Susobhan Sarkar

Tenascin-C Stimulates Glioma Cell Invasion through Matrix Metalloproteinase-12

The capacity of glioma cells to invade extensively within the central nervous system is a major cause of the high morbidity rate of primary malignant brain tumors. Glioma cell invasion involves the attachment of tumor cells to extracellular matrix (ECM), degradation of ECM components, and subsequent penetration into adjacent brain structures. These processes are accomplished in part by matrix metalloproteinases (MMP) within a three-dimensional milieu of the brain parenchyma. As the majority of studies have used a two-dimensional monolayer culture system, we have used a three-dimensional matrix of collagen type I gel to address glioma-secreted proteases, ECM, and invasiveness of glioma cells. We show that in a three-dimensional collagen type I matrix, the presence of tenascin-C, commonly elevated in high-grade gliomas, increased the invasiveness of glioma cells. The tenascin-C-mediated invasiveness was blocked by metalloproteinase inhibitors, but this did not involve the gelatinases (MMP-2 and MMP-9) commonly implicated in two-dimensional glioma growth. A thorough analysis of 21 MMPs and six members of a disintegrin and metalloproteinase domain showed that MMP-12 was increased in gliomas by tenascin-C in three-dimensional matrix. Furthermore, examinations of resected specimens revealed high MMP-12 levels in the high-grade glioblastoma multiforme tumors. Finally, a function-blocking antibody as well as small interfering RNA to MMP-12 attenuated the tenascin-C-stimulated glioma invasion. These results identify a new factor, MMP-12, in regulating glioma invasiveness through interaction with tenascin-C.

Therapeutic activation of macrophages and microglia to suppress brain tumor-initiating cells

Brain tumor initiating cells (BTICs) contribute to the genesis and recurrence of gliomas. We examined whether the microglia and macrophages that are abundant in gliomas alter BTIC growth. We found that microglia derived from non-glioma human subjects markedly mitigated the sphere-forming capacity of glioma patient-derived BTICs in culture by inducing the expression of genes that control cell cycle arrest and differentiation. This sphere-reducing effect was mimicked by macrophages, but not by neurons or astrocytes. Using a drug screen, we validated amphotericin B (AmpB) as an activator of monocytoid cells and found that AmpB enhanced the microglial reduction of BTIC spheres. In mice harboring intracranial mouse or patient-derived BTICs, daily systemic treatment with non-toxic doses of AmpB substantially prolonged life. Notably, microglia and monocytes cultured from glioma patients were inefficient at reducing the sphere-forming capacity of autologous BTICs, but this was rectified by AmpB. These results provide new insights into the treatment of gliomas.Brain tumor initiating cells (BTICs) contribute to the genesis and recurrence of gliomas. We examined whether the microglia and macrophages that are abundant in gliomas alter BTIC growth. We found that microglia derived from non-glioma human subjects markedly mitigated the sphere-forming capacity of glioma patient–derived BTICs in culture by inducing the expression of genes that control cell cycle arrest and differentiation. This sphere-reducing effect was mimicked by macrophages, but not by neurons or astrocytes. Using a drug screen, we validated amphotericin B (AmpB) as an activator of monocytoid cells and found that AmpB enhanced the microglial reduction of BTIC spheres. In mice harboring intracranial mouse or patient-derived BTICs, daily systemic treatment with non-toxic doses of AmpB substantially prolonged life. Notably, microglia and monocytes cultured from glioma patients were inefficient at reducing the sphere-forming capacity of autologous BTICs, but this was rectified by AmpB. These results provide new insights into the treatment of gliomas.

A dialog between glioma and microglia that promotes tumor invasiveness through the CCL2/CCR2/interleukin-6 axis

Glioma cells in situ are surrounded by microglia, suggesting the potential of glioma-microglia interactions to produce various outcomes. As chemokines are important mediators of cell-cell communication, we sought first to identify commonly expressed chemokines in 16 human glioma lines. We found CCL2 (macrophage chemoattractant protein-1) messenger RNA to be expressed by the majority of glioma lines. However, these lines did not express the CCL2 receptor, CCR2, which was found on microglia. Next, we overexpressed CCL2 in the U87 glioma line, which has low basal level of CCL2, to investigate the hypothesis that glioma-secreted CCL2 interacts with microglia to affect glioma growth. Stable clones with 10- to 12-fold elevation of CCL2 have similar growth rate and invasive capacity as vector controls when cultured in isolation. However, in coculture with microglia in a three-dimensional collagen gel matrix, the invasiveness of CCL2-overexpressing clones was increased. Gene array analyses were then undertaken and they revealed that interleukin (IL)-6 was consistently increased in the coculture. Recombinant IL-6 enhanced the invasiveness of glioma cells when these were cultured alone, whereas a neutralizing antibody to IL-6 attenuated the microglia-stimulated glioma invasiveness. Finally, we found that human glioma specimens in situ contained IL-6 immunoreactivity that was expressed on CD68+ cells. This study has uncovered a mechanism by which glioma cells exploit microglia for increased invasiveness. Specifically, glioma-derived CCL2 acts upon CCR2-bearing microglia, which then produces IL-6 to stimulate gliomas. The CCL2/CCR2/IL-6 loop is a potential therapeutic target for the currently incurable malignant gliomas.

