Perinecrotic Areas Research Articles

EPEN-16. BULK AND SINGLE-CELL SPATIAL CHARACTERIZATION OF THE TUMOR IMMUNE MICROENVIRONMENT OF POSTERIOR FOSSA GROUP A EPENDYMOMAS

Abstract BACKGROUND Despite advancements in molecular classification of ependymomas, the tumor immune microenvironment of posterior fossa group A (PFA) ependymomas and the spatial interplay between tumor and immune cells have not been elucidated. Here, we aim to comprehensively characterize the tumor immune microenvironment of pediatric PFA ependymomas, which could ultimately uncover leads for immunotherapy for these children. METHODS We collected bulk RNA-sequencing data of 28 PFA ependymoma samples and defined two subgroups based on the expression of immune-related genes (n = 716). Furthermore, we analyzed single-cell RNA-sequencing data of PFA ependymomas to dissect myeloid and lymphocyte heterogeneity, which we subsequently used to deconvolute bulk data. Lastly, we performed both high-dimensional cyclical immunofluorescence of 20 protein markers on a tissue microarray containing 60 cores from 20 tumor samples and immunohistochemistry on whole tissue slides. RESULTS Unsupervised hierarchical clustering of bulk RNA expression of immune-related genes revealed the presence of an immune-enriched subgroup. Furthermore, analysis of single-cell RNA-sequencing data identified multiple distinct myeloid cell subpopulations, with gene expression signatures reflective of inflammation, angiogenesis/hypoxia response and proliferation. Deconvolution of the bulk RNA-sequencing data using the newly annotated single-cell data as a reference showed enrichment of both myeloid cells and lymphocytes in the immune-enriched subgroup. Lastly, spatial immune profiling enabled detailed mapping of the organization of the tumor immune microenvironment and unveiled a heterogeneous presence of myeloid cells, with enrichment of infiltrating myeloid and T-cells in peri-necrotic areas compared to areas with high tumor cell density. CONCLUSIONS Our multi-faceted approach has shed unprecedented light on the cellular and spatial heterogeneity within the PFA ependymoma immune microenvironment. Further studies are warranted to unravel the functional differences between the two subgroups and the potential impact on response to future immunotherapeutic approaches/interventions.

Abstract 393: RAS and PI3K activation promote glioblastoma progression and ischemia-associated tumor cell death

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor in adults. Despite the association of tumor necrosis and a poor prognosis in GBM, the mechanisms underlying tumor necrosis formation remain unclear, likely due to the lack of appropriate experimental models to examine the development of tumor necrosis. In our study, we developed mouse models with tumor necrosis by intracranially injecting human GBM cells expressing KRAS or PI3K active mutants into immunodeficient mice. Surprisingly, despite all tumors displayed similar sizes, the control tumors expressing an empty vector (EV) did not exhibit tumor necrosis. This intriguing observation led us to explore the potential role of KRAS and PI3K in promoting cell death within the tumor microenvironment. Ischemia, which is characterized by insufficient nutrients and hypoxia, is often found in GBM. Under an in vitro ischemia mimetic condition, we found that the KRAS or PI3K activated GBM cells showed more cell death compared to EV cells. Mechanistically, ischemia activates the p38 MAPK-MK2 pathway to induce cell death, a process further enhanced by KRAS or PI3K. Notably, we observed elevated expression of p-MK2 in the peri-necrotic areas of GBMs harboring KRAS or PI3K mutants compared to the cellular tumor zones. Moreover, we revealed that ischemia induces the unfolded protein response (UPR) pathway. Among UPR-related molecules, KRAS and PI3K upregulate ERN1, which encodes the transmembrane protein kinase IRE1, in ischemic conditions. Inhibition of ERN1 not only prevented ischemia-induced cell death but also inhibited the p38 MAPK-MK2 pathway. Furthermore, we observed an increase in the expression of pro-inflammatory cytokine IL-8 under ischemic conditions, a response that was prevented by inhibitors of p38 MAPK or MK2. Lastly, we found that ischemia induces ATP release and calreticulin exposure, indicating the cell death possessing certain immunogenic cell death features. In summary, our findings revealed a pivotal role of KRAS and PI3K in inducing cell death in the ischemic GBM tumor microenvironment through activating the IRE1-p38 MAPK-MK2 pathway. The cell death under this circumstance displays pro-inflammatory and immunogenic properties. Therefore, the IRE1-p38 MAPK-MK2 pathway may be exploited for the treatment of GBM in which RAS or PI3K is activated. Citation Format: Soo Yeon Kim, Miaolu Tang, Stephen Chih, Jessica Thorpe, Tong Lu, Hong-Gang Wang, Wei Li. RAS and PI3K activation promote glioblastoma progression and ischemia-associated tumor cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 393.

Serum Interleukin-38 and Tumor-Infiltrating Lymphocytes in Primary Brain Tumors.

