bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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.
TMIC-60. BRATS-PATH: ASSESSING HETEROGENEOUS HISTOPATHOLOGIC REGIONS IN GLIOBLASTOMA
Authors: Thakur Siddhesh , Faghani Shahriar , Moassefi Mana , Baid Ujjwal , Chung Verena , Pati Sarthak , Innani Shubham , Baheti Bhakti , Albrecht Jacob , Karargyris Alexandros , Kassem Hasan , Nasrallah Maclean , Ahrendsen Jared , Barresi Valeria , Gubbiotti Maria , Lopez Giselle , Lucas Calixto-Hope , Miller Michael , Cooper Lee , Huse Jason , Bell William , Bakas Spyridon
Published: November 2024

Glioblastoma, the most common malignant primary adult brain tumor, poses significant diagnostic and treatment challenges due to its heterogeneous molecular and micro-environmental profiles. To this end, we organize the BraTS-Path challenge to provide a public benchmarking environment and a comprehensive dataset to develop and validate AI models for identifying distinct histopathologic glioblastoma sub-regions in H&E-stained digitized tissue sections. We identified 188 multi-institutional diagnostic slides of glioblastoma (IDH-wt, Gr.4) cases, from the TCGA-GBM and TCGA-LGG data collections, following their reclassification according to the 2021 WHO classification criteria. Sub-regions were selected according to distinctive morphology of histopathologic features and included aggressive tumor biology and areas consistent with potential treatment effect. Selected sub-region annotations included cellular tumor, geographic necrosis, cortical infiltration, pseudopalisading necrosis, microvascular proliferation, white matter penetration, regions dense with macrophages, leptomeningeal infiltration, and presence of lymphocytes. We obtained 107,340 patches of size 512x512 from the 9 sub-regions. A global network of board-certified expert neuropathologists defined and followed a systematic annotation protocol based on clinical definitions and only delineated sub-regions with high confidence, thus ensuring high-quality standardized data. Each tissue section was assigned to an annotator-approver pair, with the annotator delineating sub-regions and the approver ensuring the consistency of the annotations. By crowdsourcing annotations, the BraTS-Path challenge harnesses the collective expertise of clinical neuropathologists and fosters a collaborative environment to advance the neuro-oncology field. The anticipated developed algorithms are expected to integrate state-of-the-art computational methods, achieving high accuracy in identifying diverse histopathologic features and advancing clinical decision-making processes. The BraTS-Path challenge aims to bridge the gap between research and clinical practice by promoting the development of AI-driven tools for precise tumor characterization. This collaborative effort can significantly enhance our understanding of glioblastoma, improve diagnostic accuracy, and inform treatment strategies, thereby contributing to better patient outcomes.
TMIC-61. DEVELOPMENT OF A NOVEL NOTCH-TARGETING ONCOLYTIC HSV FOR GBM TUMORS
Authors: Vazquez-Arreguin Karina , Otani Yoshihiro , Rivera-Caraballo Kimberly , Kaur Balveen
Published: November 2024

Glioblastoma (GBM) is the most aggressive brain malignancy, which despite continuing worldwide efforts to develop new therapies, remains a deadly disease with limited treatment options. The NOTCH signaling pathway is often hyperactive in GBM tumors, and its activity contributes to tumor progression and resistance to 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 following virotherapy contributes to tumor regrowth and induces recruitment of monocytic MDSC cells into the tumor, which limit optimal immune activation to kill tumor cells. For this study, we have discovered a NOTCH interfering intracellular ligand (NIIL) that blocks NOTCH activation from inside glioma cells. RNA sequencing shows the expression of NIIL downregulates NOTCH signaling in vitro. To evaluate the effect of NIIL on virotherapy, we generated an oncolytic herpes simplex virus (oHSV) that also encodes for NIIL. This virus showed a slower spread in vitro but improved therapeutic efficacy in vivo in multiple models of intracranial tumors. In immune competent mice there was a significant improvement in the survival of mice treated with the NIIL-encoding virus, with a fraction of mice showing a complete response after a single treatment. Single-cell RNA sequencing analysis of immune infiltrates is ongoing to understand how NIIL impacts tumor microenvironment and anti-tumor immune therapy.
TMIC-81. AN IMMUNOSUPPRESSIVE MACROPHAGE NICHE MAY DRIVE T CELL DYSFUNCTION AND IMMUNOTHERAPY RESISTANCE IN MELANOMA BRAIN METASTASES
Authors: Sun Lu , Ding Lizhong , Shiuan Eileen , Lee Alexander , Kim Won , Trembath Dimitri , Moschos Stergios , Hugo Willy , Prins Robert
Published: November 2024

