Velociraptor: Cross-Platform Quantitative Search Using Hallmark Cell Features.

A key challenge for single cell discovery analysis is to identify new cell types, describe them quantitatively, and seek these novel cells in new studies often using a different platform. Over the last decade, tools were developed to address identification and quantitative description of cells in human tissues and tumors. However, automated validation of populations at the single cell level has struggled due to the cytometry field's reliance on hierarchical, ordered use of features and on platform-specific rules for data processing and analysis. Here we present Velociraptor, a workflow that implements Marker Enrichment Modeling in three cross-platform modules: 1) identification of cells specific to disease states, 2) description of hallmark features for each cell and population, and 3) searching for cells matching one or more hallmark feature sets in a new dataset. A key advance is that Velociraptor registers cells between datasets, including between flow cytometry and quantitative imaging using different, overlapping feature sets. Four datasets were used to challenge Velociraptor and reveal new biological insights. Working at the individual sample level, Velociraptor tracked the abundance of clinically significant glioblastoma brain tumor cell subsets and characterized the cells that predominate in recurrent tumors as a close match for rare, negative prognostic cells originally observed in matched pre-treatment tumors. In patients with inborn errors of immunity, Velociraptor identified genotype-specific cells associated with GATA2 haploinsufficiency. Finally, in cross-platform analysis of immune cells in multiplex imaging of breast cancer, Velociraptor sought and correctly identified memory T cell subsets in tumors. Different phenotypic descriptions generated by algorithms or humans were shown to be effective as search inputs, indicating that cell identity need not be described in terms of per-feature cutoffs or strict hierarchical analyses. Velociraptor thus identifies cells based on hallmark feature sets, such as protein expression signatures, and works effectively with data from multiple sources, including suspension flow cytometry, imaging, and search text based on known or theoretical cell features.

IL-8 Instructs Macrophage Identity in Lateral Ventricle Contacting Glioblastoma.

Adult IDH-wildtype glioblastoma (GBM) is a highly aggressive brain tumor with no established immunotherapy or targeted therapy. Recently, CD32+ HLA-DRhi macrophages were shown to have displaced resident microglia in GBM tumors that contact the lateral ventricle stem cell niche. Since these lateral ventricle contacting GBM tumors have especially poor outcomes, identifying the origin and role of these CD32+ macrophages is likely critical to developing successful GBM immunotherapies. Here, we identify these CD32+ cells as M_IL-8 macrophages and establish that IL-8 is sufficient and necessary for tumor cells to instruct healthy macrophages into CD32+ M_IL-8 M2 macrophages. In ex vivo experiments with conditioned medium from primary human tumor cells, inhibitory antibodies to IL-8 blocked the generation of CD32+ M_IL-8 cells. Finally, using a set of 73 GBM tumors, IL-8 protein is shown to be present in GBM tumor cells in vivo and especially common in tumors contacting the lateral ventricle. These results provide a mechanistic origin for CD32+ macrophages that predominate in the microenvironment of the most aggressive GBM tumors. IL-8 and CD32+ macrophages should now be explored as targets in combination with GBM immunotherapies, especially for patients whose tumors present with radiographic contact with the ventricular-subventricular zone stem cell niche.

Non-canonical functions of a mutant TSC2 protein in mitotic division.

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. TSC is caused by mutations in the TSC1 or TSC2 genes, which encode the hamartin/tuberin proteins respectively. These proteins function as a heterodimer that negatively regulates the mechanistic Target of Rapamycin Complex 1 (mTORC1). TSC research has focused on the effects of mTORC1, a critical signaling hub, on regulation of diverse cell processes including metabolism, cell growth, translation, and neurogenesis. However, non-canonical functions of TSC2 are not well studied, and the potential disease-relevant biological mechanisms of mutations affecting these functions are not well understood. We observed aberrant multipolar mitotic division, a novel phenotype, in TSC2 mutant iPSCs. The multipolar phenotype is not meaningfully affected by treatment with the inhibitor rapamycin. We further observed dominant negative activity of the mutant form of TSC2 in producing the multipolar division phenotype. These data expand the knowledge of TSC2 function and pathophysiology which will be highly relevant to future treatments for patients with TSC.

