Uncovering the expression of circPVT1 in the extracellular vesicles of acute myeloid leukemia patients.

Extracellular vesicles (EVs) act as molecular mediators in the tumor microenvironment, by shuttling information contained within malignant cells and functioning as regulators of the immune system. Circular (circ)RNAs are characterized by a closed loop-like structure that makes them more stable in the extracellular milieu and suitable to be packaged inside EVs. circPVT1 (hsa_circ_0001821) showed an oncogenic role in several cancer types and immunosuppressive properties in myeloid and lymphoid cell subsets. In this study, we characterized EVs from acute myeloid leukemia (AML) patients in terms of size, concentrations, surface markers and circPVT1 cargo. We showed that circPVT1 is overexpressed by primary blast cells from newly-diagnosed AML patients compared with hematopoietic stem-progenitor cells and is released as cell-free RNA in the plasma. We isolated EVs from the plasma of AML patients and healthy subjects by size exclusion chromatography and characterized them by nanoparticle tracking analysis. EVs from patients' plasma are larger compared with those from healthy subjects and their surface profile is characterized by higher levels of the leukemic cell markers CD133, CD105, CD49e and other immune-related epitopes, with differences according to AML molecular profile. Moreover, digital PCR analysis revealed that circPVT1 is more abundant inside EVs from the plasma of AML patients compared with healthy subjects. Our findings provide new insights on the features and content of AML EVs and suggest a role of circPVT1 in the crosstalk between AML cells and the tumor microenvironment.

Fair Max-Min Diversity Maximization in Streaming and Sliding-Window Models.

Diversity maximization is a fundamental problem with broad applications in data summarization, web search, and recommender systems. Given a set X of n elements, the problem asks for a subset S of k≪n elements with maximum diversity, as quantified by the dissimilarities among the elements in S. In this paper, we study diversity maximization with fairness constraints in streaming and sliding-window models. Specifically, we focus on the max-min diversity maximization problem, which selects a subset S that maximizes the minimum distance (dissimilarity) between any pair of distinct elements within it. Assuming that the set X is partitioned into m disjoint groups by a specific sensitive attribute, e.g., sex or race, ensuring fairness requires that the selected subset S contains ki elements from each group i∈[m]. Although diversity maximization has been extensively studied, existing algorithms for fair max-min diversity maximization are inefficient for data streams. To address the problem, we first design efficient approximation algorithms for this problem in the (insert-only) streaming model, where data arrive one element at a time, and a solution should be computed based on the elements observed in one pass. Furthermore, we propose approximation algorithms for this problem in the sliding-window model, where only the latest w elements in the stream are considered for computation to capture the recency of the data. Experimental results on real-world and synthetic datasets show that our algorithms provide solutions of comparable quality to the state-of-the-art offline algorithms while running several orders of magnitude faster in the streaming and sliding-window settings.

Analysis of EVs from patients with advanced pancreatic cancer identifies antigens and miRNAs with predictive value.

The identification of predictive factors for treatment of pancreatic cancer (PC) is an unmet clinical need. In the present work, we analyzed blood-derived extracellular vesicles (EVs) from patients with advanced PC in order to find a molecular signature predictive of response to therapy. We analyzed samples from 21 patients with advanced PC, all receiving first-line treatment with gemcitabine + nab-paclitaxel. Isolated EVs have been analyzed, and the results of laboratory have been matched with clinical data in order to investigate possible predictive factors. EV concentration and size were similar between responder and non-responder patients. Analysis of 37 EV surface epitopes showed a decreased expression of SSEA4 and CD81 in responder patients. We detected more than 450 expressed miRNAs in EVs. A comparative survey between responder and non-responder patients showed that at least 44 miRNAs were differently expressed. Some of these miRNAs have already been observed in relation to the survival and gemcitabine sensitivity of tumor cells. In conclusion, we showed the ability of our approach to identify EV-derived biomarkers with predictive value for therapy response in PC. Our findings are worthy of further investigation, including the analysis of samples from patients treated with different schedules and in different settings.

