Quality Control for Single Cell Analysis of High-plex Tissue Profiles using CyLinter.

Tumors are complex assemblies of cellular and acellular structures patterned on spatial scales from microns to centimeters. Study of these assemblies has advanced dramatically with the introduction of high-plex spatial profiling. Image-based profiling methods reveal the intensities and spatial distributions of 20-100 proteins at subcellular resolution in 103-107 cells per specimen. Despite extensive work on methods for extracting single-cell data from these images, all tissue images contain artefacts such as folds, debris, antibody aggregates, optical aberrations and image processing errors that arise from imperfections in specimen preparation, data acquisition, image assembly, and feature extraction. We show that these artefacts dramatically impact single-cell data analysis, obscuring meaningful biological interpretation. We describe an interactive quality control software tool, CyLinter, that identifies and removes data associated with imaging artefacts. CyLinter greatly improves single-cell analysis, especially for archival specimens sectioned many years prior to data collection, such as those from clinical trials.

2D and 3D multiplexed subcellular profiling of nuclear instability in human cancer.

Nuclear atypia, including altered nuclear size, contour, and chromatin organization, is ubiquitous in cancer cells. Atypical primary nuclei and micronuclei can rupture during interphase; however, the frequency, causes, and consequences of nuclear rupture are unknown in most cancers. We demonstrate that nuclear envelope rupture is surprisingly common in many human cancers, particularly glioblastoma. Using highly-multiplexed 2D and super-resolution 3D-imaging of glioblastoma tissues and patient-derived xenografts and cells, we link primary nuclear rupture with reduced lamin A/C and micronuclear rupture with reduced lamin B1. Moreover, ruptured glioblastoma cells activate cGAS-STING-signaling involved in innate immunity. We observe that local patterning of cell states influences tumor spatial organization and is linked to both lamin expression and rupture frequency, with neural-progenitor-cell-like states exhibiting the lowest lamin A/C levels and greatest susceptibility to primary nuclear rupture. Our study reveals that nuclear instability is a core feature of cancer, and links nuclear integrity, cell state, and immune signaling.

Addressing persistent challenges in digital image analysis of cancerous tissues.

The National Cancer Institute (NCI) supports many research programs and consortia, many of which use imaging as a major modality for characterizing cancerous tissue. A trans-consortia Image Analysis Working Group (IAWG) was established in 2019 with a mission to disseminate imaging-related work and foster collaborations. In 2022, the IAWG held a virtual hackathon focused on addressing challenges of analyzing high dimensional datasets from fixed cancerous tissues. Standard image processing techniques have automated feature extraction, but the next generation of imaging data requires more advanced methods to fully utilize the available information. In this perspective, we discuss current limitations of the automated analysis of multiplexed tissue images, the first steps toward deeper understanding of these limitations, what possible solutions have been developed, any new or refined approaches that were developed during the Image Analysis Hackathon 2022, and where further effort is required. The outstanding problems addressed in the hackathon fell into three main themes: 1) challenges to cell type classification and assessment, 2) translation and visual representation of spatial aspects of high dimensional data, and 3) scaling digital image analyses to large (multi-TB) datasets. We describe the rationale for each specific challenge and the progress made toward addressing it during the hackathon. We also suggest areas that would benefit from more focus and offer insight into broader challenges that the community will need to address as new technologies are developed and integrated into the broad range of image-based modalities and analytical resources already in use within the cancer research community.

Comparison of chest radiograph findings in ambulatory and hospitalized children with pulmonary tuberculosis.

