In recent years, the arrival of the immunotherapy industry has introduced the possibility of providing transformative, durable, and potentially curative outcomes for various forms of malignancies. However, further research has shown that there are a number of issues that significantly reduce the effectiveness of immunotherapy, especially in solid tumors. First of all, these problems are related to the protective mechanisms of the tumor and its microenvironment. Currently, major efforts are focused on overcoming protective mechanisms by using different adoptive cell therapy variants and modifications of genetically engineered constructs. In addition, a complex workforce is required to develop and implement these treatments. To overcome these significant challenges, innovative strategies and approaches are necessary to engineer more powerful variations of immunotherapy with improved antitumor activity and decreased toxicity. In this review, we discuss recent innovations in immunotherapy aimed at improving clinical efficacy in solid tumors, as well as strategies to overcome the limitations of various immunotherapies.

ONC201, the anticancer drug, targets and activates mitochondrial ATP-dependent caseinolytic peptidase P (ClpP), a serine protease located in the mitochondrial matrix. Given the promise of ONC201 in cancer treatment, we evaluated its effects on the breast ductal carcinoma cell line (BT474). We showed that the transient single-dose treatment of BT474 cells by 10 µM ONC201 for a period of less than 48 h induced a reversible growth arrest and a transient activation of an integrated stress response indicated by an increased expression of CHOP, ATF4, and GDF-15, and a reduced number of mtDNA nucleoids. A prolonged exposure to the drug (>48 h), however, initiated an irreversible loss of mtDNA, persistent activation of integrated stress response proteins, as well as cell cycle arrest, inhibition of proliferation, and suppression of the intrinsic apoptosis pathway. Since Natural Killer (NK) cells are quickly gaining momentum in cellular anti-cancer therapies, we evaluated the effect of ONC201 on the activity of the peripheral blood derived NK cells. We showed that following the ONC 201 exposure BT474 cells demonstrated enhanced sensitivity toward human NK cells that mediated killing. Together our data revealed that the effects of a single dose of ONC201 are dependent on the duration of exposure, specifically, while short-term exposure led to reversible changes; long-term exposure resulted in irreversible transformation of cells associated with the senescent phenotype. Our data further demonstrated that when used in combination with NK cells, ONC201 created a synergistic anti-cancer effect, thus suggesting its possible benefit in NK-cell based cellular immunotherapies for cancer treatment.

Activities of the tumour necrosis factor (TNF) family members are associated with their targeting to lipid rafts, specialised regions of the plasma membrane. Herein, we investigated the physical association of TNF and its family members cluster of differentiation 40 ligand (CD40L) and tumour necrosis factor-related apoptosis-inducing ligand with caveolin-1, a lipid raft resident protein. We discovered that the intracellular domains of TNF and CD40L interact with caveolin-1, and the membrane proximal region of TNF is required for the binding of caveolin-1 domains. Full-length TNF can form a complex with caveolin-1 in membrane rafts of HeLa cells, and caveolin-1 knockdown leads to impaired TNF transport to rafts. These findings provide the first evidence of a direct interaction between TNF, CD40L and caveolin-1 and suggest that caveolin-1 may be responsible for recruiting TNF to lipid rafts.

GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1-edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative-real time PCR (q-rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer-related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT-61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage-specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.

Fas-ligand/CD178 belongs to the TNF family proteins and is the well-characterized inducer of cell death. We showed previously that the interaction of Fas-ligand and caveolin-1 is necessary for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. Both molecules can undergo phosphorylation, however the role of the phosphorylation state of Fas-ligand and caveolin-1 in their physical association, and consequently in of Fas - mediated cell death induction is currently unknown. In this study, we show that in control cells Fas-ligand interaction with caveolin-1 is not observed, and both molecules are phosphorylated. The intracellular part of Fas-ligand was shown to form a complex with p59Fyn-kinase. Upon cell death activation, the expression and activity of p59Fyn-kinase decreases substantially, leading to the disruption of Fas-ligand - p59Fyn-kinase association, dephosphorylation of Fas-ligand and caveolin-1, and formation of a complex between them (Fas-ligand - caveolin-1). The analysis of the effects of kinase and phosphatase inhibitors revealed that phosphorylation of Fas-ligand and caveolin-1 at tyrosine residues suppressed Fas-mediated cell death. Thus, dephosphorylation of Fas-ligand and caveolin-1 is critical for triggering Fas-ligand-mediated apoptotic pathway and cell death execution.