Nearly half of metastatic melanoma patients develop brain metastases (MBM). Local therapies like surgery and radiotherapy provide limited survival benefit. While targeted therapies and immune checkpoint inhibitors (ICI) improved outcomes in patients with asymptomatic MBM, the prognosis is still grim for the majority of patients. To decipher resistance mechanism to immunotherapy in MBM, we integrated datasets from both single-cell RNAseq (scRNAseq) and spatial transcriptomics (Visium). Our scRNAseq analysis of 25 MBM patients (7 treated pre-surgery with ICI) showed that lymphoid cells increased by 8.1-fold post-ICI (p=0.006). Notably, 34% of these were CXCL13+CXCR6+ putative tumor-reactive T cells (TR-T), known for their critical role as tumor-killing effectors. This led us to investigate why, despite their prevalence, TR-T don’t translate to effective immunotherapy in MBM. Our scRNAseq interactome analysis revealed that ICI intensified IFN-II interactions between TR-T and interferon-stimulated macrophage/microglia (Mφ.ISG). However, it also boosted immunosuppressive interactions, like the TIGIT-NECTIN2 interaction, which increased 20 fold, potentially diminishing TR-T’s effectiveness. To overcome the lack of spatial context in scRNAseq, we applied spatial transcriptomics on 27 MBM sections, including 12 post-ICI treatments. Remarkably, TR-T were primarily found (63%) in proximity with Mφ.ISG cells—not with tumor cells. These TR-T-Mφ.ISG niches were 1.7 times more prevalent in ICI-treated samples; these niches are rich in chemokine receptors like CCR1 and immunosuppressive molecules like NECTIN2. This suggests that Mφ.ISG cells are chemotactically attracted to TR-T, forming a barrier that impedes TR-T’s tumor interaction and impairs their functionality. Our current efforts focus on pinpointing key pathways enriched within TR-T-Mφ.ISG niches, particularly in ICI-treated MBM. We are also assessing the clinical impact of these niches in relation to patient survival. Our goal is to elucidate how specific immunosuppressive subsets and molecules within the MBM microenvironment restrict TR-T effectiveness, with the ultimate objective of enhancing ICI efficacy for these patients.
TMOD-01. EGFR AMPLIFICATION ACCOMPANIES ADAPTATION TO MITOGEN-INDUCED DEFECTIVE MITOSIS IN PDGFA
Authors: Meode Mathieu , Omairi Hiba , Sait Sarah , Black Sophie , Cairncross J Gregory
Published: November 2024

Using a newly described in vitro murine model of GBM [Bohm et al., Neuro-Oncol 2020 and Omairi et al., Neuro-Oncol 2023] in which P53 null neural progenitor cells (NPCs) divide abnormally and evolve a GBM-like genome during exposure to Platelet-Derived Growth Factor-AA (PDGFA), we asked how a brain-abundant mitogen could transform NPCs. By analyzing gene and protein expression over time, we found that PDGFA fails to induce the transcription of kinetochore and spindle assembly checkpoint genes, while simultaneously driving NPCs to enter mitosis. These dual effects caused chromosome miss-segregation in continuously dividing NPCs; moreover, they occurred in both WT and null NPCs, although only null cells survived defective mitosis. These surviving cells gradually expanded in PDGFA accumulating both random and clonal chromosomal re-arrangements. Transcriptome analysis of NPCs in PDGFA revealed significant under-expression of Foxm1, the major regulator of kinetochore transcription, together with over-expression and phosphorylation of the immediate early response gene, FOS. Analysis of signalling downstream of PDGFRα identified the Ras-MAPK pathway, especially ERK, as responsible for FOS activation. As surviving null cells gradually expanded, they accumulated random and recurrent chromosomal rearrangements. Expansion and subsequent PDGFA-independent proliferation and tumorigenicity were associated with re-expression of Foxm1 and kinetochore proteins, and accompanied by over-expression of Egfr, an RTK-signature that defines human GBM. By stimulating proliferation without setting the stage for error-free mitosis, exposure to PDGFA transforms p53 null NPCs and generates Egfr amplified GBM-like cancer cells.
TMOD-02. PATIENT-DERIVED GLIOBLASTOMA ORGANOIDS AS REAL-TIME AVATARS FOR ASSESSING RESPONSES TO CLINICAL CAR-T CELL THERAPY
Authors: Wang Xin , Logun Meghan , Sun Yusha , Bagley Stephen J , Li Nannan , Desai Arati , Zhang Daniel Y , Nasrallah MacLean P , Pai Emily Ling-Lin , Oner Bike Su , Plesa Gabriela , Siegel Donald , Binder Zev A , Ming Guo-li , Song Hongjun , O’Rourke Donald M
Published: November 2024

Patient-derived tumor organoids have been increasingly leveraged for disease modeling and preclinical studies, but rarely in real-time to aid with the interpretation of patient treatment responses in the clinical setting. Whether tumor organoids can be applied to mirror clinical activity in patients and even to predict responses for personalized medicine remains uncertain. We recently demonstrated promising early efficacy signals in a first-in-human, phase 1 study of dual-targeting chimeric antigen receptor T cells (EGFR-IL13Rα2 CAR-T cells) in patients with recurrent glioblastoma (clinical trial identifier: NCT05168423). Determination of meaningful clinical efficacy, in particular for CAR-T cell therapy, may take months to manifest. Given this challenge, real-time analysis of patient-derived glioblastoma organoids (GBOs) could help to provide early assessment of clinical efficacy and guide patient management. We analyzed six sets of GBOs generated on the same timeline as the production of patients’ CAR-T cell products. GBOs demonstrated the presence and retention of both targeted tumor-associated antigens EGFR and IL13Rα2 by immunohistochemistry. These GBOs were then treated with the same autologous CAR-T cell products received by patients in our phase 1 study in parallel with patient treatment. CAR-T cell treatment led to target antigen reduction assessed by immunohistochemistry and flow cytometry. Furthermore, we confirmed cytolysis of tumor cells in GBOs by cleaved caspase-3 immunostaining and by co-culture in the Axion Maestro Cellular Impedance platform. Importantly, the degree of cytolysis ex vivo significantly correlated with CAR-T cell engraftment detected in patients’ cerebrospinal fluid (CSF) for all six patients. Furthermore, cytotoxic activity of CAR-T cells as measured by TNFα, IL-2, and IFNγ release kinetics in GBOs mirrored cytokine levels in patient CSF samples over time. Our findings highlight a unique trial design and suggest GBOs as a valuable platform for real-time assessment of CAR-T cell bioactivity and for insights into immunotherapy efficacy.
TMOD-04. SAFETY AND EFFICACY DATA FROM A CLINICAL TRIAL OF SONODYNAMIC THERAPY WITH 5-ALA HCL ORAL SOLUTION AND CV-01 DELIVERED WHOLE HEMISPHERIC LOW-INTENSITY NON-ABLATIVE ULTRASOUND IN PET FRENCH BULLDOGS WITHDE NOVO NATURALLY OCCURRING HIGH-GRADE GLIOMA
Authors: Elizabeth Pluhar G , Arnold Susan , Ling Jeremy , Hunt Matthew , Agarwal Vijay
Published: November 2024

