SRRM4-mediated REST to REST4 dysregulation promotes tumor growth and neural adaptation in breast cancer leading to brain metastasis.

BACKGROUND: Effective control of brain metastasis remains an urgent clinical need due a limited understanding of the mechanisms driving it. Although the gain of neuro-adaptive attributes in breast-to-brain metastases (BBMs) has been described, the mechanisms that govern this neural acclimation and the resulting brain metastasis competency are poorly understood. Herein, we define the role of neural-specific splicing factor Serine/Arginine Repetitive Matrix Protein 4 (SRRM4) in regulating microenvironmental adaptation and brain metastasis colonization in breast cancer cells. METHODS: Utilizing pure neuronal cultures and brain-naive and patient-derived BM tumor cells, along with in vivo tumor modeling, we surveyed the early induction of mediators of neural acclimation in tumor cells. RESULTS: When SRRM4 is overexpressed in systemic breast cancer cells, there is enhanced BBM leading to poorer overall survival in vivo. Concomitantly, SRRM4 knockdown expression does not provide any advantage in central nervous system metastasis. In addition, reducing SRRM4 expression in breast cancer cells slows down proliferation and increases resistance to chemotherapy. Conversely, when SRRM4/REST4 levels are elevated, tumor cell growth is maintained even in nutrient-deprived conditions. In neuronal coculture, decreasing SRRM4 expression in breast cancer cells impairs their ability to adapt to the brain microenvironment, while increasing SRRM4/RE-1 Silencing Transcription Factor (REST4) levels leads to greater expression of neurotransmitter and synaptic signaling mediators and a significant colonization advantage. CONCLUSIONS: Collectively, our findings identify SRRM4 as a regulator of brain metastasis colonization, and a potential therapeutic target in breast cancer.

Epigenetic dysregulation in meningiomas.

Background: Meningiomas are the most common primary brain tumor. Though typically benign with a low mutational burden, tumors with benign histology may behave aggressively and there are no proven chemotherapies. Although DNA methylation patterns distinguish subgroups of meningiomas and have higher predictive value for tumor behavior than histologic classification, little is known about differences in DNA methylation between meningiomas and surrounding normal dura tissue. Methods: Whole-exome sequencing and methylation array profiling were performed on 12 dura/meningioma pairs (11 WHO grade I and 1 WHO grade II). Single-nucleotide polymorphism (SNP) genotyping and methylation array profiling were performed on an additional 19 meningiomas (9 WHO grade I, 5 WHO grade II, 4 WHO grade III). Results: Using multimodal studies of meningioma/dura pairs, we identified 4 distinct DNA methylation patterns. Diffuse DNA hypomethylation of malignant meningiomas readily facilitated their identification from lower-grade tumors by unsupervised clustering. All clusters and 12/12 meningioma-dura pairs exhibited hypomethylation of the gene promoters of a module associated with the craniofacial patterning transcription factor FOXC1 and its upstream lncRNA FOXCUT. Furthermore, we identified an epigenetic continuum of increasing hypermethylation of polycomb repressive complex target promoters with increasing histopathologic grade. Conclusion: These findings support future investigations of the role of epigenetic dysregulation of FOXC1 and cranial patterning genes in meningioma formation as well as studies of the utility of polycomb inhibitors for the treatment of malignant meningiomas.

Cortisol promotes breast-to-brain metastasis through the blood-cerebrospinal fluid barrier.

BACKGROUND: Elevated basal cortisol levels are present in women with primary and metastatic breast cancer. Although cortisol's potential role in breast-to-brain metastasis has yet to be sufficiently studied, prior evidence indicates that it functions as a double-edged sword-cortisol induces breast cancer metastasis in vivo, but strengthens the blood-brain-barrier (BBB) to protect the brain from microbes and peripheral immune cells. AIMS: In this study, we provide a novel examination on whether cortisol's role in tumor invasiveness eclipses its supporting role in strengthening the CNS barriers. We expanded our study to include the blood-cerebrospinal fluid barrier (BCSFB), an underexamined site of tumor entry. METHODS AND RESULTS: Utilizing in vitro BBB and BCSFB models to measure barrier strength in the presence of hydrocortisone (HC). We established that lung tumor cells migrate through both CNS barriers equally while breast tumors cells preferentially migrate through the BCSFB. Furthermore, HC treatment increased breast-to-brain metastases (BBM) but not primary breast tumor migratory capacity. When examining the transmigration of breast cancer cells across the BCSFB, we demonstrate that HC induces increased traversal of BBM but not primary breast cancer. We provide evidence that HC increases tightness of the BCSFB akin to the BBB by upregulating claudin-5, a tight junction protein formerly acknowledged as exclusive to the BBB. CONCLUSION: Our findings indicate, for the first time that increased cortisol levels facilitate breast-to-brain metastasis through the BCSFB-a vulnerable point of entry which has been typically overlooked in brain metastasis. Our study suggests cortisol plays a pro-metastatic role in breast-to-brain metastasis and thus caution is needed when using glucocorticoids to treat breast cancer patients.

