Publications by authors named "Paul A Clark"

48 Publications

Low-Dose Radiation Potentiates the Propagation of Anti-Tumor Immunity against Melanoma Tumor in the Brain after In Situ Vaccination at a Tumor outside the Brain.

Radiat Res 2021 Apr 7. Epub 2021 Apr 7.

Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.

Brain metastases develop in over 60% of advanced melanoma patients and negatively impact quality of life and prognosis. In a murine melanoma model, we previously showed that an in situ vaccination (ISV) regimen, combining radiation treatment and intratumoral (IT) injection of immunocytokine (IC: anti-GD2 antibody fused to IL2), along with the immune checkpoint inhibitor anti-CTLA-4, robustly eliminates peripheral flank tumors but only has modest effects on co-occurring intracranial tumors. In this study, we investigated the ability of low-dose radiation to the brain to potentiate anti-tumor immunity against a brain tumor when combined with ISV + anti-CTLA-4. B78 (GD2+, immunologically "cold") melanoma tumor cells were implanted into the flank and the right striatum of the brain in C57BL/6 mice. Flank tumors (50-150 mm3) were treated following a previously optimized ISV regimen [radiation (12 Gy × 1, treatment day 1), IT-IC (50 mg daily, treatment days 6-10), and anti-CTLA-4 (100 mg, treatment days 3, 6, 9)]. Mice that additionally received whole-brain radiation treatment (WBRT, 4 Gy × 1) on day 15 demonstrated significantly increased survival compared to animals that received ISV + anti-CTLA-4 alone, WBRT alone or no treatment (control) (P < 0.001, log-rank test). Timing of WBRT was critical, as WBRT administration on day 1 did not significantly enhance survival compared to ISV + anti-CTLA-4, suggesting that the effect of WBRT on survival might be mediated through immune modulation and not just direct tumor cell cytotoxicity. Modest increases in T cells (CD8+ and CD4+) and monocytes/macrophages (F4/80+) but no changes in FOXP3+ regulatory T cells (Tregs), were observed in brain melanoma tumors with addition of WBRT (on day 15) to ISV + anti-CTLA-4. Cytokine multiplex immunoassay revealed distinct changes in both intracranial melanoma and contralateral normal brain with addition of WBRT (day 15) to ISV + anti-CTLA-4, with notable significant changes in pro-inflammatory (e.g., IFNg, TNFa and LIX/CXCL5) and suppressive (e.g., IL10, IL13) cytokines as well as chemokines (e.g., IP-10/CXCL10 and MIG/CXCL9). We tested the ability of the alkylphosphocholine analog, NM600, to deliver immunomodulatory radiation to melanoma brain tumors as a targeted radionuclide therapy (TRT). Yttrium-86 (86Y) chelated to NM600 was delivered intravenously by tail vein to mice harboring flank and brain melanoma tumors, and PET imaging demonstrated specific accumulation up to 72 h at each tumor site (∼12:1 brain tumor/brain and ∼8:1 flank tumor/muscle). When NM600 was chelated to therapeutic b particle-emitting 90Y and administered on treatment day 13, T-cell infiltration and cytokine profiles were altered in melanoma brain tumor, like that observed for WBRT. Overall, our results demonstrate that addition of low-dose radiation, timed appropriately with ISV administration to tumors outside the brain, significantly increases survival in animals co-harboring melanoma brain tumors. This observation has potentially important translational implications as a treatment strategy for increasing the response of tumors in the brain to systemically administered immunotherapies.
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http://dx.doi.org/10.1667/RADE-20-00237.1DOI Listing
April 2021

A multipurpose brachytherapy catheter to enable intratumoral injection.

Brachytherapy 2021 Mar 27. Epub 2021 Mar 27.

Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI.

Purpose: To create and test a multipurpose brachytherapy catheter prototype enabling intratumoral injection and brachytherapy after a single catheter insertion.

Methods And Materials: The design of the prototype consists of an outer tube and an inner syringe tube that can be filled with injectable agent. The outer sheath and inner syringe tube were constructed using polytetrafluoroethylene tubing, and the other components were 3D printed using dental resin and polylactic acid material. To demonstrate functionality, we injected in vitro phantoms with dyed saline. For proof of concept, we demonstrated the potential for the prototype to deliver cell therapy, enhance tumor delineation, deliver tattoo ink for pathology marking, avoid toxicity through local delivery of chemotherapy, and facilitate combination brachytherapy and immunotherapy.

Results: The prototype enables accurate injection in vitro and in vivo without altering dosimetry. To illustrate the potential for delivery of cell therapies, we injected luciferase-expressing splenocytes and confirmed their delivery with bioluminescence imaging. To demonstrate feasibility of radiographically visualizing injected material, we delivered iohexol contrast intratumorally and confirmed tumor retention using Faxitron x-ray imaging. In addition, we show the potential of intratumoral administration to reduce toxicity associated with cyclophosphamide compared with systemic administration. To demonstrate feasibility, we treated tumor-bearing mice with brachytherapy (Ir source, 2 Gy to 5 mm) in combination with intratumoral injection of 375,000 U of interleukin 2 and observed no increased toxicity.

Conclusions: These results demonstrate that a prototype multipurpose brachytherapy catheter enables accurate intratumoral injection and support the feasibility of combining intratumoral injection with brachytherapy.
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http://dx.doi.org/10.1016/j.brachy.2020.10.012DOI Listing
March 2021

Development and characterization of patient-derived xenografts from non-small cell lung cancer brain metastases.

Sci Rep 2021 Jan 28;11(1):2520. Epub 2021 Jan 28.

Department of Human Oncology, School of Medicine and Public Health, University of Wisconsin, 600 Highland Avenue, K4/B100-0600, Madison, WI, 53792, USA.

Non-small cell lung cancer (NSCLC) brain metastasis cell lines and in vivo models are not widely accessible. Herein we report on a direct-from patient-derived xenograft (PDX) model system of NSCLC brain metastases with genomic annotation useful for translational and mechanistic studies. Both heterotopic and orthotopic intracranial xenografts were established and RNA and DNA sequencing was performed on patient and matching tumors. Morphologically, strong retention of cytoarchitectural features was observed between original patient tumors and PDXs. Transcriptome and mutation analysis revealed high correlation between matched patient and PDX samples with more than more than 95% of variants detected being retained in the matched PDXs. PDXs demonstrated response to radiation, response to selumetinib in tumors harboring KRAS G12C mutations and response to savolitinib in a tumor with MET exon 14 skipping mutation. Savolitinib also demonstrated in vivo radiation enhancement in our MET exon 14 mutated PDX. Early passage cell strains showed high consistency between patient and PDX tumors. Together, these data describe a robust human xenograft model system for investigating NSCLC brain metastases. These PDXs and cell lines show strong phenotypic and molecular correlation with the original patient tumors and provide a valuable resource for testing preclinical therapeutics.
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http://dx.doi.org/10.1038/s41598-021-81832-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843608PMC
January 2021

Dapsone-induced DRESS after infliximab-induced vasculitis: a case of cerebral infarction in the context of multiple drug reactions.