To date, no animal model has been able to accurately test emerging therapeutics for high-grade glioma. Pet dogs’ high-grade gliomas closely mimic human tumors histopathologically and radiologically as well as with poor clinical survival. Dogs with these tumors have notably shorter survival than humans, with French Bulldogs showing the shortest survival of all breeds affected, aiding swift therapeutic evaluations. We report the first use of the novel CV-01 device delivering non-ablative low-intensity diffuse ultrasound (LIDU) with oral 5-ALA as a recurrent high-grade glioma treatment in a veterinary safety and efficacy trial. This Phase 1 study focused on safety, with secondary outcomes assessing histopathological efficacy markers (CC3, Iba1) and overall survival. After one SDT treatment pre-resection, treatments every 3-4 weeks were administered to eight French Bulldogs, totaling 60 administrations (average of 7.5 total treatments/patient). The control group matched breed, age, and histopathologic diagnosis. Average age was 6.35 years (range: 4.8 – 7.8 years), slightly older than controls (5.9 years, range: 3.0 - 9.9 years). No complications or toxicities were noted, and MRI scans showed no damage to the normal brain. Histopathologic analysis from four canine tumor samples revealed increased apoptosis and immune activation markers post-SDT (e.g. cleaved caspase 3 [CC3] and ionized calcium-binding adaptor molecule 1 [Iba1]), indicating selective tumor cell death. Median overall survival in the treatment group reached 205.5 days (6.8 months) compared to 55 days (1.8 months) in controls, with the longest survival at 587 days (versus 213 in controls). Although preliminary, these results affirm the safety of 5-ALA-mediated SDT in canine glioma patients. A multi-center, First-In-Human trial in recurrent high-grade glioma recently completed enrollment, aiming to further validate canine trials for emerging high-grade glioma therapeutics.
TMOD-06. ESTABLISHMENT OF PITUITARY NEUROENDOCRINE TUMOR (PITNET) ORGANOID MODELS
Authors: Maeda Sachi , Ohka Fumiharu , Kanamori Fumiaki , Okumura Yuka , Takeuchi Kazuhito , Nagata Yuichi , Yamaguchi Junya , Harada Hideyuki , Shimizu Hiroki , Takido Yuhei , Saito Ryuta
Published: November 2024

Pituitary neuroendocrine tumors (PitNETs) are tumors that originate from the anterior pituitary gland. While they are mostly benign, they can cause various clinical symptoms due to hormonal secretion abnormalities. PitNET can be broadly categorized into three lineages, each of which has its differentiation controlled by specific transcription factors. Treatment options for PitNET include surgical resection and pharmacotherapy, but available drugs are limited, and their efficacy markers remain unclear. To develop novel drugs for PitNET and identify their efficacy markers, it is necessary to develop new research models. In recent years, organoid models have garnered attention as novel research models for various cancer types. In this study, we established novel organoid models derived from PitNET patients and analyzed their histological and endocrinological characteristics. Surgical specimens from 4 cases of somatotroph adenoma, 1 case of double adenoma, 1 case of gonadotroph adenoma, and 1 case of corticotroph adenoma were minced in dissection medium and embedded in Matrigel basement membrane matrix respectively. The organoids were cultured under 5% CO2 at 37°C using media supplemented with various growth factors. After 28 days of culture, formalin-fixed paraffin-embedded sections were prepared to examine histological homology with patient tumors. Hematoxylin and eosin staining results showed that all organoids maintained cell morphology and tissue structure similar to the original tumors. Immunohistochemical staining for markers specific to each PitNET revealed similar staining patterns between the organoids and the original tumors. Furthermore, analysis of hormone levels in the culture medium showed that hormone production capabilities, such as growth hormone and follicle stimulating hormone, were maintained even after long-term culture. In this study, we successfully established PitNET organoid models that retain histological and endocrinological characteristics. These models are expected to serve as useful research tools for elucidating the molecular mechanisms of PitNET and developing novel therapies targeting critical molecular abnormalities.
TMOD-10. ESTABLISHMENT OF ADULT AND PEDIATRIC BRAZILIAN PRE-CLINICAL MODELS FOR HIGH-GRADE GLIOMAS
Authors: Baroni Mirella , Megid Raquel , Tshikawa Leticia , Martin Ana Carolina , Duarte Terence , Escremim Flavia , Gomes Izabela , Moreno Daniel , Garcia Felipe , Teixeira Gustavo , Lopes Luiz Fernando , Mançano Bruna , Polverini Allan , Lombardi Ismael , Júnior Carlos , Reis Rui , Teixeira Silvia
Published: November 2024

