The tumor microenvironment and its contribution to tumorigenesis has been a focal highlight in recent years. A two-way communication between the tumor and the surrounding microenvironment sustains and contributes to the growth and metastasis of tumors. Progression and metastasis of hepatocellular carcinoma (HCC) have been reported to be exceedingly influenced by diverse microenvironmental cues. In this study, we present a 3D-culture model of liver cancer to better mimic in vivo tumor settings. By creating novel 3D co-culture model that combines free-floating and scaffold-based 3D-culture techniques of liver cancer cells and fibroblasts, we aimed to establish a simple albeit reproducible ex vivo cancer microenvironment model that captures tumor-stroma interactions. The model presented herein exhibited unique gene expression and protein expression profiles when compared to 2D and 3D mono-cultures of liver cancer cells. Our results showed that in vivo like conditions cannot be mimicked by simply growing cancer cells as spheroids, but by co-culturing them with 3D fibroblast with which they were able to crosstalk. This was evident by the upregulation of several pathways involved in HCC, and the increase in secreted factors by co-cultured cancer cells, many of which are also involved in tumor-stroma interactions. Compared to the conventional 2D culture, the proposed model exhibits an increase in the expression of genes associated with development, progression, and poor prognosis of HCC. Our results correlated with an aggressive outcome that better mirrors in vivo HCC, and therefore, a more reliable platform for molecular understanding of HCC.

For decades since the central dogma, cancer biology research has been focusing on the involvement of genes encoding proteins. It has been not until more recent times that a new molecular class has been discovered, named non-coding RNA (ncRNA), which has been shown to play crucial roles in shaping the activity of cells. An extraordinary number of studies has shown that ncRNAs represent an extensive and prevalent group of RNAs, including both oncogenic or tumor suppressive molecules. Henceforth, various clinical trials involving ncRNAs as extraordinary biomarkers or therapies have started to emerge. In this review, we will focus on the prognostic and diagnostic role of ncRNAs for breast cancer.

Melanomas are highly immunogenic tumors that have been shown to activate the immune response. Nonetheless, a significant portion of melanoma cases are either unresponsive to immunotherapy or relapsed due to acquired resistance. During melanomagenesis, melanoma and immune cells undergo immunomodulatory mechanisms that aid in immune resistance and evasion. The crosstalk within melanoma microenvironment is facilitated through the secretion of soluble factors, growth factors, cytokines, and chemokines. In addition, the release and uptake of secretory vesicles known as extracellular vesicles (EVs) play a key role in shaping the tumor microenvironment (TME). Melanoma-derived EVs have been implicated in immune suppression and escape, promoting tumor progression. In the context of cancer patients, EVs are usually isolated from biofluids such as serum, urine, and saliva. Nonetheless, this approach neglects the fact that biofluid-derived EVs reflect not only the tumor, but also include contributions from different organs and cell types. For that, isolating EVs from tissue samples allows for studying different cell populations resident at the tumor site, such as tumor-infiltrating lymphocytes and their secreted EVs, which play a central anti-tumor role. Herein, we outline the first instance of a method for EV isolation from frozen tissue samples at high purity and sensitivity that can be easily reproduced without the need for complicated isolation methods. Our method of processing the tissue not only circumvents the need for hard-to-acquire freshly isolated tissue samples, but also preserves EV surface proteins which allows for multiplex surface markers profiling. Tissue-derived EVs provide insight into the physiological role of EVs enrichment at tumor sites, which can be overlooked when studying circulating EVs coming from different sources. Tissue-derived EVs could be further characterized in terms of their genomics and proteomics to identify possible mechanisms for regulating the TME. Additionally, identified markers could be correlated to overall patient survival and disease progression for prognostic purposes.

