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.

In resected non-small cell lung cancer (NSCLC), postsurgical recurrence is the major factor affecting long-term survival. The identification of biomarkers in extracellular vesicles (EV) obtained from serial blood samples after surgery could enhance early detection of relapse and improve NSCLC outcome. Since EV cargo contains long non-coding RNAs (lncRNAs), we aimed to analyze whether the oncogenic lncRNA HOTTIP, which higher expression in tumor tissue was related to worse outcome in NSCLC, could be detected in EV from NSCLC patients and serve as recurrence biomarker. After purification of EVs by ultracentrifugation in 52 serial samples from 18 NSCLC patients, RNA was isolated and HOTTIP was quantified by Real time PCR. We observed that patients that relapsed after surgery displayed increased postsurgical EV HOTTIP levels in comparison with presurgical levels. In the relapsed patients with several samples available between surgery and relapse, we observed an increment in the EV HOTTIP levels when approaching to relapse, which indicated its potential utility for monitoring disease recurrence. When we focused in EV HOTTIP levels in the first post-surgical sample, we observed that the detection of an increment of the expression levels in comparison to presurgical sample, predicted recurrence with high sensitivity (85.7%) and specificity (90.9%) and that patients had shorter time to relapse and shorter overall survival. In conclusion, our pilot study showed that EV HOTTIP is a potential biomarker for monitoring disease recurrence after surgery in NSCLC.

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.

Background: When genes responsible for normal embryonic development are abnormally expressed in adults, it can lead to tumor development. This can suggest that the same mechanism that controls embryonic differentiation can also control tumor differentiation. We hypothesize that the malignant phenotype of lung cancer cells could acquire benign characteristics when in contact with an embryonic lung microenvironment. We cultured two lung cancer cell lines in embryonic lung mesenchyme-conditioned medium and evaluated morphological, functional and molecular changes. Methods: The human embryonic mesenchymal lung-conditioned medium (hEML-CM) was obtained by culturing lung cells from embryos in the pseudoglandular stage of development. The NSCLC cell lines A549 and H1299 we cultured in the hEML-CM and in a tumor-conditioned medium. Morphological changes were analyzed with optical and transmission electron microscopy. To evaluate the functional effect of conditioned medium in tumor cells, we analyzed cell proliferation, migration, colony formation capacity in 2D and 3D and in vivo tumor growth capacity. The expression of the pluripotency genes OSKM, the adenocarcinoma marker NKX2-1, the lung surfactant proteins SFTP, the myofibroblast marker MYH and DNMT3A/3B was analyzed with qRT-PCR and the presence of the myofibroblast markers vimentin and α-SMA with immunofluorescence. Transcriptomic analysis was performed using Affymetrix arrays. Results: The A549 and H1299 cells cultured in hEML-CM lost their epithelial morphology, acquired mesodermal characteristics, and decreased proliferation, migration, and colony formation capacity in 2D and 3D, as well as reduced its capacity to growth in vivo. The expression of OSKM, NKX2-1 and SFTP decreased, while that of DNMT3A/3B, vimentin, α-SMA and MYH increased. Distant matrix analysis based on transcriptomic profile showed that conditioned cells were closer to myoblast and human lung fibroblast than to normal epithelial immortalized lung cells. A total of 1631 for A549 and 866 for H1299 differentially expressed genes between control and conditioned cells were identified. Conclusions: To the best of our knowledge, this is the first study to report that stimuli from the embryonic lung can modulate the malignant phenotype of lung cancer cells, control their growth capacity and activate their differentiation into myofibroblasts. These findings could lead to new strategies for lung cancer management.

Preoperative chemoradiotherapy (CRT) is a standard treatment for locally advanced rectal cancer (RC) patients, but its use in non-responders can be associated with increased toxicities and resection delay. LincRNA-p21 is a long non-coding RNA involved in the p53 pathway and angiogenesis regulation. We aimed to study whether lincRNA-p21 expression levels can act as a predictive biomarker for neoadjuvant CRT response. We analyzed RNAs from pretreatment biopsies from 70 RC patients treated with preoperative CRT. Pathological response was classified according to the tumor regression grade (TRG) Dworak classification. LincRNA-p21 expression was determined by RTqPCR. The results showed that lincRNA-p21 was upregulated in stage III tumors (p = 0.007) and in tumors with the worst response regarding TRG (p = 0.027) and downstaging (p = 0.016). ROC curve analysis showed that lincRNA-p21 expression had the capacity to distinguish a complete response from others (AUC:0.696; p = 0.014). LincRNA-p21 was shown as an independent marker of preoperative CRT response (p = 0.047) and for time to relapse (TTR) (p = 0.048). In conclusion, lincRNA-p21 is a marker of advanced disease, worse response to neoadjuvant CRT, and shorter TTR in locally advanced RC patients. The study of lincRNA-p21 may be of value in the individualization of pre-operative CRT in RC.

Purpose: TIGIT blockade in our ex vivo model of bone marrow (BM) reduced the number of malignant plasma cells (PC) in only half of patients with multiple myeloma. Here, we wanted to investigate whether increased expression of TIGIT ligands may inhibit T-cell immune response promoting resistance to TIGIT blockade. Experimental Design: We first characterized the number and phenotype of BM macrophages in different stages of the disease by multiparameter flow cytometry. We assessed the effect of TIGIT ligands on PC survival by performing experiments in the ex vivo BM model and analyzed changes in gene expression by using NanoString technology and real-time PCR. Results: The frequency of BM macrophages was significantly decreased in multiple myeloma, which was accompanied by changes in their immunophenotype. Moreover, we found a higher number of malignant PC in ex vivo BM cells cultured onto the poliovirus receptor (PVR) and nectin-2 compared with control, suggesting that both ligands may support PC survival. In addition, the presence of PVR, but not nectin-2, overcame the therapeutic effect of TIGIT blockade or exogenous IL2. Furthermore, exogenous IL2 increased TIGIT expression on both CD4+ and CD8+ T cells and, indirectly, PVR on BM macrophages. Consistently, PVR reduced the number of cytotoxic T cells and promoted a gene signature with reduced effector molecules. Conclusions: IL2 induced TIGIT on T cells in the BM, in which increased PVR expression resulted in cytotoxic T-cell inhibition, promoting PC survival and resistance to TIGIT blockade.