James S. Hale

Laminin alpha 2 enables glioblastoma stem cell growth.

Glioblastomas (GBMs) are lethal cancers that display cellular hierarchies parallel to normal brain. At the apex are GBM stem cells (GSCs), which are relatively resistant to conventional therapy. Interactions with the adjacent perivascular niche are an important driver of malignancy and self‐renewal in GSCs. Extracellular matrix (ECM) cues instruct neural stem/progenitor cell–niche interactions, and the objective of our study was to elucidate its composition and contribution to GSC maintenance in the perivascular niche.

Chemokine CXCL12 in neurodegenerative diseases: an SOS signal for stem cell-based repair.

The dynamic relation between stem cells and their niche governs self-renewal and progenitor cell deployment. The chemokine CXCL12 (C-X-C motif ligand 12) and its signaling receptor CXCR4 (C-X-C motif receptor 4) represent an important pathway that regulates homing and maintenance of stem cells in neural niches. Neural stem cells (NSCs) reside in specific niches where communication with blood vessels is regulated by CXCL12. In neurodegenerative diseases and brain tumors, reactive vasculature forms in response to diseased tissues to create new niches that secrete CXCL12, enhancing the recruitment of neural progenitor cells (NPCs) to lesion sites via long-range migration. These observations suggest that the CXCL12-CXCR4 axis maintains NSCs and serves as an emergent salvage signal for initiating endogenous stem cell-based tissue repair.

Cancer Stem Cell-Specific Scavenger Receptor CD36 Drives Glioblastoma Progression

Glioblastoma (GBM) contains a self‐renewing, tumorigenic cancer stem cell (CSC) population which contributes to tumor propagation and therapeutic resistance. While the tumor microenvironment is essential to CSC self‐renewal, the mechanisms by which CSCs sense and respond to microenvironmental conditions are poorly understood. Scavenger receptors are a broad class of membrane receptors well characterized on immune cells and instrumental in sensing apoptotic cellular debris and modified lipids. Here, we provide evidence that CSCs selectively use the scavenger receptor CD36 to promote their maintenance using patient‐derived CSCs and in vivo xenograft models. CD36 expression was observed in GBM cells in addition to previously described cell types including endothelial cells, macrophages, and microglia. CD36 was enriched in CSCs and was able to functionally distinguish self‐renewing cells. CD36 was coexpressed with integrin alpha 6 and CD133, previously described CSC markers, and CD36 reduction resulted in concomitant loss of integrin alpha 6 expression, self‐renewal, and tumor initiation capacity. We confirmed oxidized phospholipids, ligands of CD36, were present in GBM and found that the proliferation of CSCs, but not non‐CSCs, increased with exposure to oxidized low‐density lipoprotein. CD36 was an informative biomarker of malignancy and negatively correlated to patient prognosis. These results provide a paradigm for CSCs to thrive by the selective enhanced expression of scavenger receptors, providing survival, and metabolic advantages. Stem Cells 2014;32:1746–1758

Differential Connexin Function Enhances Self-Renewal in Glioblastoma

SUMMARY The coordination of complex tumor processes requires cells to rapidly modify their phenotype and is achieved by direct cell-cell communication through gap junction channels composed of connexins. Previous reports have suggested that gap junctions are tumor suppressive based on connexin43 (Cx43), but this does not take into account differences in connexin-mediated ion selectivity and intercellular communication rate that drive gap junction diversity. We find that glioblastoma cancer stem cells (CSCs) possess functional gap junctions that can be targeted using clinically relevant compounds to reduce self-renewal and tumor growth. Our analysis reveals that CSCs express Cx46, while Cx43 is predominantly expressed in non-CSCs. During differentiation, Cx46 is reduced, while Cx43 is increased, and targeting Cx46 compromises CSC maintenance. The difference between Cx46 and Cx43 is reflected in elevated cell-cell communication and reduced resting membrane potential in CSCs. Our data demonstrate a pro-tumorigenic role for gap junctions that is dependent on connexin expression.

