In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in ...hGBM TPCs. Cytofluorimetric sorting into EphA2High and EphA2Low populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications.
► Stem-like TPCs in hGBMs highly express the EphA2 receptor ► High EphA2 expression supports the undifferentiated state and self-renewal in TPCs ► TPC content and tumorigenicity are higher in EphA2High than EphA2Low hGBM cells ► EphrinA1-Fc treatment or EPHA2 silencing lessen TPC self-renewal and tumorigenicity
Metabolic reprogramming has been described in rapidly growing tumors, which are thought to mostly contain fast‐cycling cells (FCCs) that have impaired mitochondrial function and rely on aerobic ...glycolysis. Here, we characterize the metabolic landscape of glioblastoma (GBM) and explore metabolic specificities as targetable vulnerabilities. Our studies highlight the metabolic heterogeneity in GBM, in which FCCs harness aerobic glycolysis, and slow‐cycling cells (SCCs) preferentially utilize mitochondrial oxidative phosphorylation for their functions. SCCs display enhanced invasion and chemoresistance, suggesting their important role in tumor recurrence. SCCs also demonstrate increased lipid contents that are specifically metabolized under glucose‐deprived conditions. Fatty acid transport in SCCs is targetable by pharmacological inhibition or genomic deletion of FABP7, both of which sensitize SCCs to metabolic stress. Furthermore, FABP7 inhibition, whether alone or in combination with glycolysis inhibition, leads to overall increased survival. Our studies reveal the existence of GBM cell subpopulations with distinct metabolic requirements and suggest that FABP7 is central to lipid metabolism in SCCs and that targeting FABP7‐related metabolic pathways is a viable therapeutic strategy.
Synopsis
Transcriptomic and metabolomic profiling of primary brain tumor cells demonstrate that functionally different glioblastoma (GBM) cell subpopulations depend on distinct metabolic pathways for their growth and survival. More invasive slow cycling tumor cells rely on oxidative phosphorylation and lipid metabolism, suggesting targetable candidates for the inhibition of treatment‐resistant tumors.
Patient‐derived GBM cells contain fast‐cycling cells (FCCs) relying on aerobic glycolysis and slow‐cycling cells (SCCs) depending on mitochondrial oxidative phosphorylation in vivo.
SCCs show increased resistance, invasion, and metabolic gene signatures characteristic of recurrent tumors.
SCCs show increased levels of metabolites and components involved in lipid metabolism, storage, and transport.
Block of FABP7‐dependent exogenous fatty acid uptake decreases resistance of SCCs to chemotherapy and glucose deprivation.
Oxidative phosphorylation and lipid metabolism specify distinct energetic set‐up of invasive brain tumor cells.
Cancer is a disease of single cells that expresses itself at the population level. The striking similarities between initiation and growth of tumors and dynamics of biological populations, and ...between metastasis and ecological invasion and community dynamics suggest that oncology can benefit from an ecological perspective to improve our understanding of cancer biology. Tumors can be viewed as complex, adaptive, and evolving systems as they are spatially and temporally heterogeneous, continually interacting with each other and with the microenvironment and evolving to increase the fitness of the cancer cells. We argue that an eco‐evolutionary perspective is essential to understand cancer biology better. Furthermore, we suggest that ecologically informed therapeutic approaches that combine standard of care treatments with strategies aimed at decreasing the evolutionary potential and fitness of neoplastic cells, such as disrupting cell‐to‐cell communication and cooperation, and preventing successful colonization of distant organs by migrating cancer cells, may be effective in managing cancer as a chronic condition.
We argue that an eco‐evolutionary perspective is essential to understand cancer biology better. Furthermore, we suggest that ecologically‐informed therapeutic approaches that combine standard of care treatments with strategies aimed at decreasing the evolutionary potential and fitness of neoplastic cells, such as disrupting cell‐to‐cell communication and cooperation and preventing successful colonization of distant organs by migrating cancer cells, may be effective in managing cancer as a chronic condition.
Glioblastoma (GBM) remains the most pervasive and lethal of all brain malignancies. One factor that contributes to this poor prognosis is the highly invasive character of the tumor. GBM is ...characterized by microscopic infiltration of tumor cells throughout the brain, whereas non-neural metastases, as well as select lower grade gliomas, develop as self-contained and clearly delineated lesions. Illustrated by rodent xenograft tumor models as well as pathological human patient specimens, we present evidence that one fundamental switch between these two distinct pathologies--invasion and noninvasion--is mediated through the tumor extracellular matrix. Specifically, noninvasive lesions are associated with a rich matrix containing substantial amounts of glycosylated chondroitin sulfate proteoglycans (CSPGs), whereas glycosylated CSPGs are essentially absent from diffusely infiltrating tumors. CSPGs, acting as central organizers of the tumor microenvironment, dramatically influence resident reactive astrocytes, inducing their exodus from the tumor mass and the resultant encapsulation of noninvasive lesions. Additionally, CSPGs induce activation of tumor-associated microglia. We demonstrate that the astrogliotic capsule can directly inhibit tumor invasion, and its absence from GBM presents an environment favorable to diffuse infiltration. We also identify the leukocyte common antigen-related phosphatase receptor (PTPRF) as a putative intermediary between extracellular glycosylated CSPGs and noninvasive tumor cells. In all, we present CSPGs as critical regulators of brain tumor histopathology and help to clarify the role of the tumor microenvironment in brain tumor 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.
