Stereotactic Microdialysis for Metabolic Assessment and Experimental Treatment of Malignant Glioma

Stereotactic microdialysis for metabolic assessment and experimental treatment of malignant glioma

Pedram Tabatabaei Shafiei

Department of Clinical Sciences / Neurosciences Umeå 2020 This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD ISBN print: 978-91-7855-402-7 ISBN PDF: 978-91-7855-403-4 ISSN: 0346-6612 New Series No. 2104 Electronic version available at: http://umu.diva-portal.org/ Printed by: CityPrint i Norr AB Umeå, Sweden 2020

“People calculate too much and think too little.”

Charles T. Munger

2 TABLE OF CONTENTS

ABBREVIATIONS ...... 7 LIST OF ORIGINAL PUBLICATIONS ...... 10 POPULÄRVETENSKAPLIG SAMMANFATTNING PÅ SVENSKA ...... 11 GLIOBLASTOMA MULTIFORME ...... 14 BACKGROUND ...... 14 DIAGNOSIS ...... 14 Symptoms and signs ...... 14 Radiology ...... 15 Pathology ...... 15 Molecular Genetic Markers ...... 16 TREATMENT OF GLIOBLASTOMA MULTIFORME ...... 17 Surgery ...... 17 Radiochemotherapy and adjuvant chemotherapy ...... 17 Response evaluation and follow-up ...... 17 Second-line treatment ...... 18 Prognostic factors ...... 19 LOCAL DELIVERY SYSTEMS FOR TREATMENT OF BRAIN TUMORS ...... 20 BLOOD BRAIN BARRIER (BBB) ...... 20 I. Enhancing drug permeability ...... 20 II. BBB disruption ...... 21 III. Interstitial delivery of drugs, catheter injection or infusion ...... 21 IV. Convection-enhanced delivery of drugs to the CNS ...... 21 V. Direct delivery with polymers ...... 22 VI. Drug-impregnated microchip delivery ...... 22 THE MICROENVIRONMENT OF GLIOBLASTOMA MULTIFORME ...... 23 SUPPORTING TUMOR MICROENVIRONMENT ...... 23 HETEROGENEITY ...... 24 THE IMMUNE MICROENVIRONMENT OF GBM ...... 24 THE METABOLISM OF GLIOBLASTOMA MULTIFORME ...... 27 Metabolism of normal cells ...... 27 Metabolism of cancerous cells ...... 30 Metabolic changes in GBM ...... 31 IN VIVO TECHNIQUES FOR INVESTIGATION OF TUMOR METABOLISM ...... 35 MASS SPECTROMETRY ...... 36 MAGNETIC RESONANCE SPECTROSCOPY ...... 37 POSITRON EMISSION TOMOGRAPHY ...... 37 STABLE ISOTOPE-RESOLVED METABOLOMICS ...... 38

3 MICRODIALYSIS ...... 39 History ...... 39 Recovery ...... 40 Limitations of the microdialysis technique ...... 41 AIMS ...... 43 MATERIALS AND METHODS ...... 44 PATIENTS ...... 44 Study I ...... 44 Study II ...... 44 Study III and IV ...... 44 MICRODIALYSIS CATHETER IMPLANTATION AND SAMPLING ...... 45 Study I ...... 45 Study II ...... 45 RETROGRADE MICRODIALYSIS ...... 45 Study III and IV ...... 45 TREATMENTS ...... 46 Radiotherapy ...... 46 Loco-regional interstitial treatment with Cisplatin ...... 46 PATIENT FOLLOW-UP ...... 47 ANALYTICAL METHODS ...... 47 Assessment of glucose metabolites, glutamate, and glycerol (Study I–III) ...... 47 Cytokine analysis (study II) ...... 47 Immunohistochemistry (Study II) ...... 48 Detection of platinum and Cisplatin (study III) ...... 48 Metabolomic analysis (IV) ...... 49 STATISTICAL ANALYSES ...... 49 Wilcoxon signed rank test (study I, II and III) ...... 49 Analysis of variance (Study I and II) ...... 50 Correlation (study I, II, III) ...... 50 Orthogonal projections to latent structures discriminant analysis (OPLS-DA) and Orthogonal projections to latent structures effect projections (OPLS-EP) (study 4) ...... 50 ETHICS ...... 52 RESULTS AND DISCUSSION ...... 53 FEASIBILITY OF MICRODIALYSIS (STUDY I-II) ...... 53 FEASIBILITY OF RETROGRADE MICRODIALYSIS (STUDY III AND IV) ...... 53 CHANGES DURING FRACTIONATED RADIOTHERAPY (STUDY I AND II) ...... 54 Analysis of glucose metabolites, glutamate, and glycerol ...... 54 Cytokine expression (study II) ...... 55 Immunohistochemistry (study II) ...... 56 RETROGRADE MICRODIALYSIS WITH CISPLATIN (STUDY III AND IV) ...... 56 Pharmacokinetics ...... 56 Cytotoxicity assessed by glutamate and glycerol ...... 57 Quality of life, functional status, and survival ...... 58 Metabolic differences in tumor versus BAT at baseline (study IV) ...... 58 Treatment effects on metabolites ...... 58 Treatment response markers and survival ...... 59

4 CONCLUSIONS ...... 62 ACKNOWLEDGEMENTS ...... 63 REFERENCES ...... 65

5 ABSTRACT

Glioblastoma multiforme, the most common primary brain tumor, has a dire prognosis despite multimodal treatments that include surgery and radio-chemotherapy. To improve the outcome of this destructive disease, we need to improve our understanding of its tumor biology. Furthermore, the development of new treatment strategies will improve with a better understanding of the interplay between malignant cells and their direct surrounding microenvironment.

This thesis aims to increase the understanding of the processes within high-grade glioma and its microenvironment during normal conditions as well as during the distress associated with treatment. Specifically, we have investigated the metabolic response to radiotherapy (study I and II), the immunologic response to radiotherapy (study II), and the metabolic response pattern to loco-regional treatment with cisplatin (study III and IV). Using microdialysis, we collected samples from the extracellular space in both normal brain and tumor tissue during radiotherapy (study I and II) and loco-regional cisplatin treatment (study III and IV). Theses samples were analyzed for glucose metabolites, glycerol, and glutamate (study I, II, and III) and for cytokines (study II). In addition, we analyzed the global metabolism with mass spectrometry to identify and assess the response pattern of malignant glioma cells to loco-regional cisplatin treatment (study IV).

In study I and II, we found that malignant glioma cells used glucose at a higher rate than normal cells and preferred glycolysis for glucose metabolism. The given radiation dose (2 Gray (Gy) daily for five days) did not significantly affect glucose metabolism, glycerol levels, or glutamate levels in tumor tissue or the microenvironment. However, in study II, we observed an induced inflammatory effect due to the given radiation dose as several of the cytokines investigated showed significantly increased levels during radiotherapy. In study IV, we observed a complex and strong metabolic response to the loco-regional cisplatin therapy. At baseline, we found a metabolic pattern corresponding well with highly proliferating tumor tissue–i.e., high levels of amino acids, their metabolites, and other metabolic end products and low levels of sugar derivatives, antioxidants, and nucleotides. During the loco-regional therapy, we observed a clearly localized cytotoxic effect within the tumor and a metabolic response pattern corresponding with cisplatin’s complex mechanism of action, affecting several metabolic pathways within the malignant cell. Glutamate and glycerol also increased in tumor tissue following loco-regional treatment, a finding that further supported the observation of local toxicity.

In study III, we investigated microdialysis as a method to assess the microenvironment in high- grade glioma and as a method for drug delivery (retrograde microdialysis). All studies demonstrated the usefulness of microdialysis as a tool for in vivo real-time assessment of molecular events in malignant glioma tissue. Although the method is invasive, no complications related to the surgical procedure or assessment were noted. In study III, we also demonstrated that retrograde microdialysis is a feasible method for locally delivering clinically significant doses of drugs such as cisplatin to tumor tissue in the brain. However, in addition to having a cytotoxic effect on tumor cells, cisplatin may induce clinically significant edema.

ABBREVIATIONS

ACL = ATP citrate lyase ACSS2 = Acetyl-CoA synthesize short chance 2 ADP = Adenosine diphosphate AKT = Protein kinase b AMP = Adenosine monophosphate ANOVA = Analysis of variance ATP = adenosine triphosphate B7-H1 = B7 homolog BAT = Brain adjacent to tumor BBB = Blood brain barrier BCNU = Carmustine CED = Convection enhanced delivery CNA = Central nerve system CSF-1 = Colony stimulating factor 1 D-2-HG = D-2-hydroxyglutarate ECM = Extracellular matrix EGFR = Epidermal growth factor receptor F2,6BP = Fructose 2, 6 biphosphate FAS = Fatty acid dehydrogenase FasL = Fas ligand FDG = [18F] fluorodeoxyglucose G6P = Glucose 6 phosphate G6PD = Glucose 6 phosphate dehydrogenase GBM = Glioblastoma multiforme GC = Gas chromatography GLS = Glutaminase 1 GLUT 1 = Glucose transporter 1 Gy = Gray HIF 1 = Hypoxia inducible factor 1 HILIC = Hydrophilic interaction chromatography Hk 2 = Hexokinase 2 ICPMS = Inductively coupled plasma mass spectrometry IDH = Isocitrate dehydrogenase

7 IL = Interleukin LDHA = Lactate dehydrogenase A LDHB = Lactate dehydrogenase B MCP - 1 = Monocyte chemoattractant protein 1 MDSC = Myeloid-derived suppressor cells MGMT = 06-Methylguanine-DNA-methyltransferase MIP - 1 = Macrophage inflammatory protein MRI = Magnetic resonance imaging MRS = Magnetic resonance spectrometry MS = Mass spectrometry NAD = Nicotinamide adenine dinucleotide NADP= Nicotinamide adenine dinucleotide phosphate NaF = [18F] sodium fluoride NF1 = Neurofibroma 1 NMR = Nuclear magnetic resonance OPLS - DA = Orthogonal projections to latent structures discriminant analysis OPLS - EP = Orthogonal projections to latent structures effect projections OXPHOS = Oxidative phosphorylation PCPP-SA = Poly-carboxyphenoxypropane-co-sebasic acid PDGFRA = platelet derived growth factor alpha PDH = Pyruvate dehydrogenase PDH = Pyruvate dehydrogenase PET = Positron emission tomography PFK 1 = Phosphofructokinas 1 PFKFB = 6-phosphofructose-2-kinase/fructose2,6-biphosphatase PGE2 = Prostaglandin E2 PGI = Phosphoglucose isomeras PGM = Phosphoglycerate mutase PHH3 = Phosphorylated histone H3 PI3K = Phosphatidylinositol-3-kinas PKM 1 = Pyruvate kinase isomer 1 PLS = Partial least square PTEN = T homolog ROS = Reactive oxygen species SC = Subcutaneous SIRM = Stable isotope-resolved metabolomics STAT-3 = Signal transducer and activator of transcription 3 T-reg = Regulatory T-cell TAM = Tumor associated macrophages TCA cycle = Tricarboxylic cycle TGF-ß = Transforming growth factor-ß

8 TIGAR = T53 expression-induced glycolysis and apoptosis regulator WHO = World Health Organization α-KG = α-ketoglutarate [18F]-FET = O-(2-[18F]-fluoro-Ethyl)-L-tyrosine [18F] sodium fluoride = (18F-NaF) [18F]-FLT = [18F]-3′-fluoro-3′-deoxy-L-thymidine

9 LIST OF ORIGINAL PUBLICATIONS

Study I Glucose metabolites, glutamate and glycerol in malignant glioma tumors during radiotherapy Pedram Tabatabaei, Per Bergström, Roger Henriksson and A. Tommy Bergenheim J Neurooncol (2008) 90:35–39, DOI 10.1007/s11060-008-9625-2

Study II Radiotherapy induces an immediate inflammatory reaction in malignant glioma: a clinical microdialysis study Pedram Tabatabaei, Eward Visse, Per Bergström, Thomas Brännström, Peter Siesjö and A. Tommy Bergenheim J Neurooncol (2017) 131:83–92, DOI 10.1007/s11060-016-2271-1

Study III Intratumoral Retrograde Microdialysis Treatment of High-Grade Glioma with Cisplatin Pedram Tabatabaei, Thomas Asklund, Per Bergström, Erik Björn, Mikael Johansson and A. Tommy Bergenheim Acta Neurochirugica (2020), In press, DOI: 10.1007/s00701-020-04488-2

Study IV Metabolic response patterns in brain microdialysis ﬂuids and serum during interstitial cisplatin treatment of high-grade glioma Benny Björkblom, Pär Jonsson, Pedram Tabatabaei, Per Bergström, Mikael Johansson, Thomas Asklund, A. Tommy Bergenheim and Henrik Antti British Journal of Cancer (2020)122:221–232; DOI:10.1038/s41416-019-0652-x

POPULÄRVETENSKAPLIG SAMMANFATTNING PÅ SVENSKA

Glioblastoma multiforme är den vanligaste och dödligaste primära hjärntumören tillhörande tumörfamiljen som benämns gliom. Rutinbehandlingen av dessa tumörer består idag av kirurgi, där man har som målsättning att avlägsna mer än 95% av synlig tumör, följd av en sex veckors kombinationsbehandling med cytostatika och fraktionerad strålbehandling där patienten får mindre stråldoser dagligen under vardagar. Därefter följer behandling med enbart cytostatikabehandling i fem dagar, var 28:de dag i sex omgångar. Trots den omfattande behandlingsregimen är medelöverlevnaden för patienter med glioblastoma multiforme tyvärr inte längre än dryga 10 månader från diagnos. För att utveckla mer effektiva och bättre behandlingsstrategier är det av största vikt att öka vår förståelse för dessa tumörer. Trots all forskning inom området är kunskapen om tumörernas mikromiljö, d.v.s. den miljö som tumörcellerna vistas i, fortfarande begränsad. Mer kunskap om dessa tumörers biologi behövs för att skapa en bättre förståelse om deras grundläggande karaktäristiska, en kunskap som sedan kan användas för att utveckla bättre behandlingar.

Ett grundläggande problem med behandlingsstrategier i hjärnan är tillgängligheten. Hjärnan omges av ett hårt skyddande hölje, skallbenet och där under flera lager av hinnor. Hjärnan skyddas även av den så kallade blod-hjärnbarriären som skyddar hjärnan från potentiellt giftiga ämnen som nått blodet via födan, tarmfloran och infektioner i kroppen. Dessa olika skyddsmekanismer gör det svårare att leverera tillräckliga mängder läkemedel vid sjukdomar i hjärnan. Det optimala vid tumörsjukdomar hade varit att utveckla en behandlingsmetod för tillförsel av specifikt valda ämnen lokalt till tumören.

Mikrodialys är en metod där man via ett borrhål kan lägga in en tunn kateter i hjärnan. Längst ut på denna kateter finns ett halvgenomsläppligt membran. Detta membran är genomsläppligt för mindre molekyler. Koncentrationsskillnader över ett sådant membran medför att molekyler vandrar in och ut genom membranet via en passiv process som kallas diffusion, där riktningen alltid är från högre till lägre koncentration. Med denna metod kan man följa molekylära förändringar i tex hjärnvävnaden på patienter i realtid. Man kan också tillföra molekyler till hjärnvävnaden via en process som vi kallar för retrograd mikrodialys.

