bluuebunny/crossref_metadata_2025 huggingface dataset:
SURG-49. INVESTIGATING LITT-BASED COMBINATION THERAPIES IN PRECLINICAL GLIOMA MODELS
Authors: Woodiwiss Timothy , Giles David , Han Rowland , Li Derek , McCornack Colin , Beck Loren , Fu Yiwei , Leidig Will , Anzaldua-Campos Markus , Cleary Ryan , Kim Albert
Published: November 2024

BACKGROUND

Laser interstitial thermal therapy (LITT) is a minimally invasive means of tumor cyto-destruction with the potential for additional clinical benefits, such as altering the blood-brain barrier or the brain tumor microenvironment (TME). Using a murine model of GBM, we utilized a clinically relevant laser ablation model to identify and exploit immune TME changes after LITT to generate therapies that improve mouse survival, with the potential for rapid clinical translation.

METHODS

Right frontal lobe implantation of the immune “cold” SB28 murine glioma cells were performed in C57BL/6J mice. Tumor location was verified by MRI followed by stereotactic insertion of a paired fiberoptic laser and thermocouple to perform real-time hyperthermic ablation with temperature monitoring. Pulsed laser application maintained a peri-lesional temperature of 44°C for 150-180 seconds. Following LITT (or sham surgery), spectral flow cytometry was used to profile the immune TME. Survival studies were performed using a combination of LITT and immune modulating therapies.

RESULTS

Laser ablation alone provided a survival benefit compared to sham surgery (median survival: 29 vs 22.5 days, P<0.0001) in in glioma-bearing mice. Survival studies combining LITT with anti-PD1 lacked a significant response in the SB28 model but showed some long-term survivors. Flow cytometry performed on brain tissue post-LITT days 3 and 7 demonstrated little to no change in CD4+/CD8+ T cell responses but did show an increase in neutrophils, which expressed TLR2 and 4. The TME showed negligible numbers of phagocytic cells with obvious tumor cell uptake post-LITT. Using these results, we generated strategies for LITT-based combination immune therapies. Survival studies are ongoing using combinations of LITT/anti-PD1 with neutrophil depletion, inhibition of anti-CD47, and addition of broad TLR2/4 agonists as a neutrophil stimulus.

CONCLUSIONS

A clinically relevant immunocompetent murine LITT model enables the generation of novel treatment strategies with potential for rapid clinical translation.
SURG-51. DECOMPRESSIVE HEMICRANIECTOMY IN HIGH-GRADE GLIOMA PATIENTS: CLINICAL AND RADIOGRAPHIC OUTCOMES
Authors: Diaz Maria , Joanta-Gomez Adela , Welch Mary , McKhann Guy , Sisti Michael , Lassman Andrew
Published: November 2024

BACKGROUND

Decompressive hemicraniectomy (DHC) is an uncommonly needed treatment in high-grade gliomas (HGG), most used for refractory increased intracranial pressure (ICP) from vasogenic edema, mass effect, or related complications. Outcomes and imaging following DHC are not well described.

METHODS

We retrospectively identified cases of DHC for HGG at our institution since 2019.

RESULTS

Three patients with HGG underwent DHC (2 females, ages 39-70) for newly diagnosed (2 patients) or recurrent tumor (1 patient). Causes of increased ICP were tumor/vasogenic edema (2 patients) or abscess (1 patient). Subsequent cranioplasty was performed in 2 patients: at 1.8 months after DHC in one patient, and at 8.6 months in another patient who developed sinking skin flap syndrome (SSFS). SSFS presented as progressive worsening of tumor-related neurologic deficits after completion of radiation therapy with no change in imaging, followed by frank signs of increased ICP 8 months after DHC. Two patients died of disease progression (5.3 months and 9.1 months after DHC), and one patient remains alive at 13.5 months (patient with SSFS; neurologic deficits virtually resolved after cranioplasty repair). Imaging will be described for all patients.

CONCLUSION

DHC can be a life-saving intervention in HGG. When feasible, early cranioplasty should be considered to avoid complications, including SSFS, which may present with neurological deficits without corresponding imaging change.
SURG-58. COLD ATMOSPHERIC PLASMA (CAP) CAUSES SELECTIVE TUMOR CELL DEATH VIA APOPTOSIS AND FERROPTOSIS IN MALIGNANT GLIOMA CELLS
Authors: Peeters Sophie , Kim Wi , Armellini Amber , Haist Blake , Ge Weihong , Ayad George , Chen Zhitong , Obenchain Richard , Yilgor Can , Owens Geoffrey , Tamayo Andrea , Nathanson David , Prins Robert , Bhaduri Aparna , Wirz Richard , Wang Anthony
Published: November 2024

BACKGROUND

High-grade gliomas (HGGs) are primary brain tumors associated with significant morbidity and mortality, and despite advances in neuro-oncology, the prognosis remains poor. Cold atmospheric plasma, a novel experimental anti-cancer therapeutic tool, is a near-room temperature ionized gas generated at atmospheric pressure. It has many promising results in vitro and in vivo with a variety of cancers. CAP cytotoxicity appears to be selective to cancer cells without significant damage to surrounding healthy tissues. We aim to evaluate the efficacy of CAP in generating tumor-selective cytotoxicity against high-grade glioma.

METHODS

B16, U87, GL261, patient derived high grade glioma, human neural stem cells (LM-NSC008) and astrocytes (NHA hTERT) were treated with CAP. Proliferation and propidium iodide (PI)/annexin V flow cytometry assays were employed to quantify cytotoxicity, cell cycle phases and apoptosis. Cells were pre-treated with ferroptosis inhibitors Ferrostatin-1 and Deferoxamine (DFO) in rescue assays. Cerebral organoids were transplanted with fluorescent tumor cell lines and stained with cleaved caspase-3, ki67 and transferrin receptor.

RESULTS

CAP induces dose dependent cell death in all glioma cell lines tested. CAP induces an accumulation of intracellular ROS, most prominent in glioma cells. CAP-mediated cell death involves apoptosis and ferroptosis. CAP selectively leads to apoptosis in tumor implanted organoids and leads to decreased proliferation in malignant and non-malignant cells. CAP leads to a shift into G0 phase of all treated cells, malignant and non-malignant.

