bluuebunny/crossref_metadata_2025 huggingface dataset:
QOL-16. POSTOPERATIVE MOTOR FUNCTION AND PROCESSING SPEED PREDICT QUALITY OF LIFE IN PATIENTS WITH GLIOBLASTOMA AT CHRONIC-PHASE
Authors: Nakada Mitsutoshi , Nakajima Riho , Kinoshita Masashi
Published: November 2024

The quality of life (QOL) in patients with glioblastoma (GBM) should be paid attention. The aim of this study is to identify brain functions that influence the QOL of patients with GBM by objectively assessing neurocognitive functions. Data from 40 patients with newly diagnosed GBMs were analyzed. All patients completed the assessment of various neurological and neurocognitive functions and QOL at 6 months postoperatively. QOL was assessed using the 36-Item Short Form Survey (SF-36). In the SF-36, the physical, mental, and role and social component summary (PCS, MCS, and RCS, respectively) scores were calculated. Multiple logistic regression analyses and chi-square tests were used to evaluate the association between SF-36 scores and brain functions. The MCS was maintained, whereas the PCS and RCS scores were significantly lower in patients with GBMs than in healthy controls (p=0.0081 and p<0.0001, respectively). Among a variety of brain functions, motor function and processing speed were significantly correlated with PCS and RCS scores, respectively (p=0.0048 and p=0.030, respectively). Patients whose PCS or RCS remained stable had a higher probability of motor function or processing speed preservation than those whose RCS or PCS decreased (p=0.0026). Taken together, motor function and processing speed may be predictors of QOL in patients with GBMs.
SURG-14. PATTERNS OF PROGRESSION OF TEMPORAL LOBE GLIOBLASTOMAS: EVIDENCE FOR THE ROLE OF TEMPORAL LOBECTOMY
Authors: Price Stephen , Gurung Binay , Nazar Thaaqib , Mair Richard , Joannides Alexis , Santarius Thomas , Matys Tomasz
Published: November 2024

Glioblastomas extend beyond the contrast-enhancing tumor, and supramaximal resections into non-enhancing (NE) tumor improves survival. Yet, supramaximal resection is difficult to achieve in most cases. For temporal lobe tumors, supramaximal anterior temporal lobe resection is feasible and safe. Case series have shown a survival advantage by resecting the anterior temporal lobe. This study aims to explore patterns of tumor progression in temporal lobe glioblastomas and whether the presence of NE tumor in the temporal tip could be used as a biomarker to predict the site of progression. We searched a single institution tumor database looking for patients who had glioblastomas in the temporal lobe, who underwent complete resection of enhancing tumor, and went on to have chemoradiotherapy. Patients needed to have imaging demonstrating first progression. We determined pre-operatively if NE tumor had extended into the temporal tip. At progression, the direction of tumor progression was recorded and assessed if it would have been resected should a lobectomy have been performed. We identified 55 patients that fulfilled our entry criteria (mean age 57.7 years, range 19.6-72.9; 37 male). In 16 cases, the anterior temporal lobe was resected; these patients were not used in further assessments. In 25 cases (64.1%), the tumor progressed into the residual anterior temporal lobe. In 16 cases (41.0%), a lobectomy would have encompassed the area of progression. NE tumor was seen in the tip in 28 cases (71.8%). This could predict progression into the anterior temporal lobe with a sensitivity of 76.2% (95%CI: 54.9-90.6%) and specificity of 33.1% (95%CI: 11.8-61.6%). The likelihood ratio of tumor progression was 1.14 (95%CI: 0.75-1.74). We conclude that temporal lobe glioblastomas most commonly progress into the anterior temporal lobe, but NE tumor is not a suitable predictive biomarker. Developing suitable biomarkers and biomarker-driven trials of temporal lobectomy for glioblastomas are needed.
SURG-50. CLINICAL EXPERIENCE AND PATHOLOGICAL FEATURES IN AUTOPSY CASES TREATED WITH TUMOR TREATING FIELDS
Authors: Tamai Sho , Kinoshita Masashi , Nambu Kosuke , Tanaka Shingo , Nakada Mitsutoshi
Published: November 2024

BACKGROUND

Since 2017 Tumor Treating Fields (TTF) was approved for primary glioblastoma in Japan. In this study, we reviewed clinical experience and the pathological characteristics of autopsy cases treated with TTF.

METHODS

From March 2018 to April 2022, 67 patients (36 males and 31 females, median age 68 years) with primary glioblastoma, IDH-wild type treated at our institution were included in the analysis.

RESULTS

Twenty-eight patients (19 males and 9 females, median age 66 years) met the TTF indication criteria for induction, 19 cases (67.9%) were 65 years or older, and 14 cases (50.0%) indicated MGMT promoter methylation. The mean duration of TTF treatment was 7.2 months. The main reasons for discontinuation were tumor recurrence in 11 patients (40.7%), skin symptoms in 8 patients (29.6%), and psychological distress in 6 patients (22.2%). Only 12 patients (42.9%) underwent TTF treatment over 18 hours per day, because of discomfort of wearing pad and concern about srrounding. Median PFS and OS in the used patients were 9.9 and 17.7 months, respectively. Autopsies were performed in 3 of the 16 deaths. Histopathological examination of tumor specimen indicate that the Ki-67 labeling index was higher in brainstem (33.6%) than in the supratentorial primary lesion (23.6%).

DISCUSSION

Comparing to the previous report (Stupp R et al., JAMA. 2017), our cases contained a large number of patients over 65 years old with shorter mean duration of TTF treatment, and shorter OS. After TTF treatment, the Ki-67 labeling index was higher on infratentorial than on supratentorial in the autopsy specimens, suggesting the anti-tumor effect of TTF for the supratentorial lesion.

