Hsp70-containing extracellular vesicles are capable of activating of adaptive immunity in models of mouse melanoma and colon carcinoma.

The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.

Coelomocyte replenishment in adult Asterias rubens: the possible ways.

The origin of cells involved in regeneration in echinoderms remains an open question. Replenishment of circulatory coelomocytes-cells of the coelomic cavity in starfish-is an example of physiological regeneration. The coelomic epithelium is considered to be the main source of coelomocytes, but many details of this process remain unclear. This study examined the role of coelomocytes outside circulation, named marginal coelomocytes and small undifferentiated cells of the coelomic epithelium in coelomocyte replenishment in Asterias rubens. A qualitative and quantitative comparison of circulatory and marginal coelomocytes, as well as changes of circulatory coelomocyte concentrations in response to injury at different physiological statuses, was analysed. The presence of cells morphologically similar to coelomocytes in the context of coelomic epithelium was evaluated by electron microscopy. The irregular distribution of small cells on the surface and within the coelomic epithelium was demonstrated and the origin of small undifferentiated cells and large agranulocytes from the coelomic epithelium was suggested. Two events have been proposed to mediate the replenishment of coelomocytes in the coelom: migration of mature coelomocytes of the marginal cell pool and migration of small undifferentiated cells of the coelomic epithelium. The proteomic analysis of circulatory coelomocytes, coelomic epithelial cells and a subpopulation of coelomic epithelial cells, enriched in small undifferentiated cells, revealed proteins that were common and specific for each cell pool. Among these molecules were regulatory proteins, potential participants of regenerative processes.

Gold Nanoparticle Mediated Multi-Modal CT Imaging of Hsp70 Membrane-Positive Tumors.

Imaging techniques such as computed tomographies (CT) play a major role in clinical imaging and diagnosis of malignant lesions. In recent years, metal nanoparticle platforms enabled effective payload delivery for several imaging techniques. Due to the possibility of surface modification, metal nanoparticles are predestined to facilitate molecular tumor targeting. In this work, we demonstrate the feasibility of anti-plasma membrane Heat shock protein 70 (Hsp70) antibody functionalized gold nanoparticles (cmHsp70.1-AuNPs) for tumor-specific multimodal imaging. Membrane-associated Hsp70 is exclusively presented on the plasma membrane of malignant cells of multiple tumor entities but not on corresponding normal cells, predestining this target for a tumor-selective in vivo imaging. In vitro microscopic analysis revealed the presence of cmHsp70.1-AuNPs in the cytosol of tumor cell lines after internalization via the endo-lysosomal pathway. In preclinical models, the biodistribution as well as the intratumoral enrichment of AuNPs were examined 24 h after i.v. injection in tumor-bearing mice. In parallel to spectral CT analysis, histological analysis confirmed the presence of AuNPs within tumor cells. In contrast to control AuNPs, a significant enrichment of cmHsp70.1-AuNPs has been detected selectively inside tumor cells in different tumor mouse models. Furthermore, a machine-learning approach was developed to analyze AuNP accumulations in tumor tissues and organs. In summary, utilizing mHsp70 on tumor cells as a target for the guidance of cmHsp70.1-AuNPs facilitates an enrichment and uniform distribution of nanoparticles in mHsp70-expressing tumor cells that enables various microscopic imaging techniques and spectral-CT-based tumor delineation in vivo.

Suppression of mTORC1 activity in senescent Ras-transformed cells neither restores autophagy nor abrogates apoptotic death caused by inhibition of MEK/ERK kinases.