BACKGROUND: The diagnosis of childhood tuberculosis (TB) is, in many instances, solely reliant on chest radiographs (CXRs), as they are often the only diagnostic tool available, especially in TB-endemic areas. Accuracy and reliability of CXRs for detecting TB lymphadenopathy may vary between groups depending on severity of presentation and presence of parenchymal disease, which may obscure visualization. OBJECTIVE: To compare CXR findings in ambulatory versus hospitalized children with laboratory confirmed pulmonary TB versus other lower respiratory tract infections (LRTI) and test inter-rater agreement for these findings. MATERIALS AND METHODS: Retrospective review, by two pediatric radiologists, of CXRs performed on children < 12 years old referred for evaluation of LRTI with clinical suspicion of pulmonary TB in inpatient and outpatient settings. Each radiologist commented on imaging findings of parenchymal changes, lymphadenopathy, airway compression and pleural effusion. Frequency of imaging findings was compared between patients based on location and diagnosis and inter-rater agreement was determined. Accuracy of radiographic diagnosis was compared to laboratory testing which served as the gold standard. RESULTS: The number of enrolled patients was 181 (54% males); 69 (38%) were ambulatory and 112 (62%) were hospitalized. Of those enrolled, 87 (48%) were confirmed to have pulmonary TB, while 94 (52%) were other LRTI controls. Lymphadenopathy and airway compression were more common in TB patients than other LRTI controls, regardless of patient location. Parenchymal changes and pleural effusion were more common in hospitalized than ambulatory patients, regardless of patient diagnosis. Agreement for parenchymal changes was higher in the hospitalized group (kappa [κ] = 0.75), while agreement for lymphadenopathy (κ = 0.65) and airway compression (κ = 0.68) was higher in the ambulatory group. The specificity of CXRs for TB diagnosis (> 75%) was higher than the sensitivity (< 50%) for both ambulatory and hospitalized groups. CONCLUSION: Higher frequency of parenchymal changes among hospitalized children may conceal specific imaging findings of TB such as lymphadenopathy, contributing to the poor reliability of CXRs. Despite this, the high specificity of CXRs shown in our results is encouraging for continued use of radiographs for TB diagnosis in both settings.

Antigen presentation deficiency, mesenchymal differentiation, and resistance to immunotherapy in the murine syngeneic CT2A tumor model.

Background: The GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to immunotherapy. Methods: To decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics. Results: The transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM. Both models exhibited striking hypermutation, with clonal hotspot mutations in RAS genes (Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc). CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery (e.g. Tap1 p.Y488C and Psmb8 p.A275P mutations) and interferon response pathways (e.g. copy number losses of loci including IFN genes and reduced phosphorylation of JAK/STAT pathway members). The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, which play important roles as myeloid chemoattractants. Conclusion: Although the clinical contexts that can be modeled by GL261 and CT2A for huGBM are limited, CT2A may be an informative model of immunotherapy resistance due to its deficits in antigen presentation machinery and interferon response pathways.

Oncometabolite d-2HG alters T cell metabolism to impair CD8+ T cell function.

Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d-2-hydroxyglutarate (d-2HG), an oncometabolite that promotes tumorigenesis through epigenetic alterations. The cancer cell-intrinsic effects of d-2HG are well understood, but its tumor cell-nonautonomous roles remain poorly explored. We compared the oncometabolite d-2HG with its enantiomer, l-2HG, and found that tumor-derived d-2HG was taken up by CD8+ T cells and altered their metabolism and antitumor functions in an acute and reversible fashion. We identified the glycolytic enzyme lactate dehydrogenase (LDH) as a molecular target of d-2HG. d-2HG and inhibition of LDH drive a metabolic program and immune CD8+ T cell signature marked by decreased cytotoxicity and impaired interferon-γ signaling that was recapitulated in clinical samples from human patients with IDH1 mutant gliomas.

Single cell spatial analysis reveals the topology of immunomodulatory purinergic signaling in glioblastoma.

How the glioma immune microenvironment fosters tumorigenesis remains incompletely defined. Here, we use single-cell RNA-sequencing and multiplexed tissue-imaging to characterize the composition, spatial organization, and clinical significance of extracellular purinergic signaling in glioma. We show that microglia are the predominant source of CD39, while tumor cells principally express CD73. In glioblastoma, CD73 is associated with EGFR amplification, astrocyte-like differentiation, and increased adenosine, and is linked to hypoxia. Glioblastomas enriched for CD73 exhibit inflammatory microenvironments, suggesting that purinergic signaling regulates immune adaptation. Spatially-resolved single-cell analyses demonstrate a strong spatial correlation between tumor-CD73 and microglial-CD39, with proximity associated with poor outcomes. Similar spatial organization is present in pediatric high-grade gliomas including H3K27M-mutant diffuse midline glioma. These data reveal that purinergic signaling in gliomas is shaped by genotype, lineage, and functional state, and that core enzymes expressed by tumor and myeloid cells are organized to promote adenosine-rich microenvironments potentially amenable to therapeutic targeting.

