Differential binding cell-SELEX method to identify cell-specific aptamers using high-throughput sequencing.

Aptamers have in recent years emerged as a viable alternative to antibodies. High-throughput sequencing (HTS) has revolutionized aptamer research by increasing the number of reads from a few (using Sanger sequencing) to millions (using an HTS approach). Despite the availability and advantages of HTS compared to Sanger sequencing, there are only 50 aptamer HTS sequencing samples available on public databases. HTS data in aptamer research are primarily used to compare sequence enrichment between subsequent selection cycles. This approach does not take full advantage of HTS because the enrichment of sequences during selection can be due to inefficient negative selection when using live cells. Here, we present a differential binding cell-SELEX (systematic evolution of ligands by exponential enrichment) workflow that adapts the FASTAptamer toolbox and bioinformatics tool edgeR, which are primarily used for functional genomics, to achieve more informative metrics about the selection process. We propose a fast and practical high-throughput aptamer identification method to be used with the cell-SELEX technique to increase the aptamer selection rate against live cells. The feasibility of our approach is demonstrated by performing aptamer selection against a clear cell renal cell carcinoma (ccRCC) RCC-MF cell line using the RC-124 cell line from healthy kidney tissue for negative selection.

Effects of malvidin, cyanidin and delphinidin on human adipose mesenchymal stem cell differentiation into adipocytes, chondrocytes and osteocytes.

BACKGROUND: Anthocyanidins are plant phytochemicals found at high concentrations in berries, vegetables and flowers. Anthocyanidins have been extensively investigated due to their antioxidative, antidiabetic and anti-inflammatory effects. Few studies show that anthocyanidins decrease obesity and improve bone density. However, the effects of anthocyanidins on tissue regeneration have not been sufficiently clarified. Human mesenchymal stem cells (MSCs) are multipotent adult stem cells responsible for the regeneration of fat, bone and cartilage. Although MSCs are often used for screening of biologically active compounds, so far, the effect of anthocyanidins on MSC differentiation has not been addressed. PURPOSE: The aim of this study was to analyse the effect of anthocyanidins malvidin, cyanidin and delphinidin on adipose tissue-derived MSC differentiation into adipocytes, osteocytes and chondrocytes. STUDY DESIGN AND METHODS: Differentiation into adipocytes, osteocytes and chondrocytes was carried out in the defined cell culture conditions in the presence or absence of malvidin, cyanidin and delphinidin. The differentiation was confirmed by cytochemical staining and tissue-specific gene and protein expression. Antiobesity and anti-diabetes drug liraglutide was used as a reference drug in this study. RESULTS: Delphinidin inhibited MSC adipogenesis and downregulated FABP4 and adiponectin genes. Malvidin induced a significantly higher accumulation of calcium deposits in MSCs comparing to untreated MSCs, as well as upregulated the osteocyte-specific gene BMP-2 and Runx-2 expression and induced BMP-2 secretion. Cyanidin and delphinidin demonstrated a chondrogenesis stimulating effect by upregulation of Col2a1 and aggrecan. CONCLUSION: Altogether, our data show that anthocyanidins malvidin, cyanidin and delphinidin exert favourable effects on MSC osteogenesis and chondrogenesis whereas delphinidin inhibits adipogenesis. These results suggest that anthocyanidin effects on tissue regeneration could be further analysed in depth in vivo.

Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages.

BACKGROUND: Macrophages are one of the most important players in the tumor microenvironment. The polarization status of tumor associated macrophages into a pro-inflammatory type M1 or anti-inflammatory type M2 may influence cancer progression and patient survival. Extracellular vesicles (EVs) are membrane-bound vesicles containing different biomolecules that are involved in cell to cell signal transfer. Accumulating evidence suggests that cancer-derived EVs are taken up by macrophages and modulate their phenotype and cytokine profile. However, the interactions of cancer-derived EVs with monocytes and macrophages at various differentiation and polarization states are poorly understood. In the current study, we have analyzed the uptake and functional effects of primary (SW480) and metastatic (SW620) isogenic colorectal cancer (CRC) cell line-derived EVs on monocytes (M), inactive macrophages (M0) and M1 and M2 polarized macrophages. METHODS: THP-1 monocytes were differentiated into M0 macrophages by addition of phorbol-12-myristate-13-acetate. Then M0 macrophages were further polarized into M1 and M2 macrophages in the presence of LPS, IFN- γ, IL-4, and IL-13 respectively. Internalization of SW480 and SW620-derived EVs was analyzed by flow cytometry and fluorescence microscopy. Changes in monocyte and macrophage immunophenotype and secretory profile upon EV exposure were analyzed by flow cytometry, quantitative PCR and Luminex assays. RESULTS: THP-1 monocytes and M0 macrophages efficiently take up SW480 and SW620-derived EVs, and our results indicate that dynamin-dependent endocytic pathways may be implicated. Interestingly, SW480 and SW620-derived EVs increased CD14 expression in M0 macrophages whereas SW480-derived EVs decreased HLA-DR expression in M1 and M2 polarized macrophages. Moreover, SW480-derived EVs significantly increased CXCL10 expression in monocytes and M0 macrophages. In contrast, SW620-derived EVs induced secretion of IL-6, CXCL10, IL-23 and IL-10 in M0 macrophages. However, addition of CRC cell line-derived EVs together with LPS, IFN- γ (M1) and IL-4, IL-13 (M2) stimuli during macrophage polarization had no additional effect on cytokine expression in M1 and M2 macrophages. CONCLUSION: Our results suggest that CRC cell line-derived EVs are internalized and reprogram the immunophenotype and secretory profile in monocytes and inactive macrophages inducing mixed M1 and M2 cytokine response. Although CRC EVs decreased HLA-DR expression in M1, M2 polarized macrophages, their effect on the secretory profile of M1 and M2 polarized macrophages was negligible.

