Ciamarone, Sarah Meister, Jennifer Furkel, Maximilian Knoll, Christian Schwager, Jovana Bojcevski, Yannik Streibel, Christel Herold-Mende, Michael Breckwoldt, Juergen Debus, Ivana Dokic, Amir Abdollahi, Katharina Schregel. Carbon ion radiotherapy eradicates glioblastoma via tumor immune environment reprograming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1105.

Clinical feasibility of miniaturized Lissajous scanning confocal laser endomicroscopy for indocyanine green-enhanced brain tumor diagnosis.

Intraoperative real-time confocal laser endomicroscopy (CLE) is an alternative modality for frozen tissue histology that enables visualization of the cytoarchitecture of living tissues with spatial resolution at the cellular level. We developed a new CLE with a "Lissajous scanning pattern" and conducted a study to identify its feasibility for fluorescence-guided brain tumor diagnosis. Conventional hematoxylin and eosin (H&E) histological images were compared with indocyanine green (ICG)-enhanced CLE images in two settings (1): experimental study with in vitro tumor cells and ex vivo glial tumors of mice, and (2) clinical evaluation with surgically resected human brain tumors. First, CLE images were obtained from cultured U87 and GL261 glioma cells. Then, U87 and GL261 tumor cells were implanted into the mouse brain, and H&E staining was compared with CLE images of normal and tumor tissues ex vivo. To determine the invasion of the normal brain, two types of patient-derived glioma cells (CSC2 and X01) were used for orthotopic intracranial tumor formation and compared using two methods (CLE vs. H&E staining). Second, in human brain tumors, tissue specimens from 69 patients were prospectively obtained after elective surgical resection and were also compared using two methods, namely, CLE and H&E staining. The comparison was performed by an experienced neuropathologist. When ICG was incubated in vitro, U87 and GL261 cell morphologies were well-defined in the CLE images and depended on dimethyl sulfoxide. Ex vivo examination of xenograft glioma tissues revealed dense and heterogeneous glioma cell cores and peritumoral necrosis using both methods. CLE images also detected invasive tumor cell clusters in the normal brain of the patient-derived glioma xenograft model, which corresponded to H&E staining. In human tissue specimens, CLE images effectively visualized the cytoarchitecture of the normal brain and tumors. In addition, pathognomonic microstructures according to tumor subtype were also clearly observed. Interestingly, in gliomas, the cellularity of the tumor and the density of streak-like patterns were significantly associated with tumor grade in the CLE images. Finally, panoramic view reconstruction was successfully conducted for visualizing a gross tissue morphology. In conclusion, the newly developed CLE with Lissajous laser scanning can be a helpful intraoperative device for the diagnosis, detection of tumor-free margins, and maximal safe resection of brain tumors.

Using a zebrafish xenograft tumor model to compare the efficacy and safety of VEGFR-TKIs.

We constructed a zebrafish xenograft tumor model to compare and quantify the antiangiogenic efficacy and safety of nine vascular endothelial growth factor receptor-tyrosine kinase inhibitors (VEGFR-TKIs), axitinib, lenvatinib, pazopanib, apatinib, cabozantinib, sunitinib, semaxanib, sorafenib, and regorafenib, in parallel. CT26 and GL261 tumor cells were implanted into the perivitelline space of Tg (flk1: eGFP) zebrafish to construct a xenograft tumor model. VEGFR-TKIs' antiangiogenic efficacy was quantified using AngioTool software, and the median effective dose (ED50) was calculated. The toxicity was evaluated by calculating the median lethal dose (LD50) and gross morphological changes. Cardiac toxicity was further assessed by heart rate, heart rhythm, the distance between the sinus venosus (SV) and bulbus arteriosus (BA), and pericardial edema. Using the zebrafish xenograft tumor model, we found that all nine VEGFR-TKIs exhibited antiangiogenic abilities, but the effectiveness of semaxanib was worse than that of other VEGFR-TKIs. Meanwhile, the zebrafish toxicity assay showed that all tested VEGFR-TKIs were associated with cardiac-related toxicity, especially apatinib and axitinib, which caused serious pericardial edema in zebrafish at relatively low concentrations. A narrow therapeutic window was found for most VEGFR-TKIs, and the simultaneous occurrence of toxic effects of semaxanib was recognized. Our findings showed the potential of using a zebrafish xenograft tumor model to accelerate VEGFR-TKI screening and further the development of more efficient and less toxic VEGFR-TKIs.

Dicer deficiency impairs proliferation but potentiates anti-tumoral effect of macrophages in glioblastoma.