An immunosuppressed microenvironment distinguishes lateral ventricle-contacting glioblastomas.

Radiographic contact of glioblastoma (GBM) tumors with the lateral ventricle and adjacent stem cell niche correlates with poor patient prognosis, but the cellular basis of this difference is unclear. Here, we reveal and functionally characterize distinct immune microenvironments that predominate in subtypes of GBM distinguished by proximity to the lateral ventricle. Mass cytometry analysis of isocitrate dehydrogenase wild-type human tumors identified elevated T cell checkpoint receptor expression and greater abundance of a specific CD32+CD44+HLA-DRhi macrophage population in ventricle-contacting GBM. Multiple computational analysis approaches, phospho-specific cytometry, and focal resection of GBMs validated and extended these findings. Phospho-flow quantified cytokine-induced immune cell signaling in ventricle-contacting GBM, revealing differential signaling between GBM subtypes. Subregion analysis within a given tumor supported initial findings and revealed intratumor compartmentalization of T cell memory and exhaustion phenotypes within GBM subtypes. Collectively, these results characterize immunotherapeutically targetable features of macrophages and suppressed lymphocytes in GBMs defined by MRI-detectable lateral ventricle contact.

Jedi-1/MEGF12-mediated phagocytosis controls the pro-neurogenic properties of microglia in the ventricular-subventricular zone.

Microglia are the primary phagocytes in the central nervous system and are responsible for clearing dead cells generated during development or disease. The phagocytic process shapes the phenotype of the microglia, which affects the local environment. A unique population of microglia reside in the ventricular-subventricular zone (V-SVZ) of neonatal mice, but how they influence this neurogenic niche is not well-understood. Here, we demonstrate that phagocytosis creates a pro-neurogenic microglial phenotype in the V-SVZ and that these microglia phagocytose apoptotic cells via the engulfment receptor Jedi-1. Deletion of Jedi-1 decreases apoptotic cell clearance, triggering the development of a neuroinflammatory phenotype, reminiscent of neurodegenerative and-age-associated microglia, that reduces neural precursor proliferation via elevated interleukin (IL)-1ß signaling; inhibition of IL-1 receptor rescues precursor proliferation in vivo. Together, these results reveal a critical role for Jedi-1 in connecting microglial phagocytic activity to a phenotype that promotes neurogenesis in the developing V-SVZ.

Alignment, segmentation and neighborhood analysis in cyclic immunohistochemistry data using CASSATT.

Cyclic immunohistochemistry (cycIHC) uses sequential rounds of colorimetric immunostaining and imaging for quantitative mapping of location and number of cells of interest. Additionally, cycIHC benefits from the speed and simplicity of brightfield microscopy, making the collection of entire tissue sections and slides possible at a trivial cost compared to other high dimensional imaging modalities. However, large cycIHC datasets currently require an expert data scientist to concatenate separate open-source tools for each step of image pre-processing, registration, and segmentation, or the use of proprietary software. Here, we present a unified and user-friendly pipeline for processing, aligning, and analyzing cycIHC data - Cyclic Analysis of Single-Cell Subsets and Tissue Territories (CASSATT). CASSATT registers scanned slide images across all rounds of staining, segments individual nuclei, and measures marker expression on each detected cell. Beyond straightforward single cell data analysis outputs, CASSATT explores the spatial relationships between cell populations. By calculating the log odds of interaction frequencies between cell populations within tissues and tissue regions, this pipeline helps users identify populations of cells that interact-or do not interact-at frequencies that are greater than those occurring by chance. It also identifies specific neighborhoods of cells based on the assortment of neighboring cell types that surround each cell in the sample. The presence and location of these neighborhoods can be compared across slides or within distinct regions within a tissue. CASSATT is a fully open source workflow tool developed to process cycIHC data and will allow greater utilization of this powerful staining technique.