High-grade gliomas (HGG) are the most aggressive brain tumors, necessitating robust and representative preclinical models for therapeutic research. We established primary cultures and patient-derived xenograft models from three pediatric high-grade gliomas (pHGG) and one adult IDH wild-type glioblastoma (GBM IDHwt) Brazilian patients. The first pHGG case, from an 11-year-old with PDGFRA (c.1977C>G) and TP53 (c.637C>T) mutations, led to the development of the HCB570 primary culture, PDX and patient-derived orthotopic xenograft (PDOX) OXP9. The second pHGG, from an 11-year-old with CMMRD syndrome and MSH6 germline mutation, resulted in the HCB589 primary culture, PDX and PDOX OXP22. The third pHGG case, from a 14-year-old with diffuse midline glioma harboring an H3F3A mutation, established the HCB604 primary culture and PDX OXP32. The HCB612 primary culture and PDX GBM1, were established from an adult patient with GBM IDHwt, TERT mutated, with no MGMT promoter methylated. The primary cultures showed varied proliferative rates, with an average doubling time of 55 hours. All cultures demonstrated colony-formating capability, with an average IC50 of 750µM for temozolomide (TMZ) exposure. Immunohistochemical and EPIC methylation profiles confirmed that the PDX models closely resemble the patient features. Our establishment of diverse preclinical models, including those derived from patients with rare genetic conditions such as CMMRD, provides valuable insights into HGG pathogenesis and potential therapeutic strategies. The present Brazilian patient-derived models enrich glioma research inclusion diversity and foster future personalized treatment approaches.
TMOD-11. ADVANCED MRI ANALYSIS TO ASSESS CHEMO-RADIATION RESPONSE IN PEDIATRIC EPENDYMOMA MODELS
Authors: Eggers Lucas , Song Jane , Steiner Jenna , Pierce Angela , Griesinger Andrea , Foreman Nicholas , Serkova Natalie
Published: November 2024

Pediatric Ependymoma (EPN) is an aggressive brain tumor for which the benefits of chemo-radiation (CR) therapy have yet to be established. Our team has previously reported behavior patterns of EPN, including high tumor cellularity, cytological anaplasia, high mitotic index, tumor necrosis, and the presence of inflammatory cells such as M2-type macrophages. Recently, we have established advanced MRI protocols and computational algorithms for cell- and vessel-size imaging. The goal of this study is to develop advanced MRI analysis for CR response biomarkers (including tumor size, cell- and vessel-size correlates, and texture analysis) in mice with patient-derived xenografts (PDX) of pediatric EPN. Female severely immune deficient (SCID) mice were used for intracranial orthotopical inoculation of disaggregated tumors from pediatric EPN patients. All multi-parametric MRI sequences (fast spin echo, FLAIR, diffusion weighted, quantitative T2- and r2*-maps) were acquired on a Bruker 9.4 Tesla MRI scanner prior to CR, followed by 2 days and 2 weeks after CR treatment. All radiation treatment was performed using an animal image-guided precision XRAD irradiator (2Gyx5days), using MRI and CT guided EPN localization with added 30 mg/kg 5-fluoracil. All MRI analysis was performed using Bruker ParaVision software and MATLAB based modeling. The sensitivity of T2w-MRI scans was 0.2 mm for the smallest tumor detected; the median tumor volume at baseline was 2.5 mm3. Untreated mice showed rapid progression of tumor growth from small to intermediate to large EPN. The increased tumor sizes in untreated EPN were characterized by low ADC, highly elevated blood vessel density and large tumor cell size. A significant decrease in vessel size density and an increase in inflammatory cells were seen as soon as 2 days after CR therapy. The late response (2 weeks post CR) is characterized by increased ADC values and decreased cell size, resulting in significantly decreased tumor volumes.
TMOD-13. IDENTIFYING MOLECULAR SIGNATURES OF PPM1D-MUTANT DIPGS
Authors: Kundu Ipsita , Kebede Adam , Morin Eric , Langhnoja Jaldeep , Arounleut Phonepasong , Bandopadhayay Pratiti , Phoenix Timothy
Published: November 2024

Diffuse Intrinsic Pontine Glioma (DIPGs) are universally fatal pediatric tumors with a median survival of only 9-12 months. PPM1D-truncating mutations (PPM1Dtr), which promote its phosphatase activity and suppress TP53 function, are found in approximately 15% of DIPG tumors. PPM1Dtr are mutually exclusive to TP53 mutations, and present unique therapeutic opportunities to reactivate wild-type TP53. Another distinct feature of PPM1Dtr DIPGs is the co-occurrence of PI3K/AKT/mTOR pathway alterations, such as PIK3CA. There is an unmet need to elucidate the underlying molecular mechanisms of DIPG and identify novel therapeutic vulnerabilities that will improve outcomes of this devastating tumor. We hypothesize that PPM1D-mutant DIPGs can be therapeutically targeted by reactivating the p53 signaling pathway and other complementary pathways which cooperate with PPM1Dtr. To investigate this, we have generated Trp53- and Ppm1d-mutant isogenic DIPG murine models to determine how specific oncogenic alterations function in DIPG formation, growth and treatment sensitivity. Current studies aim to examine differences in sensitivity to standard of care treatment modalities and leverage next generation sequencing techniques to uncover additional vulnerabilities. We found notable differences upon addition of PIK3CA mutations in the context of PPM1Dtr DIPG tumor formation, growth, survival, and lineage identity. Ongoing studies using whole genome transcriptomics to compare tumor models will provide insight into the molecular signals and cell state changes driven by individual genetic alterations. Furthermore, by comparing isogenic TP53 and PPM1Dtr mutant DIPG models, we aim to further delineate the molecular mechanisms that drive differential sensitivity of TP53 wild-type tumors. The immune-competent nature of our murine models also provides us with a unique opportunity to delineate tumor immune microenvironment changes. Leveraging this information and our novel murine models, our long-term goal is to develop combinatorial therapeutic strategies to improve outcomes for this deadly disease.
TMOD-15. TOWARD BRAIN CANCER ORGANOID-INFORMED PRECISION MEDICINE FOR GLIOBLASTOMA
Authors: Storey Claire , Gately Lucy , Drummond Kate , Gogos Andrew , Mouradov Dmitri , Christie Michael , Brasier Helen , Priest Emma , Fakhri Alana , Gibbs Peter , Sieber Oliver
Published: November 2024