Lysophosphatidic acid suppresses endothelial cell CD36 expression and promotes angiogenesis via a PKD-1–dependent signaling pathway

In pathologic settings including retinal ischemia and malignant tumors, robust angiogenesis occurs despite the presence in the microenvironment of antiangiogenic proteins containing thrombospondin structural homology (TSR) domains. We hypothesized that antiangiogenesis mediated by TSR-containing proteins could be blunted by localized down-regulation of their cognate receptor on microvascular endothelial cells (MVECs), CD36. Through screening a panel of endothelial cell agonists, we found that lysophosphatidic acid (LPA) dramatically down-regulated CD36 surface expression on primary MVECs. LPA is a lipid-signaling mediator known to have proangiogenic activity, but the mechanisms are largely unknown. We observed that LPA caused CD36 down-regulation in a dose- and time-dependent manner and was long lasting. Down-regulation occurred at the transcriptional level via a signaling pathway involving specific LPA receptors and protein kinase D. LPA-induced MVEC CD36 repression significantly attenuated in vitro antiangiogenic responses to thrombospondin-1, including blockade of migration, tube formation, and VEGFR-2 signaling in response to fibroblast growth factor-2. In vivo relevance was demonstrated by showing that LPA abrogated thrombospondin-1-mediated inhibition of neovascularization of Matrigel plugs implanted in mice. Our data thus indicate that the proangiogenic mechanism of LPA may in part be via switching off the antiangiogenic switch mediated by TSR proteins and CD36.

Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer.

Advanced cancers display cellular heterogeneity driven by self‐renewing, tumorigenic cancer stem cells (CSCs). The use of cell lines to model CSCs is challenging due to the difficulty of identifying and isolating cell populations that possess differences in self‐renewal and tumor initiation. To overcome these barriers in triple‐negative breast cancer (TNBC), we developed a CSC system using a green fluorescent protein (GFP) reporter for the promoter of the well‐established pluripotency gene NANOG. NANOG‐GFP+ cells gave rise to both GFP+ and GFP− cells, and GFP+ cells possessed increased levels of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 and elevated self‐renewal and tumor initiation capacities. GFP+ cells also expressed mesenchymal markers and demonstrated increased invasion. Compared with the well‐established CSC markers CD24−/CD44+, CD49f, and aldehyde dehydrogenase (ALDH) activity, our NANOG‐GFP reporter system demonstrated increased enrichment for CSCs. To explore the utility of this system as a screening platform, we performed a flow cytometry screen that confirmed increased CSC marker expression in the GFP+ population and identified new cell surface markers elevated in TNBC CSCs, including junctional adhesion molecule‐A (JAM‐A). JAM‐A was highly expressed in GFP+ cells and patient‐derived xenograft ALDH+ CSCs compared with the GFP− and ALDH− cells, respectively. Depletion of JAM‐A compromised self‐renewal, whereas JAM‐A overexpression induced self‐renewal in GFP− cells. Our data indicate that we have defined and developed a robust system to monitor differences between CSCs and non‐CSCs in TNBC that can be used to identify CSC‐specific targets for the development of future therapeutic strategies. Stem Cells. Stem Cells 2015;33:2114–2125

Cancer Stem Cell-Secreted Macrophage Migration Inhibitory Factor Stimulates Myeloid Derived Suppressor Cell Function and Facilitates Glioblastoma Immune Evasion.

Shifting the balance away from tumor‐mediated immune suppression toward tumor immune rejection is the conceptual foundation for a variety of immunotherapy efforts currently being tested. These efforts largely focus on activating antitumor immune responses but are confounded by multiple immune cell populations, including myeloid‐derived suppressor cells (MDSCs), which serve to suppress immune system function. We have identified immune‐suppressive MDSCs in the brains of GBM patients and found that they were in close proximity to self‐renewing cancer stem cells (CSCs). MDSCs were selectively depleted using 5‐flurouracil (5‐FU) in a low‐dose administration paradigm, which resulted in prolonged survival in a syngeneic mouse model of glioma. In coculture studies, patient‐derived CSCs but not nonstem tumor cells selectively drove MDSC‐mediated immune suppression. A cytokine screen revealed that CSCs secreted multiple factors that promoted this activity, including macrophage migration inhibitory factor (MIF), which was produced at high levels by CSCs. Addition of MIF increased production of the immune‐suppressive enzyme arginase‐1 in MDSCs in a CXCR2‐dependent manner, whereas blocking MIF reduced arginase‐1 production. Similarly to 5‐FU, targeting tumor‐derived MIF conferred a survival advantage to tumor‐bearing animals and increased the cytotoxic T cell response within the tumor. Importantly, tumor cell proliferation, survival, and self‐renewal were not impacted by MIF reduction, demonstrating that MIF is primarily an indirect promoter of GBM progression, working to suppress immune rejection by activating and protecting immune suppressive MDSCs within the GBM tumor microenvironment. Stem Cells 2016;34:2026–2039

