Myeloid-specific KDM6B inhibition sensitizes glioblastoma to PD1 blockade.

Glioblastoma (GBM) tumors are enriched in immune-suppressive myeloid cells and are refractory to immune checkpoint therapy (ICT). Targeting epigenetic pathways to reprogram the functional phenotype of immune-suppressive myeloid cells to overcome resistance to ICT remains unexplored. Single-cell and spatial transcriptomic analyses of human GBM tumors demonstrated high expression of an epigenetic enzyme-histone 3 lysine 27 demethylase (KDM6B)-in intratumoral immune-suppressive myeloid cell subsets. Importantly, myeloid cell-specific Kdm6b deletion enhanced proinflammatory pathways and improved survival in GBM tumor-bearing mice. Mechanistic studies showed that the absence of Kdm6b enhances antigen presentation, interferon response and phagocytosis in myeloid cells by inhibition of mediators of immune suppression including Mafb, Socs3 and Sirpa. Further, pharmacological inhibition of KDM6B mirrored the functional phenotype of Kdm6b-deleted myeloid cells and enhanced anti-PD1 efficacy. This study thus identified KDM6B as an epigenetic regulator of the functional phenotype of myeloid cell subsets and a potential therapeutic target for enhanced response to ICT.

Immune checkpoint therapy-current perspectives and future directions.

Immune checkpoint therapy (ICT) has dramatically altered clinical outcomes for cancer patients and conferred durable clinical benefits, including cure in a subset of patients. Varying response rates across tumor types and the need for predictive biomarkers to optimize patient selection to maximize efficacy and minimize toxicities prompted efforts to unravel immune and non-immune factors regulating the responses to ICT. This review highlights the biology of anti-tumor immunity underlying response and resistance to ICT, discusses efforts to address the current challenges with ICT, and outlines strategies to guide the development of subsequent clinical trials and combinatorial efforts with ICT.

Translation and validation of Cerebral Palsy Quality of Life Questionnaire-Teen in Hong Kong Chinese population [CP QoL-Teen (HK)].

Cerebral palsy (CP) is an early onset, non-progressive, neuromotor disorder. Adolescence is the transition from childhood to adulthood when changes in physical and emotional aspects and self-perception occur further imposing an impact to quality of life (QoL) in individuals with CP. Cerebral Palsy Quality of Life (CP QoL) Teen is a questionnaire examining different domains of QoL for adolescents with CP. This study is aimed at translating and validating self-report and proxy-report CP QoL-Teen (HK). Prior approval of translation has been obtained. Forward and backward translations were performed following standardized translation procedures. Participants and their caregivers were asked to complete self-report and proxy-report CP QoL-Teen (HK), and Child Health Questionnaire (CHQ). Internal consistency and test-retest reliability were assessed by Cronbach's alpha and intraclass correlation coefficient (ICC), respectively. Concurrent validity was evaluated by Spearman's rank correlation between subscales of CP QoL-Teen (HK) and CHQ as well as expanded and revised version of Gross Motor Function Classification System (GMFCS-E&R). Ninety-six participants completed the study. Of these, twenty participants completed CP QoL-Teen (HK) twice. Cronbach's α of CP QoL-Teen (HK) ranged from 0.84 to 0.95 suggesting excellent internal consistency. Moderate to excellent test-retest reliability were demonstrated in all subscales of CP QoL-Teen (HK) (self-report: ICC = 0.46-0.8; proxy-report: ICC = 0.40-0.72, p < 0.05). Weak to moderate association between subscales of CP QoL-Teen (HK) and CHQ (self-report: rs = 0.24-0.61; proxy-report: rs = - 0.41-0.60) was reported. CONCLUSION: This study showed that CP QoL-Teen (HK) has good psychometric properties. It is a valid and reliable tool to assess quality of life of adolescents with CP. WHAT IS KNOWN: • Cerebral Palsy Quality of life-Teen (CP QoL-Teen) is a validated tool with strong psychometric properties and clinical utility in gauging the QoL in adolescents with CP during their transition from childhood to adulthood when changes in physical and emotional aspects and self-perception occur. Yet, a locally validated tool is lacking in measuring the QoL for adolescents with CP in Hong Kong. WHAT IS NEW: • The Chinese translated version CP QoL-Teen (HK) is a valid and reliable tool to assess quality of life of adolescents with CP tailoring to the local cultural and social background with good psychometric properties being demonstrated.

Control of brain tumor growth by reactivating myeloid cells with niacin.