Nanoparticle delivery to metastatic breast cancer cells by nanoengineered mesenchymal stem cells.

We created a 3D cell co-culture model by combining nanoengineered mesenchymal stem cells (MSCs) with the metastatic breast cancer cell line MDA-MD-231 and primary breast cancer cell line MCF7 to explore the transfer of quantum dots (QDs) to cancer cells. First, the optimal conditions for high-content QD loading in MSCs were established. Then, QD uptake in breast cancer cells was assessed after 24 h in a 3D co-culture with nanoengineered MSCs. We found that incubation of MSCs with QDs in a serum-free medium provided the best accumulation results. It was found that 24 h post-labelling QDs were eliminated from MSCs. Our results demonstrate that breast cancer cells efficiently uptake QDs that are released from nanoengineered MSCs in a 3D co-culture. Moreover, the uptake is considerably enhanced in metastatic MDA-MB-231 cells compared with MCF7 primary breast cancer cells. Our findings suggest that nanoengineered MSCs could serve as a vehicle for targeted drug delivery to metastatic cancer.

Skin-derived mesenchymal stem cells as quantum dot vehicles to tumors.

PURPOSE: Cell-mediated delivery of nanoparticles is emerging as a new method of cancer diagnostics and treatment. Due to their inherent regenerative properties, adult mesenchymal stem cells (MSCs) are naturally attracted to wounds and sites of inflammation, as well as tumors. Such characteristics enable MSCs to be used in cellular hitchhiking of nanoparticles. In this study, MSCs extracted from the skin connective tissue were investigated as transporters of semiconductor nanocrystals quantum dots (QDs). MATERIALS AND METHODS: Cytotoxicity of carboxylated CdSe/ZnS QDs was assessed by lactate dehydrogenase cell viability assay. Quantitative uptake of QDs was determined by flow cytometry; their intracellular localization was evaluated by confocal microscopy. In vitro tumor-tropic migration of skin-derived MSCs was verified by Transwell migration assay. For in vivo migration studies of QD-loaded MSCs, human breast tumor-bearing immunodeficient mice were used. RESULTS: QDs were found to be nontoxic to MSCs in concentrations no more than 16 nM. The uptake studies showed a rapid QD endocytosis followed by saturating effects after 6 h of incubation and intracellular localization in the perinuclear region. In vitro migration of MSCs toward MDA-MB-231 breast cancer cells and their conditioned medium was up to nine times greater than the migration toward noncancerous breast epithelial cells MCF-10A. In vivo, systemically administered QD-labeled MSCs were mainly located in the tumor and metastatic tissues, evading most healthy organs with the exception being blood clearance organs (spleen, kidneys, liver). CONCLUSION: Skin-derived MSCs demonstrate applicability in cell-mediated delivery of nanoparticles. The findings presented in this study promise further development of a cell therapy and nanotechnology-based tool for early cancer diagnostics and therapy.

Nano-engineered skin mesenchymal stem cells: potential vehicles for tumour-targeted quantum-dot delivery.

Nanotechnology-based drug design offers new possibilities for the use of nanoparticles in imaging and targeted therapy of tumours. Due to their tumour-homing ability, nano-engineered mesenchymal stem cells (MSCs) could be utilized as vectors to deliver diagnostic and therapeutic nanoparticles into a tumour. In the present study, uptake and functional effects of carboxyl-coated quantum dots QD655 were studied in human skin MSCs. The effect of QD on MSCs was examined using a cell viability assay, Ki67 expression analysis, and tri-lineage differentiation assay. The optimal conditions for QD uptake in MSCs were determined using flow cytometry. The QD uptake route in MSCs was examined via fluorescence imaging using endocytosis inhibitors for the micropinocytosis, phagocytosis, lipid-raft, clathrin- and caveolin-dependent endocytosis pathways. These data showed that QDs were efficiently accumulated in the cytoplasm of MSCs after incubation for 6 h. The main uptake route of QDs in skin MSCs was clathrin-mediated endocytosis. QDs were mainly localized in early endosomes after 6 h as well as in late endosomes and lysosomes after 24 h. QDs in concentrations ranging from 0.5 to 64 nM had no effect on cell viability and proliferation. The expression of MSC markers, CD73 and CD90, and hematopoietic markers, CD34 and CD45, as well as the ability to differentiate into adipocytes, chondrocytes, and osteocytes, were not altered in the presence of QDs. We observed a decrease in the QD signal from labelled MSCs over time that could partly reflect QD excretion. Altogether, these data suggest that QD-labelled MSCs could be used for targeted drug delivery studies.