Anne C. Solga

RNA Sequencing of Tumor-Associated Microglia Reveals Ccl5 as a Stromal Chemokine Critical for Neurofibromatosis-1 Glioma Growth

Solid cancers develop within a supportive microenvironment that promotes tumor formation and growth through the elaboration of mitogens and chemokines. Within these tumors, monocytes (macrophages and microglia) represent rich sources of these stromal factors. Leveraging a genetically engineered mouse model of neurofibromatosis type 1 (NF1) low-grade brain tumor (optic glioma), we have previously demonstrated that microglia are essential for glioma formation and maintenance. To identify potential tumor-associated microglial factors that support glioma growth (gliomagens), we initiated a comprehensive large-scale discovery effort using optimized RNA-sequencing methods focused specifically on glioma-associated microglia. Candidate microglial gliomagens were prioritized to identify potential secreted or membrane-bound proteins, which were next validated by quantitative real-time polymerase chain reaction as well as by RNA fluorescence in situ hybridization following minocycline-mediated microglial inactivation in vivo. Using these selection criteria, chemokine (C-C motif) ligand 5 (Ccl5) was identified as a chemokine highly expressed in genetically engineered Nf1 mouse optic gliomas relative to nonneoplastic optic nerves. As a candidate gliomagen, recombinant Ccl5 increased Nf1-deficient optic nerve astrocyte growth in vitro. Importantly, consistent with its critical role in maintaining tumor growth, treatment with Ccl5 neutralizing antibodies reduced Nf1 mouse optic glioma growth and improved retinal dysfunction in vivo. Collectively, these findings establish Ccl5 as an important microglial growth factor for low-grade glioma maintenance relevant to the development of future stroma-targeted brain tumor therapies.

RNA-sequencing reveals oligodendrocyte and neuronal transcripts in microglia relevant to central nervous system disease

Expression profiling of distinct central nervous system (CNS) cell populations has been employed to facilitate disease classification and to provide insights into the molecular basis of brain pathology. One important cell type implicated in a wide variety of CNS disease states is the resident brain macrophage (microglia). In these studies, microglia are often isolated from dissociated brain tissue by flow sorting procedures [fluorescence‐activated cell sorting (FACS)] or from postnatal glial cultures by mechanic isolation. Given the highly dynamic and state‐dependent functions of these cells, the use of FACS or short‐term culture methods may not accurately capture the biology of brain microglia. In the current study, we performed RNA‐sequencing using Cx3cr1+/GFP labeled microglia isolated from the brainstem of 6‐week‐old mice to compare the transcriptomes of FACS‐sorted versus laser capture microdissection (LCM). While both isolation techniques resulted in a large number of shared (common) transcripts, we identified transcripts unique to FACS‐isolated and LCM‐captured microglia. In particular, ∼50% of these LCM‐isolated microglial transcripts represented genes typically associated with neurons and glia. While these transcripts clearly localized to microglia using complementary methods, they were not translated into protein. Following the induction of murine experimental autoimmune encephalomyelitis, increased oligodendrocyte and neuronal transcripts were detected in microglia, while only the myelin basic protein oligodendrocyte transcript was increased in microglia after traumatic brain injury. Collectively, these findings have implications for the design and interpretation of microglia transcriptome‐based investigations. GLIA 2015;63:531–548

NG2-cells are not the cell of origin for murine neurofibromatosis-1 (Nf1) optic glioma.

Low-grade glial neoplasms (astrocytomas) represent one of the most common brain tumors in the pediatric population. These tumors frequently form in the optic pathway (optic pathway gliomas, OPGs), especially in children with the neurofibromatosis type 1 (NF1)-inherited tumor predisposition syndrome. To model these tumors in mice, we have previously developed several Nf1 genetically-engineered mouse strains that form optic gliomas. However, there are three distinct macroglial cell populations in the optic nerve (astrocytes, NG2+ (nerve/glial antigen 2) cells and oligodendrocytes). The presence of NG2+ cells in the optic nerve raises the intriguing possibility that these cells could be the tumor-initiating cells, as has been suggested for adult glioma. In this report, we used a combination of complementary in vitro and novel genetically-engineered mouse strains in vivo to determine whether NG2+ cells could give rise to Nf1 optic glioma. First, we show that Nf1 inactivation results in a cell-autonomous increase in glial fibrillary acidic protein+ (GFAP+), but not in NG2+, cell proliferation in vitro. Second, similar to the GFAP-Cre transgenic strain that drives Nf1 optic gliomagenesis, NG2-expressing cells also give rise to all three macroglial lineages in vivo. Third, in contrast to the GFAP-Cre strain, Nf1 gene inactivation in NG2+ cells is not sufficient for optic gliomagenesis in vivo. Collectively, these data demonstrate that NG2+ cells are not the cell of origin for mouse optic glioma, and support a model in which gliomagenesis requires Nf1 loss in specific neuroglial progenitors during embryogenesis.