BMJ Case Rep 2020 Dec 17;13(12). Epub 2020 Dec 17.

Department of Medicine, Inova Fairfax Hospital, Falls Church, Virginia, USA.

Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a life-threatening condition characterised by peripheral eosinophilia, rash and multi-organ failure arising several weeks after exposure to the culprit medication. Although rare, DRESS syndrome triggered by specific agents has been associated with specific genetic polymorphisms more prevalent in different ethnic groups, including an association between dapsone-induced DRESS and Human Leukocyte Antigen (HLA)-B:13*01, a single nucleotide polymorphism more prevalent in those of Asian descent. DRESS and drug-related vasculitis may affect any organ system including the central nervous system (CNS), usually manifesting as encephalitis, meningitis or embolic cerebrovascular accidents related to eosinophilic cardiac disease and thrombosis. CNS vasculitis is a much rarer complication of drug reactions that may manifest as multifocal ischemia on neuroimaging. In circumstances of drug-related vasculitides, treatment with high-dose corticosteroids may lead to rapid improvement and, ultimately, resolution of associated focal neurologic deficits.
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http://dx.doi.org/10.1136/bcr-2020-237560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7747542PMC
December 2020

Tumor-Specific Antibody, Cetuximab, Enhances the Vaccine Effect of Radiation in Immunologically Cold Head and Neck Squamous Cell Carcinoma.

Front Immunol 2020 12;11:591139. Epub 2020 Nov 12.

Department of Human Oncology, University of Wisconsin, Madison, WI, United States.

In head and neck squamous cell carcinoma (HNSCC) tumors that over-expresses huEGFR, the anti-EGFR antibody, cetuximab, antagonizes tumor cell viability and sensitizes to radiation therapy. However, the immunologic interactions between cetuximab and radiation therapy are not well understood. We transduced two syngeneic murine HNSCC tumor cell lines to express human EGFR (MOC1- and MOC2-huEGFR) in order to facilitate evaluation of the immunologic interactions between radiation and cetuximab. Cetuximab was capable of inducing antibody-dependent cellular cytotoxicity (ADCC) in MOC1- and MOC2-huEGFR cells but showed no effect on the viability or radiosensitivity of these tumor cells, which also express muEGFR that is not targeted by cetuximab. Radiation enhanced the susceptibility of MOC1- and MOC2-huEGFR to ADCC, eliciting a type I interferon response and increasing expression of NKG2D ligands on these tumor cells. Co-culture of splenocytes with cetuximab and MOC2-huEGFR cells resulted in increased expression of IFNγ in not only NK cells but also in CD8+ T cells, and this was dependent upon splenocyte expression of FcγR. In MOC2-huEGFR tumors, combining radiation and cetuximab induced tumor growth delay that required NK cells, EGFR expression, and FcγR on host immune cells. Combination of radiation and cetuximab increased tumor infiltration with NK and CD8+ T cells but not regulatory T cells. Expression of PD-L1 was increased in MOC2-huEGFR tumors following treatment with radiation and cetuximab. Delivering anti-PD-L1 antibody with radiation and cetuximab improved survival and resulted in durable tumor regression in some mice. Notably, these cured mice showed evidence of an adaptive memory response that was not specifically directed against huEGFR. These findings suggest an opportunity to improve the treatment of HNSCC by combining radiation and cetuximab to engage an innate anti-tumor immune response that may prime an effective adaptive immune response when combined with immune checkpoint blockade. It is possible that this approach could be extended to any immunologically cold tumor that does not respond to immune checkpoint blockade alone and for which a tumor-specific antibody exists or could be developed.
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http://dx.doi.org/10.3389/fimmu.2020.591139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689006PMC
November 2020

Effective Use of Angiotensin II in Coronavirus Disease 19-Associated Mixed Shock State: A Case Report.

A A Pract 2020 Apr;14(6):e01221

From the Departments of Internal Medicine.

The rapid spread of Coronavirus Disease 2019 (COVID-19) has sparked a search for effective therapies. The discovery that the virus binds the angiotensin-converting enzyme 2 (ACE2) receptor has led to investigation of the renin-angiotensin system for possible therapeutic targets. We present a case of an elderly woman with multiple comorbidities who developed severe acute respiratory distress syndrome (ARDS), a cardiomyopathy, and vasodilatory shock secondary to COVID-19 and was treated with exogenous angiotensin II. She rapidly demonstrated significant hemodynamic improvement without noted adverse effects. Thus, we propose further investigation into possible benefits of angiotensin II in shock secondary to COVID-19.
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http://dx.doi.org/10.1213/XAA.0000000000001221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7172437PMC
April 2020

In situ vaccination at a peripheral tumor site augments response against melanoma brain metastases.

J Immunother Cancer 2020 07;8(2)

Department of Human Oncology, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin, USA

Background: Immune checkpoint inhibition (ICI) alone is not efficacious for a large number of patients with melanoma brain metastases. We previously established an in situ vaccination (ISV) regimen combining radiation and immunocytokine to enhance response to ICIs. Here, we tested whether ISV inhibits the development of brain metastases in a murine melanoma model.

Methods: B78 (GD2) melanoma 'primary' tumors were engrafted on the right flank of C57BL/6 mice. After 3-4 weeks, primary tumors were treated with ISV (radiation (12 Gy, day 1), α-GD2 immunocytokine (hu14.18-IL2, days 6-10)) and ICI (α-CTLA-4, days 3, 6, 9). Complete response (CR) was defined as no residual tumor observed at treatment day 90. Mice with CR were tested for immune memory by re-engraftment with B78 in the left flank and then the brain. To test ISV efficacy against metastases, tumors were also engrafted in the left flank and brain of previously untreated mice. Tumors were analyzed by quantitative reverse transcription-PCR, immunohistochemistry, flow cytometry and multiplex cytokine assay.

Results: ISV+α-CTLA-4 resulted in immune memory and rejection of B78 engraftment in the brain in 11 of 12 mice. When B78 was engrafted in brain prior to treatment, ISV+α-CTLA-4 increased survival compared with ICI alone. ISV+α-CTLA-4 eradicated left flank tumors but did not elicit CR at brain sites when tumor cells were engrafted in brain prior to ISV. ISV+α-CTLA-4 increased CD8 and CD4 T cells in flank and brain tumors compared with untreated mice. Among ISV + α-CTLA-4 treated mice, left flank tumors showed increased CD8 infiltration and CD8:FOXP3 ratio compared with brain tumors. Flank and brain tumors showed minimal differences in expression of immune checkpoint receptors/ligands or . Cytokine productions were similar in left flank and brain tumors in untreated mice. Following ISV+α-CTLA-4, production of immune-stimulatory cytokines was greater in left flank compared with brain tumor grafts.