Modulation of the Circulating Extracellular Vesicles in Response to Different Exercise Regimens and Study of Their Inflammatory Effects.

Exercise-released extracellular vesicles (EVs) are emerging as a novel class of exerkines that promotes systemic beneficial effects. However, slight differences in the applied exercise protocols in terms of mode, intensity and duration, as well as the need for standardized protocols for EV isolation, make the comparison of the studies in the literature extremely difficult. This work aims to investigate the EV amount and EV-associated miRNAs released in circulation in response to different physical exercise regimens. Healthy individuals were subjected to different exercise protocols: acute aerobic exercise (AAE) and training (AT), acute maximal aerobic exercise (AMAE) and altitude aerobic training (AAT). We found a tendency for total EVs to increase in the sedentary condition compared to trained participants following AAE. Moreover, the cytofluorimetric analysis showed an increase in CD81+/SGCA+/CD45- EVs in response to AAE. Although a single bout of moderate/maximal exercise did not impact the total EV number, EV-miRNA levels were affected as a result. In detail, EV-associated miR-206, miR-133b and miR-146a were upregulated following AAE, and this trend appeared intensity-dependent. Finally, THP-1 macrophage treatment with exercise-derived EVs induced an increase of the mRNAs encoding for IL-1ß, IL-6 and CD163 using baseline and immediately post-exercise EVs. Still, 1 h post-exercise EVs failed to stimulate a pro-inflammatory program. In conclusion, the reported data provide a better understanding of the release of circulating EVs and their role as mediators of the inflammatory processes associated with exercise.

The Cytotoxic Effect of Curcumin in Rhabdomyosarcoma Is Associated with the Modulation of AMPK, AKT/mTOR, STAT, and p53 Signaling.

Approximately 7% of cancers arising in children and 1% of those arising in adults are soft tissue sarcomas (STS). Of these malignancies, rhabdomyosarcoma (RMS) is the most common. RMS survival rates using current therapeutic protocols have remained largely unchanged in the past decade. Thus, it is imperative that the main molecular drivers in RMS tumorigenesis are defined so that more precise, effective, and less toxic therapies can be designed. Curcumin, a common herbal supplement derived from plants of the Curcuma longa species, has an exceptionally low dietary biotoxicity profile and has demonstrated anti-tumorigenic benefits in vitro. In this study, the anti-tumorigenic activity of curcumin was assessed in rhabdomyosarcoma cell lines and used to identify the major pathways responsible for curcumin's anti-tumorigenic effects. Curcumin treatment resulted in cell cycle arrest, inhibited cell migration and colony forming potential, and induced apoptotic cell death. Proteome profiler array analysis demonstrated that curcumin treatment primarily influenced flux through the AKT-mammalian target of rapamycin (mTOR), signal transducer and activator of transcription (STAT), AMP-dependent kinase (AMPK), and p53 associated pathways in a rhabdomyosarcoma subtype-specific manner. Thus, the strategic, combinational therapeutic targeting of these pathways may present the best option to treat this group of tumors.

From phenotypical investigation to RNA-sequencing for gene expression analysis: A workflow for single and pooled rare cells.

Combining phenotypical and molecular characterization of rare cells is challenging due to their scarcity and difficult handling. In oncology, circulating tumor cells (CTCs) are considered among the most important rare cell populations. Their phenotypic and molecular characterization is necessary to define the molecular mechanisms underlying their metastatic potential. Several approaches that require cell fixation make difficult downstream molecular investigations on RNA. Conversely, the DEPArray technology allows phenotypic analysis and handling of both fixed and unfixed cells, enabling a wider range of applications. Here, we describe an experimental workflow that allows the transcriptomic investigation of single and pooled OE33 cells undergone to DEPArray analysis and recovery. In addition, cells were tested at different conditions (unfixed, CellSearch fixative (CSF)- and ethanol (EtOH)-fixed cells). In a forward-looking perspective, this workflow will pave the way for novel strategies to characterize gene expression profiles of rare cells, both single-cell and low-resolution input.