Glioblastoma is a lethal primary brain tumor with abundant immune-suppressive glioblastoma-associated macrophage (GAM) infiltration. Skewing immune suppressive GAMs towards an immune-activating phenotype represents a promising immunotherapeutic strategy against glioblastoma. Herein, we reported that genetic deletion of miRNA-processing enzyme Dicer in macrophages inhibited the growth of GL261 murine glioblastoma xenografts and prolonged survival of tumor-bearing mice. Single cell RNA sequencing (scRNA-seq) of the tumor-infiltrating immune cells revealed that Dicer deletion in macrophages reduced the proportion of cell-cycling GAM cluster and reprogramed the remaining GAMs towards a proinflammatory activation state (enhanced phagocytotic and IFN-producing signature). Dicer-deficient GAMs showed reduced level of cyclin-dependent kinases (CDK1 and CDK2) and increased expression of CDK inhibitor p27 Kip1, thus manifesting impaired proliferation. Dicer knockout enhanced phagocytotic activity of GAMs to eliminate GL261 tumor cells. Increased proinflammatory GAM clusters in macrophage Dicer-deficient mice actively interacted with tumor-infiltrating T cells and NK cells through TNF paracrine signaling to create a pro-inflammatory immune microenvironment for tumor cell elimination. Our work identifies the role of Dicer deletion in macrophages in generating an immune-activating microenvironment, which could be further developed as a potential immunotherapeutic strategy against glioblastoma.

In Vivo Evaluation of Near-Infrared Fluorescent Probe for TIM3 Targeting in Mouse Glioma.

Current checkpoint inhibitor immunotherapy strategies in glioblastoma are challenged by mechanisms of resistance including an immunosuppressive tumor microenvironment. T cell immunoglobulin domain and mucin domain 3 (TIM3) is a late-phase checkpoint receptor traditionally associated with T cell exhaustion. We apply fluorescent imaging techniques to explore feasibility of in vivo visualization of the immune state in a glioblastoma mouse model. TIM3 monoclonal antibody was conjugated to a near-infrared fluorescent dye, IRDye-800CW (800CW). The TIM3 experimental conjugate and isotype control were assessed for specificity with immunofluorescent staining and flow cytometry in murine cell lines (GL261 glioma and RAW264.7 macrophages). C57BL/6 mice with orthotopically implanted GL261 cells were imaged in vivo over 4days after intravenous TIM3-800CW injection to assess tumor-specific uptake. Cell-specific uptake was then assessed on histologic sections. The experimental TIM3-800CW, but not its isotype control, bound to RAW264.7 macrophages in vitro. Specificity to RAW264.7 macrophages and not GL261 tumor cells was quantitatively confirmed with the corresponding clone of TIM3 on flow cytometry. In vivo fluorescence imaging of the 800CW signal was localized to the intracranial tumor and significantly higher for the TIM3-800CW cohort, relative to non-targeting isotype control, immediately after tail vein injection and for up to 48h after injection. Resected organs of tumor bearing mice showed significantly higher uptake in the liver and spleen. TIM3-800CW was seen to co-stain with CD3 (13%), CD11b (29%), and CD206 (26%). We propose fluorescent imaging of immune cell imaging as a potential strategy for monitoring and localizing immunologically relevant foci in the setting of brain tumors. Alternative markers and target validation will further clarify the temporal relationship of immunosuppressive effector cells throughout glioma resistance.

IMMU-44. AN IMMUNOTHERAPEUTIC VACCINE COMPOSED OF IRRADIATED WHOLE TUMOR CELLS PULSED WITH MANNAN-BAM, TLR LIGANDS AND ANTI-CD40 ANTIBODY INDUCES POTENT IMMUNE RESPONSE IN PRECLINICAL GBM ANIMAL MODEL