Med detta som bakgrund har vi i denna avhandling, genom fyra arbeten, haft det övergripande syftet att skapa en bättre förståelse för processer i tumörers mikromiljö under normala betingelser samt under cellgifts och strålbehandling. Målsättningen har också varit att utvärdera om mikrodialys som metod kan användas för realtidsmonitorering av förändringar i glukosmetabolism, cytokinuttryck, markörer och metabola förändringar i maligna gliom. Slutligen har även retrograd mikrodialys utvärderats som metod för lokal tillförsel av Cisplatin, ett ytterst potent cytostatikum vid behandling av höggradiga gliom.

I studie I har förändringar i hjärnans mikromiljö monitorerats med hjälp av mikrodialyskatetrar med fokus på glukosmetabolismen i de maligna tumörcellerna före och under de första fem dagarna av sedvanlig strålbehandling, så kallad fraktionerad strålbehandling. Vi har tittat på glukosnivåer, men även på dess nedbrytningsprodukter i cellen, laktat och pyruvat, för att på så sätt få information om hur cellen utnyttjar glukos för att utvinna energi till sin överlevnad.

11 Vidare har även nivåerna för glutamat och glycerol följts då dessa två molekyler tros vara markörer för cellskada och därigenom skulle kunna bidra med information om hur effektiv den strålbehandling som vi ger är. Samma upplägg som i studie I har använts i studie II med tillägget att vi nu även har undersökt förändringar i tumörens mikromiljö ur ett immunologiskt perspektiv med fokus mot cytokinuttryck. Cytokiner är små molekyler som uttrycks av flertalet celler i kroppen och som orkestrerar kroppens immunförsvar. Dessa molekyler används för att påverka styrningen av den immunologiska responsen mot främmande antigener som t ex bakterier och virus, men också vid vävnadsskada som tex efter strålbehandling.

I studie III och IV har istället mikrodialyskatetrar använts för att tillföra Cisplatin lokalt till hjärntumören. Denna process kallas för retrograd mikrodialys. Fördelen med denna metod är att samtidigt som mikrodialys utnyttjas för att tillföra en aktiv substans till vävnaden kan man följa vad som händer i samma vävnad och därigenom erhålla information om hur den behandling som ges verkar. I studie III har istället fokus varit på metoden med syfte att visa huruvida retrograd mikrodialys är en metod som är säker och pålitlig för tillförsel av läkemedel för lokal behandling. I studie IV har fokus skiftats mot effekten av den givna behandlingen där vi specifikt tittat på metabola förändringar som sker i den tumörvävnad där cisplatin ges.

I studie I sågs en lägre koncentration av glukos i tumör jämfört med frisk hjärna före behandlingsstart. Vidare sågs lägre koncentrationer av pyruvat och även tendenser till högre laktat nivåer. Detta resulterade i att den så kallade laktat/pyruvat-kvoten var högre i tumör jämfört med normal vävnad. En slutsats av detta kan vara att tumörceller har en högre glukosförbränning. Detta korresponderar väl med tidigare forskningsresultat där man ser att tumörceller behöver mer energi och gärna utvinner denna energi från glukos genom en process som kallas för glykolys. Glykolysens slutprodukter är just pyruvat som metaboliseras vidare till laktat. Vad vi däremot inte kunde se var någon behandlingsrelaterad förändring i koncentrationerna av glukos, pyruvat, laktat eller cellskademarkörerna glutamat och glycerol under de fem dagar patienterna fick strålbehandling och samtidigt följdes med mikrodialys. Slutsatsen från detta är att stråldosen antingen var för låg för att inducera en cellskada i vävnaden, detekterat med de av oss analyserade metaboliterna, eller att effekten av strålbehandlingen kommer senare i förloppet och därför inte visades då patienterna bara följdes under de första 5 dagarna av sin strålbehandling.

I studie II såg resultaten vad gäller glukosförbränningen i tumörceller före och under strålbehandling identiska ut jämfört med studie I, med den lilla skillnaden att man kunde se en klart högre koncentration av laktat i tumörceller. I studie I kunde vi endast se sådana tendenser. Vidare kunde vi se en klar stegring av ett flertal cytokiner i tumörvävnad jämfört med normal hjärnvävnad. Påtagligt var att den givna stråldosen har en inflammatorisk effekt och aktiverar immunologisk aktiva celler som i sin tur producerar dessa cytokiner. Den stora frågan är om den inflammatoriska effekten är till tumörens fördel eller nackdel. Detta är en kontroversiell fråga. Det finns idag forskningsresultat som visar att den immunologiska reaktionen i tumören kan motverka tumörcellers framfart, men också en övertygande mängd nya forskningsrön som visar på det faktum att mycket av de immunologiska reaktionerna som ses i tumörvävnaden orkestreras av tumörcellerna själv och har som syfte att skapa en för tumören gynnsam mikromiljö.

I studie I och II påvisades det att mikrodialys är en utmärkt metod för att, i realtid, följa metabola samt immunologiska händelser i patienternas hjärna och i den avsedda tumörvävnaden.

12 I studie III användes retrograd mikrodialys för att administrera Cisplatin till tumörvävnad i hjärnan. Vi kunde demonstrera teknikens användbarhet och dessutom påvisa en klar celltoxisk effekt av behandlingen lokalt i tumörvävnaden. Ingen toxisk effekt noterades i kringliggande normal hjärnvävnad eller i kroppen i övrigt. Således kan retrograd mikrodialys vara ett alternativ för lokal administrering av vissa substanser i hjärnan.

I studie IV analyserades de prover som har samlats i samband med den lokala cisplatinbehandlingen i tumörvävnad (i studie III). Dessa prover analyserades avseende ämnesomsättning och dess mönster innan behandlingsstart i tumörvävnad jämfört med normal vävnad och efter behandlingsstart för att undersöka effekten av behandlingen på vävnadernas ämnesomsättning. Vi fann tydliga skillnader mellan normal vävnad och tumörvävnad före behandlingsstart. I tumörvävnad sågs ett metabolt mönster talande för en snabbväxande tumör som förbrukar molekylära byggstenar för celldelning och tillväxt samtidigt som det även fanns ett mönster som talade för ett högre energiutnyttjande i tumörvävnad jämfört med normal vävnad. Vidare kunde två distinkta grupper hos de behandlade patienterna relativt tydligt urskiljas. En grupp som kallades korttidsöverlevare där medelöverlevnaden inte var mer än ca 1 månad efter påbörjad behandling och en grupp som kom att kallas långtidsöverlevare där medelöverlevnaden var drygt 9 månader. Efter behandlingsstart noterades en klar effekt av den givna behandlingen i form av förändringar i ämnesomsättningen som talade om en ökad celldöd, ökad toxicitet och cellsönderfall. Denna effekt var distinkt mer uttalad bland långtidsöverlevarna vilket kan tolkas som att dessa hade bättre effekt av den givna behandlingen. I dessa mönster kunde vi dessutom urskilja ett flertal ämnen som skulle kunna utgöra markörer för behandlingseffekt i framtiden. Exempelvis kan fosfat nämnas som var den mest framträdande av dessa metaboliter och som påtagligt ökade i både tumörvävnad och i serum hos de patienter som levde längre.

Sammanfattningsvis kan vi med denna avhandling dra slutsatserna:

• Att mikrodialys både är en användbar metod för att följa metabola och immunologiska processer i hjärnvävnad i realtid. • Att tumörceller föredrar glykolys för att förbränna glukos för sin energiframställning. • Att den stråldos som gavs i våra studier, under de första fem dagarna av patienternaas strålbehandling sannolikt inte var tillräckligt stor för att påverka sockermetabolismen i tumörcellerna eller ge utslag i form av ökade koncentrationer av cellskademarkörer. • Att den givna stråldosen var tillräcklig för att inducera en inflammatorisk respons i tumörvävnaden. • Att retrograd mikrodialys är en användbar metod för administrering av Cisplatin lokalt till maligna gliom. • Att lokal behandling med Cisplatin tydligt förändrar metabola mönster i tumörceller. Flera metaboliter som tex fosfat ökade särskilt i tumörceller hos patienter som levde längre. Det förändrade mönstret i ämnesomsättningen skulle kunna användas i framtiden för att utvärdera behandlingseffekter.

13 GLIOBLASTOMA MULTIFORME

BACKGROUND Glioblastoma multiforme (GBM), the most common malignant primary brain tumor, has an incidence of five cases per 100,000 and a peak incidence age of 50-60 years, although GBM may be diagnosed in all ages, from early childhood through old age. As GBM is invasive, it is impossible to eradicate with localized treatments such as surgery or radiotherapy. Moreover, these tumors reside within the brain protected by the blood brain barrier (BBB), so they are virtually inaccessible for systemic therapies. Finally, their genetic instability and cellular heterogeneity make them hard to target with one treatment modality. Most physicians agree that the best hope for long-term control lies within the development of novel adjuvant therapies. However, despite adjuvant treatment, including radiotherapy and chemotherapy, the median survival is no more than ten months in unselected populations in Northern Sweden (1).

Although the results from the treatment of GBM are discouraging, in the last several decades some progress has been made in the treatment of GBM. For example, in 2005, a trial conducted by the EORTC/National Cancer Institute of Canada Clinical Trials found that postoperative radiochemotherapy and adjuvant temozolomide produced promising results. In addition, during the last decade, neuronavigation and 5-aminolevulinic acid (5-ALA; Gliolan®) has been found to improve resection grade and surgical outcomes (1, 2).

DIAGNOSIS SYMPTOMS AND SIGNS The symptoms of intracranial tumors can roughly be divided in two categories: symptoms related to increased intracranial pressure and symptoms related to the local effect of the tumor on the surrounding tissue. Intracranial pressure often presents as headaches (predominantly in the mornings), nausea, vomiting, visual defect (often blurriness and/or diplopia), and influence on consciousness. The symptoms due to local effect of the tumor on its surrounding can both be diffuse or focal. The diffuse symptoms include mainly changes in personality and cognitive disfunction. The focal symptoms are, of course, related to the localization of the tumor and the specific function of the affected brain area, including paresis, sensory deficits, dysphasia or aphasia, visual symptoms, and convulsions, which is by far the most common debut symptom of intracranial tumors (3).

RADIOLOGY During the last two decades, magnetic resonance imaging (MRI) technology has evolved into the most powerful tool for brain tumor diagnosis, prognostication, treatment response evaluation, disease progression monitoring, and surgical strategy planning. Today, MRI studies not only provide high-resolution anatomical images but also functional quantitative information on tumoral pathophysiological characteristics and its effect on the brain. When visualized with MRI, GBM appears as ring-enhanced lesions on gadolinium enhanced T1 weighted sequences. This enhancement is a sign of the BBB disruption; in many cases, this disruption is typical although not specific and definitive enough for the diagnosis. Other MRI methodologies of clinical importance are diffusion and perfusion MRI. Diffusion imaging represents the thermally induced random molecular motions of water molecules in tissue, and perfusion imaging is used to describe and quantify microvascular blood flow feeding a particular area. Both these imaging modalities give important information about the tumor, such as edema development, invasiveness, and progress due to neovascularization (4). In spite of the usefulness of MRI and the development of the technique, diagnosis can only be made after biopsy or resection and pathologic confirmation based on the current World Health Organization (WHO) classification.

PATHOLOGY Today, glioma classification is mainly based on the 2016 WHO grading scale, which is based both on histological and molecular findings. The grading scale uses an integrated phenotypic and genotypic approach to increase the objectivity in the diagnostic process (5). The neuropathological diagnosis should be based on morphological type and malignancy grading, in addition to diagnostic, prognostic and predictive biomarkers analyzed with immunohistochemistry or/and molecular genetical analysis (3).

The histological analysis is based on haematoxylin and eosin staining of tumor tissue classifying gliomas with regards to cytological features and degree of malignancy. This classification is based on microscopic findings of cell atypia, morphology of the cell nuclei, cell density, amount of mitosis, vascular proliferation and tumor necrosis. The histological analysis enables the identification of the group of tumors called diffuse gliomas, which is divided further on the basis of their molecular genetic characteristics in order to make the diagnosis more reliable and objective and therefore clinically more useful. Genetic subgrouping of gliomas creates a more accurate and reproducible diagnostic criteria (3, 5).

In the 2016 WHO classification diffuse gliomas include the WHO grade II and grade III astrocytic tumors, the grade II and grade III oligodendrogliomas, the grade IV glioblastomas and also other diffuse gliomas of childhood. The WHO grade II diffuse astrocytoma and WHO grade III anaplastic astrocytoma are divided into IDH-mutant, IDH wild type and NOS (no otherwise specified) categories, where the great majority falls into the IDH-mutant category. The diagnosis of oligodendroglioma and anaplastic oligodendroglioma requires both the findings of IDH-mutation and the combined whole-arm losses of 1p and 19 q. Glioblastomas are divided into glioblastoma, IDH-wildtype (90%) and glioblastoma IDH-mutant (10%). The glioblastoma IDH-wildtype corresponds often with the clinically defined de novo glioblastoma an predominates in older patients. Glioblastoma IDH-mutant on the other hand often corresponds with malignified diffuse gliomas of lower grade and usually arise in younger patients (5).

Diffuse growing gliomas

Histology Astrocytoma (Oligoastrocytoma) Oligodendroglioma Glioblastoma

IDH status IDH-mutant IDH-wildtype ? IDH-mutant IDH-wildtype

1p/19q status yes no

Genetic testing not done or inconclusive Oligodendroglioma (IDH mutant and 1p/19q co-deleted)

Diffuse astrocytoma, IDH mutant Diffuse astrocytoma, NOS Oligodendroglioma, NOS After exclusion of entities: Diffuse astrocytoma, IDH wild-type Oligoastrocytoma, NOS Oligodendroglioma, NOS Glioblastoma, NOS

160212

Figure 1. An algorithm of the classification of diffuse growing gliomas using an integrated phenotypic and genotypic approach. Based on the 2016 WHO classification.

MOLECULAR GENETIC MARKERS

MGMT 06-Methylguanine-DNA-methyltransferase (MGMT) can repair 06-alkylguanine lesions. Methylation of the promotor to the gene coding for MGMT silences the expression of the enzyme. GBM with a methylated MGMT-promotor has better prognosis and responds more favorably to alkylating chemotherapy compared to tumors without this methylation (6). MGMT methylation status is the first clinically useful predictive prognostic factor for GBM (3).

IDH MUTATIONS IDH, an enzyme used in the tricarboxylic cycle (TCA cycle), mainly functions to catalyze the oxidative decarboxylation of isocitrate into α-ketoglutarate. Mutations of the gene coding for the enzyme isocitrate dehydrogenase (IDH)-1 and -2 are common in grade II and III gliomas. This mutation, often found in a GBM developed from a low-grade glioma (secondary GBM), is associated with better outcome, but does not influence the selection of treatment (3, 5).

16 CO-DELETION OF CHROMOSOME 1p AND 19q This double co-deletion is strongly correlated with the diagnosis oligodendrogliomas and anaplastic oligodendrogliomas, especially together with IDH-1 or -2 mutations. The presence of this deletion is important for the choice of chemotherapy for grade II and III tumors, as these tumors have higher sensitivity to the combination therapy with procarbazin, CCNU (lomustin), and vinkristin (PCV). Furthermore, tumors with chromosome 1p/19q deletion are associated with improved chances for favorable effect of chemotherapy and better prognosis of the disease. The double co-deletion of course also excludes the diagnosis GBM (3, 5).