Neuronal exposure induces neurotransmitter signaling and synaptic mediators in tumors early in brain metastasis.

BACKGROUND: Brain metastases (BM) are responsible for neurological decline and poor overall survival. Although the pro-metastatic roles of glial cells, and the acquisition of neuronal attributes in established BM tumors have been described, there are no studies that investigate the initial interplay between neurons and brain-seeking tumor cells. The aim of this study was to characterize early tumor-neuron interactions and the induced CNS-adaptive changes in tumor cells prior to macro-colonization. METHODS: Utilizing pure neuronal cultures and brain-naïve and patient-derived BM tumor cells, we surveyed the early induction of mediators of neurotransmitter (NT) and synaptic signaling in breast and lung tumor cells. Reliance on microenvironmental GABA in breast-to-brain metastatic cells (BBMs) was assessed in vitro and in vivo. RESULTS: Coculture with neurons induces early expression of classical NT receptor genes (HTR4, GRIA2, GRIN2B, GRM4, GRM8, DRD1) and neuronal synaptic mediators (CNR1, EGR2, ARC, NGFR, NRXN1) in breast and lung cancer cells. NT-dependent classification of tumor cells within the neuronal niche shows breast cancer cells become GABAergic responsive brain metastases (GRBMs) and transition from relying on autocrine GABA, to paracrine GABA from adjacent neurons; while autocrine Dopaminergic breast and lung tumor cells persist. In vivo studies confirm reliance on paracrine GABA is an early CNS-acclimation strategy in breast cancer. Moreover, neuronal contact induces early resurgence in Reelin expression in tumor cells through epigenetic activation, facilitating CNS adaptation. CONCLUSION: Tumor-neuron interactions allow for CNS adaptation early in the course of brain metastasis.

Medulloblastoma uses GABA transaminase to survive in the cerebrospinal fluid microenvironment and promote leptomeningeal dissemination.

Medulloblastoma (MB) is a malignant pediatric brain tumor arising in the cerebellum. Although abnormal GABAergic receptor activation has been described in MB, studies have not yet elucidated the contribution of receptor-independent GABA metabolism to MB pathogenesis. We find primary MB tumors globally display decreased expression of GABA transaminase (ABAT), the protein responsible for GABA metabolism, compared with normal cerebellum. However, less aggressive WNT and SHH subtypes express higher ABAT levels compared with metastatic G3 and G4 tumors. We show that elevated ABAT expression results in increased GABA catabolism, decreased tumor cell proliferation, and induction of metabolic and histone characteristics mirroring GABAergic neurons. Our studies suggest ABAT expression fluctuates depending on metabolite changes in the tumor microenvironment, with nutrient-poor conditions upregulating ABAT expression. We find metastatic MB cells require ABAT to maintain viability in the metabolite-scarce cerebrospinal fluid by using GABA as an energy source substitute, thereby facilitating leptomeningeal metastasis formation.

Utilization of Discarded Surgical Tissue from Ultrasonic Aspirators to Establish Patient-Derived Metastatic Brain Tumor Cells: A Guide from the Operating Room to the Research Laboratory.

Patient-derived cells from surgical resections are of paramount importance to brain tumor research. It is well known that there is cellular and microenvironmental heterogeneity within a single tumor mass. Thus, current established protocols for propagating tumor cells in vitro are limiting because resections obtained from conventional singular samples limit the diversity in cell populations and do not accurately model the heterogeneous tumor. Utilization of discarded tissue obtained from cavitron ultrasonic surgical aspirator (CUSA) of the whole tumor mass allows for establishing novel cell lines in vitro from the entirety of the tumor, thereby creating an accurate representation of the heterogeneous population of cells originally present in the tumor. Furthermore, while others have described protocols for establishing patient tumor lines once tissue has arrived in the research lab, a primer from the operating room (OR) to the research lab has not been described before. This is integral, as basic research scientists need to understand the surgical environment of the OR, including the methods utilized to obtain a patient's tumor resection, in order to more accurately model cancer biology in laboratory. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Establishment of brain tumor cell lines from patient-derived CUSA samples: processing brain tumor sample from the OR to the lab Support Protocol 1: Sterilization of microsurgical tools in preparation for dissection Support Protocol 2: Collagen coating of tissue culture flasks Basic Protocol 2: Selection of tumor cells in vitro Support Protocol 3: FACS sorting tumor sample to isolate cancer cells from heterogeneous cell population.

