Gioiella, Filomena (2016) A new bioengineered 3D tumor platform in vitro to replicate tumor-stroma interaction and investigate anti-cancer drug delivery. [Tesi di dottorato]

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Nowadays, cancer is still a second leading cause of death after cardiovascular disease in the world. One of the main factors that lead the failure of cancer therapy is related to the fact that little is known about the interaction of cancer cells with microenvironment. Indeed, as hypothesized in the "seed" and "soil" theory by Paget over a century ago, tumor progression is determined not only by tumor cells but also by the surrounding stromal milieu. For these reasons in the last years, increasing attention is focused on the importance of tumor microenvironment in an effort to develop successful strategies in cancer disease treatment. In traditional two-dimensional in vitro models the absence of 3D architecture generates misleading and contradictory results. Hence emerges the need to have an in vitro versatile platform that closely recapitulates pathophysiological features of the native tumor tissue and its surrounding microenvironment. In this PhD thesis a microtissue precursors assembling strategy (µTP), was used and translated to produce 3D tumor engineered models composed by tumor and/or stromal cells. In contrast with the classical spheroid model, the µTP we proposed presents the production of extracellular matrix directly synthesized by stromal cells. First of all, in the chapter 1 a stat of art overview was presented which highlights the importance of tumor microenvironment in cancer research, the existing models for studying tumor development and the nanotechnology contribution in cancer treatment. Then the chapter 2 is focalized on the realization of stromal microtissues fabricated seeding normal or activated fibroblasts on microporous beads, in order to monitor their dynamic evolution in terms of metabolic activity, mechanical properties and ECM composition. In particular it is demonstrated how the microtissue configuration is able to keep phenotypic differences between normal and activated fibroblasts in all the aspects investigated compared to the classical 3D spheroidal model. In the chapter 3 the cross talk between epithelial tumor and the surrounding stroma in a microfluidic device is investigated. Thanks to the combination of 3D microtissues with microfluidic technology, it is possible to detect in real time the modification occurring at cellular and ECM level during the activation period. In the second part of work, the tumor microtissue model is validated as a potential drug-screening platform. In particular, in chapter 4 a commonly drug used in chemotherapy (Doxorubicin) is tested in order to detect the difference in chemoresistance between microtissues and spheroid models, both in monoculture and coculture. Finally, a stimuli-responsive nanoparticles are tested on normal and tumor 3D heterotypic microtissues to demonstrate their significant selectively. At last, the microtissue system may be a useful \emph{in vitro} screening tool for testing innovative approaches of drug delivery, reducing expensive and time-consuming protocol nowadays used in preclinical studies.

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