Dephosphorylation of 4EBP1/2 Induces Prenatal Neural Stem Cell Quiescence.

A limiting factor in the regenerative capacity of the adult brain is the abundance and proliferative ability of neural stem cells (NSCs). Adult NSCs are derived from a subpopulation of embryonic NSCs that temporarily enter quiescence during mid-gestation and remain quiescent until postnatal reactivation. Here we present evidence that the mechanistic/mammalian target of rapamycin (mTOR) pathway regulates quiescence entry in embryonic NSCs of the developing forebrain. Throughout embryogenesis, two downstream effectors of mTOR, p-4EBP1/2 T37/46 and p-S6 S240/244, were mutually exclusive in NSCs, rarely occurring in the same cell. While 4EBP1/2 was phosphorylated in stem cells undergoing mitosis at the ventricular surface, S6 was phosphorylated in more differentiated cells migrating away from the ventricle. Phosphorylation of 4EBP1/2, but not S6, was responsive to quiescence induction in cultured embryonic NSCs. Further, inhibition of p-4EBP1/2, but not p-S6, was sufficient to induce quiescence. Collectively, this work offers new insight into the regulation of quiescence entry in embryonic NSCs and, thereby, correct patterning of the adult brain. These data suggest unique biological functions of specific posttranslational modifications and indicate that the preferential inhibition of such modifications may be a useful therapeutic approach in neurodevelopmental diseases where NSC numbers, proliferation, and differentiation are altered.

GBMdeconvoluteR accurately infers proportions of neoplastic and immune cell populations from bulk glioblastoma transcriptomics data.

BACKGROUND: Characterizing and quantifying cell types within glioblastoma (GBM) tumors at scale will facilitate a better understanding of the association between the cellular landscape and tumor phenotypes or clinical correlates. We aimed to develop a tool that deconvolutes immune and neoplastic cells within the GBM tumor microenvironment from bulk RNA sequencing data. METHODS: We developed an IDH wild-type (IDHwt) GBM-specific single immune cell reference consisting of B cells, T-cells, NK-cells, microglia, tumor associated macrophages, monocytes, mast and DC cells. We used this alongside an existing neoplastic single cell-type reference for astrocyte-like, oligodendrocyte- and neuronal progenitor-like and mesenchymal GBM cancer cells to create both marker and gene signature matrix-based deconvolution tools. We applied single-cell resolution imaging mass cytometry (IMC) to ten IDHwt GBM samples, five paired primary and recurrent tumors, to determine which deconvolution approach performed best. RESULTS: Marker-based deconvolution using GBM-tissue specific markers was most accurate for both immune cells and cancer cells, so we packaged this approach as GBMdeconvoluteR. We applied GBMdeconvoluteR to bulk GBM RNAseq data from The Cancer Genome Atlas and recapitulated recent findings from multi-omics single cell studies with regards associations between mesenchymal GBM cancer cells and both lymphoid and myeloid cells. Furthermore, we expanded upon this to show that these associations are stronger in patients with worse prognosis. CONCLUSIONS: GBMdeconvoluteR accurately quantifies immune and neoplastic cell proportions in IDHwt GBM bulk RNA sequencing data and is accessible here: https://gbmdeconvoluter.leeds.ac.uk.

Learning cell identity in immunology, neuroscience, and cancer.

