La Rocca, Alessia (2022) Tumoral bioengineered microtissues in microfluidic platforms to investigate on chip Colorectal cancer liver metastasis. [Tesi di dottorato]

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Colorectal cancer (CRC) is the third most common tumor and the second leading cause of cancer-related deaths in developed countries with liver metastasis as the main cause of CRC mortality. Therefore, there is a growing need to understand the mechanisms underlying liver metastases to improve drug screening. Several phenomena, which occur in the Tumor microenvironment (TME), such as the extracellular matrix (ECM) remodeling, activation of the host cells, and Epithelial-Mesenchymal Transition (EMT), play a key role in cancer progression and metastatic process. While 2D in vitro models lack at all the TME providing an oversimplified view of tumor biology, on the other side the animal model also presents several limitations linked with the difficulty of growing a human tumor subcutaneously in mice and of replicating the initiation of the metastatic cascade. Recently, in vitro 3D models of CRC and liver, and multi-organ-on-a-chip, have become potential tools to reproduce in vivo conditions, in which it is possible to mimic the cross-talk between primary tumor and the distant metastatic growth in secondary site of the human body. However, these models often do not correctly recapitulate the complexity of the TME and the mechanisms underlying the metastasis that occurs in native tissue, owing to the use of exogenous matrix as native ECM’s surrogate. To address this dearth, this thesis focuses on the fabrication of new 3D in vitro colorectal cancer microtissues (3D CRC μTs), which reproduce more faithfully the TME in vitro. Specifically, 3D CRC μTs were fabricated using a 2-step dynamic tissue engineering strategy. Firstly, normal human dermal fibroblasts (NF) were seeded on porous biodegradable gelatin microbeads (GPMs) – in which the cells were continuously induced to synthesize and assemble their own ECM – to fabricate a 3D Stroma microtissues (3D NF μTs). Secondly, Human colon cancer cells (HCT-116 cells) were dynamically seeded on 3D NF μTs to achieve 3D CRC μTs. Deeply morphological characterization of the 3D CRC μTs was performed to assess the presence of complex different macromolecular components featuring in vivo-ECM. The results showed 3D CRC μTs recapitulated the complexity of the TME in terms of ECM production, remodeling, cell growth, and bidirectional cross-talk between cancer cells and fibroblasts. Moreover, the activation of normal fibroblasts toward activated phenotype was assessed. Moreover, the microtissues were used to evaluate the synergistic effect of Curcumin-loaded nanoemulsions (CT-NE-Curc) and 5 Fluorouracil (5-FU). The results of the combination treatments of CT-NE-Curc and 5-FU showed a protective effect of curcumin on 3D NF μTs and enhanced cytotoxic effect on 3D CRC μTs. Then, multi-organ-on-a-chip platforms to investigate the hepatic metastasis from colorectal cancer were developed. A microfabrication strategy was used to design and fabricate a platform – in which primary tumor-like (3D CRC μTs) and target organ (3D liver microtissues named 3D HepG2 µTs) were respectively loaded in a dedicated chamber, to monitor the migration of cancer cells from the primary tumor site towards the target organ site. The metastasis-on-a-chip platforms experiments proved the bidirectional cross-talk between cancer cells and fibroblasts during the cancer invasion and migration both when 3D CRC μTs were loaded alone and when 3D CRC μTs together with 3D HepG2 µTs were housed into the corresponding chambers. Furthermore, we observed the presence of few cancer cells in the target organ chamber when 3D HepG2 µTs were present. In summary, the results described the high capability of 3D CRC μTs to reproduce the TME of colorectal cancer and to be capable to be used as models for drug and/or nutraceuticals testing. Moreover, these models may be used to further investigate the metastatic cascade through metastasis-on-a-chip platforms aiming at addressing further research questions in cancer investigation and drug discovery.

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