Brain and Breast Cancer Cells with PTEN Loss of Function Reveal Enhanced Durotaxis and RHOB Dependent Amoeboid Migration Utilizing 3D Scaffolds and Aligned Microfiber Tracts

SIMPLE SUMMARY: Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations are associated with brain tumor spreading and poor patient outcomes. GBM, and possibly TNBC, migrate on axons and blood vessels to disseminate in the brain; however, the mechanism is unresolved. There is a need for new therapeutic targets to blunt brain tumor spreading. Using 3D aligned printed microfibers mimicking brain structures proved that RHOB, in addition to ROCK and PTEN signaling, were essential for GBM and TNBC 3D cell migration. GBM and TNBC cell lines with PTEN loss of function and high RHOB expression exhibited amoeboid morphology with increased durotaxis, binding and migration speed on 3D microfibers, in contrast to the PTEN wildtype. Depending on the PTEN genotype, RHO-ROCK-PTEN inhibitors or PTEN rescue significantly regulated these properties. Regarding GBM and brain metastasizing TNBC, we conclude that RHOB inhibitors could play a novel role for improved therapy response and patient outcome. ABSTRACT: Background: Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with poor patient outcome, thus new therapeutic targets are needed. To understand signaling, controlling the dynamics and mechanics of brain tumor cell migration, we implemented GBM and TNBC cell lines and designed 3D aligned microfibers and scaffolds mimicking brain structures. Methods: 3D microfibers and scaffolds were printed using melt electrowriting. GBM and TNBC cell lines with opposing PTEN genotypes were analyzed with RHO-ROCK-PTEN inhibitors and PTEN rescue using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while scanning electron microscopy and confocal microscopy addressed cell morphology. Results: In contrast to the PTEN wildtype, GBM and TNBC cells with PTEN loss of function yielded enhanced durotaxis, topotaxis, adhesion, amoeboid migration on 3D microfibers and significant high RHOB expression. Functional studies concerning RHOB-ROCK-PTEN signaling confirmed the essential role for the above cellular processes. Conclusions: This study demonstrates a significant role of the PTEN genotype and RHOB expression for durotaxis, adhesion and migration dependent on 3D. GBM and TNBC cells with PTEN loss of function have an affinity for stiff brain structures promoting metastasis. 3D microfibers represent an important tool to model brain metastasizing tumor cells, where RHO-inhibitors could play an essential role for improved therapy.