Conclusion: ISV augmented response to ICIs in murine melanoma at brain and extracranial tumor sites. Although baseline tumor-immune microenvironments were similar at brain and extracranial tumor sites, response to ISV+α-CTLA-4 was divergent with reduced infiltration and activation of immune cells in brain tumors. Additional therapies may be needed for effective antitumor immune response against melanoma brain metastases.
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http://dx.doi.org/10.1136/jitc-2020-000809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371368PMC
July 2020

Identification of variable lymphocyte receptors that can target therapeutics to pathologically exposed brain extracellular matrix.

Sci Adv 2019 05 15;5(5):eaau4245. Epub 2019 May 15.

Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, USA.

Diseases that lead to blood-brain barrier (BBB) disruption will pathologically expose normally inaccessible brain extracellular matrix (ECM) to circulating blood components. Therefore, we hypothesized that brain ECM-targeting moieties could specifically target the disrupted BBB and potentially deliver therapies. Variable lymphocyte receptors (VLRs) that preferentially associate with brain ECM were identified from an immune VLR library via yeast surface display biopanning coupled with a moderate throughput ECM screen. Brain ECM binding of VLR clones to murine and human brain tissue sections was confirmed. After systemic administration, P1C10, the lead brain ECM-targeting VLR candidate, specifically accumulated in brains with mannitol-disrupted BBB and at disrupted BBB regions in two different intracranial glioblastoma models. We also demonstrate P1C10's ability to deliver doxorubicin-loaded liposomes, leading to significantly improved survival in glioblastoma-bearing mice. Thus, VLRs can be used to selectively target pathologically exposed brain ECM and deliver drug payloads.
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http://dx.doi.org/10.1126/sciadv.aau4245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520025PMC
May 2019

Diagnosis and Management of Rhabdomyolysis in the Absence of Creatine Phosphokinase: A Medical Record Review.

Mil Med 2019 12;184(11-12):820-825

Department of Critical Care, Walter Reed National Military Medical Center, 8901 Rockville Pike, Bethesda, MD 20889.

Introduction: Rhabdomyolysis is often encountered in austere environments where the diagnosis can be challenging due to the expense or unavailability of creatine phosphokinase (CPK) testing. CPK concentration ≥5,000 U/L has previously been found to be a sensitive marker for progression to renal failure. This study sought to propose a model utilizing an alternate biomarker to allow for the diagnosis and monitoring of clinically significant rhabdomyolysis in the absence of CPK.

Materials And Methods: We performed a retrospective chart review of 77 patients admitted to a tertiary medical center with a primary diagnosis of rhabdomyolysis. A linear regression model with aspartate aminotransferase (AST) as the independent variable was developed and used to predict CPK ≥5,000 U/L on admission and CPK values on subsequent hospital days. The study was approved and monitored by the Institutional Review Board at Walter Reed National Military Medical Center.

Results: Ln(AST) explained over 80% of the variance in ln(CPK) (adjusted R2 = 0.802). The diagnostic accuracy to predict CPK ≥5,000 U/L was high (AUC 0.959; 95% CI: 0.921-0.997, P < 0.001). A cut point of AST ≥110 U/L in our study population had a 97.1% sensitivity and an 85.7% specificity for the detection of a CPK value ≥5,000 U/L. The agreement between actual CPK and predicted CPK for subsequent days of hospitalization was fair with an intraclass correlation coefficient of 0.52 (95% CI: 0.38-0.63). The developed model based on day 1 data tended to overpredict CPK values on subsequent hospital days.

Conclusions: We propose a threshold concentration of AST that has an excellent sensitivity for detecting CPK concentration ≥5,000 U/L on day of admission in a patient population with a diagnosis of rhabdomyolysis. A formula with a fair ability to predict CPK levels based on AST concentrations on subsequent hospital days was also developed.
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http://dx.doi.org/10.1093/milmed/usz101DOI Listing
December 2019

Docking of Alkylphosphocholine Analogs to Human Serum Albumin Predicts Partitioning and Pharmacokinetics.

Mol Pharm 2019 08 8;16(8):3350-3360. Epub 2019 Jul 8.

Cellectar Biosciences Inc. , Madison , Wisconsin , United States.

Alkylphosphocholine (APC) analogs are a novel class of broad-spectrum tumor-targeting agents that can be used for both diagnosis and treatment of cancer. The potential for clinical translation for APC analogs will strongly depend on their pharmacokinetic (PK) profiles. The aim of this work was to understand how the chemical structures of various APC analogs impact binding and PK. To achieve this aim, we performed docking analysis, in vitro and in vivo partitioning experiments, and PK studies. Our results have identified 7 potential high-affinity binding sites of these compounds on human serum albumin (HSA) and suggest that the size of the functional group directly influences the albumin binding, partitioning, and PK. Namely, the bulkier the functional groups, the weaker the agent binds to albumin, the more the agent partitions onto lipoproteins, and the less time the agent spends in circulation. The results of these experiments provide novel molecular insights into the binding, partitioning, and PK of this class of compounds and similar molecules as well as suggest pharmacological strategies to alter their PK profiles. Importantly, our methodology may provide a way to design better drugs by better characterizing the PK profile for lead compound optimization.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b01301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989153PMC
August 2019

Potential CAR T Cell Immunotherapy Against Diffuse Midline Gliomas.

Neurosurgery 2018 11;83(5):E198-E199

Department of Neurosurgery Dell Medical School University of Texas at Austin Austin, Texas.

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http://dx.doi.org/10.1093/neuros/nyy399DOI Listing
November 2018

Gallium Maltolate Disrupts Tumor Iron Metabolism and Retards the Growth of Glioblastoma by Inhibiting Mitochondrial Function and Ribonucleotide Reductase.

Mol Cancer Ther 2018 06 28;17(6):1240-1250. Epub 2018 Mar 28.

Department of Radiology, Medical College of Wisconsin, Milwaukee, Wisconsin.

Gallium, a metal with antineoplastic activity, binds transferrin (Tf) and enters tumor cells via Tf receptor1 (TfR1); it disrupts iron homeostasis leading to cell death. We hypothesized that TfR1 on brain microvascular endothelial cells (BMEC) would facilitate Tf-Ga transport into the brain enabling it to target TfR-bearing glioblastoma. We show that U-87 MG and D54 glioblastoma cell lines and multiple glioblastoma stem cell (GSC) lines express TfRs, and that their growth is inhibited by gallium maltolate (GaM) After 24 hours of incubation with GaM, cells displayed a loss of mitochondrial reserve capacity followed by a dose-dependent decrease in oxygen consumption and a decrease in the activity of the iron-dependent M2 subunit of ribonucleotide reductase (RRM2). IHC staining of rat and human tumor-bearing brains showed that glioblastoma, but not normal glial cells, expressed TfR1 and RRM2, and that glioblastoma expressed greater levels of H- and L-ferritin than normal brain. In an orthotopic U-87 MG glioblastoma xenograft rat model, GaM retarded the growth of brain tumors relative to untreated control ( = 0.0159) and reduced tumor mitotic figures ( = 0.045). Tumors in GaM-treated animals displayed an upregulation of TfR1 expression relative to control animals, thus indicating that gallium produced tumor iron deprivation. GaM also inhibited iron uptake and upregulated TfR1 expression in U-87 MG and D54 cells We conclude that GaM enters the brain via TfR1 on BMECs and targets iron metabolism in glioblastoma thus inhibiting tumor growth. Further development of novel gallium compounds for brain tumor treatment is warranted. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-1009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984712PMC
June 2018

Yeast display biopanning identifies human antibodies targeting glioblastoma stem-like cells.

