Impact of Double-Stranded RNA Internalization on Hematopoietic Progenitors and Krebs-2 Cells and Mechanism.

It is well-established that double-stranded RNA (dsRNA) exhibits noticeable radioprotective and radiotherapeutic effects. The experiments conducted in this study directly demonstrated that dsRNA was delivered into the cell in its native form and that it induced hematopoietic progenitor proliferation. The 68 bp synthetic dsRNA labeled with 6-carboxyfluorescein (FAM) was internalized into mouse hematopoietic progenitors, c-Kit+ (a marker of long-term hematopoietic stem cells) cells and CD34+ (a marker of short-term hematopoietic stem cells and multipotent progenitors) cells. Treating bone marrow cells with dsRNA stimulated the growth of colonies, mainly cells of the granulocyte-macrophage lineage. A total of 0.8% of Krebs-2 cells internalized FAM-dsRNA and were simultaneously CD34+ cells. dsRNA in its native state was delivered into the cell, where it was present without any signs of processing. dsRNA binding to a cell was independent of cell charge. dsRNA internalization was related to the receptor-mediated process that requires energy from ATP. Synthetic dsRNA did not degrade in the bloodstream for at least 2 h. Hematopoietic precursors that had captured dsRNA reinfused into the bloodstream and populated the bone marrow and spleen. This study, for the first time, directly proved that synthetic dsRNA is internalized into a eukaryotic cell via a natural mechanism.

The Molecular Aspects of Functional Activity of Macrophage-Activating Factor GcMAF.

Group-specific component macrophage-activating factor (GcMAF) is the vitamin D3-binding protein (DBP) deglycosylated at Thr420. The protein is believed to exhibit a wide range of therapeutic properties associated with the activation of macrophagal immunity. An original method for GcMAF production, DBP conversion to GcMAF, and the analysis of the activating potency of GcMAF was developed in this study. Data unveiling the molecular causes of macrophage activation were obtained. GcMAF was found to interact with three CLEC10A derivatives having molecular weights of 29 kDa, 63 kDa, and 65 kDa. GcMAF interacts with high-molecular-weight derivatives via Ca2+-dependent receptor engagement. Binding to the 65 kDa or 63 kDa derivative determines the pro- and anti-inflammatory direction of cytokine mRNA expression: 65 kDa-pro-inflammatory (TNF-α, IL-1ß) and 63 kDa-anti-inflammatory (TGF-ß, IL-10). No Ca2+ ions are required for the interaction with the canonical 29 kDa CLEC10A. Both forms, DBP protein and GcMAF, bind to the 29 kDa CLEC10A. This interaction is characterized by the stochastic mRNA synthesis of the analyzed cytokines. Ex vivo experiments have demonstrated that when there is an excess of GcMAF ligand, CLEC10A forms aggregate, and the mRNA synthesis of analyzed cytokines is inhibited. A schematic diagram of the presumable mechanism of interaction between the CLEC10A derivatives and GcMAF is provided. The principles and elements of standardizing the GcMAF preparation are elaborated.

Analysis of the Biological Properties of Blood Plasma Protein with GcMAF Functional Activity.

The main problem related to the studies focusing on group-specific component protein-derived macrophage-activating factor (GcMAF) is the lack of clarity about changes occurring in different types of macrophages and related changes in their properties under the effect of GcMAF in various clinical conditions. We analyzed the antitumor therapeutic properties of GcMAF in a Lewis carcinoma model in two clinical conditions: untreated tumor lesion and tumor resorption after exposure to Karanahan therapy. GcMAF is formed during site-specific deglycosylation of vitamin D3 binding protein (DBP). DBP was obtained from the blood of healthy donors using affinity chromatography on a column with covalently bound actin. GcMAF-related factor (GcMAF-RF) was converted in a mixture with induced lymphocytes through the cellular enzymatic pathway. The obtained GcMAF-RF activates murine peritoneal macrophages (p < 0.05), induces functional properties of dendritic cells (p < 0.05) and promotes in vitro polarization of human M0 macrophages to M1 macrophages (p < 0.01). Treatment of whole blood cells with GcMAF-RF results in active production of both pro- and anti-inflammatory cytokines. It is shown that macrophage activation by GcMAF-RF is inhibited by tumor-secreted factors. In order to identify the specific antitumor effect of GcMAF-RF-activated macrophages, an approach to primary reduction of humoral suppressor activity of the tumor using the Karanahan therapy followed by macrophage activation in the tumor-associated stroma (TAS) was proposed. A prominent additive effect of GcMAF-RF, which enhances the primary immune response activation by the Karanahan therapy, was shown in the model of murine Lewis carcinoma. Inhibition of the suppressive effect of TAS is the main condition required for the manifestation of the antitumor effect of GcMAF-RF. When properly applied in combination with any chemotherapy, significantly reducing the humoral immune response at the advanced tumor site, GcMAF-RF is a promising antitumor therapeutic agent that additively destroys the pro-tumor properties of macrophages of the tumor stroma.

