Characterization of the MicroRNA Cargo of Extracellular Vesicles Isolated from a Pulmonary Tumor-Draining Vein Identifies miR-203a-3p as a Relapse Biomarker for Resected Non-Small Cell Lung Cancer.

In resected non-small cell lung cancer (NSCLC), post-surgical recurrence occurs in around 40% of patients, highlighting the necessity to identify relapse biomarkers. An analysis of the extracellular vesicle (EV) cargo from a pulmonary tumor-draining vein (TDV) can grant biomarker identification. We studied the pulmonary TDV EV-miRNAome to identify relapse biomarkers in a two-phase study (screening and validation). In the screening phase, a 17-miRNA relapse signature was identified in 18 selected patients by small RNAseq. The most expressed miRNA from the signature (EV-miR-203a-3p) was chosen for further validation. Pulmonary TDV EV-miR-203a-3p was studied by qRT-PCR in a validation cohort of 70 patients, where it was found to be upregulated in relapsed patients (p = 0.0194) and in patients with cancer spread to nearby lymph nodes (N+ patients) (p = 0.0396). The ROC curve analysis showed that TDV EV-miR-203a-3p was able to predict relapses with a sensitivity of 88% (AUC: 0.67; p = 0.022). Moreover, patients with high TDV EV-miR-203a-3p had a shorter time to relapse than patients with low levels (43.6 vs. 97.6 months; p = 0.00703). The multivariate analysis showed that EV-miR-203a-3p was an independent, predictive and prognostic post-surgical relapse biomarker. In conclusion, pulmonary TDV EV-miR-203a-3p is a promising new relapse biomarker for resected NSCLC patients.

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.

Endovascular image-guided sampling of tumor-draining veins provides an enriched source of oncological biomarkers.

Introduction: Circulating tumor-derived biomarkers can potentially impact cancer management throughout the continuum of care. This small exploratory study aimed to assess the relative levels of such biomarkers in the tumor-draining vascular beds in patients with solid tumors compared to levels in their peripheral veins. Methods: Using an endovascular image-guided approach, we obtained blood samples from peripheral veins and other vascular compartments-including the most proximal venous drainage from solid tumors-from a set of nine oncology patients with various primary and metastatic malignancies. We then interrogated these samples for a panel of oncological biomarkers, including circulating tumor cells (CTCs), exosome-derived microRNAs (miRNAs), circulating tumor DNA (ctDNA) mutations, and certain cancer-related proteins/biochemical markers. Results: We found substantially higher levels of CTCs, certain miRNAs, and specific ctDNA mutations in samples from vascular beds closer to the tumor compared with those from peripheral veins and also noted that some of these signals were altered by treatment procedures. Discussion: Our results indicate that tumor-proximal venous samples are highly enriched for some oncological biomarkers and may allow for more robust molecular analysis than peripheral vein samples.

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.

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.

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.