Tumor- and Fibroblast-Derived Cell-Free DNAs Differently Affect the Progression of B16 Melanoma In Vitro and In Vivo.

It is widely postulated that the majority of pathologically elevated extracellular or cell-free DNA (cfDNA) in cancer originates from tumor cells; however, evidence has emerged regarding the significant contributions of other cells from the tumor microenvironment. Here, the effect of cfDNA originating from murine B16 melanoma cells and L929 fibroblasts on B16 cells was investigated. It was found that cfDNAL929 increased the viability and migration properties of B16 cells in vitro and their invasiveness in vivo. In contrast, cfDNAB16 exhibited a negative effect on B16 cells, reducing their viability and migration in vitro, which in vivo led to decreased tumor size and metastasis number. It was shown that cell treatment with both cfDNAs resulted in an increase in the expression of genes encoding DNases and the oncogenes Braf, Kras, and Myc. cfDNAL929-treated cells were shown to experience oxidative stress. Gene expression changes in the case of cfDNAB16 treatment are well correlated with the observed decrease in proliferation and migration of B16 cells. The obtained data may indicate the possible involvement of fibroblast DNA in the tumor microenvironment in tumor progression and, potentially, in the formation of new tumor foci due to the transformation of normal cells.

A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen.

Neutrophils are considered as the main player in innate immunity. In the last few years, it has been shown that they are involved in different physiological conditions and diseases. However, progress in the field of neutrophil biology is relatively slow due to existing difficulties in neutrophil isolation and maintenance in culture. Here we compare four protocols based on density-gradient and immunomagnetic methods for isolation of murine neutrophils from bone marrow and spleen. Neutrophil isolation was performed using Ficoll 1.077/1.119 g/mL density gradient, Ficoll 1.083/1.090/1.110 g/mL density gradient and immunomagnetic method of negative and positive selection. The different protocols were compared with respect to sample purity, cell viability, yield, and cost. The functionality of isolated neutrophils was checked by NETosis analysis and neutrophil oxidative burst test. Obtained data revealed that given purity/yield/viability/cost ratio the protocol based on cell centrifugation on Ficoll 1.077/1.119 g/mL density gradient is recommended for isolation of neutrophils from bone marrow, whereas immunomagnetic method of positive selection using Dynabeads is recommended for isolation of splenic neutrophils.

The Effect of Cell-Free DNA from Blood Serum of Mice with Metastatic Melanoma on Enhancement of Oncogenic Properties of Melanoma Cells.

Currently, a significant increase in the levels of circulating cell-free DNA (cfDNA) in the blood of patients is considered as a generally recognized marker of the development of oncological diseases. Although the tumor-associated cfDNA has been well studied, its biological functions remain unclear. In this work, we investigated the effect of cfDNA isolated from the blood serum of the mice with B16-F10 metastatic melanoma on the properties of the B16-F10 melanoma cells in vitro. It was found that the profile of cfDNA isolated from the blood serum of mice with melanoma differs significantly from the cfDNA isolated from the blood serum of healthy mice, and is similar to the genomic DNA of B16 cells with regards to abundance of oncogenes and mobile genetic elements (MGE). It was shown that the cfDNA of mice with melanoma penetrated into B16 cells, resulting in the increase in abundance of oncogenes and MGE fragments, and caused 5-fold increase of the mRNA level of the secreted DNase Dnase1l3 and a slight increase of the mRNA level of the Jun, Fos, Ras, and Myc oncogenes. cfDNA of the healthy mice caused increase of the mRNA level of intracellular regulatory DNase EndoG and 4-fold increase of the mRNA level of Fos and Ras oncogenes, which are well-known triggers of a large number of signal cascades, from apoptosis inhibition to increased tumor cell proliferation. Thus, it is obvious that the circulating cfDNA of tumor origin is able to penetrate into the cells and, despite the fact that no changes were found in the level of viability and migration activity of the tumor cells, cfDNA, even with a single exposure, can cause changes at the cellular level that increase oncogenicity of the recipient cells.

Diverse Neutrophil Functions in Cancer and Promising Neutrophil-Based Cancer Therapies.