TREATMENT OF GLIOBLASTOMA MULTIFORME SURGERY Surgery, often the first line of treatment, aims to debulk the tumor and to obtain tissue for diagnosis. Grade of resection influences prognosis and the therapeutic value of the surgery. The goal should be total or subtotal resection (>95%) without compromising neurological function (7, 8). However, a meaningful difference for the patient is achieved at 70-80% resection grade (7, 9). During the last two decades, several technological advances such as the implementation of neuronavigation and the introduction of intraoperative 5-ALA fluorescent microscope (10) have improved surgical results regarding both the rate of complications and grade of resection. Higher resection grade, ideally over 85%, is associated with improved survival (11-13). After surgery, there should be a healing period of three to four weeks before postoperative radiochemotherapy. This period is also important in order for the postoperative edema and eventual brain shift to recede so as to improve target definition for radiotherapy.

RADIOCHEMOTHERAPY AND ADJUVANT CHEMOTHERAPY Postoperative radiotherapy is usually delivered using high-energetic photons from a standard linear accelerator. Gross tumor volume (GTV) is defined as the contrast enhanced volume on T1 weighted MRI. As GBMs often recur locally, the clinical target volume (CTV) is defined as the GTV with a 2-cm margin. The final planning target volume (PTV) to which the prescribed dose is delivered is the CTV with a 3-5-mm setup margin for positioning errors (14, 15). Fractionated radiotherapy, 60 Gy given in 30 fractions, is the standard treatment after resection or biopsy. The doses are given five days a week, Monday-Friday, for a total radiotherapy period of six weeks. As an alternative to fractionated radiotherapy, hypofractionated therapy can be used with higher doses per fraction but fewer fractions. This alternative is especially favorable for treating individuals > 70 years old with newly diagnosed GBM (16).

During radiotherapy, the patient receives daily concomitant treatment with the alkylating chemotherapeutic agent temozolomide (75 mg/m2). Temozolomide alkylates the DNA of dividing cells, inhibiting DNA repair mechanisms. Readily able to pass the BBB, temozolomide can be targeted in high concentrations in the target tumor tissue (2). Starting one month after completion of radiochemotherapy, adjuvant temozolomide is administered 150-200 mg/m2 day 1-5 in six 28 day cycles (2, 8).

RESPONSE EVALUATION AND FOLLOW-UP MRI is the standard method for radiological response evaluation. As a result of improved understanding of the tumor biology of gliomas and the increased importance of residual non- enhancing tumor, glioma treatment response assessment has undergone numerous revisions

17 over the last two decades. Today, MRI is the preferred radiological method for evaluation of radiological response in glial tumors. The most widely used system to assess first-line treatment combining radiological and clinical factors is the Response Assessment in Neuro-Oncology Criteria (RANO criteria). RANO criteria categorizes the response into four responses based on MRI imaging and clinical features (use of corticosteroids and neurological function): complete response, partial response, stable disease, and progression (4, 17).

Criteria Complete Partial response Stable disease Progress response (>12 weeks after completed radiochemotherapy) T1-contrast None >50% decrease 25-50% decrease >25% increase enhancement (during at least 4 w) (during at least 4 w) T2 FLAIR Stable or decreased Stable or decreased Stable or decreased Increased

New lesion No No No Yes

Corticosteroid No Stable or decreased Stable or decreased Not relevant treatment Clinical status Stable or improved Stable or improved Stable or improved Deterioration

Response/progress All criteria All criteria All criteria At least one of the requirement above

Table 1. A summary of the RANO-criteria for assessment of high-grade glioma (3).

Assessment is made within 4–8 weeks after completion of radiochemotherapy. Since it can be difficult to differentiate progression from changes due to the treatment itself (pseudoprogression), re-evaluation with a new MRI is done four weeks after the first assessment (4). This delay makes radiological assessment less efficient for assessing the effect of treatment in the clinical setting as in many cases it may be too late to make any changes in the therapy when the treatment failure is evident. A therapeutic marker would have been ideal to identify treatment effect and eventual failures earlier in the process. However, no such reliable markers are available.

SECOND-LINE TREATMENT Before pursuing a second-line treatment, it is important to consider patient and tumor characteristics. Prognostic factors correlated with better outcome after second line treatment include good clinical performance, younger age and intact neurological function. While the goal of the treatment is longer overall survival, a preserved or even improved quality of life of the patients must be prioritized. In case of recurrence, re-operation can be an option if a total or close to total removal is possible. In addition to surgery, other treatment options such as second- line systemic chemotherapy, re-irradiation, or combined modality therapy can be considered (18, 19).

Several options for chemotherapy are available for second-line treatment, but no standard of treatment has been established. The different options include mono or combination therapy involving temozolomide, bevacizumab, or nitrosourea alkylating agents such as lomustine. Bevacizumab, a monoclonal antibody, binds to circulating VEGF-A and inhibits its biological activity, reducing endothelial proliferation and vascular growth within the tumor. Nitrosourea

18 are highly lipophilic molecules with DNA alkylating properties. As these molecules easily pass through the BBB, they are useful in the treatment of brain tumors (18, 19).

Second-line treatment may combine chemotherapy with re-irradiation. As this combined approach is likely to increase side effects, especially if radiosensitizing drugs are used, the approach is highly restricted and controversial. However, in carefully selected cases, this approach has been reported to be safe and effective (20). For example, bevacizumab, when combined with other treatments, can be effective as these antibodies block hypoxia factor mediated angiogenesis, which is upregulated by radiotherapy. In addition, bevacizumab, when used to treat radionecrosis, may reduce the risks associated with re-irradiation (18, 19).

PROGNOSTIC FACTORS Patients with GBM, according to the literature, have a median overall survival varying from 10 to 16 months and a median progression free survival of 6-12 months, a variation probably reflecting a selection bias in the studied populations. Although median survival is dire, the individual survival also varies considerably. Factors that consistently have shown to be correlated with longer survival are total or subtotal tumor resection (more than 95%), younger age (below 50), absence of inflammatory disease, absence of diabetes or metabolic disease, IDH-1 mutation, MGMT methylation, good WHO performance status and intact neurological function, and completion of postoperative chemoradiotherapy (1, 6, 8, 21-23).

19 LOCAL DELIVERY SYSTEMS FOR TREATMENT OF BRAIN TUMORS

BLOOD BRAIN BARRIER (BBB) Only a few effective methods are available for delivering drugs to the brain. The BBB and the blood-cerebrospinal fluid barrier surround most of the CNS, effectively isolating the brain. The BBB is composed of tight junctions between capillary endothelial cells in the CNS blood vessels. These tight junctions and low endothelial endocytic activity restrict transport of molecules across the BBB. The BBB prevents hydrophilic molecules and larger molecules (>400Da) from entering the CNS (24). This restriction, of course, complicates chemotherapeutic treatments as most of chemotherapeutics use larger molecules. Furthermore, the BBB is mainly intact at the tumor periphery in primary brain tumors. Therefore, conventional delivery methods of systemic treatments such as oral ingestion or intravenous injections are often ineffective treatments for brain tumors (25-28).

A successful delivery system should deliver a significant amount of the active agent to the tumor without any systemic toxicity. During the last decade, several strategies have been used to deliver active substances to the brain. Today, there are basically six approaches for delivering drugs to the CNS (25, 27):

I. Enhancing drug permeability through the BBB II. Temporarily disruption of BBB III. Interstitial delivery of drugs via catheters IV. Convection-enhanced delivery of drugs to the CNS V. Direct delivery with polymers VI. Drug impregnated microchips

I. ENHANCING DRUG PERMEABILITY Enhancing drug permeability involves chemically and/or structurally altering the drug to make it more lipophilic and therefore more easily able to pass through the lipid layers of the BBB. Drug permeability can be enhanced using liposomal drug delivery. Liposomal drug delivery encapsulates a drug in a sphere of lipids that carry the drug across lipid membranes, including the BBB. Similarly, carrier-mediated and receptor-mediated transport can be used to move a drug across the BBB (27).

20 II. BBB DISRUPTION Strategies that directly disrupt the BBB by opening endothelial intercellular gaps include hyperosmolar solutions based on molecules such as mannitol and bradykinin agonists such as RMP-7. Although these strategies are theoretically effective and experimental studies have showed increased penetration rates of drugs into the targeted tissue, these strategies have not been translated into better clinical outcome (27).

III. INTERSTITIAL DELIVERY OF DRUGS, CATHETER INJECTION OR INFUSION Lesniak et al. (2004) has reviewed several strategies for delivering interstitial drugs to brain tumors (27), including injections or infusions using implanted catheters. Injections or infusions of chemotherapeutics using repeated injections or implanted catheters bypass the BBB by delivering the active agent directly to the tumor site and/or ventricles. These methods distribute chemotherapeutics using the concentration gradient and the permeability of the tissue (25, 27).

Interstitial delivery with injections or catheters is a simple and repeatable method that can deliver large volumes of chemotherapeutics at a specific site. However, repeated injections can damage the local tissue at the injection site, which can result in complications such as infections and hemorrhages. However, the most striking limitation with this method is the poor drug distribution into the tissue. The drugs distribute only a few millimeters into the tissue from the injection site, often resulting in a very high concentration at the injection site and low concentrations farther from the injection site. In addition, this method’s bolus-based nature is a limitation (25). Interstitial delivery with injections or catheters has been used in several studies to deliver both chemotherapeutics and biological agents. Although simple, this method has not produced any meaningful effect on patient outcomes (25).

IV. CONVECTION-ENHANCED DELIVERY OF DRUGS TO THE CNS In convection-enhanced delivery (CED), a pressure gradient is used to deliver a high concentration of drugs to a large region of the brain without causing functional or structural damage. As with direct injections, CED depends on concentration gradients; however, CED also uses an external device (pump) to create a pressure gradient. This created pressure gradient increases the distribution of the delivered drug beyond the injection site. Compared to direct injections, CED drug distribution is often ten times greater, and a spherical distribution of 2-3 cm can often be achieved with a linear relationship between the volume infused and the volume distributed (29). Furthermore, this method is especially useful for delivering drugs to sensitive areas in the brain such as the brainstem (25, 27).

This technique has several limitations: the reservoir of the system must be refilled continuously, and drug distribution is affected by molecular size, surface characteristics, and half-life of the delivered molecule. Infusion characteristics of the CED device and catheter dimensions also need to be considered. As with all technical equipment, malfunction can be a source of error. In addition, CED technology requires proper location and monitoring for backflow and clogging of catheters. Overall, CED seems to be a better alternative than direct injections for local drug delivery (25). However, no studies have shown that CED is more beneficial than current treatment modalities (30).

21 V. DIRECT DELIVERY WITH POLYMERS Interstitial drug delivery can also be accomplished using drug-impregnated polymers: a matrix of hydrophobic polymers that slowly degrade to release a drug at a constant rate. The chemical composition of the polymer determines the rate of the drug release, ranging from days to years. In addition, the polymer’s shape can be engineered to fit a specific purpose as the polymer is totally biodegradable. Moreover, the breakdown products of the polymer are non-mutagenic, non-cytotoxic, and non-teratogenic (25, 27, 31).

Today, Gliadel®, a poly-carboxyphenoxypropane-co-sebacic acid (PCPP-SA) polymer with the chemotherapeutic carmustine (BCNU) incorporated within it, is the only local drug delivery technique approved by FDA. Gliadel® shows improved survival in randomized control trails for both newly diagnosed and recurrent malignant gliomas (32-34). Carmustine is a low- molecular mass alkylating agent that has potential efficacy against brain tumors. The molecule is lipophilic and has a relatively short half-life. Furthermore, when administrated intravenously, carmustine shows severe toxicity such as myelosuppression and pulmonary fibrosis (25, 27, 35).

Compared to CED, polymer technology has several advantages. Polymer technology does not depend on catheters or external devices, so the technology is not limited by the issues and sources of error associated with catheters or external devices. Polymers are instead placed at the time of surgery so that they cover the borders of the resection cavity. In addition, polymers enable a sustained continuous release of the drug. The technique, however, has some limitations, primarily involving dosing. To deliver a significant dose of carmustine, up to eight wafers must be used within the resection cavity, a requirement not always possible in the eloquent cortex or when only needle biopsies have been performed. In addition, wafers are only effective as a local therapy as they cannot be placed beyond the resection cavity. Implanted wafers are also contraindicated if the ventricles have been opened during surgery. In addition, implanted wafers present issues regarding the timing of the release of the chemotherapeutics as the half-life of the drug and the pace of degradation of the polymer need to be synced. Finally, Gliadel® has been correlated with higher risks for infection, edema, and seizures (25, 31, 32).

VI. DRUG-IMPREGNATED MICROCHIP DELIVERY Drug-impregnated microchips can be seen as an extension of the polymer technology. As noted above, polymer technology cannot control the release of drugs in a time-dependent fashion, an issue that drug-impregnated microchips resolve (36, 37). To ensure a specific time release of drugs, drug-impregnated microchips use valves, channels, and micrometer pumps. The actual release of single or multiple agents is controlled by biodegradation or electrochemical dissolution.

Compared to the polymer technique, drug-impregnated microchips seem to have some clear advantages. However, the technique also has some disadvantages such as the need to refill the device and the potential for technical malfunctions. In summary, more work needs to be done to ensure efficacy and safety of microchips before they can be used routinely for local administration of chemotherapeutics in the human brain (25, 27, 36).

THE MICROENVIRONMENT OF GLIOBLASTOMA MULTIFORME

Cancer is often regarded as a chaotic aggregation of uncontrolled cells dividing without internal cell cycle control. This view, however, is over-simplified as cancer cells interact with their environment in complex ways. As described below, the microenvironment of GBM is highly specific and poorly accessible for systemic drug delivery. There are several obstacles making these tumors hard to treat using conventional methods. With this as a background, a logical strategy would be not only to target the actual cancer cells but also to modulate the tumor microenvironment with the objective to break down the different barriers around the tumor and enhance death of cancerous cells.

SUPPORTING TUMOR MICROENVIRONMENT In spite of the highly aggressive nature of GBM, these tumors rarely metastasize outside the CNS. Local recurrences are common after standard treatment within a 2.5-cm margin of the initial resection cavity, suggesting that this tumor mainly spreads locally at the site of origin. This limited spread to the original site of origin has several possible explanations. The inability of GBM to colonize extracranial tissue could be due to low concentrations of infiltrated cells in the periphery of the tumor, the tumor’s exclusive homing properties, and a systemic immunity outside the CNS. Furthermore, within the CNS, GBMs seem to prefer following axonal tracks rather than perivascular spaces. Again, this suggests a well-developed interaction between the tumor and its microenvironment rather than random growth (38).

Within the frame of the extracellular matrix (ECM), the fluids and macromolecules are the main composition of the noncellular substrates. Malignant glioma cells, residing and infiltrating immune cells, vascular endothelial cells and pericytes are the cellular composition in the tumor microenvironment. The relative composition between the tumor cells and substrates within the ECM defines different niches and displays heterogeneity within the same tumor. Different parts of the tumor are dynamically reshaped by these different niches, forming a variety of microenvironments within the same tumor, varying from solid parts with densely packed viable cells to necrotic and peri-necrotic areas, perivascular areas with endothelial proliferation and hypoxic and peri-hypoxic areas. In spite of their differences, all these niches contribute to supporting the growing tumor (38).