CONCLUSIONS

CAP treatment induces dose-dependent increases in ROS and apoptosis in HGG lines tested more significantly than in NHAs and hNSCs. CAP induces decreased proliferation and G0 phase cell cycle arrest in all CAP-treated cells. CAP-mediated cytotoxicity was significantly mitigated with DFO pre-treatment in HGG cells, suggesting that ferroptosis plays a critical role in the mechanism of CAP treatment in HGG. CAP led to selective glioma cell death in tumor implanted cerebral organoids.
SURG-59. A RARE CASE OF COMBINED OCCURRENCE OF GLIOBLASTOMA MULTIFORME AND PHEOCHROMOCYTOMA: A CASE REPORT AND LITERATURE REVIEW
Authors: Lin YaJui
Published: November 2024

Glioblastoma is most common and malignant primary brain tumors. However, combined other solid organ malignancies sometimes occurred. To our knowledge, the combination of glioblastoma multiforme and pheochromocytoma is an exceedingly rare occurrence. As a result, we present the case of a 61-year-old male patient who suffered from slurred speech and unsteady gait due to left frontal glioblastoma with concurrent pheochromocytoma. The clinical course was retrospectively collected from medical records, including the lab data, imaging, surgical procedure and neurological examination results. We also provide a comprehensive review and discussion of the association between pheochromocytoma and glioblastoma, including genetic disorder the management strategy. The fluctuated blood pressure during brain surgery implied suspicious concurrent endocrine tumor in this patient. The patient was ultimately diagnosed with adrenal pheochromocytoma after left adrenalectomy and radical nephrectomy by urologist. No other malignancies or genetic disorders were found in this patient. Subsequently, this patient recovered well without any neurologic deficit and with effectively controlled blood pressure. The presence of a concomitant pheochromocytoma may precipitate paroxysmal catecholamine surges, fluctuations in cerebral perfusion, and an elevated risk of significant intra-cranial blood loss during neurosurgery. Once patient of brain tumors presented with uncontrolled or fluctuated blood pressure occurred, concurrent endocrine tumor should be considered.
TMET-04. METABOLOMICS ANALYSIS IN FUNCTIONING AND SILENT CORTICOTROPH ADENOMAS USING A TWO-DIMENSIONAL NANOMATERIAL HIGH-THROUGHPUT PLATFORM
Authors: Guo Xiaopeng , Liang Weiqiang , Ma Chunxia , Zhao Yuanli , Xing Bing
Published: November 2024

Functioning pituitary corticotroph adenomas (CAs) originated from T-PIT lineage with an ACTH-positive staining cause Cushing’s Disease. However, silent CAs (SCAs) baring the same histological features present clinically non-functioning with more aggressive courses. Metabolites within the tumor microenvironment play critical roles in regulating tumor behaviors and responses to therapy. Understanding their distribution and concentration provides valuable insights for diagnosis and treatment. This study investigated metabolite patterns in functioning and silent CAs. We developed a novel two-dimensional nanomaterial platform for enhanced surface-assisted laser desorption/ionization mass spectrometry. Utilizing this platform, we analyzed the metabolomes of three functioning CAs and three SCAs. Our platform exhibited exceptional sensitivity and stability in detecting metabolite molecules. Analysis of the metabolomes revealed distinct spatial distributions and peak intensity of various metabolites, including carbohydrates, amino acids, organic acids, nucleotides, and their precursors. Notably, certain metabolites such as tryptophol, clopidogrel carboxylic acid, DG(20:4(8Z,11Z,14Z,17Z)-2OH(5S,6R)/0:0/i-16:0), S-(-)-ureidoglycolate, and PC(20:4(5Z,7E,11Z,14Z)-OH(9)/14:1(9Z)) were significantly enriched in functioning CAs compared to SCAs, while others, including Cer(d18:1/16:0), Dihydrocytochalasin B, and LysoPC(O-18:0/0:0), exhibited higher peak intensity in SCAs. Overall, our findings underscored the existence of unique metabolomic signatures distinguishing functioning CAs and SCAs. Heterogeneous metabolite distributions among functioning CAs and SCAs were revealed by our novel nanomaterial high-throughput platform, and unique metabolic signatures were identified. Metabolomics analysis holds promise for precise biomedical diagnostics and individualized therapeutic strategies in functioning and silent CAs.
TMET-07. RADIORESISTANT GLIOBLASTOMA EVADES MITOCHONDRIAL LIPOTOXICITY THROUGH ALTERED DGKB AND DGAT1
Authors: Lee Haksoo , Kim Dahye , Kim Byeongsoo , Yoo Gaeun , Cha JiYong , Kang Hyunkoo , Jeon Jaewan , Youn HyeSook , Youn BuHyun
Published: November 2024

Glioblastoma (GBM) is the most common and aggressive primary brain tumor, with a median survival of only 15 months despite multimodal therapy. Radiotherapy is a conventional therapy of GBM treatment, but the development of radioresistance often leads to tumor recurrence and treatment failure. To elucidate the molecular mechanisms underlying radioresistance in GBM, we established radioresistant GBM cell lines through repeated exposure to ionizing radiation. Transcriptomic analysis revealed that diacylglycerol kinase beta (DGKB), an enzyme that converts diacylglycerol (DAG) to phosphatidic acid, was significantly downregulated in radioresistant GBM cells. Functional studies demonstrated that knockdown of DGKB or expression of a kinase-dead DGKB mutant resulted in DAG accumulation and reduced fatty acid oxidation. Conversely, overexpression of DGKB activated fatty acid oxidation, increased reactive oxygen species (ROS) production, and sensitized GBM cells to radiation. Interestingly, we found that radiation upregulated the expression of diacylglycerol acyltransferase 1 (DGAT1), which catalyzes the conversion of DAG to triglycerides. Knockdown of DGAT1 using shRNA or miR-3918 mimic reduced lipid droplet formation and enhanced radiosensitivity both in vitro and in vivo. Furthermore, using Connectivity Map, we identified cladribine as a potential radiosensitizing agent that increases DGKB and decreases DGAT1. Cladribine effectively sensitized GBM cells to radiation and significantly prolonged the survival of mice bearing GBM xenografts. In conclusion, our study reveals a novel mechanism by which radioresistant GBM cells maintain lipid homeostasis through downregulation of DGKB and upregulation of DGAT1 to evade lethal mitochondrial lipotoxicity. Targeting the DGKB-DGAT1 axis represents a promising therapeutic strategy to overcome radioresistance and improve the outcomes of GBM patients.
TMET-20. A METABOLIC BIOMARKER IN GLIOBLASTOMA: A PROMISING APPLICATION OF CO-POLARIZED [1-13C]PYRUVATE AND [1-13C]DEHYDROASCORBATE
Authors: Coffee Elizabeth , Porcari Paola , Kim Nathaniel , Patel Saket , Berishaj Marjan , Deh Kofi , Rahimi-Keshari Kayvan
Published: November 2024

BACKGROUND

Noninvasive biomarkers for treatment response in glioblastoma are needed to improve patient outcomes. Our understanding of redox biology in treatment resistance is rapidly evolving, and interrogation of tumor metabolism represents an ideal opportunity to meet this need. In this study, the utility of co-hyperpolarized [1-13C]pyruvate and [1-13C]dehydroascorbate (HP PA/DHA) to simultaneously evaluate brain tumor metabolism and oxidative stress in orthotopically implanted mice is investigated.

METHODS

To characterize HP PA/DHA, mice were treated with 0-4mM diethyl maleate and 1D slab dynamic acquisitions were performed over the mouse brain. As an initial evaluation of the utility of HP DHA/PA in brain tumors, athymic nude mice (n=8) underwent orthotopic U87 glioblastoma implantation. After treatment with 8Gy in one fraction, mice were injected with HP DHA/PA dissolved in D2O. T2-weighted and 13C Chemical Shift Imaging sequences were obtained on a 3T MRI using a quadrature double-tuned 1H/13C volume coil.

RESULTS

HP DHA/PA detected differential lactate and vitamin C generation over the mouse brain. Treatment with diethyl maleate, which depletes the cofactor glutathione, diminished fractional vitamin C generation (0.1 vs 0.03, p=0.0009). Elevation in tumor lactate generation was seen in untreated (125.1±17.6µM, p=0.005) and radiated mice (149.4±32.6µM, p=0.01). Treatment with radiation generated higher vitamin C concentrations in tumors relative to contralateral brain (46.8±4.8µM, p=0.002). Radiated tumors were distinguished by higher ratio of lactate to vitamin C compared to untreated tumor controls (3.7 vs 2.5, p=0.07), and the same relationship was preserved between treated and untreated non-tumor brain.

CONCLUSION

The ratio of vitamin C/lactate detects the metabolic effects of radiation in brain tumors, inspiring its’ potential as a biomarker for treatment response. To further characterize these findings, examination of survival and modulation of radiosensitivity are ongoing.
TMET-22. OVERCOMING RESISTANCE: PANOBINOSTAT SENSITIZES 2DG-RESISTANT PEDIATRIC HIGH-GRADE GLIOMAS WITH H3.3 K27M/G34R MUTATIONS TO APOPTOSIS
Authors: Miller Tracy , Jane Esther , Gatesman Taylor , Halbert Matthew , Sneiderman Chiam , Raphael Itay , Mullett Steven , Gelhaus Stacy Lynn , Agnihotri Sameer , Steinman Richard , Premkumar Daniel , Pollack Ian
Published: November 2024

BACKGROUND

Pediatric high-grade gliomas (pHGG) harboring H3.3 K27M/G34R mutations present formidable challenges in treatment due to their aggressiveness and resistance to standard chemotherapy. Our preliminary investigations have shown promising results, indicating that combining 2-deoxyglucose (2DG), a glycolytic inhibitor, with DNA-damaging agents synergistically inhibits pHGG growth. However, prolonged exposure to 2DG induces resistance, necessitating the exploration of alternative therapies. This study aims to investigate the potential of panobinostat, a histone deacetylase inhibitor, in sensitizing 2DG-resistant pHGG with H3.3 K27M/G34R mutations to apoptosis.

METHODS

Transcriptomic analysis via Illumina NovaSeq 6000 and metabolomic profiling employing Liquid Chromatography-High-Resolution Mass Spectrometry was used to delineate the molecular landscape of naive and 2DG-resistant pHGG cell lines. Functional assays, including the Agilent Seahorse Mito Stress Test and flow cytometry, assessed bioenergetics and mitochondrial dynamics. Intracellular NAD+ and NADH levels were quantified utilizing NAD+/NADH Glo assays, while apoptotic markers and metabolic stress were measured using the Annexin V-FITC Conjugates for Apoptosis Detection kit and western blot.

RESULTS

Comparative analysis revealed increased transketolase activity in 2DG-resistant cells, augmenting the interaction between the pentose phosphate pathway and glycolysis, resulting in elevated levels of NADPH and ATP. Additionally, these resistant cells displayed enhanced reliance on mitochondrial energy metabolism, as evidenced by substantial increases in mitochondrial membrane potential and mass, coupled with elevated NAD+ levels. Subsequent treatment with panobinostat induced a significant decrease in NAD+ biosynthesis and ATP generation. Furthermore, this intervention triggered the upregulation of pro-apoptotic BCL-2 family proteins, particularly BAX and BAK, concomitant with a notable reduction in mitochondrial membrane potential and the accumulation of reactive oxygen species. Consequently, heightened caspase-3 activity ensued, leading to significantly elevated cell death rates in 2DG-resistant gliomas.