MCMICRO: a scalable, modular image-processing pipeline for multiplexed tissue imaging.

Highly multiplexed tissue imaging makes detailed molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of large multichannel images poses a substantial computational challenge. Here, we describe a modular and open-source computational pipeline, MCMICRO, for performing the sequential steps needed to transform whole-slide images into single-cell data. We demonstrate the use of MCMICRO on tissue and tumor images acquired using multiple imaging platforms, thereby providing a solid foundation for the continued development of tissue imaging software.

A novel miR1983-TLR7-IFNß circuit licenses NK cells to kill glioma cells, and is under the control of galectin-1.

Although pharmacological stimulation of TLRs has anti-tumor effects, it has not been determined whether endogenous stimulation of TLRs can lead to tumor rejection. Herein, we demonstrate the existence of an innate anti-glioma NK-mediated circuit initiated by glioma-released miR-1983 within exosomes, and which is under the regulation of galectin-1 (Gal-1). We demonstrate that miR-1983 is an endogenous TLR7 ligand that activates TLR7 in pDCs and cDCs through a 5'-UGUUU-3' motif at its 3' end. TLR7 activation and downstream signaling through MyD88-IRF5/IRF7 stimulates secretion of IFN-ß. IFN-ß then stimulates NK cells resulting in the eradication of gliomas. We propose that successful immunotherapy for glioma could exploit this endogenous innate immune circuit to activate TLR7 signaling and stimulate powerful anti-glioma NK activity, at least 10-14 days before the activation of anti-tumor adaptive immunity.

Concurrent Dexamethasone Limits the Clinical Benefit of Immune Checkpoint Blockade in Glioblastoma.

PURPOSE: Dexamethasone, a uniquely potent corticosteroid, is frequently administered to patients with brain tumors to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in patients with glioblastoma (GBM), particularly in the context of immunotherapy. EXPERIMENTAL DESIGN: We evaluated the dose-dependent effects of dexamethasone when administered with programmed cell death 1 (PD-1) blockade and/or radiotherapy in immunocompetent C57BL/6 mice with syngeneic GL261 and CT-2A GBM tumors. Clinically, the effect of dexamethasone on survival was evaluated in 181 patients with isocitrate dehydrogenase (IDH) wild-type GBM treated with PD-(L)1 blockade, with adjustment for relevant prognostic factors. RESULTS: Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti-PD-1 therapy reduced survival in a dose-dependent manner. Concurrent dexamethasone also abrogated survival following anti-PD-1 therapy with or without radiotherapy in immune-resistant CT-2A models. Dexamethasone decreased T-lymphocyte numbers by increasing apoptosis, in addition to decreasing lymphocyte functional capacity. Myeloid and natural killer cell populations were also generally reduced by dexamethasone. Thus, dexamethasone appears to negatively affect both adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive patients with IDH wild-type GBM treated with PD-(L)1 blockade revealed poorer survival among those on baseline dexamethasone. Upon multivariable adjustment with relevant prognostic factors, baseline dexamethasone administration was the strongest predictor of poor survival [reference, no dexamethasone; <2 mg HR, 2.16; 95% confidence interval (CI), 1.30-3.68; P = 0.003 and ≥2 mg HR, 1.97; 95% CI, 1.23-3.16; P = 0.005]. CONCLUSIONS: Our preclinical and clinical data indicate that concurrent dexamethasone therapy may be detrimental to immunotherapeutic approaches for patients with GBM.

Obesity Shapes Metabolism in the Tumor Microenvironment to Suppress Anti-Tumor Immunity.

Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8+ T cell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8+ T cells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8+ T cells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8+ T cell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8+ T cell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.

Receptor-Driven ERK Pulses Reconfigure MAPK Signaling and Enable Persistence of Drug-Adapted BRAF-Mutant Melanoma Cells.

Targeted inhibition of oncogenic pathways can be highly effective in halting the rapid growth of tumors but often leads to the emergence of slowly dividing persister cells, which constitute a reservoir for the selection of drug-resistant clones. In BRAFV600E melanomas, RAF and MEK inhibitors efficiently block oncogenic signaling, but persister cells emerge. Here, we show that persister cells escape drug-induced cell-cycle arrest via brief, sporadic ERK pulses generated by transmembrane receptors and growth factors operating in an autocrine/paracrine manner. Quantitative proteomics and computational modeling show that ERK pulsing is enabled by rewiring of mitogen-activated protein kinase (MAPK) signaling: from an oncogenic BRAFV600E monomer-driven configuration that is drug sensitive to a receptor-driven configuration that involves Ras-GTP and RAF dimers and is highly resistant to RAF and MEK inhibitors. Altogether, this work shows that pulsatile MAPK activation by factors in the microenvironment generates a persistent population of melanoma cells that rewires MAPK signaling to sustain non-genetic drug resistance.

SYLARAS: A Platform for the Statistical Analysis and Visual Display of Systemic Immunoprofiling Data and Its Application to Glioblastoma.

Accurately profiling systemic immune responses to cancer initiation and progression is necessary for understanding tumor surveillance and, ultimately, improving therapy. Here, we describe the SYLARAS software tool (systemic lymphoid architecture response assessment) and a dataset collected with SYLARAS that describes the frequencies of immune cells in primary and secondary lymphoid organs and in the tumor microenvironment of mice engrafted with a standard syngeneic glioblastoma (GBM) model. The data resource involves profiles of 5 lymphoid tissues in 48 mice and shows that GBM causes wide-spread changes in the local and systemic immune architecture. We use SYLARAS to identify a subset of CD45R/B220+ CD8+ T cells that is depleted from circulation but accumulates in the tumor mass and confirm this finding using multiplexed immunofluorescence microscopy. SYLARAS is freely available for download at (https://github.com/gjbaker/sylaras). A record of this paper's transparent peer review process is included in the Supplemental Information.

Investigating the Relationship Between a Clinical Science Composite Score and USMLE Step 2 Clinical Knowledge and Step 3 Performance.

Previous research has found a moderate relationship between performance on individual clinical science subject examinations and USMLE performance. Given the widespread use of the clinical science subject examinations and the need for measures of clinical knowledge that help predict performance on Steps 2 CK and 3 and performance in residency training, this study explores the use of composite scores based on clinical science subject examinations to predict clinical knowledge outcome measures. The data set included students who took all of the five most widely used clinical science subject examinations (medicine, obstetrics and gynecology, pediatrics, psychiatry, and surgery) between January 1, 2013, and December 31, 2017 (N = 65,516). Composite scores were calculated based on average equated percent correct scores across various combinations of clinical science subject examinations. Stepwise linear regression analyses were performed with composite score and Step 1 score as predictor variables and Step 2 CK score or Step 3 score as the dependent variable. In all cases, the proportion of variance explained (R 2 ) by the composite score (0.62-0.65 for Step 2 CK score and 0.45-0.48 for Step 3 score) was greater than R 2 for Step 1 by itself (0.52 for Step 2 CK score and 0.37 for Step 3 score). Logistic regression analyses found that higher composite scores were associated with a greater probability of passing Steps 2 CK and 3. Composite scores can be used alone or in conjunction with Step 1 to identify students at risk of failing Step 2 CK and/or Step 3 to facilitate remediation.

Graft-versus-host disease propagation depends on increased intestinal epithelial tight junction permeability.