Glioblastomas are generally incurable partly because monocytes, macrophages, and microglia in afflicted patients do not function in an antitumor capacity. Medications that reactivate these macrophages/microglia, as well as circulating monocytes that become macrophages, could thus be useful to treat glioblastoma. We have discovered that niacin (vitamin B3) is a potential stimulator of these inefficient myeloid cells. Niacin-exposed monocytes attenuated the growth of brain tumor-initiating cells (BTICs) derived from glioblastoma patients by producing anti-proliferative interferon-α14. Niacin treatment of mice bearing intracranial BTICs increased macrophage/microglia representation within the tumor, reduced tumor size, and prolonged survival. These therapeutic outcomes were negated in mice depleted of circulating monocytes or harboring interferon-α receptor-deleted BTICs. Combination treatment with temozolomide enhanced niacin-promoted survival. Monocytes from glioblastoma patients had increased interferon-α14 upon niacin exposure and were reactivated to reduce BTIC growth in culture. We highlight niacin, a common vitamin that can be quickly translated into clinical application, as an immune stimulator against glioblastomas.

Demeclocycline Reduces the Growth of Human Brain Tumor-Initiating Cells: Direct Activity and Through Monocytes.

Myeloid cells that infiltrate into brain tumors are deactivated or exploited by the tumor cells. We previously demonstrated that compromised microglia, monocytes, and macrophages in malignant gliomas could be reactivated by amphotericin-B to contain the growth of brain tumorinitiating cells (BTICs). We identified meclocycline as another activator of microglia, so we sought to test whether its better-tolerated derivative, demeclocycline, also stimulates monocytes to restrict BTIC growth. Monocytes were selected for study as they would be exposed to demeclocycline in the circulation prior to entry into brain tumors to become macrophages. We found that demeclocycline increased the activity of monocytes in culture, as determined by tumor necrosis factor-α production and chemotactic capacity. The conditioned medium of demeclocycline-stimulated monocytes attenuated the growth of BTICs generated from human glioblastoma resections, as evaluated using neurosphere and alamarBlue assays, and cell counts. Demeclocycline also had direct effects in reducing BTIC growth. A global gene expression screen identified several genes, such as DNA damage inducible transcript 4, frizzled class receptor 5 and reactive oxygen species modulator 1, as potential regulators of demeclocycline-mediated BTIC growth reduction. Amongst several tetracycline derivatives, only demeclocycline directly reduced BTIC growth. In summary, we have identified demeclocycline as a novel inhibitor of the growth of BTICs, through direct effect and through indirect stimulation of monocytes. Demeclocycline is a candidate to reactivate compromised immune cells to improve the prognosis of patients with gliomas.

Differential microglia and macrophage profiles in human IDH-mutant and -wild type glioblastoma.

Microglia and macrophages are the largest component of the inflammatory infiltrate in glioblastoma (GBM). However, whether there are differences in their representation and activity in the prognostically-favorable isocitrate dehydrogenase (IDH)-mutated compared to -wild type GBMs is unknown. Studies on human specimens of untreated IDH-mutant GBMs are rare given they comprise 10% of all GBMs and often present at lower grades, receiving treatments prior to dedifferentiation that can drastically alter microglia and macrophage phenotypes. We were able to obtain large samples of four previously untreated IDH-mutant GBM. Using flow cytometry, immunofluorescence techniques with automated segmentation protocols that quantify at the individual-cell level, and comparison between single-cell RNA-sequencing (scRNA-seq) databases of human GBM, we discerned dissimilarities between GBM-associated microglia and macrophages (GAMMs) in IDH-mutant and -wild type GBMs. We found there are significantly fewer GAMM in IDH-mutant GBMs, but they are more pro-inflammatory, suggesting this contributes to the better prognosis of these tumors. Our pro-inflammatory score which combines the expression of inflammatory markers (CD68/HLA-A, -B, -C/TNF/CD163/IL10/TGFB2), Iba1 intensity, and GAMM surface area also indicates that more pro-inflammatory GAMMs are associated with longer overall survival independent of IDH status. Interrogation of scRNA-seq databases demonstrates microglia in IDH-mutants are mainly pro-inflammatory, while anti-inflammatory macrophages that upregulate genes such as FCER1G and TYROBP predominate in IDH-wild type GBM. Taken together, these observations are the first head-to-head comparison of GAMMs in treatment-naïve IDH-mutant versus -wild type GBMs. Our findings highlight biological disparities in the innate immune microenvironment related to IDH prognosis that can be exploited for therapeutic purposes.

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.

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System.

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

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.

