Triple-Negative Breast Cancer EVs Modulate Growth and Migration of Normal Epithelial Lung Cells.

Breast cancer is the most common cancer amongst women worldwide. Recently, owing to screening programs and new technologies, the survival rate has increased significantly. Breast cancer can potentially develop metastases, and, despite them, lung metastases generally occur within five years of breast cancer diagnosis. In this study, the objective was to analyze the effect of breast cancer-derived EVs on a lung epithelial cell line. BEAS-2B cells were treated with extracellular vesicles (EVs) derived from triple-negative breast cancer cells (TNBCs), e.g., MDA-MB-231 and HS578T, separated using differential ultracentrifugation. We observed an increased growth, migration, and invasiveness of normal epithelial lung cells over time in the presence of TNBC EVs compared to the control. Therefore, these data suggest that EVs released by tumor cells contain biological molecules capable of influencing the pro-tumorigenic activity of normal cells. Exploring the role of EVs in oncology research and their potential cargo may be novel biomarkers for early cancer detection and further diagnosis.

Guardians and Mediators of Metastasis: Exploring T Lymphocytes, Myeloid-Derived Suppressor Cells, and Tumor-Associated Macrophages in the Breast Cancer Microenvironment.

Breast cancers (BCs) are solid tumors composed of heterogeneous tissues consisting of cancer cells and an ever-changing tumor microenvironment (TME). The TME includes, among other non-cancer cell types, immune cells influencing the immune context of cancer tissues. In particular, the cross talk of immune cells and their interactions with cancer cells dramatically influence BC dissemination, immunoediting, and the outcomes of cancer therapies. Tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs) represent prominent immune cell populations of breast TMEs, and they have important roles in cancer immunoescape and dissemination. Therefore, in this article we review the features of TILs, TAMs, and MDSCs in BCs. Moreover, we highlight the mechanisms by which these immune cells remodel the immune TME and lead to breast cancer metastasis.

Goat milk extracellular vesicles: Separation comparison of natural carriers for theragnostic application.

Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is an interesting source of extracellular vesicles (EVs). Despite the countless potential applications that they offer in many biological fields, is not easy to compare the different proposed systems, and this is a major limitation for the real translatability of these natural nanoplatforms for theragnostic purposes. Thus, it is useful to further investigate reproducible methods to separate goat milk EVs. The choice of methods but also the preprocessing of milk has an immense impact on the separation, quality, and yield of EVs. Here, we tested four protocols to separate EVs from unpasteurised goat milk: two based on differential ultracentrifugation (DUC) and two on size-exclusion chromatography (SEC). Moreover, we assessed two different approaches of pre-treatment (acidification and precipitation) to facilitate milk protein discharge. To the best of our knowledge, a similar comparison of all performed protocols on raw goat milk has never been published before. Therefore, enriched EV samples were successfully obtained from goat milk using both DUC and SEC. Taken together, our results may be helpful to obtain natural carriers for future theragnostic applications in personalised medicine.

Effect of In Vitro Enzyme Digestion and Bile Treatment on Milk Extracellular Vesicles Stability.

SCOPE: Milk extracellular vesicles (EVs) are nanosized particles with potential immune bioactivities. This study examines their fate during in vitro infant gastrointestinal digestion (GI). METHODS AND RESULTS: Bovine milk is digested using the in vitro INFOGEST method, adjusted for the infant. To unravel the contribution of digestive enzymes from bile, milk is treated with digestive enzymes, bile, or a combination of both. EVs are collected posttreatment using differential ultracentrifugation. EVs characterization includes electrophoresis, immunoblotting, nanoparticle tracking analysis, and atomic force microscopy. EVs protein markers programmed cell death 6-interacting protein (ALIX), tumor susceptibility gene 101 (TSG101), cluster of differentiation 9 (CD9), and xanthine dehydrogenase (XDH) are detected after gastric digestion (G60), but their signal intensity is significantly reduced by intestinal conditions (p < 0.05). Enzyme digestion, compared to bile treatment (I60 + bile), results in a significant reduction of signal intensities for TSG101 and CD9 (p < 0.05). Nanoparticle tracking analysis shows a significant reduction (p < 0.05) of EV numbers at the end of the intestinal phase. EVs are detected by atomic force microscopy at the end of the intestinal phase, showing that intact EVs can survive upper gut digestion. CONCLUSION: Intact EVs can be found at the end of the intestinal phase. However, digestive enzymes and bile reduce the quantity and characteristics of EVs, with digestive enzymes playing a larger role.

An investigation of extracellular vesicles in bovine colostrum, first milk and milk over the lactation curve.

Extracellular vesicles (EVs) in milk have claimed benefits ranging from conveying immunological privilege to infants to being suitable as natural delivery vehicles for therapeutic drugs. However, a longitudinal study of bovine EVs quantities and characteristics in colostrum (COL), first milk (FM) and throughout the lactation curve of mature milk (MM) had never been performed and so was our aim. COL, FM and 9 months of MM samples were collected. Caseins -overlapping size with EVs- were removed. EVs were collected by density gradient ultracentrifugation and characterised by SDS-PAGE, Bradford assay, nanoparticle tracking analysis, immunoblotting, imaging flow cytometry analysis, and transmission electron microscopy. COL and FM had substantially more EVs than MM, with COL enriched in small EVs. No significant differences were observed between months 1-9 of MM. Altogether, although COL and FM are particularly rich sources of EVs, mature milk throughout the lactation curve is also an abundant source of intact EVs.

Influence of Breast Cancer Extracellular Vesicles on Immune Cell Activation: A Pilot Study.

Breast cancer is the leading cause of cancer-related death in women worldwide. It is well known that breast cancer shows significant alterations in the tumor microenvironment (TME), which is composed of a variety of immune cells, including natural killer (NK) cells, that have a key role in tumor development or anti-tumor responses in breast cancer patients. Luminal B (BT474) and triple-negative breast cancer (HS578T) cell lines were cultured in 2D and 3D model systems. PMBCs from healthy donors were isolated and treated with extracellular vesicles (EVs) from monolayer and spheroids of BT474 and HS578T and analyzed using cytofluorimetric approaches. We observed that EVs can alter the activation and presence of CD335+/CD11b+ NK cells. EVs derived from BT474 and HS578T cells trigger the activation and, simultaneously, a reduction in the percentage of CD335+/CD11b+ NK cells. In addition, EVs derived from BT474 also significantly reduce CD39+ T-regulatory (T-reg) cells. Our preliminary data suggest that using EVs to treat tumors could potentially alter components of the immune system, which causes hyperactivation of specific cell types and can lead to aggressive growth. These data will guide the designing of new personalized diagnostic approaches based on in-depth study of the TME.

Extracellular vesicle separation from milk and infant milk formula using acid precipitation and ultracentrifugation.

Separation of highly enriched extracellular vesicles (EVs) fractions from milk is desirable for quantification, cargo analysis, functional characterization, and investigation as delivery vehicles for nutrients and/or therapeutics. However, a rigorous, reproducible protocol is lacking. This protocol considers a crucial aspect typically overlooked, i.e., that caseins are of similar size to, but more abundant than, EVs in milk. Our protocol combines acid pre-treatment and gradient ultracentrifugation, producing EV-enriched fractions suitable for downstream orthogonal characterization approaches. For complete details on the use and execution of this protocol, please refer to Mukhopadhya et al. (2021).

Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk.

Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM.