Transcriptomic analysis reveals that BMP4 sensitizes glioblastoma tumor-initiating cells to mechanical cues.

The poor prognosis of glioblastoma (GBM) is associated with a highly invasive stem-like subpopulation of tumor-initiating cells (TICs), which drive recurrence and contribute to intra-tumoral heterogeneity through differentiation. These TICs are better able to escape extracellular matrix-imposed mechanical restrictions on invasion than their more differentiated progeny, and sensitization of TICs to extracellular matrix mechanics extends survival in preclinical models of GBM. However, little is known about the molecular basis of the relationship between TIC differentiation and mechanotransduction. Here we explore this relationship through a combination of transcriptomic analysis and studies with defined-stiffness matrices. We show that TIC differentiation induced by bone morphogenetic protein 4 (BMP4) suppresses expression of proteins relevant to extracellular matrix signaling and sensitizes TIC spreading to matrix stiffness. Moreover, our findings point towards a previously unappreciated connection between BMP4-induced differentiation, mechanotransduction, and metabolism. Notably, stiffness and differentiation modulate oxygen consumption, and inhibition of oxidative phosphorylation influences cell spreading in a stiffness- and differentiation-dependent manner. Our work integrates bioinformatic analysis with targeted molecular measurements and perturbations to yield new insight into how morphogen-induced differentiation influences how GBM TICs process mechanical inputs.

Constitutive activation of myosin-dependent contractility sensitizes glioma tumor-initiating cells to mechanical inputs and reduces tissue invasion.

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.

Matrix regulation of tumor-initiating cells.

The recognition that the progression of many tumors may be driven by specific subpopulations of cells with stem/progenitor-like properties (tumor-initiating cells or TICs, a.k.a. cancer stem cells) represents an important recent paradigm shift in cancer biology and therapeutics. TICs in solid tissues are expected to interface with the extracellular matrix (ECM), which can strongly influence cell behavior through a variety of biochemical and biophysical mechanisms. Understanding ECM regulation of TIC behavior is important for developing strategies to isolate, expand, and characterize TICs in a laboratory setting and for understanding the roles ECM-based inputs may play in disease progression and therapy. In this chapter, we discuss how the ECM regulates TICs, starting with a brief overview of TIC biology, isolation, and characterization, molecular mechanisms through which TICs may be regulated by ECM-based signals, and the potential importance of these signals to TIC-driven tumor progression and metastasis.