The New General Biological Property of Stem-like Tumor Cells (Part II: Surface Molecules, Which Belongs to Distinctive Groups with Particular Functions, Form a Unique Pattern Characteristic of a Certain Type of Tumor Stem-like Cells).

An ability of poorly differentiated cells of different genesis, including tumor stem-like cells (TSCs), to internalize extracellular double-stranded DNA (dsDNA) fragments was revealed in our studies. Using the models of Krebs-2 murine ascites carcinoma and EBV-induced human B-cell lymphoma culture, we demonstrated that dsDNA internalization into the cell consists of several mechanistically distinct phases. The primary contact with cell membrane factors is determined by electrostatic interactions. Firm contacts with cell envelope proteins are then formed, followed by internalization into the cell of the complex formed between the factor and the dsDNA probe bound to it. The key binding sites were found to be the heparin-binding domains, which are constituents of various cell surface proteins of TSCs-either the C1q domain, the collagen-binding domain, or domains of positively charged amino acids. These results imply that the interaction between extracellular dsDNA fragments and the cell, as well as their internalization, took place with the involvement of glycocalyx components (proteoglycans/glycoproteins (PGs/GPs) and glycosylphosphatidylinositol-anchored proteins (GPI-APs)) and the system of scavenger receptors (SRs), which are characteristic of TSCs and form functional clusters of cell surface proteins in TSCs. The key provisions of the concept characterizing the principle of organization of the "group-specific" cell surface factors of TSCs of various geneses were formulated. These factors belong to three protein clusters: GPs/PGs, GIP-APs, and SRs. For TSCs of different tumors, these clusters were found to be represented by different members with homotypic functions corresponding to the general function of the cluster to which they belong.

Identification of the xenograft and its ascendant sphere-forming cell line as belonging to EBV-induced lymphoma, and characterization of the status of sphere-forming cells.

BACKGROUND: We have characterized the human cell line arised from the Epstein-Barr virus (EBV) positive multiple myeloma aspirate subjected to the long-term cultivation. This cell line has acquired the ability to form free-floating spheres and to produce a xenograft upon transplantation into NOD/SCID mice. METHODS: Cells from both in vitro culture and developed xenografts were investigated with a number of analytical approaches, including pathomorphological analysis, FISH analysis, and analysis of the surface antigens and of the VDJ locus rearrangement. RESULTS: The obtained results, as well as the confirmed presence of EBV, testify that both biological systems are derived from B-cells, which, in turn, is a progeny of the EBV-transformed B-cellular clone that supplanted the primordial multiple myeloma cells. Next we assessed whether cells that (i) were constantly present in vitro in the investigated cell line, (ii) were among the sphere-forming cells, and (iii) were capable of internalizing a fluorescent TAMRA-labeled DNA probe (TAMRA+ cells) belonged to one of the three types of undifferentiated bone marrow cells of a multiple myeloma patient: CD34+ hematopoietic stem cells, CD90+ mesenchymal stem cells, and clonotypic multiple myeloma cell. CONCLUSION: TAMRA+ cells were shown to constitute the fourth independent subpopulation of undifferentiated bone marrow cells of the multiple myeloma patient. We have demonstrated the formation of ectopic contacts between TAMRA+ cells and cells of other types in culture, in particular with CD90+ mesenchymal stem cells, followed by the transfer of some TAMRA+ cell material into the contacted cell.

Ultrastructural analysis of the Krebs-2 ascites cancer cells treated with extracellular double-stranded DNA preparation.

