Tumor treating fields suppress tumor cell growth and neurologic decline in models of spinal metastases.

Spinal metastases can result in severe neurologic compromise and decreased overall survival. Despite treatment advances, local disease progression is frequent, highlighting the need for novel therapies. Tumor treating fields (TTFields) impair tumor cell replication and are influenced by properties of surrounding tissue. We hypothesized that bone's dielectric properties will enhance TTFields-mediated suppression of tumor growth in spinal metastasis models. Computational modeling of TTFields intensity was performed following surgical resection of a spinal metastasis and demonstrated enhanced TTFields intensity within the resected vertebral body. Additionally, luciferase-tagged human KRIB osteosarcoma and A549 lung adenocarcinoma cell lines were cultured in demineralized bone grafts and exposed to TTFields. Following TTFields exposure, the bioluminescence imaging (BLI) signal decreased to 10%-80% of baseline, while control cultures displayed a 4.48- to 9.36-fold increase in signal. Lastly, TTFields were applied in an orthotopic murine model of spinal metastasis. After 21 days of treatment, control mice demonstrated a 5-fold increase in BLI signal compared with TTFields-treated mice. TTFields similarly prevented tumor invasion into the spinal canal and development of neurologic symptoms. Our data suggest that TTFields can be leveraged as a local therapy within minimally conductive bone of spinal metastases. This provides the groundwork for future studies investigating TTFields for patients with treatment-refractory spinal metastases.

Glial progenitor heterogeneity and key regulators revealed by single-cell RNA sequencing provide insight to regeneration in spinal cord injury.

Recent studies have revealed the heterogeneous nature of astrocytes; however, how diverse constituents of astrocyte-lineage cells are regulated in adult spinal cord after injury and contribute to regeneration remains elusive. We perform single-cell RNA sequencing of GFAP-expressing cells from sub-chronic spinal cord injury models and identify and compare with the subpopulations in acute-stage data. We find subpopulations with distinct functional enrichment and their identities defined by subpopulation-specific transcription factors and regulons. Immunohistochemistry, RNAscope experiments, and quantification by stereology verify the molecular signature, location, and morphology of potential resident neural progenitors or neural stem cells in the adult spinal cord before and after injury and uncover the populations of the intermediate cells enriched in neuronal genes that could potentially transition into other subpopulations. This study has expanded the knowledge of the heterogeneity and cell state transition of glial progenitors in adult spinal cord before and after injury.

Coding and long non-coding gene expression changes in the CNS traumatic injuries.

Traumatic brain injury (TBI) and spinal cord injury (SCI) are two main central nervous system (CNS) traumas, caused by external physical insults. Both injuries have devastating effects on the quality of life, and there is no effective therapy at present. Notably, gene expression profiling using bulk RNA sequencing (RNA-Seq) and single-cell RNA-Seq (scRNA-Seq) have revealed significant changes in many coding and non-coding genes, as well as important pathways in SCI and TBI. Particularly, recent studies have revealed that long non-coding RNAs (lncRNAs) with lengths greater than 200 nucleotides and without protein-coding potential have tissue- and cell type-specific expression pattern and play critical roles in CNS injury by gain- and loss-of-function approaches. LncRNAs have been shown to regulate protein-coding genes or microRNAs (miRNAs) directly or indirectly, participating in processes including inflammation, glial activation, cell apoptosis, and vasculature events. Therefore, lncRNAs could serve as potential targets for the diagnosis, treatment, and prognosis of SCI and TBI. In this review, we highlight the recent progress in transcriptome studies of SCI and TBI and insights into molecular mechanisms.

The Many Faces of Astrocytes in Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and is the most common cause of dementia in an aging population. The majority of research effort has focused on the role of neurons in neurodegeneration and current therapies have limited ability to slow disease progression. Recently more attention has been given to the role of astrocytes in the process of neurodegeneration. Specifically, reactive astrocytes have both advantageous and adverse effects during neurodegeneration. The ability to isolate and depict astrocyte phenotype has been challenging. However, with the recent development of single-cell sequencing technologies researchers are provided with the resource to delineate specific biomarkers associated with reactive astrocytes in AD. In this review, we will focus on the role of astrocytes in normal conditions and the pathological development of AD. We will further review recent developments in the understanding of astrocyte heterogeneity and associated biomarkers. A better understanding of astrocyte contributions and phenotypic changes in AD can ultimately lead to more effective therapeutic targets.

Angiogenic gene networks are dysregulated in opioid use disorder: evidence from multi-omics and imaging of postmortem human brain.