Sci Rep 2017 Nov 20;7(1):15840. Epub 2017 Nov 20.

Neuroscience Training Program, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53792-8660, USA.

Glioblastoma stem-like cells (GSC) are hypothesized to evade current therapies and cause tumor recurrence, contributing to poor patient survival. Existing cell surface markers for GSC are developed from embryonic or neural stem cell systems; however, currently available GSC markers are suboptimal in sensitivity and specificity. We hypothesized that the GSC cell surface proteome could be mined with a yeast display antibody library to reveal novel immunophenotypes. We isolated an extensive collection of antibodies that were differentially selective for GSC. A single domain antibody VH-9.7 showed selectivity for five distinct patient-derived GSC lines and visualized orthotopic GBM xenografts in vivo after conjugation with a near-infrared dye. These findings demonstrate a previously unexplored high-throughput strategy for GSC-selective antibody discovery, to aid in GSC isolation, diagnostic imaging, and therapeutic targeting.
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http://dx.doi.org/10.1038/s41598-017-16066-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696472PMC
November 2017

Human Cytomegalovirus-Infected Glioblastoma Cells Display Stem Cell-Like Phenotypes.

mSphere 2017 May-Jun;2(3). Epub 2017 Jun 21.

Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. Human cytomegalovirus (HCMV) genomes are present in GBM tumors, yielding hope that antiviral treatments could prove therapeutic and improve the poor prognosis of GBM patients. We discovered that GBM cells infected with HCMV display properties of cancer stem cells. HCMV-infected GBM cells grow more slowly than mock-infected controls, demonstrate a higher capacity for self-renewal determined by a sphere formation assay, and display resistance to the chemotherapeutic drug temozolomide. Our data suggest that HCMV, while present in only a minority of the cells within a tumor, could contribute to the pathogenesis of GBMs by promoting or prolonging stem cell-like phenotypes, thereby perpetuating tumors in the face of chemotherapy. Importantly, we show that temozolomide sensitivity is restored by the antiviral drug ganciclovir, indicating a potential mechanism underlying the positive effects observed in GBM patients treated with antiviral therapy. A role for HCMV in GBMs remains controversial for several reasons. Some studies find HCMV in GBM tumors, while others do not. Few cells within a GBM may harbor HCMV, making it unclear how the virus could be contributing to the tumor phenotype without infecting every cell. Finally, HCMV does not overtly transform cells . However, tumors induced by other viruses can be treated with antiviral remedies, and initial results indicate that this may be true for anti-HCMV therapies and GBMs. With such a poor prognosis for GBM patients, any potential new intervention deserves exploration. Our work here describes an evidence-based model for how HCMV could contribute to GBM biology while infecting very few cells and without transforming them. It also illuminates why anti-HCMV treatments may be beneficial to GBM patients. Our observations provide blueprints for future studies examining how HCMV manipulates stem cell-specific pathways and future clinical studies of anti-HCMV measures as GBM therapeutics.
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http://dx.doi.org/10.1128/mSphere.00137-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5480031PMC
June 2017

I-TRACH: Validating A Tool for Predicting Prolonged Mechanical Ventilation.

J Intensive Care Med 2018 Oct 30;33(10):567-573. Epub 2016 Nov 30.

1 Department of Pulmonary, Critical Care and Sleep Medicine, Walter Reed National Military Medical Center, Bethesda, MD, USA.

Purpose: We previously developed a bedside model (I-TRACH), which used commonly obtained data at the time of intubation to predict the duration of mechanical ventilation (MV). We now sought to validate this in a prospective trial.

Methods: A prospective, observational study of 225 consecutive adult medical intensive care unit patients requiring MV. Utilizing the original 6 variables used in the I-TRACH model (Intubation in the ICU, Tachycardia [heart rate > 110], Renal dysfunction [blood urea nitrogen > 25], Acidemia [pH < 7.25], Creatinine [>2.0 or >50% increase from baseline values], and decreased HCO [<20]), we (1) confirmed that these were still predictive of length of MV by multivariate analysis and (2) assessed the correlation between the number of criteria met and the subsequent duration of MV. In addition, we compared the performance of I-TRACH to Acute Physiology Age Chronic Health Evaluation-II and III, Sequential Organ Failure Assessment, and Acute Physiology Score as predictors of length of MV.

Results: Mean age was 62.6 ± 18.7 years, with a mean duration of MV of 5.8 ± 5.7 days. The number of I-TRACH criteria met directly correlated with the duration of MV. Individuals with ≥4 criteria were significantly more likely to require MV >7 and >14 days. Similarly, those who remained on ventilators for both >7 and >14 days met significantly more I-TRACH criteria than those requiring shorter durations of MV (1.7 ± 1.3 vs 2.8 ± 1.3 vs 3.8 ± 1.3 criteria, P < .001). I-TRACH performed better than all other models used to predict the duration of MV.

Conclusion: Similar to our previous retrospective study, these findings validate I-TRACH in determining the subsequent need for MV >7 and >14 days at the time of intubation.
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http://dx.doi.org/10.1177/0885066616679974DOI Listing
October 2018

The effects of tumor treating fields and temozolomide in MGMT expressing and non-expressing patient-derived glioblastoma cells.

J Clin Neurosci 2017 Feb 16;36:120-124. Epub 2016 Nov 16.

University of Wisconsin Carbone Cancer Center, UWSMPH, United States; Division of Hematology and Oncology, Department of Medicine, UWSMPH, United States; William S Middleton Memorial Veterans Hospital, Madison, WI, United States; Department of Neurology, UWSMPH, United States; Department of Human Oncology, UWSMPH, United States. Electronic address:

A recent Phase 3 study of newly diagnosed glioblastoma (GBM) demonstrated the addition of tumor treating fields (TTFields) to temozolomide (TMZ) after combined radiation/TMZ significantly increased survival and progression free survival. Preliminary data suggested benefit with both methylated and unmethylated O-6-methylguanine-DNA methyl-transferase (MGMT) promoter status. To date, however, there have been no studies to address the potential interactions of TTFields and TMZ. Thus, the effects of TTFields and TMZ were studied in vitro using patient-derived GBM stem-like cells (GSCs) including MGMT expressing (TMZ resistant: 12.1 and 22GSC) and non-MGMT expressing (TMZ sensitive: 33 and 114GSC) lines. Dose-response curves were constructed using cell proliferation and sphere-forming assays. Results demonstrated a ⩾10-fold increase in TMZ resistance of MGMT-expressing (12.1GSCs: IC=160μM; 22GSCs: IC=44μM) compared to MGMT non-expressing (33GSCs: IC=1.5μM; 114GSCs: IC=5.2μM) lines. TTFields inhibited 12.1 GSC proliferation at all tested doses (50-500kHz) with an optimal frequency of 200kHz. At 200kHz, TTFields inhibited proliferation and tumor sphere formation of both MGMT GSC subtypes at comparable levels (12.1GSC: 74±2.9% and 38±3.2%, respectively; 22GSC: 61±11% and 38±2.6%, respectively; 33GSC: 56±9.5% and 60±7.1%, respectively; 114 GSC: 79±3.5% and 41±4.3%, respectively). In combination, TTFields (200kHz) and TMZ showed an additive anti-neoplastic effect with equal efficacy for TTFields in both cell types (i.e., ± MGMT expression) with no effect on TMZ resistance. This is the first demonstration of the effects of TTFields on cancer stem cells. The expansion of such studies may have clinical implications.
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http://dx.doi.org/10.1016/j.jocn.2016.10.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215614PMC
February 2017

Administration of Non-Torsadogenic human Ether-à-go-go-Related Gene Inhibitors Is Associated with Better Survival for High hERG-Expressing Glioblastoma Patients.

