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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.

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 kāraṇa ('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.

Background/aim We previously have described the "3+1" tumors cure approach consisting of individual time schedule of cyclophosphamide and dsDNA preparation administrations. The aim of the study was to adapt the "3+1" approach based on eradication of cancer stem cells to the model of murine ascitic cyclophosphamide-resistant lymphosarcoma (RLS). Materials and methods Adaptation of the "3+1" approach includes the identification of the timing to disrupt the tumorigenic potential of a certain tumor. Results The proposed therapeutic scheme allowed complete reduction of primary RLS ascites in experimental animals. However, reduction of primary ascites due to the complementary action of cyclophosphamide and dsDNA was inevitably followed by the development of a secondary one, most likely arising from a solid carcinomatous formation in the peritoneal wall. Conclusion The "3+1" approach resulted in the elimination of cancer stem cells, and, as a consequence, in the complete reduction of RLS ascites.

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.

BACKGROUND/AIM Oncolytic adenoviruses are promising therapeutic agents against both the bulk of tumor cells and cancer stem cells. The present study intended to test the oncolytic capability of adenovirus serotype 6 (Ad6), which has a lower seroprevalence and hepatotoxicity relatively to adenovirus 5 (Ad5), against the glioblastoma and its cancer stem cells. MATERIALS AND METHODS Oncolytic efficacy of Ad6 was compared to widespread Ad5 both in vitro and in vivo, using the U87 and U251 human glioblastoma cell lines and subcutaneously transplanted U87 cells in SCID mice, respectively. RESULTS Ad6 had a dose-dependent cytotoxicity toward glioblastoma cells in vitro and its intratumoral injections lead to a significant (p

The analysis of conditions and possible mechanisms of activation of 96 genes providing a malignant/pluripotent phenotype of Krebs-2 cancer stem cells have been performed. Three stress factors combined into the single concept of "generalized cellular stress", which are supposed to regulate the expression of these genes, are determined. Additionally, for these genes, the presence of binding sites for transcription factors that are being activated in response to factors of generalized cellular stress has been established. The data obtained suggest the existence of a mechanism for the de novo formation of a pluripotent/stem-like phenotype of tumor cells under conditions of generalized cellular stress.

The effectiveness of the new therapeutic approach aimed at the destruction of the cancer cell community was evaluated on the immortalized human glioblastoma cell line U87. Initially, the reference elements of a new strategy for eradication of tumor-initiating stem cells were characterized. The main points of the strategy were as follows. 1) Evaluation of the TAMRA+ (tumor-initiating stem cells) presence in the population of U87 culture cells. 2) Determination of the reference time points of the DNA interstrand cross-links repair, induced by cross-linking cytostatics mitomycin C. 3) Determination of the day after initiation of therapy when TAMRA+ cells accumulate and are synchronously present in the G1/S sensitive for the treatment phase of the cell cycle. Based on the data obtained, a therapeutic regimen aimed at eradicating TAMRA+ cells (tumor-initiating stem cells) was identified. Treatment of the culture was carried out by cross-linking cytostatic mitomycin C and complex DNA preparation. Transplantation experiments showed that high experimental doses of mitomycin C (20 μg/ml) totally destroy transplantation potential of U87 cells. Diminution of mitomycin C to therapeutic doses (5 μg/ ml) clearly demonstrated effect of complex double-stranded DNA preparation to reducing of U87 tumorigenic potential. These fully confirmed the concept of developed cancer treatment technology.

Functional analysis of 167 genes overexpressed in Krebs-2 tumor initiating cells was performed. Genes were assigned to the three functional groups that determine the malignant phenotype of cancer cells. These groups represent the following features of tumor cells: proliferative self-sufficiency, invasive growth and metastasis, and multiple drug resistance. Malignancy of cancer stem cells was found to be provided by the same genes that provide the stemness of normal pluri-/multipotent stem cells. These results suggest that the malignancy is just the ability to maintain the stem cell specific genes expression profile, and, as a consequence, the stemness itself regardless of the controlling effect of stem niches.

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 kāraṇa ('source') + han ('to kill')] based on the combined injection of cyclophosphamide and double-stranded DNA to eradicate cancer stem cells.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.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."Karanahan" therapy showed higher efficacy relative to in situ vaccination with CpG oligonucleotides and anti-OX40 antibodies.