Electron-microscopic analysis of the ultrastructure of the Krebs-2 carcinoma ascites cells in the first 90 min immediately after their exposure to fragmented double-stranded DNA has been performed. Morphological attributes of the treated cancer cells indicate the induction in these cells of destructive processes of presumably apoptotic type. The predominance of dystrophic-destructive changes in cells after the addition of DNA is supposed to be a consequence of the disturbance in metabolic processes caused by the experimental action.

Five-year disease-free survival among stage II-IV breast cancer patients receiving FAC and AC chemotherapy in phase II clinical trials of Panagen.

BACKGROUND: We report on the results of a phase II clinical trial of Panagen (tablet form of fragmented human DNA preparation) in breast cancer patients (placebo group n = 23, Panagen n = 57). Panagen was administered as an adjuvant leukoprotective agent in FAC and AC chemotherapy regimens. Pre-clinical studies clearly indicate that Panagen acts by activating dendritic cells and induces the development of adaptive anticancer immune response. METHODS: We analyzed 5-year disease-free survival of patients recruited into the trial. RESULTS: Five-year disease-free survival in the placebo group was 40 % (n = 15), compared with the Panagen arm - 53 % (n = 51). Among stage III patients, disease-free survival was 25 and 52 % for placebo (n = 8) and Panagen (n = 25) groups, respectively. Disease-free survival of patients with IIIB + C stage was as follows: placebo (n = 6)-17 % vs Panagen (n = 18)-50 %. CONCLUSIONS: Disease-free survival rate (17 %) of patients with IIIB + C stage breast cancer receiving standard of care therapy is within the global range. Patients who additionally received Panagen demonstrate a significantly improved disease-free survival rate of 50 %. This confirms anticancer activity of Panagen. TRIAL REGISTRATION: ClinicalTrials.gov NCT02115984 from 04/07/2014.

Combination of cyclophosphamide and double-stranded DNA demonstrates synergistic toxicity against established xenografts.

BACKGROUND: Extracellular double-stranded DNA participates in various processes in an organism. Here we report the suppressive effects of fragmented human double-stranded DNA along or in combination with cyclophosphamide on solid and ascites grafts of mouse Krebs-2 tumor cells and DNA preparation on human breast adenocarcinoma cell line MCF-7. METHODS: Apoptosis and necrosis were assayed by electrophoretic analysis (DNA nucleosomal fragmentation) and by measurements of LDH levels in ascitic fluid, respectively. DNA internalization into MCF-7 was analyzed by flow cytometry and fluorescence microscopy. RESULTS: Direct cytotoxic activity of double-stranded DNA (along or in combination with cyclophosphamide) on a solid transplant was demonstrated. This resulted in delayed solid tumor proliferation and partial tumor lysis due to necrosis of the tumor and adjacent tissues. In the case of ascites form of tumor, extensive apoptosis and secondary necrosis were observed. Similarly, MCF-7 cells showed induction of massive apoptosis (up to 45%) as a result of treatments with double-stranded DNA preparation. CONCLUSIONS: Double-stranded DNA (along or in combination with cyclophosphamide) induces massive apoptosis of Krebs-2 ascite cells and MCF-7 cell line (DNA only). In treated mice it reduces the integrity of gut wall cells and contributes to the development of systemic inflammatory reaction.

Results of multicenter double-blind placebo-controlled phase II clinical trial of Panagen preparation to evaluate its leukostimulatory activity and formation of the adaptive immune response in patients with stage II-IV breast cancer.