Neutrophils represent the most abundant cell type of leukocytes in the human blood and have been considered a vital player in the innate immune system and the first line of defense against invading pathogens. Recently, several studies showed that neutrophils play an active role in the immune response during cancer development. They exhibited both pro-oncogenic and anti-tumor activities under the influence of various mediators in the tumor microenvironment. Neutrophils can be divided into several subpopulations, thus contradicting the traditional concept of neutrophils as a homogeneous population with a specific function in the innate immunity and opening new horizons for cancer therapy. Despite the promising achievements in this field, a full understanding of tumor-neutrophil interplay is currently lacking. In this review, we try to summarize the current view on neutrophil heterogeneity in cancer, discuss the different communication pathways between tumors and neutrophils, and focus on the implementation of these new findings to develop promising neutrophil-based cancer therapies.

Dendritic Cell-Derived Artificial Microvesicles Inhibit RLS40 Lymphosarcoma Growth in Mice via Stimulation of Th1/Th17 Immune Response.

Cell-free antitumor vaccines represent a promising approach to immunotherapy of cancer. Here, we compare the antitumor potential of cell-free vaccines based on microvesicles derived from dendritic cells (DCs) with DC- and cationic-liposome-based vaccines using a murine model of drug-resistant lymphosarcoma RLS40 in vivo. The vaccines were the following: microvesicle vaccines­cytochalasin B-induced membrane vesicles (CIMVs) obtained from DCs loaded with total tumor RNA using cholesterol/spermine-containing cationic liposomes L or mannosylated liposomes ML; DC vaccines­murine DCs loaded with total tumor-derived RNA using the same liposomes; and liposomal vaccines­lipoplexes of total tumor-derived RNA with liposomes L or ML. Being non-hepatotoxic, CIMV- and DC-based vaccines administered subcutaneously exhibited comparable potential to stimulate highly efficient antitumor CTLs in vivo, whereas liposomal vaccines were 25% weaker CTL inducers. Nevertheless, the antitumor efficiencies of the different types of the vaccines were similar: sizes of tumor nodes and the number of liver metastases were significantly decreased, regardless of the vaccine type. Notably, the booster vaccination did not improve the overall antitumor efficacy of the vaccines under the study. CIMV- and DC- based vaccines more efficiently than liposome-based ones decreased mitotic activity of tumor cells and induced their apoptosis, stimulated accumulation of neutrophil inflammatory infiltration in tumor tissue, and had a more pronounced immunomodulatory activity toward the spleen and thymus. Administration of CIMV-, DC-, and liposome-based vaccines resulted in activation of Th1/Th17 cells as well as the induction of positive immune checkpoint 4-1BBL and downregulation of suppressive immune checkpoints in a raw PD-1 >>> TIGIT > CTLA4 > TIM3. We demonstrated that cell-free CIMV-based vaccines exhibited superior antitumor and antimetastatic activity in a tumor model in vivo. The obtained results can be considered as the basis for developing novel strategies for oncoimmunotherapy.

Bovine Pancreatic RNase A: An Insight into the Mechanism of Antitumor Activity In Vitro and In Vivo.

In this investigation, we extensively studied the mechanism of antitumor activity of bovine pancreatic RNase A. Using confocal microscopy, we show that after RNase A penetration into HeLa and B16 cells, a part of the enzyme remains unbound with the ribonuclease inhibitor (RI), resulting in the decrease in cytosolic RNAs in both types of cells and rRNAs in the nucleoli of HeLa cells. Molecular docking indicates the ability of RNase A to form a complex with Ku70/Ku80 heterodimer, and microscopy data confirm its localization mostly inside the nucleus, which may underlie the mechanism of RNase A penetration into cells and its intracellular traffic. RNase A reduced migration and invasion of tumor cells in vitro. In vivo, in the metastatic model of melanoma, RNase A suppressed metastases in the lungs and changed the expression of EMT markers in the tissue adjacent to metastatic foci; this increased Cdh1 and decreased Tjp1, Fn and Vim, disrupting the favorable tumor microenvironment. A similar pattern was observed for all genes except for Fn in metastatic foci, indicating a decrease in the invasive potential of tumor cells. Bioinformatic analysis of RNase-A-susceptible miRNAs and their regulatory networks showed that the main processes modulated by RNase A in the tumor microenvironment are the regulation of cell adhesion and junction, cell cycle regulation and pathways associated with EMT and tumor progression.

Antisense oligonucleotide gapmers containing phosphoryl guanidine groups reverse MDR1-mediated multiple drug resistance of tumor cells.