CONCLUSION

Our findings highlight panobinostat as a promising therapy to resensitize 2DG-resistant pHGG cells to apoptosis, offering hope for treating these aggressive tumors with H3.3 K27M/G34R mutations.
TMET-23. MASS SPECTROMETRY-BASED METABOLIC PROFILING AND IMAGING OF ASTROCYTE AND GLIOBLASTOMA INVASIVE MARGIN CELLS
Authors: Kontrimaite Une , McCrorie Phoebe , Martinez-Jarquin Sandra , Porter Harry , Wong Kei F Carver , Smith Stuart , Kim Dong-Hyun , Rahman Ruman
Published: November 2024

Despite multimodal treatment, 90% of patients diagnosed with isocitrate dehydrogenase (IDH) wild-type glioblastoma experience tumour recurrence within 5 years. Poor prognosis is in considerable part attributed to the ability of cancer cells to infiltrate a healthy brain. We hypothesise that metabolic changes underpinning cellular crosstalk between astrocytes and glioblastoma cells isolated from the invasive margin within a physiologically representative in-vitro tumour microenvironment may reveal therapeutic targets to prevent or delay glioblastoma recurrence. This study used advanced analytical techniques to establish baseline metabolomic profiles for eGFP-tagged primary glioblastoma cells isolated from the invasive margin and healthy human cortical astrocytes. Liquid chromatography-mass spectrometry (LC-MS) enabled detailed analysis of biochemical pathways, and Atmospheric Pressure-Matrix Assisted Laser Desorption/Ionisation (AP-MALDI) imaging offered spatial localization of metabolites, critical for mapping their distribution within tumour microenvironments. Metabolites were extracted using a biphasic method (methanol:chloroform:water) for LC-MS analysis, and metabolic profiling was conducted using a ZIC-pHILIC column coupled with a Q-Exactive Orbitrap Mass Spectrometer. Additionally, AP-MALDI imaging facilitated the generation of high-resolution spatial distribution maps of selected metabolites, achieving a resolution of 10 µm. Both multivariate and univariate statistical analyses were applied to identify significant metabolic alterations. Metabolic profiling identified distinct pathways altered in glioblastoma cells compared to astrocytes, including alterations in aspartate, glycine and serine, and lipid metabolism. High-resolution imaging outlined the spatial distribution of metabolites involved in these pathways, revealing regions of metabolic heterogeneity. Furthermore, this methodology facilitated an expanded identification range of essential biomolecules, notably lipids and amino acids, thereby augmenting our understanding of their dynamic functions across diverse cellular contexts. By leveraging the complementary strengths of LC-MS and AP-MALDI imaging, this research enhances our understanding of spatial metabolite distribution in glioblastoma cells isolated from the invasive margin and suggests potential applications in patient tissue, offering a deeper exploration of disease mechanisms.
TMIC-07. TRIPLE COCKTAIL IMMUNOMODULATION OF TUMOR DRAINING LYMPH NODES RESULTS IN SYNERGISTIC REVERSAL OF IMMUNOSUPPRESSION AND IMPROVED OVERALL SURVIVAL IN A MURINE GLIOBLASTOMA MODEL
Authors: Choi John , Klich John , Kim Lily , Schonfeld Ethan , Verma Rohit , Cho Kwang Bog , Li Gordon , Appel Eric , Lim Michael
Published: November 2024

Clinical trials involving anti-PD-1 fail to improve survival compared to standard of care in glioblastoma (GBM). Despite the advent of newer immunotherapies such as STING agonists and anti-CSF-1R, combination of these strategies is limited by necessitation of intracranial delivery (STING) or off-target side effects with systemic exposure (anti-CSF-1R + anti-PD-1). We have previously shown that delivery of anti-PD-1 to tumor draining lymph nodes via hydrogels decreases systemic exposure of anti-PD-1 and results in improved therapeutic response compared to systemic delivery. This study assesses the efficacy of combining STING agonist (STING), anti-CSF-1R (aCSF1r), and anti-PD-1 (aPD1) into a hydrogel nanoparticle that targets different components of immunosuppression in tumor draining lymph nodes. CT-2A murine glioma cells were orthotopically implanted into C57BL/6J mice with treatment on day 7 via deep cervical injection of hydrogel polymers loaded with either PBS (control), STING, aCSF1r, aPD1, or a combination of all three immunomodulators. Flow cytometry was used for immunophenotyping and Kaplan-Meier survival curves were generated to assess therapeutic efficacy. Each treatment strategy targeted a different component of immunosuppression. Compared to controls, aCSF1r resulted in the greatest decrease of immunosuppressive myeloid cells in the brain (P=0.0017) and cervical lymph nodes (P=0.0008); STING resulted in the greatest activation of intracranial dendritic cells (P=0.0001); and aPD1 resulted in the highest level of T cell activation of the monotherapies (P=0.0008) but also caused compensatory upregulation of regulatory T cells in the brain (P=0.0176). Combination therapy resulted in the greatest decrease in immunosuppression with increased T cell IFN-y production and dendritic cell activation, with resultant improvement in median and overall survival (P=0.0006). Triple combination therapy to tumor draining lymph nodes that specifically targets multiple components of immunosuppression is a safe and efficacious treatment that can be translated to clinical application.
TMIC-08. PATTERNS OF PSTAT3 EXPRESSION IN REACTIVE ASTROCYTES OF BRAIN METASTASES FROM NON-SMALL CELL LUNG CANCER AND CORRELATIONS WITH OUTCOME
Authors: Pellerino Alessia , Wasilewski David , Bertero Luca , Ricci Alessia Andrea , Mangherini Luca , Bianconi Andrea , Garbossa Diego , Cassoni Paola , Priego Neibla , Valiente Manuel , Soffietti Riccardo , Rudà Roberta
Published: November 2024

BACKGROUND

pSTAT3 expression in peritumoral reactive astrocytes (RA) of brain metastases (BM) from breast cancer may favor a pro-metastatic environment and negatively affect the intracranial progression-free survival (i-PFS) (Pellerino, 2023). We aim to evaluate whether pSTAT3 plays the same role also in BM from NSCLC.

MATERIAL AND METHODS

Seventy-two BM specimens from NSCLC were identified from the biobank of Pathology Unit of University of Turin and Berlin. STAT3 expression was scored in RA of peritumoral tissue according to Priego et al. (Nat Med 2018). Clinical and molecular data, and i-PFS were retrospectively retrieved.