Trisomy 21 (T21), known as Down syndrome (DS), is a widely studied chromosomal abnormality. Previous studies have shown that DS individuals have a unique cancer profile. While exhibiting low solid tumor prevalence, DS patients are at risk for hematologic cancers, such as acute megakaryocytic leukemia and acute lymphoblastic leukemia. We speculated that endothelial cells are active players in this clinical background. To this end, we hypothesized that impaired DS endothelial development and functionality, impacted by genome-wide T21 alterations, potentially results in a suboptimal endothelial microenvironment with the capability to prevent solid tumor growth. To test this hypothesis, we assessed molecular and phenotypic differences of endothelial cells differentiated from Down syndrome and euploid iPS cells. Microarray, RNA-Seq, and bioinformatic analyses revealed that most significantly expressed genes belong to angiogenic, cytoskeletal rearrangement, extracellular matrix remodeling, and inflammatory pathways. Interestingly, the majority of these genes are not located on Chromosome 21. To substantiate these findings, we carried out functional assays. The obtained phenotypic results correlated with the molecular data and showed that Down syndrome endothelial cells exhibit decreased proliferation, reduced migration, and a weak TNF-α inflammatory response. Based on this data, we provide a set of genes potentially associated with Down syndrome's elevated leukemic incidence and its unfavorable solid tumor microenvironment-highlighting the potential use of these genes as therapeutic targets in translational cancer research.

Fas-ligand/CD178 belongs to the TNF family proteins and can induce apoptosis through death receptor Fas/CD95. The important requirement for Fas-ligand-dependent cell death induction is its localization to rafts, cholesterol- and sphingolipid-enriched micro-domains of membrane, involved in regulation of different signaling complexes. Here, we demonstrate that Fas-ligand physically associates with caveolin-1, the main protein component of rafts. Experiments with cells overexpressing Fas-ligand revealed a FasL N-terminal pre-prolin-rich region, which is essential for the association with caveolin-1. We found that the N-terminal domain of Fas-ligand bears two caveolin-binding sites. The first caveolin-binding site binds the N-terminal domain of caveolin-1, whereas the second one appears to interact with the C-terminal domain of caveolin-1. The deletion of both caveolin-binding sites in Fas-ligand impairs its distribution between cellular membranes, and attenuates a Fas-ligand-induced cytotoxicity. These results demonstrate that the interaction of Fas-ligand and caveolin-1 represents a molecular basis for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. A possibility of a similar association between other TNF family members and caveolin-1 is discussed.

A sensitive and reliable technique for meat species identification is required to prevent food adulteration, particularly in meat production. This work developed an optimized multiplex PCR assay for simultaneous identification of five commonly consumed and five commonly banned meat species in meat products. We designed primers that specifically amplified mitochondrial ATPase subunit 8 gene regions of different lengths of bovine, ovine, swine, chicken, turkey, cat, dog, mouse and human DNAs. The developed multiplex PCR assay proved to be specific, sensitive down to 30pg DNA per reaction, reproducible and economical. It could be used with a variety of raw meats and processed food samples and is easily applicable in a routine laboratory analysis without specific sophisticated equipment.

Therapeutic antibodies are implicated into the very promising and fast growing area of pharmaceutics. Human hybridoma technology, allowing generation of natural human antibodies in a native form, seems to be the most direct way that require no additional modifications for production of therapeutic antibodies. However, technical difficulties in human hybridoma creation discovered in the 80s of the last century have switched the mainstream therapeutic antibody development into new directions like display and transgenic mice techniques. These approaches have provided remarkable achievements in antibody engineering within last 15 years, but also revealed other limitations. Thus, it is time to turn back to forgotten human hybridoma technology. In this review, we describe new advances in all components of human hybridoma technology and discuss challenges in generating novel therapeutic mABs based on hybridoma technologies.