BACKGROUND

Improved preclinical models for glioblastoma are urgently needed to enhance the translation of research to clinical applications. Patient-Derived Tumor Organoids (PDTOs) offer a promising model that can be generated quickly. Here we explored their feasibility.

METHODS

Primary (n=16) and recurrent (n=2) PDTOs were established using the method described in Jacob et al. Cell. 2020 by dissecting tumours into small fragments, preserving their cytoarchitecture. Histological features of the parent tumour were compared to the matched PDTOs by a neuropathologist. To assess the molecular fidelity, comprehensive whole genome sequencing compared matched tumor tissue, PDTOs, and corresponding normal blood samples (n=11), along with RNA sequencing of matched tumor tissue and PDTOs (n=10). We assessed PDTO responses to regorafenib (n=18) and conventional TMZ (n=9), lomustine (n=9) and 10 Gy of ionizing radiation (IR) (n=6) using the automated imaging system IncuCyte S3® to evaluate whole spheroid brightfield and invasion areas following drug/IR treatment and assess viability.

RESULTS

We achieved a 100% establishment rate of primary GBMs using same-day procedures and an 80% rate if held under 3 days. Histological features were retained in the PDTO. Molecular overlap was greater than 90%, along with a high concordance in gene expression. PDTOs exhibited notable resistance to temozolomide and lomustine, however, showed a very modest response to IR and a moderate response to regorafenib. IncuCyte S3® analysis revealed discernible changes in whole spheroid brightfield and invasion area for regorafenib-treated PDTOs. Clinically relevant concentrations of regorafenib reduced GBM cell proliferation (Ki67 staining). Beyond this concentration, regorafenib exhibited efficacy in eliminating GBM cells.

CONCLUSION

PDTOs recapitulate the molecular and histological features of the original tumor and may enable the prediction of individual patient responses to therapies and guide more effective treatment selection. We are now applying our established methodology to assess a new set of promising drug candidates.
TMOD-24. DEVELOPMENT OF A 3D ORGANOTYPIC EXPLANT PLATFORM TO STUDY ONCOLYTIC VIRUS TREATMENT RESPONSE IN GLIOBLASTOMA
Authors: Abou-Mrad Zaki , Yu Kenny , Tabar Viviane
Published: November 2024

BACKGROUND

Oncolytic viruses (OVs) are a promising strategy to induce an anti-tumor immune response in the lymphocyte-depleted environment of glioblastoma (GBM), however the mechanism of their oncolytic function is not well understood. Immune-intact experimental systems would be highly valuable in dissecting immune versus direct viral contribution to oncolysis. Here, we report on the development of an immune-intact organotypic explant (OE) model, amenable to perturbations and T-cell activity monitoring.

METHODS

OEs were made from fresh tumor samples and cultured in groups of 4-5. CAN-3310 OVs and T-cells were injected into each OE under a dissecting microscope. Cultured OEs were either fixed for immunofluorescence (IF) or dissociated into single-cell suspensions for flow cytometry and 5’ single-cell RNA (scRNA) and TCR sequencing.

RESULTS

Cultured OEs maintain the cellular density of the original tumor in both short- and long-term cultures. Ki67 IF shows that tumor cells retain their proliferative potential, allowing for renewal and repopulation. The TME, mainly microglia and macrophages, is maintained throughout culture. IF imaging of OV-treated OEs, 7 days post-treatment, shows significant GFP positivity in tumor cells demonstrating the feasibility of using OEs as an ex vivo model with OV. Additionally, autologous T-cells from the patient’s blood can survive in OEs, completing the TME with the equivalent of circulating T-cells. Finally, scRNA-seq of the original tumor and cultured OEs showed that the different cell types were represented at all time points. Tumor cell meta-module analysis showed an increase in hypoxic signatures recapitulating features of in vivo tumor cell growth.

CONCLUSIONS

OEs maintain tumor architecture and TME. OE co-culture with autologous T-cells allows us to better understand the oncolytic mechanisms of OVs. Future endeavors will include identifying expanded T-cell clones to create an OV-specific TCR repertoire. This OE system provides a new tool as an ex vivo platform to dissect glioma responses to therapy in a purely human system.
TMOD-26. GENERATION OF A CHROMOSOME 8 GAIN;NF1-/- HIPSC-DERIVED SCHWANN CELL PRECURSOR MODEL
Authors: Oztosun Gorkem , Acar Simge , Bhatia Himanshi , Neilson Amber , Cui Xiaoxia , Hirbe Angela
Published: November 2024