Antisense gapmer oligonucleotides containing phosphoryl guanidine (PG) groups, e.g., 1,3-dimethylimidazolidin-2-imine, at three to five internucleotidic positions adjacent to the 3' and 5' ends were prepared via the Staudinger chemistry, which is compatible with conditions of standard automated solid-phase phosphoramidite synthesis for phosphodiester and, notably, phosphorothioate linkages, and allows one to design a variety of gapmeric structures with alternating linkages, and deoxyribose or 2'-O-methylribose backbone. PG modifications increased nuclease resistance in serum-containing medium for more than 21 days. Replacing two internucleotidic phosphates by PG groups in phosphorothioate-modified oligonucleotides did not decrease their cellular uptake in the absence of lipid carriers. Increasing the number of PG groups from two to seven per oligonucleotide reduced their ability to enter the cells in the carrier-free mode. Cationic liposomes provided similar delivery efficiency of both partially PG-modified and unmodified oligonucleotides. PG-gapmers were designed containing three to four PG groups at both wings and a central "window" of seven deoxynucleotides with either phosphodiester or phosphorothioate linkages targeted to MDR1 mRNA providing multiple drug resistance of tumor cells. Gapmers efficiently silenced MDR1 mRNA and restored the sensitivity of tumor cells to chemotherapeutics. Thus, PG-gapmers can be considered as novel, promising types of antisense oligonucleotides for targeting biologically relevant RNAs.

Human Recombinant DNase I (Pulmozyme®) Inhibits Lung Metastases in Murine Metastatic B16 Melanoma Model That Correlates with Restoration of the DNase Activity and the Decrease SINE/LINE and c-Myc Fragments in Blood Cell-Free DNA.

Tumor-associated cell-free DNAs (cfDNA) play an important role in the promotion of metastases. Previous studies proved the high antimetastatic potential of bovine pancreatic DNase I and identified short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)and fragments of oncogenes in cfDNA as the main molecular targets of enzyme in the bloodstream. Here, recombinant human DNase I (commercial name Pulmozyme®), which is used for the treatment of cystic fibrosis in humans, was repurposed for the inhibition of lung metastases in the B16 melanoma model in mice. We found that Pulmozyme® strongly reduced migration and induced apoptosis of B16 cells in vitro and effectively inhibited metastases in lungs and liver in vivo. Pulmozyme® was shown to be two times more effective when administered intranasally (i.n.) than bovine DNase I, but intramuscular (i.m.) administration forced it to exhibit as high an antimetastatic activity as bovine DNase I. Both DNases administered to mice either i.m. or i.n. enhanced the DNase activity of blood serum to the level of healthy animals, significantly decreased cfDNA concentrations, efficiently degraded SINE and LINE repeats and c-Myc fragments in the bloodstream and induced apoptosis and disintegration of neutrophil extracellular traps in metastatic foci; as a result, this manifested as the inhibition of metastases spread. Thus, Pulmozyme®, which is already an approved drug, can be recommended for use in the treatment of lung metastases.

Role of Cell-Free DNA and Deoxyribonucleases in Tumor Progression.

Many studies have reported an increase in the level of circulating cell-free DNA (cfDNA) in the blood of patients with cancer. cfDNA mainly comes from tumor cells and, therefore, carries features of its genomic profile. Moreover, tumor-derived cfDNA can act like oncoviruses, entering the cells of vulnerable organs, transforming them and forming metastatic nodes. Another source of cfDNA is immune cells, including neutrophils that generate neutrophil extracellular traps (NETs). Despite the potential eliminative effect of NETs on tumors, in some cases, their excessive generation provokes tumor growth as well as invasion. Considering both possible pathological contributions of cfDNA, as an agent of oncotransformation and the main component of NETs, the study of deoxyribonucleases (DNases) as anticancer and antimetastatic agents is important and promising. This review considers the pathological role of cfDNA in cancer development and the role of DNases as agents to prevent and/or prohibit tumor progression and the development of metastases.

Antitumour Activity of the Ribonuclease Binase from Bacillus pumilus in the RLS40 Tumour Model Is Associated with the Reorganisation of the miRNA Network and Reversion of Cancer-Related Cascades to Normal Functioning.

