Upregulation of circulating cancer stem cell marker, DCLK1 but not Lgr5, in chemoradiotherapy-treated colorectal cancer patients

Cancer stem cell (CSC) markers have attracted considerable attention in tumor diagnostic, prognostic, and therapeutic implications. Detection of cancer stem cells in circulating blood using cancer stem cell markers has received remarkable attention recently. In this study, we aimed to investigate the messenger RNA (mRNA) expression level of Lgr5 and DCLK1 as most proposed colorectal CSC markers in blood circulation also determine the subsequent association to patients� clinical and pathological findings. Peripheral blood mononuclear cells (PBMCs) of 58 patients with colorectal cancer at stage I�IV with 33 out of 58 patients undergoing preoperative chemoradiotherapy (CRT), as well as 58 healthy controls have been isolated and the extracted RNAs were analyzed using real-time PCR. The mRNA expression pattern of CSC markers of patients and controls was compared using ��Ct method. The expression level of Lgr5 was significantly higher in colorectal cancer (CRC) patients comparing to healthy group (4.8-fold change, p < 0.001). Also there was a significant increase in expression level of Lgr5 in patients at stages III and IV comparing to stages I and II (p = 0.031) and higher grades (p = 0.039) of CRC. The expression of DCLK1 was also elevated in patients significantly (2.7-fold change, p < 0.001) and the related expression was increased by increasing disease stage (p = 0.025). Combination of DCLK1 and Lgr5 markers was analyzed by logistic regression and proved to be a slightly better marker compared to each marker alone. Interestingly the DCLK1 expression level was significantly higher in patients undergoing preoperative CRT (p = 0.041); however, no association to neoadjuvant CRT was observed for Lgr5. Considering the over-expression of DCLK1 and Lgr5 in circulating blood of CRC patients comparing to controls, our results might emphasize on the presence of CSCs in blood of these patients which might be attributed to their clinical and pathological characteristics and may lead to apply in future clinical implications. Moreover, the higher expression level of DCLK1 in patients undergoing CRT can propose it as a more relevant candidate among CSC markers comparing to Lgr5 for CRC patients. © 2015, International Society of Oncology and BioMarkers (ISOBM)

Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma

Purpose: Methylated cell-free fetal DNA (cffDNA) in maternal plasma can potentially be used as a biomarker for accurate noninvasive prenatal testing (NIPT) of fetal disorders. Recovery and purification of cffDNA are key steps for downstream applications. In this study, we aimed to developed and evaluated different aspects of an optimized method and compared its efficiency with common methods used for extraction of methylated cffDNA. Methods: Single factor experiments, Plackett-Burman (PB) design, and response surface methodology (RSM) were conducted for conventional Triton/Heat/Phenol (cTHP) method optimization. The total cell-free DNA (cfDNA) was extracted from pooled maternal plasma using the optimized method called the Triton/Heat/Phenol/Glycogen (THPG), cTHP method, a column-based kit, and a magnetic bead-based kit. In the next step, methylated cfDNA from the extracted total cfDNA was enriched using a methylated DNA immunoprecipitation (MeDIP) kit. Real-time quantitative polymerase chain reaction was performed on the RASSF1 gene and hyper region to determine the genomic equivalents per milliliter (GEq/ml) values of the methylated cfDNA and cffDNA, respectively. Results: The optimum values of the significant factors affecting cfDNA extraction from 200 μl of plasma were 3% SDS, 1% Triton X-100, 0.9 μg/μl glycogen, and 0.3 M sodium acetate. The GEq/ml values of methylated cffDNA extracted using the THPG method were significantly higher than for the tested extraction methods (p < 0.001). Conclusions: Our results indicate that the THPG method is more efficient than the other tested methods for extraction of low copy number methylated cffDNA from a small volume of maternal plasm

A new insight into cancer stem cell markers: Could local and circulating cancer stem cell markers correlate in colorectal cancer?

Cancer stem cell (CSC) markers could serve as potential prognostic procedure. This study is aimed to investigate the local expression of doublecortin-like kinase 1 (DCLK1) and Lgr5 in colorectal cancer tissues (CRC) at both protein and messenger RNA (mRNA) level, followed by providing a comparison of the local and circulating expression pattern of these markers, based on our present and previous study. The mRNA expression level of DCLK1 and Lgr5 was evaluated using comparative real-time PCR method applying 58 fresh tumor tissues and their correspondent normal margins. Immunohistochemistry was applied to analyze the protein expression level of DCLK1 and Lgr5 in paraffin-embedded CRC tissues. The correlation of DCLK1 and Lgr5 expression pattern with clinicopathological characteristics was assessed. A higher mRNA expression level of DCLK1 (3.28-fold change, p < 0.001) and Lgr5 (2.29-fold change, p < 0.001) was observed in CRC fresh tissues compared to the normal adjacent margins, and the expression level was higher in patients with higher grade and stages of disease and patients who underwent neoadjuvant chemoradiotherapy (CRT). The protein expression level of DCLK1 and Lgr5 was also increased significantly in tumor tissues compared to normal colon tissues which were positively correlated to tumor stage and grade and neoadjuvant CRT. Taken together, the results of protein analysis were in accordance with mRNA assessment. The local expression pattern of DCLK1 and Lgr5 was also in accordance with their expression level in circulation. However, some minor inconsistencies were observed which may be attributed to several factors including the possible effect of CRT on CSC reprogramming. © 2015, International Society of Oncology and BioMarkers (ISOBM)