Neurofibromatosis type 1 (NF1) is an autosomal dominant cancer predisposition syndrome caused by a germline mutation in the NF1 gene. The loss of the second copy of NF1 within Schwann cell precursors (SCPs) contributes to the formation of plexiform neurofibromas (PNs) found in 30-50% of NF1 patients. PNs can progress into malignant peripheral sheath tumors (MPNSTs), an aggressive form of cancer and a leading cause of mortality in NF1 patients. Our lab previously demonstrated frequent chromosome 8 (Chr8) gain in NF1-MPNST patient-derived xenografts (PDX) and patient tumors. To investigate Chr8 gain’s role, we created isogenic NF1-/- iPSC cell lines with and without Chr8 gain. Trisomy 8 fibroblasts were obtained and single-cell cloned to establish wildtype (WT) and Chr8 gain populations. Washington University’s Genome Engineering & Stem Cell Center (GESC@MGI) reprogrammed these cells into human induced pluripotent stem cells (hiPSCs). Using synthetic gRNA and CRISPR/Cas9, NF1 knockout lines were generated. Four iPSC cell lines were differentiated into SCPs, and cell survival and proliferation were assessed using Incucyte cell survival and CellTiter-Glo® Assay. Fluorescence in situ hybridization (FISH) and karyotyping validated the human iPSCs. qPCR revealed high expression of iPSC markers OCT3/4 and SOX2 in all iPSC lines. After differentiation, SCP markers (GAP43 and ITGA4) were upregulated in WT, NF1-/-, and Chr8 gain;NF1-/- SCP lines, demonstrating successful differentiation. Furthermore, Chr8 gain; NF1-/- SCPs displayed enhanced proliferation and increased cell survival compared to WT and NF1-/- SCPs, suggesting improved characteristics in the presence of Chr8 gain. In summary, our research provides insights into Chr8 gain and NF1 loss effects on SCP differentiation, proliferation, and survival. Understanding these molecular mechanisms may contribute to developing targeted therapies for NF1.
TMOD-29. BETA INTEGRINS REGULATE MITOCHONDRIALLY-MEDIATED CELL MIGRATION IN H3K27M-DIFFUSE MIDLINE GLIOMA
Authors: Cartaxo Rodrigo , Nieblas-Bedolla Edwin , Niculcea Michael , Natarajan Siva , Adam Tiffany , Clausen Madison , Miclea Madeline , Siada Ruby , Schwark Kallen , Ji Sunjong , Gera Ananya , Doherty Robert , Galban Stefanie , Nuechterlein Nicholas , Filbin Mariella , Venneti Sriram , Hara Toshiro , Koschmann Carl
Published: November 2024

Infiltration into the brain tissue is one of the main features of diffuse midline gliomas (DMG), also characterized by the histone mutation H3K27M and overall survival of 12-15 months from diagnosis. To study which genes are critical for H3K27M-DMG tumor cell migration in the brain, we established a novel two-step pooled whole genome CRISPR-Migration screen of metastatic H3K27M-DMG (n=3) and glioblastoma (GBM, n=2) stem cells by using serum-free conditions and laminin-dependent cell attachment, mimicking cell migration on brain vasculature scaffolds. Genes involved in focal adhesion (ITGB1, CRKL, PARVA, PTK2, FERMT2) were identified as significantly restricting migration in all H3K27M-DMG lines. From those, only knockout of ITGB1 (ITGB1-KO) fully ablated in vitro migration in all models. In human tumor RNAseq, expression of ITGB1 correlated with higher glioma grade and worse survival specifically in H3K27M-DMG across pediatric brain tumors. In H3K27M-DMG scRNAseq, increased ITGB1 expression was found in cells with OPC-like/Mesenchymal cell signature associated with leading edge and microvasculature. Unexpectedly, ITGB1-KO led to the decreased expression of multiple mitochondrial NADH dehydrogenase genes, and reduced levels of glycolysis and oxidative phosphorylation (OXPHOS) activity. Preliminary mechanistic data linked this phenotype to increased mitochondrial fusion. Genetic (ITGB1-KO) and pharmacologic (anti-ITGB1 antibody AIIB2, CNS delivered) targeting of ITGB1 in a novel orthotopic model of metastatic H3K27M-DMG (UMPED83) led to reduced proliferation and significant extension of survival (p=0.0003 ITGB1-KO, and p=0.0006 AIIB2). Interestingly, ITGB1-KO in another orthotopic H3K27M-DMG model (SVZ) led to upregulation of other integrins (ITGB3 and ITGB5) without change in survival, resulting in susceptibility to treatment with the ITGB3/5 inhibitor Cilengitide (p=0.006). Treatment with AIIB2 and Cilengitide alone or in combination was ineffective in an H3WT adult GBM orthotopic model suggesting unique H3K27M-DMG targetability. In summary, targeting of integrin pathways offers untapped therapeutic opportunities in H3K27M-DMG by impairing tumor cell survival through altered mitochondrial dynamics.
TMOD-32. MURINE MODELLING OF THE STING PATHWAY IN GLIOMAS
Authors: Low Justin , Lang Patrick , Bowie Michelle , Baker Eric , Wertman Virginia , Fuller Rebecca , Gibson Emiley , Ashley David
Published: November 2024