Tumor-infiltrating lymphocytes (TILs) and brain stromal cells produce immunosuppressive cytokines, contributing to an immunosuppressive tumor microenvironment (TME). Interleukin-38 (IL-38) is a novel anti-inflammatory cytokine and a natural modulator of the innate and adaptive immune system. However, its biological roles in brain tumors are not well defined. To assess the serum levels of IL-38 and the percentages of TILs in the tumor tissues of patients with primary brain tumors and to determine their associations with the pathological features of the disease. IL-38 was evaluated in sera using the enzyme-linked immunosorbent assay (ELISA). Hematoxylin and eosin (H&E)-stained sections were scored to determine the percentages of TILs in four different areas: the invasive margin, central tumor, perivascular and perinecrotic areas. IL-38 serum levels were significantly higher in low- and high-grade tumors than in healthy individuals, meanwhile, its levels remained consistent between these two grades. Although no significant difference was found in IL-38 serum levels between different histological subtypes of brain tumors, its levels were significantly higher in intra-axial brain tumors than in extra-axial ones. Additionally, a significant positive correlation was observed between serum levels of IL-38 and tumor size in patients with low-grade tumors. TILs were detected in at least one of the four examined areas; however, no statistically significant correlation was found between IL-38 levels and TILs. Our data may suggest a connection between IL-38 and immune suppression and tumor progression in primary brain tumors. Further investigation is needed to uncover the role of IL-38 in the brain tumor microenvironment.

Presence of Activated (Phosphorylated) STAT3 in Radiation Necrosis Following Stereotactic Radiosurgery for Brain Metastases.

Brain radiation necrosis (RN) is a subacute or late adverse event following radiotherapy, involving an exacerbated inflammatory response of the brain tissue. The risk of symptomatic RN associated with stereotactic radiosurgery (SRS) as part of the treatment of brain metastases (BMs) has been a subject of recent investigation. The activation of the signal transducer and activator of transcription 3 (STAT3) was shown in reactive astrocytes (RA) associated with BMs. Given that the pathophysiological mechanisms behind RN are not fully understood, we sought to investigate the role of STAT3 among other inflammatory markers in RN development. A mouse model of RN using clinical LINAC-based SRS was designed to induce brain necrosis with the administration of 50 Gy in a single fraction to the left hemisphere using a circular collimator of 5 mm diameter. Immunohistochemistry and multiplex staining for CD4, CD8, CD68, GFAP, and STAT3 were performed. For validation, eleven patients with BMs treated with SRS who developed symptomatic RN and required surgery were identified to perform staining for CD68, GFAP, and STAT3. In the mouse model, the RN and perinecrotic areas showed significantly higher staining for F4/80+ and GFAP+ cells, with a high infiltration of CD4 and CD8 T-lymphocytes, when compared to the non-irradiated cerebral hemisphere. A high number of GFAP+pSTAT3+ and F4/80+pSTAT3+ cells was found in the RN areas and the rest of the irradiated hemisphere. The analysis of human brain specimens showed that astrocytes and microglia were actively phosphorylating STAT3 in the areas of RN and gliosis. Phosphorylated STAT3 is highly expressed in the microglia and RA pertaining to the areas of brain RN. Targeting STAT3 via inhibition represents a promising strategy to ameliorate symptomatic RN in BM patients undergoing SRS.

Multi-Omics Analysis of Glioblastoma and Glioblastoma Cell Line: Molecular Insights Into the Functional Role of GPR56 and TG2 in Mesenchymal Transition.

G protein-coupled receptor 56 (GPR56/ADGRG1) is an adhesion GPCR with an essential role in brain development and cancer. Elevated expression of GPR56 was observed in the clinical specimens of Glioblastoma (GBM), a highly invasive primary brain tumor. However, we found the expression to be variable across the specimens, presumably due to the intratumor heterogeneity of GBM. Therefore, we re-examined GPR56 expression in public domain spatial gene expression data and single-cell expression data for GBM, which revealed that GPR56 expression was high in cellular tumors, infiltrating tumor cells, and proliferating cells, low in microvascular proliferation and peri-necrotic areas of the tumor, especially in hypoxic mesenchymal-like cells. To gain a better understanding of the consequences of GPR56 downregulation in tumor cells and other molecular changes associated with it, we generated a sh-RNA-mediated GPR56 knockdown in the GBM cell line U373 and performed transcriptomics, proteomics, and phospho-proteomics analysis. Our analysis revealed enrichment of gene signatures, pathways, and phosphorylation of proteins potentially associated with mesenchymal (MES) transition in the tumor and concurrent increase in cell invasion and migration behavior of the GPR56 knockdown GBM cells. Interestingly, our analysis also showed elevated expression of Transglutaminase 2 (TG2) - a known interactor of GPR56, in the knockdown cells. The inverse expression of GPR56 and TG2 was also observed in intratumoral, spatial gene expression data for GBM and in GBM cell lines cultured in vitro under hypoxic conditions. Integrating all these observations, we propose a putative functional link between the inverse expression of the two proteins, the hypoxic niche and the mesenchymal status in the tumor. Hypoxia-induced downregulation of GPR56 and activation of TG2 may result in a network of molecular events that contribute to the mesenchymal transition of GBM cells, and we propose a putative model to explain this functional and regulatory relationship of the two proteins.