RESULTS

High proliferative activity of infratentrial lesion was suggested as a typical form of progression for TTF treatment.
SURG-57. FLUORESCENCE LIFETIME IMAGING (FLIM) PROVIDES HIGHLY ACCURATE, DYE-FREE TUMOR MARGIN DETECTION DURING GLIOMA RESECTIONS
Authors: Bloch Orin , Anbunesan Silvia , Alfonso-Garcia Alba , Lee Han , Riestenberg Robert , Marcu Laura
Published: November 2024

INTRODUCTION

Fluorescence guided tumor margin detection is an important tool to enhance the extent of resection for gliomas. Existing dyes, such as 5-ALA, require planned administration, surgical resection in dark conditions, and are only effective for high-grade tumors. We have developed a novel laser scanning probe to assess tissue autofluorescence decay (lifetime) which allows tumor margin detection without exogenous dyes and under normal room light conditions. This technology has been optimized for margin detection in low-grade and high-grade gliomas.

METHODS

Patients undergoing craniotomy for resection of gliomas were enrolled. During resection of the final tumor margin, multiple 1x1 mm regions were scanned with FLIm and biopsied for evaluation by a neuropathologist. FLIm data and ground truth pathology were processed by a machine-learning algorithm to develop classifiers for the FLIm data. Classifiers were then applied to the full data set to determine the accuracy of FLIm predictions.

RESULTS

12 patients with non-enhancing (LGG) and 25 patients with enhancing (HGG) tumors were enrolled, with a total of 228 biopsies obtained. For LGG, sensitivity of FLIm for detection of residual tumor was 0.89 (95% CI 0.8-0.98) with a specificity of 0.92 (95% CI 0.86-0.97), resulting in a positive predictive value of 0.76 and negative predictive value of 0.96 with an overall accuracy of 0.91. For HGG, sensitivity of FLIm for detection of residual tumor was 0.92 (95% CI 0.84-1.0) with a specificity of 0.95 (95% CI 0.91-0.99), resulting in a positive predictive value of 0.90 and negative predictive value of 0.96 with an overall accuracy of 0.94, exceeding reported values for 5-ALA.

CONCLUSION

FLIm can distinguish between residual low and high-grade glioma and normal brain at resection margins with very high sensitivity and specificity. This quantitative technology does not require administration of exogenous dyes and can be used under normal room light illumination.
TMET-01. LIPID METABOLIC PROGRAMS DEFINE GLIOMA STATE IDENTITY, PLASTICITY AND DEPENDENCIES
Authors: Minami Jenna , Bayley Nicholas , Tse Christopher , Andrasz Cassidy , Cadet Dimitri , Salinas Jennifer , Yan Weihong , Borujerdpur Amir , Liau Linda , Cloughesy Timothy , Williams Kevin , Bensinger Steven , Bhaduri Aparna , Graeber Thomas , Nathanson David
Published: November 2024

Gliomas are lethal malignancies comprised of heterogeneous and dynamic subpopulations of cell states resembling normal neurodevelopmental cell types (radial glia (RG), oligodendrocyte progenitor cell (OPC), neuron progenitor cell (NPC), neuron, etc). While both intrinsic (genetics) and extrinsic (brain environment) cues are coupled to glioma state identity, the functional programs governing glioma cell state and plasticity are unknown. Here we performed a multi-omic interrogation of a large library (n=392) of glioma patient tumors, in vivo orthotopic xenografts and in vitro gliomasphere cultures. Comparisons of matched glioma samples across environments revealed the non-native in vitro environment constrains glioma state diversity specifically enriching for stem-like glioma cell states (RG, immune, vascular). This enrichment was linked to lipid metabolic flexibility, enabling tumors enriched for stem-like states to adapt to various tumor microenvironments. By contrast, “lineage-committed” cell states (OPC, NPC, and neuron) demonstrate restricted lipid metabolism, consequently having a dependence on lipid scavenging from the brain microenvironment for survival. These results connect intra-tumoral heterogeneity and plasticity of glioma to lipid metabolism, leveraging the functional diversity of cellular states to reveal potential therapeutic opportunities.
TMET-18. MANNOSE INHIBITS GLIOMA CELL PROLIFERATION AND POTENTIATES TEMOZOLOMIDE EFFICACY
Authors: Cho HyeJoung , Yoon Yeo Jun , Hoon Roh Tae , Kang Seok-Gu
Published: November 2024

Gliomas are among the most aggressive types of brain tumors, often displaying resistance to standard therapies such as temozolomide (TMZ). Recent research suggests that metabolic modulation may influence tumor growth and therapeutic response. This study explores the effects of mannose, a monosaccharide, on glioma cell growth and its potential to enhance the efficacy of TMZ. In vitro experiments were conducted to evaluate the impact of mannose and its combination with TMZ on glioma cells. Cell viability assays, ATP measurements, and flow cytometry (FACS) analysis were performed to assess cell cycle and apoptosis post-treatment with various monosaccharides, including mannose. The synergistic effects of the combined treatment with mannose and TMZ on cell viability were evaluated using Bliss score analysis and FACS. The impact on cancer stem cell characteristics was investigated through neurosphere formation assays, while tumor invasiveness was assessed using 3D invasion assays. Expression of stemness and invasion markers was analyzed by western blot. Differentially expressed gene (DEG) analysis was conducted to assess changes in gene expression related to stemness and invasiveness. Mannose treatment significantly reduced glioma cell viability and ATP levels, indicating decreased metabolic activity. Combined treatment with mannose and TMZ demonstrated a synergistic effect, enhancing cell death compared to either treatment alone, as evidenced by Bliss score and FACS analysis. Co-treatment reduced neurosphere formation and 3D invasion capabilities, with western blot results showing downregulation of stemness and invasion markers. DEG analysis revealed significant changes in gene expression related to stemness and invasiveness with the combined treatment. Mannose significantly impairs glioma cell growth and enhances the therapeutic efficacy of TMZ in vitro. Combining mannose with standard chemotherapy offers a promising strategy for improving glioma treatment outcomes. Further investigation in clinical settings is warranted to validate these findings.
TMET-30. ONCOLYTIC HSV REWIRES METABOLISM TO PROMOTE ANTITUMOR IMMUNITY THROUGH REDUCTIVE CARBOXYLATION AND FERROPTOSIS
Authors: Sahu Upasana , Mullarkey Matthew P , Murphy Sara , Diaz Miguel , Park Junhyoung , Kamal Abu Hena Mustafa , Kaiparettu Benny Abraham , Lee Tae Jin , Anderson Joshua Charles , Sharma Ashok , Putluri Nagireddy , Wenzel Pamela , Wiley Christopher , Markert James M , Kaur Balveen
Published: November 2024