Autophagy is conservative catabolic process that degrades organelles, in particular, mitochondria, and misfolded proteins within the lysosomes, thus maintaining cellular viability. Despite the close relationship between mitochondrial dysfunction and cellular senescence, it is unclear how mitochondria damage can induce autophagy in senescent cells. We show that MEK/ERK suppression induces mitochondria damage followed by apoptosis of senescent Ras-expressing cells. To understand the role of persistent mTORC1 signaling in breaking the cAMPK-induced autophagy caused by mitochondrial damage, we inhibited mTORС1 with low concentrations of pp242. mTORC1 suppression neither restores the AMPK-induced autophagy nor decreases the level of apoptosis upon MEK/ERK inhibition. We discovered the existence of an alternative autophagy-like way that partially increases the viability of senescent cells under suppressed mTORC1. The pp242-treated cells survive due to formation of the non-autophagous LC3-negative vacuoles, which contain the damaged mitochondria and lysosomes with the following excretion the content from the cell. MEK/ERK activity is required to implement this process in senescent cells. Senescent cells exhibit distinctive spatial distribution of organelles and proteins that provides uncoupling of final participants of autophagy. We show that this feature stops the process of cytoprotective autophagy in response to MEK/ERK suppression, thus allowing selective elimination of senescent Ras-expressing cells.

Targeted elimination of senescent Ras-transformed cells by suppression of MEK/ERK pathway.

The Ras-Raf-MEK-ERK pathway plays a central role in tumorigenesis and is a target for anticancer therapy. The successful strategy based on the activation of cell death in Ras-expressing cells is associated with the suppression of kinases involved in Ras pathway. However, activation of cytoprotective autophagy overcomes antiproliferative effect of the inhibitors and develops drug resistance. We studied whether cellular senescence induced by HDAC inhibitor sodium butyrate in E1a+cHa-Ras-transformed rat embryo fibroblasts (ERas) and A549 human Ki-Ras mutated lung adenocarcinoma cells would enhance the tumor suppressor effect of MEK/ERK inhibition. Treatment of control ERas cells with PD0325901 for 24 h results in mitochondria damage and apoptotic death of a part of cellular population. However, the activation of AMPK-dependent autophagy overcomes pro-apoptotic effects of MEK/ERK inhibitor and results in restoration of the mitochondria and rescue of viability. Senescent ERas cells do not develop cytoprotective autophagy upon inhibition of MEK/ERK pathway due to spatial dissociation of lysosomes and autophagosomes in the senescent cells. Senescent cells are unable to form the autophagolysosomes and to remove the damaged mitochondria resulting in apoptotic death. Our data show that suppression of MEK/ERK pathway in senescent cells provides a new strategy for elimination of Ras-expressing cells.

mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells.

mTOR is a critical target for controlling cell cycle progression, senescence and cell death in mammalian cancer cells. Here we studied the role of mTOR-dependent autophagy in implementating the antiprolifrative effect of mTORC1-specific inhibitor rapamycin and ATP-competitive mTOR kinase inhibitor pp242. We carried out a comprehensive analysis of pp242- and rapamycin-induced autophagy in ERas tumor cells. Rapamycin exerts cytostatic effect on ERas tumor cells, thus causing a temporary and reversible cell cycle arrest, activation of non-selective autophagy not accompanied by cell death. The rapamycin-treated cells are able to continue proliferation after drug removal. The ATP-competitive mTORC1/mTORC2 kinase inhibitor pp242 is highly cytotoxic by suppressing the function of mTORC1-4EBP1 axis and mTORC1-dependent phosphorylation of mTORC1 target--ULK1-Ser757 (Atg1). In contrast to rapamycin, pp242 activates the selective autophagy targeting mitochondria (mitophagy). The pp242-induced mitophagy is accompanied by accumulation of LC3 and conversion of LC3-I form to LC3-II. However reduced degradation of p62/SQSTM indicates abnormal flux of autophagic process. According to transmission electron microscopy data, short-term pp242-treated ERas cells exhibit numerous heavily damaged mitochondria, which are included in single membrane-bound autophagic/autolysophagic vacuoles (mitophagy). Despite the lack of typical for apoptosis features, ERas-treated cells with induced mitophagy revealed the activation of caspase 3, 9 and nucleosomal DNA fragmentation. Thus, pp242 activates autophagy with suppressed later stages, leading to impaired recycling and accumulation of dysfunctional mitochondria and cell death. Better understanding of how autophagy determines the fate of a cell--survival or cell death, can help to development of new strategy for cancer therapy.