Suspension and imaging cytometry techniques that simultaneously measure hundreds of cellular features are powering a new era of cell biology and transforming our understanding of human tissues and tumors. However, a central challenge remains in learning the identities of unexpected or novel cell types. Cell identification rubrics that could assist trainees, whether human or machine, are not always rigorously defined, vary greatly by field, and differentially rely on cell intrinsic measurements, cell extrinsic tissue measurements, or external contextual information such as clinical outcomes. This challenge is especially acute in the context of tumors, where cells aberrantly express developmental programs that are normally time, location, or cell-type restricted. Well-established fields have contrasting practices for cell identity that have emerged from convention and convenience as much as design. For example, early immunology focused on identifying minimal sets of protein features that mark individual, functionally distinct cells. In neuroscience, features including morphology, development, and anatomical location were typical starting points for defining cell types. Both immunology and neuroscience now aim to link standardized measurements of protein or RNA to informative cell functions such as electrophysiology, connectivity, lineage potential, phospho-protein signaling, cell suppression, and tumor cell killing ability. The expansion of automated, machine-driven methods for learning cell identity has further created an urgent need for a harmonized framework for distinguishing cell identity across fields and technology platforms. Here, we compare practices in the fields of immunology and neuroscience, highlight concepts from each that might work well in the other, and propose ways to implement these ideas to study neural and immune cell interactions in brain tumors and associated model systems.

Pembrolizumab alters the tumor immune landscape in a patient with dMMR glioblastoma.

Congenital DNA mismatch repair defects (dMMR), such as Lynch Syndrome, predispose patients to a variety of cancers and account for approximately 1% of glioblastoma cases. While few therapeutic options exist for glioblastoma, checkpoint blockade therapy has proven effective in dMMR tumors. Here we present a case study of a male in their 30s diagnosed with dMMR glioblastoma treated with pembrolizumab who experienced a partial response to therapy. Using a multiplex IHC analysis pipeline on archived slide specimens from tumor resections at diagnosis and after therapeutic interventions, we quantified changes in the frequency and spatial distribution of key cell populations in the tumor tissue. Notably, proliferating (KI67+) macrophages and T cells increased in frequency as did other KI67+ cells within the tumor. Therapeutic intervention remodeled the cellular spatial distribution in the tumor leading to a greater frequency of macrophage/tumor cell interactions and T cell/T cell interactions, highlighting impacts of checkpoint blockade on tumor cytoarchitecture and revealing spatial patterns that may indicate advantageous immune interactions in glioma and other solid tumors treated with these agents.

Rescue of Impaired Blood-Brain Barrier in Tuberous Sclerosis Complex Patient Derived Neurovascular Unit.

Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene. Dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB). We generated disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. Using these microphysiological systems, we demonstrate that the BBB generated from TSC2 heterozygous mutant cells shows increased permeability which can be rescued by wild type astrocytes and with treatment with rapamycin, an mTOR kinase inhibitor. Our results further demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of the cell lineages contributing to TSC pathogenesis.

GLI3 Is Required for OLIG2+ Progeny Production in Adult Dorsal Neural Stem Cells.

The ventricular-subventricular zone (V-SVZ) is a postnatal germinal niche. It holds a large population of neural stem cells (NSCs) that generate neurons and oligodendrocytes for the olfactory bulb and (primarily) the corpus callosum, respectively. These NSCs are heterogeneous and generate different types of neurons depending on their location. Positional identity among NSCs is thought to be controlled in part by intrinsic pathways. However, extrinsic cell signaling through the secreted ligand Sonic hedgehog (Shh) is essential for neurogenesis in both the dorsal and ventral V-SVZ. Here we used a genetic approach to investigate the role of the transcription factors GLI2 and GLI3 in the proliferation and cell fate of dorsal and ventral V-SVZ NSCs. We find that while GLI3 is expressed in stem cell cultures from both dorsal and ventral V-SVZ, the repressor form of GLI3 is more abundant in dorsal V-SVZ. Despite this high dorsal expression and the requirement for other Shh pathway members, GLI3 loss affects the generation of ventrally-, but not dorsally-derived olfactory interneurons in vivo and does not affect trilineage differentiation in vitro. However, loss of GLI3 in the adult dorsal V-SVZ in vivo results in decreased numbers of OLIG2-expressing progeny, indicating a role in gliogenesis.

Modeling tuberous sclerosis with organoids.

[Figure: see text].

Histological Studies of the Ventricular-Subventricular Zone as Neural Stem Cell and Glioma Stem Cell Niche.