BACKGROUND: We performed a multicenter, double-blind, placebo-controlled, phase II clinical trial of human dsDNA-based preparation Panagen in a tablet form. In total, 80 female patients with stage II-IV breast cancer were recruited. METHODS: Patients received three consecutive FAC (5-fluorouracil, doxorubicin and cyclophosphamide) or AC (doxorubicin and cyclophosphamide) adjuvant chemotherapies (3 weeks per course) and 6 tablets of 5 mg Panagen or placebo daily (one tablet every 2-3 hours, 30 mg/day) for 18 days during each chemotherapy course. Statistical analysis was performed using Statistica 6.0 software, and non-parametric analyses, namely Wilcoxon-Mann-Whitney and paired Wilcoxon tests. To describe the results, the following parameters were used: number of observations (n), median, interquartile range, and minimum-maximum range. RESULTS: Panagen displayed pronounced leukostimulatory and leukoprotective effects when combined with chemotherapy. In an ancillary protocol, anticancer effects of a tablet form of Panagen were analyzed. We show that Panagen helps maintain the pre-therapeutic activity level of innate antitumor immunity and induces formation of a peripheral pool of cytotoxic CD8+ perforin + T-cells. Our 3-year follow-up analysis demonstrates that 24% of patients who received Panagen relapsed or died after the therapy, as compared to 45% in the placebo cohort. CONCLUSIONS: The data collected in this trial set Panagen as a multi-faceted "all-in-one" medicine that is capable of simultaneously sustaining hematopoiesis, sparing the innate immune cells from adverse effects of three consecutive rounds of chemotherapy and boosting individual adaptive immunity. Its unique feature is that it is delivered via gastrointestinal tract and acts through the lymphoid system of intestinal mucosa. Taken together, maintenance of the initial levels of innate immunity, development of adaptive cytotoxic immune response and significantly reduced incidence of relapses 3 years after the therapy argue for the anticancer activity of Panagen. TRIAL REGISTRATION: ClinicalTrials.gov NCT02115984 from 04/07/2014.

The Macrophage Activator GcMAF-RF Enhances the Antitumor Effect of Karanahan Technology through Induction of M2-M1 Macrophage Reprogramming.

Macrophages are the immune cells of high-immunological plasticity, which can exert both pro- and anti-inflammatory activity, as well as repolarize their phenotype to the opposite or neutral one. In this regard, M2 macrophages of the tumor-associated stroma (TAS) are a promising therapeutic target in treating malignant neoplasms. Using FACS assay, we have estimated the CD11b+/Ly-6G+/Ly-6C+ fraction of macrophages from the peritoneum and TAS in intact healthy mice and those with developed Lewis carcinoma, both untreated and treated according to Karanahan technology in combination with group-specific macrophage activator (GcMAF-RF). As well, the pattern of pro- and anti-inflammatory cytokines mRNA expression in different groups of experimental and tumor-bearing animals was assessed. It was found that: (i) exposure of intact mice to GcMAF-RF results in the increased number of CD11b+/Ly-6C+ peritoneal macrophages and, at the same time, the expression pattern of cytokines in peritoneal macrophages switches from that characteristic of the mixed M1/M2 phenotype to that characteristic of the neutral M0 one; (ii) combination of Karanahan technology and GcMAF-RF treatment results in M0/M1 repolarization of TAS macrophages; (iii) in tumor-bearing mice, the response of peritoneal macrophages to such a treatment is associated with the induction of anti-inflammatory reaction, which is opposite to that in TAS macrophages.

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.

Effects of human exogenous DNA on production of perforin-containing CD8+ cytotoxic lymphocytes in laboratory setting and clinical practice.

We investigated the influence of Panagen DNA preparations on laboratory animals and IFN-induced human dendritic cells, as well as analyzed the data from a phase II clinical trial in the therapy of breast cancer. It was shown that this treatment resulted in increased number of CD8+/perforin+ T cells in peripheral lymphoid organs of experimental animals, in mixed lymphocyte culture population and in peripheral blood of breast cancer patients. Moreover, we demonstrated that when Panagen DNA preparations are used in combination with the standard FAC-based breast cancer therapies, non-specific immune response activity remains at the same levels as observed prior to therapy, whereas in FAC-placebo patients, non-specific immunity is greatly diminished.

Karanahan: A Potential New Treatment Option for Human Breast Cancer and Its Validation in a Clinical Setting.