A functional role of S100A4/non-muscle myosin IIA axis for pro-tumorigenic vascular functions in glioblastoma

BackgroundGlioblastoma (GBM) is the most aggressive form of brain tumor and has vascular-rich features. The S100A4/non-muscle myosin IIA (NMIIA) axis contributes to aggressive phenotypes in a variety of human malignancies, but little is known about its involvement in GBM tumorigenesis. Herein, we examined the role of the S100A4/NMIIA axis during tumor progression and vasculogenesis in GBM.MethodsWe performed immunohistochemistry for S100A4, NMIIA, and two hypoxic markers, hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase 9 (CA9), in samples from 94 GBM cases. The functional impact of S100A4 knockdown and hypoxia were also assessed using a GBM cell line.ResultsIn clinical GBM samples, overexpression of S100A4 and NMIIA was observed in both non-pseudopalisading (Ps) and Ps (-associated) perinecrotic lesions, consistent with stabilization of HIF-1α and CA9. CD34(+) microvascular densities (MVDs) and the interaction of S100A4 and NMIIA were significantly higher in non-Ps perinecrotic lesions compared to those in Ps perinecrotic areas. In non-Ps perinecrotic lesions, S100A4(+)/HIF-1α(−) GBM cells were recruited to the surface of preexisting host vessels in the vascular-rich areas. Elevated vascular endothelial growth factor A (VEGFA) mRNA expression was found in S100A4(+)/HIF-1α(+) GBM cells adjacent to the vascular-rich areas. In addition, GBM patients with high S100A4 protein expression had significantly worse OS and PFS than did patients with low S100A4 expression. Knockdown of S100A4 in the GBM cell line KS-1 decreased migration capability, concomitant with decreased Slug expression; the opposite effects were elicited by blebbistatin-dependent inhibition of NMIIA.ConclusionS100A4(+)/HIF-1α(−) GBM cells are recruited to (and migrate along) preexisting vessels through inhibition of NMIIA activity. This is likely stimulated by extracellular VEGF that is released by S100A4(+)/HIF-1α(+) tumor cells in non-Ps perinecrotic lesions. In turn, these events engender tumor progression via acceleration of pro-tumorigenic vascular functions.-mzznLaNnzTzVPc8r2eojtVideo abstract

Macrophage/microglia-derived IL-1β induces glioblastoma growth via the STAT3/NF-κB pathway.

Glioblastoma is a glioma characterized by highly malignant features. Numerous studies conducted on the relationship between glioblastoma and the microenvironment have indicated the significance of tumor-associated macrophages/microglia (TAMs) in glioblastoma progression. Since interleukin (IL)-1β secreted by TAMs has been suggested to promote glioblastoma growth, we attempted to elucidate the detailed mechanisms of IL-1β in glioblastoma growth in this study. A phospho-receptor tyrosine kinase array and RNA-sequencing studies indicated that IL-1β induced the activation of signal transducer and activator of transcription-3 and nuclear factor-kappa B signaling. Glioblastoma cells stimulated by IL-1β induced the production of IL-6 and CXCL8, which synergistically promoted glioblastoma growth via signal transducer and activator of transcription-3 and nuclear factor-kappa B signaling. By immunohistochemistry, IL-1β expression was seen on TAMs, especially in perinecrotic areas. These results suggest that IL-1β might be a useful target molecule for anti-glioblastoma therapy.

Targeting the Urotensin II/UT G Protein-Coupled Receptor to Counteract Angiogenesis and Mesenchymal Hypoxia/Necrosis in Glioblastoma.