Aim/Background: Interleukin-8 (IL-8), a proinflammatory CXC chemokine, via its cognate receptors, CXCR1 and CXCR2, is able to stimulate angiogenesis, enhance the proliferation and survival of endothelial and tumor cells as well as tumor cell migration (metastasis). Accordingly, the development of therapeutic agents, targeting IL-8 signalling, seems an urgent task. The anti-cancer potential of various small molecule antagonists and humanized monoclonal antibodies to IL-8 is currently actively investigated. In this study we present the results of investigation of the anti-tumor activity of recombinant soluble protein in which the N-terminal domain of CXCR2 is juxtaposed on the Fc-fragment of human IgG1. Methods: Recombinant human CXCR2-Fc protein produced by HEK 293 was isolated and purified by affinity chromatography with protein A-Sepharose followed by two-step ion-changed chromatography. The physical and chemical properties of recombinant protein were studied using gel-filtration chromatography and SDS-PAGE-electrophoresis. The IL-8 binding activity of CXCR2-Fc protein was analyzed in ELISA-tests with recombinant IL-8. The cytotoxic activity of CXCR2-Fc protein was estimated in experiments with human breast adenocarcinoma MDA-MD-231, highly expressing IL-8, and the IL-8-expressing primary tumor cell cultures from breast cancer patients by MTT-test and Annexin V staining. The content of IL-8 in the supernatant of primary breast tumor cell cultures was determined using the sandwich ELISA. Results: The electrophoretic and chromatographic analyses show that recombinant human CXCR2-Fc protein is present mostly as dimers effectively binding IL-8. The elevated level of the secreted IL-8 (up to 70 ng/ml) is found in supernatants of some primary tumor cell cultures from breast cancer patients. The MTT assay and Annexin V staining show that CXCR2-Fc protein is capable of inducing the death of MDA-MD-231 cells as well as some primary tumor cells expressing IL-8. Conclusions: The results obtained indicate that recombinant human CXCR2-Fc protein may be regarded as the promising anti-cancer agent. This work was supported by a grant from the Ministry of Education and Science of the Russian Federation (# 14.604.21.0025, ID RFMEFI60414X0025). Legal entity responsible for the study: Institute of Theoretical and Experimental Biophysics of RAS Funding: Ministry of Education and Science of the Russian Federation (grant # 14.604.21.0025, ID RFMEFI60414X0025) Disclosure: All authors have declared no conflicts of interest.

The translocator protein (TSPO; 18 kDa) is a high-affinity cholesterol-binding protein located in the outer membrane of mitochondria. A domain in the C-terminus of TSPO was characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide may participate in the regulation of mitochondrial membrane permeability. Herein, we report the effect of the synthetic CRAC peptide, VLNYYVW, on mitochondrial permeability transition pore (mPTP) opening. It was found that the CRAC peptide alone prevents the mPTP from opening, as well as the release of apoptotic factors (cytochrome c, AIF, and EndoG) in rat brain mitochondria (RBM). Co-incubation of CRAC, together with the TSPO drug ligand, PK 11195, resulted in the acceleration of mPTP opening and in the increase of apoptotic factor release. VLNYYVW did not induce swelling in rat liver mitochondria (RLM). 3,17,19-androsten-5-triol (19-Atriol; an inhibitor of the cholesterol-binding activity of the CRAC peptide) alone and in combination with the peptide was able to stimulate RLM swelling, which was Ca2+- and CsA-sensitive. Additionally, a combination of 19-Atriol with 100 nM PK 11195 or with 100 µM PK 11195 displayed the opposite effect: namely, the addition of 19-Atriol with 100 µM PK 11195 in a suspension of RLM suppressed the Ca2+-induced swelling of RLM by 40%, while the presence of 100 nM PK 11195 with 19-Atriol enhanced the swelling of RLM by 60%. Taken together, these data suggest the participation of the TSPO's CRAC domain in the regulation of permeability transition.

Aim/Background: The role of soluble Her2 (sHer2) in Her2-dependent oncogenicity and in conditioning the response to HER2-mediated therapies has remained elusive. In such studies, the concentrations level of sHer2 and alternative splicing form(s) of Her2 in oncological patients are in the focus of attention. Both the oligomeric status of sHer2 generated by tumor cells and its possible prognostic value have not been examined so far. In this study we have tried to analyze the oligomeric status of sHer2 in breast cancer patients. Methods: Recombinant human sHer2 produced by HEK 293 and sHer2 isolated from the supernatant of the primary tumor cell culture from breast cancer patients was used in the experiments. The oligomeric status was estimated using the method of gel-filtration chromatography and electrophoresis. The stability of Her2 was determined by the change in the protein secondary structure under variation of temperature and pH values by means of CD spectroscopy. The inhibitory activity of sHer2 was analyzed in tests with Her2-expressing cell cultures. The content of sHer2 in the supernatant of primary tumor cell cultures from breast cancer patients was determined using the sandwich ELISA. Amplification of the HER2 gene in the primary tumor cells was verified employing the FISH. Results: The electrophoretic and chromatographic analyses show that sHer2 is present mostly as a tetramer capable of inhibiting proliferation of Her2-expressing cell cultures. sHer2 is stable upon heating the solution to 50°C in the pH range from 7 to 9. It should be noted that the high level of sHer2 is found in supernatants of the primary tumor cell cultures from breast cancer patients not only with amplified Her, but also in triple negative breast cancer samples (ER - 0; PR - 0; Her-2/neu negative (0 + 1); Ki 67 - 27 - 87%). Conclusions: In our opinion, the results obtained show that the oligomeric status of sHer2 can be regarded as a prognostic factor. This work was supported by the grant from Ministry of Education and Science of the Russian Federation (№ 14.604.21.0025, ID RFMEFI60414X0025). Disclosure: All authors have declared no conflicts of interest.