INTRODUCTION: Karanahan, a cancer treatment technology aimed at eradicating tumor-initiating stem cells, has already proven effective in 7 tumor models. Karanahan comprises the following procedures: (1) collecting surgical specimens, (2) determining the duration of the DNA repair process in tumor cells exposed to a cross-linking cytostatic agent, and (3) determining the time point, when cells, including tumor-initiating stem cells, are synchronized in the certain phase of the cell cycle after triple exposure to the cytostatic, becoming vulnerable for the terminal treatment, which is supposed to completely eliminate the rest of survived tumor-initiating stem cells. Determining these basic tumor properties allows to design the schedule for the administration of a cross-linking cytostatic and a complex composite DNA preparation. Being conducted in accordance with the schedule designed, Karanahan results in the large-scale apoptosis of tumor cells with elimination of tumor-initiating stem cells. METHODS: Breast tumor specimens were obtained from patients, and basic tumor properties essential for conducting Karanahan therapy were determined. RESULTS: We report the first use of Karanahan in patients diagnosed with breast cancer. Technical details of handling surgical specimens for determining the essential Karanahan parameters (tumor volume, cell number, cell proliferation status, etc) have been worked out. The terminally ill patient, who was undergoing palliative treatment and whose tumor specimen matched the required criteria, received a complete course of Karanahan. CONCLUSIONS: The results of the treatment conducted indicate that Karanahan technology has a therapeutic potency and can be used as a breast cancer treatment option.

Antitumor efficacy of multi-target in situ vaccinations with CpG oligodeoxynucleotides, anti-OX40, anti-PD1 antibodies, and aptamers.

To overcome immune tolerance to cancer, the immune system needs to be exposed to a multi-target action intervention. Here, we investigated the activating effect of CpG oligodeoxynucleotides (ODNs), mesyl phosphoramidate CpG ODNs, anti-OX40 antibodies, and OX40 RNA aptamers on major populations of immunocompetent cells ex vivo. Comparative analysis of the antitumor effects of in situ vaccination with CpG ODNs and anti-OX40 antibodies, as well as several other combinations, such as mesyl phosphoramidate CpG ODNs and OX40 RNA aptamers, was conducted. Antibodies against programmed death 1 (PD1) checkpoint inhibitors or their corresponding PD1 DNA aptamers were also added to vaccination regimens for analytical purposes. Four scenarios were considered: a weakly immunogenic Krebs-2 carcinoma grafted in CBA mice; a moderately immunogenic Lewis carcinoma grafted in C57Black/6 mice; and an immunogenic A20 B cell lymphoma or an Ehrlich carcinoma grafted in BALB/c mice. Adding anti-PD1 antibodies (CpG+αOX40+αPD1) to in situ vaccinations boosts the antitumor effect. When to be used instead of antibodies, aptamers also possess antitumor activity, although this effect was less pronounced. The strongest effect across all the tumors was observed in highly immunogenic A20 B cell lymphoma and Ehrlich carcinoma.

The new general biological property of stem-like tumor cells Part I. Peculiarities of the process of the double-stranded DNA fragments internalization into stem-like tumor cells.

Stem-like tumor cells of ascites carcinoma Krebs-2 and Epstein-Barr virus-induced B-lymphoma were shown to possess the innate capability of binding and internalizing the TAMRA-labeled double-stranded DNA (dsDNA) probe. The process of binding and internalizing is rather complicated and composed of the following successive stages: 1) initiating electrostatic interaction and contact of a negatively charged dsDNA molecule with a positively charged molecule(s) on the surface of a stem-like tumor cell; 2) binding of the dsDNA probe to a tumor stem cell surface protein(s) via the formation of a strong chemical/molecular bond; and 3) the very internalization of dsDNA into the cell. Binding of DNA to cell surface proteins is determined by the presence of heparin/polyanion-binding sites within the protein structure, which can be competitively blocked by heparin and/or dextran sulfate, wherein heparin blocks only the binding, while dextran sulfate abrogates both binding and internalization. The abrogation of internalization by dextran sulfate implies the role of scavenger receptors in this process. Cells were shown to uptake DNA in amounts constituting ∼0.008% of the haploid genome. Inhibitors of caveolae-dependent internalization abrogate the DNA uptake in Krebs-2 cells, and inhibitors of the clathrin/caveolar mechanism block the internalization in B-lymphoma cells. In the present report, it is shown for the first time that in contrast to the majority of committed tumor cells, stem-like tumor cells of Krebs-2 and B-lymphoma carry a general positive charge on their surface.

Theoretical premises of a "three in one" therapeutic approach to treat immunogenic and nonimmunogenic cancers: a narrative review.