Glioblastomas (GBMs) are the most common primary brain tumors characterized by strong invasiveness and angiogenesis. GBM cells and microenvironment secrete angiogenic factors and also express chemoattractant G protein-coupled receptors (GPCRs) to their advantage. We investigated the role of the vasoactive peptide urotensin II (UII) and its receptor UT on GBM angiogenesis and tested potential ligand/therapeutic options based on this system. On glioma patient samples, the expression of UII and UT increased with the grade with marked expression in the vascular and peri-necrotic mesenchymal hypoxic areas being correlated with vascular density. In vitro human UII stimulated human endothelial HUV-EC-C and hCMEC/D3 cell motility and tubulogenesis. In mouse-transplanted Matrigel sponges, mouse (mUII) and human UII markedly stimulated invasion by macrophages, endothelial, and smooth muscle cells. In U87 GBM xenografts expressing UII and UT in the glial and vascular compartments, UII accelerated tumor development, favored hypoxia and necrosis associated with increased proliferation (Ki67), and induced metalloproteinase (MMP)-2 and -9 expression in Nude mice. UII also promoted a “tortuous” vascular collagen-IV expressing network and integrin expression mainly in the vascular compartment. GBM angiogenesis and integrin αvβ3 were confirmed by in vivo 99mTc-RGD tracer imaging and tumoral capture in the non-necrotic area of U87 xenografts in Nude mice. Peptide analogs of UII and UT antagonist were also tested as potential tumor repressor. Urotensin II-related peptide URP inhibited angiogenesis in vitro and failed to attract vascular and inflammatory components in Matrigel in vivo. Interestingly, the UT antagonist/biased ligand urantide and the non-peptide UT antagonist palosuran prevented UII-induced tubulogenesis in vitro and significantly delayed tumor growth in vivo. Urantide drastically prevented endogenous and UII-induced GBM angiogenesis, MMP, and integrin activations, associated with GBM tumoral growth. These findings show that UII induces GBM aggressiveness with necrosis and angiogenesis through integrin activation, a mesenchymal behavior that can be targeted by UT biased ligands/antagonists.

Hypoxia-Induced Reactivity of Tumor-Associated Astrocytes Affects Glioma Cell Properties.

Glioblastoma is characterized by extensive necrotic areas with surrounding hypoxia. The cancer cell response to hypoxia in these areas is well-described; it involves a metabolic shift and an increase in stem cell-like characteristics. Less is known about the hypoxic response of tumor-associated astrocytes, a major component of the glioma tumor microenvironment. Here, we used primary human astrocytes and a genetically engineered glioma mouse model to investigate the response of this stromal cell type to hypoxia. We found that astrocytes became reactive in response to intermediate and severe hypoxia, similarly to irradiated and temozolomide-treated astrocytes. Hypoxic astrocytes displayed a potent hypoxia response that appeared to be driven primarily by hypoxia-inducible factor 2-alpha (HIF-2α). This response involved the activation of classical HIF target genes and the increased production of hypoxia-associated cytokines such as TGF-β1, IL-3, angiogenin, VEGF-A, and IL-1 alpha. In vivo, astrocytes were present in proximity to perinecrotic areas surrounding HIF-2α expressing cells, suggesting that hypoxic astrocytes contribute to the glioma microenvironment. Extracellular matrix derived from hypoxic astrocytes increased the proliferation and drug efflux capability of glioma cells. Together, our findings suggest that hypoxic astrocytes are implicated in tumor growth and potentially stemness maintenance by remodeling the tumor microenvironment.

CBIO-09. INTRATUMORAL HETEROGENEITY OF DIELECTRIC PROPERTIES IN GLIOBLASTOMA

Abstract INTRODUCTION Recently, tumor treating fields (TTFields) were established for the treatment of newly diagnosed glioblastoma (GBM). One of the most crucial parameters defining the treatment efficacy of TTFields is the electric field intensity, which depends on the dielectric properties of the tumor tissue. In this study we determined the dielectric properties of GBM by analyzing resected tissue following a fast acquisition protocol. To account for the intratumoral heterogeneity, different regions of the tumor were analyzed separately. METHODS A cohort of 38 patients with newly diagnosed GBM were analyzed. Tissue probes were acquired from the vital tumor area and perinecrotic compartment. The tissue was measured immediately to avoid artifacts. A fragment was dissected from each tissue sample and was placed into a cylindrical cell with a known diameter. The impedance was recorded at frequencies 20Hz-1MHz using a software specifically developed for this study, which controls the LCR meter. The measured impedance was translated into dielectric properties of the sample (conductivity and relative permittivity) based on the parallel plate model, the recorded complex impedance and the geometry of the samples. Each tissue probe was fixed, and stained with H&E to visualize cellularity, luxol fast blue to analyze the myelinated fiber content and against factor VIII related antigen to assess tumor vascularity. RESULTS We found significant differences between the conductivity and permittivity of tissue samples from each individual tumor (mean conductivity [S/m]: 0.302; range: 0.607 – 0.100; mean permittivity [Farad/m]: 3519.8; range: 11182.5 – 135.7). Consistently, the perinecrotic areas displayed lower conductivity values compared to the solid tumor compartments. Histological analysis revealed significantly higher cellularity and lower myelinated fiber content in tissue samples with high conductivity and permittivity. CONCLUSION The dielectric properties of GBM show a high intratumoral heterogeneity which correlate to the extent of cellularity and myelin fiber content within the tissue.

