Search

Simple Summary: Kinin receptors have been implicated in cancer migration, invasion, angiogenesis, and metastasis. However, research on the molecular changes that occur during glioma development remains incomplete. Therefore, we assessed differences in the expression of kinin receptors and kinin-dependent genes in forty-three patients with astrocytic gliomas of various tumor grades (G2, G3, and G4). In our study, we confirmed that kinins and their receptors are involved in the development of glioma. We also found genes whose expression change may have diagnostic and therapeutic significance. Kinins are a set of peptides present in tissues that are involved in the inflammatory response and cancer progression. However, studies showing the expression of kinin receptors in human glioma samples are still incomplete and contradictory. The aim of the present study was to ascertain the expression of BDKRB1 and BDKRB2 genes, as well as the level of B1R and B2R proteins in human gliomas, depending on the degree of malignancy. Additionally, representative kinin-dependent genes with altered expression were indicated. The expression profile of kinin-dependent genes was determined using oligonucleotide microarray technique. In addition, RT-qPCR was used to assess the expression level of selected differentiating genes. The location of kinin receptors in brain gliomas was assessed using immunohistochemical methods. The oligonucleotide microarray method was used to identify 12 mRNA IDs of kinin-related genes whose expression was upregulated or downregulated in gliomas of different grades. In immunohistochemically stained samples, the concentrations of BR1 and BR2 proteins, measured by optical density, were statistically significantly higher in grade G3 vs. G2 and G4 vs. G3. Increased expression of kinin receptors BDKRB1 and BDKRB2 in brain gliomas, depending on the degree of malignancy, suggests the involvement of kinins and their receptors in the disease's pathogenesis. Quantitative assessment of mRNA BDKRB1, PRKAR1A, MAP2K, and EGFR in patients with brain tumors may hold diagnostic and therapeutic significance. [ABSTRACT FROM AUTHOR]

Simple Summary: Almost all patients with glioblastoma (GBM) eventually relapse, mainly due to adaptive and acquired resistance that results from tumour heterogeneity and its relatively immune-depleted ("cold") microenvironment. High levels of programmed death ligand-1 (PD-L1) have been associated with GBM invasiveness and immuno-resistance. Presently, there is no standardised approach for the assessment of PD-L1 expression level that would help in predicting the response to immune checkpoint inhibitors. Therefore, we investigated the ability of a radiolabelled ZPD-L1 affibody molecule to measure the expression level of PD-L1 in GBM xenograft models. There is no established method to assess the PD-L1 expression in brain tumours. Therefore, we investigated the suitability of affibody molecule (ZPD-L1) radiolabelled with F-18 (Al18F) and Ga-68 to measure the expression of PD-L1 in xenograft mouse models of GBM. Mice bearing subcutaneous and orthotopic tumours were imaged 1 h post-radioconjugate administration. Ex vivo biodistribution studies and immunohistochemistry (IHC) staining were performed. Tumoural PD-L1 expression and CD4+/CD8+ tumour-infiltrating lymphocytes were evaluated in human GBM specimens. ZPD-L1 was radiolabelled with radiochemical yields of 32.2 ± 4.4% (F-18) and 73.3 ± 1.8% (Ga-68). The cell-associated radioactivity in vitro was consistent with PD-L1 expression levels assessed with flow cytometry. In vivo imaging demonstrated that 18F-AlF-NOTA-ZPD-L1 can distinguish between PD-L1 high-expressing tumours (U87-MGvIII) and PD-L1-negative ones (H292PD-L1Ko). The radioconjugate was quickly cleared from the blood and normal tissues, allowing for high-contrast images of brain tumours as early as 1 h post-injection. 68Ga-NOTA-ZPD-L1 showed heterogeneous and diffuse accumulation that corresponded to the extensively infiltrating GCGR-E55 tumours involving contiguous lobes of the brain. Lastly, 39% of analysed GBM patient samples showed PD-L1+ staining of tumour cells that was associated with elevated levels of CD4+ and CD8+ lymphocytes. Our results suggest that the investigated radioconjugates are very promising agents with the potential to facilitate the future design of treatment regimens for GBM patients. [ABSTRACT FROM AUTHOR]