23 HETEROGENEITY One characteristic of GBMs is their heterogeneity, which can be divided into inter-tumor and intra-tumor heterogenicity. That is, the tumors within the same diagnosis differ and the tumor cells within a given tumor are composed of different clones (pleomorphism). The heterogeneity of GBM helps explain why these tumors are extremely difficult to treat (39, 40).

Three molecular subtypes of GBM have been characterized based on gene cluster analysis of abnormalities in the genes: isocitrate dehydrogenase 1 (IDH1); neurofibroma 1 (NF1); epidermal growth factor receptor (EGFR); and platelet-derived growth factor receptor alpha (PDGFRA). These subtypes are: proneural, classical, and mesenchymal (41). Additionally, six epigenic GBM subtypes of GBM can be identified using global DNA methylation profiling techniques. Moreover, more than one molecular subtype can be found within the same tumor depending on the tumor quadrant investigated. This heterogeneity makes personalizing specific treatments for a target extremely difficult and only partially effective (39).

THE IMMUNE MICROENVIRONMENT OF GBM The paradigm that the CNS have been considered as an immunologically privileged site is based on three assumptions. Firstly, the BBB’s tight junctions prevents lymphocytes from entering the CNS, secondly the lack of a lymphatic system within the CNS and finally the rarity of resident antigen-presenting cells. However, these assumptions have showed to be wrong. Inflammation and pathological conditions break down the BBB, allowing free inflow of lymphocytes into the CNS. Lymphatics have been identified in the wall of major dural sinuses, providing evidence for a connection between CNS and the systemic immune system. Finally, the role of microglia as antigen-presenting cells within the CNS has been confirmed (42). The only conclusion that is reasonable today is the fact that the CNS is not an immune privileged site, but rather a different immunologically distinct environment. The quality and quantity of the immune reaction within the CNS is highly contextual.

A great part of the viable cells within GBM are immunocompetent cells. Several studies suggest that up to 30-40% of the cells consist of monocytes/microglia, infiltrating macrophages, and, to a lesser degree, infiltrating lymphocytes. Previously, it was believed that this infiltration was proof of an immuno-activation against the tumor and therefore an advantage for the patient. However, increasing evidence suggests that these cells are under the influence of tumor cells, modulated to enhance tumor growth, immunosuppression, and tumor proliferation. GBMs are profoundly immunosuppressive both locally (within the tumor) and systemically expressing multiple immunosuppressive mediators such as transforming growth factor-ß (TGF-ß), Prostaglandin E2 (PGE2), and B7-homolog 1(B7-H1), which in turn may reflect underlying oncogenic molecular abnormalities such as mutation/deletion of phosphatase and tensin homolog (PTEN) or aberrant signal transducer and activator of transcription 3 (STAT-3) activation. GBM cells networking with the infiltrating monocytes/macrophages, circulating myeloid-derived suppressor cells, and regulatory T cells contribute significantly to both local and systemic immunosuppression in glioblastoma patients (42-44).

24 T-cell GBM apoptosis cells A

Glioma infiltrating B Activated monocytes T-cells

D Naïve T-cells Myeloid-derived supresssor cells

Regulatory T-cells

Figure 2. Schematic presentation of potential model for GBM-mediated systemic immunosuppression: (A) Inhibition of T-cell activation through decreased proliferation and apoptosis induced by the action of GBM expressed immunosuppressive molecules (TGF-_2, PGE2, B7-H1). GBM cells also secrete multiple factors that may serve to attract monocytes/microglia (IL-6, CSF-1). (B) GBMs are highly infiltrated with monocytes expressing immunosuppressive molecules (IL-8 and FasL). (C) Immature myeloid cells called Myeloid-derived suppressor cells (MDSC) are increased in GBM patient’s serum. These cells induce apoptosis in activated T-cells and stimulate proliferation of regulatory T-cells (T-reg). MDSC cell are believed to originate from glioma infiltrated monocytes. (D) Increased levels of T-reg in glioblastoma patients’ circulation reduce T-cell proliferation and activation. T-reg cell levels in glioblastoma patients might reflect the increased activity of circulating MDSC.

The local immunomodulating effect of malignant glioma cells within a tumor is mediated by several modulatory factors as discussed above. Locally, these factors shift the immunity within the tumor from a cell-mediated immunity to humoral (type 2 helper cell) responses that are less effective against solid tumors. Here, the attracted monocytic cells (monocytes, macrophages, and microglia) play a crucial role. This interplay results in a vicious cycle where tumor cells actively suppress and modulate the immune response within the microenvironment of the tumor, resulting in attraction and stimulation of cells, which in return suppresses a proper immune response and further stimulates proliferation of tumor cells. Disrupting the interplay between cancerous cells and surrounding immunocompetent cells should boost immune response both systemically and locally, creating a more favorable microenvironment for immunotherapy (42-45).

Today, it is generally accepted that immune reactions within the CNS are possible and that gliomas express tumor-specific antigens either by themselves or through interactions with neighboring microglia. These facts ensure access to both Class I and Class II MHC restricted

25 antigen presentation and their immune activating pathways. Moreover, glioma cells appear to be sensitive to Fas-mediated apoptosis as they express the Fas protein on their cell membrane. This expression is the principal pathway for cytotoxic T-cell mediated immunity, which is the most effective immunological response to solid tumors. All these factors suggest that gliomas should be able to activate a proper cell-mediated immune response with potent antitumoral effect. However, glioma cells produce and secrete several immunosuppressive factors such as TGF-ß32, prostaglandin E2, and possibly Fas-L. These factors effectively inhibit lymphocyte activation. Moreover, immune suppressive cytokines such as IL-6 and IL-10 secreted both from glioma cells and from other immunocompetent cells within the microenvironment of gliomas modulate and shift the immunity to less effective humoral responses. Finally, several glioma- derived cytokines and chemokines such as IL-6, IL-8, and colony stimulation factors appear not only to suppress the immunological antitumoral response, but also to have a tumor proliferative role in the microenvironment of the tumor, promoting angiogenesis, growth, and invasiveness (43, 44).

TH activation Antigen presented with MHC class II B7/Cd28 binding

TH TH

APC Signal 2 Signal 2 Microglia IL-2 + IL-2 + Macrophage + IFN IL-6 IL-4 IL-12 IL-10 + + TK Microglia B Macrophage + Lymphocyte + Chemoattractant Humoral immunity Signal 1 Cell mediated immunity Antigen presented with MHC class I Apoptosis via FAS/FAS-L mediated apoptosis B7/CD28 binding + - IL-6 TNF-b2 Hypoxia GLIOMA Prostaglandin E2 IL-8 Tumor specific FAS-L antigen Colony stimulating factor

Growth factors Tumor cell proliferation Angiogenetic factors Angiogenesis Proteases Invasion/metastasizing

Figure 3. Schematic presentation of the immune microenvironment of malignant glioma. Several immunosuppressive factors produced and secreted by glioma cells–e.g., TGF-ß32, prostaglandin E2, and possibly Fas-L–inhibit lymphocyte activation. Immune suppressive cytokines secreted from glioma cells and from other immunocompetent cells–e.g., IL-6 and IL-10–modulate and shift the immunity to less effective humoral responses. Several glioma-derived cytokines and chemokines–e.g., IL-6, IL-8, and colony stimulation factors–appear to not only suppress the immunological antitumoral response, but also to play a tumor proliferative role promoting angiogenesis, growth, and invasiveness

26 THE METABOLISM OF GLIOBLASTOMA MULTIFORME Metabolism of normal cells is reprogrammed during the malign transformations of normal cells. This metabolic reprogramming in malignant cells is likely the result of the multifactorial effects of genomic alterations (e.g., mutations of oncogenes and tumor suppressors), the tumor microenvironment (which imposes metabolic stress caused by compromised nutrients and oxygen availability), and other influences. To develop new strategies and find new therapeutic targets, it is essential to understand the metabolic changes that cells undergo during malignification and the causes of these changes. This section discusses the metabolism of normal cells. In addition, this section describes some important metabolic modifications in cancer cells and their potential as potential targets for therapy. Finally, this section presents an overview of the metabolic changes within GBM cells.

METABOLISM OF NORMAL CELLS The cellular content is duplicated as the cells undergo mitosis. This process, of course, requires both energy and biosynthetic intermediates (nucleotides, amino acids, and lipids). There are several metabolic pathways within the cell for this purpose (Figure 4). The glycolytic pathway describes the metabolism of glucose, the main source of cellular energy and provides macromolecular precursors such as acetyl-CoA for fatty acids, glycolytic intermediates for non- essential amino acids, and ribose for nucleotides to generate biomass. The pentose phosphate pathway generates NADPH and pentose sugars from glucose-6-phosphate (G6P). The tricarboxylic acid cycle (TCA cycle) pathway releases stored energy through acetyl-CoA derived from sugars, proteins, and fats and plays a role in lipid synthesis. Another pathway worth mentioning is glutaminolysis. After glucose, glutamine is the second most catabolized molecule, supplying the cell with free energy, carbon, nitrogen, and reducing equivalents necessary for cell growth and division. These four metabolic pathways will be discussed in more detail below (46, 47).

Glucose Cell membrane

Glucose

2 NADH

2 ATP + 6 NADH Glycolysis NUCLEOTIDE SYNTHESIS

Lactate Pyruvate Alanine

Malate Pyruvate Acetyl CoA O Oxaloacetate X P 36 ATP LIPID H Malate TCA CYCLE Citrate SYNTHESIS O S ⍺ -Ketoglutarate 4 NADH

NADH GLUTAMINOLYSIS

Figure 4. A schematic overview of the main processes of the metabolism of normal cells.

27 GLYCOLYTIC PATHWAY Glucose enter the cells via glucose transporters (GLUT) and enters the glycolysis where the molecule is metabolized to pyruvate through four enzymatic catalyzed steps (Figure 5). In normal cells, most of the pyruvate is directed into the mitochondria where pyruvate hydrogenase converts the pyruvate into acetyl-CoA or is transaminated to form alanine. The glycolysis results in two ATP and six NADH molecules per metabolized glucose molecule (46, 47).

Glucose

GLUT 1

Glucose HK 2 G Glucose-6-P L Y Fructose-6-P 2ATP + PFK 1 6 NADPH C Fructose-1,6-P O L PGM Y 2-phoshpoglycerate S GLUT 1 = Glucose transporter 1 HK 2 = Hexokinas 2 I 2-phosphoenolpyruvate PGI = Phosphoglucose isomerase PFK 1 = Phosphofructokinas 1 S PKM 1 PGM = Phosphoglycerate mutase Pyruvate PKM 1 =Pyruvate kinas isomer 1

Figure 5. A schematic overview of the glycolytic pathway in normal cells.

PENTOSE PHOSPHATE PATHWAYS This pathway is governed by G6P dehydrogenase, an enzyme regulated by its substrate and the NADP/NADPH ratio within the cell. G6P is metabolized to ribose-5 phosphate and two NADPH molecules (Figure 6). The ribose-5-phosphate is metabolized further for the purpose of nucleotide synthesis. NADPH has a central role in many of the different pathways within the cell. NADPH, an important antioxidant, maintains glutathione in a reduced state to prevent oxidative damage. In addition, as a cofactor, NADPH has a reductive role in the biosynthesis of many molecules within the cell such as fatty acids, nucleotides, and amino acids and plays its most important role in oxidative phosphorylation (OXPHOS) within the inner mitochondrial membrane (47).

Pentose Phosphate Pathway G6PD

Nucleotide Glucose-6-P Ribose-5-P synthesis

2 NADPH

G6PD = Glucose-6-phosphate dehydrogenase

Figure 6. A schematic overview of the pentose phosphate pathway in normal cells.

28 TCA CYCLE Most pyruvate that enters the mitochondria is converted into acetyl-CoA where TCA cycle releases its stored energy via oxidation. Within the TCA cycle, citrate is consumed and regenerated. The cycle consumes acetate (in the form of acetyl-CoA, which is metabolized through several steps of intermediate metabolites) and water and reduces NAD+ to NADH, releasing carbon dioxide (Figure 7). The NADH generated by the citric acid cycle is fed into the OXPHOS (electron transport chain) pathway, where enzymes oxidize nutrients, releasing the chemical energy of molecular oxygen. This energy is used to form ATP. The electron transport chain uses one glucose molecule to produce 36 ATPs. The net result of these two closely linked pathways is the oxidation of nutrients to produce usable chemical energy (47).

The TCA cycle plays also an important role in the fatty acid synthesis. The majority of the carbon used in fatty acid synthesis is derived from intramitochondrial acetyl-CoA. However, acetyl-CoA cannot pass through the mitochondrial membrane, so it needs to be converted into citrate and exported out to the cytoplasm. Here, citrate is converted back to acetyl-CoA used in a chain of enzymatic reactions for formation and elongation of fatty acids. The cytoplasmic citrate might also be converted to isocitrate and then to α-ketoglutarate (α-KG), generating another molecule of NADPH by the action of isocitrate dehydrogenase 1 (IDH1). Within the mitochondria a similar reaction is governed by IDH 2. These reactions are non-reversible steps and has to be carefully controlled (46, 47).

There are several other important metabolic events that involve the intermediate metabolites of the TCA cycle. One of these metabolites is oxaloacetate, which is used for the synthesis of non- essential amino acids. In addition, malate is produced in the TCA cycle before exiting the mitochondria and converted into pyruvate and NADPH. Also, acetyl-CoA (both cytosolic and nuclear) is a precursor of gene regulatory protein (e.g., histones) produced by acetylation (46, 47).

PDH = Pyruvate dehydrogenase IDH = Isocitrate dehydrogenase FAS = Fatty acid synthesize ACL = ATP citrate lyase Tricarboxylic acid cycle

AlanineGlycolysis

Lactate Pyruvate ⍺-ketoglutarate PDH

NADPH IDH 1 NADPH Malate Pyruvate Acetyl CoA Isocitrate Oxaloacetate 3 NADPH Malate IDH 2 Citrate Citrate

Acetyl-CoA ⍺-ketoglutarate ACL FAS Lipid GLUTAMINOLYSIS synthesis Glutamate Glutamine

Figure 7. A schematic overview of the TCA cycle and lipid synthesis in normal cells.

29 GLUTAMINOLYSIS Glutamine, as mentioned above, is an important metabolite for generation of energy and as a source for carbon and nitrogen. Glutamine also plays an essential role for the control of redox potentials through the synthesis of NADPH. Glutamine, via the intermediate glutamate, is converted to ⍺-ketoglutarate that enters the TCA cycle (Figure 8). Glutamate can also be converted to aspartate, a metabolite contributing to nucleotide synthesis. Glutaminolysis also results in alanine and ammonium production and their secretion from the cell (46, 47).

GLUTAMINOLYSIS GLS

Glutamine Glutamate ⍺-ketoglutarate

NADPH

TCA CYCLE

GLS = Glutaminase 1

Figure 8. A schematic overview of glutaminolysis in normal cells.

METABOLISM OF CANCEROUS CELLS To proliferate and grow, cancer cells need to deal with three metabolic challenges: producing energy and biosynthetic intermediates for growth; surviving in spite of nutrient and oxygen limitations; and maintaining a redox hemostasis in the face of high metabolic rates. Several mechanisms can explain the differences in metabolic pathways within cancerous cells.

