Transcriptome Changes in Glioma Cells upon Infection with the Oncolytic Virus VV-GMCSF-Lact.

Oncolytic virotherapy is a rapidly evolving approach that aims to selectively kill cancer cells. We designed a promising recombinant vaccinia virus, VV-GMCSF-Lact, for the treatment of solid tumors, including glioma. We assessed how VV-GMCSF-Lact affects human cells using immortalized and patient-derived glioma cultures and a non-malignant brain cell culture. Studying transcriptome changes in cells 12 h or 24 h after VV-GMCSF-Lact infection, we detected the common activation of histone genes. Additionally, genes associated with the interferon-gamma response, NF-kappa B signaling pathway, and inflammation mediated by chemokine and cytokine signaling pathways showed increased expression. By contrast, genes involved in cell cycle progression, including spindle organization, sister chromatid segregation, and the G2/M checkpoint, were downregulated following virus infection. The upregulation of genes responsible for Golgi vesicles, protein transport, and secretion correlated with reduced sensitivity to the cytotoxic effect of VV-GMCSF-Lact. Higher expression of genes encoding proteins, which participate in the maturation of pol II nuclear transcripts and mRNA splicing, was associated with an increased sensitivity to viral cytotoxicity. Genes whose expression correlates with the sensitivity of cells to the virus are important for increasing the effectiveness of cancer virotherapy. Overall, the results highlight molecular markers, biological pathways, and gene networks influencing the response of glioma cells to VV-GMCSF-Lact.

MicroRNA and mRNA Expression Changes in Glioblastoma Cells Cultivated under Conditions of Neurosphere Formation.

Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. The study of the pathogenesis of GBM, as well as the development of targeted oncolytic drugs, require the use of actual cell models, in particular, the use of 3D cultures or neurospheres (NS). During the formation of NS, the adaptive molecular landscape of the transcriptome, which includes various regulatory RNAs, changes. The aim of this study was to reveal changes in the expression of microRNAs (miRNAs) and their target mRNAs in GBM cells under conditions of NS formation. Neurospheres were obtained from both immortalized U87 MG and patient-derived BR3 GBM cell cultures. Next generation sequencing analysis of small and long RNAs of adherent and NS cultures of GBM cells was carried out. It was found that the formation of NS proceeds with an increase in the level of seven and a decrease in the level of 11 miRNAs common to U87 MG and BR3, as well as an increase in the level of 38 and a decrease in the level of 12 mRNA/lncRNA. Upregulation of miRNAs hsa-miR: -139-5p; -148a-3p; -192-5p; -218-5p; -34a-5p; and -381-3p are accompanied by decreased levels of their target mRNAs: RTN4, FLNA, SH3BP4, DNPEP, ETS2, MICALL1, and GREM1. Downregulation of hsa-miR: -130b-5p, -25-5p, -335-3p and -339-5p occurs with increased levels of mRNA-targets BDKRB2, SPRY4, ERRFI1 and TGM2. The involvement of SPRY4, ERRFI1, and MICALL1 mRNAs in the regulation of EGFR/FGFR signaling highlights the role of hsa-miR: -130b-5p, -25-5p, -335-3p, and -34a-5p not only in the formation of NS, but also in the regulation of malignant growth and invasion of GBM. Our data provide the basis for the development of new approaches to the diagnosis and treatment of GBM.

Transcriptome Changes in Glioma Cells Cultivated under Conditions of Neurosphere Formation.

Glioma is the most common and heterogeneous primary brain tumor. The development of a new relevant preclinical models is necessary. As research moves from cultures of adherent gliomas to a more relevant model, neurospheres, it is necessary to understand the changes that cells undergo at the transcriptome level. In the present work, we used three patient-derived gliomas and two immortalized glioblastomas, while their cultivation was carried out under adherent culture and neurosphere (NS) conditions. When comparing the transcriptomes of monolayer (ML) and NS cell cultures, we used Enrichr genes sets enrichment analysis to describe transcription factors (TFs) and the pathways involved in the formation of glioma NS. It was observed that NS formation is accompanied by the activation of five common gliomas of TFs, SOX2, UBTF, NFE2L2, TCF3 and STAT3. The sets of transcripts controlled by TFs MYC and MAX were suppressed in NS. Upregulated genes are involved in the processes of the epithelial-mesenchymal transition, cancer stemness, invasion and migration of glioma cells. However, MYC/MAX-dependent downregulated genes are involved in translation, focal adhesion and apical junction. Furthermore, we found three EGFR and FGFR signaling feedback regulators common to all analyzed gliomas-SPRY4, ERRFI1, and RAB31-which can be used for creating new therapeutic strategies of suppressing the invasion and progression of gliomas.

Efficacy of the new therapeutic approach in curing malignant neoplasms on the model of human glioblastoma.