During development and continuing into adulthood, stem cells function as a reservoir of undifferentiated cell types, whose role is to support cell genesis in several tissues and organs. In the adult, ...they play an essential homeostatic role by replacing differentiated cells that are lost due to physiological turnover, injury, or disease. The discovery of such cells in the adult mammalian central nervous system (CNS), an organ traditionally thought to have little or no regenerative capacity, has opened the door to the possibility of designing innovative regenerative therapeutics, an unexpected concept in neurobiology 15 years ago. In 1992, to detect precursor cells in the adult brain, we employed a serum-free culture system whereby the majority of primary differentiated CNS cells did not survive but a small population of EGF-responsive cells were maintained in an undifferentiated state and proliferated to form clusters, called neurospheres (Reynolds and Weiss, Science 255:1707-1710, 1992). These neurospheres could be (a) dissociated to form numerous secondary spheres or (b) induced to differentiate, generating the three major cell types of the CNS. This chapter outlines the adult mammalian NSC culture methodology and provides technical details of the neurosphere assay to achieve reproducible cultures.
Based on the clonal evolution model and the assumption that the vast majority of tumor cells are able to propagate and drive tumor growth, the goal of cancer treatment has traditionally been to kill ...all cancerous cells. This theory has been challenged recently by the cancer stem cell (CSC) hypothesis, that a rare population of tumor cells, with stem cell characteristics, is responsible for tumor growth, resistance, and recurrence. Evidence for putative CSCs has been described in blood, breast, lung, prostate, colon, liver, pancreas, and brain. This new hypothesis would propose that indiscriminate killing of cancer cells would not be as effective as selective targeting of the cells that are driving long-term growth (ie, the CSCs) and that treatment failure is often the result of CSCs escaping traditional therapies.The CSC hypothesis has gained a great deal of attention because of the identification of a new target that may be responsible for poor outcomes of many aggressive cancers, including malignant glioma. As attractive as this hypothesis sounds, especially when applied to tumors that respond poorly to current treatments, we will argue in this article that the proposal of a stemlike cell that initiates and drives solid tissue cancer growth and is responsible for therapeutic failure is far from proven. We will present the point of view that for most advanced solid tissue cancers such as glioblastoma multiforme, targeting a putative rare CSC population will have little effect on patient outcomes. This review will cover problems with the CSC hypothesis, including applicability of the hierarchical model, inconsistencies with xenotransplantation data, and nonspecificity of CSC markers.
Hedgehog (Hh) signaling is crucial for the generation and maintenance of both embryonic and adult stem cells, thereby regulating development and tissue homeostasis. In the developing neocortex, Sonic ...Hedgehog (Shh) regulates neural progenitor cell proliferation. During neurogenesis, radial glial cells of the ventricular zone (VZ) are the predominant neocortical progenitors that generate neurons through both symmetric and asymmetric divisions. Despite its importance, relatively little is known of the molecular pathways that control the switch from symmetric proliferative to differentiative/neurogenic divisions in neural progenitors.
Here, we report that conditional inactivation of Patched1, a negative regulator of the Shh pathway, in Nestin positive neural progenitors of the neocortex leads to lamination defects due to improper corticogenesis and an increase in the number of symmetric proliferative divisions of the radial glial cells. Hedgehog-activated VZ progenitor cells demonstrated a concomitant upregulation of Hes1 and Blbp, downstream targets of Notch signaling. The Notch signaling pathway plays a pivotal role in the maintenance of stem/progenitor cells and the regulation of glial versus neuronal identity. To study the effect of Notch signaling on Hh-activated neural progenitors, we inactivated both Patched1 and Rbpj, a transcriptional mediator of Notch signaling, in Nestin positive cells of the neocortex.
Our data indicate that by mid neurogenesis (embryonic day 14.5), attenuation of Notch signaling reverses the effect of Patched1 deletion on neurogenesis by restoring the balance between symmetric proliferative and neurogenic divisions. Hence, our results demonstrate that correct corticogenesis is an outcome of the interplay between the Hh and Notch signaling pathways.
Glioblastoma is the most aggressive and deadly brain cancer. There is growing interest to develop drugs that specifically target to glioblastoma tumor-initiating cells (TICs). However, the ...cost-effective production of large numbers of high quality glioblastoma TICs for drug discovery with current cell culturing technologies remains very challenging. Here, we report a new method that cultures glioblastoma TICs in microscale alginate hydrogel tubes (or AlgTubes). The AlgTubes allowed long-term culturing (~50 days, 10 passages) of glioblastoma TICs with high growth rate (~700-fold expansion/14 days), high cell viability and high volumetric yield (~3.0 × 10
cells/mL) without losing the stem cell properties, all offered large advancements over current culturing methods. This method can be applied for the scalable production of glioblastoma TICs at affordable cost for drug discovery.
Advancement in our understanding of the biology of adult stem cells and their therapeutic potential relies heavily on meaningful functional assays that can identify and measure stem cell activity in ...vivo and in vitro. In the mammalian nervous system, neural stem cells (NSCs) are often studied using a culture system referred to as the neurosphere assay. We previously challenged a central tenet of this assay, that all neurospheres are derived from a NSC, and provided evidence that it overestimates NSC frequency, rendering it inappropriate for quantitation of NSC frequency in relation to NSC regulation. Here we report the development and validation of the neural colony-forming cell assay (NCFCA), which discriminates stem from progenitor cells on the basis of their proliferative potential. We anticipate that the NCFCA will provide additional clarity in discerning the regulation of NSCs, thereby facilitating further advances in the promising application of NSCs for therapeutic use.
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