Abstract Despite numerous therapeutic advances, the treatment of glioblastoma multiforme (GBM) remains a challenge, with current 5-year survival rates estimated at 4%. Multiple characteristic elements of GBM contribute to its treatment-resistance, including its low immunogenicity and its highly immunosuppressive microenvironment that can effectively disarm adaptive immune responses. Hence, therapeutic strategies that aim to boost T-lymphocyte mediated responses against GBM are of great therapeutic value. Herein, we present a therapeutic vaccination strategy that promotes the phagocytosis of tumor cells, enhances tumor antigen presentation, and induces a tumor-specific adaptive immune response. This strategy consists of vaccinations with irradiated whole tumor cells (rWTC) pulsed with phagocytic agonists (Mannan-BAM), TLR ligands [LTA, Poly (I:C), and R-848], and anti-CD40 antibody (collectively abbreviated as rWTC-MBTA). We evaluated the therapeutic efficacy of rWTC-MBTA strategy in a mouse syngeneic GL261 orthotopic GBM tumor model. rWTC-MBTA or vehicle control were administered subcutaneously over the right foreleg three days after intracranial injection of GL261 cells. Complete regression (CR) of intracranial tumors was achieved in 70% (7/10) of rWTC-MBTA treated animals while none survived in the control group. Immunophenotyping analyses of peripheral lymph nodes and brain tumors of rWTC-MBTA treated mice demonstrated: (1) increased mature dendritic cells and MHC II+ monocytes; (2) increased effector (CD62L-CD44+) CD4-T and CD8-T cells; (3) increased cytotoxic IFNγ-, TNFα-, and granzyme B-secreting CD4-T and CD8-T cells. Of note, the therapeutic efficacy of rWTC-MBTA disappeared in CD4-T and/or CD8-T lymphocyte depleted mice. Three mice that achieved CR were rechallenged with 50k GL261 cells intracranially 14 months after the last rWTC-MBTA treatment and all rechallenged animals resisted GL261 tumor development, confirming the establishment of long-term immunological memory against GL261 tumor cells. Collectively, our study demonstrated that rWTC-MBTA strategy can effectively activate antigen presenting cells and induce more favorable T-cell signatures in the GBM tumors.

IMMU-24. TEMOZOLOMIDE INDUCED IMMUNE DYSFUNCTION REQUIRES THE PRESENCE OF INTRACRANIAL TUMORS

Abstract Temozolomide was recently shown to cause peripheral and intra-tumoral T cell dysfunction in a dosing schedule dependent fashion. Standard dose (SD) temozolomide (TMZ) resulted in T cell dysfunction precluding response to immune checkpoint inhibition that was avoided with a metronomic dosing (MD) schedule. Building on these studies, we investigated the TMZ-induced immune changes in tumor and non-tumor bearing models to understand the interaction of an intracranial tumor on host immunity. C57BL/6 mice underwent intracranial implantation of GL-261 tumor cells. Tumor bearing animals and naïve animals with no tumor were treated with standard dose (50 mg/kg x 5 days) or metronomic dose (25mg/kg x 10 days) of TMZ. Peripheral blood and spleens were collected for flow cytometry, ELISA and luciferase killing assay. Tumor bearing animals treated with SD TMZ demonstrated an increase in circulating myeloid derived suppressor cells (MDSCs), an upregulation of exhaustion markers on endogenous host CD8 T cells (TIM3, LAG3) and a decrease in IFN-gamma secretion from adoptively transferred T cells (tested via ELISA). The cell killing capability of adoptively transferred T cells was not reduced after exposure to a TMZ treated host. Non-tumor bearing animals treated with SD TMZ did not demonstrate an increase in circulating MDSCs or exhaustion markers on endogenous T cells. IFN-gamma secretion from adoptively transferred T cells was still reduced in these animals. The host immune dysfunction induced by TMZ is dependent on the presence of an intracranial malignancy. The mechanisms causing these changes are under active investigation.

CBMT-40. THE RELATIONSHIP BETWEEN GLIOMA AND THE GUT-BRAIN AXIS

Abstract Recent studies demonstrate the potential role of the microbiome in immune-oncology, revealing specific microbial taxa can augment the effects of various therapeutic modalities against tumors. Gut dysbiosis, a disequilibrium in the host’s bacterial ecosystem, can potentially lead to overrepresentation of some bacteria and favor chronic inflammation and immunosuppression. However, the effects of microbial dysbiosis on non-gastrointestinal cancers in particular gliomas are unknown. Here, we explored the effects of glioma and Temozolomide (TMZ) on the fecal microbiome (FM) in mice (n=24) and FM and metabolome in humans (n=40). Aged C57/B6 mice were implanted with Gl261 tumor cells or vehicle and were assigned to one of the following treatment (oral) groups: vehicle, 5mg/kg TMZ or 25mg/kg TMZ beginning 14 days after surgery for 3-weeks following a 5 day on/2 day off treatment. Fecal samples were collected prior to surgery, at treatment initiation and weekly thereafter until sacrifice and sequenced for 16s RNA. Fecal samples were collected from humans with newly diagnosed glioma before resection, chemoradiation, and after chemoradiation (16s RNA, metabolomic, neurotransmitter analysis). In mice, FM beta diversity was significantly altered with glioma (p=0.003) while the alpha diversity remained unchanged. At a genus and family level analysis the relative abundance of Bacteroides (p=0.01) and Bacteroidaceae (p=0.02) was increased. Beta diversity of mice receiving 5mg/kg TMZ changed from baseline (p=0.02). Collectively, this suggests that glioma alters the FM, to what consequence remains to be explored. Alpha (Observed OTUs, p=0.029) and beta diversity (p=0.034) differences in mice correlated with survival (< 25 - >25 days). In humans, norepinephrine and 5-hydroxyindoleacetic acid were significantly lower in glioma patients at diagnosis compared to controls. Our findings demonstrate for the first time the relationship between glioma and the gut-brain axis. Understanding alterations in the FM in glioma patients may allow novel interventions and should be further investigated.

