Circulating Extracellular Vesicles in Gynecological Tumors: Realities and Challenges

Although liquid biopsy can be considered a reality for the clinical management of some cancers, such as lung or colorectal cancer, it remains a promising field in gynecological tumors. In particular, circulating extracellular vesicles (cEVs) secreted by tumor cells represent a scarcely explored type of liquid biopsy in gynecological tumors. Importantly, these vesicles are responsible for key steps in tumor development and dissemination and are recognized as major players in cell-to-cell communication between the tumor and the microenvironment. However, limited work has been reported about the biologic effects and clinical value of EVs in gynecological tumors. Therefore, here we review the promising but already relatively limited data on the role of circulating EVs in promoting gynecological tumor spread and also their value as non-invasive biomarkers to improve the management of these type of tumors

EGFR-Based Immunoisolation as a Recovery Target for Low-EpCAM CTC Subpopulation

Circulating tumour cells (CTCs) play a key role in the metastasis process, as they are responsible for micrometastasis and are a valuable tool for monitoring patients in real-time. Moreover, efforts to develop new strategies for CTCs isolation and characterisation, and the translation of CTCs into clinical practice needs to overcome the limitation associated with the sole use of Epithelial Cell Adhesion Molecule (EpCAM) expression to purify this tumour cell subpopulation. CTCs are rare events in the blood of patients and are believed to represent the epithelial population from a primary tumour of epithelial origin, thus EpCAM immunoisolation is considered an appropriate strategy. The controversy stems from the impact that the more aggressive mesenchymal tumour phenotypes might have on the whole CTC population. In this work, we first characterised a panel of cell lines representative of tumour heterogeneity, confirming the existence of tumour cell subpopulations with restricted epithelial features and supporting the limitations of EpCAM-based technologies. We next developed customised polystyrene magnetic beads coated with antibodies to efficiently isolate the phenotypically different subpopulations of CTCs from the peripheral blood mononuclear cells (PBMCs) of patients with metastatic cancer. Besides EpCAM, we propose Epidermal Growth Factor Receptor (EGFR) as an additional isolation marker for efficient CTCs detection.This work was supported by Axencia Galega de Innovación (Xunta de Galicia) and InveNNta (Innovation in Nanomedicine), cofinanced by the European Union (EU) through the Operational Programme for Cross-border Cooperation: Spain-Portugal (POCTEP 2007-2013), and European Regional Development Fund (ERDF)S

Endometrial carcinoma: molecular alterations involved in tumor development and progression

In the western world, endometrial carcinoma (EC) is the most common cancer of the female genital tract. The annual incidence has been estimated at 10-20 per 100 000 women. Two clinicopathological variants are recognized: the estrogen related (type I, endometrioid) and the non-estrogen related (type II, non-endometrioid).The clinicopathological differences are paralleled by specific genetic alterations, with type I showing microsatellite instability and mutations in phosphatase and tensin homologue deleted on chromosome 70, PIK3CA, K-RAS and CTNNB1 (beta-catenin), and type II exhibiting TP53 mutations and chromosomal instability. Some non-endometrioid carcinomas probably arise from pre-existing endometrioid carcinomas as a result of tumor progression and, not surprisingly, some tumors exhibit combined or mixed features at the clinical, pathological and molecular levels. In EC, apoptosis resistance may have a role in tumor progression. Understanding pathogenesis at the molecular level is essential in identifying biomarkers for successful targeted therapies. In this review, the genetic changes of endometrial carcinogenesis are discussed in the light of the morphological features of the tumors and their precursors

ETV5 transcription program links BDNF and promotion of EMT at invasive front of endometrial carcinomas