The important role of miRNA in cell proliferation and differentiation has raised interest in exogenous ribonucleases (RNases) as tools to control tumour-associated intracellular and extracellular miRNAs. In this work, we evaluated the effects of the RNase binase from Bacillus pumilus on small non-coding regulatory RNAs in the context of mouse RLS40 lymphosarcoma inhibition. In vitro binase exhibited cytotoxicity towards RLS40 cells via apoptosis induction through caspase-3/caspase-7 activation and decreased the levels of miR-21a, let-7g, miR-31 and miR-155. Intraperitoneal injections of binase in RLS40-bearing mice resulted in the retardation of primary tumour growth by up to 60% and inhibition of metastasis in the liver by up to 86%, with a decrease in reactive inflammatory infiltration and mitosis in tumour tissue. In the blood serum of binase-treated mice, decreases in the levels of most studied miRNAs were observed, excluding let-7g, while in tumour tissue, the levels of oncomirs miR-21, miR-10b, miR-31 and miR-155, and the oncosuppressor let-7g, were upregulated. Analysis of binase-susceptible miRNAs and their regulatory networks showed that the main modulated events were transcription and translation control, the cell cycle, cell proliferation, adhesion and invasion, apoptosis and autophagy, as well as some other tumour-related cascades, with an impact on the observed antitumour effects.

Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance.

Irreversible destruction of disease-associated regulatory RNA sequences offers exciting opportunities for safe and powerful therapeutic interventions against human pathophysiology. In 2017, for the first time we introduced miRNAses-miRNA-targeted conjugates of a catalytic peptide and oligonucleotide capable of cleaving an miRNA target. Herein, we report the development of Dual miRNases against oncogenic miR-21, miR-155, miR-17 and miR-18a, each containing the catalytic peptide placed in-between two short miRNA-targeted oligodeoxyribonucleotide recognition motifs. Substitution of adenines with 2-aminoadenines in the sequence of oligonucleotide "shoulders" of the Dual miRNase significantly enhanced the efficiency of hybridization with the miRNA target. It was shown that sequence-specific cleavage of the target by miRNase proceeded metal-independently at pH optimum 5.5-7.5 with an efficiency varying from 15% to 85%, depending on the miRNA sequence. A distinct advantage of the engineered nucleases is their ability to additionally recruit RNase H and cut miRNA at three different locations. Such cleavage proceeds at the central part by Dual miRNase, and at the 5'- and 3'-regions by RNase H, which significantly increases the efficiency of miRNA degradation. Due to increased activity at lowered pH Dual miRNases could provide an additional advantage in acidic tumor conditions and may be considered as efficient tumor-selective RNA-targeted therapeutic.

Targeting Circulating SINEs and LINEs with DNase I Provides Metastases Inhibition in Experimental Tumor Models.

Tumor-associated cell-free DNAs (cfDNAs) are found to play some important roles at different stages of tumor progression; they are involved in the transformation of normal cells and contribute to tumor migration and invasion. DNase I is considered a promising cancer cure, due to its ability to degrade cfDNAs. Previous studies using murine tumor models have proved the high anti-metastatic potential of DNase I. Later circulating cfDNAs, especially tandem repeats associated with short-interspersed nuclear elements (SINEs) and long-interspersed nuclear elements (LINEs), have been found to be the enzyme's main molecular targets. Here, using Lewis lung carcinoma, melanoma B16, and lymphosarcoma RLS40 murine tumor models, we reveal that tumor progression is accompanied by an increase in the level of SINE and LINEs in the pool of circulating cfDNAs. Treatment with DNase I decreased in the number and area of metastases by factor 3-10, and the size of the primary tumor node by factor 1.5-2, which correlated with 5- to 10-fold decreasing SINEs and LINEs. We demonstrated that SINEs and LINEs from cfDNA of tumor-bearing mice are able to penetrate human cells. The results show that SINEs and LINEs could be important players in metastasis, and this allows them to be considered as attractive new targets for anticancer therapy.

Immunotherapy Based on Dendritic Cell-Targeted/-Derived Extracellular Vesicles-A Novel Strategy for Enhancement of the Anti-tumor Immune Response.

