Rejuvenating effects of young extracellular vesicles in aged rats and in cellular models of human senescence.

Rejuvenation of an old organism was achieved in heterochronic parabiosis experiments, implicating different soluble factors in this effect. Extracellular vesicles (EVs) are the secretory effectors of many cells, including cardiosphere-derived cells (CDCs) with demonstrated anti-senescent effect. 1. To determine the role of EVs (versus other blood fractions) on the rejuvenating effect of the young blood. 2. To evaluate the anti-aging properties of therapeutically administered EVs secreted by young-CDCs in an old organism. Neonatal blood fractioned in 4 components (whole blood, serum, EV-depleted serum and purified EVs) was used to treat old human cardiac stromal cells (CSPCs). CDCs were generated from neonatal rat hearts and the secreted CDC-EVs were purified. CDC-EVs were then tested in naturally-aged rats, using monthly injections over 4-months period. For validation in human samples, pediatric CDC-EVs were tested in aged human CSPCs and progeric fibroblasts. While the purified EVs reproduced the rejuvenating effects of the whole blood, CSPCs treated with EV-depleted serum exhibited the highest degree of senescence. Treatment with young CDC-EVs induce structural and functional improvements in the heart, lungs, skeletal muscle, and kidneys of old rats, while favorably modulating glucose metabolism and anti-senescence pathways. Lifespan was prolonged. EVs secreted by young CDCs exert broad-ranging anti-aging effects in aged rodents and in cellular models of human senescence. Our work not only identifies CDC-EVs as possible therapeutic candidates for a wide range of age-related pathologies, but also raises the question of whether EVs function as endogenous modulators of senescence.

miR-1227 Targets SEC23A to Regulate the Shedding of Large Extracellular Vesicles.

Cancer cells shed a heterogenous mixture of extracellular vesicles (EVs), differing in both size and composition, which likely influence physiological processes in different manners. However, how cells differentially control the shedding of these EV populations is poorly understood. Here, we show that miR-1227, which is enriched in prostate cancer EVs, compared to the cell of origin, but not in EVs derived from prostate benign epithelial cells, induces the shedding of large EVs (such as large oncosomes), while inhibiting the shedding of small EVs (such as exosomes). RNA sequencing from cells stably expressing miR-1227, a modified RISCTRAP assay that stabilizes and purifies mRNA-miR-1227 complexes for RNA sequencing, and in silico target prediction tools were used to identify miR-1227 targets that may mediate this alteration in EV shedding. The COPII vesicle protein SEC23A emerged and was validated by qPCR, WBlot, and luciferase assays as a direct target of miR-1227. The inhibition of SEC23A was sufficient to induce the shedding of large EVs. These results identify a novel mechanism of EV shedding, by which the inhibition of SEC23A by miR-1227 induces a shift in EV shedding, favoring the shedding of large EV over small EV.

Loss of CDCP1 triggers FAK activation in detached prostate cancer cells.

A major metastasis suppressing mechanism is the rapid apoptotic death of cancer cells upon detachment from extracellular matrix, a process called anoikis. Focal adhesion kinase (PTK2/FAK) is a key enzyme involved in evasion of anoikis. We show that loss of the Cub-domain containing protein-1 (CDCP1), paradoxically stimulates FAK activation in the detached state of prostate cancer cells. In CDCP1low DU145 and PC3 prostate cancer cells, detachment-activation of FAK occurs through local production of PI(4,5)P2. PI(4,5)P2 is generated by the PIP5K1c-201 splicing isoform of PIP5K1c, which contains a unique SRC phosphorylation site. In the detached state, reduced expression of CDCP1 and an alternative CDCP1-independent SRC activation mechanism triggers PIP5K1c-pY644 phosphorylation by SRC. This causes a switch of Talin binding from ß1-integrin to PIP5K1c-pY644 and leads to activation of PIP5K1c-FAK. Reduced CDCP1 expression also inactivates CDK5, a negative regulator of PIP5K1c. Furthermore, immersion of prostate cancer cells in 10% human plasma or fetal bovine serum is required for activation of PIP5K1c-FAK. The PIP5K1c induced detachment-activation of FAK in preclinical models sensitizes CDCP1low prostate cancer cells to FAK inhibitors. In patients, CDCP1High versus CDCP1low circulating tumor cells differ in expression of AR-v7, ONECUT2 and HOXB13 oncogenes and TMPRSS2 and display intra-patient heterogeneity of FAK-pY397 expression. Taken together, CDCP1low and CDCP1high detached prostate cancer cells activate distinct cytoplasmic kinase complexes and targetable transcription factors, which has important therapeutic implications.

Single-cell analysis reveals transcriptomic remodellings in distinct cell types that contribute to human prostate cancer progression.

Prostate cancer shows remarkable clinical heterogeneity, which manifests in spatial and clonal genomic diversity. By contrast, the transcriptomic heterogeneity of prostate tumours is poorly understood. Here we have profiled the transcriptomes of 36,424 single cells from 13 prostate tumours and identified the epithelial cells underlying disease aggressiveness. The tumour microenvironment (TME) showed activation of multiple progression-associated transcriptomic programs. Notably, we observed promiscuous KLK3 expression and validated the ability of cancer cells in altering T-cell transcriptomes. Profiling of a primary tumour and two matched lymph nodes provided evidence that KLK3 ectopic expression is associated with micrometastases. Close cell-cell communication exists among cells. We identified an endothelial subset harbouring active communication (activated endothelial cells, aECs) with tumour cells. Together with sequencing of an additional 11 samples, we showed that aECs are enriched in castration-resistant prostate cancer and promote cancer cell invasion. Finally, we created a user-friendly web interface for users to explore the sequenced data.

Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles.

Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers.

Low-Background Acyl-Biotinyl Exchange Largely Eliminates the Coisolation of Non-S-Acylated Proteins and Enables Deep S-Acylproteomic Analysis.

Protein S-acylation (also called palmitoylation) is a common post-translational modification whose deregulation plays a key role in the pathogenesis of many diseases. Acyl-biotinyl exchange (ABE), a widely used method for the enrichment of S-acylated proteins, has the potential of capturing the entire S-acylproteome in any type of biological sample. Here, we showed that current ABE methods suffer from a high background arising from the coisolation of non-S-acylated proteins. The background can be substantially reduced by an additional blockage of residual free cysteine residues with 2,2'-dithiodipyridine prior to the biotin-HPDP reaction. Coupling the low-background ABE (LB-ABE) method with label-free proteomics, 2 895 high-confidence candidate S-acylated proteins (including 1 591 known S-acylated proteins) were identified from human prostate cancer LNCaP cells, representing so-far the largest S-acylproteome data set identified in a single study. Immunoblotting analysis confirmed the S-acylation of five known and five novel prostate cancer-related S-acylated proteins in LNCaP cells and suggested that their S-acylation levels were about 0.6-1.8%. In summary, the LB-ABE method largely eliminates the coisolation of non-S-acylated proteins and enables deep S-acylproteomic analysis. It is expected to facilitate a much more comprehensive and accurate quantification of S-acylproteomes than previous ABE methods.

Protein Composition Reflects Extracellular Vesicle Heterogeneity.

Extracellular vesicles (EVs) are membrane-enclosed particles that are released by virtually all cells from all living organisms. EVs shuttle biologically active cargo including protein, RNA, and DNA between cells. When shed by cancer cells, they function as potent intercellular messangers with important functional consequences. Cells produce a diverse spectrum of EVs, spanning from small vesicles of 40-150 nm in diameter, to large vesicles up to 10 µm in diameter. While this diversity was initially considered to be purely based on size, it is becoming evident that different classes of EVs, and different populations within one EV class may harbor distinct molecular cargo and play specific functions. Furthermore, there are considerable cell type-dependent differences in the cargo and function of shed EVs. This review focuses on the most recent proteomic studies that have attempted to capture the EV heterogeneity by directly comparing the protein composition of different EV classes and EV populations derived from the same cell source. Recent studies comparing protein composition of the same EV class(es) derived from different cell types are also summarized. Emerging approaches to study EV heterogeneity and their important implications for future studies are also discussed.

Proteomic Characterization of Epithelial-Like Extracellular Vesicles in Advanced Endometrial Cancer.

Endometrial cancer (EC) is the most frequent gynecological cancer. Tumor dissemination affecting ∼20% of EC patients is characterized at the primary carcinoma by epithelial-to-mesenchymal transition (EMT) associated with myometrial infiltration. At distant sites, the interaction of circulating tumor cells (CTCs) with the microenvironment is crucial for metastatic colonization, with a participation of the extracellular vesicles (EVs). We comprehensively approached these primary and secondary sites to study the impact of tumor EVs on the metastatic efficiency of CTCs in EC. Tumor EVs in circulation reproduce the epithelial phenotype predominant in the primary carcinoma, whereas CTCs are characterized by an EMT phenotype. We modeled this EMT-related clinical scenario in the Hec1A endometrial cell line and characterized the epithelial-like EVs in circulation by SILAC proteome analysis. The identification of proteins involved in cell-cell and cell-matrix interaction and binding, together with in vitro evidence of an improved adhesion of CTC to a functionalized endothelium, suggests a contribution of the epithelial-like EVs in the homing of CTCs at metastatic sites. Accordingly, adhesion protein LGALS3BP was found to be significantly enriched in circulating EVs from a cohort of EC patients with a high risk of recurrence by targeted proteomics (multiple reaction monitoring), highlighting its potential in liquid biopsy in EC.

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-Fluorouracil.

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.

Molecular profiling of circulating tumor cells links plasticity to the metastatic process in endometrial cancer.

BACKGROUND: About 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in high-risk EC patients. METHODS: CTC isolation was carried out in peripheral blood samples from 34 patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls using two methodologies. Samples were subjected to EpCAM-based immunoisolation using the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal) followed by RTqPCR analysis. The phenotypic determinants of endometrial CTC in terms of pathogenesis, hormone receptor pathways, stem cell markers and epithelial to mesenchymal transition (EMT) drivers were asked. Kruskal-Wallis analysis followed by Dunn's post-test was used for comparisons between groups. Statistical significance was set at p < 0.05. RESULTS: EpCAM-based immunoisolation positively detected CTC in high-risk endometrial cancer patients. CTC characterization indicated a remarkable plasticity phenotype defined by the expression of the EMT markers ETV5, NOTCH1, SNAI1, TGFB1, ZEB1 and ZEB2. In addition, the expression of ALDH and CD44 pointed to an association with stemness, while the expression of CTNNB1, STS, GDF15, RELA, RUNX1, BRAF and PIK3CA suggested potential therapeutic targets. We further recapitulated the EMT phenotype found in endometrial CTC through the up-regulation of ETV5 in an EC cell line, and validated in an animal model of systemic dissemination the propensity of these CTC in the accomplishment of metastasis. CONCLUSIONS: Our results associate the presence of CTC with high-risk EC. Gene-expression profiling characterized a CTC-plasticity phenotype with stemness and EMT features. We finally recapitulated this CTC-phenotype by over-expressing ETV5 in the EC cell line Hec1A and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model of CTC dissemination and homing.