Tumor-initiating cells (TIC) perpetuate tumor growth, enable therapeutic resistance, and drive initiation of successive tumors. Virtually nothing is known about the role of mechanotransductive signaling in controlling TIC tumorigenesis, despite the recognized importance of altered mechanics in tissue dysplasia and the common observation that extracellular matrix (ECM) stiffness strongly regulates cell behavior. To address this open question, we cultured primary human glioblastoma (GBM) TICs on laminin-functionalized ECMs spanning a range of stiffnesses. Surprisingly, we found that these cells were largely insensitive to ECM stiffness cues, evading the inhibition of spreading, migration, and proliferation typically imposed by compliant ECMs. We hypothesized that this insensitivity may result from insufficient generation of myosin-dependent contractile force. Indeed, we found that both pharmacologic and genetic activation of cell contractility through RhoA GTPase, Rho-associated kinase, or myosin light chain kinase restored stiffness-dependent spreading and motility, with TICs adopting the expected rounded and nonmotile phenotype on soft ECMs. Moreover, constitutive activation of RhoA restricted three-dimensional invasion in both spheroid implantation and Transwell paradigms. Orthotopic xenotransplantation studies revealed that control TICs formed tumors with classical GBM histopathology including diffuse infiltration and secondary foci, whereas TICs expressing a constitutively active mutant of RhoA produced circumscribed masses and yielded a 30% enhancement in mean survival time. This is the first direct evidence that manipulation of mechanotransductive signaling can alter the tumor-initiating capacity of GBM TICs, supporting further exploration of these signals as potential therapeutic targets and predictors of tumor-initiating capacity within heterogeneous tumor cell populations.

KIF3A, a component of the kinesin-2 motor, is necessary for the progression of diverse tumor types. This is partly due to its role in regulating ciliogenesis and cell responsiveness to sonic hedgehog (SHH). Notably, primary cilia have been detected in human glioblastoma multiforme (GBM) tumor biopsies and derived cell lines. Here, we asked whether disrupting KIF3A in GBM cells affected ciliogenesis, in vitro growth and responsiveness to SHH, or tumorigenic behavior in vivo. We used a lentiviral vector to create three patient-derived GBM cell lines expressing a dominant negative, motorless form of Kif3a (dnKif3a). In all unmodified lines, we found that most GBM cells were capable of producing ciliated progeny and that dnKif3a expression in these cells ablated ciliogenesis. Interestingly, unmodified and dnKif3a-expressing cell lines displayed differential sensitivities and pathway activation to SHH and variable tumor-associated survival following mouse xenografts. In one cell line, SHH-induced cell proliferation was prevented in vitro by either expressing dnKif3a or inhibiting SMO signaling using cyclopamine, and the survival times of mice implanted with dnKif3a-expressing cells were increased. In a second line, expression of dnKif3a increased the cells' baseline proliferation while, surprisingly, sensitizing them to SHH-induced cell death. The survival times of mice implanted with these dnKif3a-expressing cells were decreased. Finally, expression of dnKif3a in a third cell line had no effect on cell proliferation, SHH sensitivity, or mouse survival times. These findings indicate that KIF3A is essential for GBM cell ciliogenesis, but its role in modulating GBM cell behavior is highly variable.