Cancer cells autonomously rewire metabolism to meet increased bioenergetic demands. Oncolytic HSVs (oHSVs) are attenuated Herpes simplex type 1 derived viruses that lyse tumor cells releasing damage-associated molecules triggering antitumor immunity. Currently one oHSV-1 derived virus is approved for melanoma treatment in the USA and another for recurrent GBM in Japan. While several other viruses are being evaluated in clinical trials for safety and efficacy, an understanding of the impact of oHSV therapy on cancer cell metabolism is largely unexplored. A detailed understanding of how oHSV impact tumor cell metabolism and oxidative stress is important to understand the unique susceptibilities that can further be exploited to improve the therapeutic index. Here, we analyzed transcriptomic changes in tumor biopsies of recurrent high-grade glioma patients treated with oHSV. RNA-seq data obtained from pre- and post-G207 treated biopsies from phase-Ib study patients (NCT00028158) revealed enrichment in pathways related to citric acid cycle, oxidative phosphorylation, and glutamate metabolism. We confirmed these changes by RNA sequencing of oHSV-infected patient derived GBM cells. Mechanistically, oHSV hijacked cellular glucose metabolism to increase oxidative phosphorylation and modulated cellular glutamine metabolism towards reductive carboxylation and reduced free glutathione, collectively contributing to increased ROS generation. Kinome profiling revealed PKC activation upon treatment with oHSV. Increased ROS combined with reduced glutathione, and PKC activation promoted ferroptotic cell death characterized by increased lipid peroxidation and smaller, ruptured mitochondria, and contributed to immunotherapy by this treatment. IDH mutant tumors that block reductive carboxylation were resistant to oHSV-induced ferroptotic cell death and did not benefit from oHSV-induced antitumor benefit. Together, this study presents avenues to enhance antitumor immunity by oHSV therapy.
TMET-39. DUAL TARGETING OF MAP KINASE SIGNALING AND METABOLISM IS A THERAPEUTIC VULNERABILITY IN PEDIATRIC DIFFUSE MIDLINE GLIOMAS
Authors: Gatesman Taylor , Casillo Stephanie , Chilukuri Akanksha , Varadharajan Srinidhi , Premkumar Daniel , Jane Esther , Johnson Brenden , Plute Tritan , Halbert Matthew , Cruz Andrea , Abel Taylor , Mack Stephen , Pollack Ian , Agnihotri Sameer
Published: November 2024

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. However, there remains a knowledge gap in identifying effectors of ERK signaling and their roles in DMG-H3K27a. Leveraging several patient multi-omic datasets, we identified a unique relationship between ERK5 and brain tumor glycolysis. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a, where ERK5 knockdown leads to decreases in glycolysis and increases in mitochondrial metabolism. We demonstrate through loss of function and overexpression studies that ERK5 mediates glycolysis through regulation of glycolytic enzyme Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 (PFKFB3). PFKFB3 is a protein that catalyzes the synthesis or degradation of fructose-2,6-bisphosphate (F2,6BP). Furthermore, we establish a novel mechanism, in which ERK5 mediates PFKFB3 expression via activation of MEF2A, an oncogenic transcription factor. DMG-H3K27a cells are sensitive to the loss or inhibition of PFKFB3 both in vitro and in orthotopic mouse models. However, monotherapy is ineffective in heterogenous diseases such as gliomas, including DMG-H3K27a. To combat this, we show multi-targeted therapy against the ERK5-PFKFB3 axis is synergistic in vitro and in vivo. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma. Moreover, we have identified resistance to our dual therapy using several systems biology approaches, which we are currently investigating and validating. Collectively, our study defines a novel, targetable axis in DMG-H3K27a and highlights the importance of tumor cell metabolism in defining therapeutic vulnerabilities.
TMET-40. UPREGULATION OF URIDINE SALVAGE IN MESENCHYMAL GLIOBLASTOMA STEM-LIKE CELLS
Authors: Baljinnyam Tuvshintugs , Sowers Mark , Chang-gu Bruce , Tang Hui , Herring Jason , Zhang Kangling , Sowers Lawrence
Published: November 2024

Glioblastoma is a heterogenous tumor comprised of multiple molecular subtypes of cancer cells and cancer stem-like cells. Of particular importance are the cancer stem-like cell niche population as they are believed to be more resistant to existing therapies and re-seed the tumor following initial response to standard-of-care treatment. To better understand glioblastoma stem-like cells we completed an untargeted proteomic analysisof 35 patient derived glioblastoma stem cell lines. This system driven analysis revealed an increase in several important proteins, in the mesenchymal subtype, involved in the uridine salvage pathway. A subset of these proteins, including ecto-5’-nucleotidase (NT5E/CD73), uridine phosphorylase (UPP1), phosphoglucomutase-2-like-1 (PGM2L1), nicotinamide phosphoribosyltransferase (NAMPT), and nicotinamide N-methyltransferase (NNMT), suggest an augmented pathway for utilization of UMP and uridine as a fuel source and to replenish NAD+. Uridine and UMP are present in human serum and are enriched in the tumor microenvironment providing a sustainable resource to resupply the tumor. The upregulation of this pathway was validated in established cell lines and orthogonal patient-derived xenograft cell lines not included in the proteomics study. Further work is underway to selectively inhibit or exploit this pathway with small molecule inhibitors.
TMIC-09. DENDRITIC CELL DYSFUNCTION RESTRICTS TUMOR ANTIGEN SPREADING IN ADOPTIVE CELLULAR TRANSFER THERAPY-ESCAPED GLIOMAS: IMPLICATIONS FOR TUMOR IMMUNE EVASION
Authors: Jin Dan
Published: November 2024

