Glioblastoma multiforme (GBM) is the most common and fatal primary brain tumor, is highly resistant to conventional radiation and chemotherapy, and is not amenable to effective surgical resection. ...The present review summarizes recent advances in our understanding of the molecular mechanisms of therapeutic resistance of GBM to already known drugs, the molecular characteristics of glioblastoma cells, and the barriers in the brain that underlie drug resistance. We also discuss the progress that has been made in the development of new targeted drugs for glioblastoma, as well as advances in drug delivery across the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB).
Among the great variety of anti-cancer therapeutic strategies, boron neutron capture therapy (BNCT) represents a unique approach that doubles the targeting accuracy due to the precise positioning of ...a neutron beam and the addressed delivery of boron compounds. We have recently demonstrated the principal possibility of using a cell-specific 2'-F-RNA aptamer for the targeted delivery of boron clusters for BNCT. In the present study, we evaluated the amount of boron-loaded aptamer inside the cell via two independent methods: quantitative real-time polymerase chain reaction and inductive coupled plasma-atomic emission spectrometry. Both assays showed that the internalized boron level inside the cell exceeds 1 × 10
atoms/cell. We have synthesized
-dodecaborate conjugates of 2'-F-RNA aptamers GL44 and Waz, with boron clusters attached either at the 3'- or at the 5'-end. The influence of cluster localization was evaluated in BNCT experiments on U-87 MG human glioblastoma cells and normal fibroblasts and subsequent analyses of cell viability via real-time cell monitoring and clonogenic assay. Both conjugates of GL44 aptamer provided a specific decrease in cell viability, while only the 3'-conjugate of the Waz aptamer showed the same effect. Thus, an individual adjustment of boron cluster localization is required for each aptamer. The efficacy of boron-loaded 2'-F-RNA conjugates was comparable to that of
B-boronophenylalanine, so this type of boron delivery agent has good potential for BNCT due to such benefits as precise targeting, low toxicity and the possibility to use boron clusters made of natural, unenriched boron.
Boron neutron capture therapy (BNCT) is a binary radiotherapeutic approach to the treatment of malignant tumors, especially glioblastoma, the most frequent and incurable brain tumor. For successful ...BNCT, a boron-containing therapeutic agent should provide selective and effective accumulation of 10B isotope inside target cells, which are then destroyed after neutron irradiation. Nucleic acid aptamers look like very prospective candidates for carrying 10B to the tumor cells. This study represents the first example of using 2′-F-RNA aptamer GL44 specific to the human glioblastoma U-87 MG cells as a boron delivery agent for BNCT. The closo-dodecaborate residue was attached to the 5′-end of the aptamer, which was also labeled by the fluorophore at the 3′-end. The resulting bifunctional conjugate showed effective and specific internalization into U-87 MG cells and low toxicity. After incubation with the conjugate, the cells were irradiated by epithermal neutrons on the Budker Institute of Nuclear Physics neutron source. Evaluation of the cell proliferation by real-time cell monitoring and the clonogenic test revealed that boron-loaded aptamer decreased specifically the viability of U-87 MG cells to the extent comparable to that of 10B-boronophenylalanine taken as a control. Therefore, we have demonstrated a proof of principle of employing aptamers for targeted delivery of boron-10 isotope in BNCT. Considering their specificity, ease of synthesis, and large toolkit of chemical approaches for high boron-loading, aptamers provide a promising basis for engineering novel BNCT agents.
Boron neutron capture therapy is a unique form of adjuvant cancer therapy for various malignancies including malignant gliomas. The conjugation of boron compounds and human serum albumin (HSA)-a ...carrier protein with a long plasma half-life-is expected to extend systemic circulation of the boron compounds and increase their accumulation in human glioma cells. We report on the synthesis of fluorophore-labeled homocystamide conjugates of human serum albumin and their use in thiol-'click' chemistry to prepare novel multimodal boronated albumin-based theranostic agents, which could be accumulated in tumor cells. The novelty of this work involves the development of the synthesis methodology of albumin conjugates for the imaging-guided boron neutron capture therapy combination. Herein, we suggest using thenoyltrifluoroacetone as a part of an anticancer theranostic construct: approximately 5.4 molecules of thenoyltrifluoroacetone were bound to each albumin. Along with its beneficial properties as a chemotherapeutic agent, thenoyltrifluoroacetone is a promising magnetic resonance imaging agent. The conjugation of bimodal HSA with undecahydro-
-dodecaborate only slightly reduced human glioma cell line viability in the absence of irradiation (~30 μM of boronated albumin) but allowed for neutron capture and decreased tumor cell survival under epithermal neutron flux. The simultaneous presence of undecahydro-
-dodecaborate and labeled amino acid residues (fluorophore dye and fluorine atoms) in the obtained HSA conjugate makes it a promising candidate for the combination imaging-guided boron neutron capture therapy.