ProteinSeq: High-Performance Proteomic Analyses by Proximity Ligation and Next Generation Sequencing

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use

Spectroscopic and microscopic analyses of rod-shaped gold nanoparticles interacting with single-stranded DNA oligonucleotides

The application of rod-shaped gold nanoparticles as probes and carriers in biological systems have recently attracted great interest. UV-vis spectroscopy, circular dichroism, Fourier transform infrared spectroscopy, scanning electron microscopy and atomic force microscopy were used to study optical and structural properties of rod-shaped gold nanoparticles when interacting with DNA oligomers in phosphate sodium salt buffer. The morphological transformation process of rod-shaped gold nanoparticles resulting from the interaction with single-stranded DNA (ssDNA) showed the role of hexadecyltrimethylammonium bromide (CTAB) in nanostructures as the main interacting agent. The obtained results confirmed that the CTAB coat of rod-shaped gold nanoparticles have powerful positive charges for conjugations with surface negative charges of phosphate groups on ssDNA oligomers. The CTAB also inhibit the formation of covalent sulphide bonds between the gold core of rod-shaped nanoparticles and alkanethiol oligonucleotides. The authors found that when the nanorods were exposed to ssDNA oligonucleotides, the gold nanorods changed their shapes and sizes, and exposed some microscopic malformations which could be used in the development of colorimetric assays of nucleic acids. © The Institution of Engineering and Technology 2013

Evaluation of circulating cellular DCLK1 protein, as the most promising colorectal cancer stem cell marker, using immunoassay based methods

BACKGROUND: DCLK1, as the most potential colorectal cancer stem cell (CSC) marker has been the core of many recent investigations. However, no study has been performed to evaluate the circulating cellular DCLK1 protein (CCDP) that might reflect the presences of colorectal CSC in circulation. OBJECTIVES: We aimed to evaluate CCDP in the peripheral blood mononuclear cells (PBMCs) of colorectal cancer (CRC) patients applying immunoassay based methods including PLA, IPCR and ELISA in order to introduce the method of choice for clinical detection of CCDP. METHODS: PBMCs were extracted from blood samples of 58 CRC patients along with 58 blood samples of tumor free controls. Total protein of PBMC was extracted and the CCDP level was evaluated. The results of three applied immunoassay tests were compared and the best approach for clinical application was introduced, accordingly. In addition, the correlation of CCD Plevel with clincopathologic findings of CRC patients was assessed. RESULTS: The results of three immuneassay methods confirmed each other. Based on our finding, ELISA could be the most judicious method for clinical evaluation of CCDP considering its simplicity for clinical implications. Our results also showed a significant higher amount of CCDP in peripheral blood of CRC patients compared to control group which was also correlated with patients' clinicopathologic finding such as stage, grade and neoadjuvant history. CONCLUSION: CCDP could be applied for monitoring purposes in CRC patients. However, its application needs to be more elucidated in future investigations implementing larger samples. © 2016 - IOS Press and the authors

Exosome loaded alginate hydrogel promotes tissue regeneration in full-thickness skin wounds: An in vivo study

Wound healing is known as one of the most complicated biological processes for injured skin caused by surgical, trauma, burns, or diabetic diseases, which causes a nonfunctioning mass of fibrotic tissue. Recent reports have suggested that exosomes (EXOs) secreted by this type of stem cells may contribute to their paracrine effect. In this study, the EXOs were isolated from the supernatant of cultured adipose-derived stem cells (ADSCs) via ultracentrifugation and filtration. The EXO loaded in the alginate-based hydrogel was used as a bioactive scaffold to preserve the EXO in the wound site in the animal model. The physical and biochemical properties of EXO loaded Alg hydrogel were characterized and results proved that fabricated structure was biodegradable and biocompatible. This bioactive wound dressing technique has significantly improved wound closure, collagen synthesis, and vessel formation in the wound area. Results offer a new viewpoint and a cell-free therapeutic strategy, for wound healing through the application of the composite structure of EXO encapsulated in alginate hydrogel. © 2019 Wiley Periodicals, Inc