The stimulator of interferon genes (STING) pathway is a critical component of innate antitumor immunity through the recognition of pathogenic cytosolic DNA and stimulating the production of type 1 interferons and pro-inflammatory cytokines. We have previously shown that STING signaling is silenced in the neoplastic compartment of the GBM tumor microenvironment, but remains intact in vascular and immune cells. In human patient derived cell lines, such silencing appears to be mediated by hypermethylation of a region of the STING promoter and can be reversed by treatment with both the DNA methyltransferase (DNMT) inhibitor Decitabine and DNMT1-selective inhibitor GSK-3685032. These results suggest that neoplastic cell STING promoter methylation represents a therapeutic vulnerability that can be exploited by epigenetic therapies to inflame the glioma tumor microenvironment. Murine models are critical tools for exploring the translational implications of STING silencing in vivo. Distinct mouse models have been developed to optimally represent specific aspects of human glioma biology. Here we characterize the STING pathway in commonly used syngeneic murine glioma models, including CT2A, GL261, SB28, and SMA560. We show that the expression of STING pathway components varies significantly across these lines. Importantly, unlike in humans, murine STING (mSTING) expression is not suppressed by promoter methylation and does not respond to decitabine treatment. Additionally, the mSTING protein is expressed in neoplastic cells in vivo. These results demonstrate a fundamental difference in epigenetic regulation of STING between human and murine glioma cells, and suggest that murine modeling of STING epigenetics requires the use of patient derived cell lines implanted in immunodeficient mice. Meanwhile, immune competent syngeneic models are appropriate for exploring the tumor immune microenvironment but would require non-epigenetic approaches (e.g. genetic knock-outs/knock-ins) for modulating STING expression. Humanized mice represent a promising emerging approach for faithfully recapitulating both epigenetic control and immune environment of human gliomas.
Evolution of NAC transcription factors from early land plants to domesticated crops
Authors: Kim Taehoon , Alvarez Javier C , Rana Divya , Preciado Jesus , Liu Tie , Begcy Kevin
Published: November 2024

NAC [NO APICAL MERISTEM (NAM), ARABIDOPSIS TRANSCRIPTION ACTIVATOR FACTOR 1/2 (ATAF1/2), and CUP-SHAPED COTYLEDON (CUC2)] transcription factors are key regulators of plant growth, development, and stress responses but were also crucial players during land plant adaptation and crop domestication. Using representative members of green algae, bryophytes, lycophytes, gymnosperms, and angiosperms, we expanded the evolutionary history of NAC transcription factors to unveil the relationships among members of this gene family. We found a massive increase in the number of NAC transcription factors from green algae to lycophytes and an even larger increase in flowering plants. Many of the NAC clades arose later during evolution since we found eudicot- and monocot-specific clades. Cis-elements analysis in NAC promoters showed the presence of abiotic and biotic stress as well as hormonal response elements, which indicate the ancestral function of NAC transcription factor genes in response to environmental stimuli and in plant development. At the transcriptional level, the expression of NAC transcription factors was low or absent in male reproduction, particularly mature pollen, across the plant kingdom. We also identified NAC genes with conserved expression patterns in response to heat stress in Marchantia polymorpha and Oryza sativa. Our study provides further evidence that transcriptional mechanisms associated with stress responses and development emerged early during plant land adaptation and are still conserved in flowering plants and domesticated crops.
Getting lucid about lucid dreaming
Authors: Cowan Robert
Published: November 2024

Lucid dreams are a distinctive and intriguing phenomenon where subjects apparently possess, inter alia, conscious knowledge that they are dreaming while they are dreaming. I here develop and defend a new model of lucid dreaming, what I call the ‘Dyadic Model’, according to which lucid dreams involve the tokening of both dreaming and non-dreaming states. The model is developed to successfully defend the Imagination Theory of dreams, according to which to dream is to imagine, against the underexplored objection that it cannot account for lucid dreams. Moreover, as I explain, a range of theories of dreaming have reason to adopt the Dyadic Model, and it has further potential applications, including to false awakening and emotional responses to dreams. Hence, getting lucid about lucid dreaming reveals general insights for dream theorizing.
Temporal Dynamics of Two Genetically Diverse Downy Mildew Mitochondrial Clades on Cucumber Cultivars in South Carolina and Florida
Authors: Mandal Mihir K. , Turechek William W. , Ikerd Jennifer L. , Wallace Emma , Quesada-Ocampo Lina , Kousik Chandrasekar S.
Published: November 2024

Downy mildew (DM, Pseudoperonospora cubensis) is an important disease that can significantly reduce yield of field-grown cucumbers in the US. Cucumber cultivars with resistance to DM were widely grown for >30 years. However, resistance broke down in 2004 resulting in severe DM epidemics. Large plot trials were conducted to characterize pathogen virulence and population dynamics on DM tolerant cultivars Peacemaker and Expedition and susceptible SMR-58 in Charleston, SC and Fort Pierce, FL in 2016 and 2017. Rapid DM development on SMR-58 was observed at both locations in 2016 and 2017. Disease progress on Expedition and Peacemaker were slow as compared to SMR-58 in Fort Pierce, FL in 2016. Very low to no DM was observed on Peacemaker or Expedition in Charleston in 2016. In 2017, all cultivars were affected in Charleston, but only SMR-58 had severe DM (95-100%) in Fort Pierce. Pathogen population analysis using DM specific mitochondrial clade (MC1 and MC2) markers showed differences between the P. cubensis populations across location and years on the cucumber cultivars. Both MC1 and MC2 were detected in cucumber plots in SC and FL. The most prevalent DM mitochondrial clade in SC during June 2016 was MC1 primarily on SMR-58 (95-100%). However, in July 2016 that population shifted towards MC2 (45-77%). This suggested that the DM populations can change relatively quickly when suitable environment favors. Mixed populations of MC1 and MC2 continued to be observed in following year in Charleston, SC in 2017. In Fort Pierce, FL a complete shift was observed from MC2 in 2016 to MC1 in 2017. Since both MCs are present in the southeastern United States either at the same time or possibly different times during the production year, cultivars with combined resistance to P. cubensis populations will be needed to reduce reliance on fungicides to manage DM.
Atrazine application shapes white leaf spot (Neopseudocercosporella capsellae) development on triazine-resistant canola
Authors: Ai Yan , You Ming Pei , Yan Guijun , Barbetti Martin John
Published: November 2024