Estrogen activation of microglia underlies the sexually dimorphic differences in Nf1 optic glioma-induced retinal pathology.

Children with neurofibromatosis type 1 (NF1) develop low-grade brain tumors throughout the optic pathway. Nearly 50% of children with optic pathway gliomas (OPGs) experience visual impairment, and few regain their vision after chemotherapy. Recent studies have revealed that girls with optic nerve gliomas are five times more likely to lose vision and require treatment than boys. To determine the mechanism underlying this sexually dimorphic difference in clinical outcome, we leveraged Nf1 optic glioma (Nf1-OPG) mice. We demonstrate that female Nf1-OPG mice exhibit greater retinal ganglion cell (RGC) loss and only females have retinal nerve fiber layer (RNFL) thinning, despite mice of both sexes harboring tumors of identical volumes and proliferation. Female gonadal sex hormones are responsible for this sexual dimorphism, as ovariectomy, but not castration, of Nf1-OPG mice normalizes RGC survival and RNFL thickness. In addition, female Nf1-OPG mice have threefold more microglia than their male counterparts, and minocycline inhibition of microglia corrects the retinal pathology. Moreover, pharmacologic inhibition of microglial estrogen receptor-&bgr; (ER&bgr;) function corrects the retinal abnormalities in female Nf1-OPG mice. Collectively, these studies establish that female gonadal sex hormones underlie the sexual dimorphic differences in Nf1 optic glioma–induced retinal dysfunction by operating at the level of tumor-associated microglial activation.

3-D imaging mass spectrometry of protein distributions in mouse Neurofibromatosis 1 (NF1)-associated optic glioma.

Neurofibromatosis type 1 (NF1) is a common neurogenetic disorder, in which affected individuals develop tumors of the nervous system. Children with NF1 are particularly prone to brain tumors (gliomas) involving the optic pathway that can result in impaired vision. Since tumor formation and expansion requires a cooperative tumor microenvironment, it is important to identify the cellular and acellular components associated with glioma development and growth. In this study, we used 3-D matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) to measure the distributions of multiple molecular species throughout optic nerve tissue in mice with and without glioma, and to explore their spatial relationships within the 3-D volume of the optic nerve and chiasm. 3-D IMS studies often involve extensive workflows due to the high volume of sections required to generate high quality 3-D images. Herein, we present a workflow for 3-D data acquisition and volume reconstruction using mouse optic nerve tissue. The resulting 3-D IMS data yield both molecular similarities and differences between glioma-bearing and wild-type (WT) tissues, including protein distributions localizing to different anatomical subregions. BIOLOGICAL SIGNIFICANCE The current work addresses a number of challenges in 3-D MALDI IMS, driven by the small size of the mouse optic nerve and the need to maintain consistency across multiple 2-D IMS experiments. The 3-D IMS data yield both molecular similarities and differences between glioma-bearing and wild-type (WT) tissues, including protein distributions localizing to different anatomical subregions, which could then be targeted for identification and related back to the biology observed in gliomas of the optic nerve.

Whole tumor RNA-sequencing and deconvolution reveal a clinically-prognostic PTEN/PI3K-regulated glioma transcriptional signature