ADAM-9 is a novel mediator of tenascin-C-stimulated invasiveness of brain tumor–initiating cells

BACKGROUND Tenascin-C (TNC), an extracellular matrix protein overexpressed in malignant gliomas, stimulates invasion of conventional glioma cell lines (U251, U87). However, there is a dearth of such information on glioma stemlike cells. Here, we have addressed whether and how TNC may regulate the invasiveness of brain tumor-initiating cells (BTICs) that give rise to glioma progenies. METHODS Transwell inserts coated with extracellular matrix proteins were used to determine the role of TNC in BTIC invasion. Microarray analysis, lentiviral constructs, RNA interference-mediated knockdown, and activity assay ascertained the role of proteases in TNC-stimulated BTIC invasion in culture. Involvement of proteases was validated using orthotopic brain xenografts in mice. RESULTS TNC stimulated BTIC invasiveness in a metalloproteinase-dependent manner. A global gene expression screen identified the metalloproteinase ADAM-9 as a potential regulator of TNC-stimulated BTIC invasiveness, and this was corroborated by an increase of ADAM-9 protein in 4 glioma patient-derived BTIC lines. Notably, RNA interference to ADAM-9, as well as inhibition of mitogen-activated protein kinase 8 (c-Jun NH2-terminal kinase), attenuated TNC-stimulated ADAM-9 expression, proteolytic activity, and BTIC invasiveness. The relevance of ADAM-9 to tumor invasiveness was validated using resected human glioblastoma specimens and orthotopic xenografts where elevation of ADAM-9 and TNC expression was prominent at the invasive front of the tumor. CONCLUSIONS This study has identified TNC as a promoter of the invasiveness of BTICs through a mechanism involving ADAM-9 proteolysis via the c-Jun NH2-terminal kinase pathway.

Glioblastoma-associated microglia and macrophages: targets for therapies to improve prognosis

Glioblastoma is the most common and most malignant primary adult human brain tumour. Diagnosis of glioblastoma carries a dismal prognosis. Treatment resistance and tumour recurrence are the result of both cancer cell proliferation and their interaction with the tumour microenvironment. A large proportion of the tumour microenvironment consists of an inflammatory infiltrate predominated by microglia and macrophages, which are thought to be subverted by glioblastoma cells for tumour growth. Thus, glioblastoma-associated microglia and macrophages are logical therapeutic targets. Their emerging roles in glioblastoma progression are reflected in the burgeoning research into therapeutics directed at their modification or elimination. Here, we review the biology of glioblastoma-associated microglia and macrophages, and model systems used to study these cells in vitro and in vivo. We discuss translation of results using these model systems and review recent advances in immunotherapies targeting microglia and macrophages in glioblastoma. Significant challenges remain but medications that affect glioblastoma-associated microglia and macrophages hold considerable promise to improve the prognosis for patients with this disease.

Reduction of protein kinase C delta attenuates tenascin-C stimulated glioma invasion in three-dimensional matrix

The invasiveness of glioma cells, a major cause of mortality in malignant brain tumors, is mediated in part by the cellular microenvironment. We have reported that in a three-dimensional matrix of type 1 collagen (3D-CL) gel, the extracellular matrix protein tenascin-C (TN) increased the invasiveness of glioma cells through the downstream production of matrix metalloproteinase (MMP)-12. In the present study, we have investigated the signaling mechanisms involved in the TN-stimulated glioma invasiveness. We found that the pan protein kinase C (PKC) inhibitor, bisindolylmaleimide I, decreased TN-enhanced glioma invasion in 3D-CL. Calphostin C, an inhibitor of conventional and novel PKC isozymes, and the relatively selective PKCdelta inhibitor rottlerin decreased TN-stimulated glioma invasiveness in a concentration- and time-dependent manner. These findings of the possible involvement of PKCdelta was supported by its translocation from the cytosol to membrane fraction in 3D-CL gel supplemented with TN as detected by western blot assays and immunofluorescence microscopy and by elevation of PKCdelta enzyme activity. Moreover, pharmacological blockade of PKCdelta decreased MMP-12 levels and glioma invasiveness. Finally, small interfering RNA to PKCdelta reduced TN-stimulated glioma invasiveness concurrent with decreased MMP-12 production. Our results implicate PKCdelta as a therapeutic target to reduce MMP-12 expression and glioma invasiveness when tumor cells are stimulated by the TN-enriched glioma microenvironment.