The malignant social network: Cell-cell adhesion and communication in cancer stem cells

Tumors contain a vastly complicated cellular network that relies on local communication to execute malignant programs. The molecular cues that are involved in cell-cell adhesion orchestrate large-scale tumor behaviors such as proliferation and invasion. We have recently begun to appreciate that many tumors contain a high degree of cellular heterogeneity and are organized in a cellular hierarchy, with a cancer stem cell (CSC) population identified at the apex in multiple cancer types. CSCs reside in unique microenvironments or niches that are responsible for directing their behavior through cellular interactions between CSCs and stromal cells, generating a malignant social network. Identifying cell-cell adhesion mechanisms in this network has implications for the basic understanding of tumorigenesis and the development of more effective therapies. In this review, we will discuss our current understanding of cell-cell adhesion mechanisms used by CSCs and how these local interactions have global consequences for tumor biology.

Glioblastoma Cancer Stem Cells Evade Innate Immune Suppression of Self-Renewal through Reduced TLR4 Expression

Tumors contain hostile inflammatory signals generated by aberrant proliferation, necrosis, and hypoxia. These signals are sensed and acted upon acutely by the Toll-like receptors (TLRs) to halt proliferation and activate an immune response. Despite the presence of TLR ligands within the microenvironment, tumors progress, and the mechanisms that permit this growth remain largely unknown. We report that self-renewing cancer stem cells (CSCs) in glioblastoma have low TLR4 expression that allows them to survive by disregarding inflammatory signals. Non-CSCs express high levels of TLR4 and respond to ligands. TLR4 signaling suppresses CSC properties by reducing retinoblastoma binding protein 5 (RBBP5), which is elevated in CSCs. RBBP5 activates core stem cell transcription factors, is necessary and sufficient for self-renewal, and is suppressed by TLR4 overexpression in CSCs. Our findings provide a mechanism through which CSCs persist in hostile environments because of an inability to respond to inflammatory signals.

Context Dependent Role of the CD36 - Thrombospondin - Histidine-Rich Glycoprotein Axis in Tumor Angiogenesis and Growth

The angiogenic switch is a promising therapeutic target in cancer. Work by our laboratory and others has described an important endogenous anti-angiogenic pathway mediated by interactions of CD36, a receptor on microvascular endothelial cells, with proteins containing thrombospondin (TSP) type I repeat domains (TSR). Recent studies revealed that circulating Histidine Rich Glycoprotein (HRG) inhibits the anti-angiogenic potential of the CD36-TSR pathway by functioning as a decoy receptor that binds and sequesters TSR proteins. As tumors of different origin display variable expression profiles of numerous targets, we hypothesized that the TSP-CD36-HRG axis regulates vascularization and growth in the tumor microenvironment in a context, or tumor type, dependent manner. Growth of Lewis Lung Carcinoma (LL2) and B16F1 Melanoma tumor cell implants in syngeneic wild type (WT), hrg, or cd36 null mice were used as a model to interrogate this signaling axis. LL2 tumor volumes were greater in cd36 null mice and smaller in hrg null mice compared to WT. Immunofluorescent staining showed increased vascularity in cd36 null vs. WT and WT vs. hrg null mice. No differences in tumor growth or vascularity were observed with B16F1 implants, consistent with lack of expression of TSP-1 in B16F1 cells. When TSR expression was induced in B16F1 cells by cDNA transfection, tumor growth and vascularity were similar to that seen with LL2 cells. These data show a role for CD36-mediated anti-angiogenic activity in the tumor microenvironment when TSR proteins are available and demonstrate that HRG modulates this activity. Further, they suggest a mechanism by which tumor microenvironments may regulate sensitivity to TSR containing proteins.