Clin Cancer Res 2017 Jan 15;23(1):73-80. Epub 2016 Sep 15.

Department of Neurological Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin.

Purpose: Glioblastoma is the most malignant primary brain tumor, with a median survival of less than 2 years. More effective therapeutic approaches are needed to improve clinical outcomes.

Experimental Design: Glioblastoma patient-derived cells (GPDC) were isolated from patient glioblastomas and implanted in mice to form xenografts. IHC was performed for human Ether-à-go-go-Related Gene (hERG) expression and tumor proliferation. Sphere-forming assays with the hERG blocker E-4031 were performed on a high and low hERG-expressing lines. A glioblastoma tissue microarray (TMA; 115 patients) was used to correlate hERG expression with patient survival. Clinical data were analyzed to determine whether patient survival was affected by incidental administration of hERG inhibitory drugs and the correlative effect of patient glioblastoma hERG expression levels.

Results: hERG expression was upregulated in glioblastoma xenografts with higher proliferative indices. High hERG-expressing GPDCs showed a reduction in sphere formation when treated with hERG inhibitors compared with low hERG-expressing GPDCs. Glioblastoma TMA analysis showed worse survival for glioblastoma patients with high hERG expression versus low expression-43.5 weeks versus 60.9 weeks, respectively (P = 0.022). Furthermore, patients who received at least one hERG blocker had a better survival rate compared with patients who did not (P = 0.0015). Subgroup analysis showed that glioblastoma patients with high hERG expression who received hERG blockers had improved survival (P = 0.0458). There was no difference in survival for low hERG-expressing glioblastoma patients who received hERG blockers (P = 0.4136).

Conclusions: Our findings suggest that hERG is a potential glioblastoma survival marker, and that already approved drugs with non-torsadogenic hERG inhibitory activity may potentially be repurposed as adjuvant glioblastoma therapy in high hERG-expressing glioblastoma patients. Clin Cancer Res; 23(1); 73-80. ©2016 AACRSee related commentary by Arcangeli and Becchetti, p. 3.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-3169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627655PMC
January 2017

Association of collagen architecture with glioblastoma patient survival.

J Neurosurg 2017 Jun 2;126(6):1812-1821. Epub 2016 Sep 2.

Departments of 1 Neurological Surgery and.

OBJECTIVE Glioblastoma (GBM) is the most malignant primary brain tumor. Collagen is present in low amounts in normal brain, but in GBMs, collagen gene expression is reportedly upregulated. However, to the authors' knowledge, direct visualization of collagen architecture has not been reported. The authors sought to perform the first direct visualization of GBM collagen architecture, identify clinically relevant collagen signatures, and link them to differential patient survival. METHODS Second-harmonic generation microscopy was used to detect collagen in a GBM patient tissue microarray. Focal and invasive GBM mouse xenografts were stained with Picrosirius red. Quantitation of collagen fibers was performed using custom software. Multivariate survival analysis was done to determine if collagen is a survival marker for patients. RESULTS In focal xenografts, collagen was observed at tumor brain boundaries. For invasive xenografts, collagen was intercalated with tumor cells. Quantitative analysis showed significant differences in collagen fibers for focal and invasive xenografts. The authors also found that GBM patients with more organized collagen had a longer median survival than those with less organized collagen. CONCLUSIONS Collagen architecture can be directly visualized and is different in focal versus invasive GBMs. The authors also demonstrate that collagen signature is associated with patient survival. These findings suggest that there are collagen differences in focal versus invasive GBMs and that collagen is a survival marker for GBM.
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http://dx.doi.org/10.3171/2016.6.JNS152797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386834PMC
June 2017

Analysis of Cancer-Targeting Alkylphosphocholine Analogue Permeability Characteristics Using a Human Induced Pluripotent Stem Cell Blood-Brain Barrier Model.

Mol Pharm 2016 09 4;13(9):3341-9. Epub 2016 Aug 4.

Cellectar Biosciences , Madison, Wisconsin 53716, United States.

Cancer-targeting alkylphosphocholine (APC) analogues are being clinically developed for diagnostic imaging, intraoperative visualization, and therapeutic applications. These APC analogues derived from chemically synthesized phospholipid ethers were identified and optimized for cancer-targeting specificity using extensive structure-activity studies. While they strongly label human brain cancers associated with disrupted blood-brain barriers (BBB), APC permeability across intact BBB remains unknown. Three of our APC analogues, CLR1404 (PET radiotracer), CLR1501 (green fluorescence), and CLR1502 (near-infrared fluorescence), were tested for permeability across a BBB model composed of human induced pluripotent stem cell-derived brain microvascular endothelial cells (iPSC-derived BMECs). This in vitro BBB system has reproducibly consistent high barrier integrity marked by high transendothelial electrical resistance (TEER > 1500 Ω-cm(2)) and functional expression of drug efflux transporters. The radioiodinated and fluorescent APC analogues demonstrated fairly low permeability across the iPSC-BMEC (35 ± 5.7 (CLR1404), 54 ± 3.2 (CLR1501), and 26 ± 4.9 (CLR1502) × 10(-5) cm/min) compared with BBB-impermeable sucrose (13 ± 2.5) and BBB-permeable diazepam (170 ± 29). Only the fluorescent APC analogues (CLR1501, CLR1502) underwent BCRP and MRP polarized drug efflux transport in the brain-to-blood direction of the BBB model, and this efflux can be specifically blocked with pharmacological inhibition. None of the tested APC analogues appeared to undergo substantial P-gp transport. Limited permeability of the APC analogues across an intact BBB into normal brain likely contributes to the high tumor to background ratios observed in initial human trials. Moreover, addition of fluorescent moieties to APCs resulted in greater BMEC efflux via MRP and BCRP, and may affect fluorescence-guided applications. Overall, the characterization of APC analogue permeability across human BBB is significant for advancing future brain tumor-targeted applications of these agents.
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http://dx.doi.org/10.1021/acs.molpharmaceut.6b00441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014630PMC
September 2016

Resveratrol targeting of AKT and p53 in glioblastoma and glioblastoma stem-like cells to suppress growth and infiltration.