EXTH-35. LASER INTERSTITIAL THERMAL THERAPY INCREASES BLOOD-BRAIN BARRIER AND BLOOD-TUMOR BARRIER PERMEABILITY IN A MOUSE MODEL OF GLIOBLASTOMA

Abstract INTRODUCTION A central challenge in glioblastoma treatment is the presence of the blood-brain barrier (BBB) and blood-tumor barrier (BTB), which prevent access of drugs to the brain and tumor respectively. Recent evidence in patients suggests laser interstitial thermal therapy (LITT), used clinically for tumor ablation, locally disrupts BBB integrity, potentially creating a therapeutic window to deliver otherwise brain-impermeant agents. METHODS A mouse model for LITT, established using a Nd-YAG laser coupled to a 600 mm fiber optic and thermocouple probe, was inserted via burrhole to target the somatosensory cortex. Syngeneic GL261 tumor cells were stereotactically implanted prior to LITT. BBB and BTB permeability were assessed through measurement of fluorescein and doxorubicin after IV injection. Permeability of IV dextran (10 and 70 kDa) and human IgG was monitored by immunohistochemistry (IHC) analysis. Mechanisms of BBB breakdown in vivo were explored utilizing electron microscopy and IHC. RESULTS By fluorescein assay, LITT-induced BBB and BTB permeability began one day post-treatment and was sustained for at least 2 weeks. Additionally, both normal brain and brain tumors demonstrated an increase in Dextran 10 kDa, Dextran 70 kDa, and human IgG extravasation after IV injection in vivo. Mechanistically, we provide evidence that LITT triggers both a decrease in tight junction integrity and an increase in brain endothelial cell transcytosis. As proof-of-concept that LITT can enhance tumor delivery of systemic drugs, LITT increased IV doxorubicin permeability in brain in vivo. Moreover, LITT plus doxorubicin significantly increased survival in brain tumor-bearing mice compared to doxorubicin or LITT alone. CONCLUSIONS Our data suggest that LITT increases BBB and BTB permeability over a defined time window to large molecular weight agents, including antibodies, through multiple cellular mechanisms. Our preclinical results with LITT plus doxorubicin, which mirror a current clinical trial, indicate LITT can enhance the efficacy of systemically delivered drugs.

Laser Therapy Enhances Blood-Brain Barrier and Blood-Tumor Barrier Permeability in a Mouse Model of Glioblastoma

Abstract INTRODUCTION A main challenge in the treatment of glioblastoma is the blood-brain barrier (BBB). In this study, we established a laser interstitial thermotherapy (LITT) platform in mice to test the hypothesis that LITT can increase BBB and blood-tumor barrier (BTB) permeability, which could potentially be exploited to enhance delivery of antitumor agents. METHODS We established a preclinical mouse model for LITT using an Nd-YAG laser coupled to a 600 μm fiber optic and thermocouple probe in C57BL/6 mice. Both the fiber optic and temperature probes were inserted through a small burrhole to target somatosensory cortex. In other experiments, mice were stereotactically implanted with syngeneic GL261 tumor cells prior to LITT. Post-LITT, brain tissue was assessed for BBB and BTB permeability through quantitative measurement of brain fluorescein (FL) after intraperitoneal injection and immunohistochemistry (IHC) after intravenously injected dextran (MWs 10 and 70 kDa) and human IgG (MW 150 kDa) to define spatial characteristics. Electron microscopy and IHC was used to delineate the mechanisms of BBB break down. RESULTS Using BBB permeability tracers, noninjected mice and mice orthotopically implanted with GL261 tumor cells were used to characterize the effect of laser therapy on the BBB and BTB, respectively. We found LITT substantially disrupts the BBB in the first day post-treatment and is sustained for 4 wk. Additionally, tumor-implanted mice demonstrated an increase in Dextran 10 and 70 kDa and human IgG extravasation after IV injection of these tracers. Mechanistically, we provide evidence, by IHC and electron microscopy, that LITT triggers both a decrease in tight junction integrity and an increase in brain endothelial cell transcytosis to increase overall BBB permeability. CONCLUSION Our data suggest that LITT increases BBB and BTB permeability over a defined time window in a mouse model and that large molecular size agents, including human IgG, can infiltrate brain parenchyma and brain tumors after systemic administration. Future experiments will focus on testing delivery of antitumor agents following LITT to enhance survival in brain tumor-bearing mice.