Niche-derived soluble DLK1 promotes glioma growth

Tumor cell behaviors associated with aggressive tumor growth such as proliferation, therapeutic resistance, and stem cell characteristics are regulated in part by soluble factors derived from the tumor microenvironment. Tumor-associated astrocytes represent a major component of the glioma tumor microenvironment, and astrocytes have an active role in maintenance of normal neural stem cells in the stem cell niche, in part via secretion of soluble delta-like noncanonical Notch ligand 1 (DLK1). We found that astrocytes, when exposed to stresses of the tumor microenvironment such as hypoxia or ionizing radiation, increased secretion of soluble DLK1. Tumor-associated astrocytes in a glioma mouse model expressed DLK1 in perinecrotic and perivascular tumor areas. Glioma cells exposed to recombinant DLK1 displayed increased proliferation, enhanced self-renewal and colony formation abilities, and increased levels of stem cell marker genes. Mechanistically, DLK1-mediated effects on glioma cells involved increased and prolonged stabilization of hypoxia-inducible factor 2alpha, and inhibition of hypoxia-inducible factor 2alpha activity abolished effects of DLK1 in hypoxia. Forced expression of soluble DLK1 resulted in more aggressive tumor growth and shortened survival in a genetically engineered mouse model of glioma. Together, our data support DLK1 as a soluble mediator of glioma aggressiveness derived from the tumor microenvironment.

CBMT-33. VISUALIZING THE METABOLISM OF GLIOBLASTOMA PATIENT-DERIVED ORTHOTOPIC XENOGRAFTS BY MASS SPECTROMETRY IMAGING

Abstract INTRODUCTION. Glioblastoma multiforme (GBM) has a dismal prognosis, which has been attributed to extensive inter- and intra-tumoural heterogeneity. To investigate the metabolic heterogeneity of GBM, we employed desorption electrospray ionization (DESI) imaging on snap frozen samples of GBM patient-derived orthotopic xenografts (PDOX) in rat brains, following infusion of [U-13C]glucose. METHODS. Athymic nude rats (n=3) were infused with [U-13C]glucose (0.4 mg/g body weight bolus, 0.012 mg/g/min at 300 µL/h infusion [1]) for 40, 80 and 120 min, followed by cardiac puncture to measure blood [U-13C]glucose levels using 13C NMR spectroscopy. A second set of rats (n=2) were implanted intracranially with patient-derived GBM cells. The presence of tumour was confirmed by T2-weighted MRI. One animal was infused with [U-13C]glucose for 2 h, while another with [12C]glucose. The brains were snap frozen in liquid nitrogen followed by analysis with DESI imaging in negative ion mode (35 µm resolution). RESULTS. After a 2 h infusion, 43% of the circulating glucose was 13C labelled. Unsupervised spatial clustering of the MSI data resulted in segmentation of normal brain from tumour. Levels of [12C and 13C]glucose were high in the peri-tumoural region and low within the tumour. [12C and 13C]lactate showed the opposite distribution, indicating that the rate of glucose utilization within the tumour exceeded the rate of delivery. The peri-necrotic and peri-tumoural areas had higher levels of glutamate compared to the rest of the tumour. CONCLUSIONS. The results demonstrate that the PDOX is metabolically heterogeneous and differs from surrounding brain. While the tumour was predominately glycolytic, the presence of glutamate in peri-necrotic and peri-tumoral regions indicates the presence of increased TCA cycle activity. Comparison of MSI data with histological staining of brain sections should yield additional information about the relationship between the tumour and its microenvironment.

CBMT-14. THE DIELECTRIC PROPERTIES OF BRAIN TUMOR TISSUE

Abstract INTRODUCTION Recently, tumor treating fields (TTFields) were established for the treatment of newly diagnosed GBM. One of the most crucial parameters defining the treatment efficacy of TTFields is the electric field intensity, which depends on the dielectric properties of the tumor tissue. In this study we determined the dielectric properties of brain tumors by analyzing resected tissue following a fast acquisition protocol. To account for the intratumoral heterogeneity, different regions of the tumor were analyzed separately. METHODS A cohort of 84 patients with tumors of different histology have been recruited. Tissue probes were acquired from the vital tumor area and perinecrotic compartment. The tissue was measured immediately to avoid artifacts. A fragment was dissected from each tissue sample and was placed into a cylindrical cell with a known diameter. The impedance was recorded at frequencies 20Hz-1MHz using a software specifically developed for this study, which controls the LCR meter. The measured impedance was translated into dielectric properties of the sample (conductivity and relative permittivity) based on the parallel plate model, the recorded complex impedance and the geometry of the samples. Each tissue probe was fixed, H&E stained and histologically analyzed. RESULTS We found significant differences between the conductivity of different types of tumors with meningiomas showing the lowest conductivity (mean conductivity [S/m]: 0.193; range: 0.327 – 0.113) and GBM tissue exhibiting the highest conductivity values (mean conductivity [S/m]: 0.402; range: 0.893 – 0.157). Consistently, the perinecrotic areas displayed lower conductivity values compared to the solid tumor compartments. Also, we found a significant intratumoral heterogeneity in tumors of one specific histological diagnosis. CONCLUSION The dielectric properties of intracranial tumors appear to be depending on histological class and malignancy grade and show significant intratumoral heterogeneity. These results may allow a more precise modelling of electric field intensity distribution within the tumor.