Dendritic cell (DC)-based anti-tumor vaccines have great potential for the treatment of cancer. To date, a large number of clinical trials involving DC-based vaccines have been conducted with a view to treating tumors of different histological origins. However, DC-based vaccines had several drawbacks, including problems with targeted delivery of tumor antigens to DCs and prolong storage of cellular vaccines. Therefore, the development of other immunotherapeutic approaches capable of enhancing the immunogenicity of existing DC-based vaccines or directly triggering anti-tumor immune responses is of great interest. Extracellular vesicles (EVs) are released by almost all types of eukaryotic cells for paracrine signaling. EVs can interact with target cells and change their functional activity by delivering different signaling molecules including mRNA, non-coding RNA, proteins, and lipids. EVs have potential benefits as natural vectors for the delivery of RNA and other therapeutic molecules targeted to DCs, T-lymphocytes, and tumor cells; therefore, EVs are a promising entity for the development of novel cell-free anti-tumor vaccines that may be a favourable alternative to DC-based vaccines. In the present review, we discuss the anti-tumor potential of EVs derived from DCs, tumors, and other cells. Methods of EV isolation are systematized, and key molecules carried by EVs that are necessary for the activation of a DC-mediated anti-tumor immune response are analyzed with a focus on the RNA component of EVs. Characteristics of anti-tumor immune responses induced by EVs in vitro and in vivo are reviewed. Finally, perspectives and challenges with the use of EVs for the development of anti-tumor cell-free vaccines are considered.

Surveillance of Tumour Development: The Relationship Between Tumour-Associated RNAs and Ribonucleases.

Tumour progression is accompanied by rapid cell proliferation, loss of differentiation, the reprogramming of energy metabolism, loss of adhesion, escape of immune surveillance, induction of angiogenesis, and metastasis. Both coding and regulatory RNAs expressed by tumour cells and circulating in the blood are involved in all stages of tumour progression. Among the important tumour-associated RNAs are intracellular coding RNAs that determine the routes of metabolic pathways, cell cycle control, angiogenesis, adhesion, apoptosis and pathways responsible for transformation, and intracellular and extracellular non-coding RNAs involved in regulation of the expression of their proto-oncogenic and oncosuppressing mRNAs. Considering the diversity/variability of biological functions of RNAs, it becomes evident that extracellular RNAs represent important regulators of cell-to-cell communication and intracellular cascades that maintain cell proliferation and differentiation. In connection with the elucidation of such an important role for RNA, a surge in interest in RNA-degrading enzymes has increased. Natural ribonucleases (RNases) participate in various cellular processes including miRNA biogenesis, RNA decay and degradation that has determined their principal role in the sustention of RNA homeostasis in cells. Findings were obtained on the contribution of some endogenous ribonucleases in the maintenance of normal cell RNA homeostasis, which thus prevents cell transformation. These findings directed attention to exogenous ribonucleases as tools to compensate for the malfunction of endogenous ones. Recently a number of proteins with ribonuclease activity were discovered whose intracellular function remains unknown. Thus, the comprehensive investigation of physiological roles of RNases is still required. In this review we focused on the control mechanisms of cell transformation by endogenous ribonucleases, and the possibility of replacing malfunctioning enzymes with exogenous ones.

Catalytic Knockdown of miR-21 by Artificial Ribonuclease: Biological Performance in Tumor Model.

Control of the expression of oncogenic small non-coding RNAs, notably microRNAs (miRNAs), is an attractive therapeutic approach. We report a design platform for catalytic knockdown of miRNA targets with artificial, sequence-specific ribonucleases. miRNases comprise a peptide [(LeuArg)2Gly]2 capable of RNA cleavage conjugated to the miRNA-targeted oligodeoxyribonucleotide, which becomes nuclease-resistant within the conjugate design, without resort to chemically modified nucleotides. Our data presented here showed for the first time a truly catalytic character of our miR-21-miRNase and its ability to cleave miR-21 in a multiple catalytic turnover mode. We demonstrate that miRNase targeted to miR-21 (miR-21-miRNase) knocked down malignant behavior of tumor cells, including induction of apoptosis, inhibition of cell invasiveness, and retardation of tumor growth, which persisted on transplantation into mice of tumor cells treated once with miR-21-miRNase. Crucially, we discover that the high biological activity of miR-21-miRNase can be directly related not only to its truly catalytic sequence-specific cleavage of miRNA but also to its ability to recruit the non-sequence specific RNase H found in most cells to elevate catalytic turnover further. miR-21-miRNase worked synergistically even with low levels of RNase H. Estimated degradation in the presence of RNase H exceeded 103 miRNA target molecules per hour for each miR-21-miRNase molecule, which provides the potency to minimize delivery requirements to a few molecules per cell. In contrast to the comparatively high doses required for the simple steric block of antisense oligonucleotides, truly catalytic inactivation of miRNA offers more effective, irreversible, and persistent suppression of many copy target sequences. miRNase design can be readily adapted to target other pathogenic microRNAs overexpressed in many disease states.

The systemic tumor response to RNase A treatment affects the expression of genes involved in maintaining cell malignancy.