RESULTS

Median age was 64 years (IC95% 59-67). Immunohistochemistry for GFAP and pSTAT3 was feasible in 66/72 (91,6%). 42/66 (63,6%) had BM with druggable mutations, while 24/66 (36,4%) did not show any actionable mutations. Druggable mutations were as follows: 8/42 (19,1%) KRAS G12C, 6/42 (14,3%) EGFR, 5/42 (11,9%) ALK, 1/42 (2,4%) BRAFv600E, 31/42 (73,8%) PDL1 expression. Overall, 53/66 (80,3%) showed positive staining of pSTAT3: 22/66 (33,3%) with score 3, 21/66 (31,8%) with 2, 10/66 (15,1%) with 1, and 12/66 (19.8%) with 0 (negative). High pSTAT3 expression (score 2-3) was observed in 32/42 (76,2%) BM with actionable mutations and in 12/24 (50%) without actionable mutations (p 0,031). PDL1 expression was significantly correlated with high pSTAT3 expression (29/31, 93,5%), while most of BM without PDL1 (20/35 – 57,1%) had low or absent pSTAT3 (score 0-1) (p<0,0001). Median i-PFS was 12 months (IC95% 8-23): low pSTAT3 BM had a median i-PFS of 18 months (IC95% 14-28) versus 9 months (IC95% 8-12) for high pSTAT3 BM (HR 5,3;p 0,0001).

CONCLUSION

pSTAT3 overexpression in RA is associated with PDL1 expression and may negatively impact the i-PFS in BM from NSCLC. These data support the investigation of STAT3 inhibitor silibinin in preventing intracranial recurrence in a clinical trial (NCT05689619).
TMIC-10. GENETIC LANDSCAPE OF BRAIN METASTASIS IN TRIPLE-NEGATIVE-BREAST CANCER: COMPARISON OF PAIRED PRIMARY AND METASTATIC TUMOR USING SPATIAL TRANSCRIPTOMIC ANALYSIS
Authors: Yoo Jihwan
Published: November 2024

The survival rate of individuals diagnosed with breast cancer has shown improvement over time, primarily attributed to advancements in early detection methods and the implementation of proactive treatment strategies. However, it is concerning that the incidence of breast cancer continues to rise, thereby leading to a corresponding increase in mortality rates among female breast cancer patients. Among these, the presence of brain metastasis is associated with a worse prognosis in individuals with breast cancer. Notably, triple-negative breast cancer exhibits a heightened propensity for the development of brain metastasis. Furthermore, there has been a notable rise in the duration of breast cancer survival, leading to a concomitant increase in the occurrence of metastatic cancer throughout the course of the disease. Presently, a greater number of patients are experiencing brain metastases, surpassing the figures reported in current statistical data. In instances of metastatic cancer, a distinct microenvironment that is particular to the tumor is formed in a distant location. The comprehension of the molecular genetic and immunological mechanisms involved in the several phases of brain metastasis, including tumor cell invasion, engraftment, and proliferation, has significant importance in the context of patient treatment and prognosis prediction. However, it is worth noting that a well-defined scientific foundation in this area is currently absent. The objective of this study was to analyze the molecular genetic characteristics of triple-negative breast cancer primary tumors and brain metastasis tumors. This was achieved by utilizing spatial transcriptome analysis technology to compare the differences in genetic profiles between the tumor cells and the surrounding tumor microenvironment. The findings of this study allowed for a comprehensive comparison of the tumor microenvironment at each site. In order to validate alterations in gene expression of various constituents and elucidate the mechanisms behind metastasis, it is imperative to conduct spatial confirmation.
TMIC-12. LITT RESULTS IN ROBUST T CELL INFILTRATION WITHIN THE TUMOR MICROENVIRONMENT IN A BRAIN METASTASIS MURINE MODEL
Authors: Chauhan Anjali , Thomas Nagheme , Frain Matthew , Otero Oriana Cristina , Kumar Ravi , Tomdio Macaulay Y , Packham Olivia R , Bova Frank J , Deleyrolle Loic , Takacs Gregory Pal , Harrison Jeffrey K , Rahman Maryam
Published: November 2024

INTRODUCTION

Laser interstitial thermal therapy (LITT) is a novel minimally invasive neurosurgical technique used to ablate intra-axial brain tumors with real-time thermometry. The impact of LITT on the brain metastatic tumor microenvironment (TME) is unknown in patients due to limitations of tumor tissue collection post-LITT. The primary objective of this study was to describe the kinetics of immunologic infiltration within the TME in a melanoma brain metastasis model. METHOD: C57BL6 mice underwent intra-cranial implantation of B16F10-OVA tumor cells and treated with LITT using a custom-made murine LITT system developed in our laboratory. The tumors were treated to 42°C (near infra-red 1064 nm DPSS laser system) 7 days post-surgery. Tumors were collected 2 days and 10 days post-LITT for immune-phenotyping using flow cytometry. Blood was collected 6 h and 48 h post-LITT to perform the ELISA for IFN-g and TNF-a.

RESULTS

LITT treatment triggered a cascade of local infiltration of immune cells at different time points. 2-days post-LITT resulted in increased CD8 T cells (p<0.05), macrophage (p<0.05) and microglia infiltration (p<0.01) within the TME compared to control and sham surgery animals. By 10 days after LITT, significant infiltration by CD8 T cells (p<0.0001) and M1 macrophages (p<0.0001) was observed. The CD8 T cells were T central memory (TCM) and T effector memory (TEM). Moreover, comparison between 2 days and 10 days after LITT revealed significant increase of TEM (p<0.0001) and TCM (p<0.001) 10 days post-LITT. In an ELISA assay, non-significant differences were observed in IFN-g and TNF-a level at 48 h.