Superparamagnetic iron oxide nanoparticles conjugated with epidermal growth factor (SPION-EGF) for targeting brain tumors.

Superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with recombinant human epidermal growth factor (SPION-EGF) were studied as a potential agent for magnetic resonance imaging contrast enhancement of malignant brain tumors. Synthesized conjugates were characterized by transmission electron microscopy, dynamic light scattering, and nuclear magnetic resonance relaxometry. The interaction of SPION-EGF conjugates with cells was analyzed in a C6 glioma cell culture. The distribution of the nanoparticles and their accumulation in tumors were assessed by magnetic resonance imaging in an orthotopic model of C6 gliomas. SPION-EGF nanosuspensions had the properties of a negative contrast agent with high coefficients of relaxation efficiency. In vitro studies of SPION-EGF nanoparticles showed high intracellular incorporation and the absence of a toxic influence on C6 cell viability and proliferation. Intravenous administration of SPION-EGF conjugates in animals provided receptor-mediated targeted delivery across the blood-brain barrier and tumor retention of the nanoparticles; this was more efficient than with unconjugated SPIONs. The accumulation of conjugates in the glioma was revealed as hypotensive zones on T2-weighted images with a twofold reduction in T2 relaxation time in comparison to unconjugated SPIONs (P<0.001). SPION-EGF conjugates provide targeted delivery and efficient magnetic resonance contrast enhancement of EGFR-overexpressing C6 gliomas.

Tumor targeting using magnetic nanoparticle Hsp70 conjugate in a model of C6 glioma.

BACKGROUND: Superparamagnetic iron oxide nanoparticles (SPIONs), due to their unique magnetic properties, have the ability to function both as magnetic resonance (MR) contrast agents, and can be used for thermotherapy. SPIONs conjugated to the heat shock protein Hsp70 that selectively binds to the CD40 receptor present on glioma cells, could be used for MR contrast enhancement of experimental C6 glioma. METHODS: The magnetic properties of the Hsp70-SPIONs were measured by NMR relaxometry method. The uptake of nanoparticles was assessed on the C6 glioma cells by confocal and electron microscopes. The tumor selectivity of Hsp70-SPIONs being intravenously administered was analyzed in the experimental model of C6 glioma in the MRI scanner. RESULTS: Hsp70-SPIONs relaxivity corresponded to the properties of negative contrast agents with a hypointensive change of resonance signal in MR imaging. A significant accumulation of the Hsp70-SPIONs but not the non-conjugated nanoparticles was observed by confocal microscopy within C6 cells. Negative contrast tumor enhancement in the T2-weighted MR images was higher in the case of Hsp70-SPIONs in comparison to non-modified SPIONs. Histological analysis of the brain sections confirmed the retention of the Hsp70-SPIONs in the glioma tumor but not in the adjacent normal brain tissues. CONCLUSION: The study demonstrated that Hsp70-SPION conjugate intravenously administered in C6 glioma model accumulated in the tumors and enhanced the contrast of their MR images.

Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture.

Genomic stability is critical for the clinical use of human embryonic and induced pluripotent stem cells. We performed high-resolution SNP (single-nucleotide polymorphism) analysis on 186 pluripotent and 119 nonpluripotent samples. We report a higher frequency of subchromosomal copy number variations in pluripotent samples compared to nonpluripotent samples, with variations enriched in specific genomic regions. The distribution of these variations differed between hESCs and hiPSCs, characterized by large numbers of duplications found in a few hESC samples and moderate numbers of deletions distributed across many hiPSC samples. For hiPSCs, the reprogramming process was associated with deletions of tumor-suppressor genes, whereas time in culture was associated with duplications of oncogenic genes. We also observed duplications that arose during a differentiation protocol. Our results illustrate the dynamic nature of genomic abnormalities in pluripotent stem cells and the need for frequent genomic monitoring to assure phenotypic stability and clinical safety.

The release of Hsp70 from A431 carcinoma cells is mediated by secretory-like granules.