Myometrial infiltration represents a main clinical determinant of endometrial carcinomas (EC) presenting as aggressive high-grade deeply invasive neoplasms, substantially associated with risk of recurrence and death. The up-regulation of ETV5 transcription factor linked to the promotion of epithelial to mesenchymal transition is considered as a basic mechanism underlying the initial steps of EC invasion. In this work, we aimed to investigate the transcription program of tumor invasion regulated by ETV5. We performed a comparative Chip-on-chip analysis at invasive front and superficial area of human EC. ETV5 specific binding to promoter regions of genes related to cellular migration, adhesion and invasion at deep invasion tumor areas highlighted the relevance of neural networks associated with cellular plasticity. Interestingly, brain-derived neurotrophic factor (BDNF) demonstrated a principal role orchestrating ETV5-mediated epithelial-to-mesenchymal transition in endometrial cancer. Impairment of the BDNF/tropomyosin-related kinase B (TrkB)/extracellular signal-regulated kinase axis in endometrial cancer cell lines reversed the aggressive and invasive phenotype promoted by the up-regulation of ETV5 at the invasive front of EC. Likewise, BDNF directly impacted on the efficiency of ETV5 promoted metastasis in a mice model of endometrial distant dissemination. These results translate the recognized role of BDNF/TrkB on neural plasticity into a relevant cancer metastasis event; suggest common mechanisms shared by neural development and tumor invasion; and offer new therapeutic opportunities specifically directed against disseminated disease in endometrial cancer

Global Gene Expression Characterization of Circulating Tumor Cells in Metastasic Castration-Resistant Prostate Cancer Patients

BACKGROUND: Current therapeutic options in the course of metastatic castration-resistant prostate cancers (mCRPC) reinforce the need for reliable tools to characterize the tumor in a dynamic way. Circulating tumor cells (CTCs) have emerged as a viable solution to the problem, whereby patients with a variety of solid tumors, including PC, often do not have recent tumor tissue available for analysis. The biomarker characterization in CTCs could provide insights into the current state of the disease and an overall picture of the intra-tumor heterogeneity. METHODS: in the present study, we applied a global gene expression characterization of the CTC population from mCRPC (n = 9), with the goal to better understand the biology of these cells and identify the relevant molecules favoring this tumor progression. RESULTS: This analysis allowed the identification of 50 genes specifically expressed in CTCs from patients. Six of these markers (HOXB13, QKI, MAOA, MOSPD1, SDK1, and FGD4), were validated in a cohort of 28 mCRPC, showing clinical interest for the management of these patients. Of note, the activity of this CTC signature was related to the regulation of MYC, a gene strongly implicated in the biology of mCRPC. CONCLUSIONS: Overall, our results represent new evidence on the great value of CTCs as a non-invasive biopsy to characterize PC

Selective interaction of PEGylated polyglutamic acid nanocapsules with cancer cells in a 3D model of a metastatic lymph node

Background Metastases are the most common reason of cancer death in patients with solid tumors. Lymph nodes, once invaded by tumor cells, act as reservoirs before cancer cells spread to distant organs. To address the limited access of intravenously infused chemotherapeutics to the lymph nodes, we have developed PEGylated polyglutamic acid nanocapsules (PGA-PEG NCs), which have shown ability to reach and to accumulate in the lymphatic nodes and could therefore act as nanotransporters. Once in the lymphatics, the idea is that these nanocapsules would selectively interact with cancer cells, while avoiding non-specific interactions with immune cells and the appearance of subsequent immunotoxicity. Results The potential of the PGA-PEG NCs, with a mean size of 100 nm and a negative zeta potential, to selectively reach metastatic cancer cells, has been explored in a novel 3D model that mimics an infiltrated lymph node. Our 3D model, a co-culture of cancer cells and lymphocytes, allows performing experiments under dynamic conditions that simulate the lymphatic flow. After perfusion of the nanocarriers, we observe a selective interaction with the tumor cells. Efficacy studies manifest the need to develop specific therapies addressed to treat metastatic cells that can be in a dormant state. Conclusions We provide evidence of the ability of PGA-PEG NCs to selectively interact with the tumor cells in presence of lymphocytes, highlighting their potential in cancer therapeutics. We also state the importance of designing precise in vitro models that allow performing mechanistic assays, to efficiently develop and evaluate specific therapies to confront the formation of metastasisThe authors acknowledge financial support given by the Carlos III Health Institute (ISCIII) and European Regional Development Fund (FEDER) (CP12/03150, PIE13/00024 and PI15/00828), ERA-NET EuroNanoMed 2009 (Lymphotarg PI09/2670) and EuroNanoMed 2013 (053 NICHE). The first author also acknowledges a fellowship received from the Fundación Ramón Domínguez, Spain. Abellan-Pose also acknowledges a fellowship from the Biomedical Sciences and Health Technologies Doctoral School-University of Santiago de Compostela (Spain)S