Functional equivalence of stem cell and stem cell-derived extracellular vesicle transplantation to repair the irradiated brain.

Cranial radiotherapy, although beneficial for the treatment of brain tumors, inevitably leads to normal tissue damage that can induce unintended neurocognitive complications that are progressive and debilitating. Ionizing radiation exposure has also been shown to compromise the structural integrity of mature neurons throughout the brain, an effect believed to be at least in part responsible for the deterioration of cognitive health. Past work has shown that cranially transplanted human neural stem cells (hNSCs) or their extracellular vesicles (EVs) afforded long-term beneficial effects on many of these cognitive decrements. To provide additional insight into the potential neuroprotective mechanisms of cell-based regenerative strategies, we have analyzed hippocampal neurons for changes in structural integrity and synaptic remodeling after unilateral and bilateral transplantation of hNSCs or EVs derived from those same cells. Interestingly, hNSCs and EVs similarly afforded protection to host neurons, ameliorating the impact of irradiation on dendritic complexity and spine density for neurons present in both the ipsilateral and contralateral hippocampi 1 month following irradiation and transplantation. These morphometric improvements were accompanied by increased levels of glial cell-derived growth factor and significant attenuation of radiation-induced increases in postsynaptic density protein 95 and activated microglia were found ipsi- and contra-lateral to the transplantation sites of the irradiated hippocampus treated with hNSCs or hNSC-derived EVs. These findings document potent far-reaching neuroprotective effects mediated by grafted stem cells or EVs adjacent and distal to the site of transplantation and support their potential as therapeutic agents to counteract the adverse effects of cranial irradiation.

Clinical Perspectives in Brain Metastasis.

Brain metastases (BMs) are responsible for decline in neurological function, reduction in overall quality of life, and mortality from recurrent or untreatable lesions. Advances in diagnostics and imaging have led to increased detection of central nervous system (CNS) metastases in patients with progressive cancers. Improved control of extracranial systemic disease, and the limited ability of current therapeutics to cross the blood-brain barrier (BBB) also contribute to the increase in incidence of brain metastases, as tumor cells seek refuge in the brain. Surgery, chemotherapy, and/or radiation (whole-brain radiation therapy and stereotactic radiation surgery [WBRT/SRS]) are a clinically established treatment paradigm for patients with brain metastases. With the advent of genetic and molecular characterization of tumors and their immune microenvironment, clinical trials seek to include targeted drugs into the therapeutic regimen for eligible patients. Several challenges, like treatment of multiple CNS lesions, superior uptake of chemotherapy into the brain, and trials with multidisciplinary approaches, are now being clinically addressed.

Isolation of Neural Stem Cells from Whole Brain Tissues of Adult Mice.

A population of neural stem cells exists in the adult mammalian central nervous system. Purification and characterization of neurospheres provide valuable tools to study the regulation and differentiation of neural stem cells both in vitro and in vivo. Successful stimulation and production of neurospheres can ultimately be used for therapeutic purposes. The currently available methods are limited by their poor yield and the large number of animals required to compensate for that. Here, we describe a procedure to purify neurospheres from adult mouse whole brain. We provide detailed steps on how to propagate, passage, and maintain the adult neurospheres, and how to differentiate the pure neurospheres into the lineage of interest. Using this method, neurospheres can be easily derived from adult mouse whole brain. The derived adult neurospheres maintain their homogenous undifferentiated status while retaining their differentiation potential. This new protocol facilitates adult neurospheres isolation, purification, maintenance, and differentiation. © 2019 by John Wiley & Sons, Inc.

Medulloblastoma initiation and spread: Where neurodevelopment, microenvironment and cancer cross pathways.

Medulloblastomas are the most common malignant pediatric brain tumors. Over the past several decades, a wide range of tumor-centric studies have identified genes and their regulators within signaling pathways that promote medulloblastoma growth. This review aims to raise awareness that transdisciplinary research between developmental neurobiology and cancer foundations can advance our current understanding of how the nervous system contributes to medulloblastomas. By leveraging current advances in neurodevelopment, microenvironment (including secreted neuropeptides), neurotransmitters, and axon guidance cues, we can uncover novel mechanisms used by the nervous system to promote medulloblastoma growth and spread. This will ultimately result in development of improved strategies for cancer prevention and treatment of pediatric patients with this devastating disease. © 2016 Wiley Periodicals, Inc.

