Identification of melanoma brain invasion factors and their niche-dependentregulation through CRISPR-based technologies

With this project I aim to identify key factors which permits and likely leads the malignant melanoma cells to BM formation, and I aim to do so through the use of CRISPR-based technologies which modulate the transcription of 10 defined candidate genes for brain invasion in a BM-derived melanoma cellular model. We selected these candidate genes from melanoma BM-specific expression data coming from multiple tumor samples, which was then filtered for relevance on the metastatic process, association with prognosis and targetability. The first goal of this project consists in the establishment of CRISPR, CRISPR-activation and CRISPR-inhibition systems on a brain metastatic cell line (BMC1) and on a non-brain (lymph-node) metastatic cell line (MeWo) to then perform a transcriptional repression screen of 10 brain invasion candidate genes reading for the migratory and proliferation phenotype changes derived. Then, with the goal to assess the reprogramming or regulation that the astrocytes from the brain metastatic niche exert upon the melanoma cells, I will perform co-culture studies of BMC1 cells with the human astrocytes immortalized cell line SVG. In case the contact with the extracellular matrix, the tridimensional configuration or the astrocyte reactive state would be relevant for the reprogramming of the brain metastatic characteristics, I will perform 3D co-cultures with matrigel and use two different models of astrogliosis on the SVG cells: one consisting in the BDNF overexpression and the other of the pre-treatment to trigger the reactive state. The co-culture will be tested via flow cytometry and FACS sorted followed by qPCR of the first step of validation. Afterwards, as the last important step to get insight of the signaling associated with the reprogramming, I will further interrogate the resulting changes with QuantSeq. Overall, this project would generate as outcome the identification of important genes and pathways for the development of BM, nobel data on their impact on the two cycling melanoma phenotype states in the context of BM formation and their regulation derived by astrocytes, astrogliosis and its potential dependency of BDNF. Besides, in this project I also address the stem-like invasive cellular subset likely responsible for the drug resistance and increased brain tumor formation, through the detailed study of CD271 transcriptional alteration, included in the brain invasion candidate gene list.

ÖAW DOC

Zentrum für Biologische Wissenschaften