CONCLUSIONS

LITT enhances the recruitment of cytotoxic CD8 T cells within TME in a time-dependent manner. The ability of LITT to recruit TCM and TEM to the TME strongly support its potential therapeutic benefit, especially in the context of immunotherapy.
TMIC-25. MICROGLIA IN THE GLIOBLASTOMA MICROENVIRONMENT SECRETE ACETYLATED AMINO ACIDS WHICH ASSIST IN THE REPAIR OF RADIATION INDUCED DNA DAMAGE
Authors: Palavalasa Sravya , Freeman Jack , Singer Ava , Gokul Aditri , Baker Emily , Scott Andrew , Wilder-Romans Kari , Shankar Sunita , Ravikumar Visweswaran , Andren Anthony , Wong Harrison , Sajjakulnikit Peter , Bhaduri Sagnik , Zhou Weihua , Lyssiotis Costas , Rao Arvind , Al-holou Wajd , Wahl Daniel
Published: November 2024

Rapid repair of DNA damage mediates genotoxic therapy resistance and poor prognosis in glioblastoma (GBM). The GBM tumor microenvironment (TME) is heterogenous, and we hypothesized that non-malignant cells support the repair of DNA damage in glioblastoma cells. Consistent with this hypothesis, we found that GBM tumors grown intracranially in mice had rapid DNA repair and were resistant to radiation compared to tumors of identical genotype grown extracranially in mouse flanks. Further interrogation of irradiated intracranial tumors using gamma-H2AX immunofluorescence revealed spatial heterogeneity in DNA damage repair, with spatially separate regions of rapid and slow gamma-H2AX resolution. Paired spatial transcriptomic analysis and gamma-H2AX immunofluorescence showed that tumor regions with rapid DNA repair were rich in microglia. Having nominated microglia from these results, we cocultured GBM cells and microglia in direct contact as well as in transwell coculture. We found that GBM cells cocultured with microglia repair DNA damage faster in a contact-independent manner. We then assessed the supernatant metabolome of microglia and GBM monocultures and cocultures and found that acetylated amino acids are secreted by microglia and consumed by GBM cells. Supplementation with acetylated amino acids promotes radiation resistance by speeding the repair of radiation-induced DNA damage. Mechanistically, acetylated amino acid supplementation allows glioma cells to re-route glucose utilization away from the TCA cycle and towards nucleotide synthesis. Hence, we show that microglia in the GBM microenvironment assist GBM cells to repair radiation induced DNA damage by secreting acetylated amino acids which are consumed by GBM cells and used to fuel the TCA cycle allowing utilization of glucose for nucleotide synthesis, thereby making the GBM cells resistant to radiation therapy.
TMIC-27. PDPN ACTIVATION OF CLEC5A IN THE PERI-NECROTIC NICHE DRIVES TAM POLARIZATION AND TME IMMUNOSUPPRESSION IN GLIOBLASTOMA
Authors: Li Jiabo , Wang Xuya , Shih Ling-kai , Feng Bo , Gruchala Tomasz , Yang Xuejun , Brat Daniel
Published: November 2024

Glioblastoma, IDH-wildtype (GBM, WHO grade 4) is the most common malignant glioma of adults and is characterized by a severely hypoxic and immunosuppressive tumor microenvironment (TME). CLEC5A regulates immune responses in inflammatory and infectious diseases. However, its role in GBM immune regulation remain unclear. Here, we found that CLEC5A has the strongest association with poor clinical outcomes among all immune-related genes in GBM. Tumor-associated macrophages (TAMs) account for the majority of immune cells in the GBM and CLEC5A expression in this population is highest in hypoxic, peri-necrotic zones. Both hypoxia and GBM conditioned media cause increased CLEC5A expression by THP-1 cells, and overexpression of CLEC5A enhanced TAM polarization, migration toward glioma cells, and increased secretion of cytokines that mediate immunosuppression. We show that CLEC5A is a cell surface receptor activated by podoplanin (PDPN) and induces TAM polarization through Syk-JAK-STAT3 signaling. Co-IP assay showed that CLEC5A and PDPN could be binded together using antibodies for CLEC5A, Flag-CLEC5A or PDPN when we cocultured control THP-1 cells and THP-1 cells overexpressing Flag-CLEC5A with primary GBM cells (NU02068). PDPN blocking antibody (α-PDPN) reduced the specific binding between PDPN and CLEC5A, as well as inhibited the TAM-IM polarization. Overexpression of CLEC5A significantly upregulated the expression of p-Syk, Jak2, p-Jak2, STAT3, p-STAT3, CD163, CD206 and PD-L1. Moreover, Bay 61-3606, a selective Syk inhibitor, rescued CLEC5A overexpression-mediated TAM-IM polarization and Syk-JAK-STAT3 activation in THP-1 cells. In vivo, both silencing CLEC5A in TAMs and silencing PDPN in GBM cells, as well as pharmacologically targeting Syk delayed glioma growth, prolonged survival and was associated with diminished immunosuppressive TAMs. Collectively, we found that PDPN-CLEC5A-Syk-JAK-STAT3 signaling causes TAM polarization and enhances TME immunosuppression in GBM and can be targeted to suppress growth.
TMIC-30. FOREBRAIN TO HINDBRAIN INVASION IN GLIOBLASTOMA IS DIRECTED BY ANATOMICALLY DISTINCT REACTIVE ASTROCYTES
Authors: Silver Daniel , Randall Sabre , Lathia Justin
Published: November 2024

Initial detection of glioblastoma (GBM) often reveals a large mass in the forebrain; however, the hazard for many patients lies in extensive invasion that can extend far from the primary tumor. New evidence suggests that end-stage GBM coincides with malignant infiltration of the pons and brainstem. Mechanisms that direct tumor cells from initial positions within the forebrain to terminal positions in the brainstem are largely unknown. Our analysis indicates that spatially defined, reactive astrocytes instruct GBM cells to either proliferate or invade. During development, astrocytes help establish directional gradients and boundaries that mediate proper routing of migrating neural cells and extending axons. We posit that onco-reactive astrocytes re-engage these guidance programs to direct tumor cells to the brainstem. To test this hypothesis, we exposed a series of human GBM models to media conditioned by cortical, midbrain, or brainstem reactive astrocytes. When treated with cortical conditioned media, tumor cells were driven into a state of hyper-proliferation, which mirrored early-stage forebrain tumor generation. In contrast, midbrain and brainstem conditioned medias suppressed proliferation and enhanced the rate of tumor cell migration. RNA sequencing corroborated these functional data, identifying cell division programs augmented in GBM cells exposed to cortical astrocyte conditioned media. This contrasted with axon development and guidance programs enriched in GBM cells exposed to midbrain and especially brainstem conditioned medias. Finally, mRNA and protein analysis identified region-specific engagement with the Eph-ephrin guidance system patterned along a forebrain to hindbrain trajectory. These data support a new hypothesis that GBM tumors recapitulate a form of neurodevelopment, invading specific anatomic regions based on cues generated by resident reactive astrocytes.
TMIC-38. INTEGRIN BLOCKING PEPTIDE REPROGRAMS THE IMMUNOSUPPRESSIVE MICROENVIRONMENT OF EXPERIMENTAL GLIOMAS IMPROVING ANTI-PD-1 THERAPY OUTCOME
Authors: Kaminska Bozena , Ellert-Miklaszewska Aleksandra , Pilanc-Kudlek Paulina , Lenkiewicz Anna , Poleszak Katarzyna , Cyranowski Salwador , Roura Adria-Jaume , Pasierbinska Maria , Baluszek Szymon , Hovorova Juliana , Jacek Karol , Wojnicki Kamil , Ghosh Mitrajit , Gielniewski Bartlomiej , Szadkowska Paulina
Published: November 2024