Protection from ionizing radiation is the most important component in the curing malignant neoplasms, servicing atomic reactors, and resolving the situations associated with uncontrolled radioactive pollutions. In this regard, discovering new effective radioprotectors as well as novel principles of protecting living organisms from high-dose radiation is the most important factor, determining the new approaches in medical and technical usage of radiation.Experimental animals were irradiated on the γ-emitter (CsIn this study, several biological features of the radioprotective action of double-stranded RNA are characterized. It was shown that 160 µg of the double-stranded RNA per mouse protect experimental animals from the absolutely lethal dose of γ-radiation of 9.4 Gy. In different experiments, 80-100% of irradiated animals survive and live until their natural death. Radioprotective properties of double-stranded RNA were found to be independent on its sequence, but strictly dependent on its double-stranded form. Moreover, double-stranded RNA must have 'open' ends of the molecule to exert its radioprotective activity.Experiments indicate that radioprotective effect of double-stranded RNA is tightly bound to its internalization into hematopoietic stem cells, which further repopulate the spleen parenchyma of irradiated mice. Actively proliferating progenitors form the splenic colonies, which further serve as the basis for restoration of hematopoiesis and immune function and determine the survival of animals received the lethal dose of radiation.

Primary cultures of human glioblastoma were obtained from the surgical material of patients K. (female, 61 years, Ds: relapse of glioblastoma) and Zh. (female, 60 years, Ds: relapse of glioblastoma). The effectiveness of a new therapeutic approach aimed at destroying the cancer cell community was evaluated on the primary cell lines of human glioblastoma culture by employing a new strategy of tumor-initiating stem cell synchronization and a domestic strategy of their eradication "3+1". The key elements of the strategy were the following indicator results: (1) evaluation of the presence of tumor-initiating stem cells in a population of cells from analyzed cultures by their ability to internalize double-stranded labeled DNA (TAMRA+ cells); (2) determination of the reference time points of the repair cycle of DNA interstrand cross-links induced by cross-linking cytostatic mitomycin C; (3) evaluation of cell cycle synchronization; (4) determination of the time (day after therapy initiation) when TAMRA+ cells were synchronously present in phase G1/S of the cell cycle, sensitive to the therapy; and (5) establishment of the TAMRA+ (tumor-initiating stem cells) eradication schedule. The cultures were treated with cross-linking cytostatic mitomycin C and a compositional DNA preparation. After the treatments, cell division slows down, and the cultures degrade. The K cell line completely degraded within 30 days of observation. The cell number of the Zh culture fell to nearly one-third of the starting value by day 15 of observation. On day 15, this indicator constituted 1/7.45 for mitomycin C and 1/10.28 for mitomycin C + DNA with reference to the control. The main target of the mitomycin C + DNA regimen was TAMRA+ tumor-initiating stem cells of the glioblastoma cell populations. The action of mitomycin C alone or in the combination with DNA demonstrated effective elimination of TAMRA+ tumor-initiating stem cells and the whole primary cultures of human glioblastomas.

The present study demonstrates that monocyte-derived dendritic cells (moDCs) produced in vitro using a GM-CSF and IFN-α differentiation protocol encompass a rare (∼5%) subpopulation of cells showing classical dendritic cell morphology and capable of natural internalization of extracellular self-DNA. We established that DEFB, HMGB1, LL-37 and RAGE antigens, which mediate the process of DNA internalization, are expressed on the surface of moDCs similar to plasmacytoid dendritic cells. However, in constrast to the latter subpopulation, these cells do not produce interleukin (IL)-37. Nonetheless, the process of DNA internalization was not in direct relation to the presence of the above antigens on the surface of these cells. Dendritic cells were sorted into total and non-DNA-internalizing populations and cytokine production was analyzed at 24-48 hours post-DNA treatment. We show that massive secretion of cytokines by dendritic cells is associated with the dsDNA-internalizing subpopulation. A total pool of IFN-moDCs secrete pro-inflammatory "first-wave" cytokines (IL-2, IL-6, IL-8, TNF-α) at both 24 and 48 hours time points. The anti-inflammatory cytokines IL-4 and IL-10 were found to be modestly induced, whereas GM-CSF, G-CSF, and IFN-γ production was strongly induced. Treatment of moDCs with dsDNA results in the up-regulated transcription of IFN-α, IFN-β, IFN-γ, IL-8, IL-10, and VEGF by 6 hours. Combined dsDNA + chloroquine treatment has a synergistic effect on transcription of only one of the genes tested, with the pro-inflammatory cytokine IFN-β displaying the strongest fold induction by 24 hours.