Global Gene Expression Characterization of Circulating Tumor Cells in Metastasic Castration-Resistant Prostate Cancer Patients

Background: Current therapeutic options in the course of metastatic castration-resistant prostate cancers (mCRPC) reinforce the need for reliable tools to characterize the tumor in a dynamic way. Circulating tumor cells (CTCs) have emerged as a viable solution to the problem, whereby patients with a variety of solid tumors, including PC, often do not have recent tumor tissue available for analysis. The biomarker characterization in CTCs could provide insights into the current state of the disease and an overall picture of the intra-tumor heterogeneity. Methods: in the present study, we applied a global gene expression characterization of the CTC population from mCRPC (n = 9), with the goal to better understand the biology of these cells and identify the relevant molecules favoring this tumor progression. Results: This analysis allowed the identification of 50 genes specifically expressed in CTCs from patients. Six of these markers (HOXB13, QKI, MAOA, MOSPD1, SDK1, and FGD4), were validated in a cohort of 28 mCRPC, showing clinical interest for the management of these patients. Of note, the activity of this CTC signature was related to the regulation of MYC, a gene strongly implicated in the biology of mCRPC. Conclusions: Overall, our results represent new evidence on the great value of CTCs as a non-invasive biopsy to characterize PCThis work was partially financed with the “liquid Biopsy crowdfunding, 2017”. L.M-L. is supported by AECCS

Improving zebrafish embryo xenotransplantation conditions by increasing incubation temperature and establishing a proliferation index with ZFtool

Background Zebrafish (Danio rerio) is a model organism that has emerged as a tool for cancer research, cancer being the second most common cause of death after cardiovascular disease for humans in the developed world. Zebrafish is a useful model for xenotransplantation of human cancer cells and toxicity studies of different chemotherapeutic compounds in vivo. Compared to the murine model, the zebrafish model is faster, can be screened using high-throughput methods and has a lower maintenance cost, making it possible and affordable to create personalized therapies. While several methods for cell proliferation determination based on image acquisition and quantification have been developed, some drawbacks still remain. In the xenotransplantation technique, quantification of cellular proliferation in vivo is critical to standardize the process for future preclinical applications of the model. Methods This study improved the conditions of the xenotransplantation technique – quantification of cellular proliferation in vivo was performed through image processing with our ZFtool software and optimization of temperature in order to standardize the process for a future preclinical applications. ZFtool was developed to establish a base threshold that eliminates embryo auto-fluorescence and measures the area of marked cells (GFP) and the intensity of those cells to define a ‘proliferation index’. Results The analysis of tumor cell proliferation at different temperatures (34 °C and 36 °C) in comparison to in vitro cell proliferation provides of a better proliferation rate, achieved as expected at 36°, a maintenance temperature not demonstrated up to now. The mortality of the embryos remained between 5% and 15%. 5- Fluorouracil was tested for 2 days, dissolved in the incubation medium, in order to quantify the reduction of the tumor mass injected. In almost all of the embryos incubated at 36 °C and incubated with 5-Fluorouracil, there was a significant tumor cell reduction compared with the control group. This was not the case at 34 °C. Conclusions Our results demonstrate that the proliferation of the injected cells is better at 36 °C and that this temperature is the most suitable for testing chemotherapeutic drugs like the 5-FluorouracilThis research was funded by the Fondo de Investigación Sanitaria (Instituto Carlos III) - FIS project (PI13/01388). The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing of this manuscriptS