MECHANISMS OF METABOLIC CHANGE IN CANCEROUS CELLS Metabolic change in cancerous cells can be the result of mutations and genomic alterations in the gens of metabolic enzymes that result in the accumulation of metabolites that directly contribute to more mutations and finally to malignant transformation. These metabolites, generated either by gain of neomorphic enzyme activity or loss of enzyme activity, are commonly referred to as oncometabolites. For example, D-2-hydroxyglutarate (D-2-HG) is an oncometabolite generated by increased neomorphic activity. Mutations in the IDH 1 gene results in an elevated enzymatic activity in NADPH-dependent generation of excess D-2-HG from α-KG. This enzymatic regulation is equivalent to a molecular switch that pushes metabolic processes towards more anabolic processes favoring cellular invasiveness and growth, resulting in elevated levels of cytosine and histone methylation, altering the epigenetic landscape within the cell. These enzymatic alterations lead to impaired cellular differentiation, invasion, and angiogenesis. However, the mutated form of IDH also results in lower NADPH levels and may sensitize tumors to irradiation and chemotherapy (46, 47).

30 In addition to the accumulation of metabolites, metabolic change in cancerous cells can be the result of mechanisms that reprogram cellular metabolism induced by the cancer genotype. Genetic damage or mutations can affect several metabolic pathways through post-translational protein modifications, signaling or activating transcriptional networks. Genetic alterations orchestrate a modified signaling to modulate metabolic pathways in cancerous cells. Metabolic pathways are often activated in cancer due to mutations/amplifications of key regulatory subunits and/or deletion of tumor suppressors. For example, the PTEN–the key negative regulator of the phosphatidylinositol-3-kinas (PI3K) pathway and the most frequently deleted tumor suppressor gene (chromosome 10)–is linked to the development of several tumors, including GBM. Several molecules in this pathway (e.g., EGFR, PTEN, and PI3KCA) alter GBM by inducing cell proliferation and blocking apoptosis. Furthermore, PI3K seems to affect glucose metabolism by favoring aerobic glycolysis by stimulating transcription of the glucose transporter GLUT 1 through protein kinas b (AKT), activating and promoting the activity of two important glycolysis enzymes–HK2 and PFK1 (46-48).

Cancerous cells can also undergo a metabolic re-wiring and dysregulation associated with tumor progression or aggressiveness. As the tumor progresses and infiltrates its surroundings either by metastasis or growth, the changed metabolic requirements in the cells alters metabolic pathways. Again, IDH mutations is a good example of this. As the tumor progresses, there is an increased oxidative stress within the cell. Elevated mitochondrial ROS levels require transfer of NADPH from the pentose phosphate pathways in the cytosol to the mitochondria. The mechanism of transfer involves the NADPH-dependent reductive carboxylation of α-KG by IDH1, followed by entry of the resulting isocitrate/citrate into the mitochondria where it is used in IDH2-depenent NADPH production. Basically, the metabolism re-wires and adapts in the shadow of the metabolic changes due to genetic alteration and metabolic needs due to tumor progression (46, 47, 49).

Finally, the metabolic changes in non-cancerous cells in the microenvironment of a tumor need to be considered. We have earlier discussed the importance of the interplay between the different cells in the microenvironment of a tumor. Of course, the metabolic changes in tumors affect this interplay. For example, tumor-promoting tumor associated macrophages (TAM) elevate OXPHOS. These cells have an increased reliance on glutamine metabolism and fatty acid oxidation, whereas tumor-inhibiting TAMs elevate glycolysis and pentose phosphate pathway activity (46, 50). More and more evidence suggests that both the environment and the oncogenic driver influence the metabolic phenotype of cancerous cells (49).

METABOLIC CHANGES IN GBM

ENERGY METABOLISM OF GBM The increase demand for energy requires increased use of glucose and glutamine. In 1931, Otto Warburg demonstrated that cancer cells do not metabolize glucose in the same way as normal differentiating cells. In the transition from normal cell to cancerous cell, energy metabolism is one of the main processes affected and believed to occur early in tumor genesis. The transition

31 takes place to support rapid proliferation and involves reduced use of the TCA cycle and a switch to glycolysis as the main source of energy, even in the presence of abundant oxygen, when aerobic glycolysis is preferred; this transition is known as the Warburg effect (51). As cancer cells use glycolysis, an anaerobic and less efficient process, rather than the more efficient TCA cycle, they need to produce ATP by converting two ADPs to one ATP and one adenosine monophosphate (AMP). This process helps the cancer cell maintain a viable ATP/ADP ratio and enables the cancer cells to use glucose to produce ATP and carbon atoms to produce biomass (46-49).

As discussed earlier, in normal cells pyruvate, the end product of glycolysis, is mainly transferred into the mitochondria and metabolized through the TCA cycle in the presence of oxygen. The fate of pyruvate, however, is very different in cancerous cells. In cancer cells, the PDH activity is blocked by the hypoxia-driven enzyme pyruvate dehydrogenase kinase 1 (PDK1), preventing pyruvate from entering the TCA cycle. In addition, an increase in lactate dehydrogenase A (LDHA) enzyme activity promotes the conversion of pyruvate into lactate, further contributing to the Warburg effect (46, 47).

Hypoxia inducible factor (HIF-1), a pleiotropic hypoxia induced transcription factor, is critical for metabolic adaptation to hypoxia as it increases conversion of glucose and subsequently lactate. As hypoxia is one of the main microenvironmental features of malignant solid tumors, HIF-1 plays a major role as a metabolic switch in these tumors as it shunts glucose metabolite from the mitochondria to glycolysis. HIF-1 activates GLUT 1, targets all glycolytic enzymes including phosphoglycerate mutase (PGM), and upregulates LDHA and PFK 1 through activation of 6-phosphofructose-2-kinase/fructose2,6-biphosphatase isomer 3 (PFKFB3). HIF- 1 also switches to the isoform PKM2, activates PDK1, and inhibits PDH. Overall, HIF-1 inhibits the TCA cycle, increases intracellular ATP levels, promotes glycolysis and lactate production, attenuates hypoxic reactive oxygen species (ROS) formation, and rescues these cells from hypoxia-induced apoptosis (46, 47, 52).

In normal conditions, the cells can take up lactate from the extracellular space using monocarboxylate transporter 1 and convert it back into pyruvate through the activity of lactate dehydrogenase B (LDHB). However, cancerous cells convert pyruvate to lactate, which is transported out of the cell via the monocarboxylate transporter 4. The large amount of exported lactate creates an acidic microenvironment further encouraging cancer cell invasion (47, 49).

In addition to glucose, GBM cells use other substrates for energy metabolism. Evidence suggests that only 30-70% of the acetyl-CoA used in the TCA cycle in cancerous cells are derived from glucose. Instead, GBM cells pick up acetate from their surrounding microenvironment. The acetate is converted to acetyl-CoA in the cytosol by the enzyme acetyl- CoA synthesize short chance 2 (ACSS2), highly expressed in GBM, and used in the TCA cycle (47).

Glutamine is one of the most abundant molecules in the human circulation and shuttles carbon and nitrogen between different organs and tissues. This molecule is believed to play an important metabolic role both in normal and cancerous cells as it is metabolized in appreciable quantities. Glutamine is believed to supply the cells with free energy, but even more importantly glutamine is an important carbon donor to the TCA cycle and nucleotide synthesis. Glutaminolysis is also an important generator of reducing power required for fatty acid biosynthesis by NADPH production via the activity of NADP-specific malate dehydrogenase (malic enzyme) (46, 47, 49).

Glutaminolysis seems to be especially important in transformed cells regulated by the proto oncogene family MYC. This family of regulatory genes is involved in cell control, metabolism, mitochondrial function, and regulation of apoptosis. The functions of the MYC depend on the microenvironment and needs of the cancerous cell to survive. The MYC gene can promote both glycolysis and OXPHOS depending on the cell’s situation. Furthermore, MYC genes seem to have both direct and indirect transcriptional control over glutaminolysis and involved proteins. Therefore, MYC-driven cells are sensitive to a shortage of glutamine, which rapidly can lead to loss of TCA cycle intermediates and cell death. Similar to HIF-1, MYC upregulates most glycolytic enzymes and GLUT 1, promoting the Warburg effect (46, 47, 52).

Finally, the tumor suppressor gene p53 and its effects on cellular metabolism is worth mentioning. Inactivation of p53 is observed in many types of cancers including GBM (53). The most important metabolic effect of this suppressor gene is to dampen glycolysis by direct downregulation of the expression of several glucose transporters (GLUT1, GLUT3, and GLUT4), ubiquitination, and inactivation of PGM, lowering F2,6BP by T53 expression- induced glycolysis and apoptosis regulator (TIGAR) expression. Furthermore, P53 has a regulatory role for metabolic genes, promoting OXPHOS before glycolysis and playing an important role in the induction of apoptosis. The p53 gene also has a promoting effect on the first stages of glycolysis through activation of HK2 and PGM. In addition to affecting TIGAR expression and deactivating PFK 1, activation of HK2 and PGM pushes intermediate molecules in the PPP pathway. Finally, P53 exerts a strong promoting effect on OXPHOS, maintaining mitochondrial health (46, 47, 52).

ANABOLIC PROCESSES WITHIN GBM Cellular metabolic anabolism, which enables the cancerous phenotype, fast growth, and invasion, is promoted by many of the above discussed metabolic pathways, oncogenes, and tumor suppressor genes and the environmental interplay between different cell types within the tumor microenvironment. Malignant cells need biosynthetic intermediates for their growth, so they increase their uptake of glucose, amino acids, and fatty acids from their surrounding microenvironment. Furthermore, the ATP produced by glycolysis can be used as a precursor for the synthesis of hexoamines, nucleotides, lipids, and proteins. Glutaminolysis further generates ATP and contributes to macromolecular synthesis as a source of carbon and nitrogen, and produces NADPH, ensuring reducing power for fatty acid production. As tumors grow, their need for energy and biomaterial increases. Eventually, their demand for simple nutrients such as glucose and glutamine exceed the supply in their surroundings and limits cell survival and growth. Many of the oncogenic metabolic changes that have been discussed earlier also have anabolic metabolic effects, pushing the cancerous cell to generate metabolic intermediates from pools of macromolecules within the cell such as with the PI3K/AKT pathway discussed earlier. Cancer cells rely mainly on aerobic glycolysis for their growth and survival under AKT- mediated metabolic influence. AKT activation promotes a glycolytic switch even under normoxic conditions without any effect on the OXPHOS mechanism and generation of metabolic intermediates (46, 47, 49).

33 OXIDATIVE STRESS IN GBM The high metabolic rate of cancerous tissue also gives rise to high levels of ROS and redox stress. Several of the previously discussed metabolic pathways in cancerous cells cause oxidative stress. In summary, the suppression of pyruvate available for the TCA cycle and the OXPHOS within the mitochondria often results in elevated levels of ROS. Furthermore, mutations within HIF-1, oncogene-mediated suppression, and the mitochondria genome encoding for thirteen of the electron transport proteins results in malfunction of the OXPHOS process, which inhibits the production of ATP and reduces oxygen, the main source for ROS. The high levels of generated ROS favor genetic instability, damaged macromolecules, and growth and increased tumor malignancy. To address this issue, cancerous cells engage pathways and enzymes that produce antioxidants such as the pentose phosphate pathway, mitochondrial folate pathway and NADPH-dependent isoforms of malic enzyme and isocitrate dehydrogenase (46, 47, 49).

Glucose Cell membrane GLUT 1

PI3K/ MYC AKT HIF1

HK 2 P53 PFKFB3 D-2-hydroxy-glutarate PFK 1 Fructose-2,6 P Glycolysis Mutant IDH 1 Glycolysis NUCLEOTID TIGAR ⍺-ketoglutarate E SYNTHESIS P53 IDH 1 LDHA PKM2 Isocitrate F Pyruvate Alanine A Lactate T PDK1 HIF1 T MYC Malate Y PDH LIPID Pyruvate Acetyl CoA SYNTHESIS A A O C M Oxaloacetate I I X D N S O P Malate TCA CYCLE Citrate A H C O G I D S L S ⍺ -Ketoglutarate U T

GLS MYC A M ADP + ATP =ATP + AMP Glutamate Glutamine I N GLUTAMINOLYSIS E

Figure 9. A schematic overview of the metabolic processes within malignant glioma cells.

IN VIVO TECHNIQUES FOR INVESTIGATION OF TUMOR METABOLISM

The origin of cancers is a stepwise accumulation of genomic alterations finally affecting important cellular functions. Genetic alterations such as mutations, translocations, and other genetic re-arrangements affect the transcriptome, the proteome, and finally the metabolome. Changes in the metabolome are the most downstream biological effects of the genetic alteration. Therefore, deciphering the metabolome should be a sensitive and robust approach for monitoring changes in a biological system and identifying pathways that are affected by a given genetic alteration. A more holistic understanding of the metabolic differences between cancer cells and normal cells might provide new insights into therapeutic strategies and targets (46).

Replication

G Genome DNA E N Transcription O T Y Transcriptome RNA P E Translation P H E Proteome Protein N O T Y Metabolome Metabolite P E

Figure 9. The central dogma of molecular biology. Replication, transcription, and translation are the three main processes used by cells to maintain their genetic information and to convert the genetic information encoded in DNA into gene products.

35 Although metabolic processes in tumor cells may be studied in cell cultures, this approach does not consider the factors affecting cellular metabolism that are associated with the microenvironment such as local nutrient concentrations and cell-cell interactions, phenomena difficult to model in culture. As discussed earlier, tumor biology, especially the microenvironment of GBM, is complex and largely remains a gap in our knowledge. For example, knowledge could improve by comparing tumor microenvironments under normal conditions and under distressed conditions during different treatment modalities. This approach could also improve our understanding of tumor microbiology, metabolism, and immunology and how treatment modalities affect the tumor microenvironment (49).

Methods for analyzing the metabolome in biological samples can roughly be divided into three approaches: untargeted metabolomics, metabolic profiling, and stable isotope-resolved metabolomics (SIRM). Untargeted metabolomics is an unbiased, hypothesis-free analysis of all detectable metabolites from a biological sample. Metabolic profiling identifies and quantifies a predefined list of metabolites from a biological sample. SIRM dynamically traces the fate of isotopes to identify metabolic alteration in a specific pathway (54). To determine which of these methods is most suitable for a special purpose, one must evaluate the possibilities and limitations of each method and, of course, evaluate the risk-benefit when in vivo investigations are the objective. Often, a combination of methods is needed for a comprehensive understanding of a metabolic network. For example, an untargeted metabolomic method can be used to identify a metabolic network, but to understand the characteristics of that network metabolic profiling is a better method. Finally, a SIRM method could be used to identify and understand different pathways in that metabolic network.

These three methodological strategies can be used to analyze metabolic changes. Involving thousands of metabolites, metabolomics could be seen as a snapshot of the metabolic profile of the investigated tissues. The coverage of metabolomics depends on the method of tissue extraction/sample collection and analysis. Magnetic resonance spectroscopy (MRS) and positron emission tomography (PET) are indirect non-invasive methods widely used by researchers and clinicians to assess in vivo metabolic processes. As these methods have the advantage of being non-invasive, they do not cover any wide range of metabolites within the metabolic network. Mass spectrometry (MS), on the other hand, enables the analysis of hundreds to thousands of molecules. However, this method requires samples gathered invasively, e.g., by biopsy or microdialysis (46, 49).

