Validation of a 3D culture model for toxicity studies in malignant and non-cancerous cells

Abstract

Cancer is a widespread and commonly fatal disease in constant need of new treatments. Traditionally, new pharmaceuticals have been tested in 2D models. This is not ideal, as 2D environments do not reflect the in vivo milieu of the cells. Instead, it has been increasingly suggested to implement 3D models, which are thought to be more predictive of in vivo responses to treatment. It has been shown that cancerous cells are less sensitive to anti-cancer drugs when cultured in 3D. Thus, this study aimed to compare the effect of 2D and 3D environments on the potency of the MEK inhibitor trametinib in human cancerous A549 and non-cancerous LL47 cells. The 3D model used was an electrospun poly-caprolactone membrane, on which the cells were seeded and then treated with varying trametinib concentrations. The 2D model was a traditional cell culture plate. The study also investigated the mechanism of trametinib. It was shown that trametinib affects the amount of p-ERK, in line with data published by others. No statistically significant difference in potency could be shown between the models. However, after optimisation the 3D model itself was shown to be working and giving reproducible results, suggesting further use in preclinical studies.

Popular Abstract

2D vs 3D models – the point and effects of adding a third dimension in cancer research
The battle against cancer is an ongoing one, not just for individual patients, but for the pharmaceutical industry at large. To efficiently test new drug candidates, it is important to use accurate models. In this study, one model that is thought to provide some of this accuracy – a three-dimensional electrospun membrane – was investigated. While drug potency remained the same as in a more traditional model, some other interesting differences were revealed. All-in-all, the notion that this model has the potential to be used in drug development was supported.
Long before reaching any human trials, new potential drugs are tested in so called in vitro models. This typically involves testing the drug candidates on cells grown on flat, two-dimensional (2D) surfaces. While this method has been – and continues to be – very useful, it also has several flaws. A big concern is that the 2D environment this method provides is nothing like the three-dimensional (3D) environment of the human body. This may result in the cells behaving differently, and a drug candidate that seem promising in 2D tests may be useless for actually treating the disease in a patient. To combat this issue, more and more attention has been put on newer 3D models. In such models, the cells can grow in three dimensions, more like they would do naturally. Many studies have shown that cells are less sensitive to drugs in this kind of model, when comparing to a traditional 2D one. One example of a 3D model is electrospun membranes, which consist of very thin fibres that can be oriented in different ways. These membranes aim to mimic the extracellular matrix (ECM), which can be thought of as a scaffold to which the cells are attached in the body. In our study, this was the 3D model used for comparing 2D and 3D environments.
Of course, while there is a need for new cancer drugs, there are already working ones on the market. One drug that is used against certain cancers is trametinib. Trametinib inhibits MEK, an enzyme which is a part of the so called MAPK pathway, which is involved in cell growth and proliferation. Here, trametinib was used to treat the cells to see if the potency changed when going from a 2D to a 3D model. The cells used were human cancerous A549 cells, and human non-cancerous LL47 cells. The A549 cells have a mutation that results in their MAPK pathway always being active.
No potency change could be seen, neither between the different cells, nor when going from the 2D to the 3D model. However, there were some other changes of interest. Firstly, the non-cancerous LL47 cells seemed more reliant on their MAPK pathway in 3D. This is a hint that they do indeed behave differently in the different models. Secondly, the cancerous A549 cells showed a result in 3D that might indicate that they formed a tumour-like aggregate in this model. This is an advantage of a 3D model, as it mimics the tumours found in the body. Of course, further studies investigating this possible tumour-formation would be useful to confirm or deny this theory.
Leaving the 2D vs 3D comparison behind, the 3D model itself proved to be easy to work with and give reproducible results. Given the known advantages of 3D models in general, and in line with studies done by others, this highly suggests that this model should be included in more studies of new anti-cancer drugs.

• author: Andreasson Dahlgren, Kajsa
• supervisor: Malin Lindstedt
• organization: Department of Immunotechnology
• alternative title: Validering av en 3D-odlingsmodell för toxicitetsstudier i maligna och icke-cancerösa celler
• course: KIMM05 20211
• year: 2021
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: Dose-response, potency, in vitro, 2D model, 3D model, cancer, MEK inhibitor, trametinib, A549, LL47, Western blot
• language: English
• id: 9052626
• date added to LUP: 2021-06-23 11:16:28
• date last changed: 2021-06-23 11:16:28