Knockout immune regulator FGL2 in tumor cells impairs tumor progression in the CNS by facilitating CD103+ dendritic cell differentiation

Abstract Although many studies link brain tumor progression to oncogene activation and tumor-suppressor gene inactivation in tumor cells, few studies implicate immune regulatory gene expression in tumor cells in arbitrating brain tumor progression. Here we show that fibrinogen-like protein 2 (FGL2) is highly expressed in glioma stem cells and primary glioblastoma (GBM) cells. FGL2 knockout (FGL2KO) in GL261, DBT, and LLC tumor cells did not affect tumor cell proliferation in vitro or tumor progression in immunodeficient NSG mice, but completely impaired GBM progression in immune-competent C57bl/6 mice. This impairment was reversed in mice with a defect in Batf3 (a key transcription factor for CD103+ DCs differentiation). Mechanistic studies revealed that FGL2KO in tumor cells induces CD103+ DCs differentiation in both the central nervous system (CNS) and in tumor draining lymph nodes (TDLN). The increased CD103+ DCs population in the CNS and TDLNs induce CD8+ T cells priming and activation and thereby gliomas regress. More specifically, the presence of FGL2 in tumor cells inhibited granulocyte-macrophage colony-stimulating factor (GM-CSF)–induced CD103+ DC differentiation by suppressing NF-κB, STAT1/5, and p38 activation. These findings are relevant to GBM patients because a low level of FGL2 expression with concurrent high GM-CSF expression is associated with higher CD8B expression and longer survival. These data provide a rationale for therapeutic inhibition of FGL2 in brain tumors.

Deletion of GARP on mouse regulatory T cells is not sufficient to inhibit the growth of transplanted tumors

GARP is a transmembrane protein that presents latent TGF-β1 on the surface of regulatory T cells (Tregs). Neutralizing anti-GARP monoclonal antibodies that prevent the release of active TGF-β1, inhibit the immunosuppressive activity of human Tregs in vivo. In this study, we investigated the contribution of GARP on mouse Tregs to immunosuppression in experimental tumors. Unexpectedly, Foxp3 conditional garp knockout (KO) mice challenged orthotopically with GL261 tumor cells or subcutaneously with MC38 colon carcinoma cells did not show prolonged survival or delayed tumor growth. Also, the suppressive function of KO Tregs was similar to that of wild type Tregs in the T cell transfer model in allogeneic, immunodeficient mice. In conclusion, garp deletion in mouse Tregs is not sufficient to impair their immunosuppressive activity in vivo.

Abstract 4726A: The oxygen carrier omx restores antitumor immunity and cures tumors as a single agent or in combination with checkpoint inhibitors in an intracranial glioblastoma mouse model