Comparative Analysis of Free-Circulating and Vesicle-Associated Plasma microRNAs of Healthy Controls and Early-Stage Lung Cancer Patients.

In recent years, circulating extracellular miRNAs have emerged as a useful tool for the molecular characterization and study of tumors' biological functions. However, the high heterogeneity in sample processing, isolation of circulating fraction, RNA extraction, and sequencing hamper the reproducibility and the introduction of these biomarkers in clinical practice. In this paper, we compare the content and the performance of miRNA sequencing in plasma-derived samples processed with different isolation protocols. We tested three different fractions of miRNA from healthy-donor human blood: whole plasma (WP), free-circulating (FC) and EV-associated, isolated by either column (ccEV) or size exclusion chromatography (secEV) miRNAs. An additional cohort of 18 lung cancer patients was analyzed. Protein profiles of ccEV and secEV were compared and miRNA expression profiles were assessed through sequencing. Slight differences were found between ccEV and secEV expressions of typical EV markers. Conversely, sequencing performance and the mirnome profile varied between RNA extracted using different isolation methods. Sequencing performance was better in FC samples. Higher varieties of miRNAs were identified in WP and FC with respect to ccEV and secEV. Analysis of free-circulating and EV-associated miRNA profiles in lung cancer patients demonstrated the reliability of the biomarkers identifiable on plasma with these approaches.

Extra Virgin Olive Oil (EVOO), a Mediterranean Diet Component, in the Management of Muscle Mass and Function Preservation.

Aging results in a progressive decline in skeletal muscle mass, strength and function, a condition known as sarcopenia. This pathological condition is due to multifactorial processes including physical inactivity, inflammation, oxidative stress, hormonal changes, and nutritional intake. Physical therapy remains the standard approach to treat sarcopenia, although some interventions based on dietary supplementation are in clinical development. In this context, thanks to its known anti-inflammatory and antioxidative properties, there is great interest in using extra virgin olive oil (EVOO) supplementation to promote muscle mass and health in sarcopenic patients. To date, the molecular mechanisms responsible for the pathological changes associated with sarcopenia remain undefined; however, a complete understanding of the signaling pathways that regulate skeletal muscle protein synthesis and their behavior during sarcopenia appears vital for defining how EVOO might attenuate muscle wasting during aging. This review highlights the main molecular players that control skeletal muscle mass, with particular regard to sarcopenia, and discusses, based on the more recent findings, the potential of EVOO in delaying/preventing loss of muscle mass and function, with the aim of stimulating further research to assess dietary supplementation with EVOO as an approach to prevent or delay sarcopenia in aging individuals.

Dissecting Molecular Heterogeneity of Circulating Tumor Cells (CTCs) from Metastatic Breast Cancer Patients through Copy Number Aberration (CNA) and Single Nucleotide Variant (SNV) Single Cell Analysis.

Circulating tumor cells' (CTCs) heterogeneity contributes to counteract their introduction in clinical practice. Through single-cell sequencing we aim at exploring CTC heterogeneity in metastatic breast cancer (MBC) patients. Single CTCs were isolated using DEPArray NxT. After whole genome amplification, libraries were prepared for copy number aberration (CNA) and single nucleotide variant (SNV) analysis and sequenced using Ion GeneStudio S5 and Illumina MiSeq, respectively. CTCs demonstrate distinctive mutational signatures but retain molecular traces of their common origin. CNA profiling identifies frequent aberrations involving critical genes in pathogenesis: gains of 1q (CCND1) and 11q (WNT3A), loss of 22q (CHEK2). The longitudinal single-CTC analysis allows tracking of clonal selection and the emergence of resistance-associated aberrations, such as gain of a region in 12q (CDK4). A group composed of CTCs from different patients sharing common traits emerges. Further analyses identify losses of 15q and enrichment of terms associated with pseudopodium formation as frequent and exclusive events. CTCs from MBC patients are heterogeneous, especially concerning their mutational status. The single-cell analysis allows the identification of aberrations associated with resistance, and is a candidate tool to better address treatment strategy. The translational significance of the group populated by similar CTCs should be elucidated.