Clinical trials with immune checkpoint inhibitors (ICI) have benefited many cancer patients but failed in a number of tumors, including glioblastoma (GBM), the most common and aggressive primary brain tumor in adults. The main obstacle for ICI efficacy in GBM is the continuously evolving tumor microenvironment (TME), in which antitumor immunity is inhibited or eluded by tumor-secreted factors. Accumulation and reprogramming of myeloid cells (GAMs) creates a “cold” immunosuppressive TME with a poor infiltration and exhaustion of effector T cells. We employ Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) with 40 protein markers along with spatial transcriptomics and immunophenotyping to dissect identities and functionalities of immune cells (CD45+) in experimental GL261 gliomas. The results revealed identities of immune cells instrumental for creating the immunosuppressive milieu and cell-cell communication networks. We have developed a designer RGD peptide that blocks the reprogramming of GAMs by targeting tumor-GAMs interactions. We demonstrate that RGD efficiently blocks microglia-dependent invasion of murine and human glioma cells in vitro. We explored if the intratumorally delivered RGD can revert tumor-induced changes in the TME of experimental gliomas and improve anti-PD-1 immunotherapy. While RGD alone did not reduce tumor growth in vivo, it prevented reprogramming of myeloid cells into protumoral GAMs and led to the normalization of peritumoral blood vessels. Combining RGD with anti-PD-1 antibody resulted in reduced tumor growth, increase in percentages of proliferating, interferon-ɣ producing CD8+T cells and depletion of regulatory T cells. Transcriptomic profiles of GAMs were altered by the combined treatment, consistently with the restored “hot” inflammatory TME which resulted in boosting immunotherapy responses. Intratumorally delivered RGD modified in the same way functionalities of GAMs in the TME of human U87MG gliomas in nude mice. Our results demonstrate that the integrin blockade combined with immune checkpoint inhibitors reinvigorates both innate and adaptive antitumor immunity. The results pave the way to improve responses to immunotherapy in GBM and other cancers.
TMIC-45. CYTOMEGALOVIRUS PP65 ANTIGEN EXPRESSION FOLLOWING PP65-SPECIFIC DENDRITIC CELL VACCINATION IN MATCHED PRIMARY AND RECURRENT GLIOBLASTOMA SPECIMENS
Authors: Bou Dargham Tarek , Su Zuowei , Pina-Oviedo Sergio , Brown Michael , Cook Sarah , Satterfield Diane , Threatt Stevie , Xie Weihua , Norberg Pam , Khasraw Mustafa , McLendon Roger E , Archer Gerald E , Sampson John H , Mitchell Duane A , Cobbs Charles , Lawler Sean , Batich Kristen
Published: November 2024

BACKGROUND

Human cytomegalovirus (HCMV) expression in glioblastoma (GBM) has been corroborated by multiple independent laboratories. HCMV genes have been directly linked to GBM cell proliferation, invasion, and angiogenesis. The HCMV tegument protein pp65 has served as a tumor-specific antigen in serial pp65 RNA-pulsed autologous dendritic cell (DC) vaccine trials. Here we report pp65 expression in matched primary and recurrent GBM samples from those trials following pp65-DC vaccination.

METHODS

Paired GBM specimens were collected from patients receiving pp65-DC vaccines according to ATTAC and ATTAC-GM clinical trials (NCT00639639). Of 23 treated patients, 9 cases were available for analysis. Formalin-fixed paraffin-embedded specimens were mounted on glass slides and stained for pp65 via immunohistochemistry (IHC). CD31 served as a control for tissue quality and to investigate the role of pp65 co-staining in the tumor perivascular niche.

RESULTS

Of 9 available cases, 5 were excluded due to poor tissue quality/absent CD31 staining on aged slides. Among the remaining four cases, three showed pp65 staining in non-vascular and perivascular areas within both primary and recurrent samples. However, recurrent specimens of these positive cases demonstrated a clear reduction in both intensity and distribution of pp65 staining within non-vascular regions but retained a strong positivity within the perivascular space. The fourth case showed absent pp65 staining and only weakly positive CD31 staining in both primary and recurrent specimens. Necrotic areas within pp65-positive slides showed minimal to no pp65 staining.

CONCLUSIONS

This study confirms HCMV pp65 expression in both primary and recurrent GBM following pp65-DC vaccination, with an interesting phenomenon of retained pp65 expression specifically in the perivascular niche. These findings support the role of HCMV in GBM angiogenesis and tumor progression as well as mechanisms of immune evasion following single antigen vaccine platforms. Our study reinforces the importance of tissue quality for proper HCMV detection in GBM.
TMIC-51. NEUROIMMUNE-COMPETENT ORGANOID MODELING OF MICROGLIA/TUMOR INTERACTIONS AND DRUG RESPONSIVENESS
Authors: Sarnow Katharina , Majercak Emma , Qurbonov Qurbonali , Cruzeiro Gustavo Alencastro Veiga , Silva Andrezza Do Nascimento , Haque Ishraq , Jeong Daeun , Baird Lissa , Filbin Mariella , Tang Xin
Published: November 2024

INTRODUCTION

Due to its highly aggressive and infiltrative nature, lack of effective treatment options and localization in the skull base, diffuse intrinsic pontine glioma (DIPG) is universally fatal with less than a year median survival time. Studying how DIPG cells interact with brain cells in its microenvironment, including microglia which is the primary brain immune cell type, may lead to new therapeutic strategies to halt the invasion of DIPG into brain tissue. We developed a clinically relevant human immunocompetent brain organoid-DIPG fusion model for investigating the tumor invasion process at the cellular and molecular levels.