Dendritic cells (DCs) play a pivotal role in initiating anti-tumor immune responses. Our group has developed an adoptive cellular transfer therapy (ACT) that significantly increases the infiltration of DCs and T cells into tumors, markedly enhancing survival in murine brain tumor models. However, tumors develop mechanisms to restrict the immune surveillance and the efficacy of immunotherapy, leading to tumor escape. In this study, we identified a mechanism involving DC dysfunction in ACT-escaped brain tumors. The finding that DCs are enriched after ACT treatment prompts an investigation into whether DC function is impaired in ACT-escaped tumors. Our results showed that DCs from both primary and ACT-escaped tumors were dysfunctional in their ability to activate T cells when co-cultured with tumor-reactive T cells or OT1 T cells. RNA-seq data revealed that, compared to primary tumor DCs, ACT-escaped tumor DCs exhibited a more pronounced reduction in conventional DC markers and antigen-presenting genes, alongside increased tolerance markers. Gene set enrichment analysis indicated that epithelial-mesenchymal transition (EMT) and hypoxia pathways were significantly enriched in ACT-escaped tumor DCs, suggesting divergent DC dysfunction mechanisms in primary versus escaped tumors. The anti-tumor role of DCs is primarily exerted through the activation of tumor-reactive T cells. Therefore, we assessed the cytotoxicity and exhaustion status of T cells in tumors. Surprisingly, we found that T cells from ACT-escaped tumors exhibited significantly higher levels of cytotoxicity and lower levels of T cell exhaustion compared to primary tumors. This suggests a T cell exhaustion-independent mechanism in ACT-escaped tumors. We further demonstrated that T cells in ACT-escaped tumor are mainly sourced from the adoptive transferred T cells, which recognize primary tumor antigens but not escaped tumor antigens. These results suggest that a combined mechanism of tumor antigen shifting and impaired DC function leads to a failure of antigen spreading and tumor eradication.
TMIC-42. CONQUERING SPP1+ MYELOID-DRIVEN RESISTANCE OF GLIOBLASTOMA TO IL13RA2-TARGETED CAR T CELL THERAPY
Authors: Gholamin Sharareh , Natri Heini M , Zhao Yuqi , Xu Shengchao , Aftabizadeh Maryam , Comin-Anduix Begonya , Saravanakumar Supraja , Masia Christian , Wong Robyn , Peter Lance , Chung Mei-i , Mee Evan D , Aguilar Brenda , Starr Renate , Torrejon Davis Y , Kalbasi Anusha , Alizadeh Darya , Wu Xiwei , Ribas Antoni , Forman Stephen , Badie Behnam , Banovich Nicholas E , Brown Christine E
Published: November 2024

In the evolving landscape of glioblastoma (GBM) therapy, the recent proliferation of clinical trials exploring chimeric antigen receptor (CAR) T cell interventions has garnered attention, although their therapeutic efficacy remains limited. Despite recent progress in elucidating the role of the GBM microenvironment in immunotherapy resistance, particularly regarding intercellular networks and macrophages, significant ambiguities endure, impeding a comprehensive understanding of resistance mechanisms amidst the patient-specific heterogeneity of the tumor microenvironment (TME). In this study, we utilized single-cell RNA sequencing to analyze tumors from 41 glioma patients undergoing IL13Rα2-targeted CAR T cell therapy. Our findings unveiled heightened suppressive extracellular network signatures, particularly SPP-1, predominantly observed within myeloid cells from non-responsive patients. The clinical significance of both SPP1+ myeloid cell abundance and overall expression levels revealed associations with patient outcomes. Subsequently, transcriptomic insights were translated to myeloid cell functions in patients exhibiting high versus low SPP1 expression. SPP1-expressing macrophages displayed diminished antigen presentation, phagocytosis, and cell-mediated cytotoxicity, along with augmented extracellular matrix biogenesis and degradation, iron efflux, and suppressive interleukin pathways. Moreover, SPP1-high macrophages exhibited enhanced ligand-receptor interactions, implying a predisposition towards suppressive activities. Immunometabolism pathways, including lipid and ketone body biogenesis and regeneration, were upregulated in SPP1-expressing macrophages, indicating an increased reliance on non-glycolysis-mediated ATP production. To assess translational implications, the therapeutic efficacy of SPP1 blockade was evaluated in a syngeneic glioma model. Blockade of SPP1 with an anti-SPP1 antibody prior to CAR T cell infusion effectively mitigated the immunosuppressive milieu, reversing resistance in preclinical GBM models to murine IL13Rα2-targeted CAR T cell therapy. Subsequent profiling of tumors post-treatment provided insights into TME alterations following SPP1 blockade therapy. This investigation illuminates the intricate interplay between the TME and CAR T cell therapy resistance in GBM, underscoring SPP1 as a promising therapeutic target to surmount CAR T cell resistance and enhance treatment outcomes.
TMIC-43. BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF NOVEL GLIOBLASTOMA TME SUBTYPES USING TRANSCRIPTOMIC ANALYSIS AND NETWORK MODELLING TO IDENTIFY NOVEL TARGETS OF VULNERABILITY
Authors: Dabbour Mohannad , White Kieron , Kalmykov Aleksandr , Kel Alexander , Dussman Hieko , Prehn Jochen , Byrne Annette
Published: November 2024

