Development of an in vitro murine three-dimensional tumor model to study the micro-environment ability to tune cell?s features
Creators: name: COSTABILE, FRANCESCA
Publisher: name: Universit� degli studi di Genova
Year: 2022

description: Background: The tumor microenvironment (TME) is a complex system, shaped by direct interactions among different cell types and by the interactions between cells and extracellular matrix (ECM). Given the emerging importance of the TME in modulating cells? morphology and function, more sophisticated tumor models incorporating TME features are needed to elucidate cellular, molecular, and immunologic mechanisms of tumor response or resistance. An intensive investigation of in vitro models able to study tumor biology has led to the generation of different three-dimensional (3D) culture methods that better mimic in vivo conditions compared to the usual 2D culture methods. The 3D mono- and co-cultures are able to reproduce some ?in vivo features? such as 3D cell morphology, which permits cells to better execute their function and enables them to deposit significant increased amount of ECM. 
Aim of this study is the development of an accurate in vitro 3D tumor model to study the impact of tumor ECM on the cells of the microenvironment. The understanding of the specific contributions that ECM proteins make to the tumor microenvironment became crucial due to the emergency of find alternative cures to the many cancer harboring patients resistant to the existing therapies. 
Methods: Our experimental approach can be divided in two main experimental plans: 
? the development of tumor spheroid model, helpful to understand the beginning stage of the nascent tumor; at day 7, 14 and 21 of spheroids culture we evaluated ECM deposition through immunofluorescence (IF) analysis of collagen type VI; we also analyzed how much the 3D co-culture model we generated was similar compared to the in vivo microenvironment regarding gene expression;
? the development of a scaffold obtained by murine tumor tissue decellularization, utilized for the study of the advanced tumor stage, with a more complex ECM compared to the spheroid model; some scaffolds underwent to collagen type VI IF analysis while other scaffolds were used for recellularization experiments with B16f10 or NIH/3T3 cell lines. Some scaffolds were also used for scanning electron microscopy analysis (SEM). 
Results and Discussion: Spheroids generated by the co-culture of B16f10 and NIH/3T3 cells were the only ones lasting for 21 days and the only ones where collagen type VI was detected, compared to spheroids made just by B16f10. These results let us deduce that ECM deposition is fibroblast-dependent. According to the 3D morphology classification of Kenny et al. 2007, B16f10 + NIH/3T3 derived spheroids we generated belong to the Mass class, characterized by cells organized in a regular manner around the center of the colony; B16f10 derived spheroids seems to belong instead to the Grape-like class, characterized by colonies with poor cell-cell contacts and distinguished by their grape-like appearance. B16f10-spheroid?s morphology clearly show a lack of robust cell-cell adhesion, result that has to be overlapped with the absence of collagen type VI. Therefore, fibroblasts are needed for the generation of an early tumor stage 3D model because of their major role as ECM producers and tumor micro-environment organizers. We even investigated spheroid gene expression signature. We focused on the genes that were expressed at the same level between the tumor cells derived from the 3D coculture and the actual in vivo tumor microenvironment. Clusters analysis highlights the similarity between the 2 groups showing 23.1% of the found pathways referring to cancer microenvironment, giving grand importance to the spheroid as in vitro model compared to the 2D systems. 
To define an in vitro 3D model suitable to the experimentation on advanced solid tumor phase, murine B16f10 derived tumors were processed for decellularization and studied to verify the efficacy as a natural scaffold for 3D culture system. As observed also from other research groups, cell attachment was limited to the border of the tissue, and it seems cells were not able to pass through the ECM. Control experiment with polyurethane demonstrate the capability of cells to infiltrate a synthetic matrix. To have a better understanding of the ECM structure, frozen sections of decellularized tissue were analyzed by confocal microscope for detection of collagen type VI. Interestingly, both immunofluorescence and histochemistry analysis showed an increase of ECM in decellularized tissue compared to the not decellularized one. This may be due to a slight collapse of the tumor structure since cells have been removed from it. We implemented the ECM observation with the support of SEM: after the decellularization treatment the extracellular fibres are exposed and as hypothesized, the lack of cells makes the fibres twist on themselves, generating a complex net, probably impenetrable from cells. To create a method to measure the collapse of the extracellular matrix that a decellularization treatment may provokes, we compared a fresh and a decellularized specimen of mouse pulmonary parenchyma, where structural modifications are easy to be detected. In this way we were able to quantify the collapse of the ECM through alveoli area reduction. 
Conclusions: The development of the spheroid made by tumor and fibroblast cells is a more realistic environment compared to the usual 2D culture and to the spheroid made simply by tumor cells. Fibroblasts are needed for the generation of a better 3D model because of their major role as ECM producers and tumor micro-environment organizers. The limit of the spheroid model is the time, since, to date, the system does not provide oxygen import, necessary for the tumor bulk growth. Tumor derived 3D scaffolds by decellularization most truthfully represents the real in vivo scenario of a tumor 3D model, but the recellularization lack is very often a big limit. Our data demonstrate that the decellularizzation process provokes the collapse of the matrix that does not allow the tissue to be utilized as scaffold for in vitro cell experimentation. Here we also provide, for the first time, a method to test the damage that the treatment provokes on the samples, avoiding the loss of precious materials.