Murine Krebs-2 tumor-initiating stem cells are known to natively internalize extracellular double-stranded DNA fragments. Being internalized, these fragments interfere in the repair of chemically induced interstrand cross-links. In the current investigation, 756 bp polymerase chain reaction (PCR) product containing bulky photoreactive dC adduct was used as extracellular DNA. This adduct was shown to inhibit the cellular system of nucleotide excision repair while being resistant to excision by this DNA repair system. The basic parameters for this DNA probe internalization by the murine Krebs-2 tumor cells were characterized. Being incubated under regular conditions (60 min, 24°C, 500 μL of the incubation medium, in the dark), 0.35% ± 0.18% of the Krebs-2 ascites cells were shown to natively internalize modified DNA. The saturating amount of the modified DNA was detected to be 0.37 μg per 10

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. Electronic supplementary material The online version of this article (10.1186/s12935-019-0842-x) contains supplementary material, which is available to authorized users.

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.

// Ekaterina A. Potter 1, * , Evgenia V. Dolgova 1, * , Anastasia S. Proskurina 1, * , Yaroslav R. Efremov 1, 2 , Alexandra M. Minkevich 1 , Aleksey S. Rozanov 1 , Sergey E. Peltek 1 , Valeriy P. Nikolin 1 , Nelly A. Popova 1, 2 , Igor A. Seledtsov 3 , Vladimir V. Molodtsov 2, 3 , Evgeniy L Zavyalov 1 , Oleg S. Taranov 4 , Sergey I. Baiborodin 1 , Alexander A. Ostanin 5 , Elena R. Chernykh 5 , Nikolay A. Kolchanov 1 , Sergey S. Bogachev 1 1 Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia 2 Novosibirsk State University, Novosibirsk 630090, Russia 3 Softberry Inc., New York 10549, USA 4 The State Research Center of Virology and Biotechnology VECTOR, Koltsovo, Novosibirsk 630559, Russia 5 Research Institute of Fundamental and Clinical Immunology, Novosibirsk 630099, Russia * These authors contributed equally to this work Correspondence to: Sergey S. Bogachev, email: labmolbiol@mail.ru Keywords: tumor-initiating stem cells, DNA internalization, RNAseq, Real Time PCR, TAMRA Received: August 29, 2016 Accepted: December 15, 2016 Published: December 23, 2016 ABSTRACT Using the ability of poorly differentiated cells to natively internalize fragments of extracellular double-stranded DNA as a marker, we isolated a tumorigenic subpopulation present in Krebs-2 ascites that demonstrated the features of tumor-inducing cancer stem cells. Having combined TAMRA-labeled DNA probe and the power of RNA-seq technology, we identified a set of 168 genes specifically expressed in TAMRA-positive cells (tumor-initiating stem cells), these genes remaining silent in TAMRA-negative cancer cells. TAMRA+ cells displayed gene expression signatures characteristic of both stem cells and cancer cells. The observed expression differences between TAMRA+ and TAMRA− cells were validated by Real Time PCR. The results obtained corroborated the biological data that TAMRA+ murine Krebs-2 tumor cells are tumor-initiating stem cells. The approach developed can be applied to profile any poorly differentiated cell types that are capable of immanent internalization of double-stranded DNA.

Previously, we reported on the development of a therapeutic regimen allowing eradication of primary murine Krebs-2 ascites transplants. This protocol involved multiple injections of dsDNA preparations administered during the NER and HR phases of repair of interstrand DNA cross-links induced by prior cyclophosphamide treatments. Mice treated under this protocol frequently developed secondary ascites, which indicated that some tumor-inducing cancer stem cells could survive the treatment and caused relapse. Further, we observed that animals receiving multiple dsDNA injections developed pronounced systemic inflammatory response. This prompted us to develop a more straightforward treatment regimen based on the synergistic activity of cyclophosphamide and dsDNA preparations, which would allow complete eradication of established primary Krebs-2 ascites and also be less toxic for the treated animals. This protocol relies on a precisely timed single injection of dsDNA during the NER/HR transition period of each repair cycle. Under this protocol, 8-day remission of Krebs-2 engrafted mice was achieved, which was similar to the results of the multiple-injection treatment schedule. We observed an increase in the average life span of Krebs-2- transplanted mice on a single-injection regimen, which was consistent with reduced toxicity of such treatment.

