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3D Cell Cultures As Prospective Models to Study Extracellular Vesicles in Cancer

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3D Cell Cultures As Prospective Models to Study Extracellular Vesicles in Cancer cancers Review 3D Cell Cultures as Prospective Models to Study Extracellular Vesicles in Cancer Guillermo Bordanaba-Florit 1 , Iratxe Madarieta 2 , Beatriz Olalde 2 , Juan M. Falcón-Pérez 1,3,4 and Félix Royo 1,3,* 1 Center for Cooperative Research in Biosciences (CIC bioGUNE), Exosomes Laboratory, Basque Research and Technology Alliance (BRTA), E48160 Derio, Spain; [email protected] (G.B.-F.); [email protected] (J.M.F.-P.) 2 TECNALIA Basque Research and Technology Alliance (BRTA), E20009 Donostia San Sebastian, Spain; [email protected] (I.M.); [email protected] (B.O.) 3 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), E28029 Madrid, Spain 4 Ikerbasque, Basque Foundation for Science, E48009 Bilbao, Spain * Correspondence: [email protected] Simple Summary: 3D cell cultures are a qualitative improvement in cancer research because these models preserve cancer physiological characteristics better than traditional bi-dimensional cultures. Moreover, they facilitate the study of complex 3D interactions using extracellular matrices and the co-culture of different cell types. In this manner, the cells can contact themselves in a fully physiological but also controlled arrangement. In the context of tumor interactions, extracellular vesicles are essential in number of key aspects in oncology: as major interactors with extracellular matrix, as cell-to-cell messengers, as carriers of diagnostic-valuable biomarkers, and as target-specific treatment-deliver agents. The present article aims to discuss the findings achieved using 3D culture models in oncology. We further review the involvement of extracellular vesicles in the pathogenesis of cancer as well as their potential use in diagnostics and therapeutics. Citation: Bordanaba-Florit, G.; Abstract: The improvement of culturing techniques to model the environment and physiological Madarieta, I.; Olalde, B.; Falcón-Pérez, conditions surrounding tumors has also been applied to the study of extracellular vesicles (EVs) J.M.; Royo, F. 3D Cell Cultures as Prospective Models to Study in cancer research. EVs role is not only limited to cell-to-cell communication in tumor physiology, Extracellular Vesicles in Cancer. they are also a promising source of biomarkers, and a tool to deliver drugs and induce antitumoral Cancers 2021, 13, 307. https:// activity. In the present review, we have addressed the improvements achieved by using 3D culture doi.org/10.3390/cancers13020307 models to evaluate the role of EVs in tumor progression and the potential applications of EVs in diagnostics and therapeutics. The most employed assays are gel-based spheroids, often utilized to Received: 17 December 2020 examine the cell invasion rate and angiogenesis markers upon EVs treatment. To study EVs as drug Accepted: 12 January 2021 carriers, a more complex multicellular cultures and organoids from cancer stem cell populations Published: 15 January 2021 have been developed. Such strategies provide a closer response to in vivo physiology observed responses. They are also the best models to understand the complex interactions between different Publisher’s Note: MDPI stays neu- populations of cells and the extracellular matrix, in which tumor-derived EVs modify epithelial or tral with regard to jurisdictional clai- mesenchymal cells to become protumor agents. Finally, the growth of cells in 3D bioreactor-like ms in published maps and institutio- systems is appointed as the best approach to industrial EVs production, a necessary step toward nal affiliations. clinical translation of EVs-based therapy. Keywords: 3D culture; extracellular vesicles; tumoral cells; cancer; therapy Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- 1. Introduction ditions of the Creative Commons At- In recent years, the number of scientific groups dedicated to the study of extracellular tribution (CC BY) license (https:// vesicles has grown notably, and with it the amount of published information describing creativecommons.org/licenses/by/ 4.0/). extracellular vesicles (EVs) physiology. Released by all types of cells, they are an important Cancers 2021, 13, 307. https://doi.org/10.3390/cancers13020307 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 307 2 of 16 tool to study cell’s biology, and to look for biomarkers. Cancer research is one of the main fields that can benefit of the study of EVs associated to tumors. In fact, the vesicle-mediated cell-to-cell crosstalk seems to be important in every step of cancer progression [1]. In parallel, the study of cancer biology had evolved itself along the last years towards culture models