Growth Inhibition of Retinoblastoma Cell Line by Exosome-Mediated Transfer of miR-142-3p.

Introduction: Retinoblastoma (Rb) is the most common ocular paediatric malignancy and is caused by a mutation of the two alleles of the tumor suppressor gene, RB1. The tumor microenvironment (TME) represents a complex system whose function is not yet well defined and where microvesicles, such as exosomes, play a key role in intercellular communication. Micro-RNAs (mRNAs) have emerged as important modifiers of biological mechanisms involved in cancer and been able to regulate tumor progression. Methods: Co-culture of monocytes with retinoblastoma cell lines, showed a significant growth decrease. Given the interaction between Rb cells and monocytes, we investigated the role of the supernatant in the cross-talk between cell lines, by taking the product of the co-culture and then using it as a culture medium for Rb cells. Results: miR-142-3p showed to be particularly over-expressed both in the Rb cell line and in the medium used for their culture, comparing to control cell line and the normal supernatant, respectively. Therefore, we provided evidence that miR-142-3p is released by monocytes in the co-culture medium's exosomes and that it is subsequently up-taken by Rb cells, causing the inhibition of proliferation of Rb cell line by affecting cell cycle progression. Conclusion: This study highlights the role of exosomic miR-142-3p in the TME of Rb and identifies new molecular targets, which are able to control tumor growth aiming the development of a forward-looking miR-based strategy.

Cancer Stem Cells (CSCs), Circulating Tumor Cells (CTCs) and Their Interplay with Cancer Associated Fibroblasts (CAFs): A New World of Targets and Treatments.

The importance of defining new molecules to fight cancer is of significant interest to the scientific community. In particular, it has been shown that cancer stem cells (CSCs) are a small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity; on the other side, circulating tumor cells (CTCs) seem to split away from the primary tumor and appear in the circulatory system as singular units or clusters. It is becoming more and more important to discover new biomarkers related to these populations of cells in combination to define the network among them and the tumor microenvironment. In particular, cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment with different functions, including matrix deposition and remodeling, extensive reciprocal signaling interactions with cancer cells and crosstalk with immunity. The settings of new markers and the definition of the molecular connections may present new avenues, not only for fighting cancer but also for the definition of more tailored therapies.

Case Report: Analysis of Plasma Extracellular Vesicles in a Triple Negative Spindle-Cell Metaplastic Breast Cancer Patient.

Metaplastic breast cancer (MpBC) is a rare tumor representing 1% of all breast malignancies. The prognosis of this histologic subtype is actually poor and there are no current clear-cut therapeutic guidelines. Hence, despite its uniqueness, its aggressive prognostic profile strongly encourages further studies to identify new markers and therapeutic targets. Herein, we report a case of 32-years-old patient affected with of triple negative spindle-shaped MpBC. The research of molecular targets on the primary tumor did not allow performing an effective therapeutic choice. Extracellular Vesicles (EVs) are under intense study as new potential pathophysiological markers and targets for therapeutic applications, in different tumors for their role in tumor onset, progression and aggressiveness. Here, we examined the involvement of EVs in this case, to look into the MpBC microenvironment willing to identify new potential molecular targets, pathways of aggressiveness, and markers of prognosis and therapeutic efficacy. Firstly, we characterized MpBC patient EV dimensions and surface proteins. Moreover, we analyzed the EV RNA cargo supposed to be delivered to nearby and distant recipient cells. Interestingly, we observed a dysregulation EV-contained miRNAs, which could determine an increased expression of oncogenes in the tumor microenvironment, probably enabling cancer progression. These data suggest that the characterization of miRNA cargo of EVs could be important for the identification of new markers and for the application of future new target therapies.