Cumulative evidence obtained in this series of studies has guided the logic behind the development of a novel composite dsDNA-based preparation whose therapeutic application according to the specific regimen completely cures the mice engrafted with otherwise lethal Krebs-2 ascites. The likely mechanism involves elimination of TAMRA+ tumor-inducing stem cells (TISCs) from Krebs-2 tumors. We performed quantitative analysis of TISC dynamics in Krebs-2 ascites following treatment with the cytostatic drug cyclophosphamide (CP) and untreated control cells. In intact ascites, TISC percentage oscillates around a certain value. Following CP treatment and massive apoptosis of committed cancer cell subpopulation, we observed relative increase in TISC percentage, which is consistent with reduced susceptibility of TISCs to CP. Nonetheless, this treatment apparently synchronizes TISCs in a cell cycle phase when they become sensitive to further drug treatments. We describe the regimen of synergistic DNA + CP activity against Krebs-2 ascites. This protocol results in a complete cure of 50 % of Krebs-2 engrafted mice and involves three metronomic injections of CP exactly at the timepoints when repair cycles are about to finish combined with dsDNA injections 18 hours following each CP injection. The “final shot” uses CP + DNA treatment, which targets the surviving yet highly synchronized and therefore treatmentsensitive cells. The first three CP/DNA injections appear to arrest Krebs-2 cells in late S-G2-M phase and result in their simultaneous progression into G1-S phase. The timing of the “final shot” is crucial for the successful treatment, which eradicates tumorigenic cell subpopulation from Krebs-2 ascites. Additionally, we quantified the changes in several biochemical, cellular and morphopathological parameters in mice throughout different treatment stages.

In our earlier studies, we observed that when mice are treated with cyclophosphamide and fragmented exogenous dsDNA (18-30 h post cytostatic treatment), they develop a very characteristic set of symptoms and 80-90% of such animals succumb within 6-25 days. This was called "delayed death" phenomenon, and the gap between cyclophosphamide and DNA injections required for such phenotype to develop was termed "death window". We established that mice succumbed to multi-organ failure, which was caused by systemic inflammation and sepsis. These processes unfolded along with accidental involution of lymphoid organs, which resulted from the failure of CD34(+) hematopoietic stem cells to differentiate into lymphoid lineage progenitors. Here we compare SPF and non-SPF animals, and demonstrate that the major cause of systemic inflammation and sepsis observed upon such treatments is activation of an opportunistic infection. Mice of the same strain (CBA) housed under SPF conditions do not develop the characteristic symptoms, nor do they become moribund. Yet, regardless of the breeding conditions, upon synergistic action of cyclophosphamide and dsDNA, CD34(+) hematopoietic stem cells consistently fail to give rise to lymphoid lineage progenitors. We demonstrate that this differentiation defect is reversible and that population of lymphoid progenitors is restored by day 29 after cyclophosphamide injection.

Krebs-2 solid carcinoma was cured using a new “3+1” strategy for eradication of Krebs-2 tumor-initiating stem cells. This strategy was based on synchronization of these cells in a treatment-sensitive phase of the cell cycle. The synchronization mechanism, subsequent destruction of Krebs-2 tumor-initiating stem cells, and cure of mice from a solid graft were found to depend on the temporal profile of the interstrand cross-link repair cycle. Also, the temporal profile of the Krebs-2 interstrand repair cycle was found to have a pronounced seasonal cyclicity at the place of experiments (Novosibirsk, Russia). As a result, the therapeutic effect that is based on application of the described strategy, originally developed for the “winter repair cycle” (November−April), is completely eliminated in the summer period (June−September). We conclude that оne of the possible and the likeliest reasons for our failure to observe the therapeutic effects was the seasonal cyclicity in the duration of the interstrand repair cycle, the parameter that is central to our strategy.

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.

