If you’re planning to give a child a present of a jigsaw puzzle of the Eiffel Tower, would you choose a two-dimensional or three-dimensional puzzle and which do you think the child would prefer? The more realistic and exciting way of building a replica of such renowned monuments is obviously in 3D. It captures the imposing stature, the characteristic relations of height, length and width and adds that extra touch of authenticity – namely by adding that extra dimension, bringing it closer to real life.

Not surprisingly, when trying to replicate the setting of a tumour in the human body in a laboratory, the aim is to do this as accurately as possible. Historically most cell culture experiments were performed by culturing the cells in a so-called monolayer, which means the cells grow in a single layer on a flat surface at the bottom of a flask or dish. As you can imagine, this isn’t the best way to represent how the cells are arranged at the tumour site within the body. There they are aggregated to a tumour mass surrounded by different types of other cells, blood vessels, macromolecules, tissue and supporting scaffolds, the extra-cellular matrix. All of these components together form the tumour microenvironment and this plays an active part in tumour development and progression. Hence, mimicking the disease as a simple monolayer of one cell type bears little similarity to the true situation. Needless to say, it has its advantages, such as fairly easy procedures, high reproducibility and cost-effectiveness. But despite that, the aim of creating a more realistic model of the tumour setting and environment led to the increased use of 3D models to better recreate the dynamic nature of a living tumour. Another layer of complexity is added by including scaffolds such as gels that mimic the extracellular matrix and tissue of the organ of origin, and it all is taken even further with techniques such as 3D-bioprinting, organ-on-a-chip models and microfluidic devices – all with the aim of creating the most representative model of the tumour setting in a living body (in vivo) without the necessity of performing animal experiments and before translating the results into clinical trials.

My research focusses on the development of 3D models of triple-negative breast cancer (TNBC) to analyse characteristics and molecular events in this rare and aggressive subtype of breast cancer. TNBC accounts for 15% of breast cancer diagnoses and has a higher chance of metastasis and recurrence than other types. The absence of the three typical targets for treatment – the estrogen receptor, the progesterone receptor and the HER2 receptor – makes this type of breast cancer difficult to manage and specific therapies are still missing. Generating a 3D co-culture of TNBC cells surrounded by other cell types such as fibroblasts and immune cells allows for the study of the interplay of these cells, the molecular interactions and reciprocal influences they have on each other. Furthermore, the 3D models can act as a platform for testing possible new drug compounds for the treatment of TNBC. The more realistic the testing models are, the more likely the results can be confirmed in the human setting as part of first clinical trials.

The cells are cultured in such a way that they form spheroids – balls of cells that have comparable interactions among themselves and with the surrounding media and surfaces as in vivo. The polarity of the cells is equivalent to those found in the body, as is their accessibility to oxygen and nutrients provided by the media around them. In vivo, cells typically experience different gradients of oxygen and numerous soluble factors and thus zones of different cell characteristics – outer proliferating layer, middle quiescent layer and inner hypoxic core – are formed. This dynamic setting of the tumour bulk in vivo cannot be adequately represented in 2D. Even in 3D it is difficult enough to consider as many influencing parameters as possible.

As mentioned before, the cancer cells forming the tumour are surrounded by the extra-cellular matrix (ECM) consisting of various macromolecules such as collagen, enzymes or glycoproteins providing structural and biochemical support and fulfilling cell adhesion, cell-cell communication and cell differentiation purposes. To imitate this in the lab, the tumour spheroid is not only surrounded by cell culture media, but by the addition of scaffolding matrices such as collagen gels, the stiffness of and interaction with the ECM in vivo is replicated. I add collagen or Matrigel, a different type of hydrogel containing various macromolecules, to the cell culture experiments and thereby aim to assess the influence of these on cell characteristics such as proliferation, migration, invasion and drug response.

I am especially interested in the mechanisms of action of an enzyme called iNOS (inducible Nitric Oxide Synthase) that produces the gaseous signalling molecule NO (Nitric Oxide). It has been shown that high expression levels of iNOS in estrogen-receptor negative breast cancers, to which TNBC belongs, is a predictor for poor outcome with the survival rate decreasing correspondingly. The aim is to understand how iNOS or its product NO act, how they drive the tumour development and progression and what pathways or other molecules are affected by them. The tricky thing with iNOS is that is has very different effects depending on its concentration in vivo – ranging from proliferative, migratory and generally pro-tumorigenic to stress-related and anti-cancer effects. This makes it very hard to imitate its impact in the laboratory setting as the concentration ranges for certain effects are very small and difficult to hit. To achieve this, we work with NO-donating agents as well as with cells that stably express iNOS after we transferred the corresponding gene into the cells.

The overall aim is to increase our understanding of the role of the tumour microenvironment in the context of cancer generation and progression and more specifically the role of iNOS / NO in the pathogenesis of TNBC. This would enable us to discern specific targets for drug treatment and thereby help the outcomes and life quality of women diagnosed with TNBC.