Prognostic and therapeutic implications of migrating tumor cells in glioblastomas

Glioblastom er den hyppigste og mest ondartede form for primær hjernetumor, og glioblastompatienter har enmeget dårlig prognose med en median overlevelse på omtrentligt 15 måneder. Næsten alle glioblastompatienter får recidiv på trods af standardbehandlingen bestående af maksimal kirurgisk tumor resektion og adjuverende stråle- og kemoterapi med det alkylerende kemoterapeutikum temozolomid. Årsagen til recidiv findes iglioblastomers udtalt infiltrative vækstmønster. Migrerende tumorceller infiltrerer ind i det omkringliggendehjerneparenkym, og undslipper dermed kirurgisk resektion. Ydermere opnår disse migrerende tumorceller endelvis beskyttelse mod kemoterapi qua den mere intakte blod-hjerne-barriere, og deres placering i periferienaf strålefeltet medfører lavere stråledosis. De migrerende tumorceller som overlever den initiale behandlingdanner således grundlaget for recidiv, hvilket i sidste ende fører til patientens død. Det overordnede formålmed denne afhandling var at undersøge migrerende tumorceller i en prognostisk kontekst, med inkorporationaf biomarkør udtrykket i migrerende celler, samt at undersøge disse migrerende celler i en terapeutisk kontekst. Ydermere var formålet at undersøge kræftbehandlingspotentialet af en ny kinasehæmmer, SB747651A,som hæmmer flere kinaser der udtrykkes i migrerende tumorceller, og at identificere potentielle fænotypiskeforandringer i migrerende tumorceller efter terapeutisk intervention i form af kirurgisk tumor resektion.Studie I undersøgte tilstedeværelsen og den prognostiske værdi af to transkriptionsfaktorer, EGR1 ogEGR3, i en velkarakteriseret gliomkohorte med 207 patienter. Begge transkriptionsfaktorer har vist prognostisk værdi i flere forskellige kræftformer. En undergruppe bestående af 21 glioblastompatienter med P53udtryk, der blev anvendt som tumorcelle markør, og samtidig tilstedeværelse af både centrale og periferetumorområder blev undersøgt for EGR1 og EGR3 udtrykket i de forskellige tumorområder. Både EGR1 ogEGR3 protein var tilstede i alle undersøgte tumorer, men med betydelig inter-tumor variation. EGR1 proteinudtrykket viste en positiv korrelation med WHO tumor grad, men havde ingen prognostisk værdi efter justering for potentielle kliniske confoundere. EGR3 proteinet var udtrykt i højere grad i perifere migrerendetumorceller sammenlignet med centrale ikke-migrerende celler. Højt EGR3 proteinniveau var associeret meddårlig patientoverlevelse i en undergruppe af glioblastompatienter med methyleret MGMT-promoter. Fremtidige studier bør undersøge en potentiel funktionel rolle af EGR3 proteinet i tumorcelle migration og validereden potentielle prognostiske værdi.Studie II fokuserede på den første basale og prækliniske undersøgelse af multi-kinasehæmmerenSB747651A i glioblastomer. SB747651A hæmmer bl.a. proteiner som er involveret i tumorcellers migration,og kan således potentielt være en kinasehæmmer, der kan ramme de migrerende tumorceller, som fører tilrecidiv. Vi undersøgte virkningen af SB747651A i tre forskellige velkarakteriserede patient-deriveredeglioblastom cellekulturer og i mus med orthotope glioblastom xenografts. Koncentrationer på 5-10 µM inducerede koncentrations- og tidsafhængig celledød i all tre glioblastom kulturer, og kombinationsbehandlingmed SB747651A og temozolomid medførte en additiv effekt på graden af celledød. Tumorceller behandletmed SB747651A viste nedsat sphæroidedannelse, reduceret vækst og migration, mens fosforyleringsgradenaf mTOR, CREB, GSK3 og GYS1 kinaserne samt mængden af SOX2 protein blev reduceret. Behandlede celler akkumulerede glycogen intracellulært og var mere tilbøjelige til at danne reaktive iltradikaler. Musmed orthotope glioblastom xenografts behandlet med 25 mg/kg SB747651A 5 dage/uge i 8 uger levedesignifikant længere end mus fra kontrolgruppen, og ingen bivirkninger blev observeret i de behandlede dyr.Formålet med studie III var at undersøge den isolerede indflydelse som kirurgisk tumor resektion har påde tilbageværende migrerende tumorceller i resektionskaviteten. Dette undersøgte vi ved at etablere en præklinisk glioblastom resektionsmodel. Modellen blev etableret i immundefekte rotter og integrerede PET/CTscanninger med radioisotopen 18F-FET for at detektere vækst af både primær- og recidiv tumorer. Tumorceller fra primær og recidivtumorer blev RNA sekventeret, og deregulerede gener samt signaleringsveje blevidentificeret og yderligere undersøgt i vævssnit fra hhv. resektionsmodellen og fra glioblastompatienter medparrede primær/recidiv tumorer. Vi foretog yderligere funktionelle undersøgelser in vitro med flere forskellige patient-deriverede glioblastom cellekulturer. 