// Ekaterina A. Potter 1 , Evgenia V. Dolgova 1 , Anastasia S. Proskurina 1 , Alexandra M. Minkevich 1 , Yaroslav R. Efremov 1, 2 , Oleg S. Taranov 3 , Vladimir V. Omigov 3 , Valeriy P. Nikolin 1 , Nelly A. Popova 1, 2 , Sergey I. Bayborodin 1, 2 , Alexander A. Ostanin 4 , Elena R. Chernykh 4 , Nikolay A. Kolchanov 1 , Mikhail A. Shurdov 5 , Sergey S. Bogachev 1 1 Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia 2 Department of Natural Sciences, Novosibirsk State University, Novosibirsk 630090, Russia 3 The State Research Center of Virology and Biotechnology VECTOR, Novosibirsk 630559, Russia 4 Institute of Clinical Immunology, Siberian Branch of the Russian Academy of Medical Sciences, Novosibirsk 630099, Russia 5 LLC Panagen, Gorno-Altaisk 649000, Russia Correspondence to: Sergey S. Bogachev, e-mail: labmolbiol@mail.ru Keywords: ascites Krebs-2, cyclophosphamide, extracellular dsDNA, tumor-initiating stem cells, repair Received: December 18, 2015 Accepted: January 26, 2016 Published: February 10, 2016 ABSTRACT We describe the strategy, which allows curing experimental mice engrafted with Krebs-2 ascites. The strategy is based on the facts that i) Krebs-2 tumor-initiating stem cells (TISCs) are naturally capable of internalizing fragments of extracellular double-stranded DNA (dsDNA); ii) upon delivery into TISCs, these dsDNA fragments interfere with the on-going DNA repair process so that TISCs either die or lose their tumorigenic potential. The following 3-step regimen of therapeutic procedures leading to eradication of Krebs-2 ascites is considered. Firstly, three timed injections of cyclophosphamide (CP) exactly matching the interstrand cross-link (ICL) repair phases that lead to synchronization of ascites cells in late S/G2/M. Secondly, additional treatment of ascites 18 hours post each CP injection (at NER/HR transition timepoint) with a composite dsDNA-based preparation interfering with the NER and HR repair pathways, so that tumorigenic properties of ascites cells are compromised. Thirdly, final treatment of mice with a combination of CP and dsDNA injections as ascites cells undergo apoptotic destruction, and the surviving TAMRA+ TISCs arrested in late S/G2/M phases massively enter into G1/S, when they regain sensitivity to CP+dsDNA treatment. Thus, this regimen assures that no viable cells, particularly Krebs-2 TISCs, remain.

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).

Background Previously, we demonstrated that poorly differentiated cells of various origins, including tumor-initiating stem cells present in the ascites form of mouse cancer cell line Krebs-2, are capable of naturally internalizing both linear double-stranded DNA and circular plasmid DNA. Methods The method of co-incubating Krebs-2 cells with extracellular plasmid DNA (pUC19) or TAMRA-5’-dUTP-labeled polymerase chain reaction (PCR) product was used. It was found that internalized plasmid DNA isolated from Krebs-2 can be transformed into competent Escherichia coli cells. Thus, the internalization processes taking place in the Krebs-2 cell subpopulation have been analyzed and compared, as assayed by E. coli colony formation assay (plasmid DNA) and cytofluorescence (TAMRA-DNA). Results We showed that extracellular DNA both in the form of plasmid DNA and a PCR product is internalized by the same subpopulation of Krebs-2 cells. We found that the saturation threshold for Krebs-2 ascites cells is 0.5 μg DNA/106 cells. Supercoiled plasmid DNA, human high-molecular weight DNA, and 500 bp PCR fragments are internalized into the Krebs-2 tumor-initiating stem cells via distinct, non-competing internalization pathways. Under our experimental conditions, each cell may harbor 340–2600 copies of intact plasmid material, or up to 3.097 ± 0.044×106 plasmid copies (intact or not), as detected by quantitative PCR. Conclusion The internalization dynamics of extracellular DNA, copy number of the plasmids taken up by the cells, and competition between different types of double-stranded DNA upon internalization into tumor-initiating stem cells of mouse ascites Krebs-2 have been comprehensively analyzed. Investigation of the extracellular DNA internalization into tumor-initiating stem cells is an important part of understanding their properties and possible destruction mechanisms. For example, a TAMRA-labeled DNA probe may serve as an instrument to develop a target for the therapy of cancer, aiming at elimination of tumor stem cells, as well as developing a straightforward test system for the quantification of poorly differentiated cells, including tumor-initiating stem cells, in the bulk tumor sample (biopsy or surgery specimen).