Abstract Background: Hypoxia, a common feature in solid tumors such as glioblastoma (GB), is associated with resistance to chemo- and radio-therapies and poor patient outcomes. In addition, hypoxia promotes the immune escape of tumors. Therefore, reversing tumor hypoxia to create an immunopermissive microenvironment can improve antitumor response, and combined with immunotherapy approaches such as checkpoint inhibitors (CPI), may increase therapeutic efficacy. OMX is an oxygen carrier well tolerated in small (rats and mice) and large (sheep and dogs) animals. Following intravenous administration, OMX extravasates through leaky tumor vasculature and efficiently accumulates in orthotopic rodent GB and spontaneous canine brain tumors. Consequently, OMX significantly reduces hypoxia and improves the efficacy of radiotherapy and CPI. Methods: We used in vivo bioluminescence imaging of tumor, immunohistochemistry, flow cytometry, and cytokine multiplex assays to evaluate OMX's ability to immunosensitize the GL261 brain tumor microenvironment and promote tumor cures. Results: A single dose of OMX in brain tumor-bearing mice reduces tumor hypoxia, enhances the recruitment and infiltration of tumor-specific CX3CR1+ CD8 T cells into the tumor (using the EphA2 as a GL261-specific tumor antigen), decreases Tregs and increases activation and proliferation of cytotoxic T lymphocytes (CTLs). Specifically, OMX increases the Teff/Treg ratio by ~3-fold, indicating a switch from an immunosuppressive to an immunopermissive microenvironment. Similarly, when combined with anti-PD-1, OMX decreases Tregs, increases CTL infiltration, proliferation and cytotoxic activity, and modulates IFNg and IFNg-inducible cytokines that polarize T cells towards a Th1 phenotype. Treatment with OMX alone resulted in a 55% tumor cure rate, comparable to anti-PD-1 treatment. Furthermore, in late-stage tumor-bearing mice, we observed a 40% tumor cure rate for the combination of OMX with anti-PD-1, while anti-PD-1 alone resulted only in 5% tumor cures. In symptomatic mice with very high tumor burden, in which the combination of anti-PD-1 with anti-CTLA4 does not provide tumor cures, the addition of OMX resulted in a 20% tumor cure rate. Following rechallenge with GL261 tumor cells injected on the other side of the brain, all mice treated with OMX alone or in combination with CPI survived, indicating the presence of long-term immunologic memory against glioma cells. The survival benefit observed with OMX could be predicted with an identified circulating chemokine biomarker signature (post-hoc test). Conclusion: By delivering oxygen specifically to the hypoxic tumor microenvironment, OMX may restore anticancer immune responses in GB patients and synergize with radiotherapy and immunotherapy to enhance tumor control and improve patient outcomes. Citation Format: Natacha Le Moan, Philberta Leung, Sarah Ng, Tina Davis, Carol Liang, Jonathan Winger, Stephen P. Cary, Nicholas Butowski, Ana Krtolica. The oxygen carrier omx restores antitumor immunity and cures tumors as a single agent or in combination with checkpoint inhibitors in an intracranial glioblastoma mouse model [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 4726A.

OS7 - 145 Combination Immunotherapy for Glioma: Beyond PD 1 Inhibition

Programmed Cell Death – 1 (PD1) inhibition activates tumor-specific T-lymphocytes and is an effective clinical therapy against some cancers. Preclinical data regarding immune checkpoint inhibitors against malignant glioma is scant, and interim analyses of clinical trials suggest modest effect in patients as single agents. We examined PD-1 inhibition in murine glioblastoma models in combination with other immunomodulatory agents. Methods – Syngeneic glioma tumors (GL261 and CT2A) were implanted intracranially in C57/Bl6 mice. In separate experiments, PD-1 inhibition was combined with antibody blockade of t-cell immunoglobulin and mucin protein (TIM3), ligation of OX40 on T-lymphocytes, or vaccination with irradiated GM-CSF expressing tumor cells. Systemic antitumor immunity and tumor infiltrating lymphocytes were analyzed by ELISPOT assay and flow cytometry, respectively. Results - In both syngeneic glioma models, day 3,6, and 9 systemic delivery of a monoclonal antibody against PD-1 led to increased survival vs. controls. In animals with GL261 intracranial tumors, survival was improved by combination of PD-1 blockade with subcutaneous injection of irradiated GM-CSF expressing GL261 tumor cells, with antibody blockade of t-cell immunoglobulin and mucin protein 3 (TIM3), or binding of OX40 on T-lymphocytes by an activating antibody. In most cases, ELISPOT analyses demonstrated enhanced Th1 immunity by combination immunotherapies. Vaccination was associated with an increased intratumoral CD8+ T lymphocyte / FoxP3+ T lymphocyte ratio. Conclusion –Blockade of PD-1 on T lymphocytes in glioma-bearing mice is active. Both antitumor immunity and survival can be enhanced by combination of PD-1 inhibition with agents that activate antitumor immunity by complementary mechanisms.

Abstract 542: Assessing and augmenting the immune response to glioblastoma using repurposed pharmaceuticals