Development of crizotinib, a rationally designed tyrosine kinase inhibitor for non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the number one cause of global mortality. Despite aggressive treatment, the prognosis is dismal. Patients with advanced NSCLC have a median survival of 4 months from the time of diagnosis. Fortunately, molecularly based approaches to drug discovery have yielded a tyrosine kinase inhibitor, crizotinib, which significantly prolongs median progression-free survival in a subset of patients. Although initial clinical trial results demonstrate crizotinib has a promising role to play in NSCLC treatment, development of resistance leaves much to be elucidated about how to effectively combat this deadly disease. In this review, we follow the discovery and development of crizotinib from bench to bedside and provide an example of successful bottom-up drug design. Then, we explore the clinical trial results that fast-tracked its eventual use as a frontline therapy for sensitive NSCLC patients and the development of resistance. Lastly, we discuss the potential for future uses of crizotinib both within and beyond NSCLC.

Comparative genomic and genetic analysis of glioblastoma-derived brain tumor-initiating cells and their parent tumors.

BACKGROUND: Glioblastoma (GBM) is a fatal cancer that has eluded major therapeutic advances. Failure to make progress may reflect the absence of a human GBM model that could be used to test compounds for anti-GBM activity. In this respect, the development of brain tumor-initiating cell (BTIC) cultures is a step forward because BTICs appear to capture the molecular diversity of GBM better than traditional glioma cell lines. Here, we perform a comparative genomic and genetic analysis of BTICs and their parent tumors as preliminary evaluation of the BTIC model. METHODS: We assessed single nucleotide polymorphisms (SNPs), genome-wide copy number variations (CNVs), gene expression patterns, and molecular subtypes of 11 established BTIC lines and matched parent tumors. RESULTS: Although CNV differences were noted, BTICs retained the major genomic alterations characteristic of GBM. SNP patterns were similar between BTICs and tumors. Importantly, recurring SNP or CNV alterations specific to BTICs were not seen. Comparative gene expression analysis and molecular subtyping revealed differences between BTICs and GBMs. These differences formed the basis of a 63-gene expression signature that distinguished cells from tumors; differentially expressed genes primarily involved metabolic processes. We also derived a set of 73 similarly expressed genes; these genes were not associated with specific biological functions. CONCLUSIONS: Although not identical, established BTIC lines preserve the core molecular alterations seen in their parent tumors, as well as the genomic hallmarks of GBM, without acquiring recurring BTIC-specific changes.

Selective dorsal rhizotomy in Hong Kong: multidimensional outcome measures.

We prospectively case series study evaluated the short-term effectiveness of selective dorsal rhizotomy plus physiotherapy. Twenty children with spastic cerebral palsy, selected for selective dorsal rhizotomy (mean age, 8.57 years; range, 5.96-11.18 years), were assessed before, and 6 and 12 months after, selective dorsal rhizotomy. Main outcome measures included the Modified Ashworth Scale, passive range of joint movement, the Gross Motor Function Measure, the Pediatric Evaluation of Disability Inventory, the Canadian Occupational Performance Measure, and three-dimensional gait analysis. The results confirmed that selective dorsal rhizotomy plus physiotherapy provided a statistically significant reduction of spasticity, functional improvements in mobility and self-care performance, and increased participation in social situations in our study group (85% exhibited normal intelligence, and 90% belonged to Gross Motor Function Classification System levels I-III). The Gross Motor Function Measure proved to be sensitive in documenting motor functional changes, except for children at Gross Motor Function Classification System level I. Instrumental three-dimensional gait analysis with kinematics and kinetics data analysis confirmed gait improvements in children of higher motor function. The Canadian Occupational Performance Measure indicated improvements in social participation.

Intracerebral hemorrhage models in rat: comparing collagenase to blood infusion.

Many therapies have shown promise in preclinical stroke studies, but few benefit patients. A greater understanding of stroke pathophysiology is needed to successfully develop therapies, and this depends on appropriate animal models. The collagenase and blood infusion models of intracerebral hemorrhage (ICH) are widely used; yet, investigators often prefer using one model for a variety of reasons. Thus, we directly compared these to highlight advantages and limitations of each as well as the assessment approach. An ICH was created by infusing blood or bacterial collagenase into the rats' striatum. We matched initial hematoma volume in each model (Experiment 1) and assessed the time course of bleeding (Experiment 2). Functional deficits and the progression of injury were tracked over 6 weeks using behavior, magnetic resonance imaging, and histology (Experiment 3). Despite similar initial hematoma volumes, collagenase-induced ICH resulted in a greater blood-brain barrier breakdown and more damage to the striatum, substantia nigra, white matter, and cortex. Magnetic resonance imaging revealed faster hematoma resolution in the blood model, and little increase in the volume of tissue lost from 1 to 6 weeks. In contrast, tissue loss continued over 4 weeks in the collagenase model. Finally, functional deficits recovered more quickly and completely in the blood model. This study highlights key differences between these models and that neither closely replicates the human condition. Thus, both should be used whenever possible taking into account the significant differences between these models and their limitations. Furthermore, this work illustrates significant weaknesses with several outcome measures.