In our earlier work we have demonstrated that the treatment of squamous carcinoma cell line A431 with a pharmacological inhibitor of phospholipase C activity, U73122, resulted in fast release of stress-inducible heat shock protein 70 (Hsp70) into the extracellular medium (Evdonin et al., Cancer Cell Int., 4, 2, 2004). The purpose of the present study was to identify cellular organelles involved in the release of Hsp70 from A431 cells. We determined that Hsp70 is present in granules located at the periphery of cells, which had been treated with U73122 or subjected to heat shock. An inhibitor of the classical protein export pathway, brefeldin A was found to prevent the U73122-induced appearance of Hsp70 in the extracellular medium and in the peripheral granules. These findings suggest that vesicular transport is involved in Hsp70 release. The Hsp70-containing granules did not carry markers specific for lipid bodies, endosomes, or lysosomes. However, they were positive for a marker of secretory granules, i.e. chromogranin A. The levels of extracellular Hsp70 and chromogranin A were found to increase simultaneously. The secretory-like granule-dependent transport of Hsp70 was also studied in minimally transformed human HaCaT keratinocytes. We found that after U73122 and heat stress treatment, HaCaT cells secreted Hsp70 in a manner similar to A431 cells. Collectively our results suggest that human keratinocyte-derived cells release Hsp70 in the extracellular medium through a pathway involving secretory-like granules.

Heart and the peritoneal cover of the gut sinus in the polychaete Arenicola marina: an ultrastructural and autoradiographic study.

To elucidate the cellular mechanism underlying the growth of the peritoneal cover of the gut sinus and the heart in the polychaete Arenicola marina, cellular organization of these structures and proliferative potential of their cells were investigated using electron microscopy and electron microscopic autoradiography. Arenicola has a pair of dorsolaterally situated hearts connected to the gut sinus via a short duct and composed of two muscular layers separated by a layer of the extracellular matrix (ECM). The peritoneal cover of the gut sinus and the outer muscular layer of the heart present a myoepithelial layer resting on the ECM. The inner muscular layer of the heart is composed of myofibril-containing cells lacking well-defined polarity in arrangement of organelles. However, their persistent connection to branches of the ECM and the adherens-like intercellular junctions allow for considering the inner layer a modified myoepithelium. In the peritoneal cover of the gut sinus and in both myoepithelial layers of the heart, noncontractile epithelial cells have been observed. As determined by thymidine labeling, these epithelial cells are capable of DNA synthesis, while myoepithelial cells are not. Some suggestions are made about the myogenic nature of the epithelial cells in the investigated structures of A. marina.

Undifferentiated cells in the snail myocardium are capable of DNA synthesis and myodifferentiation.

Cellular mechanisms of heart-muscle growth in the snail Achatina fulica have been studied using cytophotometry and electron microscopic autoradiography. Cytophotometric DNA measurements showed that the snail cardiomyocytes are mononucleated cells with diploid nuclei. Ultrastructural analysis of the snail myocardium revealed that, in addition to mature myocytes, it contains small roundish undifferentiated cells (UCs) and poorly differentiated muscle cells. EM autoradiography detected silver grains over the nuclei of UCs 2 h after injection of tritiated thymidine ([(3)H]Tdr), while the nuclei of both mature and poorly differentiated myocytes remained unlabeled. In EM autographs of the myocardial tissue fixed 14 days after [(3)H]Tdr administration, labeled myonuclei were evident, which may suggest some myodifferentiation of prelabeled UCs. Many labeled UCs persist for 14 days after a single [(3)H]Tdr injection, suggesting that not all UCs undergo myodifferentiation after passing through the cell cycle, and that those that do not can enter the next cycle. UCs in the snail myocardium presumably provide not only reserve but also stem cells for myocytes. Thus, the heart muscle of the adult snail consists of mononucleated diploid myocytes with blocked proliferative activity and a renewable population of precursor myogenic cells. The results obtained suggest that the growth of this muscle involves a myoblastic mechanism of myogenesis; this mechanism differs from that of vertebrate cardiac muscle growth, which is non-myoblastic-that is, based on proliferation or polyploidization of cardiomyocytes. Evolutionary aspects of cellular mechanisms of the heart-muscle growth are discussed.