LncRNA Quantification from Extracellular Vesicles Isolated from Blood Plasma or Conditioned Media.

During the last years, the study of extracellular vesicles (EVs) and its cargo has gained interest in the scientific media. EVs have been found in all biofluids and it is postulated that all cells are capable to secrete a wide variety of these vesicles, which play a key role in different cell-to-cell communication processes as well as in the microenvironment modulation. In the EV cargo, DNA, protein, and RNA molecules can be found, including long noncoding RNAs (lncRNAs). Several authors consider the study of EV lncRNAs an ideal source of biomarkers due to the easy sampling of EVs in different biofluids and the high specificity of the lncRNA expression pattern.In the present chapter, a detailed explanation of the EV isolation workflow followed by RNA isolation and lncRNA gene expression study is provided for two sample sources: blood plasma and cell culture conditioned media. EVs from both plasma samples and cell cultured media are isolated using sequential ultracentrifugation method (UC), which has been reported as one of the best methods available to date in terms of purity. UC is followed by RNA extraction based on the combination of phenol/guanidine-based lysis of samples with silica-membrane-based purification of total RNA. LncRNA quantification is performed by qRT-PCR. This chapter includes detailed discussion on lncRNA quantification using hydrolysis probes, recommended housekeeping genes and evaluation of methods for comparing lncRNA levels between EVs and its parental cells. In summary, we describe here the main steps for a successful isolation of the EVs-lncRNA cargo, paying attention to how overcome the different challenges found in the experimental procedure and in the data analysis of lncRNA expression from this source.

LincRNA-p21 Levels Relates to Survival and Post-Operative Radiotherapy Benefit in Rectal Cancer Patients.

LincRNA-p21 is a long non-coding RNA involved in the p53 pathway and angiogenesis regulation that acts as prognostic marker in several tumors. In the present study, we aimed to analyze the clinical value of lincRNA-p21 in 177 resected stage I-III colorectal cancer (CRC) patients. Tumor and normal paired tissue and plasma samples from tumor-draining mesenteric veins and paired peripheral veins were analyzed. LincRNA-p21 expression was determined by RTqPCR and correlated with disease-free (DFS) and overall survival (OS). LincRNA-p21 was downregulated in tumor versus normal tissue (p = 0.0012). CRC patients with high lincRNA-p21 expression had shorter DFS (p = 0.0372) and shorter OS (p = 0.0465). Of note, the major prognostic impact was observed in the subset of rectal cancer patients where patients with high lincRNA-p21 levels had worse DFS (p = 0.0226) and OS (p = 0.0457). Interestingly, rectal cancer patients with high lincRNA-p21 benefited from post-operative chemoradiotherapy, as indicated by a longer OS in the group of high lincRNA-p21 patients receiving post-operative chemoradiotherapy (p = 0.04). Finally, patients with high lincRNA-p21 levels in mesenteric vein (MV) had shorter OS (p = 0.0329). LincRNA-p21 is a marker of advanced disease and worse outcome in CRC. Moreover, rectal cancer patients with high lincRNA-p21 levels could benefit from post-operative chemoradiotherapy, and plasmatic-lincRNA-p21 is a promising liquid biopsy biomarker.

Extracellular Vesicle lincRNA-p21 Expression in Tumor-Draining Pulmonary Vein Defines Prognosis in NSCLC and Modulates Endothelial Cell Behavior.