Using ion chromatography and gel filtration we showed that at acidic pH values a caspase-3 substrate may be cleaved by different proteases of the brain. Previously, we showed that one of these enzymes is cathepsin B. Here, we used substrate analysis and Western blotting and found that the proteasome has the capacity to cleave a caspase-3 substrate at acidic pH values.

The efficiency of hybridization analysis with oligonucleotide microarrays depends heavily on the method of detection. Conventional methods based on labeling nucleic acids with fluorescent, chemiluminescent, enzyme, or radioactive reporters suffer from a number of serious drawbacks which demand development of new detection techniques. Here, we report two new approaches for detection of hybridization with oligonucleotide microarrays employing magnetic beads as active labels. In the first method streptavidin-coated magnetic beads are used to discover biotin-labeled DNA molecules hybridized with arrayed oligonucleotide probes. In the second method biotin-labeled DNA molecules are bound first to the surface of magnetic beads and then hybridized with arrayed complementary strands on bead-array contacts. Using a simple low-power microscope with a dark-field illumination and a pair of complementary primers as a model hybridization system we evaluated sensitivity, speed, and cost of the new detection method and compared its performance with the detection techniques employing enzyme and fluorescent labels. It was shown that the detection of microarray-hybridized DNA with magnetic beads combines low cost with high speed and enhanced assay sensitivity, opening a new way to routine hybridization assays which do not require precise measurements of DNA concentration.

Selon l'invention, dans un dispositif multifonctions de mesure de fluorescence, de luminescence et de passage de lumiere dans des buts diagnostiques, un support de l'echantillon a examiner se presente comme une biopuce, une cellule, une cuvette ou une micro-carte imprimee. Le dispositif comprend aussi un premier et un deuxieme groupes d'ecrans. Les ecrans sont montes derriere la surface arriere du support solide de l'echantillon, et les dispositifs d'eclairage de l'echantillon a examiner sont dotes d'elements absorbants pour absorber la lumiere reflechie depuis la surface avant du support d'echantillons et des surfaces des ecrans. Les supports d'ecrans permettent d'installer en alternance des ecrans reflechissant ou renvoyant la lumiere de maniere a assurer un signal de fluorescence ou de luminescence maximal. L'ecran de diffusion assure un regime de mesure du passage de lumiere a travers l'echantillon a examiner. Les ecrans d'absorption de lumiere, disposees derriere la surface arriere de l'echantillon, en combinaison avec les elements d'absorption de lumiere, disposes sur les sources de lumiere, du cote superieur de l'echantillon, permettent d'ameliorer le rapport signal / bruit. Le dispositif permet d'effectuer la mesure des signaux a la surface des biopuces et dans des solutions lors des reactions d'hybridation ou d'amplification. Le dispositif et le procede de traitement des donnees de diagnostic peuvent s'utiliser pour un criblage en masse d'echantillons de materiaux biologiques.

We show that an enzyme exists in rat brain capable of cleaving the caspase-3 specific peptide substrate Ac-DEVD-AMC at low pH. The enzyme shows properties of a cysteine protease and is localized, predominantly, in lysosomes. We have purified this enzyme from rat brain and identified it by MALDI-TOF MS. The enzyme possessing "acidic" DEVDase activity in rat brain appears to be cathepsin B. It remains obscure, whether cathepsin B participates in cleavage of caspase-3 substrates in vivo. We suggest that under certain conditions (e.g. in hypoxia) cathepsin B participates in cleavage of caspase-3 substrates in brain cells.