Recently, pancreatic RNase A was shown to inhibit tumor and metastasis growth that accompanied by global alteration of miRNA profiles in the blood and tumor tissue (Mironova et al., 2013). Here, we performed a whole transcriptome analysis of murine Lewis lung carcinoma (LLC) after treatment of tumor-bearing mice with RNase A. We identified 966 differentially expressed transcripts in LLC tumors, of which 322 were upregulated and 644 were downregulated after RNase A treatment. Many of these genes are involved in signaling pathways that regulate energy metabolism, cell-growth promoting and transforming activity, modulation of the cancer microenvironment and extracellular matrix components, and cellular proliferation and differentiation. Following RNase A treatment, we detected an upregulation of carbohydrate metabolism, inositol phosphate cascade and oxidative phosphorylation, re-arrangement of cell adhesion, cell cycle control, apoptosis, and transcription. Whereas cancer-related signaling pathways (e.g., TGF-beta, JAK/STAT, and Wnt) were downregulated following RNase A treatment, as in the case of the PI3K/AKT pathway, which is involved in the progression of non-small lung cancer. RNase A therapy resulted in the downregulation of genes that inhibit the biogenesis of some miRNAs, particularly the let-7 miRNA family. Taken together, our data suggest that the antitumor activity and decreased invasion potential of tumor cells caused by RNase A are associated with enhanced energy cascade functioning, rearrangement of cancer-related events regulating cell growth and dissemination, and attenuation of signaling pathways having tumor-promoting activity. Thus, RNase A can be proposed as a potential component of anticancer therapy with multiple modes of action.

Alteration of the exDNA profile in blood serum of LLC-bearing mice under the decrease of tumour invasion potential by bovine pancreatic DNase I treatment.

Taking into account recently obtained data indicating the participation of circulating extracellular DNA (exDNA) in tumorigenesis, enzymes with deoxyribonucleic activity have again been considered as potential antitumour and antimetastatic drugs. Previously, using murine Lewis lung carcinoma and hepatocellular carcinoma A1 tumour models, we have shown the antimetastatic activity of bovine DNase I, which correlates with an increase of DNase activity and a decrease of exDNA concentration in the blood serum of tumour-bearing mice. In this work, using next-generation sequencing on the ABS SOLiD™ 5.500 platform, we performed a search for molecular targets of DNase I by comparing the exDNA profiles of healthy animals, untreated animals with Lewis lung carcinoma (LLC) and those with LLC treated with DNase I. We found that upon DNase I treatment of LLC-bearing mice, together with inhibition of metastasis, a number of strong alterations in the patterns of exDNA were observed. The major differences in exDNA profiles between groups were: i) the level of GC-poor sequences increased during tumour development was reduced to that of healthy mice; ii) levels of sequences corresponding to tumour-associated genes Hmga2, Myc and Jun were reduced in the DNase I-treated group in comparison with non-treated mice; iii) 224 types of tandem repeat over-presented in untreated LLC-bearing mice were significantly reduced after DNase I treatment. The most important result obtained in the work is that DNase I decreased the level of B-subfamily repeats having homology to human ALU repeats, known as markers of carcinogenesis, to the level of healthy animals. Thus, the obtained data lead us to suppose that circulating exDNA plays a role in tumour dissemination, and alteration of multiple molecular targets in the bloodstream by DNase I reduces the invasive potential of tumours.

miRNases: Novel peptide-oligonucleotide bioconjugates that silence miR-21 in lymphosarcoma cells.

MicroRNAs (miRNAs) are active regulators in malignant growth and constitute potential targets for anticancer therapy. Consequently, considerable effort has focused on identifying effective ways to modulate aberrant miRNA expression. Here we introduce and assess a novel type of chemically engineered biomaterial capable of cleaving specific miRNA sequences, i.e. miRNA-specific artificial ribonucleases (hereafter 'miRNase'). The miRNase template presented here consists of the catalytic peptide Acetyl-[(LeuArg)2Gly]2 covalently attached to a miRNA-targeting oligonucleotide, which can be linear or hairpin. The peptide C-terminus is conjugated to an aminohexyl linker located at either the 3'- or 5'-end of the oligonucleotide. The cleavage efficacy, structural aspects of cleavage and biological relevance of a set of these designed miRNases was assayed with respect to highly oncogenic miR-21. Several miRNases demonstrated effective site-selective cleavage of miR-21 exclusively at G-X bonds. One of the most efficient miRNase was shown to specifically inhibit miR-21 in lymphosarcoma cells and lead to a reduction in their proliferative activity. This report provides the first experimental evidence that metallo-independent peptide-oligonucleotide chemical ribonucleases are able to effectively and selectively down-regulate oncogenic miRNA in tumour cells, thus suggesting their potential in development of novel therapeutics aimed at overcoming overexpression of disease-related miRNAs.