Hypoxia-induced upregulation of lincRNA-p21 in tumor tissue was previously shown by our group to be related to poor prognosis in resected non-small cell lung cancer (NSCLC) patients. In the present study, we have evaluated the presence of lincRNA-p21 in extracellular vesicles (EVs) from NSCLC patients and assessed its potential as a prognostic biomarker. High EV lincRNA-p21 levels in blood from the tumor-draining vein were associated with shorter time to relapse and shorter overall survival. Moreover, the multivariate analysis identified high lincRNA-p21 levels as an independent prognostic marker. In addition, lincRNA-p21 was overexpressed in H23 and HCC44 NSCLC cell lines and their derived EVs under hypoxic conditions. Functional assays using human umbilical vein endothelial cells (HUVECs) showed that tumor-derived EVs enriched in lincRNA-p21 affected endothelial cells by promoting tube formation and enhancing tumor cell adhesion to endothelial cells. Additionally, the analysis of selected EV microRNAs related to angiogenesis and metastasis showed that the microRNAs correlated with EV lincRNA-p21 levels in both patients and cell lines. Finally, EV co-culture with HUVEC cells increased the expression of microRNAs and genes related to endothelial cell activation. In conclusion, EV lincRNA-p21 acts as a novel prognosis marker in resected NSCLC patients, promoting angiogenesis and metastasis.

KRAS mutations by digital PCR in circulating tumor cells isolated from the mesenteric vein are associated with residual disease and overall survival in resected colorectal cancer patients.

PURPOSE: In colorectal cancer (CRC), circulating tumor cells (CTCs) are released into the mesenteric veins (MV). We chose to determine whether KRAS mutations detected in CTCs from blood obtained at the time of surgery could be a marker of survival. METHODS: From 52 surgically resected CRC patients who later relapsed, samples of tumor tissue, normal tissue, and blood from the peripheral vein (PV) and MV were obtained from each patient at the time of surgery. KRAS mutations were assessed by Sanger sequencing and digital PCR (DGPCR) in tissue samples and by DGPCR in CTCs. Mutant KRAS copy number was assessed in CTCs. Results were correlated with overall survival (OS). RESULTS: Sanger sequencing detected KRAS mutations in ten tumor samples (19.2%), while DGPCR detected mutations in 30 (58%). Mutations were detected in CTCs in 21 MV samples (40.4%) and 18 PV samples (34.6%). Patients with G13D mutations in CTCs from the MV had shorter OS than those with G12D mutations (28.1 vs 54.6 months; p = 0.025). Patients with a high mutant KRAS copy number in CTCs had shorter OS than those with a low mutant KRAS copy number (MV: 20.5 vs 43.7 months; p = 0.002; PV: 15.1 vs 38.2 months; p = 0.027). CONCLUSION: DGPCR is more efficient than Sanger sequencing for detecting KRAS mutations. KRAS G13D mutations and high mutant KRAS copy number are associated with shorter OS. The analysis of KRAS mutations in CTCs from blood obtained at the time of surgery can identify patients with a higher risk of relapse.

Clinical significance of long non-coding RNA HOTTIP in early-stage non-small-cell lung cancer.

BACKGROUND: HOTTIP, a long non-coding RNA located in the HOXA cluster, plays a role in the patterning of tissues with mesodermal components, including the lung. Overexpression of HOXA genes, including HOTTIP, has been associated with a more aggressive phenotype in several cancers. However, the prognostic impact of HOTTIP has not yet been explored in non-small-cell lung cancer (NSCLC). We have correlated HOTTIP expression with time to relapse (TTR) and overall survival (OS) in early-stage NSCLC patients. METHODS: Ninety-nine early-stage NSCLC patients who underwent surgical resection in our center from June 2007 to November 2013 were included in the study. Mean age was 66; 77.8% were males; 73.7% had stage I disease; and 55.5% had adenocarcinoma. A validation data set comprised stage I-II patients from The Cancer Genome Atlas (TCGA) Research Network. RESULTS: HOTTIP was expressed in all tumor samples and was overexpressed in squamous cell carcinoma (p = 0.007) and in smokers (p = 0.018). Patients with high levels of HOTTIP had shorter TTR (78.3 vs 58 months; p = 0.048) and shorter OS (81.2 vs 61 months; p = 0.023) than those with low levels. In the multivariate analysis, HOTTIP emerged as an independent prognostic marker for TTR (OR: 2.05, 95%CI: 1-4.2; p = 0.05), and for OS (OR: 2.31, 95%CI: 1.04-5.1; p = 0.04). HOTTIP was validated as a prognostic marker for OS in the TCGA adenocarcinoma cohort (p = 0.025). Moreover, we identified a 1203-mRNA and a 61-miRNA signature that correlated with HOTTIP expression. CONCLUSIONS: The lncRNA HOTTIP can be considered a prognostic biomarker in early-stage NSCLC.