18F-FET PET/CT scanningerne kunne detektere væksten afortotope xenografts 3-4 uger efter tumorcelle implantation. Efter valideret tumorvækst, etablerede vi succesfuldt selve tumor resektionsproceduren, som i meget høj grad afspejler resektion af glioblastomer i patienterne, hvor der efterlades få tumorceller i resektionskaviteten. Sammenligning af primære og recidiv tumorer fraresektionsmodellen viste at recidiv tumorerne havde højere grad af tumorcelle proliferation, mere udtaltinfiltration af mikroglia/makrofager og en opregulering af flere stamcelle gener, såsom SOX2, POU3F2,OLIG2 og Notch1 sammenlignet med primærtumorerne. Denne stamcelle opregulering blev valideret påproteinniveau både i vævssnit fra resektionsmodellen og i vævssnit fra glioblastompatienter, hvilket indikererhøj overførbarhed af disse resultater. Blandt de signifikant opregulerede gener i recidivtumorerne var pleiotrophin genet og den primære pleiotrophin receptor PTPRZ1. Mekanistisk, påviste vi at pleiotrophin var i standtil at inducere tumorcelle proliferation og selvfornyelse i flere forskellige patient-deriverede glioblastomkulturer. Blokering af pleiotrophins virkning med neutraliserende antistoffer stoppede denne pleiotrophinmedierede selvfornyelse af tumorceller. Pleiotrophin kunne inducere udtryk af stamcelle proteinerne SOX2,POU3F2 og i mindre grad OLIG2 i tumorcellerne, og pleiotrophin er således en plausibel årsag bag promoveringen af glioblastom stamceller observeret i recidiv tumorerne. Yderligere var høje niveauer af pleiotrophinprotein associeret med dårlig patientoverlevelse.Denne afhandling viser samlet set, at inkorporation af specifikt biomarkør udtryk i migrerende tumorcellerkan generere nye hypoteser vedrørende glioblastomers patofysiologi, men at en afklaring af disse cellerspotentielle prognostiske værdi besværliggøres af manglen på tumor-specifikke markører og begrænsninger iantallet af inkluderbare patienter. Migrerende tumorceller udgør en cellepopulation med betydelige terapeutiske implikationer, og disse celler bør således være integreret i fremtidig translationel glioblastomforskning.Afhandlingen præsenterer nye indsigter i konsekvenserne af kirurgisk glioblastom resektion, og antyder atresektion i sig selv fremmer glioblastom stamcellepopulationen blandt de tilbageblivende, tidligere migrerende, tumorceller i recidiverne. Brugen af mere avancerede prækliniske modeller der også omfatter kirurgisktumor resektion, som anvendt i denne afhandling, vil formentligt øge den translationelle succesrate for nye lovende fund. Dette vil i sidste ende gavne glioblastompatienter via udvikling af bedre og mere effektivebehandlingstilbud.Glioblastoma is the most common and malignant type of primary brain tumor with a dismal prognosis and median survival of approximately 15 months. Inevitably, virtually all glioblastomas recur after standard-ofcare therapy consisting of maximal safe tumor resection and adjuvant radio- and chemotherapy with thealkylating agent temozolomide. The origin of tumor recurrence is found in the highly infiltrative growthpattern that glioblastomas notoriously exhibit, combined with high intrinsic resistance to therapy. As migrating tumor cells infiltrate the surrounding brain parenchyma, they escape both surgical resection and are betterguarded against adjuvant radio- and chemotherapy by a more intact blood-brain-barrier in the tumor periphery, and by localization at the outer margins of the radiation field. These migrating tumor cells that survivetherapeutic interventions therefore lay the foundation for eventual tumor recurrence, which ultimately leadsto patient death.The overall aim of this thesis was to investigate migrating tumor cells in the context of prognostic biomarkers, considering specific expression in migrating tumor cells, and to investigate these cells in a therapeutic context. Further, this thesis aimed to investigate the anticancer effects of a novel drug compound,SB747651A, with affinity for protein kinases expressed in migrating glioblastoma cells, and to uncoverpotential phenotypic changes in migrating tumor cells following therapeutic intervention in the form of surgical tumor resection.Study I in this thesis investigated the expression and prognostic value of two transcription factors, EGR1and EGR3, in a well-annotated glioma cohort including 207 patients. These two transcription factors haveshown prognostic value in multiple cancer types. A subset of 21 glioblastomas with P53 expression, whichwas utilized as a tumor cell marker, and inclusion of both central and peripheral tumor regions were investigated for intra-tumoral EGR1 and EGR3 staining patterns. Protein expression was assessed both as meanexpression in total tumor area, and specifically in migrating tumor cells. Relevant findings were elucidated byin silico analysis using publicly available glioma datasets. EGR1 and EGR3 protein expression was identifiedin all investigated tumors, but considerable inter-tumor variation was found. EGR1 protein expression positively correlated with WHO grade, but did not show prognostic value after adjusting for potential clinicalconfounders in Cox-regression analysis. EGR3 expression in peripheral migrating tumor cells was higherthan in central non-migrating cells, and high EGR3 