MASS SPECTROMETRY Mass spectrometry (MS) is a collective term for techniques used to determine the masses of atoms and molecules by measuring the mass-to-charge ratio of ions. MS basically consists of three parts: an ion source, a mass analyzer, and a detector. The MS procedure used (i.e., the arrangement of these three components) depends on what atoms or molecules are to be analyzed and the purpose of the analysis (55). MS generates many ions from the ion source, separates the ions in the mass analyzer according to their specific mass-to-charge ratio (m/z), and records the relative abundance of each ion type in the detector. Because the mass-to charge ratio is specific for each molecule, the ratio can be used to identify and quantify specific molecules. The results are typically presented as a mass spectrogram. The amplitude of the signal is proportional to the number of ions that reach the detector at any given moment, producing a peak on the spectrogram. Therefore, the amplitude (area under the peak) can be used to quantify an ion species (55).

MS is used in many research fields. It can be applied to both pure samples and complex mixtures. MS’s advantages include its sensitivity and specificity of detection, ability to provide analyte structural information, and (potentially) provide unambiguous detection and quantitation of analytes. Furthermore, the results are highly reproducible and accurate over several orders of magnitude, making MS a reliable analysis technique in the research as well as clinical setting (55).

MAGNETIC RESONANCE SPECTROSCOPY The physical process of recording the stimulated absorption and emission of energy from nuclei placed within a magnetic field is referred to as nuclear magnetic resonance (NMR). When imaging methods using the NMR signal were first developed, the term NMR imaging was used. However, as the word “nuclear” had an alarming effect on patients, the name was changed to magnetic resonance imaging (MRI). In current use, the term NMR is preferable when describing the physical phenomenon itself or when referring to the measurements of the nuclear induction signal in physics or chemistry laboratories. When referring to in vivo investigations, the term MRS is preferred, which should be understood as a method using the physical phenomena of nuclear magnetic resonance (56).

MRS, an in vivo and non-invasive method, uses high-power magnetic fields to detect the spatial resolution of metabolites and to quantify chemical compounds within a tissue. Most MRI scanners used in everyday clinical practice are capable of performing at least simple MRS analysis of metabolites such as lactate and choline. The basic principle behind MRS is based on the difference of the distribution of electrons within the atoms of different molecules, giving slightly different magnetic fields and resulting in slightly different resonant frequencies and detectable signal. These signals are then analyzed for metabolites–e.g., lactate, lipids, creatine, glycine, alanine, N-acetylasparatate, glutamine/glutamate, GABA citrate, and choline. Some of these metabolites are diagnostically useful when screening for cancers. MRS is widely used both as a scientific tool and in the clinical setting, especially for diagnostic purposes. MRS is often used to complement MRI: MRI is used for the structural and anatomical mapping of a tumor and MRS is used to gather functional information about the metabolism of a tumor (49, 57).

MRS has several limitations. MRS is limited by number of metabolites it can detect and by its lack of sensitivity to detect low concentrations of metabolites. Moreover, MRS provides the metabolic composition within a voxel, which only represents a small area of a total investigated tissue, so this voxel may not be representative for the whole tissue, a problem that is further exacerbated when the investigated voxel includes more than one type of tissue. Finally, MRS only represents a snapshot of the metabolic processes in a voxel, making this method impractical for longitudinal assessments (57).

POSITRON EMISSION TOMOGRAPHY Positron emission tomography (PET) can image the biodistribution and tissue localization of small amounts of radio-labelled biomolecules or drugs. It provides functional images that can be used to map physiological and/or pharmacological processes in vivo such as blood flow to

37 the heart, glucose metabolism in the brain, and ligand binding to specific receptors or binding sites. As with MRS, PET is a non-invasive, indirect method used to visualize metabolic processes in vivo. The most common isotopes used are 11C, 13N, 15O, and 18F. These isotopes are incorporated into a tracer substance of interest before being administered. The uptake and retention of the tracer substance is evaluated and registered using a detector in the PET scanner. PET is usually a part of a hybrid imaging strategy that uses computerized tomography (CT) or MRI to obtain morphological information. Hence, PET reports discrete metabolic activities rather than static metabolic levels, making PET a valuable method for diagnosis, staging, and monitoring of drug treatments for several medical conditions and malignancies (58-60).

Of course, the tracer selected depends on the purpose of the PET scan. For example, [18F] fluorodeoxyglucose (FDG) is used to detect tumors, [18F] sodium fluoride (NaF) is used to 15 18 detect bone formation, and [ O] H2O is used to measure blood flow. [ F] FDG, which is especially important in the clinical setting for early detection of metastases, is metabolized by hexokinase (the first step of the glycolysis) as it enters cells. However, as the tracer cannot be metabolized further, it is trapped in this stage of glycolysis. Therefore, [18F] FDG is very good for intense radiolabeling of tissues with high glucose uptake such as the normal brain, liver, kidneys, and most cancers, which have elevated glucose uptake due to the Warburg effect (61). Other important tracers in the field of neuro-oncology are O-(2-[18F]-fluoro-Ethyl)-L-tyrosine ([18F]-FET) and [18F]-3′-fluoro-3′-deoxy-L-thymidine ([18F]-FLT). [18F]-FET, an amino acid tracer, is often used to assess cell density, proliferation rate, and microvascular density within glial brain tumors. Several clinical studies support the practical value of [18F]-FET PET in addition to other imaging modalities (61, 62). In addition, [18F]-FLT is useful in clinical practice as it correlates with Ki67 levels in newly-diagnosed high grade gliomas (63).

In spite of the above-mentioned advantages, PET has some major limitations. PET’s imperfect specificity and sensitivity can give false positives as benign processes such as inflammation, which can produce metabolic patterns similar to malignant tissues. Furthermore, PET can be difficult to use for assessing tumors with low glucose utilization or when the benign surrounding tissue has a high basal level of glucose metabolism. In addition, altered blood sugar or blood insulin levels may adversely affect the test results of diabetic patients or patients who have eaten a few hours before the exam (58, 59).

Although PET’s image resolution is lower compared to other modern radiological methods, superimposing a PET image onto a CT or MRI image produces high resolution morphological images. Because PET is a functional imaging method that displays ongoing metabolic processes rather than just structural information, the sensitivity for PET/CT or PET/MRI is higher compared to CT or MRI alone for a variety of indications. The functional information PET yields are often unobtainable by other imaging techniques.

PET has some other practical limitations. PET imaging can take up to several hours to perform and radiotracers can take several hours to days to accumulate in the area of interest. Moreover, if the examinations are performed too early or too late, the results will not be useful (58, 59).

STABLE ISOTOPE-RESOLVED METABOLOMICS There is a long history of metabolic studies using isotope tracing. For example, isotope deciphering was one the cornerstones for uncovering the mechanisms involved in the TCA cycle. Traditionally, studies preferred radioisotopes because they were easy to obtain and easy

38 to detect. Today, studies rely on stable isotope-resolved metabolomics (SIRM) as stable isotopes are non-hazardous, amenable for human studies, and can be analyzed by NMR and MS (64). Successful SIRM analysis requires choosing the appropriate tracers. SIRM analysis calculates intracellular and extracellular metabolic fluxes–e.g., carbon (13C), nitrogen (15N), deuterium (2H), and oxygen (18O)–to generate metabolic datasets. However, brain cancer studies typically use 13C-glucose, 13C-glutamine, and 13C-acetate to generate datasets as these stable isotopes are simply detected by both MRS and MS (65).

NMR and MS can be used to cross validate each other because they quantify metabolites using different physical principles. Moreover, NMR and MS are complementary as they provide different information about the same samples analyzed. NMR provides better information on positional isotopomers (isomers with isotopic atoms) than MS, whereas MS provides better information on isotopologues (molecules that differ only in their isotopic composition) of given metabolites than NMR. Furthermore, NMR has the unique ability to detect metabolic transformations and their rates (fluxes) in live tissues or organisms in situ. That is, more specific, versatile, and reliable results can be obtained by using both NMR and MS (64, 65).

MICRODIALYSIS Microdialysis is an established method for monitoring the microenvironment in the interstitial tissue based on sampling of soluble molecules from the interstitial space using a semipermeable membrane at the tip of a microdialysis probe. This technique enables continuous analysis of the concentrations of a drug or biomolecule in the extracellular fluid of body tissues without significantly disturbing the function of these tissues. Microdialysis is also used to monitor changes in neurochemistry during acute brain injury and in intracerebral hematomas, subarachnoid hematomas, and different brain tumors. During the last decade, microdialysis has been used to monitor drug levels in brain tissue as well as macromolecules such as cytokines and other small proteins (66, 67).

Microdialysis, a minimally invasive technique, only requires the insertion of a 1-mm catheter and this catheter can be inserted into any tissue to continuously measure free, unbound analyte concentrations in the extracellular space of the tissue. Therefore, microdialysis can be used to assess biochemical processes and the distribution of exogenous substances and pharmaceuticals in the extracellular space (66, 67).

HISTORY In the early 1960s, the first microdialysis studies implanted push-pull cannulas and dialysis sacs into rodent brain. In 1972, continuously perfused dialytrodes were introduced, enabling the continuous measurement of extracellular events. In 1974, the “hollow fiber” was invented. This tubular semipermeable membrane led to the design of the probe used today: a sterile microdialysis catheter fitted with a simple microdialysis pump and a bedside biochemical analyzer (CMA Microdialysis, Stockholm, Sweden; present manufacturer M Dialysis, Stockholm, Sweden). Today, the CMA 70 catheter enables stereotactic implantation of microdialysis catheters intrathecally, an apparatus jointly developed with the Department of Neurosurgery at Umeå University (68). This work has made possible high precision implantation of microdialysis catheters within the brain and integrated the microdialysis method as a bedside routine for multi-modality monitoring. The microdialysis technique has evolved

39 during the past years and is today used for both assessment of endogenous and exogenous molecules in most tissues. Some examples of the endogenous molecules assessed by microdialysis are glucose metabolites, neurotransmitters, neuromodulators, cytokines, amino acids, and smaller proteins. The exogenous molecules assessed by microdialysis are primarily pharmaceuticals such as antibiotics, antidepressants, and antipsychotics. During the last decade, microdialysis has also been used to deliver exogenous molecules such as butyric acid and cisplatin. The delivery of cisplatin via microdialysis was developed at our center in Umeå (67, 69, 70).

Originally, microdialysis was intended to mimic the function of a blood capillary: a microdialysis catheter, a shaft with a semipermeable hollow fiber membrane tip, has inlet and outlet tubing perfused continuously with perfusate resembling the ionic composition of the extracellular matrix. When perfusate is perfused through the catheter, solutes passively diffuse across the semipermeable membrane. Diffusion is a passive process occurring due to the kinetic energy of the molecules, without the need of any reactions with other molecules The direction of diffusion is determined by the concentration gradients over the semipermeable membrane. Therefore, this technique can be used to collect samples and to deliver molecules. The dialysate, the perfusate obtained from the probe, is collected into micro vials for analysis (66, 67, 70).

Microvial Perfusion pump

S E Dialysate Perfusate M I P E R M E A B L E Diffusion

Figure 10. A schematic overview of microdialysis system consisting of a perfusion pump, micro vial, and microdialysis catheter with the distal part made of a semipermeable membrane. The molecular exchange across the semipermeable membrane is accomplished by diffusion.

RECOVERY As the microdialysis probe is constantly perfused with fresh perfusate, it is difficult to reach an equilibrium between the dialysate and the sampling site. The degree (ratio) of exchange of molecules between the two compartments is referred to as recovery and depends on the following conditions (67, 69):

• The probe area; • The molecular charge; • The probe’s location;

40 • The temperature; • The molecular weight (size) of the molecules; • The semipermeable membrane cut-off–i.e., the membrane permeability; • The flow velocity of the perfusate through the probe–i.e., the flow-rate; • The concentration gradient over the semipermeable membrane at the tip of the probe; and • The local conditions at the sampling site (diffusion in the surrounding interstitial fluid).

Recovery is defined as the ratio between the loss/gain of analyte during its passage through the probe (Cin−Cout) and the difference in concentration between perfusate and distant sampling site (Cin−Csample) (67):

Cin-Cout/Cin-Csample.

In reality, there are two ways to define the recovery of a microdialysis system:

• changing the measured molecules concentration while keeping the flow rate constant, and • changing the flow rate while keeping the respective molecules concentrations constant.

At steady state, the same recovery is obtained whether the analyte is enriched or depleted in the perfusate. The process of defining the recovery of a microdialysis system is referred to as calibration. Calibration can be achieved both by measuring the loss of molecules from the perfusate or by measuring the gain of molecules from the sample solution (66, 67). Before using a specific microdialysis system in vivo, it is essential to consider the factors affecting the recovery of the system. Considering these factors during the design phase of a microdialysis experiment will improve the chances of success (67, 70).

LIMITATIONS OF THE MICRODIALYSIS TECHNIQUE

PRACTICAL LIMITATIONS Implantation procedures can alter tissue morphology involved in microcirculation, rate of metabolism, and integrity of physiological barriers. Furthermore, implantation trauma can give rise to acute reactions in tissues involved in necrosis, inflammatory responses, and wound healing processes. These effects on tissues need to be considered. In addition, sufficient recovery time for molecular stabilization must be allowed before start of sampling (68).

Microdialysis also has several technical issues and problems associated with sampling. For example, catheters can be obstructed, the semipermeable membrane can be damaged, the pump can malfunction, and the implanted system can be a source of infection. If these problems are not dealt with as they occur, the volume and quality of the fluid collected during the sampling period will be compromised. As microdialysis presents a risk of infection and catheters have limited durability, the length of microdialysis studies are often restricted to 10 to 14 days (67, 68).

Recovery of the molecules needs to be considered. The four factors that have the greatest effect on in vivo recovery are the area and characteristics of the semi-permeable membrane, the perfusion rate, the molecules to be analyzed, and the diffusion properties of the surrounding

41 interstitial fluid. The diffusion of molecules varies depending on the molecular weight, solubility, and eventual charge of the analytes as well as the volume and physical properties of the interstitium. Recoveries can vary between different tissues and pathophysiological conditions, resulting in considerable interindividual variation within a study population. This limitation, however, is not an issue as long as the study’s focus is on trends and longitudinal changes of the analytes. However, a quantitative study needs to deal with the diffusion limitation of the surrounding interstitial space (66-68).

Finally, the implantation of the catheters at the desired location can be a challenge, especially when implanting intracranially. A surgeon using a stereotactical technique or neuronavigation can place a catheter with high precision. However, some parts of the brain are more difficult to target using these techniques–e.g., lateral temporal areas and areas in the posterior fossa (68).

ETHICAL LIMITATIONS Microdialysis is an invasive technique as a surgeon must implant catheters, a procedure that necessarily involves the risks such as intraoperative or postoperative hemorrhage and infection. The procedure may also result in a slightly longer time for postoperative recovery. Additionally, microdialysis requires patients to be hospitalized. These factors must be considered especially when dealing with patients with malignant diseases and co-morbidities.

42 AIMS

This thesis aims to improve the understanding of the microenvironment of high-grade glioma during normal conditions and during treatment. Specifically, this thesis investigates the following:

1. The usefulness of stereotactic microdialysis in investigating the molecular microenvironment in high-grade glioma; 2. The metabolic response to radiotherapy; 3. The immunologic response to radiotherapy; 4. The feasibility of retrograde microdialysis of cisplatin in high-grade glioma; and 5. The metabolic response pattern to loco-regional treatment with cisplatin.

MATERIALS AND METHODS

This thesis is based on three prospective patient series that involved implanting microdialysis catheters in brain tumor tissue. Results from the three series are presented in four papers. In all studies microdialysis catheters were also implanted in brain adjacent tumor (BAT) and in two studies (Papers II, III and IV) reference catheters were implanted in abdominal subcutaneous tissue. In the third study microdialysis catheters were implanted for administration of cisplatin through the process of retrograde microdialysis. Additionally, clinical information was obtained from clinical investigation of the patients according to study protocols and from patient records.

PATIENTS STUDY I This study included 13 patients–nine men and four women with an average age of 65 (range 50–79)–who had radiologically highly suspicious malignant glioma (10 GBM and three WHO grade III anaplastic astrocytoma) located centrally and not suitable for resection. Biopsies were performed for diagnosis before implantation of microdialysis catheters.

STUDY II This study included eleven patients–eight men and three women with an average age of 63 (range 50–81 years). These patients had radiologically suspicious malignant glioma not suitable for resection. The suspicious malignant glioma (10 GBM and 1 WHO grade III anaplastic astrocytoma) were confirmed by biopsy before the implantation of microdialysis catheters.

STUDY III AND IV These two studies included the same nine patients–five men and five women with an average age of 54 years (range 40–80 years)–with previously histopathological verified GBM. One patient had a WHO grade III anaplastic astrocytoma. All patients received first-line treatment with surgery (nine patients subtotal/total resection and one patient biopsy) followed by concomitant radiochemotherapy and up to six cycles of adjuvant temozolomide. All patients except patient 6 and 7 also received second-line treatment with bevacizumab in combination with chemotherapy. At the beginning of the study, none of the patients were suitable candidates for further resection or any other conventional chemotherapy and had an estimated survival of at least three months based on their clinical and radiological status. Two patients had several surgeries and a secondary transformation from WHO grade II to grade III (patient 10) and grade IV glioma (patient 5). Patients 3 and 10 had isocitrate dehydrogenase 1 (IDH-1) mutations. After receiving an experimental locoregional treatment with cisplatin using microdialysis, two of the patients with long survival received additional treatment with irinotecan/bevacizumab (patient 4, ten months after cisplatin treatment) or re-irradiation of 3.4 Gy X 10 (patient 10, seven months after cisplatin treatment).

MICRODIALYSIS CATHETER IMPLANTATION AND SAMPLING STUDY I Stereotactic serial biopsy was first performed to confirm the diagnosis before microdialysis catheters were stereotactically introduced. One catheter was placed with its semipermeable membrane into the biopsy target area within the radiologically contrast enhancing defined tumor. A second catheter was placed approximately 10-mm outside the contrast-enhancing tumor in the BAT. The catheters were tunneled away from the burr hole and fixated in the head dressing, allowing the patients to move freely about the ward. Catheters with a 10-mm long 20kDa semipermeable membrane (CMA 70) were used for intracranial sampling. Ringer was used as perfusate with a flow rate of 0.3 ul/min. A reference catheter (CMA 60) was implanted subcutaneously in the abdomen. In the afternoon after surgery, a CT was performed to confirm the position of the catheters.

Sampling started after confirmation of the catheter position by CT, and a minimum of 20 hours of sampling was performed before the first radiotherapy fraction. Samples were collected in micro vials every two hours. After collection, the samples were frozen and kept at -80 °C. The samples collected before the radiotherapy were considered baseline samples. Sampling continued for the first five days of therapy. The sampling was aborted, and the catheters explanted in the morning after the fifth fraction of radiotherapy. Samples collected between 00:00-05:00 a.m. were analyzed using the CMA 600 analyzer.

STUDY II The implantation of the catheters was carried out similarly to study I and the study protocol was close to identical (see above). The major differences were related to the catheters and perfusion fluid used. In this study, catheters with a 10-mm long 100kDa semipermeable membrane were used for intracranial sampling. To avoid ultrafiltration, Ringer/Dextran (Perfusion fluid T1: CMA Microdialysis mixed with 30g Dextran 60 1000/mL) was used as perfusate with a flow rate of 0.3 ul/min. For the analysis of glucose metabolites, glutamate and glycerol fasting samples collected between 00:00-05:00 a.m. were analyzed with the CMA 600 analyzer. The samples analyzed for cytokines were from the same time points, but only on the days before the start of the radiotherapy (baseline) and after the first, third, and fifth fraction of radiation.

RETROGRADE MICRODIALYSIS STUDY III AND IV

IN VITRO STUDY An in vitro study was performed to test whether the administration of cisplatin is possible using microdialysis catheters. For this purpose, we used microdialysis catheters with a 30-mm semipermeable membrane with a 20 kDa and 100 kDa cut-off. The catheters were placed in a buffer solution with similar molecular composition as brain extracellular matrix (NaCl 147 mmol/L, KCl 2,7 mmol/L, CaCl2 1,2 mmol/L, MgCl2 0,85 mmol/L). The catheters were perfused with a perfusate containing three known concentrations of cisplatin (0.1, 0.5, and 1.0 mg/mL) and three flow rates (0.5, 2.0, and 5uL/min). In addition, Dextran-60 (Plasmodex®)

45 was used as a perfusate in one catheter to investigate whether Dextran-60 could minimize problems with ultrafiltration. Cisplatin concentration were analyzed in the buffer solutions to quantify the amount of cisplatin transferred to the buffer solution through microdialysis.

IN VIVO STUDY Microdialysis catheters were implanted in tumor tissue and BAT either by stereotactic technique (eight patients) or neuronavigation (two patients). Patients 1-3 were implanted with Dyphylon Cardiac® catheters (100 kDa), patient 4 was implanted with the CMA 60® catheter (100kDa), and the remaining patients were implanted with the CMA 71® catheter (20 kDa). Two systemic reference catheters were implanted in subcutaneous abdominal fat: CMA 71® catheter (100 kDa) for patients 1-3 and CMA 60® catheter (20 kDa) for the rest of the patients. All catheters had the same semipermeable polyamide membrane. In the afternoon following surgery, a CT was performed to confirm the position of the catheters.

Sampling was started directly after surgery to establish baseline levels for the analyzed metabolites. On day one after surgery, cisplatin treatment was started using catheter A (usually the most centrally located catheter). The sampling from the other surrounding catheters allowed pharmacokinetic analysis of cisplatin. On day two after surgery, the treatment was started using the other catheters within the tumor.

Samples were obtained daily between 04:00 and 06:00 a.m. and 04:00 and 06:00 p.m. In addition, the disposal of the samples was the same as in study II and III. Fasting samples taken between 04:00 and 06:00 a.m. were analyzed with the CMA 600 analyzer for potential cytotoxicity markers, glutamate and glycerol. Total platinum content was determined using inductively coupled plasma mass spectrometry (ICPMS). If cisplatin concentration needed to be quantified, hydrophilic interaction chromatography (HILIC) coupled to ICPMS was used.

TREATMENTS RADIOTHERAPY

STUDY I AND II All patients received fractionated radiotherapy with 2 Gy/daily to a total dose of 60 Gy except for three elderly patients in study II. These patients received hypofractionated radiotherapy– two patients received 3 Gy/day for 13 days and one patient received 3.4 Gy/day for 10 days.

LOCO-REGIONAL INTERSTITIAL TREATMENT WITH CISPLATIN

STUDY III AND IV Cisplatin inhibits DNA replication through ionic bind to DNA and this binding interferes with mitosis. The damaged DNA activates DNA repair mechanisms, which in turn activates apoptosis when repair proves impossible. Cisplatin was used because it has a strong cytotoxic effect on glioma cells in vitro but does not easily reach therapeutically significant concentrations in brain tumors without resulting in unacceptable systemic toxicity (71). Cisplatin is a positively charged ion that do not cross the BBB in therapeutic concentrations. Furthermore, the molecular size and properties of cisplatin are such that it is believed to be a suitable substance for administration via retrograde microdialysis.

Designed as a phase I trial, the clinical study monitored toxicity of cisplatin administered by retrograde microdialysis. Cisplatin was administered in a stepwise dose escalated manner. The first four patients were administered a cisplatin dose of 1 mg/day for 12 days. The remaining patients were administered a cisplatin dose of 3 mg/day and later 5 mg/day. The perfusate concentration and the flow rate were set based on results from the in vitro study and were adjusted according to the planned daily dose and the number of implanted catheters. All the patients were administered commercially prepared cisplatin–i.e., 1 mg/ml diluted in NaCl. The exception, patient no. 10, was administered a cisplatin solution of 1 mg/ml with 1mg/ml of mannitol added by the manufacturer. At the time of the trial, these were the only commercially available preparations.

PATIENT FOLLOW-UP All patients in all four studies spent the first 24 hours post-surgery at the neuro-intensive care unit for monitoring. The patients underwent daily clinical examinations for their neurological status. Fasting blood samples were collected the morning before treatment and after 3, 6, and 12 days of treatment and when clinically needed. The analysis of the samples included blood cell count, electrolytes, liver enzymes, creatinine, and C-reactive protein (CRP). During the whole treatment, blood glucose was monitored and kept below 8 mmol/l, using insulin if needed.

At 1- and 3-months post-procedure, all patients in study III and IV were to receive a clinical evaluation, MRI, EORTC-QLQC30, and EORTC-BN20. Further follow-up was performed if the treatment or the condition of the patient warranted.

ANALYTICAL METHODS ASSESSMENT OF GLUCOSE METABOLITES, GLUTAMATE, AND GLYCEROL (STUDY I–III) The CMA 600 analyzer (CMA Microdialysis) was developed to analyze microdialysis samples using small sample volumes. The analyzer, intended only for microdialysate, uses a colorimeter to measure the absorbance of particular wavelengths of light of a specific solution. The CMA 600 analyzer incorporates a spectrometer that uses an enzymatic technique. For the substances in question, the rate of formation of the colored substance, quinonimine, is measured in a filter photometer at 546 nm. This technique is commonly used to determine the concentration of a known solute in a given solution by the application of the Beer-Lambert law: the concentration of solute is proportional to its absorbance. In study I, II, and III, the CMA 600 analyzer was used to analyze the microdialysis samples for glucose metabolites (glucose, pyruvate, and lactate), glutamate, and glycerol.

CYTOKINE ANALYSIS (STUDY II) The CBA Cytokine Bead Array (CBA, BD, Stockholm) was used to identify and quantify cytokines in the microdialysis samples. This bead array is a cytometric bead array assay that allows for the detection of multiple antigens in a solution. The array uses two kinds of antibodies: capture antibodies (conjugated to the polystyrene beads) and detection antibodies (detected via fluorescent signal). Each bead in the array is conjugated to one specific capture

47 antibody specific for a single antigen and fluoresce at a specific wavelength, an arrangement that distinguishes this bead from other beads and ultimately can be used to identify a specific antigen (cytokine). The detection antibodies, which have a fluorescent marker, are used to quantify an antigen (72, 73).

The AccuriC6 (BD) flow cytometer is used to detect and quantify the fluorescence of the arrays: a fluorescence bead array determines the location of specific antigens (in their specific bead) and quantifies the antigens within the beads as the expression level in each bead correlates with the amount of bound detection antibodies and that antibody’s specific antigen. The obtained fluorescence intensity is compared to a standard for quantification of antigens (72, 73).

Unlike ordinary ELISA, this technique can analyze many substances simultaneously, saving time and resources. Study II used Flex-sets (in advance prepared antibody-coated beads) for the cytokines IL-4, IL-6, IL-8, IL-10, TNF-α, IFN-γ, GM-CSF, MCP-1, MIP-1α, and MIP-1β.

IMMUNOHISTOCHEMISTRY (STUDY II) Blocks of the stereotactic biopsies were cut into 4-um thick sections that were prepared and stained with heamatoxylin/eosin and immunostained according to the manufacturer’s recommendations (Benchmark Ultra). Benchmark Ultra is a fully automated immunohistochemistry and in situ hybridization slide staining system. Micrographs were taken with an Olympus BX53 microscope equipped with a DP73 camera (Olympus, Hamburg, Germany) and using the Cellsens dimension software (Olympus).

The following primary antibodies were used: anti-GFAP (code Z 0334; Dakocytomation, Glostrup, Denmark; 1:5000); anti-vimentin (catalog number 790–2917; Ventana Medical Systems; 1:1); anti- IDH1(R132H) (clone H09; Dianova, Hamburg, Germany; 1:50); anti-Ki- 67 (clone 30-9; Ventana Medical Systems; 1:50); anti-p53 (clone DO-7; Novocastra™, Newcastle-upon- Tyne, England; 1:25); anti-EGFR (clone 3C6; Ventana Medical Systems; 1:100); anti-phosphohistone-H3 (catalog number 369A; Cell marque, Rocklin, CA, USA; 1:300); anti-human CD31 (clone JC70A; Dako, Glostrup, Denmark; 1:10); anti-human CD68 (clone KP1; Dako; 1:2000; CC1 pretreatment); anti-CD163 (clone 10D6; Novocastra; CC2 pretreatment); anti-MCP1 (catalog number ab9669; Abcam, Cambridge, England; 1:100; CC2 pretreatment); anti-IL6 (catalog number ab6672; Abcam; 1:200; CC1 pretreatment); anti-IL8 (catalog number 17038-1-AP; Proteintech, Chicago, USA; 1:25; CC1 pretreatment).

DETECTION OF PLATINUM AND CISPLATIN (STUDY III) Using inductively coupled plasma mass spectrometry (ICPMS), we determined the total content of platinum. ICPMS produces ions with argon gas flowing through a torch apparatus charged with an electromagnetic coil to create a plasma of ionized argon. Next, a gas flow is passed through the middle of the plasma, creating a channel colder than the surrounding plasma. Samples to be analyzed are released into this central channel, generally as a mist of liquid created by driving the liquid sample through a nebulizer. When the sample passes through the central conduit, it evaporates, and molecules fall apart. The atoms within the sample are ionized as they have lost loosely linked electrons, creating a single-charged ion. The sample ions travel through the mass analyzer before being registered by the detector (74). The sample concentrations can be established via calibration of the ICPMS system with an inorganic PtCi3 standard.

The cisplatin concentration was quantified using an ICPMS coupled with hydrophilic interaction chromatography (HILIC). HILIC, a liquid chromatography technique, is used to

48 separate moderate to highly hydrophilic and polar compounds based on their retention on a hydrophilic stationary phase versus the hydrophobic aqueous polar organic mobile phase. HILIC is highly compatible with mass spectrometry. The high polar organic solvent component of the HILIC mobile phase increases the efficiency of ionization in the ICP process. Therefore, HILIC coupled to ICPMS can provide a highly sensitive quantitative analysis of a wide range of complex solutions containing polar components (75).

METABOLOMIC ANALYSIS (IV) The microdialysis samples and serum samples were prepared and analyzed with a time-of-flight mass spectrometer coupled to a gas chromatograph (Leco Pegasus HT time-of-flight mass spectrometer equipped with an Agilent 7890 A gas chromatograph). Metabolites were identified and quantified using an in-house developed software for raw data analysis, transforming raw data into chromatographic profiles for each compound in each sample with a common spectral profile. The integrated area under the resolved chromatographic profile was used for quantification. The identities of the resolved peaks were determined by comparing mass spectra and retention indices with data from Swedish Metabolomics Center in-house spectral library.