Exosome Analysis in Tumor-Draining Pulmonary Vein Identifies NSCLC Patients with Higher Risk of Relapse after Curative Surgery.

Since tumor-draining pulmonary vein blood (PV) is enriched in tumor-secreted products, we hypothesized that it would also be enriched in tumor-derived exosomes, which would be important in the metastasis process. We characterized exosomes from PV of 61 resected non-small cell lung cancer (NSCLC) patients to evaluate its potential as relapse biomarkers. Exosomes were characterized using transmission electron microscopy, western blot and nanoparticle tracking analysis and we examined time to relapse (TTR) and overall survival (OS). Differences between PV and peripheral vein were found. PV was enriched in smaller exosomes than the paired peripheral vein (p = 0.01). Moreover, PV exosome size mode was able to identify relapsed patients (Area under the curve [AUC] = 0.781; 95%CI: 0.6641⁻0.8978), in whom exosome size was smaller (<112 nm; p < 0.001). The combination of PV exosome size and N (lymph node involvement) showed an AUC of 0.89 (95%CI: 0.80⁻0.97). Moreover, smaller PV exosome size was associated with shorter TTR (28.3 months vs. not reached, p < 0.001) and OS (43.9 months vs. not reached, p = 0.009). Multivariate analyses identified PV exosome size and stage as independent prognostic markers for TTR and OS. PV exosome size is a promising relapse biomarker after surgery that can add valuable information to clinical variables.

Prognostic Impact of miR-200 Family Members in Plasma and Exosomes from Tumor-Draining versus Peripheral Veins of Colon Cancer Patients.

OBJECTIVE: To evaluate the prognostic potential of expression levels of miR-200 family members (miR-200a, miR-200b, miR-200c, miR-429, miR-141) in plasma and exosomes from the tumor-draining vein (mesenteric vein [MV]) and peripheral vein (PV) of colon cancer (CC) patients. METHODS: We analyzed the expression of miR-200 family members in matched samples of MV and PV plasma from 50 resected patients with CC and correlated our findings with overall survival (OS). We also examined the content of these microRNAs in MV and PV exosomes. RESULTS: Expression levels were higher in MV than in PV (miR-200a, p < 0.001; miR-200b, p < 0.001; miR-429, p = 0.01; miR-200c, p = 0.05; miR-141, p = 0.05). Low levels of both miR-200c and miR-141 in MV plasma were associated with longer OS (p = 0.02). This association was maintained for the MV exosome cargo of miR-200c and miR-141 (p = 0.02). CONCLUSION: Our findings provide the first indication that expression levels of miR-200c and miR-141 in MV plasma can identify CC patients with poor prognosis. In addition, our results lend further support to the premise that tumor-draining veins constitute a better source of biomarkers than do PVs.

Proteomic Analysis of Liquid Biopsy from Tumor-Draining Vein Indicates that High Expression of Exosomal ECM1 Is Associated with Relapse in Stage I-III Colon Cancer.

BACKGROUND: The analysis of exosomes in blood obtained from the tumor-draining mesenteric vein (MV) can identify tumor biomarkers before they reach target organs and form the premetastatic niche where circulating tumor cells can anchor. Our group has recently shown that microRNAs in plasma from the MV-but not the peripheral vein (PV)-have been related to liver metastases in colon cancer (CC) patients. Here we examine the exosomal protein cargo in plasma from the MV and paired PV in 31 CC patients. PATIENTS AND METHODS: The study included patients who were initially diagnosed with stage I-III CC and 10 healthy controls. Exosomes from the MV and PV of all patients and controls were isolated by ultracentrifugation and confirmed by cryogenic transmission electron microscopy. High-throughput proteomic analysis by mass spectrometry was used to identify expression levels of exosomal proteins. Findings were confirmed by Western blot. RESULTS: Exosomal ECM1 protein was more highly expressed in patients than in controls and was 13.55 times higher in MV from relapsed than relapse-free patients. High exosomal ECM1 expression was associated with liver metastases. Patients with high exosomal ECM1 expression in MV-but not PV-plasma had shorter time to relapse than those with low ECM1 expression (P = .04). CONCLUSION: High levels of exosomal ECM1 protein can identify CC patients with a higher risk of relapse. The analysis of exosomes isolated from the tumor-draining MV is a promising method for the identification of biomarkers before they reach the target organ.