Emerging Roles of Aldehyde Dehydrogenase Isoforms in Anti-cancer Therapy Resistance.

Aldehyde dehydrogenases (ALDHs) are a family of detoxifying enzymes often upregulated in cancer cells and associated with therapeutic resistance. In humans, the ALDH family comprises 19 isoenzymes active in the majority of mammalian tissues. Each ALDH isoform has a specific differential expression pattern and most of them have individual functional roles in cancer. ALDHs are overexpressed in subpopulations of cancer cells with stem-like features, where they are involved in several processes including cellular proliferation, differentiation, detoxification and survival, participating in lipids and amino acid metabolism and retinoic acid synthesis. In particular, ALDH enzymes protect cancer cells by metabolizing toxic aldehydes in less reactive and more soluble carboxylic acids. High metabolic activity as well as conventional anticancer therapies contribute to aldehyde accumulation, leading to DNA double strand breaks (DSB) through the generation of reactive oxygen species (ROS) and lipid peroxidation. ALDH overexpression is crucial not only for the survival of cancer stem cells but can also affect immune cells of the tumour microenvironment (TME). The reduction of ROS amount and the increase in retinoic acid signaling impairs immunogenic cell death (ICD) inducing the activation and stability of immunosuppressive regulatory T cells (Tregs). Dissecting the role of ALDH specific isoforms in the TME can open new scenarios in the cancer treatment. In this review, we summarize the current knowledge about the role of ALDH isoforms in solid tumors, in particular in association with therapy-resistance.

Appropriateness and Economic Analysis of Conventional Circulating Biomarkers Assessment in Early Breast Cancer: A Real-World Experience from the E.Pic.A Study.

The risk of relapse for early breast cancer (BC) patients persists even after decades and to date, no specific and sensitive effective circulating biomarker for recurrence prediction has been identified yet. The international guidelines do not recommend the assessment of the serum tumor markers CEA and CA15-3 in the follow-up of asymptomatic early BC patients. In our institute, IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori", as part of the E.Pic.A study, which was designed to assess the economic appropriateness of integrated care pathways in early BC, the use of CEA and CA15-3 as circulating tumor biomarkers in early BC patients was evaluated in 1502 patients one year after surgery, from 2015 to 2018, with an overall expense of EUR 51,764. A total of EUR 47,780 (92%) was used for execution of circulating tumor markers in early BC patients with stage 0, I and II tumors, neglecting the current guidelines and considered inappropriate by our professional board. We found that no patients with stage I BC experienced relapse in the 365 days after surgery, and in any case examination of the circulating markers CEA and CA15-3 was considered crucial for diagnosis of relapse. Our findings suggest that this inadequacy is a low-value area, supporting the reallocation of economic resources for interventions of a higher value for patients.

Overexpression of ultraconserved region 83- induces lung cancer tumorigenesis.

The expression of non-coding RNAs (ncRNAs) is dysregulated in human cancers. The transcribed ultraconserved regions (T-UCRs) express long ncRNAs involved in human carcinogenesis. T-UCRs are non-coding genomic sequence that are 100% conserved across humans, rats and mice. Conservation of genomic sequences across species intrinsically implies an essential functional role and so we considered the expression of T-UCRs in lung cancer. Using a custom microarray we analyzed the global expression of T-UCRs. Among these T-UCRs, the greatest variation was observed for antisense ultraconserved element 83 (uc.83-), which was upregulated in human lung cancer tissues compared with adjacent non cancerous tissues. Even though uc.83- is located within the long intergenic non-protein coding RNA 1876 (LINC01876) gene, we found that the transcribed uc.83- is expressed independently of LINC01876 and was cloned as a 1143-bp RNA gene. In this study, functional analysis confirmed important effects of uc.83- on genes involved in cell growth of human cells. siRNA against uc.83- decreased the growth of lung cancer cells while the upregulation through a vector overexpressing the uc.83- RNA increased cell proliferation. We also show the oncogenic function of uc.83- is mediated by the phosphorylation of AKT and ERK 1/2, two important biomarkers of lung cancer cell proliferation. Based on our findings, inhibition against uc.83- could be a future therapeutic treatment for NSCLC to achieve simultaneous blockade of pathways involved in lung carcinogenesis.

