bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract

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&lt;0.05), macrophage (p&lt;0.05) and microglia infiltration (p&lt;0.01) within the TME compared to control and sham surgery animals. By 10 days after LITT, significant infiltration by CD8 T cells (p&lt;0.0001) and M1 macrophages (p&lt;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&lt;0.0001) and TCM (p&lt;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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract

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

Abstract

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

Abstract
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

Abstract
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.
TMIC-56. LONGITUDINAL SINGLE CELL RNA SEQUENCING UNVEILS PHENOTYPIC CO-OPTION AND LINEAGE TRAITS OF MESENCHYMAL GLIOBLASTOMA AND GLIOMA-ASSOCIATED FIBROBLASTS
Authors: Liang Jiyong , Huo Longfei , Xu Jihong , Saidane Madhuri , Chen Xiaohua , Zhang Katherine , Sharma Pratibha , Kuo Truc , Norberg Lisa , Buchold Gregory , Wathoo Chetna , McDonald Malcolm , Lang Frederick , Rao Ganesh , Puduvalli Vinay 
Published: November 2024

Abstract

BACKGROUND
Adult gliomas exhibit a high level of heterogeneity presenting a substantial challenge hampering development of targeted therapies. Bulk RNAseq studies have identified a mesenchymal gene expression signature across the glioma continuum, based on which a mesenchymal subtype of glioblastoma (MES-GBM) has been described. However, the mesenchymal phenotype of gliomas remains inconsistently defined and the origin of the mesenchymal signature has been debated). Single cell transcriptomic profiling has allowed resolution of gene expression profiles of tumor and non-tumor cells, leading to recent identification of cancer-associated fibroblasts in gliomas and improved understanding of the mesenchymal gene expression signature.

METHODS
We performed longitudinal scRNAseq analysis of organotypic human glioma slices from 8 patients cultured ex vivo for up to 3 weeks.

RESULTS
We found 2 of the 6 IDHwt glioblastomas exhibit the mesenchymal phenotype based on Consensus Methylation Profiling (NIH) and scRNAseq analysis, annotated as Mes-1 and Mes-2. The gene expression profiles of mesenchymal glioblastomas show limited concordance with that of either the proneural or classical glioblastoma signatures or of cell types in physiological human brain, with Mes-2 deviating more extensively from non-mesenchymal gliomas. Instead, single cell transcriptomics suggests a hyper-secretory phenotype and overlaps broadly with that of cancer-associated fibroblasts (CAFs) and both express FGF2, other growth factors, and cytokines. Notably, Mes-2 cells express minimal to no levels of genes associated with brain cell lineages, such as PTPRZ1 and SCG3, which are abundantly expressed in other glioma subtypes.

CONCLUSIONS
Taken together, our data reveals extended depth of glioma heterogeneity that distinguishes MES-GBM from other glioma subtypes and suggests a phenotypic and lineage linkage between MES-GBM and glioma-associated fibroblasts. Thus, new diagnostic and treatment strategies leveraging auto- and paracrine mechanisms underpinning tumor and non-tumor interactions including disruption of the FGF2-FGFR1 circuitry may apply specifically to MES-GBM.