New precision medicine therapies are urgently required for glioblastoma (GBM). Recently, we developed a novel GBM classification system, identifying three patient clusters uniquely characterized by tumor microenvironment (TME) composition: TMELow, TMEMedium, and TMEHigh1. Our objective now is to further investigate these subtypes employing transcriptomic analysis and network modelling approaches to identify novel subtype-specific targets of vulnerability. We analyzed transcriptomic data from >600 GBM samples from publicly available and in-house datasets. All samples underwent TME subtyping. Next, we performed differential gene expression (using DESeq2, edgeR) and pathway analysis (using PROGENy). The ‘TRANSFAC’ tool 2 was used to identify potential transcription factors enriched in each TME subtype. TMELow tumours manifested highly upregulated NLGN3 (P=5.777e-5) and HES5 (P=2.233e-3) expression compared to TMEHigh. Moreover, TMEHigh compared to TMELow tumours were enriched for genes associated with tissue remodelling, and showed elevated MMP7 (P=3.051e-6), CLCL13 (P= 3.843e-4), and CXCL5 expression (P= 1.592e-6). PROGENy analysis suggested that the WNT, TRAIL, and JAK/STAT pathways were significantly enriched in TMEHigh, while the PI3K pathway was significantly upregulated in TMEMedium. Additionally, VEGF pathway activity was significantly upregulated in TMELow. TRANSFAC analysis measured by regulatory score (a measure of a transcription factor effect on gene expression) revealed that in TMELow, key transcription factors includes ETS2, NANOG, and MECP2 (regulatory score: 1.97, 1.45, and 1.4, respectively). In TMEMedium, transcription factors HIF1A, PPARA, and CDX2 were identified (regulatory score: 2.52, 2.08 and 1.96, respectively). In TMEHigh, TRANSFAC indicated activiation of transcription factors SMAD3, ETS2, and NFKB1 regulatory score: 2.56, 2.41, 2.24, respectively). Overall these findings highlight distinct molecular signatures across TME subtypes, and provide a deeper understanding of associated molecular mechanisms. Ongoing work is focused on validating key subtype specific genes and associated pathways using spatial proteomics. Prioritised targets will ultimately be interrogated in vivo, as novel subtype-specific treatments. 1. White et al. Ann Oncol. 2023;34(3):300–314. 2. Matys et al. Nucleic Acids Res. 2003;31.
TMIC-48. THREE DIMENSIONAL CANCER CELL-MONOCYTE CO-CULTURE SPHEROIDS RECAPITULATE IMMUNOSUPPRESSIVE AND PRO-TUMORIGENIC MICROENVIRONMENTS IN BRAIN METASTATIC BREAST CANCER
Authors: Gurung Sumiran , Berger Kyra , Rao Shreyas
Published: November 2024

Brain metastasis occurs in 15 % of breast cancer patients and is one of the major causes of breast cancer related morbidity. Brain metastatic breast cancer (BMBC) microenvironment is a heterogeneous niche of cellular and acellular components of which the innate immune cells, microglia, and macrophages, comprise the major fraction. However, the role of these innate immune cells in the tumor microenvironment (TME) in mediating cancer progression and response to therapy is not well understood. To address this need, herein, we developed three dimensional (3D) co-culture spheroids using brain metastatic triple negative breast cancer (TNBC) cell line MDA-MB-231Br or patient derived brain metastatic TNBC cell line F2-7 with THP1 monocytes. Under co-culture conditions MDA-MB-231Br co-culture spheroids assumed highly protrusive morphology with enhanced mesenchymal and proliferative characteristics. Additionally, phenotypic determination of cocultured macrophages revealed elevated levels of markers associated with alternatively activated macrophages i.e. CD163, CD206, and c-Myc. Furthermore, we found that under coculture conditions immunosuppressive and evasive markers PD1, PDL1, CD47 and CD24 were significantly upregulated. Similarly, F2-7 and monocyte cocultures also led to an immunosuppressive environment with elevated levels of M2 type markers and associated anti-inflammatory cytokines. We further found that the presence of M2 type macrophages in the microenvironment of BMBC spheroids led to acquired resistance against Paclitaxel, a clinically relevant chemotherapeutic drug against BMBC. In sum, in our coculture conditions, brain metastatic TNBC cells polarized monocytes into immunosuppressive and pro-tumorigenic tumor associated macrophages leading to a pro-tumoral microenvironment. Thus, 3D co-culture spheroids of BMBC cells and monocytes can serve as robust platform for studying cancer cell and innate immune cell interactions occurring in the BMBC environment.
TMIC-58. LEVERAGING VIRO-IMMUNOTHERAPY BY TARGETING IGF2-IGF1R SIGNALING
Authors: Noh MinHye , Kang Jin Muk , Nguyen Grace , Shim Ji Seon , Bueso-Perez Alberto J , Murphy Sara , Rivera-Caraballo Kimberly A , Otani Yoshihiro , Kim EunJu , Yoo Seung-Hee , Yan Yuanqing , Banasavadi-Siddegowda Yeshavanth , Nakashima Hiroshi , Chiocca E Antonio , Kaur Balveen , Zhao Zhongming , Lee Tae Jin , Yoo Ji Young
Published: November 2024