Lineage-specific mechanisms and drivers of breast cancer chemoresistance revealed by 3D biomimetic culture.

To improve the success rate of current preclinical drug trials, there is a growing need for more complex and relevant models that can help predict clinical resistance to anticancer agents. Here, we present a three-dimensional (3D) technology, based on biomimetic collagen scaffolds, that enables the modeling of the tumor hypoxic state and the prediction of in vivo chemotherapy responses in terms of efficacy, molecular alterations, and emergence of resistance mechanisms. The human breast cancer cell lines MDA-MB-231 (triple negative) and MCF-7 (luminal A) were treated with scaling doses of doxorubicin in monolayer cultures, 3D collagen scaffolds, or orthotopically transplanted murine models. Lineage-specific resistance mechanisms were revealed by the 3D tumor model. Reduced drug uptake, increased drug efflux, and drug lysosomal confinement were observed in triple-negative MDA-MB-231 cells. In luminal A MCF-7 cells, the selection of a drug-resistant subline from parental cells with deregulation of p53 pathways occurred. These cells were demonstrated to be insensitive to DNA damage. Transcriptome analysis was carried out to identify differentially expressed genes (DEGs) in treated cells. DEG evaluation in breast cancer patients demonstrated their potential role as predictive biomarkers. High expression of the transporter associated with antigen processing 1 (TAP1) and the tumor protein p53-inducible protein 3 (TP53I3) was associated with shorter relapse in patients affected by ER+ breast tumor. Likewise, the same clinical outcome was associated with high expression of the lysosomal-associated membrane protein 1 LAMP1 in triple-negative breast cancer. Hypoxia inhibition by resveratrol treatment was found to partially re-sensitize cells to doxorubicin treatment. Our model might improve preclinical in vitro analysis for the translation of anticancer compounds as it provides: (a) more accurate data on drug efficacy and (b) enhanced understanding of resistance mechanisms and molecular drivers.

An Abnormal Host/Microbiomes Signature of Plasma-Derived Extracellular Vesicles Is Associated to Polycythemia Vera.

Polycythemia Vera (PV) is a myeloproliferative neoplasm with increased risk of thrombosis and progression to myelofibrosis. Chronic inflammation is commonly observed in myeloproliferative neoplasms including PV. The inflammatory network includes the extracellular vesicles (EVs), which play a role in cell-cell communication. Recent evidence points to circulating microbial components/microbes as potential players in hemopoiesis regulation. To address the role of EVs in PV, here we investigated phenotype and microbial DNA cargo of circulating EVs through multidimensional analysis. Peripheral blood and feces were collected from PV patients (n=38) and healthy donors (n=30). Circulating megakaryocyte (MK)- and platelet (PLT)-derived EVs were analyzed by flow cytometry. After microbial DNA extraction from feces and isolated EVs, the 16S rDNA V3-V4 region was sequenced. We found that the proportion of circulating MK-derived EVs was significantly decreased in PV patients as compared with the healthy donors. By contrast, the proportion of the PLT-derived EVs was increased. Interestingly, PV was also associated with a microbial DNA signature of the isolated EVs with higher diversity and distinct microbial composition than the healthy counterparts. Of note, increased proportion of isolated lipopolysaccharide-associated EVs has been demonstrated in PV patients. Conversely, the gut microbiome profile failed to identify a distinct layout between PV patients and healthy donors. In conclusion, PV is associated with circulating EVs harbouring abnormal phenotype and dysbiosis signature with a potential role in the (inflammatory) pathogenesis of the disease.