Simple Summary: The extent of tumour oxygenation is crucial for glioblastoma progression and the effectiveness of radio- and chemotherapy. Patient-derived in vitro cell cultures are the mainstay of molecular biology research, the discovery of new therapeutic targets, and drug testing. Therefore, mirroring as many aspects of in vivo settings as possible, including oxygen concentration, is desired. Here we analyse the effect of oxygen tension on the transcriptome of numerous patient-derived GBM cells and demonstrate that cells cultured in lowered oxygen tension express more genes indicative of higher levels of hypoxia, metabolic adaptation, stemness and tumour progression than cells growing in standard, atmospheric oxygen concentration. The same transcriptomic pattern was also found in primary GBM samples. Its specificity for GBMs was confirmed using the public TCGA dataset. Our data strongly argue for the benefit of lower oxygen tension during culturing of patient-derived GBM cells to preserve oxygen-sensitive pathways in GBM. The proposed approach better mimics certain aspects of GBM pathophysiology than traditional cultures and may advance GBM research in finding a cure. Despite numerous efforts aiming to characterise glioblastoma pathology (GBM) and discover new therapeutic strategies, GBM remains one of the most challenging tumours to treat. Here we propose the optimisation of in vitro culturing of GBM patient-derived cells, namely the establishment of GBM-derived cultures and their maintenance at oxygen tension mimicking oxygenation conditions occurring within the tumour. To globally analyse cell states, we performed the transcriptome analysis of GBM patient-derived cells kept as spheroids in serum-free conditions at the reduced oxygen tension (5% O2), cells cultured at atmospheric oxygen (20% O2), and parental tumour. Immune cells present in the tumour were depleted, resulting in the decreased expression of the immune system and inflammation-related genes. The expression of genes promoting cell proliferation and DNA repair was higher in GBM cell cultures when compared to the relevant tumour sample. However, lowering oxygen tension to 5% did not affect the proliferation rate and expression of cell cycle and DNA repair genes in GBM cell cultures. Culturing GBM cells at 5% oxygen was sufficient to increase the expression of specific stemness markers, particularly the PROM1 gene, without affecting neural cell differentiation markers. GBM spheroids cultured at 5% oxygen expressed higher levels of hypoxia-inducible genes, including those encoding glycolytic enzymes and pro-angiogenic factors. The genes up-regulated in cells cultured at 5% oxygen had higher expression in parental GBMs compared to that observed in 20% cell cultures, suggesting the preservation of the hypoxic component of GBM transcriptome at 5% oxygen and its loss in standard culture conditions. Evaluation of expression of those genes in The Cancer Genome Atlas dataset comprising samples of normal brain tissue, lower-grade gliomas and GBMs indicated the expression pattern of the indicated genes was specific for GBM. Moreover, GBM cells cultured at 5% oxygen were more resistant to temozolomide, the chemotherapeutic used in GBM therapy. The presented comparison of GBM cultures maintained at high and low oxygen tension together with analysis of tumour transcriptome indicates that lowering oxygen tension during cell culture may more allegedly reproduce tumour cell behaviour within GBM than standard culture conditions (e.g., atmospheric oxygen tension). Low oxygen culture conditions should be considered as a more appropriate model for further studies on glioblastoma pathology and therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Numerous molecular changes are observed during tumor progression. Genes associated with TGFβ isoforms are involved in many cancers, including brain cancer. Using molecular techniques to evaluate 43 brain tumor sections from patients at different stages of astrocytic brain tumor, we assessed differences in the expression patterns of genes associated with TGFβ isoforms and quantified the mRNA of three TGFβ isoforms. Our study confirmed significant differences in the expression of genes associated with TGFβ isoforms as well as differences in isoform expression and the identification of 16 genes that differentiate between disease grades. Database analysis revealed interactions between the products of these genes, some of which are associated with tumorigenesis. Differences in the expression patterns of transcripts associated with TGFβ isoforms confirm that they are involved in astrocytic brain tumor transformation. Quantitative assessment of TGFβ2 mRNA may be a useful method in the future to facilitate the diagnosis of disease grade. Genes associated with the TGFβ isoforms are involved in a number of different cancers, and their effect on the progression of brain tumors is also being discussed. Using an oligonucleotide microarray method, we assessed differences in expression patterns of genes in astrocytic brain tumor sections from 43 patients at different stages of disease. Quantitative mRNA assessment of the three TGFβ isoforms was also performed by real-time RT-qPCR. Oligonucleotide microarray data were analyzed using the PL-Grid Infrastructure. The microarray analysis showed a statistically significant (p < 0.05) increase in TGFβ1 and TGFβ2 expression in G3/G4 stage relative to G2, whereas real-time RT-qPCR validation confirmed this change only for the TGFβ2 isoform (p < 0.05). The oligonucleotide microarray method allowed the identification of 16 differential genes associated with TGFβ isoforms. Analysis of the STRING database showed that the proteins encoded by the analyzed genes form a strong interaction network (p < 0.001), and a significant number of proteins are involved in carcinogenesis. Differences in expression patterns of transcripts associated with TGFβ isoforms confirm that they play a role in astrocytic brain tumor transformation. Quantitative assessment of TGFβ2 mRNA may be a valuable method to complement the diagnostic process in the future. [ABSTRACT FROM AUTHOR]