protein expression was associated with poor overallpatient survival in a subgroup of glioblastoma patients with methylated MGMT-promoters. Future investigations should functionally assess a potential role of EGR3 in the process of tumor cell migration and validateits role as a prognostic biomarker in GBM.Study II focused on the first basic and preclinical investigation of the small-molecule kinase inhibitorSB747651A in glioblastomas. With its multi-target affinity, this inhibitor also targets proteins associated withtumor cell migration, and may therefore be a mean to combat the migrating tumor cells responsible for tumorrecurrence. We investigated the anticancer efficacy of SB747651A in three well-characterized patient-derivedglioblastoma cell cultures and in a murine orthotopic xenograft model. SB747651A concentrations between 5-10 µM induced cell death in a concentration- and time dependent manner in all three investigated cellcultures, and addition of temozolomide yielded an additive effect on cell death. Treated tumor cells showedimpaired spheroid formation, reduced growth and cell migration, while phosphorylation of mTOR, CREB,GSK3 and GYS1 kinases as well as SOX2 protein expression was reduced after SB747651A treatment.Further, exposed cells accumulated intracellular glycogen and had increased generation of reactive oxygenspecies. Orthotopically xenografted mice lived significantly longer when treated with SB747651A for 5days/weekly with 25 mg/kg throughout 8 weeks compared to vehicle treated controls, and no adverse effectswere observed in vivo.In study III, we aimed to investigate the isolated impact of surgical tumor resection on the phenotype ofresidual migrating tumor cells left behind in the resection cavity. To investigate this aspect, we developed apreclinical glioblastoma resection model using immunocompromised nude rats combined with small animalPET/CT imaging utilizing the radioisotope 18F-FET to monitor tumor growth and recurrence. Tumor cellsfrom primary and recurrent tumors were isolated and subject to total mRNA sequencing. Significantly deregulated genes and pathways were identified and further investigated in tissue specimens from the resectionmodel and from glioblastoma patients with paired primary/recurrent tumors. Functional investigations wereperformed in vitro with several patient-derived glioblastoma cell cultures. 18F-FET PET/CT scans couldsuccessfully detect growth of orthotopic glioblastoma xenografts 3-4 weeks after tumor cell implantation.Following detection of primary tumor growth, we successfully established preclinical glioblastoma resection,which closely mimics resection of patient glioblastomas, with few residual tumor cells left behind in thecavity. Comparisons of primary and recurrent tumors from our preclinical resection model revealed thatrecurrent tumors were more proliferative, showed increased infiltration of microglia/macrophages and upregulated multiple glioblastoma stem cell genes including SOX2, POU3F2, OLIG2 and Notch1 compared toprimary tumors. This upregulation was validated at the protein level both in recurrent xenografts and in earlyrecurrent patient glioblastomas indicating high translatability. Among the upregulated genes in recurrenttumors, pleiotrophin and its primary receptor PTPRZ1 were identified. Mechanistically, pleiotrophin couldinduce tumor cell proliferation and self-renewal in several patient-derived glioblastoma cell cultures, andinhibition of pleiotrophin with neutralizing antibodies abolished the pleiotrophin-mediated increase of tumorcell self-renewal. Further, pleiotrophin was able to induce expression of stem cell proteins SOX2, POU3F2and to a lesser extent OLIG2 in tumor cells, thereby making it a plausible mediator of the glioblastoma stemcell enrichment observed in recurrent tumors. Additionally, high pleiotrophin expression was associated withpoor overall patient survival.In conclusion, this thesis shows that incorporating specific biomarker expression in migrating tumor cellscan be used to generate novel hypotheses regarding glioblastoma pathophysiology, however, unraveling thepotential prognostic value of biomarker expression in migrating tumor cells is complicated by lack of optimaltumor specific markers and sample size limitations. Migrating tumor cells pose a cell population with profound therapeutic implications, and should therefore be incorporated into future translational glioblastoma research to a greater extent. We provide novel insights into the implications of surgical tumor resection, andpropose that resection itself is an iatrogenic driver of glioblastoma stem cell enrichment among residual,formerly migrating, tumor cells in recurrent tumors. Application of more advanced preclinical models thatalso include tumor resection, as applied in this thesis, will most likely increase the translational success rateof novel findings to ultimately benefit glioblastoma patients through development of better and more efficienttherapeutic approaches