Prophylactic Dendritic Cell-Based Vaccines Efficiently Inhibit Metastases in Murine Metastatic Melanoma.

Recent data on the application of dendritic cells (DCs) as anti-tumor vaccines has shown their great potential in therapy and prophylaxis of cancer. Here we report on a comparison of two treatment schemes with DCs that display the models of prophylactic and therapeutic vaccination using three different experimental tumor models: namely, Krebs-2 adenocarcinoma (primary tumor), melanoma (B16, metastatic tumor without a primary node) and Lewis lung carcinoma (LLC, metastatic tumor with a primary node). Dendritic cells generated from bone marrow-derived DC precursors and loaded with lysate of tumor cells or transfected with the complexes of total tumor RNA with cationic liposomes were used for vaccination. Lipofectamine 2000 and liposomes consisting of helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and cationic lipid 2D3 (1,26-Bis(1,2-de-O-tetradecyl-rac-glycerol)-7,11,16,20-tetraazahexacosan tetrahydrocloride) were used for RNA transfection. It was shown that DCs loaded with tumor lysate were ineffective in contrast to tumor-derived RNA. Therapeutic vaccination with DCs loaded by lipoplexes RNA/Lipofectamine 2000 was the most efficient for treatment of non-metastatic Krebs-2, where a 1.9-fold tumor growth retardation was observed. Single prophylactic vaccination with DCs loaded by lipoplexes RNA/2D3 was the most efficient to treat highly aggressive metastatic tumors LLC and B16, where 4.7- and 10-fold suppression of the number of lung metastases was observed, respectively. Antimetastatic effect of single prophylactic DC vaccination in metastatic melanoma model was accompanied by the reductions in the levels of Th2-specific cytokines however the change of the levels of Th1/Th2/Th17 master regulators was not found. Failure of double prophylactic vaccination is explained by Th17-response polarization associated with autoimmune and pro-inflammatory reactions. In the case of therapeutic DC vaccine the polarization of Th1-response was found nevertheless the antimetastatic effect was less effective in comparison with prophylactic DC vaccine.

Multicomponent mannose-containing liposomes efficiently deliver RNA in murine immature dendritic cells and provide productive anti-tumour response in murine melanoma model.

Here we demonstrate the ability of mannosylated liposomes (ML) targeted to mannose receptors (MR) to perform the targeted delivery of model plasmid DNA encoding EGFP and total tumour RNA into murine bone-marrow-derived dendritic cells (DCs) and enhance the efficiency of anti-tumour response triggered by these DCs in murine melanoma model. ML consist of cationic lipid 2X3 (1,26-Bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and 2.5, 5 or 10% mol. of novel mannosylated lipoconjugates. In the structure of lipoconjugates D-mannose was attached to ditetradecylglycerol residue via succinyl (lipoconjugate 1) or diethylsquarate (lipoconjugate 2) linker groups. ML spontaneously form complexes with plasmid DNA and RNA due to electrostatic interaction between positively charged lipid amino group and negatively charged phosphate of nucleic acids. ML demonstrated the benefit in transfection efficiency (TE) of pDNA into DC progenitors and immature DCs in comparison with the control liposomes at low N/P (nitrogen to phosphate) ratios (1/1 and 2/1) but not at high N/P ratios where the TE was comparable with control liposomes. Moreover, ML at low N/P were more effective in RNA delivery into immature DCs in comparison with DC progenitors. At high N/P ratios liposomal formulations containing 5 and 10% mol. of mannosylated lipoconjugate 2 with diethylsquarate linker were the most effective (up to 50% of transfected cells). DCs transfected ex vivo with ML/melanoma B16 RNA complexes after i.v. injection into mice caused five- to six-fold inhibition of melanoma lung metastasis number. Moreover, the i.v. injection of ML/melanoma B16 RNA complexes into mice induced generation of the melanoma B16-specific cytotoxic T-lymphocytes, which were two-fold more efficient in B16 cell killing than those from control liposome group.