The concept that solid tumors are maintained by a productive interplay between neoplastic and non-neoplastic elements has gained traction with the demonstration that stromal fibroblasts and immune system cells dictate cancer development and progression. While less studied, brain tumor (glioma) biology is likewise influenced by non-neoplastic immune system cells (macrophages and microglia) which interact with neoplastic glioma cells to create a unique physiological state (glioma ecosystem) distinct from that found in the normal tissue. To explore this neoplastic ground state, we leveraged several preclinical mouse models of neurofibromatosis type 1 (NF1) optic glioma, a low-grade astrocytoma whose formation and maintenance requires productive interactions between non-neoplastic and neoplastic cells, and employed whole tumor RNA-sequencing and mathematical deconvolution strategies to characterize this low-grade glioma ecosystem as an aggregate of cellular and acellular elements. Using this approach, we demonstrate that optic gliomas generated by altering the germline Nf1 gene mutation, the glioma cell of origin, or the presence of co-existing genetic alterations represent molecularly-distinct tumors. However, these optic glioma tumors share a 25-gene core signature, not found in normal optic nerve, that is normalized by microglia inhibition (minocycline), but not conventional (carboplatin) or molecularly-targeted (rapamycin) chemotherapy. Lastly, we identify a genetic signature conferred by Pten reduction and corrected by PI3K inhibition. This signature predicts progression-free survival in patients with either low-grade or high-grade glioma. Collectively, these findings support the concept that gliomas are composite ecological systems whose biology and response to therapy may be best defined by examining the tumor as a whole.The concept that solid tumors are maintained by a productive interplay between neoplastic and non-neoplastic elements has gained traction with the demonstration that stromal fibroblasts and immune system cells dictate cancer development and progression. While less studied, brain tumor (glioma) biology is likewise influenced by non-neoplastic immune system cells (macrophages and microglia) which interact with neoplastic glioma cells to create a unique physiological state (glioma ecosystem) distinct from that found in the normal tissue. To explore this neoplastic ground state, we leveraged several preclinical mouse models of neurofibromatosis type 1 (NF1) optic glioma, a low-grade astrocytoma whose formation and maintenance requires productive interactions between non-neoplastic and neoplastic cells, and employed whole tumor RNA-sequencing and mathematical deconvolution strategies to characterize this low-grade glioma ecosystem as an aggregate of cellular and acellular elements. Using this approach, we demonstrate that optic gliomas generated by altering the germline Nf1 gene mutation, the glioma cell of origin, or the presence of co-existing genetic alterations represent molecularly-distinct tumors. However, these optic glioma tumors share a 25-gene core signature, not found in normal optic nerve, that is normalized by microglia inhibition (minocycline), but not conventional (carboplatin) or molecularly-targeted (rapamycin) chemotherapy. Lastly, we identify a genetic signature conferred by Pten reduction and corrected by PI3K inhibition. This signature predicts progression-free survival in patients with either low-grade or high-grade glioma. Collectively, these findings support the concept that gliomas are composite ecological systems whose biology and response to therapy may be best defined by examining the tumor as a whole.

The cell of origin dictates the temporal course of neurofibromatosis-1 (Nf1) low-grade glioma formation

Low-grade gliomas are one of the most common brain tumors in children, where they frequently form within the optic pathway (optic pathway gliomas; OPGs). Since many OPGs occur in the context of the Neurofibromatosis Type 1 (NF1) cancer predisposition syndrome, we have previously employed Nf1 genetically-engineered mouse (GEM) strains to study the pathogenesis of these low-grade glial neoplasms. In the light of the finding that human and mouse low-grade gliomas are composed of Olig2+ cells and that Olig2+ oligodendrocyte precursor cells (OPCs) give rise to murine high-grade gliomas, we sought to determine whether Olig2+ OPCs could be tumor-initiating cells for Nf1 optic glioma. Similar to the GFAP-Cre transgenic strain previously employed to generate Nf1 optic gliomas, Olig2+ cells also give rise to astrocytes in the murine optic nerve in vivo. However, in contrast to the GFAP-Cre strain where somatic Nf1 inactivation in embryonic neural progenitor/stem cells (Nf1flox/mut; GFAP-Cre mice) results in optic gliomas by 3 months of age in vivo, mice with Nf1 gene inactivation in Olig2+ OPCs (Nf1flox/mut; Olig2-Cre mice) do not form optic gliomas until 6 months of age. These distinct patterns of glioma latency do not reflect differences in the timing or brain location of somatic Nf1 loss. Instead, they most likely reflect the cell of origin, as somatic Nf1 loss in CD133+ neural progenitor/stem cells during late embryogenesis results in optic gliomas at 3 months of age. Collectively, these data demonstrate that the cell of origin dictates the time to tumorigenesis in murine optic glioma.

