Discovery of NRG1-VII: the myeloid-derived class of NRG1.

The growth factor Neuregulin-1 (NRG1) has pleiotropic roles in proliferation and differentiation of the stem cell niche in different tissues. It has been implicated in gut, brain and muscle development and repair. Six isoform classes of NRG1 and over 28 protein isoforms have been previously described. Here we report a new class of NRG1, designated NRG1-VII to denote that these NRG1 isoforms arise from a myeloid-specific transcriptional start site (TSS) previously uncharacterized. Long-read sequencing was used to identify eight high-confidence NRG1-VII transcripts. These transcripts presented major structural differences from one another, through the use of cassette exons and alternative stop codons. Expression of NRG1-VII was confirmed in primary human monocytes and tissue resident macrophages and induced pluripotent stem cell-derived macrophages (iPSC-derived macrophages). Isoform switching via cassette exon usage and alternate polyadenylation was apparent during monocyte maturation and macrophage differentiation. NRG1-VII is the major class expressed by the myeloid lineage, including tissue-resident macrophages. Analysis of public gene expression data indicates that monocytes and macrophages are a primary source of NRG1. The size and structure of class VII isoforms suggests that they may be more diffusible through tissues than other NRG1 classes. However, the specific roles of class VII variants in tissue homeostasis and repair have not yet been determined.

CREB: A multifaceted transcriptional regulator of neural and immune function in CNS tumors.

Cancers of the central nervous system (CNS) are unique with respect to their tumor microenvironment. Such a status is due to immune-privilege and the cellular behaviors within a highly networked, neural-rich milieu. During tumor development in the CNS, neural, immune and cancer cells establish complex cell-to-cell communication networks which mimic physiological functions, including paracrine signaling and synapse-like formations. This crosstalk regulates diverse pathological functions contributing to tumor progression. In the CNS, regulation of physiological and pathological functions relies on various cell signaling and transcription programs. At the core of these events lies the cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), a master transcriptional regulator in the CNS. CREB is a kinase inducible transcription factor which regulates many CNS functions, including neurogenesis, neuronal survival, neuronal activation and long-term memory. Here, we discuss how CREB-regulated mechanisms operating in diverse cell types, which control development and function of the CNS, are co-opted in CNS tumors.

Repurposing FDA-approved drugs as inhibitors of therapy-induced invadopodia activity in glioblastoma cells.

Glioblastoma (GBM) is the most prevalent primary central nervous system tumour in adults. The lethality of GBM lies in its highly invasive, infiltrative, and neurologically destructive nature resulting in treatment failure, tumour recurrence and death. Even with current standard of care treatment with surgery, radiotherapy and chemotherapy, surviving tumour cells invade throughout the brain. We have previously shown that this invasive phenotype is facilitated by actin-rich, membrane-based structures known as invadopodia. The formation and matrix degrading activity of invadopodia is enhanced in GBM cells that survive treatment. Drug repurposing provides a means of identifying new therapeutic applications for existing drugs without the need for discovery or development and the associated time for clinical implementation. We investigate several FDA-approved agents for their ability to act as both cytotoxic agents in reducing cell viability and as 'anti-invadopodia' agents in GBM cell lines. Based on their cytotoxicity profile, three agents were selected, bortezomib, everolimus and fludarabine, to test their effect on GBM cell invasion. All three drugs reduced radiation/temozolomide-induced invadopodia activity, in addition to reducing GBM cell viability. These drugs demonstrate efficacious properties warranting further investigation with the potential to be implemented as part of the treatment regime for GBM.

IL-10 in glioma.

The prognosis for patients with glioblastoma (GBM), the most common and malignant type of primary brain tumour, is very poor, despite current standard treatments such as surgery, radiotherapy and chemotherapy. Moreover, the immunosuppressive tumour microenvironment hinders the development of effective immunotherapies for GBM. Cytokines such as interleukin-10 (IL-10) play a major role in modulating the activity of infiltrating immune cells and tumour cells in GBM, predominantly conferring an immunosuppressive action; however, in some circumstances, IL-10 can have an immunostimulatory effect. Elucidating the function of IL-10 in GBM is necessary to better strategise and improve the efficacy of immunotherapy. This review discusses the immunostimulatory and immunosuppressive roles of IL-10 in the GBM tumour microenvironment while considering IL-10-targeted treatment strategies. The molecular mechanisms that underlie the expression of IL-10 in various cell types are also outlined, and how this resulting information might provide an avenue for the improvement of immunotherapy in GBM is explored.

Toward precision immunotherapy using multiplex immunohistochemistry and in silico methods to define the tumor immune microenvironment.

Recent developments in cancer immunotherapy promise better outcomes for cancer patients, although clinical trials for difficult to treat cancers such as malignant brain cancer present special challenges, showing little response to first generation immunotherapies. Reasons for differences in immunotherapy response in some cancer types are likely due to the nature of tumor microenvironment, which harbors multiple cell types which interact with tumor cells to establish immunosuppression. The cell types which appear to hold the key in regulating tumor immunosuppression are the tumor-infiltrating immune cells. The current standard treatment for difficult to treat cancer, including the most malignant brain cancer, glioblastoma, continues to offer a bleak outlook for patients. Immune-profiling and correlation with pathological and clinical data will lead to a deeper understanding of the tumor immune microenvironment and contribute toward the selection, optimization and development of novel precision immunotherapies. Here, we review the current understanding of the tumor microenvironmental landscape in glioblastoma with a focus on next-generation technologies including multiplex immunofluorescence and computational approaches to map the brain tumor microenvironment to decipher the role of the immune system in this lethal malignancy.