Graft-versus-host disease (GVHD) is a complication of hematopoietic stem cell transplantation (HSCT) that affects multiple organs. GVHD-associated intestinal damage can be separated into two distinct phases, initiation and propagation, which correspond to conditioning-induced damage and effector T cell activation and infiltration, respectively. Substantial evidence indicates that intestinal damage induced by pretransplant conditioning is a key driver of GVHD initiation. Here, we aimed to determine the impact of dysregulated intestinal permeability on the subsequent GVHD propagation phase. The initiation phase of GVHD was unchanged in mice lacking long MLCK (MLCK210), an established regulator of epithelial tight junction permeability. However, MLCK210-deficient mice were protected from sustained barrier loss and exhibited limited GVHD propagation, as indicated by reduced histopathology, fewer CD8+ effector T cells in the gut, and improved overall survival. Consistent with these findings, intestinal epithelial MLCK210 expression and enzymatic activity were similarly increased in human and mouse GVHD biopsies. Intestinal epithelial barrier loss mediated by MLCK210 is therefore a key driver of the GVHD propagation. These data suggest that inhibition of MLCK210-dependent barrier regulation may be an effective approach to limiting GVHD progression.

Flavorings in Tobacco Products Induce Endothelial Cell Dysfunction.

OBJECTIVE: Use of alternative tobacco products including electronic cigarettes is rapidly rising. The wide variety of flavored tobacco products available is of great appeal to smokers and youth. The flavorings added to tobacco products have been deemed safe for ingestion, but the cardiovascular health effects are unknown. The purpose of this study was to examine the effect of 9 flavors on vascular endothelial cell function. APPROACH AND RESULTS: Freshly isolated endothelial cells from participants who use nonmenthol- or menthol-flavored tobacco cigarettes showed impaired A23187-stimulated nitric oxide production compared with endothelial cells from nonsmoking participants. Treatment of endothelial cells isolated from nonsmoking participants with either menthol (0.01 mmol/L) or eugenol (0.01 mmol/L) decreased A23187-stimulated nitric oxide production. To further evaluate the effects of flavoring compounds on endothelial cell phenotype, commercially available human aortic endothelial cells were incubated with vanillin, menthol, cinnamaldehyde, eugenol, dimethylpyrazine, diacetyl, isoamyl acetate, eucalyptol, and acetylpyrazine (0.1-100 mmol/L) for 90 minutes. Cell death, reactive oxygen species production, expression of the proinflammatory marker IL-6 (interleukin-6), and nitric oxide production were measured. Cell death and reactive oxygen species production were induced only at high concentrations unlikely to be achieved in vivo. Lower concentrations of selected flavors (vanillin, menthol, cinnamaldehyde, eugenol, and acetylpyridine) induced both inflammation and impaired A23187-stimulated nitric oxide production consistent with endothelial dysfunction. CONCLUSIONS: Our data suggest that short-term exposure of endothelial cells to flavoring compounds used in tobacco products have adverse effects on endothelial cell phenotype that may have relevance to cardiovascular toxicity.

Evaluation of the respiratory tract toxicity of ortho-phthalaldehyde, a proposed alternative for the chemical disinfectant glutaraldehyde.

ortho-Phthalaldehyde (OPA) is a high-level chemical disinfectant that is commonly used for chemical sterilization of dental and medical instruments as an alternative to glutaraldehyde, a known skin and respiratory sensitizer. Concern for safe levels of human exposure remains due to a lack of toxicity data as well as human case reports of skin and respiratory sensitization following OPA exposure. The present study evaluated the inhalational toxicity of OPA in Harlan Sprague-Dawley rats and B6C3F1/N mice. Groups of 10 male and female rats and mice were exposed to OPA by whole-body inhalation for 3 months at concentrations of 0 (control), 0.44, 0.88, 1.75, 3.5, or 7.0 ppm. Rats and mice developed a spectrum of lesions at sites of contact throughout the respiratory tract (nose, larynx, trachea, lung), as well as in the skin and eye, consistent with a severe irritant response. In general, histologic lesions (necrosis, inflammation, regeneration, hyperplasia and metaplasia) occurred at deeper sites within the respiratory tract with increasing exposure concentration. As a first site of contact, the nose exhibited the greatest response to OPA exposure and resulted in an increased incidence, severity and variety of lesions compared to a previous study of glutaraldehyde exposure at similar exposure concentrations. This increased response in the nasal cavity, combined with extensive lesions throughout the respiratory tract, provides concern for use of OPA as a replacement for glutaraldehyde as a high-level disinfectant.