OBJECTIVE: Glioma is a highly invasive tumor, frequently disposed in essential areas of the brain, which makes its surgical excision extremely difficult; meanwhile adjuvant therapy remains quite ineffective. METHODS: In the current report, a new therapeutic approach in curing malignant neoplasms has been performed on the U87 human glioblastoma model. This approach, termed "Karanahan", is aimed at the eradication of cancer stem cells (CSCs), which were recently shown to be capable of internalizing fragments of extracellular double-stranded DNA. After being internalized, these fragments interfere in the process of repairing interstrand cross-links caused by exposure to appropriate cytostatics, and such an interference results either in elimination of CSCs or in the loss of their tumorigenic potency. Implementation of the approach requires a scheduled administration of cytostatic and complex composite double-stranded DNA preparation. RESULTS: U87 cells treated in vitro in accordance with the Karanahan approach completely lost their tumorigenicity and produced no grafts upon intracerebral transplantation into immunodeficient mice. In SCID mice with developed subcutaneous grafts, the treatment resulted in reliable slowing down of tumor growth rate (P < 0.05). In the experiment with intracerebral transplantation of U87 cells followed by surgical excision of the developed graft and subsequent therapeutic treatment, the Karanahan approach was shown to reliably slow down the tumor growth rate and increase the median survival of the mice twofold relative to the control. CONCLUSIONS: The effectiveness of the Karanahan approach has been demonstrated both in vitro and in vivo in treating developed subcutaneous grafts as well as orthotopic grafts after surgical excision of the tumor.

6-(4'-Aryl-1',2',3'-triazolyl)-spirostan-3,5-diols and 6-(4'-Aryl-1',2',3'-triazolyl)-7-hydroxyspirosta-1,4-dien-3-ones: Synthesis and analysis of their cytotoxicity.

In an endeavour to develop potent anti-tumor agents from diosgenin, a series of C-6 derived 1,2,3-triazolyl derivatives were designed and synthesized by employing Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction of novel azides - (22R,25R)-6ß-azidospirostan-3ß,5α-diol and 6ß-azido-7α-hydroxyspirosta-1,4-dien-3-one with aryl(hetaryl)alkynes. All the derivatives were evaluated for cytotoxic activity by MTT assay against eight different human cancer cell lines: T-cellular leucosis (CEM-13), human monocytes (U-937), breast (MDA-MB-231, BT-474), prostate (DU-145) and glioblastoma (U-87MG, SNB-19, T98G). The results of this study suggested that 6-(4'-aryl-1',2',3'-triazolyl)spirostan-3,5-diols 2, 3, 4, 5 and 6 possessed a promising cytotoxic potential. The corresponding 6-substituted 7-hydroxy-1,4-spirostadien-3-ones shown less cytotoxity on the human cancer cells. Compounds 2, 3, 4, and 5 which demonstrated high grown inhibition against glioma cancer cells U-87 and T98G, and also on the human-derived N118669 primary glioblastoma cell line (with GI50 values in the range of 5-9 µM), were not affected the growth of SNB-19 cells. The data revealed that phenyl, 4-methoxyphenyl, 4-fluorophenyl, 3,4,5-trimethoxyphenyl or 2-pyridinyl substituent in the triazole moiety at the C-6 position significantly improved the anti-tumor activity. The mentioned position at the spirostan core may be favourable for the synthesis of potent anticancer leads from diosgenin.

Novel Cancer Stem Marker and Its Applicability for Grading Primary Human Gliomas.

Poorly differentiated cell populations including tumor-initiating stem cells have been demonstrated to display a unique ability to natively internalize fragmented double-stranded DNA. Using this feature as a marker, we show that 0.1% to 6% of human glioblastoma cells from the bioptates can effectively internalize a fluorescently labeled DNA probe. Of these, using samples from 3 patients, 66% to 100% cells are also positive for CD133, a well-established surface marker of tumor-initiating glioma stem cells. Using the samples from primary malignant brain lesions (33 patients), we demonstrate that tumor grading significantly correlates ( R = .71) with the percentage of DNA-internalizing cells. No such correlation is observed for relapse samples (18 patients).

Identification of cancer stem cells and a strategy for their elimination.

It has been established previously that up to 40% of mouse CD34(+) hematopoietic stem cells are capable of internalizing exogenous dsDNA fragments both in vivo and ex vivo. Importantly, when mice are treated with a combination of cyclophosphamide and dsDNA, the repair of interstrand crosslinks in hematopoietic progenitors is attenuated, and their pluripotency is altered. Here we show for the first time that among various actively proliferating mammalian cell populations there are subpopulations capable of internalizing dsDNA fragments. In the context of cancer, such dsDNA-internalizing cell subpopulations display cancer stem cell-like phenotype. Furthermore, using Krebs-2 ascites cells as a model, we found that upon combined treatment with cyclophosphamide and dsDNA, engrafted material loses its tumor-initiating properties which we attribute to the elimination of tumor-initiating stem cell subpopulation or loss of its tumorigenic potential.

Cytotoxic activity of ex-vivo generated IFNα-induced monocyte-derived dendritic cells in brain glioma patients.

Recent studies have revealed that besides the important role in triggering the adoptive antitumor immunity, dendritic cells (DCs) possess direct cytotoxic antitumor activity. Here, we investigated brain glioma patient monocyte-derived DCs generated in the presence of IFNα and GM-CSF (IFN-DCs). These DCs were characterized by reduced cytotoxic activity against TRAIL-resistant HEp-2 cells. The impairment of DC cytotoxic function was observed mainly in high-grade glioma patients and associated with poor survival. The dysfunction of patient DC cytotoxicity was partially restored under in vitro pretreatment of DCs with double-stranded human DNA as well as rIL-2. In contrast to healthy donors, IFN-DCs in a part of high-grade glioma patients also failed to lyse primary autologous or allogeneic glioma cells. Our findings point to possible contribution of DC impairment in tumor pathogenesis in brain glioma and justify the necessity to evaluate and correct DC cytotoxic function when exploring DCs as cancer vaccines in glioma.