Cranial grafting of stem cell-derived microvesicles improves cognition and reduces neuropathology in the irradiated brain.

Cancer survivors face a variety of challenges as they cope with disease recurrence and a myriad of normal tissue complications brought on by radio- and chemotherapeutic treatment regimens. For patients subjected to cranial irradiation for the control of CNS malignancy, progressive and debilitating cognitive dysfunction remains a pressing unmet medical need. Although this problem has been recognized for decades, few if any satisfactory long-term solutions exist to resolve this serious unintended side effect of radiotherapy. Past work from our laboratory has demonstrated the neurocognitive benefits of human neural stem cell (hNSC) grafting in the irradiated brain, where intrahippocampal transplantation of hNSC ameliorated radiation-induced cognitive deficits. Using a similar strategy, we now provide, to our knowledge, the first evidence that cranial grafting of microvesicles secreted from hNSC affords similar neuroprotective phenotypes after head-only irradiation. Cortical- and hippocampal-based deficits found 1 mo after irradiation were completely resolved in animals cranially grafted with microvesicles. Microvesicle treatment was found to attenuate neuroinflammation and preserve host neuronal morphology in distinct regions of the brain. These data suggest that the neuroprotective properties of microvesicles act through a trophic support mechanism that reduces inflammation and preserves the structural integrity of the irradiated microenvironment.

Stem cell transplantation reverses chemotherapy-induced cognitive dysfunction.

The frequent use of chemotherapy to combat a range of malignancies can elicit severe cognitive dysfunction often referred to as "chemobrain," a condition that can persist long after the cessation of treatment in as many as 75% of survivors. Although cognitive health is a critical determinant of therapeutic outcome, chemobrain remains an unmet medical need that adversely affects quality of life in pediatric and adult cancer survivors. Using a rodent model of chemobrain, we showed that chronic cyclophosphamide treatment induced significant performance-based decrements on behavioral tasks designed to interrogate hippocampal and cortical function. Intrahippocampal transplantation of human neural stem cells resolved all cognitive impairments when animals were tested 1 month after the cessation of chemotherapy. In transplanted animals, grafted cells survived (8%) and differentiated along neuronal and astroglial lineages, where improved cognition was associated with reduced neuroinflammation and enhanced host dendritic arborization. Stem cell transplantation significantly reduced the number of activated microglia after cyclophosphamide treatment in the brain. Granule and pyramidal cell neurons within the dentate gyrus and CA1 subfields of the hippocampus exhibited significant reductions in dendritic complexity, spine density, and immature and mature spine types following chemotherapy, adverse effects that were eradicated by stem cell transplantation. Our findings provide the first evidence that cranial transplantation of stem cells can reverse the deleterious effects of chemobrain, through a trophic support mechanism involving the attenuation of neuroinflammation and the preservation host neuronal architecture.

Defining the optimal window for cranial transplantation of human induced pluripotent stem cell-derived cells to ameliorate radiation-induced cognitive impairment.

Past preclinical studies have demonstrated the capability of using human stem cell transplantation in the irradiated brain to ameliorate radiation-induced cognitive dysfunction. Intrahippocampal transplantation of human embryonic stem cells and human neural stem cells (hNSCs) was found to functionally restore cognition in rats 1 and 4 months after cranial irradiation. To optimize the potential therapeutic benefits of human stem cell transplantation, we have further defined optimal transplantation windows for maximizing cognitive benefits after irradiation and used induced pluripotent stem cell-derived hNSCs (iPSC-hNSCs) that may eventually help minimize graft rejection in the host brain. For these studies, animals given an acute head-only dose of 10 Gy were grafted with iPSC-hNSCs at 2 days, 2 weeks, or 4 weeks following irradiation. Animals receiving stem cell grafts showed improved hippocampal spatial memory and contextual fear-conditioning performance compared with irradiated sham-surgery controls when analyzed 1 month after transplantation surgery. Importantly, superior performance was evident when stem cell grafting was delayed by 4 weeks following irradiation compared with animals grafted at earlier times. Analysis of the 4-week cohort showed that the surviving grafted cells migrated throughout the CA1 and CA3 subfields of the host hippocampus and differentiated into neuronal (∼39%) and astroglial (∼14%) subtypes. Furthermore, radiation-induced inflammation was significantly attenuated across multiple hippocampal subfields in animals receiving iPSC-hNSCs at 4 weeks after irradiation. These studies expand our prior findings to demonstrate that protracted stem cell grafting provides improved cognitive benefits following irradiation that are associated with reduced neuroinflammation.

Targeted overexpression of mitochondrial catalase prevents radiation-induced cognitive dysfunction.