While an FDA-approved oncolytic herpes simplex-1 virus (oHSV) has shown therapeutic promise with superior safety, accumulating clinical data revealed that its therapeutic efficacy is observed in a small subset of patients. Here, we show that NFκB-mediated Insulin-like Growth Factor 2 (IGF2)/Insulin-like Growth Factor-1 Receptor (IGF1R) signaling pathway as a key modulator for oHSV therapy-mediated tumor resistance. RNA sequencing on the oHSV-infected primary glioblastoma (GBM) and breast cancer (BC) cells identified IGF2 as one of the top 10 upregulated secretomes. Transcriptomic analysis also revealed significant upregulation/enrichment of genes related to IGF2-IGF1R-MAPK pathway in oHSV-treated tumor cells. Infection with oHSV in GBM and BC cells up-regulated IGF2 transcription via direct binding of NFκB in the IGF2 promoter 3. Compromising the IGF2-IGF1R signaling pathway using IGF2 neutralizing antibody significantly increased oHSV-mediated tumor cell killing in vitro and mice survival in vivo. To circumvent this and promptly translate our findings in clinical settings, we have developed a novel oHSV, oHSV-D11mt, by incorporating modified IGF2R domain 11 into the parental oHSV genome to function as IGF2 decoy receptor as a single-agent modality. oHSV-D11mt specifically bind to IGF2 not IGF1, blocks oHSV-induced IGF2-IGF1R signaling, increases tumor cell killing, decreases oHSV-induced neutrophils/PMN-MDSCs infiltration, immune suppressive/proangiogenic cytokine secretion, and increases CD8+ cytotoxic T lymphocytes (CTLs) activation, resulting in increased virus propagation and mice survival. These findings suggest that IGF2-IGF1R signaling is a novel target to overcome oHSV therapy resistance and oHSV-D11mt could be used for improved viro-immunotherapy.
TMIC-59. ENTANGLED: THE INTERPLAY OF CELLULAR PLASTICITY AND NEURONAL SIGNALING IN BRAIN CANCER PROGRESSION
Authors: Senga Sasi S
Published: November 2024

The interplay between two recently proposed hallmarks of cancer, namely dedifferentiation/transdifferentiation and altered neuronal signaling, may dictate the spatiotemporal coupling between neurons and astrocytes. Synaptic activity correlates with brain cancer progression and further influences the directionality of metabolites. Through phenotypic plasticity, brain parenchymal cells may dedifferentiate in a manner that determines the directionality of metabolites, thereby altering plasticity and exerting selective pressure on the immune microenvironment within the brain. Therapeutic advancements in brain cancers, including maximizing surgical resection margins and Chimeric Antigen Receptor (CAR) T-cell therapy, have their own limitations. Thus, understanding the interplay between dedifferentiation/transdifferentiation and its role in dictating altered neuronal signaling to modulate the brain tumor microenvironment may provide novel targets for improving prognosis and limiting therapeutic toxicity.
TMIC-64. EXPLORING ULTRASTRUCTURAL PATHOLOGY IN GLIOMAS: INSIGHTS FROM RECENT STUDIES
Authors: Sulman Muhammad , Aboud Orwa
Published: November 2024

Glioma tumors arise from the glial cells making up to 40% of the brain tumors, and contributing up to 85% of the malignant tumors of the brain. Gliomas are a heterogeneous group of tumors classified based on various factors such as genetics, molecular profiles, etc. Glioblastoma, an aggressive form of gliomas, has incredibly low survival of only 15 months suggesting that thorough research, improved diagnostics, and therapeutics development is needed to improve the outcomes for patients with gliomas. These advancements require a critical analysis of the microenvironment of gliomas to understand the pathophysiology at play. Ultrastructural analysis (UA) using high-resolution electron microscopy offers crucial insights into the cellular and subcellular characteristics of gliomas, revealing organelle abnormalities and confirming molecular alterations. In this review, we focus on the recent developments exploring the ultrastructural pathology of gliomas. UA shows a detailed microenvironment of gliomas to reveal the abnormal presence of lipids in the intra- and extracellular environment of gliomas accompanied by the disrupted structure of the cellular membranes and organelles. The disruptions were noted in the blood vessels of the tumors displaying intriguing details regarding the formation of neovasculature and the absence of typical contents of vessel walls like endothelial cells. A comparative survey of the mitochondrial ultrastructure of gliomas with or without IDH1 mutations also reveals the role of UA in deciphering the impact of different mutations on the microenvironment level. Here, we also discuss the major implications of these findings regarding the research direction, diagnostic modalities, and therapeutic options.
TMIC-65. USING BIOINFORMATICS TO INVESTIGATE THE TUMOR MICROENVIRONMENT OF DIFFUSE MIDLINE GLIOMA
Authors: Kalpatthi Havisha , Cruz Andrea , Agnihotri Sameer
Published: November 2024

Diffuse midline gliomas, H3K27-altered (DMG) are highly malignant brain tumors with a 2-year survival rate of less than 10 percent. These tumors are primarily localized to the pons and thalamus regions of the brain. The cellular compartment of the tumor microenvironment (TME) which surrounds a tumor is composed of tumor and non-tumor cells, such as immune cells and oligodendrocytes. Communication between tumor cells and non-tumor cells in the TME can promote tumor growth and targeting communication networks could improve patient outcomes. Using a bioinformatics approach and publicly available pediatric DMG single-cell RNA-sequence

(scRNA-seq) data, several methods of communication (secreted signaling, cell-cell contact, and extracellular matrix) within the DMG TME were identified. Based on the high communication probability, outgoing and incoming networks from non-tumor cells and tumor cells were inferred, such as the Complement, Galectin, and CD96 pathways. Specific ligand-receptor interactions between non-tumor and tumor cells were also found, including LGALS9-HAVCR2, PDGFB/PDGFRA, and COL4A5/ITGAV. A subsequent literature review was performed to validate these identified pathways in other tumors, including gliomas, and inform future in vitro experiments with primary DMG cell lines.
TMIC-66. A NEW MOUSE MODEL THAT COMBINES DRIVER MUTATIONS TO ACCURATELY RECAPITULATE THE DIVERSE PHENOTYPES OF HUMAN GLIOBLASTOMA
Authors: Forsythe Devlin , Burn Olivia , Hermans Ian , J McConnell Melanie
Published: November 2024