OBJECTIVE: We describe experimental and theoretical premises of a powerful cancer therapy based on the combination of three approaches. These include (I) in situ vaccination (intratumoral injections of CpG oligonucleotides and anti-OX40 antibody); (II) chronometric or metronomic low-dose cyclophosphamide (CMLD CP)-based chemotherapy; (III) cancer stem cell-eradicating therapy referred to as Karanahan (from the Sanskrit karana ["source"] + han ["to kill"]). BACKGROUND: In murine models, the first two approaches are particularly potent in targeting immunogenic tumors for destruction. In situ vaccination activates a fully fledged anticancer immune response via an intricate network of ligand-receptor-cytokine interactions. CMLD CP-based chemotherapy primarily targets the suppressive tumor microenvironment and activates tumor-infiltrating effectors. In contrast, Karanahan technology, being aimed at replicative machinery of tumor cells (both stem-like and committed), does not depend on tumor immunogenicity. With this technology, mice engrafted with ascites and/or solid tumors can be successfully cured. There is a significant degree of mechanistic and therapeutic overlap between these three approaches. For instance, the similarities shared between in situ vaccination and Karanahan technology include the therapeutic procedure, the cell target [antigen-presenting cells (APC) and dendritic cells (DC)], and the use of DNA-based preparations (CpG and DNAmix). Features shared between CMLD CP-based chemotherapy and Karanahan technology are the timing and the dose of the cytostatic drug administration, which lead to tumor regression. METHODS: The following keywords were used to search PubMed for the latest research reporting successful eradication of transplantable cancers in animal models that relied on approaches distinct from those used in the Karanahan technology: eradication of malignancy, cure cancer, complete tumor regression, permanently eradicating advanced mouse tumor, metronomic chemotherapy, in situ vaccination, immunotherapy, and others. CONCLUSION: We hypothesize, therefore, that very potent anticancer activity can be achieved once these three therapeutic modalities are combined into a single approach. This multimodal approach is theoretically curative for any type of cancer that depends on the presence of tumor-inducing cancer stem cells, provided that the active therapeutic components are efficiently delivered into the tumor and the specific biological features of a given patient's tumor are properly addressed. We expect this multimodal approach to be primarily applicable to late-stage or terminal cancer patients who have exhausted all treatment options as well as patients with inoperable tumors.

Experimental Comparison of the In Vivo Efficacy of Two Novel Anticancer Therapies.

BACKGROUND/AIM: We compared the therapeutic efficacy of two recently developed experimental anticancer technologies: 1) in situ vaccination based on local immunotherapy with CpG oligonucleotides and anti-OX40 antibodies to activate antitumor immune response and 2) "Karanahan" technology [from the Sanskrit karana ('source') + han ('to kill')] based on the combined injection of cyclophosphamide and double-stranded DNA to eradicate cancer stem cells. MATERIALS AND METHODS: The anticancer approaches were compared on three types of mouse malignant tumors with different grades of immunogenicity: weakly immunogenic carcinoma Krebs-2, moderately immunogenic Lewis carcinoma, and highly immunogenic A20 В-cellular lymphoma. RESULTS: Our results indicated that in situ vaccination was the most effective against the highly immunogenic tumor А20. In addition, "Karanahan" demonstrated high efficiency in all types of tumors, regardless of their immunogenicity or size. CONCLUSION: "Karanahan" therapy showed higher efficacy relative to in situ vaccination with CpG oligonucleotides and anti-OX40 antibodies.

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.

Effect of double-stranded DNA on maturation of dendritic cells in vitro.

A preparation of human genomic fragmented double-stranded DNA (dsDNA) was used as maturation stimulus in cultures of human dendritic cells (DCs) generated in compliance with the interferon protocol. Culturing of the DCs in medium with 5µg/ml of the DNA preparation was associated with a decrease in the relative proportion of CD14 + cells and an increase in that of CD83 + cells. These changes are markers of DC maturation. The efficiency with which the DNA preparation was able to elicit DC maturation was commensurate with that of lypopolysaccharide from bacterial cell, the standard inducer of DC maturation. Generated ex vivo, matured in the presence of the human DNA preparation, pulsed with tumor antigens mouse DCs were used as a vaccine in biological tests for its antitumor activity. The experimental results demonstrate that reinfusion of mature pulsed with tumor antigens DCs cause a statistically significant suppression of tumor graft growth.