AIMS: Radiation-induced disruption of mitochondrial function can elevate oxidative stress and contribute to the metabolic perturbations believed to compromise the functionality of the central nervous system. To clarify the role of mitochondrial oxidative stress in mediating the adverse effects of radiation in the brain, we analyzed transgenic (mitochondrial catalase [MCAT]) mice that overexpress human catalase localized to the mitochondria. RESULTS: Compared with wild-type (WT) controls, overexpression of the MCAT transgene significantly decreased cognitive dysfunction after proton irradiation. Significant improvements in behavioral performance found on novel object recognition and object recognition in place tasks were associated with a preservation of neuronal morphology. While the architecture of hippocampal CA1 neurons was significantly compromised in irradiated WT mice, the same neurons in MCAT mice did not exhibit extensive and significant radiation-induced reductions in dendritic complexity. Irradiated neurons from MCAT mice maintained dendritic branching and length compared with WT mice. Protected neuronal morphology in irradiated MCAT mice was also associated with a stabilization of radiation-induced variations in long-term potentiation. Stabilized synaptic activity in MCAT mice coincided with an altered composition of the synaptic AMPA receptor subunits GluR1/2. INNOVATION: Our findings provide the first evidence that neurocognitive sequelae associated with radiation exposure can be reduced by overexpression of MCAT, operating through a mechanism involving the preservation of neuronal morphology. CONCLUSION: Our article documents the neuroprotective properties of reducing mitochondrial reactive oxygen species through the targeted overexpression of catalase and how this ameliorates the adverse effects of proton irradiation in the brain.

Long-term cognitive effects of human stem cell transplantation in the irradiated brain.

PURPOSE: Radiotherapy remains a primary treatment modality for the majority of central nervous system tumors, but frequently leads to debilitating cognitive dysfunction. Given the absence of satisfactory solutions to this serious problem, we have used human stem cell therapies to ameliorate radiation-induced cognitive impairment. Here, past studies have been extended to determine whether engrafted cells provide even longer-term benefits to cognition. MATERIALS AND METHODS: Athymic nude rats were cranially irradiated (10 Gy) and subjected to intrahippocampal transplantation surgery 2 days later. Human embryonic stem cells (hESC) or human neural stem cells (hNSC) were transplanted, and animals were subjected to cognitive testing on a novel place recognition task 8 months later. RESULTS: Grafting of hNSC was found to provide long lasting cognitive benefits over an 8-month post-irradiation interval. At this protracted time, hNSC grafting improved behavioral performance on a novel place recognition task compared to irradiated animals not receiving stem cells. Engrafted hESC previously shown to be beneficial following a similar task, 1 and 4 months after irradiation, were not found to provide cognitive benefits at 8 months. CONCLUSIONS: Our findings suggest that hNSC transplantation promotes the long-term recovery of the irradiated brain, where intrahippocampal stem cell grafting helps to preserve cognitive function.

Impaired cognitive function and hippocampal neurogenesis following cancer chemotherapy.

PURPOSE: A substantial proportion of breast cancer survivors report significant, long-lasting impairments in cognitive function, often referred to as "chemobrain." Advances in detection and treatment mean that many more patients are surviving long-term following diagnosis of invasive breast cancer. Thus, it is important to define the types, extent, and persistence of cognitive impairments following treatment with cytotoxic cancer drugs. EXPERIMENTAL DESIGN: We examined the effects of chronic treatment with two agents commonly used in patients with breast cancer, cyclophosphamide and doxorubicin (Adriamycin). Athymic nude rats were given 50 mg/kg cyclophosphamide, 2 mg/kg doxorubicin, or saline injections once per week for 4 weeks. A novel place recognition task and contextual and cued fear conditioning were used to characterize learning and memory ability. Immunofluorescence staining for immature and mature neurons and activated microglia was used to assess changes in neurogenesis and neuroinflammation. RESULTS: Cyclophosphamide- and doxorubicin-treated rats showed significantly impaired performance on the novel place recognition task and the contextual fear conditioning task compared with untreated controls, suggesting disrupted hippocampal-based memory function. Chemotherapy-treated animals showed a significant decline in neurogenesis [80%-90% drop in bromodeoxyuridine (BrdUrd)-labeled cells expressing NeuN]. Activated microglia (ED1-positive) were found after cyclophosphamide but not doxorubicin treatment. CONCLUSIONS: Our results show that chronic treatment with either of two commonly used chemotherapeutic agents impairs cognitive ability and suggest that strategies to prevent or repair disrupted hippocampal neurogenesis may be effective in ameliorating this serious side effect in cancer survivors.