To effectively beat glioblastoma, new pre-clinical models are needed: to dissect the biology of processes like migration and therapy resistance; to understand immune suppression and immunotherapy; and to use as a model for testing in therapeutic development. We have developed a new panel of glioblastoma mouse models, which engineer well-characterised glioblastoma driver mutations into an immortalised eGFP murine astrocyte-like cell. These “GEM-CLeM” are Genetically Engineered Mouse Cell Line Models. When transplanted intra-cranially into immunocompetent mice, these lines formed tumors with the key characteristics of glioblastoma – migratory and invasive, with rapid proliferation and infiltration of suppressive immune cells. The eGFP expression allowed detection of glioblastoma cells in situ and ex vivo, and the ability to readily distinguish tumor and stromal components of the tumors. Importantly, different combinations of driver mutations led to diverse tumor phenotypes. Loss of Pten with over-expression of mutant RAS generated tumors with 100% efficiency and the same time to tumor onset as the commonly used GL261 cell line. However, unlike GL261 tumors the Pten-RASV12 tumors had a highly invasive edge and extensive migration, and were full of pro-tumorigenic macrophages and monocytes, with very few T-cells or dendritic cells. Together, this recapitulated the phenotype of human wildtype IDH1 glioblastoma. In contrast, the very common EGFRvIII mutant did not drive tumor formation in combination with Pten loss. We also modelled mutant IDH glioblastoma by combining loss of p53 with overexpression of IDH1R132H. These were slow growing, densely packed tumors with extensive microglial involvement in addition to pro-tumorigenic macrophages. This GEM-CLeM system combines the benefits of the traditional genetically engineered mouse models with accessible, rapid, reproducible tumor formation. The GEM-CLeM lines are amenable to further genetic manipulation, so cells can be customised with other mutations to ask important biological questions in the presence of an intact tumor immune microenvironment.
TMIC-69. HUMAN ORGANOID TUMOR TRANSPLANTATION IDENTIFIES FUNCTIONAL GLIOBLASTOMA MICROENVIRONMENTAL COMMUNICATION MEDIATED BY PTPRZ1
Authors: Ge Weihong , Kan Ryan , Yilgor Can , Fazzari Elisa , Nano Patricia , Azizad Daria , Li Matthew , Ito Joyce , Tse Christopher , Tum Andrew , Scholes Jessica , Patel Kunal , Nathanson David , Bhaduri Aparna
Published: November 2024

Glioblastoma, the most aggressive form of primary brain cancer, is driven by both intrinsic cellular properties and extrinsic factors from the tumor microenvironment (TME). Here, we leverage our novel human organoid tumor transplantation (HOTT) system to explore how external cues modulate glioblastoma cell type specification, heterogeneity, and migration. Comparing the single-cell transcriptomes of HOTT and matched primary tumors, we found that HOTT recapitulates not only core features of major patient tumor cell types but also those of peritumor non-neoplastic cell types. This enables us to explore tumor-TME interactions and identify PTPRZ1, a receptor tyrosine phosphatase implicated in brain tumor migration, as a key mediator in intercellular communication. This finding was further confirmed by interrogation of published datasets from patients containing non-neoplastic microenvironmental cells. PTPRZ1 activation, resulting from the reactivation of developmental programs in both the tumor and its TME, highlights the relevance of the organoid system for modeling the GBM microenvironment. Moreover, HOTT uniquely allows for perturbations in either patient tumors or their microenvironments, or both, to verify PTPRZ1 function. Tumor knockdown of PTPRZ1 confirmed its role in promoting tumor migration and maintaining progenitor identity. Surprisingly, environmental PTPRZ1 knockdown in human cortical organoids before tumor transplantation inhibited tumor migration and promoted differentiation, even without direct tumor manipulation. Overall, this fundamental result demonstrates that a single treatment delivered to the tumor and its TME can exert opposite effects on the tumor. Consequently, it underscores the necessity of studying human glioblastoma within a human microenvironment context, such as HOTT, especially when evaluating therapeutic efficacy in GBM, as effective treatments must address both the tumor and its complex TME, rather than the tumor alone.
TMIC-79. IDH1-R132H REWIRES AUTOPHAGY IN THE GLIOMA MICROENVIRONMENT TO FACILITATE TUMOR PROGRESSION AND RESPONSE TO RADIATION
Authors: Banerjee Kaushik , Nunez Felipe , Mujeeb Anzar , Mausar Ava , Tronrud Claire , Zhu Ziwen , Taher Ayman , Kadiyala Padma , Carney Stephen , Garcia-Fabiani Maria Belen , Comba Andrea , Alghamri Mahmoud , McClellan Brandon , Faisal Syed , Nwosu Zeribe , Hong Hanna , Qin Tingting , Sartor Maureen , Ljungman Mats , Cheng Shi-Yuan , Appleman Henry , Lowenstein Pedro , Lahann Joerg , Lyssiotis Costas , Castro Maria
Published: November 2024

Gliomas are primary brain tumors with a poor prognosis. IDH1R132H exhibits a gain of function mutation leading to 2-hydroxyglutarate (2HG) production. We previously demonstrated, 2-HG mediated epigenetic rewiring is associated with better prognosis and its presence impacts several cellular functions including immune responses and enhanced DNA-damage response in IDH1R132H gliomas harboring p53 and ATRX loss-of-function mutations. Also, IDH1R132H elicits metabolic reprogramming which implies that IDH1R132H could modify the energetic state of gliomas with consequences in tumor biology and therapeutic responses. In this study, RNA-seq and ChIP-seq data revealed human and mouse IDH1R132H gliomas have downmodulated gene ontologies related to mitochondrial metabolism. This was paralleled by a decrease in glycolysis, rendering autophagy as a source of energy for IDH1R132H glioma cells. We also found that IDH1R132H-neurospheres have decreased levels of oxygen consumption and extracellular acidification rates, characteristic of an autophagic/quiescent state when compared to IDH1WT-neurospheres. Analysis of the autophagy pathway revealed that both human and mouse IDH1R132H gliomas have increased expression of pULK1-S555, pATG4b-S383, enhanced conversion of LC3I to LC3II, and decreased expression of pULK1-S757, p62 indicating that IDH1R132H cells have augmented autophagy activity compared to IDH1WT cells. Blocking autophagy selectively impairs the growth of IDH1R132H glioma cells in vitro. Targeting autophagy by systemic administration of synthetic protein nanoparticles packaged with siRNA targeting Atg7 sensitized IDH1R132H glioma cells to radiation-induced cell death, resulting in tumor regression, long-term survival, and immunological memory when used in combination with irradiation. Our results thus indicate that the metabolic changes in IDH1R132H glioma cells lead to compromised mitochondrial functions and increased autophagic activity which contributes to their radioresistance, representing a novel therapeutic target for IDH1R132H cells. *Corresponding author: Maria G. Castro: mariacas@med.umich.edu Equal contributions: KB-FN-AM-AM-CT Funding: This work was supported by National Institutes of Health/National Institute of Neurological Disorders & Stroke (NIH/NINDS) Grants: [R37-NS094804, R01-NS122165, R21-NS123879-01 awarded to MGC]; [R01-NS122378, R01-NS122234 awarded to PRL], Ian’s Friends & Chad Tough Foundation.
TMIC-82. CHARACTERIZATION OF GENETIC MARKERS AND BIOLOGICAL PROCESSES UNDERLYING THE TRANSITION OF GLIOBLASTOMA CELLS FROM A DIFFERENTIATED TO A STEM-CELL-LIKE STATE
Authors: Mekonnen Mahlet , Chandla Anubhav , Suresh Meera , Aguirre Brian , Gur Shir , Wadehra Madhuri , Yang Isaac
Published: November 2024