that reflect the biological complexity of tumoral cells and their interactions with the extracellular matrix. The reason is that the traditional bidimensional (2D) cultures differ from tridimensional (3D) cultures in their morphological characteristics, proliferation rate and degree of differentiation, the level of cell-to-cell interaction and cell-to-matrix, as well as their resistance to drugs [2,3]. However, the application of complex culture models to unravel the role of EVs in cancer research has not been yet popularized among EVs research, given the difficulties that this type of cultures presents, both technically and in terms of cost. Nevertheless, several studies have highlighted the importance of 3D cultures in the study of EVs in cancer research [4–6]. In this article, we aim to emphasize the contribution of those studies as a fundamental path to understand the involvement of EVs in cancer physiology and to pinpoint possible applications to the clinical oncology. To help to understand the background of this review, we are providing a short introduction to the different roles that EVs play in cancer and cancer therapy, and a brief description of the different 3D cultures employed to study tumoral cells. Afterwards, the review summarizes different studies that employ 3D culture systems to elucidate the role of EVs in cancer biology, diagnosis and therapy. 2. The 3D Cultures as a Physiological Model of Tumoral Cells For many years, in vitro models were based on 2D monolayers of immortalized human cancer-derived cell lines. The popularization of 3D culturing has come with the observation that this type of cell cultures often retain heterogeneity. This feature allows the study of tumor evolution. Moreover, 3D cultures offer advantages over conventional monolayered cell cultures including preservation of the topology and cell-to-matrix interactions [7,8]. On the other hand, the application of 3D cultures is also challenging, given the difficulties to stabilize the cultures, and the requirement of specific material to perform the culture. In Table1, we present a comparative between 2D and 3D cultures characteristics. In spite of the difficulties, 3D cultures become a great model to study the interplay between cancer and non-cancer cells in order to unveil biological mechanisms involved in cancers initiation and progression [9]. Spheroids are probably the type of 3D culture most commonly used. Spheroid formation methodologies can be divided into two categories: scaffold-based models, either incorporating materials which are components of the matrix (collagen, fibronectin, agarose, laminin, and gelatin) [10], or synthetic materials that provide cell support [11], and scaffold-free models that comprise non-adherent and in suspension cells, which are forced to aggregate and form spheroids [12]. Table 1. Main advantages and limitations of the different cellular models in cancer research [13,14]. Model Advantages Limitations Easy and cost effective Reduced cell-to-cell interactions Large amount of data available 2D Monolayers Different sensitivity to drugs Reproducible cultures, easy to work for downstream Loss of biological characteristics over time applications and imaging Cell–ECM interactions Difficult to dispense cells Possible to incorporate different factors in the gel, Gel based 3D Cultures Change of growth media could be irregular extending release over time Difficult to retrieve cells and downstream analysis Uniform spheroids/organoids Simpler and cheaper when compared to gel Time consuming and low yield achieved Low-attachment plates based systems Heterogenous spheroids Long-term culture Possible chemical gradients Expensive commercial devices or not Control of fluid rates Microfluidic systems well-characterized “in house” build devices Convenient for multicellular cultures controlling Fluidic problems related to bubbles and clogging cell locations Cancers 2021, 13, 307 3 of 16 One of the first applications of 3D cultures was the study of tumorigenesis. Typically, the cells are cultured in a mouse sarcoma-derived gel (i.e., Matrigel®). Other alternatives ex- ist, such human leiomyoma discs and their matrix (Myogel). This has been commercialized for in vitro assays such IncuCyte®, spheroid and sandwich assays [15]. 3D culture models grown in vitro from cancer stem directly or from primary tissues are a more evolved form of organoids [16]. The latter option has an attractive potential for personalized medicine. For instance, when comparing organoids derived from primary colorectal tumors and metastatic lesions isolated from the same patients, it has been observed that they share common mutations. This implies that the driver alterations preceded metastatic dissemination [17]. Organoids display greater number of features and functions
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