Exosomal microRNAs isolated from plasma of mesenteric veins linked to liver metastases in resected patients with colon cancer.

Before reaching a peripheral vein (PV), miRNAs released by the tumor are diluted and dispersed throughout the body or even retained in a specific organ. We hypothesized that blood drawn from the tumor-draining vein could provide more homogeneous information than blood drawn from the PV as that blood would contain all the biomarkers released by the tumor before they reach a potential metastatic site. We have profiled 754 miRNAs in 15 colon cancer plasma samples from the tumor-draining vein, the mesenteric vein (MV), identifying 13 microRNAs associated with relapse. The prognostic impact of these miRNAs were validated in 50 MV and 50 paired PV plasma samples of stage I-III colon cancer patients. Four miRNAs, let-7g, miR-15b, miR-155 and miR-328, were found overexpressed in MV compared to PV, and patients with high levels of those miRNAs in MV plasma had shorter time to relapse. Interestingly, in patients developing liver metastases, the exosomal cargo of miR-328 was much greater in MV than in PV plasma indicating a possible role of miR-328 in the development of liver metastases. Our results indicate that in colon cancer, the primary tumor releases high concentrations of miRNAs through the MV, and some of them are contained in tumor derived exosomes.

LincRNA-p21 Impacts Prognosis in Resected Non-Small Cell Lung Cancer Patients through Angiogenesis Regulation.

INTRODUCTION: Long intergenic noncoding RNA-p21 (lincRNA-p21) is a long noncoding RNA transcriptionally activated by tumor protein p53 (TP53) and hypoxia inducible factor 1 alpha subunit (HIF1A). It is involved in the regulation of TP53-dependent apoptosis and the Warburg effect. We have investigated the role of lincRNA-p21 in NSCLC. METHODS: LincRNA-p21 expression was assessed in tumor and normal tissue from 128 patients with NSCLC and correlated with time to relapse and cancer-specific survival (CSS). H23, H1299, and HCC-44 cell lines were cultured in hypoxic conditions after silencing of lincRNA-p21. The TaqMan human angiogenesis array was used to explore angiogenesis-related gene expression. Levels of the protein vascular endothelial growth factor A were measured by enzyme-linked immunosorbent assay in the cell supernatants. Angiogenic capability was measured by human umbilical vein endothelial cell tube formation assay. Microvascular density in tumor samples was analyzed by immunohistochemistry. RESULTS: LincRNA-p21 was down-regulated in tumor tissue, but no association was observed with TP53 mutational status. High lincRNA-p21 levels were associated with poor CSS in all patients (p = 0.032). When patients were classified according to histological subtypes, the impact of lincRNA-p21 was confined to patients with adenocarcinoma in both time to relapse (p = 0.006) and CSS (p < 0.001). To explain the poor outcome of patients with high lincRNA-p21 expression, we studied the role of lincRNA-p21 in angiogenesis in vitro and observed a global downregulation in the expression of angiogenesis-related genes when lincRNA-p21 was inhibited. Moreover, supernatants from lincRNA-p21-inhibited cells were significantly less angiogenic and had lower levels of secreted vascular endothelial growth factor A than controls did. Finally, tumor samples with high lincRNA-p21 levels had higher microvascular density. CONCLUSIONS: Our findings suggest that lincRNA-p21 affects outcome in patients with NSCLC adenocarcinoma through the regulation of angiogenesis.