P11.50 The dielectric properties of brain tumor tissue

Abstract BACKGROUND Recently, tumor treating fields (TTFields) were established for the treatment of newly diagnosed GBM. One of the most crucial parameters defining the treatment efficacy of TTFields is the electric field intensity. The dielectric properties of the normal intracranial compartments are well established, allowing the prediction of the electric field distribution. In contrast, there is no data available about the dielectric properties of brain tumor tissue. In this study we determined the dielectric properties of brain tumors by analyzing resected tissue following a fast acquisition protocol. To account for the intratumoral heterogeneity, different regions of the tumor were analyzed separately. MATERIAL AND METHODS A cohort of 84 patients with tumors of different histology and malignancy grade have been recruited (meningioma: n=26; brain metastases n=18; low grade glioma n=6; glioblastoma n=34). Tissue probes were acquired whenever possible from the vital tumor area, and perinecrotic compartment identified intraoperatively using neuronavigation, intraoperative ultrasound and fluorescence guidance. After acquisition, the tissue was measured immediately to avoid artifacts induced by temperature change, differences of fluid composition as well as post resection ischemia. A fragment was dissected from each tissue sample and was placed into a cylindrical cell with a known diameter. Two parallel electrodes were placed on both sides of the sample and the thickness of the tissue was measured using a micrometer. The impedance was recorded at frequencies 20Hz-1MHz using a software specifically developed for this study, which controls the LCR meter (Keysight Technologies, Santa Rosa, USA). The measured impedance was translated into dielectric properties of the sample (conductivity and relative permittivity) based on the parallel plate model, the recorded complex impedance and the geometry of the samples. Each tissue probe was fixed, H&E stained and histologically analyzed. RESULTS We found significant differences between the conductivity of different types of tumors with meningiomas showing the lowest conductivity (mean conductivity [S/m]: 0.193; range: 0.327 - 0.113) and GBM tissue exhibiting the highest conductivity values (mean conductivity [S/m]: 0.402; range: 0.893 - 0.157). Consistently, the perinecrotic areas of tumors displayed lower conductivity values compared to the solid tumor compartments. Also, we found a significant intratumoral heterogeneity in tumors of one specific histological diagnosis. CONCLUSION The dielectric properties of intracranial tumors appear to be depending on histological class and malignancy grade and show significant intratumoral heterogeneity. These results may allow a more precise modelling of electric field intensity distribution within the tumor.

Tumor Heterogeneity in Gliomas – a Histopathology-Targeted Proteomic Pilot Study

Intra- and inter-tumor heterogeneity underlies both tumor evolution and response to treatment. Our aim was to explore the protein expression of infiltrative gliomas (grades II to IV) with high throughput protein-based mass spectrometry (MS), comparing different histopathologic areas of glioblastomas (GBM) and lower grade gliomas (LGG), to further characterize gliomas pathways, and identify druggable targets and protein signatures to be used as diagnostic and prognostic biomarkers. We studied 12 patients with diffuse gliomas (1 diffuse astrocytoma, 1 anaplastic astrocytoma, 3 anaplastic oligodendrogliomas, and 7 glioblastomas). Each tumor was reclassified according to the 2016 WHO classification of tumors of CNS (IDHmutant-1p19q Codeleted; IDHmutant-1p19q intact; IDH wild type glioma), and mapped using H&E stained slides to identify 2-3 areas with different histopathologic phenotypes, different cellularity, and perinecrotic areas. The selected areas with at least 80% of tumor cells were cored with 1.0mm needle using a semi-automated tissue microarray platform, in a total of 35 samples – hemorrhagic and necrotic portions of the tumors were avoided in all cases. Total protein was extracted from the FFPE tissue and analyzed with 4 hour liquid chromatography tandem MS (LC-MS-MS) for label-free expression proteomics. We compared the average of protein expression in different tumor areas from each patient to identify associations with Wilcoxon rank-sum test. The results were further interpreted following a top-bottom approach and compared in silico with TCGA expression data (mRNA). The statistical analysis was performed using R software. Overall, 9,222 peptides were mapped to 1758 non-redundant proteins in the samples, 320 of which had a significant differential expression in GBM versus LGG. The PCA analysis showed that according to the molecular signatures, the samples clustered well by disease and by patient. Samples also clustered by IDH1 status and WHO subgroup. One patient had non-neoplastic brain adjacent to a GBM available for analysis and this sample clustered with the LGGs. Among the top 35 proteins most differentially expressed in the 2 groups (raw p-value= 0.00252; FDR=0.12684), 14 had higher expression in GBM, and 21 in LGG. This included one protein previously explored by our group in previous studies (TAGLN2), validating our findings with different approach. Our results showed that LC-MS-MS analysis of morphologically different glioma areas is feasible and was able to identify clusters with different patterns of protein expression. Further correlation of proteome profiles with clinical data, histological features, pathway analysis, and druggable target analysis are ongoing. FUNDING: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01 (NCI), Brain Tumor Funders Collaborative Grant, and the Ohio State University CCC (all to AC).