High-grade gliomas (HGGs), the most common and aggressive primary brain tumors in adults, inevitably recur due to incomplete surgery or resistance to therapy. Intratumoral genomic and cellular heterogeneity of HGGs contributes to therapeutic resistance, recurrence, and poor clinical outcomes. Transcriptomic profiles of HGGs at recurrence have not been investigated in detail. Using targeted sequencing of cancer-related genes and transcriptomics, we identified single nucleotide variations, small insertions and deletions, copy number aberrations (CNAs), as well as gene expression changes and pathway deregulation in 16 pairs of primary and recurrent HGGs. Most of the somatic mutations identified in primary HGGs were not detected after relapse, suggesting a subclone substitution during the tumor progression. We found a novel frameshift insertion in the ZNF384 gene which may contribute to extracellular matrix remodeling. An inverse correlation of focal CNAs in EGFR and PTEN genes was detected. Transcriptomic analysis revealed downregulation of genes involved in messenger RNA splicing, cell cycle, and DNA repair, while genes related to interferon signaling and phosphatidylinositol (PI) metabolism are upregulated in secondary HGGs when compared to primary HGGs. In silico analysis of the tumor microenvironment identified M2 macrophages and immature dendritic cells as enriched in recurrent HGGs, suggesting a prominent immunosuppressive signature. Accumulation of those cells in recurrent HGGs was validated by immunostaining. Our findings point to a substantial transcriptomic deregulation and a pronounced infiltration of immature dendritic cells in recurrent HGG, which may impact the effectiveness of frontline immunotherapies in the GBM management. Key messages: Most of the somatic mutations identified in primary HGGs were not detected after relapse. Focal CNAs in EGFR and PTEN genes are inversely correlated in primary and recurrent HGGs. Transcriptomic changes and distinct immune-related signatures characterize HGG recurrence. Recurrent HGGs are characterized by a prominent infiltration of immature dendritic and M2 macrophages. [ABSTRACT FROM AUTHOR]

Simple Summary: Human RecQ helicases participate in DNA replication, repair and transcription. Due to important roles in many cellular processes, deregulation of these helicases in cancer could be tumour promoting. We found upregulated RECQL4 expression in highly malignant brain tumours (glioblastomas) associated with poor survival of patients. While RECQL4 depletion in human glioma cells with genetic tools slightly impaired cell viability and DNA replication, it induced gross changes in gene expression and increased sensitivity of glioma cells and glioma stem cells to chemotherapeutics. Therefore, targeting RECQL4 in deadly brain tumours could be a new strategy to improve eradication of tumour cells by chemotherapeutics. Anti-tumour therapies eliminate proliferating tumour cells by induction of DNA damage, but genomic aberrations or transcriptional deregulation may limit responses to therapy. Glioblastoma (GBM) is a malignant brain tumour, which recurs inevitably due to chemo- and radio-resistance. Human RecQ helicases participate in DNA repair, responses to DNA damage and replication stress. We explored if a helicase RECQL4 contributes to gliomagenesis and responses to chemotherapy. We found upregulated RECQL4 expression in GBMs associated with poor survival of GBM patients. Increased levels of nuclear and cytosolic RECQL4 proteins were detected in GBMs on tissue arrays and in six glioma cell lines. RECQL4 was detected both in cytoplasm and mitochondria by Western blotting and immunofluorescence. RECQL4 depletion in glioma cells with siRNAs and CRISPR/Cas9 did not affect basal cell viability, slightly impaired DNA replication, but induced profound transcriptomic changes and increased chemosensitivity of glioma cells. Sphere cultures originated from RECQL4-depleted cells had reduced sphere forming capacity, stronger responded to temozolomide upregulating cell cycle inhibitors and pro-apoptotic proteins. RECQL4 deficiency affected mitochondrial network and reduced mitochondrial membrane polarization in LN18 glioblastoma cells. We demonstrate that targeting RECQL4 overexpressed in glioblastoma could be a new strategy to sensitize glioma cells to chemotherapeutics. [ABSTRACT FROM AUTHOR]