METHODS

Neuronal progenitor and GFP-labeled myeloid precursor cells were derived from human embryonic stem cells and combined to self-assemble and co-develop over two months into microglia-containing brain organoids (MiCBOs). The development of the MiCBOs as well as the distribution and motility of the microglia within the brain organoid was characterized by confocal and multiphoton microscopy, immunofluorescence staining and Western Blots. Confocal live-cell imaging was used to investigate the invasion of TdTomato-labeled tumor cells into MiCBO. We further performed single-cell RNA-Sequencing (scRNA-Seq) analysis to gain insight into the cell-specific gene expression changes after the confrontation of the MiCBOs with tumor spheroids.

RESULTS

MiCBOs contain viable, widely distributed and highly motile microglia as well as functionally mature neurons, highly resembling those in the human brain. scRNA-Seq showed distinct gene expression patterns in both microglia and tumor cells after confronting MiCBOs with DIPG spheroids, suggesting candidate signaling pathways between those two cell types.

CONCLUSION

Our novel MiCBO-DIPG fusion model provides a pathophysiologically-relevant system to elucidate molecular mechanisms underlying cell-cell interactions during tumor invasion in a human brain tissue context. This scalable model can be adapted to drug screening applications to identify therapeutics that modulate neuroimmune/tumor interactions and to establish the therapeutic index for anti-cancer medications.
TMIC-53. USE OF CEREBRAL OPEN-FLOW MICROPERFUSION TO IDENTIFY POTENTIAL ONCOMETABOLITE, L-2-HYDROXYGLUTARATE, IN GLIOBLASTOMA CHEMOTHERAPY ADAPTATION
Authors: Olson Ian , Stepniak Amelia , Chen Rebecca , Chojak Rafal , Miska Jason , Ahmed Atique
Published: November 2024

Glioblastoma (GBM) is among the most prevalent and lethal brain tumors, comprising nearly half of all central nervous system malignancies and imparting a median overall survival of only 18-20 months. One major challenge in curing this disease is the limited understanding of how GBM cells biologically respond to treatments like radiotherapy and chemotherapy, including temozolomide (TMZ), in real-time. A significant challenge is observing changes within the tumor interstitial fluid (ISF), which mediates adaptive intercellular signaling between tumor cells and their surrounding tumoral and stromal neighbors. Microfluidic sampling methods, such as cerebral microdialysis and microperfusion, have started to provide valuable insights into the ISF signaling dynamics. These methods enable real-time biomolecular monitoring of this often-neglected component of the tumor microenvironment (TME). We have performed in vivo cerebral open-flow microperfusion (cOFM) pilot studies to identify metabolomic and proteomic changes within the tumor ISF resulting from differential treatment with either TMZ or vehicle control. Among these metabolite and protein changes, we identified an increase in 2-hydroxyglutarate (2-HG). While D-2-HG is a hallmark metabolite of isocitrate dehydrogenase mutant (IDH-mut) tumors, our model utilized the IDH wild-type (IDH-WT) CT2A mouse-syngeneic glioma model. Subsequent analysis of enantiomeric confirmation characterized this to be predominantly L-2-HG. Follow-up in vitro studies have shown that elevated L-2-HG plays a functional role in IDH-WT patient-derived glioma cells via non-canonical HIF1α stabilization and subsequent expression of HRE genes. This suggested that L-2-HG may play a similar role in IDH-WT GBM as D-2-HG plays in IDH-mutant tumors. This study demonstrates the potential of L-2-HG to function as a chemo-responsive oncometabolite in the TME of highly aggressive IDH-WT glioblastoma. By targeting the metabolic and signaling properties of L-2-HG, it may be possible to prevent chemo-resistant adaptations mounted by GBM cells when treated with TMZ.
TMIC-54. OHSV-MEDIATED NOTCH BLOCKADE FOR BRAIN TUMORS
Authors: Rivera Caraballo Kimberly , Hong Bangxing , Lee Tae Jin , Vázquez-Arreguín Karina , Murphy Sara , Sharma Ashok , Kaur Balveen
Published: November 2024

Primary brain tumors such as glioblastoma (GBM) are refractory to current standard-of-care, and so there is an urgent need to develop novel therapies to treat brain tumors. NOTCH is a cell-cell communication pathway that is exploited by GBM stem cells to bypass treatment. The role of NOTCH signaling in tumor growth and resistance is well studied, but the significance of this pathway for immunotherapy of GBM is not very well understood. We have recently shown that NOTCH activation with viro-immunotherapy contributes to tumor regrowth and induces recruitment of monocytic MDSC cells into the tumor, which limit optimal immune activation to kill tumor cells. Herein, we evaluated the impact of blocking NOTCH signaling using two different peptides: NIX1 and NIX2 that can interfere with NOTCH ligand binding with their receptors. Both NIX1 and NIX2 could effectively block the ligation of NOTCH ligands with Notch receptors in the tumor microenvironment. Secretion of both NIX1 and NIX2 was confirmed by western blot (WB) and ELISA. Functionality of NIX1 and NIX2 to block NOTCH signaling was assessed by western blot analysis for NOTCH cleaved intracellular domain (NICD). RNA sequencing and PCR based assays were utilized to evaluate the impact of NIX1 and NIX2 on global pathways that were enriched. Gene therapy using oncolytic immunotherapy vectors revealed that both NIX1 and NIX2 could suppress NOTCH signaling. In vitro, NIX1 and NIX2 did not increase the benefit of viro-immunotherapy. Treatment of mice bearing intracranial tumors with NIX1 or NIX2 armed viro-immunotherapy revealed a significant increase in therapeutic benefit with some long-term survivors. Both NIX1 and NIX2-expressing viruses induced macrophage migration relative to unarmed viro-immunotherapy in two different glioma models. Role of macrophages in guiding therapeutic benefit is currently being explored.