Abstract 4031: The dielectric properties of brain tumor tissue

Abstract Introduction: Recently, tumor treating fields (TTFields) have been established as for the treatment of newly diagnosed GBM1. One of the most crucial parameters defining the treatment efficacy of TTFields is the electric field intensity. The electric properties of the normal intracranial compartments are well established, allowing the prediction of the electric field distribution. In contrast, there is no data available about the electric properties of tumor tissue. In this study we determined the dieclectric properties of brain tumors by analyzing resected tissue following a fast acquisition protocol. To account for the intratumoral heterogeneity, different regions of the tumor were sampled and analyzed separately. Methods: A cohort of thirty patients with tumors of different histology and malignancy grade have been recruited (meningioma: n=11; brain metastases n=7; low grade glioma n=6; glioblastoma n=6). Tissue probes were acquired whenever possible from the vital tumor area, and perinecrotic compartment identified intraoperatively using neuronavigation, intraoperative ultrasound and fluorescence guidance. From each region, five tissue probes were sampled. After acquisition, the tissue was measured immediately to avoid artifacts induced by temperature change, differences of fluid composition as well as post resection ischemia. A fragment was dissected from each tissue sample and was placed into a cylindrical cell with a known diameter. Two parallel electrodes were placed on both sides of the sample and the thickness of the tissue was measured using a micrometer. The impedance was recorded at frequencies 20Hz-1MHz using a software specifically developed for this study, which controls the LCR meter (Keysight Technologies, Santa Rosa, USA). The measured impedance was translated into dielectric properties of the sample (conductivity and relative permittivity) based on the parallel plate model, the recorded complex impedance and the geometry of the samples. Each tissue probe was fixed, H&E stained and histologically analyzed for tumor cell count and specific tissue features. Results: After receiving a positive ethics votum, the first 30 patients were recruited. As a reference, grey and white matter tissue samples from mouse brains were used. We found significant differences between the tumor entities with meningiomas showing the lowest and GBM tissue the highest conductivity values. Consistently, the perinecrotic areas displayed lower conductivity values compared to the solid tumor compartment. Also, we found a significant heterogeneity in the dielectric properties within one tumor. Conclusion: The dielectric properties of intracranial tumors appear to be depending on histological class and malignancy grade and show significant intratumoral heterogeneity. These results may allow a more precise modelling of electric field intensity distribution within the Tumor. Citation Format: Martin A. Proescholdt, Amer Haj, Christian Doenitz, Alexander Brawanski, Zeev Bomzon, Hadas Sara Hershkovich. The dielectric properties of brain tumor tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4031.

Downregulation of DNA repair proteins and increased DNA damage in hypoxic colon cancer cells is a therapeutically exploitable vulnerability.

Surgical removal of colorectal cancer (CRC) liver metastases generates areas of tissue hypoxia. Hypoxia imposes a stem-like phenotype on residual tumor cells and promotes tumor recurrence. Moreover, in primary CRC, gene expression signatures reflecting hypoxia and a stem-like phenotype are highly expressed in the aggressive Consensus Molecular Subtype 4 (CMS4). Therapeutic strategies eliminating hypoxic stem-like cells may limit recurrence following resection of primary tumors or metastases.Here we show that expression of DNA repair genes is strongly suppressed in CMS4 and inversely correlated with hypoxia-inducible factor-1 alpha (HIF1α) and HIF-2α co-expression signatures. Tumors with high expression of HIF signatures and low expression of repair proteins showed the worst survival. In human tumors, expression of the repair proteins RAD51, KU70 and RIF1 was strongly suppressed in hypoxic peri-necrotic tumor areas. Experimentally induced hypoxia in patient derived colonospheres in vitro or in vivo (through vascular clamping) was sufficient to downregulate repair protein expression and caused DNA damage. Hypoxia-induced DNA damage was prevented by expressing the hydroperoxide-scavenging enzyme glutathione peroxidase-2 (GPx2), indicating that reactive oxygen species mediate hypoxia-induced DNA damage. Finally, the hypoxia-activated prodrug Tirapazamine greatly augmented DNA damage and reduced the fraction of stem-like (Aldefluorbright) tumor cells in vitro, and in vivo following vascular clamping.We conclude that decreased expression of DNA repair proteins and increased DNA damage in hypoxic tumor areas may be therapeutically exploited with hypoxia-activated prodrugs, and that such drugs reduce the fraction of Aldefluorbright (stem-like) tumor cells.

ALK signaling cascade confers multiple advantages to glioblastoma cells through neovascularization and cell proliferation.