Abstract In order to improve the therapeutic responses to immunotherapies in glioblastoma (GBM) patients, a known pharmaceutical, ritanserin, was repurposed to augment cytotoxic T cell responses and suppress tumor growth. To determine the kinetics of the immune response to GBM, the transplantable syngeneic glioma cell line GL261 was used in C57BL/6 mice to assess the tumor immune response in the brain, spleen, and cervical lymph node via flow cytometry weekly for one month. After identifying the optimal T cell response temporally, mice with established tumors were treated with 100mg/kg of Ritanserin for one week and harvested post treatment. Ritanserin is able to inhibit the activity of diacylglycerol kinase alpha (DGKA), an enzyme that converts diacylglycerol to phosphatidic acid, and can inhibit tumor growth in mice. In addition, DGKA is expressed in T cells, and is more highly expressed in those that have entered an unresponsive (anergic) state, characteristic of dysfunctional T cell responses to tumors. To determine if ritanserin can counteract T cell anergy, T cell responses were determined by assessment of T cell number by subset (CD8+, CD4+, CD4+FoxP3+), by activation (CD62L-CD44+), and by tumor-specific response to ovalbumin-expressing GL261 tumor cells in the brain, spleen, and cervical lymph nodes. In comparison to mock-injected mice, tumor-bearing mice showed a robust T cell response to tumors in the brain, peaking at days 21 and 28 post injection. Treatment with ritanserin resulted in a trend toward increased number and activation of T cells in the brain compared to vehicle-treated mice. In addition, there was a significant increase in OVA-specific T cells in the brain with treatment. In conclusion, T cells respond robustly to brain tumors in mice, and ritanserin treatment appears to increase this response by increased activation and priming of tumor-specific T cells. This indicates that ritanserin may have combinatorial effects on T cells when combined with checkpoint inhibitors such as ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1). Future experiments will focus on combining ritanserin and other repurposed drugs with these checkpoint inhibitors and further characterizing the effect of ritanserin on T cells. Citation Format: Breanna R. Brenneman, Desiree H. Floyd, Tajie Harris, Benjamin Purow. Assessing and augmenting the immune response to glioblastoma using repurposed pharmaceuticals. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 542.

Intratumoral INF-γ triggers an antiviral state in GL261 tumor cells: a major hurdle to overcome for oncolytic vaccinia virus therapy of cancer

Oncolytic vaccinia virus (VACV) therapy is an alternative treatment option for glioblastoma multiforme. Here, we used a comparison of different tumor locations and different immunologic and genetic backgrounds to determine the replication efficacy and oncolytic potential of the VACV LIVP 1.1.1, an attenuated wild-type isolate of the Lister strain, in murine GL261 glioma models. With this approach, we expected to identify microenvironmental factors, which may be decisive for failure or success of oncolytic VACV therapy. We found that GL261 glioma cells implanted subcutaneously or orthotopically into Balb/c athymic, C57BL/6 athymic, or C57BL/6 wild-type mice formed individual tumors that respond to oncolytic VACV therapy with different outcomes. Surprisingly, only Balb/c athymic mice with subcutaneous tumors supported viral replication. We identified intratumoral IFN-γ expression levels that upregulate MHCII expression on GL261 cells in C57BL/6 wild-type mice associated with a non-permissive status of the tumor cells. Moreover, this IFN-γ-induced tumor cell phenotype was reversible.

IT-16 * COMBINATION TREATMENT WITH DELTA24-RGD AND TEMOZOLOMIDE IMPROVES SURVIVAL AND AFFECTS INTRATUMORAL IMMUNE CELL SUBSETS IN A MURINE MALIGNANT GLIOMA MODEL

INTRODUCTION: The oncolytic adenovirus Delta24-RGD specifically lyses tumor cells and is currently tested in clinical phase I/II trials for recurrent glioma. We recently showed that the efficacy of Delta24-RGD is mainly dependent on an induced anti-tumor immune response. As known, TMZ has pronounced effects on the immune system, which could hamper the viral-induced anti-tumor immune response. To assess the combined effects, we tested different treatment regimens in an immune competent glioma mouse model. METHODS: C57BL/6 mice were injected intracranially with GL261 cells. TMZ was given for 5 days before (pre-treatment regimen) or after (post-treatment regimen) intratumoral injection of Delta24-RGD. Mice were followed for survival or sacrificed at earlier time-points to assess intratumoral influx of immune cells. T-cell response directed to the tumor cells was assessed by co-culture of splenocytes with GL261 tumor cells and analysis of IFNgamma production. Furthermore, to assess the type of cell death induced by the treatments, western blotting for LC3 (autophagy) and caspase 3/7 analysis (apoptosis) was performed. RESULTS: In vitro, apoptosis is highly induced in virus and combination treated cells, whereas autophagy is induced by TMZ monotherapy. In vivo, both combination treatment regimens significantly improved survival compared to TMZ alone. The post-treatment regimen also prolonged survival significantly compared to virus only. In the pre-treatment group, influx of dendritic cells is reduced, followed by a diminished influx of CD8+ T-cells. Interestingly, splenocytes from mice of virus and both combination treatment groups recognize GL261 cells, indicating that the diminished influx of immune cells did not hamper T-cell activation. CONCLUSION: The addition of TMZ to Delta24-RGD treatment in immune competent mice improves survival compared to Delta24-RGD or TMZ alone. Contrary to the post-treatment regimen, administering TMZ prior to virus injection hampers the intratumoral influx of immune cells; however, with both regimens an effective anti-tumor immune response is elicited.