Opioid use disorder (OUD) is a public health crisis in the U.S. that causes over 50 thousand deaths annually due to overdose. Using next-generation RNA sequencing and proteomics techniques, we identified 394 differentially expressed (DE) coding and long noncoding (lnc) RNAs as well as 213 DE proteins in Brodmann Area 9 of OUD subjects. The RNA and protein changes converged on pro-angiogenic gene networks and cytokine signaling pathways. Four genes (LGALS3, SLC2A1, PCLD1, and VAMP1) were dysregulated in both RNA and protein. Dissecting these DE genes and networks, we found cell type-specific effects with enrichment in astrocyte, endothelial, and microglia correlated genes. Weighted-genome correlation network analysis (WGCNA) revealed cell-type correlated networks including an astrocytic/endothelial/microglia network involved in angiogenic cytokine signaling as well as a neuronal network involved in synaptic vesicle formation. In addition, using ex vivo magnetic resonance imaging, we identified increased vascularization in postmortem brains from a subset of subjects with OUD. This is the first study integrating dysregulation of angiogenic gene networks in OUD with qualitative imaging evidence of hypervascularization in postmortem brain. Understanding the neurovascular effects of OUD is critical in this time of widespread opioid use.

OLIG2 regulates lncRNAs and its own expression during oligodendrocyte lineage formation.

BACKGROUND: Oligodendrocytes, responsible for axon ensheathment, are critical for central nervous system (CNS) development, function, and diseases. OLIG2 is an important transcription factor (TF) that acts during oligodendrocyte development and performs distinct functions at different stages. Previous studies have shown that lncRNAs (long non-coding RNAs; > 200 bp) have important functions during oligodendrocyte development, but their roles have not been systematically characterized and their regulation is not yet clear. RESULTS: We performed an integrated study of genome-wide OLIG2 binding and the epigenetic modification status of both coding and non-coding genes during three stages of oligodendrocyte differentiation in vivo: neural stem cells (NSCs), oligodendrocyte progenitor cells (OPCs), and newly formed oligodendrocytes (NFOs). We found that 613 lncRNAs have OLIG2 binding sites and are expressed in at least one cell type, which can potentially be activated or repressed by OLIG2. Forty-eight of them have increased expression in oligodendrocyte lineage cells. Predicting lncRNA functions by using a "guilt-by-association" approach revealed that the functions of these 48 lncRNAs were enriched in "oligodendrocyte development and differentiation." Additionally, bivalent genes are known to play essential roles during embryonic stem cell differentiation. We identified bivalent genes in NSCs, OPCs, and NFOs and found that some bivalent genes bound by OLIG2 are dynamically regulated during oligodendrocyte development. Importantly, we unveiled a previously unknown mechanism that, in addition to transcriptional regulation via DNA binding, OLIG2 could self-regulate through the 3' UTR of its own mRNA. CONCLUSIONS: Our studies have revealed the missing links in the mechanisms regulating oligodendrocyte development at the transcriptional level and after transcription. The results of our research have improved the understanding of fundamental cell fate decisions during oligodendrocyte lineage formation, which can enable insights into demyelination diseases and regenerative medicine.

Systematic analysis of purified astrocytes after SCI unveils Zeb2os function during astrogliosis.

Spinal cord injury (SCI) is one of the most devastating neural injuries without effective therapeutic solutions. Astrocytes are the predominant component of the scar. Understanding the complex contributions of reactive astrocytes to SCI pathophysiologies is fundamentally important for developing therapeutic strategies. We have studied the molecular changes in the injury environment and the astrocyte-specific responses by astrocyte purification from injured spinal cords from acute to chronic stages. In addition to protein-coding genes, we have systematically analyzed the expression profiles of long non-coding RNAs (lncRNAs) (>200 bp), which are regulatory RNAs that play important roles in the CNS. We have identified a highly conserved lncRNA, Zeb2os, and demonstrated using functional assays that it plays an important role in reactive astrogliosis through the Zeb2os/Zeb2/Stat3 axis. These studies provide valuable insights into the molecular basis of reactive astrogliosis and fill the knowledge gap regarding the function(s) of lncRNAs in astrogliosis and SCI.

Human CAFs promote lymphangiogenesis in ovarian cancer via the Hh-VEGF-C signaling axis.