BACKGROUND

The main drivers of Glioblastoma (GBM) tumor growth are elevated angiogenesis and heightened characteristics of stemness. These driving factors make GBM an extremely invasive and aggressive tumor. While the GBM tumor microenvironment can be classified into several molecular subtypes, one common characteristic is the atypical induction of various signal transduction pathways including the receptor tyrosine kinases (RTKs), pro-angiogenic, and stem-cell-determining transcription factors, which all play important roles in GBM tumor formation, growth, and progression.

OBJECTIVE

To investigate the regulations of RTKs, pro-angiogenic, and stem-cell–determining transcription factors in two different tumor environments - monolayer cultures that induce a more differentiated state and neurosphere cultures that select for stem-like cells.

METHODS

Neurosphere- and monolayer-forming cell samples were cultured from four primary GBM cell lines including HK-336, HK-374, U87, and GS-154. RNA sequencing was performed at the Technology Center for Genomics and Bioinformatics at the University of California, Los Angeles. The transcriptomic data findings were validated by qPCR and western blot assays.

RESULTS

Our transcriptomic data revealed the significant upregulation of EGFR, PDGFR-α, VEGFA, and SOX2 in the neurosphere-forming HK-336, HK-374, U87, and GS-154 primary GBM cells. Consistent with the transcriptomic data, western blot, and qPCR assays demonstrated significantly higher expressions of these genes in the neurosphere-forming compared to the monolayer-forming GBM cells. Neurosphere-forming GBM cells possess stem-cell-like characteristics with the ability to self-renew and differentiate via the induction of various genes and disease progression pathways.

CONCLUSIONS

Neurosphere-forming GBM cells demonstrate upregulation of SOX2, EGFR, PDGFR-α, and VEGFA in RNA sequencing, western blot, and qPCR assays. Multipotent drug therapies designed to target multiple gene regulation pathways involving RTKs, pro-angiogenic, and stem-cell-determining transcription factors may be necessary to effectively treat GBM. Understanding the underlying mechanisms driving these aberrant cellular pathways could provide valuable insights for developing targeted therapies against this aggressive brain cancer.
TMIC-83. ABERRANT EGFR ACTIVATION SUPPORTS ENHANCED TUMOR-MYELOID CROSSTALK AND IMPAIRS EFFECTOR T CELL INFILTRATION AND FUNCTION IN GLIOBLASTOMA
Authors: Pioso Marissa , Li Marissa , Sanchez Julio , Tamayo Andrea , Grausam Katie , Breunig Joshua , Nathanson David , Prins Robert
Published: November 2024

Glioblastoma (GBM) is known to display a highly suppressive tumor immune microenvironment (TIME) dominated by myeloid cells with low T cell infiltration. Aberrant epidermal growth factor receptor (EGFR) activation is observed in ~50% of GBM patients via amplification and the constitutively active EGFRvIII variant, however the impact of EGFR activation on the brain TIME and T cell-mediated anti-tumor immunity remains poorly understood. Single cell transcriptomic and receptor-ligand interaction analyses of paired newly diagnosed GBM tumor and intratumoral CD45+ immune cells revealed increased crosstalk between EGFR activated tumors and EGFR ligand-expressing tumor-associated myeloid cells. Therefore, a novel orthotopic murine EGFRvIII-expressing glioma model wherein the mEGFRvIII gene is under a tetracycline-off system (MADR-mEGFRvIII) was leveraged to identify how EGFR signaling impacts overall TIME composition and the phenotype of intratumoral and systemic T cells in vivo. Temporal flow cytometric immunophenotyping following tumor or mock (saline) implantation identified no significant shift in T cell infiltration within the brain or cervical lymph nodes (cLNs). In contrast, one week of doxycycline-mediated genetic EGFR ablation conferred a significant survival benefit and was associated with enhanced intratumoral T cell infiltration as well as diminished tumor-associated F4/80+ myeloid cells compared to vehicle treated animals. Further characterization revealed genetic EGFR ablation enhanced recruitment of CXCR3+, TNFα+, and TIGIT+ CD4 T cells to the tumor, while the prevalence of these populations contracted in CD4 and CD8 T cells within the draining cLNs. In vitro, pharmacologic EGFR inhibition enhanced antigen-specific Pmel-1 T cell killing of gp100-expressing MADR-mEGFRvIII tumor cells and was associated with increased IFNγ secretion by ELISA. Collectively, our work demonstrates that aberrant EGFR signaling promotes an immunosuppressive brain TIME with reduced T cell infiltration potentially via tumor-myeloid signaling. Targeting this oncogenic axis may therefore reprogram the TIME and directly enable enhanced trafficking of effector T cells to the tumor.