MARCKS as a mediator and target for pathological tunneling nanotubes between patient-derived glioblastoma cells and astrocytes.

During glioblastoma (GBM) progression, therapeutic resistance is influenced by a heterogeneous network of tumor- and tumor-promoting subpopulations within the tumor microenvironment. Therapeutic interventions against tumor cells, such as chemoradiotherapy, can further induce adaptive defense mechanisms mediated by sophisticated intercellular communication and aberrant signaling pathways. Tunneling nanotubes (TNTs) have emerged as key mediators of this process, promoting tumor cell survival through metabolic rescue while enabling the recruitment and reprogramming of surrounding normal cells into tumor-supportive phenotypes. This dynamic is exemplified in GBM, where TNT-mediated interactions between brain tumor-initiating cells (BTICs) and normal human astrocytes (NHAs) highlight the need to elucidate the molecular mediators underlying this crosstalk. Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) has never been investigated as a potential regulator of TNTs despite several overlapping signaling pathways and its influence on GBM therapeutic resistance. In the present study, we demonstrate a role for the MARCKS effector domain (ED) and PKC activation in the formation and functionality of TNTs between PTEN-null GBM BTICs and NHAs. We employ a MARCKS phosphorylation site (MPS) peptide derived from MARCKS effector domain (MED2), PKC-targeting drugs, and an inducible MARCKS ED U87 model to elucidate a potential role for MARCKS phosphorylation and PKC in TNT regulation between GBM cells (i.e., BTICs or U87s) and NHAs.