The neural stem cell niche of the ventricular-subventricular zone supports the persistence of stem and progenitor cells in the mature brain. This niche has many notable cytoarchitectural features that affect the activity of stem cells and may also support the survival and growth of invading tumor cells. Histochemical studies of the niche have revealed many proteins that, in combination, can help to reveal stem-like cells in the normal or cancer context, although many caveats persist in the quest to consistently identify these cells in the human brain. Here, we explore the complex relationship between the persistent proliferative capacity of the neural stem cell niche and the malignant proliferation of brain tumors, with a special focus on histochemical identification of stem cells and stem-like tumor cells and an eye toward the potential application of high-dimensional imaging approaches to the field.

Creation and validation of 3D-printed head molds for stereotaxic injections of neonatal mouse brains.

BACKGROUND: An increasing number of rodent model systems use injection of DNA or viral constructs in the neonatal brain. However, approaches for reliable positioning and stereotaxic injection at this developmental stage are limited, typically relying on handheld positioning or molds that must be re-aligned for use in a given laboratory. NEW METHOD: A complete protocol and open-source software pipeline for generating 3D-printed head molds derived from a CT scan of a neonatal mouse head cast, together with a universal adapter that can be placed on a standard stereotaxic stage. RESULTS: A series of test injections with adenovirus encoding red fluorescent protein, or Fluorogold, were conducted using original clay molds and newly generated 3D printed molds. Several metrics were used to compare spread and localization of targeted injections. COMPARISON WITH EXISTING METHODS: The new method of head mold generation gave comparable results to the field standard, but also allowed the rapid generation of additional copies of each head mold with standardized positioning of the head each time. CONCLUSIONS: This 3D printing pipeline can be used to efficiently develop a series of head molds with standardized injection coordinates across multiple laboratories. More broadly, this pipeline can easily be adapted to other perinatal ages or species.

Sustained response to erlotinib and rapamycin in a patient with pediatric anaplastic oligodendroglioma.

One goal of precision medicine is to identify mutations within individual tumors to design targeted treatment approaches. This report details the use of genomic testing to select a targeted therapy regimen of erlotinib and rapamycin for a pediatric anaplastic oligodendroglioma refractory to standard treatment, achieving a 33-month sustained response. Immunohistochemical analysis of total and phosphorylated protein isoforms showed abnormal signaling consistent with detected mutations, while revealing heterogeneity in per-cell activation of signaling pathways in multiple subpopulations of tumor cells throughout the course of disease. This case highlights molecular features that may be relevant to designing future targeted treatments.

Glioblastoma Distance From the Subventricular Neural Stem Cell Niche Does Not Correlate With Survival.

OBJECTIVE: To determine the relationship between survival and glioblastoma distance from the ventricular-subventricular neural stem cell niche (VSVZ). METHODS: 502 pre-operative gadolinium-enhanced, T1-weighted MRIs with glioblastoma retrieved from an institutional dataset (n = 252) and The Cancer Imaging Atlas (n=250) were independently reviewed. The shortest distance from the tumor contrast enhancement to the nearest lateral ventricular wall, the location of the VSVZ, was measured (GBM-VSVZDist). The relationship of GBM-VSVZDist with the proportion of glioblastomas at each distance point and overall survival was explored with a Pearson's correlation and Cox regression model, respectively, adjusting for the well-established glioblastoma prognosticators. RESULTS: 244/502 glioblastomas had VSVZ contact. The proportion of non-VSVZ-contacting glioblastomas correlated inversely with GBM-VSVZDist (partial Pearson's correlation adjusted for tumor volume R=-0.79, p=7.11x10-7). A fit of the Cox regression model adjusted for age at diagnosis, Karnofsky performance status score, post-operative treatment with temozolomide and/or radiotherapy, IDH1/2 mutation status, MGMT promoter methylation status, tumor volume, and extent of resection demonstrated a significantly decreased overall survival only when glioblastoma contacted the VSVZ. Overall survival did not correlate with GBM-VSVZDist. CONCLUSIONS: In the two independent cohorts analyzed, glioblastomas at diagnosis were found in close proximity or in contact with the VSVZ with a proportion that decreased linearly with GBM-VSVZDist. Patient survival was only influenced by the presence or absence of a gadolinium-enhanced glioblastoma contact with the VSVZ. These results may guide analyses to test differential effectiveness of VSVZ radiation in VSVZ-contacting and non-contacting glioblastomas and/or inform patient selection criteria in clinical trials of glioblastoma radiation.

