Funktionelle Charakterisierung des Wachstumsfaktors Midkine in Gliomzellen

Gliomas are the most common primary brain tumours. Subtypes can be divided into 4 different grades, classified by the World Health Organisation according to their progressiveness. Glioblastoma represents the most aggressive subtype, classified as WHO grade 4, with an average life expectancy of approximately 15 months after diagnosis. Due to its high invasiveness and heterogeneity, this tumour remains difficult to treat and new therapeutic approaches are increasingly being sought. Midkine (Mdk) is a heparin-binding growth factor known to be positively correlated with progression and invasion of several tumour types, including glioblastoma, and negatively correlated with survival. In this study, we aim to investigate the potential use of Mdk not only as a biomarker to facilitate diagnosis and monitoring during therapy, but also as a promising target to improve therapy itself.

So far, we have used Western blotting to determine Mdk levels in 16 different primary glioma cultures and the following glioblastoma cell lines: U87, LN229, U251, F98, A172, U343 and GL261. We also performed XTT cell viability assays with LN229 and U251 treated with 0 nM, 500 nM or 1 µM iMDK (Mdk inhibitor) for 24, 48, 72 and 96 hours. To assess the effect of Mdk expression, we developed transfectants of U87, U251 and LN229. Since LN229 and U251 showed high levels of Mdk expression, we chose these cell lines for knockout, while we chose U87 (very low levels of Mdk expression) for overexpression. The cell lines were transfected with an Mdk-pIRES Venus overexpression vector and sgRNA against Mdk cloned in a pL-CRISPR.EFS.PAC vector. Subsequently, the transfected cells were selected with Puromycin or Neomycin.

We were able to show that the different cell lines and primary cultures differ greatly in terms of Mdk protein level. Using the viability assay, we were also able to show that iMDK reduces cell growth in cell lines with initially high Mdk expression. With iMdk treatment, we observed a reduction in cell viability of glioblastoma cells up to 62.41% compared to the untreated cells after 96 hours of growth. In addition, an altered growth behaviour of the Mdk overexpression and knockout cells was detected.

The results presented suggest that Mdk promotes cell growth in glioblastoma cells and that its inhibition could reduce further progression of the disease. Its use as an additional therapeutic approach is promising and should be further investigated.