Adaptive resistance of melanoma cells to RAF inhibition via reversible induction of a slowly dividing de-differentiated state.

Treatment of BRAF-mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient benefit. We use live-cell imaging, single-cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug. Drug-adapted cells up-regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly. This phenotype is transiently stable, reverting to the drug-naïve state within 9 days of drug withdrawal. Transcriptional profiling of cell lines and human tumors implicates a c-Jun/ECM/FAK/Src cascade in de-differentiation in about one-third of cell lines studied; drug-induced changes in c-Jun and NGFR levels are also observed in xenograft and human tumors. Drugs targeting the c-Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (Emax) of RAF/MEK kinase inhibitors by promoting cell killing. Thus, analysis of reversible drug resistance at a single-cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations.

Assessing the footprint and volume of oil deposited in deep-sea sediments following the Deepwater Horizon oil spill.

The lateral and vertical extents of Macondo oil in deep-sea sediments resulting from the 2010 Deepwater Horizon oil spill were determined using chemical forensics and geostatistical kriging of data from 2397 sediment samples from 875 cores collected in 2010/2011 and 2014. The total mass of Macondo-derived hopane on the seafloor in 2010/2011 was conservatively estimated between 2.00 and 2.26metric tons, derived from 219,000 to 247,000barrels of oil; or 6.9 to 7.7% of the 3.19millionbarrels spilled. Macondo-derived hopane was deposited over 1030 to 1910km2 of the seafloor, mostly (>97%) in surface (0-1cm) and near-surface (1-3cm) sediments, which is consistent with short-term oil deposition. Although Macondo oil was still present in surface sediments in 2014, the total mass of Macondo-derived hopane was significantly lower (~80 to 90%) than in 2010/2011, affirming an acute impact from the spill and not long-term deposition from natural seeps.

CXCR4 increases in-vivo glioma perivascular invasion, and reduces radiation induced apoptosis: A genetic knockdown study.

Glioblastoma (GBM) is a highly invasive brain tumor. Perivascular invasion, autovascularization and vascular co-option occur throughout the disease and lead to tumor invasion and progression. The molecular basis for perivascular invasion, i.e., the interaction of glioma tumor cells with endothelial cells is not well characterized. Recent studies indicate that glioma cells have increased expression of CXCR4. We investigated the in-vivo role of CXCR4 in perivascular invasion of glioma cells using shRNA-mediated knock down of CXCR4. We show that primary cultures of human glioma stem cells HF2303 and mouse glioma GL26-Cit cells exhibit significant migration towards human (HBMVE) and mouse (MBVE) brain microvascular endothelial cells. Blocking CXCR4 on tumor cells with AMD3100 in-vitro, inhibits migration of GL26-Cit and HF2303 toward MBVE and HBMVE cells. Additionally, genetic down regulation of CXCR4 in mouse glioma GL26-Cit cells inhibits their in-vitro migration towards MBVE cells; in an in-vivo intracranial mouse model, these cells display reduced tumor growth and perivascular invasion, leading to increased survival. Quantitative analysis of brain sections showed that CXCR4 knockdown tumors are less invasive. Lastly, we tested the effects of radiation on CXCR4 knock down GL26-Cit cells in an orthotopic brain tumor model. Radiation treatment increased apoptosis of CXCR4 downregulated tumor cells and prolonged median survival. In summary, our data suggest that CXCR4 signaling is critical for perivascular invasion of GBM cells and targeting this receptor makes tumors less invasive and more sensitive to radiation therapy. Combination of CXCR4 knock down and radiation treatment might improve the efficacy of GBM therapy.