Prokaryotic and eukaryotic adaptive immunity differ considerably. Yet, their fundamental mechanisms of gene editing via Cas9 and activation-induced deaminase (AID), respectively, can be conveniently complimentary. Cas9 is an RNA targeted dual nuclease expressed in several bacterial species. AID is a cytosine deaminase expressed in germinal centre B cells to mediate genomic antibody diversification. AID can also mediate epigenomic reprogramming via active DNA demethylation. It is known that sequence motifs, nucleic acid structures, and associated co-factors affect AID activity. But despite repeated attempts, deciphering AID’s intrinsic catalytic activities and harnessing its targeted recruitment to DNA is still intractable. Even recent cytosine base editors are unable to fully recapitulate AID’s genomic and epigenomic editing properties. Here, we describe the first instance of a modular AID-based editor that recapitulates the full spectrum of genomic and epigenomic editing activity. Our ‘Swiss army knife’ toolbox will help better understand AID biology per se as well as improve targeted genomic and epigenomic editing.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Coronavirus disease 2019 (COVID-19) has transformed very quickly into a world pandemic with severe and unexpected consequences on human health. Concerted efforts to generate better diagnostic and prognostic tools have been ongoing. Research, thus far, has primarily focused on the virus itself or the direct immune response to it. Here, we propose extracellular vesicles (EVs) from serum liquid biopsies as a new and unique modality to unify diagnostic and prognostic tools for COVID-19 analyses. EVs are a novel player in intercellular signalling particularly influencing immune responses. We herein show that innate and adaptive immune EVs profiling, together with SARS-CoV-2 Spike S1+ EVs provide a novel signature for SARS-CoV-2 infection. It also provides a unique ability to associate the co-existence of viral and host cell signatures to monitor affected tissues and severity of the disease progression. And provide a phenotypic insight into COVID-associated EVs.

Extracellular vesicles (EVs) are increasingly being analyzed by flow cytometry. Yet their minuscule size and low refractive index cause the scatter intensity of most EVs to fall below the detection limit of most flow cytometers. A new class of devices, known as spectral flow analyzers, are becoming standards in cell phenotyping studies, largely due to their unique capacity to detect a vast panel of markers with higher sensitivity for light scatter detection. Another class of devices, known as nano-analyzers, provides high-resolution detection of sub-micron-sized particles. Here, the EV phenotyping performance between the Aurora (Cytek) spectral cell analyzer and the NanoFCM (nFCM) nanoflow analyzer are compared. These two devices are specifically chosen given their lead in becoming gold standards in their respective fields. Immune cell-derived EVs remain poorly characterized despite their clinical potential. Therefore, B- and T-cell line-derived EVs and donor-matched human biofluid-derived EVs from plasma, urine, and saliva are used in combination with a panel of established immune markers for this comparative study. A comparative evaluation of both cytometry platforms is performed, discussing their potential and suitability for different applications. It is found that nFCM can accurately i) analyze small EVs (40-200 nm) matching the size accuracy of electron microscopy; ii) measure the concentration of a single EV particle per volume; iii) identify underrepresented EV marker subsets; and iv) provide co-localization of EV surface markers. It can also be shown that human sample biofluids have unique EV marker signatures that can have future clinical relevance. Finally, nFCM and Aurora have their unique strength, preferred fashion of data acquisition, and visualization to fit different research interests.

B cell maturation is crucial for effective adaptive immunity. It requires a complex signalling network to mediate antibody diversification through mutagenesis. B cells also rely on queues from other cells within the germinal centre. Recently, a novel class of intercellular signals mediated by extracellular vesicles (EVs) has emerged. Studies have shown that B cell EV-mediated signalling is involved in immune response regulation and tumorigenesis. However, the mechanistic role of B cell EVs is not yet established. We herein study the biological properties and physiological function of B cell EVs during B cell maturation. We use emerging technologies to profile B cell EV surface marker signatures at the single particle level, molecular cargo and physiological roles in B cell maturation. EV ncRNA cargo, characterised by RNA-seq, identified an EV-mediated novel non-coding RNA (ncRNA) regulatory network for B cell maturation. We show that a previously uncharacterised micro-RNA (miR-5099) in combination with a set of long ncRNA are carried within B cell EVs and could contribute to antibody diversification. The physiological role of EVs in B cell maturation is investigated using EV blockade assays and complementation studies using diverse EV sources further confirmed the physiological role and mode of action of EVs in B cell maturation.