Project Portugal 2030

Summary

With CLICKors, we aim to control and tailor cellular organization within complex 3D tumor models. Until the present, controlling cellular organization was performed by adjusting cellular confluency within the model, together with a multistage introduction of cells to the spheroids. Still, when using such techniques, one is limited to the capacity of cells to deposit extracellular matrix, remodel it, and migrate within these models in response to biochemical signaling. In order to achieve hierarchical structures within these models, strong intercellular interactions are required among certain cell types. Within the scope of CLICKors, as a proof of concept, we have focused on invasive ductal carcinoma and have set a series of objectives to achieve during the onset of this project: 1. By using dibenzocyclooctyne conjugates of different linear and branched forms, we hope to achieve different levels of cellular interactions between the targeted cell types, therefore fine-tuning the way that these cells will aggregate and form anatomical attributes that are relevant when attempting to mimic these tissues in vitro. 2. We aim to achieve a comparable histoarchitecture within these in vitro models when compared to native tumor tissue. Within human breast tissue, the branching ductal networks are composed of two epithelial cell types: an inner layer of polarized luminal epithelial cells and an outer layer of myoepithelial cells. Typically, breast cancer arises from this luminal epithelial compartment (Sainsbury et al., 2000), while myoepithelial cells, on the other hand, may act as protectors of tissue integrity by preserving tissue polarity (Gudjonsson et al., 2002). It is this hierarchical interaction that we aim to mimic in vitro within a spheroid model comprising stroma and a vascular compartment together with diseased triple-negative luminal epithelial cells (Hs578T) and diseased myoepithelial cells (Hs578Bst). 3. Tumor-supporting cells are important as vascularization surrounding the ducts together with the distribution that these vascular endothelial cells assume contribute to the pathogenesis of this disease. Within this proposal, we aim to demonstrate that with tools such as biorthogonal chemistry, it is possible to recreate these cellular interactions within in vitro tumor models. 4. Produce IDC in vitro models following two main approaches. The first will be the production of a multicellular spheroid system in which BIEN will be used to develop duct-like structures within the multicellular aggregate. The second approach will comprehend the production of an assembloid-based system, with two distinct subunits that will be used to study cellular interactions. Interactions between both systems upon co-culture will be studied. With this, not only are we hoping to contribute to the advance of the field by creating tumor models with greater biosimilarity but also in exploring the potential that tools like BIEN offer for a new spectrum of applications.