T Cell Exhaustion in Glioblastoma: Intricacies of Immune Checkpoints

Glioblastoma is an aggressive and incurable primary brain tumor. While the blockade of immune checkpoints leads to reversal of T cell exhaustion in many cancers, the efficacy of this therapy in glioblastoma requires further consideration of the brain microenvironment beyond T cell activity. Neural cells are crucially dependent on glucose for survival, and tumor cells rabidly consume glucose; the glucose-deprived microenvironment further elevates immune checkpoint molecules to benefit tumor growth and exacerbate T cell exhaustion. We review here how immune checkpoints drive exhaustion in T cells while favoring tumor metabolism, and discuss how glucose competition in the unique CNS milieu is an important consideration to improve the outcomes of immune checkpoint blockade in glioblastoma.

Activation of NOTCH signaling by tenascin-C promotes growth of human brain tumor-initiating cells

Oncogenic signaling by NOTCH is elevated in brain tumor-initiating cells (BTIC) in malignant glioma, but the mechanism of its activation is unknown. Here we provide evidence that tenascin-C (TNC), an extracellular matrix protein prominent in malignant glioma, increases NOTCH activity in BTIC to promote their growth. We demonstrate the proximal localization of TNC and BTIC in human glioblastoma specimens and in orthotopic murine xenografts of human BTIC implanted intracranially. In tissue culture, TNC was superior amongst several extracellular matrix proteins in enhancing the sphere-forming capacity of glioma patient-derived BTIC. Exogenously applied or autocrine TNC increased BTIC growth through an α2β1 integrin-mediated mechanism that elevated NOTCH ligand Jagged1 (JAG1). Microarray analyses and confirmatory PCR and Western analyses in BTIC determined that NOTCH signaling components including JAG1, ADAMTS15, and NICD1/2 were elevated in BITC after TNC exposure. Inhibition of γ-secretase and metalloproteinase proteolysis in the NOTCH pathway, or silencing of α2β1 integrin or JAG1, reduced the proliferative effect of TNC on BTIC. Collectively, our findings identified TNC as a pivotal initiator of elevated NOTCH signaling in BTIC and define the establishment of a TN-α2β1-JAG1-NOTCH signaling axis as a candidate therapeutic target in glioma patients. Cancer Res; 77(12); 3231-43. ©2017 AACR.

MRI monitoring of monocytes to detect immune stimulating treatment response in brain tumor

Background Glioblastoma (GBM) is an aggressive brain cancer with a poor prognosis. The use of immune therapies to treat GBM has become a promising avenue of research. It was shown that amphotericin B (Amp B) can stimulate the innate immune system and suppress the growth of brain tumor initiating cells (BTICs). However, it is not feasible to use histopathology to determine immune activation in patients. We developed an MRI technique that can rapidly detect a therapeutic response in animals treated with drugs that stimulate innate immunity. Ultra-small iron oxide nanoparticles (USPIOs) are MRI contrast agents that have been widely used for cell tracking. We hypothesized that the increased monocyte infiltration into brain tumors due to Amp B can be detected using USPIO-MRI, providing an indicator of early drug response. Methods We implanted human BTICs into severe combined immunodeficient mice and allowed the tumor to establish before treating the animals with either Amp B or vehicle and then imaged them using MRI with USPIO (ferumoxytol) contrast. Results After 7 days of treatment, there was a significantly decreased T2* in the tumor of Amp B but not vehicle animals, suggesting that USPIO is carried into the tumor by monocytes. We validated our MRI results with histopathology and confirmed that Amp B-treated animals had significantly higher levels of macrophage/microglia that were colocalized with iron staining in their brain tumor compared with vehicle mice. Conclusion USPIO-MRI is a promising method of rapidly assessing the efficacy of anticancer drugs that stimulate innate immunity.

Microglia induces Gas1 expression in human brain tumor-initiating cells to reduce tumorigenecity

We reported previously that microglia decreased the growth of human brain tumor-initiating cells (BTICs). Through microarray analyses of BTICs exposed in vitro to microglia, we found the induction of several genes ascribed to have roles in cell cycle arrest, reduced cell proliferation and differentiation. Herein, we tested the hypothesis that one of these genes, growth arrest specific 1 (Gas1), is a novel growth reduction factor that is induced in BTICs by microglia. We found that microglia increased the expression of Gas1 transcript and protein in glioblastoma patient-derived BTIC lines. Using neurosphere assay we show that RNAi-induced reduction of Gas1 expression in BTICs blunted the microglia-mediated BTIC growth reduction. The role of Gas1 in mediating BTIC growth arrest was further validated using orthotopic brain xenografts in mice. When microglia-induced Gas1-expressing BTIC cells (mGas1-BTICs) were implanted intra-cranially in mice, tumor growth was markedly decreased; this was mirrored in the remarkable increase in survival of mGas1-BT025 and mGas1-BT048 implanted mice, compared to mice implanted with non-microglia-exposed BTIC cells. In conclusion, this study has identified Gas1 as a novel factor and mechanism through which microglia arrest the growth of BTICs for anti-tumor property.