NF1-Associated Optic Glioma

Gliomas are the most common central nervous system tumor encountered in individuals with NF1. These tumors are typically low-grade gliomas (pilocytic astrocytomas) involving the optic nerve, chiasm, and tracts (optic pathway gliomas), but they may also develop in the hypothalamus or brainstem of children with NF1. Over the past decade, several Nf1 genetically-engineered mouse models of optic glioma have been established. These instructive preclinical mouse strains have revealed new insights into the role of the NF1 protein (neurofibromin) in astrocyte and neural stem cell growth regulation as well as the importance of the tumor microenvironment and genomic modifiers to gliomagenesis and tumor maintenance. In this chapter, we review these new findings and discuss their implications for future therapeutic drug design.

Abstract 1074: Tumor-associated microglia secrete paracrine factors that promote Nf1-deficient optic glial cell growth

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Previous studies have demonstrated that brain tumors are composed of heterogeneous mixtures of neoplastic and non-neoplastic (stromal) brain cells. However, current brain tumor therapies are primarily focused on inhibiting the growth of the cancerous cells, largely ignoring the contribution of the surrounding stromal elements. One of these stromal cell types is an immune system-like cell (microglia), which constitutes as many as half of the cells present in human brain tumors (gliomas). The tumor microenvironment is considered to play an important role in tumor formation and maintenance by providing signals that both negatively and positively influence tumor cell growth. Since children with the neurofibromatosis type 1 (NF1) inherited cancer syndrome are prone to the development of gliomas involving the optic nerve (optic gliomas), we have employed Nf1 genetically-engineered mice as an experimental model system to examine the role of microglia in optic glioma formation and maintenance. We leveraged RNA-sequencing data from FACS-isolated, mouse optic glioma-associated microglia to identify specific paracrine factors that are expressed in tumor-associated, but not normal, microglia. These factors were validated using RNA fluorescent in situ hybridization and investigated for their ability to increase the growth of Nf1-deficient optic nerve astroglial cells in vitro. Current studies are ongoing to determine whether (1) inhibition of microglia function in Nf1 genetically engineered mice reduces the expression of these paracrine factors and (2) inhibiting these factors attenuates optic glioma growth in vivo. Together, these studies identify a novel set of paracrine factors secreted by a key glioma-associated stromal cell type (microglia) relevant to the pathogenesis of NF1-associated tumors, raising the possibility that future therapies might be directed against tumor microenvironment paracrine factors. Citation Format: Anne C. Solga, Winnie W. Pong, Patrick J. Cimino, Keun Y. Kim, Jason Walker, Todd Wylie, Vincent Magrini, Joshua B. Rubin, David Piwnica-Worms, Mark H. Ellisman, Elaine R. Mardis, David H. Gutmann. Tumor-associated microglia secrete paracrine factors that promote Nf1-deficient optic glial cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1074. doi:10.1158/1538-7445.AM2014-1074

Abstract 358: Nf1 inactivation in NG2-cells is not sufficient for murine optic glioma formation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Individuals with the tumor predisposition syndrome, neurofibromatosis 1 (NF1), are prone to development of low-grade astrocytomas (gliomas). These tumors frequently include optic pathway gliomas predominantly arising in young children. We have previously developed several Nf1 genetically-engineered mouse (GEM) strains that form optic gliomas similar to those that occur in children with NF1. However, in the optic nerve, there are two types of potential neuroglial progenitors, GFAP+ and NG2+ cells. This latter population has been shown to represent a potential cell of origin for rat malignant gliomas, suggesting that NG2+ progenitors may represent the initiating cell for Nf1 optic glioma. In this study, we employed a complementary series of in vitro and in vivo approaches to determine whether NG2+ cells could give rise to Nf1 optic glioma. First, we demonstrate that Nf1 loss in NG2+ cells does not increase glial cell proliferation in vitro. Second, NG2-Cre-expressing cells give rise to all three macroglial lineages (astrocytes, NG2+ cells, and oligodendrocytes) in vivo, similar to GFAP-Cre-expressing cells that drive Nf1 optic glioma formation. Third, in striking contrast to the GFAP-Cre strain, Nf1 loss in NG2+ progenitor cells in vivo is insufficient for optic gliomagenesis. Together, these data exclude NG2+ cells as the likely cell of origin for NF1-associated optic glioma and establish a model of gliomagenesis in which Nf1 loss occurs in specific neuroglial progenitors during embryonic development. Citation Format: Anne C. Solga, Scott M. Gianino, David H. Gutmann. Nf1 inactivation in NG2-cells is not sufficient for murine optic glioma formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 358. doi:10.1158/1538-7445.AM2013-358