Studies were undertaken on two atrazine-tolerant canola (Brassica napus) cultivars (moderately resistant Crusher TT and highly susceptible Thunder TT) to white leaf spot (WLS) (Neopseudocercosporella capsellae), to determine how atrazine application affects WLS development. There were significant main treatment effects in relation to percent leaf disease incidence (%LDI), percent leaf area diseased (%LAD) and percent leaf area collapsed (%LAC) for canola cultivar, disease development period (days-post-inoculation), atrazine spray (before or after inoculation), and %LDI for atrazine spray timing (0, 1, 3, 7 days), along with significant two- and three-way interactions between treatments. For %LDI across a 21-day period, Crusher TT showed greatest disease suppression from atrazine application 0 or 7 days before inoculation, and Thunder TT from application 0 days before and 7 days after inoculation. In terms of %LAD, Crusher TT showed greatest suppression from application at 0 and 7 days before inoculation, and Thunder TT from application 0 days before or 1 day after inoculation. In relation to %LAC, Crusher TT showed greatest suppression from application 7 days before inoculation and Thunder from application 0 days before or 1 day after inoculation. That the extent of suppression of WLS by atrazine application was substantial and that it varied with cultivar susceptibility, application timing, and that there was a strong interaction of cultivar x application timing, highlights potential opportunities for farmers to exploit better cultivar choices in conjunction with manipulating the timing of atrazine application to maximize WLS suppression and consequent canola yield.
Clinical Comparison of Endoscopic Posterior Lumbar Interbody Fusion and Open Posterior Lumbar Interbody Fusion for Treating Lumbar Spondylolisthesis
Authors: Yang Lihui , Du Peng , Zang Lei , An Likun , Liu Wei , Li Jian , Diao Wenbo , Gao Jian , Yan Ming , Zhu Wenyi , Yuan Shuo , Fan Ning
Published: November 2024

Study Design:

A retrospective case-control study.

Objective:

To compare the clinical efficacy of endoscopic (Endo) and open posterior lumbar interbody fusion (PLIF) in treating lumbar spondylolisthesis.

Background:

Endo-PLIF has emerged as a new technique for treating lumbar spondylolisthesis. We propose Endo-PLIF as an alternative method.

Materials and Methods:

Sixty-four patients with single-segment lumbar spondylolisthesis underwent Endo-PLIF (n = 39) or open PLIF (n = 25) treatment. Demographic data, perioperative parameters, and radiographic parameters were recorded. Clinical results were evaluated by Visual Analog Scale (VAS) and Oswestry Disability Index (ODI) scores. The fusion rate was evaluated by computed tomography at 12 months postoperatively. In addition, a case-control process was included to ensure unbiased comparisons.

Results:

The average operation time was longer in the Endo-PLIF group. Endo-PLIF showed advantages in reducing blood loss, shortening hospital stay, and early ambulation, but with a longer x-ray exposure time. Both VAS and ODI scores significantly improved in both groups, but the VAS for back pain was lower in the Endo-PLIF group. The radiographic results were similar in both groups. Three patients in the Endo-PLIF group had minor complications. Two patients in the open PLIF group experienced cerebrospinal fluid leakage. Both VAS and ODI scores significantly improved in both groups compared with preoperative scores, but the Endo-PLIF group showed more significant improvement at early follow-up (P < 0.05). There was no significant difference in interbody fusion rate between the two groups.

Conclusion:

Both Endo-PLIF and open PLIF are effective for treating single-segment lumbar spondylolisthesis. Endo-PLIF shows advantages in reducing blood loss, shortening hospital stays, and promoting early ambulation, with comparable fusion rates and patient satisfaction to open PLIF. Despite minor complications in the Endo-PLIF group and cerebrospinal fluid leakage in the open PLIF group, both procedures lead to significant improvements in pain and disability scores, with Endo-PLIF demonstrating more significant early improvements.
Recent advances in immunotherapy for small cell lung cancer
Authors: Ren Ziyuan , Shang Shijie , Chen Dawei
Published: November 2024

Purpose of review

This review aims to provide an overview of recent advances in immunotherapy for small cell lung cancer (SCLC), with a focus on the current status of immune checkpoint inhibitors (ICIs), novel combination strategies, and key biomarkers.

Recent findings

The integration of ICIs into standard chemotherapy has established them as the first-line treatment for extensive-stage SCLC (ES-SCLC). The ADRIATIC trial further demonstrated the efficacy of ICI maintenance therapy in limited-stage SCLC. Additionally, combining radiotherapy with ICIs has shown promising synergistic effects, including the abscopal and radscopal effects. Ongoing investigations into the combination of ICIs with targeted therapies, such as antiangiogenic agents and DNA damage response inhibitors, have yielded encouraging preliminary results. Notably, the novel therapeutic agent tarlatamab, the first bispecific DLL3-directed CD3 T-cell engager, has recently received FDA approval for second-line treatment of ES-SCLC. Advances in omics technologies have shed light on the intra-tumor and inter-tumor heterogeneity of SCLC, leading to the identification of new molecular subtypes and biomarkers, thereby paving the way for precision medicine.

Despite the improved outcomes associated with immunotherapy in SCLC, the overall clinical benefit remains modest. Further preclinical and clinical studies are essential to identify optimal treatment regimens and enhance therapeutic efficacy.