Molecular Profiling of Circulating Tumour Cells Identifies Notch1 as a Principal Regulator in Advanced Non-Small Cell Lung Cancer

Knowledge on the molecular mechanisms underlying metastasis colonization in Non-Small Cell Lung Cancer (NSCLC) remains incomplete. A complete overview integrating driver mutations, primary tumour heterogeneity and overt metastasis lacks the dynamic contribution of disseminating metastatic cells due to the inaccessibility to the molecular profiling of Circulating Tumour Cells (CTCs). By combining immunoisolation and whole genome amplification, we performed a global gene expression analysis of EpCAM positive CTCs from advanced NSCLC patients. We identified an EpCAM+ CTC-specific expression profile in NSCLC patients mostly associated with cellular movement, cell adhesion and cell-to-cell signalling mediated by PI3K/AKT, ERK1/2 and NF-kB pathways. NOTCH1 emerged as a driver connecting active signalling pathways, with a reduced number of related candidate genes (NOTCH1, PTP4A3, LGALS3 and ITGB3) being further validated by RT-qPCR on an independent cohort of NSCLC patients. In addition, these markers demonstrated high prognostic value for Progression-Free Survival (PFS). In conclusion, molecular characterization of EpCAM+ CTCs from advanced NSCLC patients provided with highly specific biomarkers with potential applicability as a “liquid biopsy” for monitoring of NSCLC patients and confirmed NOTCH1 as a potential therapeutic target to block lung cancer dissemination.This work was funded by InveNNta (Innovation in Nanomedicine); Operational Programme for Cross-border Cooperation: Spain-Portugal (POCTEP) and European Regional Development Fund (ERDF). Javier Mariscal is recipient of a fellowship from Escola de Doutoramento Internacional Campus Vida of the University of Santiago de Compostela. Laura Muinelo-Romay is supported by ISCIII as Responsible of the Liquid Biopsy Analysis UnitS

Extracellular vesicles-based biomarkers represent a promising liquid biopsy in endometrial cancer

Tumor-derived extracellular vesicles (EVs) are secreted in large amounts into biological fluids of cancer patients. The analysis of EVs cargoes has been associated with patient´s outcome and response to therapy. However, current technologies for EVs isolation are tedious and low cost-e cient for routine clinical implementation. To explore the clinical value of circulating EVs analysis we attempted a proof-of-concept in endometrial cancer (EC) with ExoGAG, an easy to use and highly e cient new technology to enrich EVs. Technical performance was first evaluated using EVs secreted by Hec1A cells. Then, the clinical value of this strategy was questioned by analyzing the levels of two well-known tissue biomarkers in EC, L1 cell adhesion molecule (L1CAM) and Annexin A2 (ANXA2), in EVs purified from plasma in a cohort of 41 EC patients and 20 healthy controls. The results demonstrated the specific content of ANXA2 in the purified EVs fraction, with an accurate sensitivity and specificity for EC diagnosis. Importantly, high ANXA2 levels in circulating EVs were associated with high risk of recurrence and non-endometrioid histology suggesting a potential value as a prognostic biomarker in EC. These results also confirmed ExoGAG technology as a robust technique for the clinical implementation of circulating EVs analysesThis research was funded by Instituto de Salud Carlos III, grant PI17/01919, co-financed by the European Regional Development Fund (FEDER), and by Fundación Científica de la Asociación Española Contra el Cáncer (AECC), Grupos Clínicos Coordinados 2018. Carolina Herrero is supported by a predoctoral i-PFIS fellowship from Instituto de Salud Carlos III (IFI17/00047); Laura Muinelo is supported by Asociación Española Contra el Cáncer (AECC)