Unexpected effects of ivermectin and selamectin on inducible CreER activity in mice.

BACKGROUND: Anti-parasitics are frequently used in research animal facilities to treat a multitude of common infections, with pinworms and fur mites being amongst the most common. Ivermectin and selamectin are common oral and topical treatments for these infections, respectively. Although commonly thought to be innocuous to both the research animals and any transgenic elements that the animals may carry, evidence exists that ivermectin is capable of activating the recombinase activity of at least one CreER. The goal of the current study was to determine if there was an effect of either anti-parasitic agent on the activity of CreER proteins in transgenic mice. CASE PRESENTATION: We analyzed the offspring of transgenic mice exposed to either ivermectin or selamectin during pregnancy and nursing. Through analysis of reporter genes co-expressed with multiple, independently generated transgenic CreER drivers, we report here that ivermectin and selamectin both alter recombinase activity and thus may have unintended consequences on gene inactivation studies in mice. CONCLUSIONS: Although the mechanisms by which ivermectin and selamectin affect CreER activity in the offspring of treated dams remain unclear, the implications are important nonetheless. Treatment of pregnant transgenic mice with these anti-parasitics has the potential to alter transgene activity in the offspring. Special considerations should be made when planning treatment of transgenic mice with either of these pharmacologics.

DEPDC5 haploinsufficiency drives increased mTORC1 signaling and abnormal morphology in human iPSC-derived cortical neurons.

Mutations in the DEPDC5 gene can cause epilepsy, including forms with and without brain malformations. The goal of this study was to investigate the contribution of DEPDC5 gene dosage to the underlying neuropathology of DEPDC5-related epilepsies. We generated induced pluripotent stem cells (iPSCs) from epilepsy patients harboring heterozygous loss of function mutations in DEPDC5. Patient iPSCs displayed increases in both phosphorylation of ribosomal protein S6 and proliferation rate, consistent with elevated mTORC1 activation. In line with these findings, we observed increased soma size in patient iPSC-derived cortical neurons that was rescued with rapamycin treatment. These data indicate that human cells heterozygous for DEPDC5 loss-of-function mutations are haploinsufficient for control of mTORC1 signaling. Our findings suggest that human pathology differs from mouse models of DEPDC5-related epilepsies, which do not show consistent phenotypic differences in heterozygous neurons, and support the need for human-based models to affirm and augment the findings from animal models of DEPDC5-related epilepsy.

Unsupervised machine learning reveals risk stratifying glioblastoma tumor cells.

A goal of cancer research is to reveal cell subsets linked to continuous clinical outcomes to generate new therapeutic and biomarker hypotheses. We introduce a machine learning algorithm, Risk Assessment Population IDentification (RAPID), that is unsupervised and automated, identifies phenotypically distinct cell populations, and determines whether these populations stratify patient survival. With a pilot mass cytometry dataset of 2 million cells from 28 glioblastomas, RAPID identified tumor cells whose abundance independently and continuously stratified patient survival. Statistical validation within the workflow included repeated runs of stochastic steps and cell subsampling. Biological validation used an orthogonal platform, immunohistochemistry, and a larger cohort of 73 glioblastoma patients to confirm the findings from the pilot cohort. RAPID was also validated to find known risk stratifying cells and features using published data from blood cancer. Thus, RAPID provides an automated, unsupervised approach for finding statistically and biologically significant cells using cytometry data from patient samples.