Anaplastic lymphoma kinase (ALK), which is a receptor tyrosine kinase, is essentially and transiently expressed in the developing nervous system. Here we examined the functional role of the ALK gene in glioblastomas (GBMs). In clinical samples of GBMs, high ALK expression without gene rearrangements or mutations was frequently observed in perivascular lesions, in contrast to the relatively low expression in the perinecrotic areas, which was positively correlated with N-myc and phosphorylated (p) Stat3 scores and Ki-67 labeling indices. ALK immunoreactivity was also found to be associated with neovascular features including vascular co-option and vascular mimicry. In astrocytoma cell lines, cells stably overexpressing full-length ALK showed an increase in expression of pStat3 and pAkt proteins, as well as hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor-A (VEGF-A) mRNAs, in contrast to cells with knockdown of endogenous ALK which showed decreased expression of these molecules. Transfection of the constitutively active form of Stat3 induced an increase in HIF-1α promoter activity, and the overexpression of HIF-1α in turn resulted in enhancement of VEGF-A promoter activity. In addition, cells with overexpression or knockdown of ALK also showed a tendency toward increased and decreased proliferation, respectively, through changes in expression of pAkt and pStat3. Finally, ALK promoter was significantly activated by transfection of Sox4 and N-myc, which are known to contribute to neuronal properties. These findings therefore suggest that N-myc/Sox4-mediated ALK signaling cascades containing Stat3, Akt, HIF-1α, and VEGF-A confer multiple advantages to tumor growth through alterations in neovascularization and cell proliferation in GBMs.

Dosage effects of extracorporeal shockwave therapy in early hip necrosis

BackgroundThis study investigated the effects of different dosages of extracorporeal shockwave therapy (ESWT) in early osteonecrosis of the femoral head (ONFH). Materials and methodsThirty-three patients (42 hips) were randomly divided into three groups. Group A (10 patients with 16 hips) received 2000 impulses of ESWT at 24 Kv to the affected hip. Group B (11 patients with 14 hips) and Group C (12 patients with 12 hips) received 4000 and 6000 impulses of ESWT respectively. The evaluations included clinical assessment, radiographs, dynamic contrast-enhanced MRI for microcirculation (Ktrans) and plasma volume (Vp), and blood tests for biomarker analysis (NO3, VEGF, BMP-2, osteocalcin, TNF-α, IL-6, substance P, CGRP, DKK-1 and IGF). ResultsSignificant differences of pain and Harris hip scores were noticed between Group A and C in 6 months after ESWT (all P < 0.05). The pain score decreased, but not Harris hip score improved over the observation time period from 6 to 24 months. Total hip arthroplasty was performed in 3 patients (4 hips) in Group A, but none in Groups B and C. Group C showed significant changes in serum biomarkers for angiogenesis, osteogenesis, anti-inflammation, pain threshold and tissue regeneration between one week and one month after treatment (all P < 0.05). However, no significant changes in the infarction volume in image studies were noted in all groups (all P > 0.05). The post-treatment Ktrans and Vp in the peri-necrotic areas of Group B and C were significantly greater than pre-treatment data (both P < 0.05). ConclusionsHigh dosage ESWT is more effective in early stage ONFH. The systemic beneficial effects of ESWT may ultimately enhance angiogenesis with improvement of microcirculation of the peri-necrotic areas, that in turn, can improve subchondral bone remodeling and prevent femoral head collapse.

MET immunolabelling is a useful predictive tool for MET gene amplification in glioblastoma.

MET gene amplification is rare in glioblastoma (GBM) and represents a potential target for MET inhibitors. An immunohistochemical screening may be useful to identify MET amplification. The aim of our study was to establish how MET immunolabelling correlates with MET amplification. Three cohorts including 108 GBM (cohort 1, prospective), 104 GBM (cohort 2, retrospective) and 52 GBM (cohort 3, prospective) were investigated for MET expression by immunohistochemistry. MET amplification was assessed by comparative genomic hybridization on microarray (CGH-array) in all cohorts and by fluorescent in situ hybridization (FISH) in cohorts 2 and 3. Active form of MET was assessed using p-MET (Y1349) immunohistochemistry. Diffuse MET amplification detectable by CGH-array was associated with diffuse, strong MET immunolabelling (four cases in cohort 1 and one case in cohort 2). Focal MET amplification detectable only by FISH was observed in small foci of strongly immunopositive cells in two GBM (cohort 2). In both cohorts, MET amplification was never detected in GBM devoid of strongly immunopositive cells. MET overexpression, observed in 23% of unamplified GBM, was associated with a predominant weak-to-moderate staining intensity and with necrosis (P < 0.005). p-MET was detected in all MET-amplified GBM and in perinecrotic areas of nonamplified GBM. A strong MET immunostaining intensity, at least focal and distant from necrosis, showed 100% sensitivity and 84% specificity for predicting MET amplification in cohort 3. MET amplification is characterized by strongly immunopositive cells. Only GBM showing strong MET immunostaining is appropriate for the assessment of MET amplification.