MicroRNA-16 Restores Sensitivity to Tyrosine Kinase Inhibitors and Outperforms MEK Inhibitors in KRAS-Mutated Non-Small Cell Lung Cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Chemotherapy, the treatment of choice in non-operable cases, achieves a dismal success rate, raising the need for new therapeutic options. In about 25% of NSCLC, the activating mutations of the KRAS oncogene define a subclass that cannot benefit from tyrosine kinase inhibitors (TKIs). The tumor suppressor miR-16 is downregulated in many human cancers, including NSCLC. The main objectives of this study were to evaluate miR-16 treatment to restore the TKI sensitivity and compare its efficacy to MEK inhibitors in KRAS-mutated NSCLC. METHODS: We performed in vitro and in vivo studies to investigate whether miR-16 could be exploited to overcome TKI resistance in KRAS-mutated NSCLC. We had three goals: first, to identify the KRAS downstream effectors targeted by mir-16, second, to study the effects of miR-16 restoration on TKI resistance in KRAS-mutated NSCLC both in vitro and in vivo, and finally, to compare miR-16 and the MEK inhibitor selumetinib in reducing KRAS-mutated NSCLC growth in vitro and in vivo. RESULTS: We demonstrated that miR-16 directly targets the three KRAS downstream effectors MAPK3, MAP2K1, and CRAF in NSCLC, restoring the sensitivity to erlotinib in KRAS-mutated NSCLC both in vitro and in vivo. We also provided evidence that the miR-16-erlotinib regimen is more effective than the selumetinib-erlotinib combination in KRAS-mutated NSCLC. CONCLUSIONS: Our findings support the biological preclinical rationale for using miR-16 in combination with erlotinib in the treatment of NSCLC with KRAS-activating mutations.

CNA Profiling of Single CTCs in Locally Advanced Esophageal Cancer Patients during Therapy Highlights Unexplored Molecular Pathways.

BACKGROUND: Here, we monitored the evolution of CTCs spread in 11 patients affected by locally advanced EC who were undergoing therapy. METHODS: In this perspective study, we designed multiple blood biopsies from individual patients: before and after neoadjuvant chemo-radio therapy and after surgery. We developed a multi-target array, named Grab-all assay, to estimate CTCs for their epithelial (EpCAM/E-Cadherin/Cytokeratins) and mesenchymal/stem (N-Cadherin/CD44v6/ABCG2) phenotypes. Identified CTCs were isolated as single cells by DEPArray, subjected to whole genome amplification, and copy number aberration (CNA) profiles were determined. Through bioinformatic analysis, we assessed the genomic imbalance of single CTCs, investigated specific focal copy number changes previously reported in EC and aberrant pathways using enrichment analysis. RESULTS: Longitudinal monitoring allowed the identification of CTCs in at least one time-point per patient. Through single cell CNA analysis, we revealed that CTCs showed significantly dynamic genomic imbalance during treatment. Individual CTCs from relapsed patients displayed a higher degree of genomic imbalance relative to disease-free patients' groups. Genomic aberrations previously reported in EC occurred mostly in post-neoadjuvant therapy CTCs. In-depth analysis showed that networks enrichment in all time-point CTCs were inherent to innate immune system. Transcription/gene regulation, post-transcriptional and epigenetic modifications were uniquely affected in CTCs of relapsed patients. CONCLUSIONS: Our data add clues to the comprehension of the role of CTCs in EC aggressiveness: chromosomal aberrations on genes related to innate immune system behave as relevant to the onset of CTC-status, whilst pathways of transcription/gene regulation, post-transcriptional and epigenetic modifications seem linked to patients' outcome.