The combination of the unique properties of cancer cells makes it possible to find specific ligands that interact directly with the tumor, and to conduct targeted tumor therapy. Phage display is one ...of the most common methods for searching for specific ligands. Bacteriophages display peptides, and the peptides themselves can be used as targeting molecules for the delivery of diagnostic and therapeutic agents. Phage display can be performed both in vitro and in vivo. Moreover, it is possible to carry out the phage display on cells pre-enriched for a certain tumor marker, for example, CD44 and CD133.
For this work we used several methods, such as phage display, sequencing, cell sorting, immunocytochemistry, phage titration.
We performed phage display using different screening systems (in vitro and in vivo), different phage libraries (Ph.D-7, Ph.D-12, Ph.D-C7C) on CD44+/CD133+ and without enrichment U-87 MG cells. The binding efficiency of bacteriophages displayed tumor-targeting peptides on U-87 MG cells was compared in vitro. We also conducted a comparative analysis in vivo of the specificity of the accumulation of selected bacteriophages in the tumor and in the control organs (liver, brain, kidney and lungs).
The screening in vivo of linear phage peptide libraries for glioblastoma was the most effective strategy for obtaining tumor-targeting peptides providing targeted delivery of diagnostic and therapeutic agents to glioblastoma.
Combination therapy is becoming an increasingly important treatment strategy because multi-drugs can maximize therapeutic effect and overcome potential mechanisms of drug resistance. A new ...albumin-based theranostic containing gemcitabine
-dodecaborate analogue has been developed for combining boron neutron capture therapy (BNCT) and chemotheraphy. An exo-heterocyclic amino group of gemcitabine was used to introduce
dodecaborate, and a 5'-hydroxy group was used to tether maleimide moiety through an acid-labile phosphamide linker. The
-trifluoroacylated homocysteine thiolactone was used to attach the gemcitabine analogue to human serum albumin (HSA) bearing Cy5 or Cy7 fluorescent dyes. The half-maximal inhibitory concentration (IC
) of the designed theranostic relative to T98G cells was 0.47 mM with the correlation coefficient R = 0.82. BNCT experiments resulted in a decrease in the viability of T98G cells, and the survival fraction was ≈ 0.4.
Aptamers are currently being investigated for their potential to improve virotherapy. They offer several advantages, including the ability to prevent the aggregation of viral particles, enhance ...target specificity, and protect against the neutralizing effects of antibodies. The purpose of this study was to comprehensively investigate an aptamer capable of enhancing virotherapy. This involved characterizing the previously selected aptamer for vaccinia virus (VACV), evaluating the aggregation and molecular interaction of the optimized aptamers with the recombinant oncolytic virus VV-GMCSF-Lact, and estimating their immunoshielding properties in the presence of human blood serum. We chose one optimized aptamer, NV14t_56, with the highest affinity to the virus from the pool of several truncated aptamers and built its 3D model. The NV14t_56 remained stable in human blood serum for 1 h and bound to VV-GMCSF-Lact in the micromolar range (Kd ≈ 0.35 μM). Based on dynamic light scattering data, it has been demonstrated that aptamers surround viral particles and inhibit aggregate formation. In the presence of serum, the hydrodynamic diameter (by intensity) of the aptamer-virus complex did not change. Microscale thermophoresis (MST) experiments showed that NV14t_56 binds with virus (EC50 = 1.487 × 10
PFU/mL). The analysis of the amplitudes of MST curves reveals that the components of the serum bind to the aptamer-virus complex without disrupting it. In vitro experiments demonstrated the efficacy of VV-GMCSF-Lact in conjunction with the aptamer when exposed to human blood serum in the absence of neutralizing antibodies (Nabs). Thus, NV14t_56 has the ability to inhibit virus aggregation, allowing VV-GMCSF-Lact to maintain its effectiveness throughout the storage period and subsequent use. When employing aptamers as protective agents for oncolytic viruses, the presence of neutralizing antibodies should be taken into account.
Oncolytic viruses are highly promising for cancer treatment because they target and lyse tumor cells. These genetically engineered vectors introduce therapeutic or immunostimulatory genes into the ...tumor. However, viral therapy is not always safe and effective. Several problems are related to oncolytic viruses' targeted delivery to the tumor and immune system neutralization in the bloodstream. Cryoprotection and preventing viral particles from aggregating during storage are other critical issues. Aptamers, short RNA, or DNA oligonucleotides may help to crawl through this bottleneck. They are not immunogenic, are easily synthesized, can be chemically modified, and are not very demanding in storage conditions. It is possible to select an aptamer that specifically binds to any target cell, oncolytic virus, or molecule using the SELEX technology. This review comprehensively highlights the most important research and methodological approaches related to oncolytic viruses and nucleic acid aptamers. Here, we also analyze possible future research directions for combining these two methodologies to improve the effectiveness of cancer virotherapy.
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.
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.
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