Cancer-associated fibroblasts (CAFs) play a pivotal role in the development and progression of many human cancers. Recent studies have shown that Hedgehog (Hh) signalling modulates the stromal microenvironment and prepares a suitable niche for tumour metastasis. However, the detailed molecular mechanisms underlying CAF-mediated lymphangiogenesis have not been fully elucidated. Therefore, our goal is to illustrate whether Hh ligands can activate Hh signalling in CAFs in a paracrine fashion and elucidate the effect of CAFs on lymphangiogenesis. We determined here that Sonic Hedgehog (SHH) secreted by ovarian cancer (OC) cells activated Hh signalling in CAFs and promoted the proliferation of CAFs. Moreover, we co-injected SHH-overexpressing OC cells and CAFs in a xenograft model and found that the CAFs accelerated tumourigenesis and lymphangiogenesis in OC. Mechanistically, we found that SHH secreted by the OC cells induced VEGF-C expression in CAFs. Inhibition of Hh signalling in CAFs decreased VEGF-C expression and diminished the positive role of CAFs in supporting tumourigenesis and lymphangiogenesis in a murine xenograft model. Our results demonstrate that CAFs constitute a supportive niche for cancer lymphangiogenesis via the Hh/VEGF-C signalling axis and provide evidence for the clinical application of Hh inhibitors in the treatment of OC.

Lung Protective Effects of Low-Volume Resuscitation and Pharmacologic Treatment of Swine Subjected to Polytrauma and Hemorrhagic Shock.

Hemorrhage is a common cause of death in the battlefield. Valproic acid (VPA) has been associated with improved outcomes in multiple models of trauma, when combined with isotonic fluid resuscitation. However, isotonic fluid administered in this setting is logistically impractical and may be associated with complications. In this study, we sought to evaluate the feasibility and immunologic impact of combining VPA treatment with low-volume hypertonic saline (HTS). In vivo: female Yorkshire swine were subjected to hemorrhage (40% total blood volume) and polytrauma (rib fracture and delayed liver injury). Animals were kept in shock for 30 minutes and resuscitated with (1) normal saline (NS, 3× hemorrhaged volume), (2) HTS (7.5% saline, 4 mL/kg), or (3) HTS + VPA (4 mg/kg; 150 mg/kg; n = 3/cohort). After 18 hours of observation, animals were euthanized and the lungs evaluated for acute injury and expression of myeloperoxidase (MPO) and caveolin-1 (Cav-1). In vitro: human umbilical vein endothelial cells (HUVECs) were exposed to anoxic conditions (5% CO2, 95% N2) for 16 hours in (1) normosmotic, (2) hyperosmotic (400 mOsm), or (3) hyperosmotic + VPA (4 mM) media. Immunohistochemistry and Western blots were performed to determine Cav-1 expression. Lungs from VPA-treated animals demonstrated decreased acute injury, MPO expression, and endothelial expression of Cav-1 when compared to lungs from animals resuscitated with NS or HTS alone. Similarly, HUVECs cultured in hyperosmotic media containing VPA demonstrated decreased expression of Cav-1. This study demonstrates that combined treatment with VPA and HTS is a viable strategy in hemorrhagic shock and polytrauma. Attenuation of lung injury following VPA treatment may be related to modulation of the inflammatory response.

The Effects of Molecular Hydrogen and Suberoylanilide Hydroxamic Acid on Paraquat-Induced Production of Reactive Oxygen Species and TNF-α in Macrophages.

The aim of this study is to investigate the effects of molecular hydrogen (H2) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on paraquat (PQ)-stimulated production of reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α) in macrophages. First, the PQ optimal concentration was determined in RAW264.7 macrophage by treating serum-starved cells with PQ at 0, 0.001, 0.01, 0.1, 1, and 10 mM. We evaluated at 1, 2 and 8 h (1) cell viability (by means of trypan blue exclusion method), (2) intracellular ROS levels (with a fluorescent DCFH-DA probe), and (3) TNF-α level in the culture media (determined by enzyme-linked immunosorbent assay, ELISA). Subsequently, mouse RAW267.4 macrophages were treated with PQ in combination with SAHA and/or H2 for 8 h. PQ exerted a significant stimulatory but nontoxic effect on RAW267.4 macrophages at 0.1 mM. This PQ concentration was used in the subsequent experiments. H2 and H2 combined with SAHA evoked a greater reduction in PQ-induced ROS production than SAHA alone, especially at 2 and 8 h. At 1 and 2 h, treatments involving H2 caused a greater decrease in PQ-induced production of TNF-α than the corresponding treatments without H2. However, at 8 h, treatment with SAHA evoked more pronounced effects on TNF-α than treatment without SAHA. H2 decreases PQ-induced ROS production and attenuates early PQ-induced TNF-α production whereas SAHA reduces the late phase of the PQ-induced TNF-α production in macrophages. The effects are enhanced by the combination of H2 and SAHA.