Oncolytic viruses induce tumor cell death and antitumor immune memory in the GL261 orthotopic mouse glioma model

Introduction and Aim: Glioblastoma Multiforme is a WHO grade IV neoplasm and is the most frequent primary brain tumor in adults. In spite of current multimodal treatment the prognosis remains poor, with a median overall survival of 14.6 months and a mortality rate of 88% within 3 years. At time of relapse (which is universal), the prognosis is particularly dismal with reports of 100% mortality within 1.5 years. In view of these discouraging results, (pre)clinical research is focused on long-term and tumor-specific treatments, such as oncolytic virotherapy. Not only do oncolytic viruses have the potential to lower tumor volume and viability considerably, they might also alter the local microenvironment within the tumor, to subsequently induce a better immune control through immunotherapeutic approaches. Here we investigate the efficacy of the oncolytic Reovirus Type 3 Dearing, Parvovirus H-1 and Newcastle disease virus (NDV) Hitchner B1 strains in the orthotopic murine GL261 glioma model. Materials and Methods: monolayers of GL261 cells and healthy astrocytes were inoculated with Reovirus, Parvovirus or NDV at different multiplicities of infection, ranging from 0,05 to 10 plague forming units/cells. 72 hours later, cell death was measured via MTT assays. C57/BL6 and Rag2-/- mice were challenged with GL261 cells orthotopically and treated intratumorally with NDV on day 3 or day 7 after tumor induction. Animals were followed for symptoms and survival. Long-term surviving animals were rechallenged with GL261 cells after 100 days. Results: We demonstrated in vitro that Reovirus, Parvovirus and NDV exhibit strong cytotoxicity towards the GL261 cell line. In vivo we showed that local injection of NDV was feasible and significantly prolonged survival of treated animals versus untreated controls. Furthermore, 45% of treated animals survived long-term versus none in the control group. Similar survival data were obtained whether NDV was injected at time of minimal disease or at time of bulky tumor. Tumor rechallenge in long-term surviving animals resulted in 80% survival, suggesting immunization in most mice after NDV therapy. In immunodeficient Rag2-/- animals, NDV failed to prolong survival after glioma challenge, further substantiating the notion that the anticancer effects of NDV can be tracked not only to direct viral oncolysis, but also to an additional immune component. Conclusion: For the first time, we describe that NDV has therapeutic activity against GL261 tumor cells, evidenced in an orthotopic mouse model. NDV therapy significantly improves median survival and induces an antitumor memory response in treated animals. This is a highly innovative finding and ongoing work is focused on further elucidating the immune component in successful oncolytic virotherapy. Furthermore, this model will allow us to study the potential synergism between NDV therapy and dendritic cell mediated immunotherapy, which thus far has not been studied in the field of glioma research

Glioma‐derived galectin‐1 regulates innate and adaptive antitumor immunity

Galectin-1 is a glycan-binding protein, which is involved in the aggressiveness of glioblastoma (GBM) in part by stimulating angiogenesis. In different cancer models, galectin-1 has also been demonstrated to play a pivotal role in tumor-mediated immune evasion especially by modulating cells of the adaptive immune system. It is yet unknown whether the absence or presence of galectin-1 within the glioma microenvironment also causes qualitative or quantitative differences in innate and/or adaptive antitumor immune responses. All experiments were performed in the orthotopic GL261 mouse high-grade glioma model. Stable galectin-1 knockdown was achieved via transduction of parental GL261 tumor cells with a lentiviral vector encoding a galectin-1-targeting miRNA. We demonstrated that the absence of tumor-derived but not of host-derived galectin-1 significantly prolonged the survival of glioma-bearing mice as such and in combination with dendritic cell (DC)-based immunotherapy. Both flow cytometric and pathological analysis revealed that the silencing of glioma-derived galectin-1 significantly decreased the amount of brain-infiltrating macrophages and myeloid-derived suppressor cells (MDSC) in tumor-bearing mice. Additionally, we revealed a pro-angiogenic role for galectin-1 within the glioma microenvironment. The data provided in this study reveal a pivotal role for glioma-derived galectin-1 in the regulation of myeloid cell accumulation within the glioma microenvironment, the most abundant immune cell population in high-grade gliomas. Furthermore, the prolonged survival observed in untreated and DC-vaccinated glioma-bearing mice upon the silencing of tumor-derived galectin-1 strongly suggest that the in vivo targeting of tumor-derived galectin-1 might offer a promising and realistic adjuvant treatment modality in patients diagnosed with GBM.