High-Throughput Multiplex Immunohistochemistry of Glioma Organoids.

The invasive capacity and progression of glioblastoma cells and neoplastic cells in other are dependent on interactions with the surrounding tumor microenvironment. In particular, cancer cells form a reciprocal relationship with noncellular dysregulated extracellular matrix in the tumors. Here, we describe a protocol that can be used to model the functional relationship between tumor cells and extracellular matrix. We demonstrate how 3D organoids, including glioma tumor organoids, can be processed, embedded, and sectioned in a high-throughput setup that enables investigation of the organoids by histopathological methods, multiplex immunohistochemistry, and spatial analysis within the same section.

Cell signaling activation and extracellular matrix remodeling underpin glioma tumor microenvironment heterogeneity and organization.

PURPOSE: Tumor cells thrive by adapting to the signals in their microenvironment. To adapt, cancer cells activate signaling and transcriptional programs and migrate to establish micro-niches, in response to signals from neighboring cells and non-cellular stromal factors. Understanding how the tumor microenvironment evolves during disease progression is crucial to deciphering the mechanisms underlying the functional behavior of cancer cells. METHODS: Multiplex immunohistochemistry, spatial analysis and histological dyes were used to identify and measure immune cell infiltration, cell signal activation and extracellular matrix deposition in low-grade, high-grade astrocytoma and glioblastoma. RESULTS: We show that lower grade astrocytoma tissue is largely devoid of infiltrating immune cells and extracellular matrix proteins, while high-grade astrocytoma exhibits abundant immune cell infiltration, activation, and extensive tissue remodeling. Spatial analysis shows that most T-cells are restricted to perivascular regions, but bone marrow-derived macrophages penetrate deep into neoplastic cell-rich regions. The tumor microenvironment is characterized by heterogeneous PI3K, MAPK and CREB signaling, with specific signaling profiles correlating with distinct pathological hallmarks, including angiogenesis, tumor cell density and regions where neoplastic cells border the extracellular matrix. Our results also show that tissue remodeling is important in regulating the architecture of the tumor microenvironment during tumor progression. CONCLUSION: The tumor microenvironment in malignant astrocytoma, exhibits changes in cell composition, cell signaling activation and extracellular matrix deposition during disease development and that targeting the extracellular matrix, as well as cell signaling activation will be critical to designing personalized therapy.

Identification and isolation of slow-cycling glioma stem cells.

Cancer stem cells are defined as low-abundance, quiescent cells and are considered a major cellular source of tumor recurrence following therapy, which identifies these cells as important therapeutic targets for difficult-to-treat cancers, including high-grade gliomas. By contrast to the highly proliferative bulk tumor cells, glioma stem cells (GSC) are slow-cycling, and therefore less sensitive to DNA damaging cytotoxic drugs. GSC are also less reliant on aerobic glycolytic metabolism, leading to inadequate clearing of GSC by chemotherapy and radiotherapy. The definition of GSC is based on the expression of specific stem cell protein markers. This method of GSC isolation is successful in isolating cell populations that can reliably recapitulate the tumor. However, cell populations that lack stem marker expression may also be capable of tumor recapitulation. Therefore, robust, reproducible methods for isolating GSC are required to identify and isolate cells with stem cell characteristics. Here, we provide a comprehensive and reproducible protocol for the isolation of slow-cycling GSC. Using this method, GSC isolated retain key characteristics of the cells in situ, including expression of genes associated with cell quiescence and invasive potential, compared to non-quiescent cell populations. Thus, isolation of GSC gated on cell proliferation offers a reliable alternative method for in vitro GSC identification, that adequately mirrors the physiological properties of GSC seen in vivo.

Inhibition of Radiation and Temozolomide-Induced Glioblastoma Invadopodia Activity Using Ion Channel Drugs.

Glioblastoma (GBM) is the most prevalent and malignant type of primary brain cancer. The rapid invasion and dissemination of tumor cells into the surrounding normal brain is a major driver of tumor recurrence, and long-term survival of GBM patients is extremely rare. Actin-rich cell membrane protrusions known as invadopodia can facilitate the highly invasive properties of GBM cells. Ion channels have been proposed to contribute to a pro-invasive phenotype in cancer cells and may also be involved in the invadopodia activity of GBM cells. GBM cell cytotoxicity screening of several ion channel drugs identified three drugs with potent cell killing efficacy: flunarizine dihydrochloride, econazole nitrate, and quinine hydrochloride dihydrate. These drugs demonstrated a reduction in GBM cell invadopodia activity and matrix metalloproteinase-2 (MMP-2) secretion. Importantly, the treatment of GBM cells with these drugs led to a significant reduction in radiation/temozolomide-induced invadopodia activity. The dual cytotoxic and anti-invasive efficacy of these agents merits further research into targeting ion channels to reduce GBM malignancy, with a potential for future clinical translation in combination with the standard therapy.