J Neurosurg 2017 May 15;126(5):1448-1460. Epub 2016 Jul 15.

Departments of 1 Neurological Surgery and.

OBJECTIVE Glioblastoma multiforme (GBM) is an aggressive brain cancer with median survival of less than 2 years with current treatment. Glioblastomas exhibit extensive intratumoral and interpatient heterogeneity, suggesting that successful therapies should produce broad anticancer activities. Therefore, the natural nontoxic pleiotropic agent, resveratrol, was studied for antitumorigenic effects against GBM. METHODS Resveratrol's effects on cell proliferation, sphere-forming ability, and invasion were tested using multiple patient-derived GBM stem-like cell (GSC) lines and established U87 glioma cells, and changes in oncogenic AKT and tumor suppressive p53 were analyzed. Resveratrol was also tested in vivo against U87 glioma flank xenografts in mice by using multiple delivery methods, including direct tumor injection. Finally, resveratrol was delivered directly to brain tissue to determine toxicity and achievable drug concentrations in the brain parenchyma. RESULTS Resveratrol significantly inhibited proliferation in U87 glioma and multiple patient-derived GSC lines, demonstrating similar inhibitory concentrations across these phenotypically heterogeneous lines. Resveratrol also inhibited the sphere-forming ability suggesting anti-stem cell effects. Additionally, resveratrol blocked U87 glioma and GSC invasion in an in vitro Matrigel Transwell assay at doses similar to those mediating antiproliferative effects. In U87 glioma cells and GSCs, resveratrol reduced AKT phosphorylation and induced p53 expression and activation that led to transcription of downstream p53 target genes. Resveratrol administration via oral gavage or ad libitum in the water supply significantly suppressed GBM xenograft growth; intratumoral or peritumoral resveratrol injection further suppressed growth and approximated tumor regression. Intracranial resveratrol injection resulted in 100-fold higher local drug concentration compared with intravenous delivery, and with no apparent toxicity. CONCLUSIONS Resveratrol potently inhibited GBM and GSC growth and infiltration, acting partially via AKT deactivation and p53 induction, and suppressed glioblastoma growth in vivo. The ability of resveratrol to modulate AKT and p53, as well as reportedly many other antitumorigenic pathways, is attractive for therapy against a genetically heterogeneous tumor such as GBM. Although resveratrol exhibits low bioavailability when administered orally or intravenously, novel delivery methods such as direct injection (i.e., convection-enhanced delivery) could potentially be used to achieve and maintain therapeutic doses in the brain. Resveratrol's nontoxic nature and broad anti-GBM effects make it a compelling candidate to supplement current GBM therapies.
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http://dx.doi.org/10.3171/2016.1.JNS152077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5237623PMC
May 2017

Glioma-mediated microglial activation promotes glioma proliferation and migration: roles of Na+/H+ exchanger isoform 1.

Carcinogenesis 2016 09 9;37(9):839-851. Epub 2016 Jun 9.

Department of Neurology, University of Pittsburgh, Pittsburgh, PA 15213, USA.

Microglia play important roles in extracellular matrix remodeling, tumor invasion, angiogenesis, and suppression of adaptive immunity in glioma. Na(+)/H(+) exchanger isoform 1 (NHE1) regulates microglial activation and migration. However, little is known about the roles of NHE1 in intratumoral microglial activation and microglia-glioma interactions. Our study revealed up-regulation of NHE1 protein expression in both glioma cells and tumor-associated Iba1(+) microglia in glioma xenografts and glioblastoma multiforme microarrays. Moreover, we observed positive correlation of NHE1 expression with Iba1 intensity in microglia/macrophages. Glioma cells, via conditioned medium or non-contact glioma-microglia co-cultures, concurrently upregulated microglial expression of NHE1 protein and other microglial activation markers (iNOS, arginase-1, TGF-β, IL-6, IL-10 and the matrix metalloproteinases MT1-MMP and MMP9). Interestingly, glioma-stimulated microglia reciprocally enhanced glioma proliferation and migration. Most importantly, inhibition of microglial NHE1 activity via small interfering RNA (siRNA) knockdown or the potent NHE1-specific inhibitor HOE642 significantly attenuated microglial activation and abolished microglia-stimulated glioma migration and proliferation. Taken together, our findings provide the first evidence that NHE1 function plays an important role in glioma-microglia interactions, enhancing glioma proliferation and invasion by stimulating microglial release of soluble factors. NHE1 upregulation is a novel marker of the glioma-associated microglial activation phenotype. Inhibition of NHE1 represents a novel glioma therapeutic strategy by targeting tumor-induced microglial activation.
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http://dx.doi.org/10.1093/carcin/bgw068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5008247PMC
September 2016

CSF1 Overexpression Promotes High-Grade Glioma Formation without Impacting the Polarization Status of Glioma-Associated Microglia and Macrophages.

Cancer Res 2016 05 24;76(9):2552-60. Epub 2016 Mar 24.

School of Pharmacy, Carbone Cancer Center and the Molecular and Cellular Pharmacology Graduate Program, University of Wisconsin-Madison, Madison, Wisconsin.

Current therapies for high-grade gliomas extend survival only modestly. The glioma microenvironment, including glioma-associated microglia/macrophages (GAM), is a potential therapeutic target. The microglia/macrophage cytokine CSF1 and its receptor CSF1R are overexpressed in human high-grade gliomas. To determine whether the other known CSF1R ligand IL34 is expressed in gliomas, we examined expression array data of human high-grade gliomas and performed RT-PCR on glioblastoma sphere-forming cell lines (GSC). Expression microarray analyses indicated that CSF1, but not IL34, is frequently overexpressed in human tumors. We found that while GSCs did express CSF1, most GSC lines did not express detectable levels of IL34 mRNA. We therefore studied the impact of modulating CSF1 levels on gliomagenesis in the context of the GFAP-V12Ha-ras-IRESLacZ (Ras*) model. Csf1 deficiency deterred glioma formation in the Ras* model, whereas CSF1 transgenic overexpression decreased the survival of Ras* mice and promoted the formation of high-grade gliomas. Conversely, CSF1 overexpression increased GAM density, but did not impact GAM polarization state. Regardless of CSF1 expression status, most GAMs were negative for the M2 polarization markers ARG1 and CD206; when present, ARG1(+) and CD206(+) cells were found in regions of peripheral immune cell invasion. Therefore, our findings indicate that CSF1 signaling is oncogenic during gliomagenesis through a mechanism distinct from modulating GAM polarization status. Cancer Res; 76(9); 2552-60. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-2386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4873447PMC
May 2016

Fluorescent cancer-selective alkylphosphocholine analogs for intraoperative glioma detection.

Neurosurgery 2015 Feb;76(2):115-23; discussion 123-4

*Department of Neurological Surgery, ‡Cellular and Molecular Biology Training Program, ¶Department of Radiology, ‖Department Medical Physics, and #Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin; §Cellectar Biosciences, Inc, Madison, Wisconsin; **Department of Surgery, National University of Singapore, Singapore.