Achieving Optimal Correction for Young Myopic Children:A Concept Study

The purpose of this article is to explore alternative ways of achieving optimal correction for myopic children who cannot cooperate to subjective manifest refraction (SR). The study included myopic children aged 9-12 years who underwent non-cycloplegic SR and autorefraction with and without cycloplegia using the Shin-Nippon Nvision-K 5001 autorefractor (AR) as well as non-cycloplegic autorefraction using the Topcon KR-800S AR. There were 21 children (mean age, 10.62 years) included. The spherical equivalent refractive error of SR was not significantly different from that of non-cycloplegic AR measurements, but it was significantly different from that of cycloplegic Shin-Nippon Nvision-K 5001 measurements (p &lt; 0.001). Compared with SR, cycloplegic Shin-Nippon Nvision-K 5001 measured a less myopic refractive error (median: -2.44 D vs. -2.88 D, p &lt; 0.001). For both ARs, the axis measurements and astigmatic dioptre values between SR and autorefraction were not significantly different. Compared with non-cycloplegic SR, cycloplegic measurements showed a lesser degree of myopic refractive error. There was no significant difference between SR and non-cycloplegic autorefraction. Therefore, the Topcon KR-800S and the Shin-Nippon Nvision-K 5001 ARs may be useful for prescribing glasses in myopic children who cannot cooperate during SR. However, caution should be taken with cylinders &lt;0.75 D because the agreement in axis between SR and AR measurement is poor. Therefore, in such cases, we suggest to add half the cylinder to the spherical component.</p

Portable runtime environments for Python-based FMUs: Adding Docker support to UniFMU

Co-simulation is a means to combine and leverage the strengths of different modeling tools, environments and formalisms and has been applied successfully in various domains. The Functional Mock-Up Interface (FMI) is the most commonly used standard for co-simulation. In this paper we extend UniFMU, a tool that allows users to build Functional Mock-Up Units (FMUs) in virtually any programming language, to support execution within Docker. As a result the generated FMUs can be distributed in an environment containing all runtime dependencies. To describe the process of creating dockerized FMUs using UniFMU, we show how to model and co-simulate a robotic arm and a controller using two Python-based FMUs

Strukturer i et mediaarkiv : analyse av hvordan NRK beskriver sine nyhetsinnslag, og hvordan det kan tenkes påvirke framtidens forståelse av vår samtid.

Our understanding of the society and the world around us is partly communicated to us through the newsmedia. The language used, both written, oral and images, affects the way that we talk about, understand and act upon certain events. In a newsbroadcast there are several interpretations and representations, all communicated in various forms of language, and this language will in turn be the basis for the language that is being used when a news segment is being described in an archive. I have analyzed how the Norwegian Broadcasting Corporation (NRK) describe their news segments in a digital archive structure. By using a critical discourse analysis, I have looked at how NRK covered two foreign events, and how that material is being described in the form of metadata. My study was on two events that gained a lot of media attention in Norway at the time. One of them is the riots that took place in Great Britain in august 2011, and the second event is the riots that happened in Sweden in may 2013. The news coverage of these events used images to communicate what happened to a large degree. The choice of images and how the news presented the stories, I suggest, have an affect on how norwegian society percieved them. By using a critical discourse analysis on the metadata for these news segments, I analyzed how the representations of the news stories are available in NRKs digital archiving structure, Programbanken. The main representations and identities that the news segments produced are recurrent in the metadata, and in turn becomes the terms that will give results on searches within the archive structure. As the news segments from 2011 was being described by metadata professionals from the metadata section, and the metadata for the segments from 2013 is a result of the journalists themselves ascribing metadata, a comparison show that the journalists describe the material in a far lesser extent than the professionals at the metadata section. My basis of this study is that the archivist have profound impact on the material, as they have an active part in how the material is being made available, by the language they use to describe any material