Pre-clinical research

Pre-clinical research forms the foundation of the four elements mentioned on the previous page. Carrying this out enables the start and progress of the actual research. In the Netherlands, clinical trials on children are not permitted, therefore this research requires animal testing. The animal model will be used for the purpose of diagnostics, also called ‘imaging’, and for the testing of medicines.

To date, little research has been done globally to develop a reliable DIPG animal model. Why is this model needed?

• Before new potential medicines can be tested on children, they must first be tested on animals. This is not only to determine if they are effective, but also to define that they are not too toxic for the rest of the body. In other words, the malignant glioma has to be eradicated without causing the person to die. It has been known to occur that the results obtained in the labs differ from those seen in a living organism. An organism is complex and there are many factors that can influence it.

• The tumor’s response to the therapy has to be tested at the brain stem. Due to its delicate and specific anatomy it differs largely from other parts of the brain, which might play a key role in the quantity of the medicine that actually reaches the malignant area. In other words, the anatomy of this part of the brain and the surroundings of the glioma can have an impact on the effect of the doses and also ultimately the effectiveness of the medicine. In fact, specific techniques are being researched to apply medicine in a targeted manner.

The researchers would rather avoid animal testing, however, there are no alternative strategies available to even research how organisms respond to medicines and surgery techniques.

In the Netherlands, researchers had to apply for permission to carry out animal testing from the Animal Ethical Committee (DEC). The research team was fully prepared and knew exactly how to best execute the tests. However, the approval process was significantly delayed due to various adaptations in the actual testing that the DEC wanted to make. The best part of 2009 was unfortunately spent waiting for a response from the DEC. Final approval from the DEC was received in March / April 2010, fortunately!

The researchers in the lab first started by keeping the cells, ‘normal’ glioma cells and later also DIPG cells, alive and to increase their number. Subsequently, they learned how to color the cells and to flag them: for instance, ‘lentivirus transduction’. With this technique it is possible to follow and examine the growth curve of the glioma cells.

See the graphics below that illustrate this:

Mice are used for the animal model, and they were first injected with U87 glioma cells. This cell line was used more often previously so the technique could be learned. What was new, was the injection of these cells in the pons.

In 2009, E98 glioma cells were injected and this resulted in the development of a DIPG mouse model, since E98 (unlike U87) has a diffuse growth pattern, also when injected in the pons.

Progression: apply CED
With CED (Convection Enhanced Delivery), medication will be injected under pressure directly into the brain stem.

The graphic below shows how CED could be applied in practice.

CED

As said earlier, clinical trials on children are not permitted. In order to accelerate the process and still offer a treatment in case of progression, the aim is to use the known medicine Carmustine. This medicine is already being used as a therapy against adult gliomas.

The steps are as follows:
• The test animal receives Carmustine in the brain stem via CED in order to test for side-effects
• It is then given to a test animal with DIPG
• If proven to be safe, it will be included in the clinical protocol for children with a progressive DIPG