Background: 5-Aminolevulinic acid (5-ALA)-induced tumor fluorescence aids brain tumor resections but is not approved for routine use in the United States. We developed and describe testing of 2 novel fluorescent, cancer-selective alkylphosphocholine analogs, CLR1501 (green) and CLR1502 (near infrared), in a proof-of-principle study for fluorescence-guided glioma surgery.

Objective: To demonstrate that CLR1501 and CLR1502 are cancer cell-selective fluorescence agents in glioblastoma models and to compare tumor-to-normal brain (T:N) fluorescence ratios with 5-ALA.

Methods: CLR1501, CLR1502, and 5-ALA were administered to mice with magnetic resonance imaging-verified orthotopic U251 glioblastoma multiforme- and glioblastoma stem cell-derived xenografts. Harvested brains were imaged with confocal microscopy (CLR1501), the IVIS Spectrum imaging system (CLR1501, CLR1502, and 5-ALA), or the Fluobeam near-infrared fluorescence imaging system (CLR1502). Imaging and quantitative analysis of T:N fluorescence ratios were performed.

Results: Excitation/emission peaks are 500/517 nm for CLR1501 and 760/778 nm for CLR1502. The observed T:N ratio for CLR1502 (9.28±1.08) was significantly higher (P<.01) than for CLR1501 (3.51±0.44 on confocal imaging; 7.23±1.63 on IVIS imaging) and 5-ALA (4.81±0.92). Near-infrared Fluobeam CLR1502 imaging in a mouse xenograft model demonstrated high- contrast tumor visualization compatible with surgical applications.

Conclusion: CLR1501 (green) and CLR1502 (near infrared) are novel tumor-selective fluorescent agents for discriminating tumor from normal brain. CLR1501 exhibits a tumor-to-brain fluorescence ratio similar to that of 5-ALA, whereas CLR1502 has a superior tumor-to-brain fluorescence ratio. This study demonstrates the potential use of CLR1501 and CLR1502 in fluorescence-guided tumor surgery.
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http://dx.doi.org/10.1227/NEU.0000000000000622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343207PMC
February 2015

Myelin-forming cell-specific cadherin-19 is a marker for minimally infiltrative glioblastoma stem-like cells.

J Neurosurg 2015 Jan;122(1):69-77

Neuroscience Training Program, 

Object: Glioblastoma stem-like cells (GSCs) exhibit stem-like properties, are highly efficient at forming tumor xenografts, and are resistant to many current therapies. Current molecular identifiers of GSCs are scarce and controversial. The authors describe differential cell-surface gene expression profiling to identify GSC-specific markers.

Methods: Independent human GSC lines were isolated and maintained in standard neural stem cell (NSC) media and were validated for self-renewal, multipotent differentiation, and tumor initiation properties. Candidate upregulated GSCspecific plasma membrane markers were identified through differential Affymetrix U133 Plus 2.0 Array gene expression profiling of GSCs, human NSCs (hNSCs), normal brain tissue, and primary/recurrent glioblastoma multiforme samples. Results were validated by using comparative quantitative reverse transcription polymerase chain reaction and Western blot analysis of GSCs, hNSCs, normal human astrocytes, U87 glioma cell line, and patient-matched serum-cultured glioblastoma multiforme samples.

Results: A candidate GSC-specific signature of 19 upregulated known and novel plasma membrane-associated genes was identified. Preferential upregulation of these plasma membrane-linked genes was validated by quantitative polymerase chain reaction. Cadherin-19 (CDH19) protein expression was enhanced in minimally infiltrative GSC lines.

Conclusions: Gene expression profiling of GSCs has shown CDH19 to be an exciting new target for drug development and study of GBM tumorigenesis.
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http://dx.doi.org/10.3171/2014.9.JNS132373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337891PMC
January 2015

MicroRNAs in cancer: glioblastoma and glioblastoma cancer stem cells.

Neurochem Int 2014 Nov 14;77:68-77. Epub 2014 Jun 14.

Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States; Department of Neurological Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States; Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States.

MicroRNAs represent an abundant class of endogenously expressed 18-25 nucleotide non-coding RNA molecules that function to silence gene expression through a process of post-transcriptional modification. They exhibit varied and widespread functions during normal development and tissue homeostasis, and accordingly their dysregulation plays major roles in many cancer types. Gliomas are cancers arising from the central nervous system. The most malignant and common glioma is glioblastoma multiforme (GBM), and even with aggressive treatment (surgical resection, chemotherapy, and radiation), average patient survival remains less than 2 years. In this review we will summarize the current findings regarding microRNAs in GBM and the biological and clinical implications of this data.
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http://dx.doi.org/10.1016/j.neuint.2014.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390175PMC
November 2014

Alkylphosphocholine analogs for broad-spectrum cancer imaging and therapy.

Sci Transl Med 2014 Jun;6(240):240ra75

Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792, USA. Department of Neurological Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792, USA.

Many solid tumors contain an overabundance of phospholipid ethers relative to normal cells. Capitalizing on this difference, we created cancer-targeted alkylphosphocholine (APC) analogs through structure-activity analyses. Depending on the iodine isotope used, radioiodinated APC analog CLR1404 was used as either a positron emission tomography (PET) imaging ((124)I) or molecular radiotherapeutic ((131)I) agent. CLR1404 analogs displayed prolonged tumor-selective retention in 55 in vivo rodent and human cancer and cancer stem cell models. (131)I-CLR1404 also displayed efficacy (tumor growth suppression and survival extension) in a wide range of human tumor xenograft models. Human PET/CT (computed tomography) and SPECT (single-photon emission computed tomography)/CT imaging in advanced-cancer patients with (124)I-CLR1404 or (131)I-CLR1404, respectively, demonstrated selective uptake and prolonged retention in both primary and metastatic malignant tumors. Combined application of these chemically identical APC-based radioisosteres will enable personalized dual modality cancer therapy of using molecular (124)I-CLR1404 tumor imaging for planning (131)I-CLR1404 therapy.
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http://dx.doi.org/10.1126/scitranslmed.3007646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336181PMC
June 2014

Upregulation of NHE1 protein expression enables glioblastoma cells to escape TMZ-mediated toxicity via increased H⁺ extrusion, cell migration and survival.

Carcinogenesis 2014 Sep 9;35(9):2014-24. Epub 2014 Apr 9.

Department of Neurological Surgery, The Second Affiliated Hospital of the Harbin Medical University, Harbin 150086, China, Department of Neurology, University of Pittsburgh, Pittsburgh, PA 15213, USA, Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA, 15213, USA

Sodium-hydrogen exchanger isoform 1 (NHE1) plays a role in survival and migration/invasion of several cancers and is an emerging new therapeutic target. However, the role of NHE1 in glioblastoma and the interaction of NHE1 expression and function in glioblastoma cells with cytotoxic temozolomide (TMZ) therapy remain unknown. In this study, we detected high levels of NHE1 protein only in primary human glioma cells (GC), glioma xenografts and glioblastoma, but not in human neural stem cells or astrocytes. GC exhibited an alkaline resting pHi (7.46±0.04) maintained by robust NHE1-mediated H(+) extrusion. GC treatment with TMZ for 2-24h triggered a transient decrease in pHi, which recovered by 48h and correlated with concurrent upregulation of NHE1 protein expression. NHE1 protein was colocalized with ezrin at lamellipodia and probably involved in GC migration. The TMZ-treated GC exhibited increased migration and invasion, which was attenuated by addition of NHE1 inhibitor HOE-642. Most importantly, NHE1 inhibition prevented prosurvival extracellular signal-regulated kinase activation and accelerated TMZ-induced apoptosis. Taken together, our study provides the first evidence that GC upregulate NHE1 protein to maintain alkaline pHi. Combining TMZ therapy with NHE1 inhibition suppresses GC migration and invasion, and also augments TMZ-induced apoptosis. These findings strongly suggest that NHE1 is an important cytoprotective mechanism in GC and presents a new therapeutic strategy of combining NHE1 inhibition and TMZ chemotherapy.
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http://dx.doi.org/10.1093/carcin/bgu089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146414PMC
September 2014

Glioblastoma cancer stem cells: Biomarker and therapeutic advances.

Neurochem Int 2014 May 19;71:1-7. Epub 2014 Mar 19.

University of Wisconsin School of Medicine and Public Health, Madison, WI, United States; Department of Neurological Surgery, Madison, WI, United States; Cellular and Molecular Biology, Madison, WI, United States; Neuroscience Training Program, Madison, WI, United States; Cellular and Molecular Pathology Training Program, Madison, WI, United States; Human Oncology, Madison, WI, United States; Carbone Cancer Center, Madison, WI, United States. Electronic address:

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans. It accounts for fifty-two percent of primary brain malignancies in the United States and twenty percent of all primary intracranial tumors. Despite the current standard therapies of maximal safe surgical resection followed by temozolomide and radiotherapy, the median patient survival is still less than 2 years due to inevitable tumor recurrence. Glioblastoma cancer stem cells (GSCs) are a subgroup of tumor cells that are radiation and chemotherapy resistant and likely contribute to rapid tumor recurrence. In order to gain a better understanding of the many GBM-associated mutations, analysis of the GBM cancer genome is on-going; however, innovative strategies to target GSCs and overcome tumor resistance are needed to improve patient survival. Cancer stem cell biology studies reveal basic understandings of GSC resistance patterns and therapeutic responses. Membrane proteomics using phage and yeast display libraries provides a method to identify novel antibodies and surface antigens to better recognize, isolate, and target GSCs. Altogether, basic GBM and GSC genetics and proteomics studies combined with strategies to discover GSC-targeting agents could lead to novel treatments that significantly improve patient survival and quality of life.
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http://dx.doi.org/10.1016/j.neuint.2014.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4119816PMC
May 2014

WNK1-OSR1 kinase-mediated phospho-activation of Na+-K+-2Cl- cotransporter facilitates glioma migration.

Mol Cancer 2014 Feb 20;13:31. Epub 2014 Feb 20.

Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA.

Background: The bumetanide (BMT)-sensitive Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) maintains cell volume homeostasis by increasing intracellular K+ and Cl- content via regulatory volume increase (RVI). Expression levels of NKCC1 positively correlate with the histological grade and severity of gliomas, the most common primary adult brain tumors, and up-regulated NKCC1 activity facilitates glioma cell migration and apoptotic resistance to the chemotherapeutic drug temozolomide (TMZ). However, the cellular mechanisms underlying NKCC1 functional up-regulation in glioma and in response to TMZ administration remain unknown.

Methods: Expression of NKCC1 and its upstream kinases With-No-K (Lysine) kinase 1 (WNK1) and oxidative stress-responsive kinase-1 (OSR1) in different human glioma cell lines and glioma specimens were detected by western blotting and immunostaining. Live cell imaging and microchemotaxis assay were applied to record glioma cell movements under different treatment conditions. Fluorescence indicators were utilized to measure cell volume, intracellular K+ and Cl- content to reflect the activity of NKCC1 on ion transportation. Small interfering RNA (siRNA)-mediated knockdown of WNK1 or OSR1 was used to explore their roles in regulation of NKCC1 activity in glioma cells. Results of different treatment groups were compared by one-way ANOVA using the Bonferroni post-hoc test in the case of multiple comparisons.

Results: We show that compared to human neural stem cells and astrocytes, human glioma cells exhibit robust increases in the activation and phosphorylation of NKCC1 and its two upstream regulatory kinases, WNK1 and OSR1. siRNA-mediated knockdown of WNK1 or OSR1 reduces intracellular K+ and Cl- content and RVI in glioma cells by abolishing NKCC1 regulatory phospho-activation. Unexpectedly, TMZ activates the WNK1/OSR1/NKCC1 signaling pathway and enhances glioma migration. Pharmacological inhibition of NKCC1 with its potent inhibitor BMT or siRNA knockdown of WNK1 or OSR1 significantly decreases glioma cell migration after TMZ treatment.

Conclusion: Together, our data show a novel role for the WNK1/OSR1/NKCC1 pathway in basal and TMZ-induced glioma migration, and suggest that glioma treatment with TMZ might be improved by drugs that inhibit elements of the WNK1/OSR1/NKCC1 signaling pathway.
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http://dx.doi.org/10.1186/1476-4598-13-31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3936893PMC
February 2014

Clinical model for predicting prolonged mechanical ventilation.

J Crit Care 2013 Oct 14;28(5):880.e1-7. Epub 2013 May 14.

Department of Medicine, Walter Reed National Military Medical Center, Bethesda, MD 20889, USA. Electronic address:

Introduction: Mechanical ventilation (MV) predisposes patients to numerous complications, which increases with longer durations of treatment. Identifying individuals more likely to require prolonged MV (PMV) may alter ventilation strategies or potentially minimize the duration of therapy and its associated complications. Our aim was to identify clinical variables at the time of intubation that could identify individuals who will require PMV.

Methods: One hundred thirty consecutive adult patients requiring MV support in a medical intensive care unit (ICU)were retrospectively assessed. Prolonged MV was defined as MV support more than 14 days.

Results: Mean age was 62.3±21.1 years, 64.6% were men, and mean duration of MV support was 11.4±11.9 days. Prolonged MV was required in 31.3%. Requiring intubation after admission to the ICU, heart rate greater than 110, blood urea nitrogen more than 25 mg/dL, serum pH less than 7.25, serum creatinine more than 2.0 mg/dL, and a HCO3 less than 20 mEq/L were the only variables independently associated with PMV. Specificity for predicting PMV was 100% with 4 or more of these variables.

Conclusion: The novel predictive model, using Intubation in the ICU, Tachycardia, Renal dysfunction, Acidemia, elevated Creatinine, and a decreased HCO3, was highly specific in identifying patients who subsequently required PMV support and performed better than Acute Physiology Age Chronic Health Evaluation III.
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http://dx.doi.org/10.1016/j.jcrc.2013.03.013DOI Listing
October 2013