Gas chromatography is a common type of chromatography used in analytical chemistry to separate and analyze compounds that can be vaporized without decomposition. In gas chromatography, the mobile phase (“moving phase”) is a carrier gas. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support inside a piece of glass or metal tubing called a column. The column is located in an oven where the temperature of the gas can be controlled. The gaseous compounds being analyzed interact with the stationary phase coated to the walls of the column. This interaction causes each compound to elute at a different time. The comparison of retention times is what gives GC its analytical information. Furthermore, the concentration of a compound in the gas phase is solely a function of the vapor pressure of the gas (76).

The data from the GC is presented as a graph of detector response (y-axis) against retention time (x-axis), which is called a chromatogram. The chromatogram provides a spectrum of peaks representing the analytes present in a sample eluting from the column at different times. A mass spectrometer is used to identify the analytes represented by the peaks. The area under a peak is proportional to the amount of analyte present in the chromatogram. The area under the peak can be used to calculate the concentration of an analyte in the original sample (76).

STATISTICAL ANALYSES WILCOXON SIGNED RANK TEST (STUDY I, II AND III) The Wilcoxon signed-rank test, a non-parametric statistical hypothesis test, is used to assess whether the population mean ranks of two related samples, matched samples, or repeated samples differed. The Wilcoxon signed-rank test was used for the analysis of data from study I, II, and III as the samples were small and not normally distributed (77). The test was used to determine whether there was any statistically significant difference between the means of concentration of metabolites in tumor compared to BAT. A P-value of < 0.05 was considered significant.

ANALYSIS OF VARIANCE (STUDY I AND II) The one-way analysis of variance (ANOVA) is used to determine whether there are any statistically significant differences between the means of two or more independent (unrelated) groups. However, in practice, this method is used when there is a minimum of three or more groups. The one-way ANOVA compares the means between the groups and determines whether any of these means are statistically significantly different from each other. The Null hypothesis in ANOVA is valid when all the sample means are equal, or they do not have any significant difference. The alternate hypothesis is valid when at least one of the sample means is different from the rest of the sample means (77). As one-way ANOVA is an omnibus test statistic, it can only determine that at least two groups were statistically significantly different from each other, but not the specific groups, which would require a post hoc test (77). ANOVA was used in study I and II to determine whether or not the given treatment–i.e., fractionated radiotherapy– induced any change in the microenvironment of the tumor. A P-value of < 0.05 was considered significant.

CORRELATION (STUDY I, II, III) The Pearson correlation coefficient measures the strength of a linear association between two variables, where r = 1 means a perfect positive correlation, r = -1 means a perfect negative correlation, and r = 0 means no correlation between the two variables (77). Sometimes, the r is squared to form a useful statistic called the coefficient of determination (r2). The coefficient of determination is a measurement used to explain how much variability of one factor can be caused by its relationship to another related factor. This correlation, known as the “goodness of fit,” is represented as a value between 0.0 and 1.0 (77).

Study I and II did not reveal any correlation between survival and the concentrations of glucose metabolites. However, study II revealed a positive correlation (baseline versus survival) between IL 6 and IL8 concentrations. In spite of these findings, we could not see any correlation between survival and the change in the cytokine levels in study II.

ORTHOGONAL PROJECTIONS TO LATENT STRUCTURES DISCRIMINANT ANALYSIS (OPLS-DA) AND ORTHOGONAL PROJECTIONS TO LATENT STRUCTURES EFFECT PROJECTIONS (OPLS-EP) (STUDY 4)

Multiple linear regression is a statistical technique that uses several explanatory variables to predict the outcome of a response variable. The goal of multiple linear regression is to model the linear relationship between the explanatory (independent) variables and response (dependent) variable. Multicollinearity is the occurrence of high intercorrelations among independent variables in a multiple regression model. Multicollinearity can lead to skewed or misleading results when a researcher or analyst attempts to determine how well each independent variable can be used most effectively to predict or understand the dependent variable in a statistical model. This issue is considered a major threat to traditional regression models in classical statistics when there are a large number of variables and a small sample size (77).

One of the most common ways of eliminating the problem of multicollinearity is to first identify collinear independent variables and then remove all but one. It is also possible to eliminate

50 multicollinearity by combining two or more collinear variables into a single variable. Statistical analysis can then be conducted to study the relationship between the specified dependent variable and a single independent variable. Partial least squares (PLS) regression analysis is known to attenuate the above-mentioned problems. At the same time, PLS consists of some limitations such as interpretability problems, multi-component results, and biased coefficients in some situations, leading to a greater risk of overlooking real correlations (78, 79).

Orthogonal projections to latent structures (OPLS), a recent modification to the PLS regression analysis method, separates the systematic variation in the X variable into two parts: one linear to Y and one orthogonal to Y. The systemic variation in X that is orthogonal to Y is removed to make the interpretation of the resulting PLS model easier and with the additional benefit that non-correlated variation itself can be analyzed further. The OPLS methodology identifies joint variation within biological samples to enable the removal of sources of variation that are non- correlated (orthogonal) to the within-sample variation. This ensures that structured variation related to the underlying biological samples is separated from the remaining bias-related sources of systematic variation. As a consequence, the methodology does not require any explicit knowledge regarding the presence or characteristics of certain biases. Basically, OPLS is a preprocessing method for multivariate data that makes the regression model more sensitive, reliable, and interpretational (Figure 11) (79).

Orginaldata set x o x o x o x o x o • Harder to interpret o x o x o x o x o x • More PLS components • Orthogonal variations x o x o x o x o x o

o o o o o o o o o o o o o o o

Orthogonal dataset • Identify source of orthogonal variation x x x x x x x x x x OPLS treated data x x x x x • Easier to interpret • Fewer components • More relevant

PLS

Figure 11. Original data set is preprocessed with OPLS before analyzed further with PLS in order to make the analyze more sensitive, reliable and relevant.

In study IV, multivariate statistical analysis was applied to identify metabolites and metabolic pattern. The collected metabolite data relating to effect of treatment, differences between short

51 and long-time survivors, as well as differences between microdialysis fluids from tumor and BAT were analyzed using OPLS-DA for independent statistical analysis of short-time survivors vs. longtime survivors and tumor vs. BAT. Discriminant analysis (DA) is statistical technique used to classify observations into non-overlapping groups based on scores on one or more quantitative predictor variables (78).

OPLS-EP was used to analyze the effect of treatments in dependent samples (pretreatment vs. day 3 or 6). Effect projections (EP) analysis can be compared to a paired t-test involving dependent samples used to determine whether the same sample population changes over time (78).

ETHICS All four studies were approved by the local ethics review board in Umeå, Sweden. The patients in all four studies were fully informed of the research projects. For study III and IV, the experimental treatment was explained both orally and in writing to ensure the participants understood the experiment and their rights as participants. All patients participated voluntarily and provided their written informed consent. In study III and IV, the patients received loco- regional interstitial cisplatin treatment, an experimental treatment with risks for potential serious side effects. These risks were put against the fact that the patients included in the study already had undergone all available standard treatment with nothing more to offer them beside the best supportive care as their disease progressed. We believe that severeness of the patients’ disease and the lack of further therapy options justified the higher risks involved with the offered therapy. Study III and IV was also approved by the Swedish Medical Products Agency (EU-nr 2010-018281-23).

RESULTS AND DISCUSSION

FEASIBILITY OF MICRODIALYSIS (STUDY I-II) In study I and II, there were no complications such as infection, hemorrhage, or increased cerebral edema in association with the microdialysis or the radiotherapy. All patients were ambulatory the day after surgery and could move freely within the neurosurgical ward with the apparatus attached to a head cloth. This demonstrates that microdialysis is a safe and feasible method for real-time direct assessment of metabolic and immunological changes in tumor microenvironment. Although risk of infection may limit monitoring time, up to ten days of monitoring, as in study I and II, seems safe. This timeline agrees with other studies using this technique (80-82).

FEASIBILITY OF RETROGRADE MICRODIALYSIS (STUDY III AND IV) In study III and IV, patients received interstitial cisplatin treatment through retrograde microdialysis. Although the implantation of the microdialysis catheters was carried out without any complications and cisplatin mainly affected the targeted glioma tissue, the treatment also affected the patient’s clinical status as all patients developed some edema around the treating catheters. The edema resulted in transient neurological deterioration in six of the ten patients. These six patients were treated with an increased dose of corticosteroids together with a decreased cisplatin dose (five of six patients). In four patients, the treatment was aborted before the planned 12 days of treatment. In all cases, the edema regressed after the treatment.

Patient no. 8 deteriorated after surgery. Before the therapy started, this patient’s condition had deteriorated, so we considered excluding the patient from the study. However, as the patient had received surgery, had catheters implanted, and both the patient and relatives had the expectation the therapy could be helpful, we concluded it would be unethical to withhold the planned therapy. Furthermore, we observed a rapid tumor progression the weeks before the treatment. At this point, we did not believe that the patient had anything to lose by starting the treatment as the prognosis of not receiving any further treatment was considered very dire. The condition of this patient progressed and deteriorated even further during the therapy. Radiological assessment of this patient did not show any other findings than edema, a condition similar to the other patients in the study. The edema regressed as the treatment was aborted and steroids were given. Unfortunately, the patient did not recover neurologically and died ten days after the start of the treatment.

Tumor tissue had concentrations of cisplatin in possibly clinically relevant levels. BAT, serum, and subcutaneous tissue had concentrations of cisplatin in very low concentrations. A local toxicity could be detected within the tumor but not in BAT and no systemic toxicity was registered. This finding suggests that retrograde microdialysis could provide an effective method for loco-regional interstitial treatment with cisplatin. Despite its invasiveness,

53 retrograde microdialysis has several important advantages that could make it an effective way to deliver drugs to the brain. Because retrograde microdialysis can deliver active substances to the brain without water molecules, edema can be avoided. Furthermore, the delivery can be targeted to a specific area within the brain, so the spread of the active substance delivered depends only on the specific molecule’s ability to diffuse into the tissue, a process influenced by the characteristics of the tumor. In addition, retrograde microdialysis makes it possible to follow patients using simultaneous sampling during the delivery of an active substance, a technique that makes it possible to assess a drug’s therapeutic effect in real time. Finally, we now also know that the method is feasible and, in most patients, safe. However, patients who suffer from pronounced edema and tumor expansion may not be suitable for this aggressive treatment. Future studies should be designed with cisplatin and other substances to create new opportunities for discovery.

CHANGES DURING FRACTIONATED RADIOTHERAPY (STUDY I AND II) ANALYSIS OF GLUCOSE METABOLITES, GLUTAMATE, AND GLYCEROL

In study I and II, the baseline samples showed a significantly lower concentration of glucose and pyruvate in tumor tissue than in BAT. In addition, the lactate/pyruvate ratio was significantly higher in tumor at baseline compared to BAT. In study I, we observed higher levels of baseline lactate, glutamate, and glycerol in tumor tissue than in BAT, but this difference was not statistically significance. In study II, significance was reached for higher lactate and glutamate levels in tumor. These results correspond well with other studies performed with indirect methods such as MRI and PET, suggesting a higher glucose metabolism and a higher dependence on both aerobic (Warburg effect) and anaerobic glycolysis in malignant cells (47, 51, 83-85).

Study I Study II Glucose Lower in tumor Lower in tumor Lactate No significance Higher in tumor Pyruvate Lower in tumor Lower in tumor Lactate/Pyruvate Higher in tumor Higher in tumor Glutamate No significance Higher in tumor Glycerol No significance No significance

Table 2. Overview of glucose metabolites at baseline in study I and study II.

We did not observe any significant change in any of the glucose metabolites, glutamate, and glycerol in both study I and study II during the five days of fractionated radiotherapy with a daily dose of 2 Gy. We also found no correlations between survival and glucose levels or the lactate/pyruvate ratio. In a previous study from our group, we demonstrated that glycerol may be a marker for toxicity following BNCT treatment of malignant glioma (86). However, this BNCT treatment delivered a total dose of radiation in one fraction, whereas in the present study the dose was given fractionated and with a dose of only 10 Gy in all but three patients, who received 15-17 Gy during microdialysis.

54 In malignant cells, glutamate is a source of energy used during aerobic glycolysis and a potential marker for cellular damage (47, 49). Although cancerous cells harvest glutamate for energy, we were able to measure increased levels of glutamate in tumor compared to BAT, suggesting an increased cellular release of glutamate in tumors possibly because of increased cell damage and high turn over time of cells in malignant tissue. Another potential marker for cellular damage is glycerol (86). As glycerol is mainly derived from the glycolytic chain via glycerol-3- phosphate and the degeneration of cell-membrane glycerophospholipids, it would be reasonable to predict that elevated glycerol would result in radiation-induced cell damage and cell death. However, as the glucose metabolites, glutamate, and glycerol levels did not change during the five days of fractionated radiotherapy, the given dose probably was too low to induce any change in metabolism or cell damage or the effect might be delayed. On the other hand, in study III, we found a distinct increase in both glutamate and glycerol as a result of the cytotoxic effect of the cisplatin treatment. Moreover, the cytotoxic effect of cisplatin treatment had profound metabolic effects. These facts strengthen our belief that the given radiation dose in study I and II was too small to induce any changes within the treated tissue. In addition, our findings suggest that the dose of cisplatin used was a clinically relevant dose.

CYTOKINE EXPRESSION (STUDY II) As microdialysis can reliably and constantly detect IL-6, IL-8, MVP-1, MIP-1a, and MIP-1b, these cytokines can be statistically analyzed. The analysis of these cytokines resulted in the following conclusions:

• There was a significant increase of IL-6 in the tumor after the fifth fraction of radiation. In BAT and SC, however, a decrease could be detected soon after the first radiation fraction. • The IL-8 levels in the tumor were significantly increased within the first 24 h following the first given fractions of radiation and increased during the whole time of radiation. The IL-8 concentrations in BAT and SC increased with a delay compared to tumor tissue. • The MCP-1 concentrations in tumor tissue significantly increased after the first fraction and kept on increasing during radiotherapy. However, a significant increase could only be observed after the fifth fraction of radiation in SC tissue. • The MIP-1a concentrations in tumor tissue also significantly increased from the first fraction of radiotherapy and kept increasing during the whole therapy. For the SC tissue, a significant increase could only be observed after the fifth fraction of radiation. • A temporary significant increase in MIP1-b levels was observed in tumor after the third fraction of radiation. The concentration in SC was also increased but not until after the fifth fraction.

Inflammation plays an important role in the regulation of both tumor progression and regression (42). However, very little is known about how different treatment modalities affect the immune and inflammatory microenvironment in the CNS. The results indicate that fractionated radiotherapy with a daily dose of 2-3.4 Gy up to five days results in an inflammatory response in malignant gliomas. This response was detected by an increase of several cytokines and chemokines measured in tumor tissue. We also found that the induced inflammatory response was more pronounced in tumor tissue compared to BAT and subcutaneous tissue. Although the radiotherapy clearly affected the immune microenvironment of the tumor in this study, it is difficult to draw any conclusions whether this induced effect exerts a protective effect in favor of the tumor or an anti-tumorous cytotoxic effect. Both IL6 and IL8 are considered to be mainly

55 derived from malignant glioma cells in GBM and have immunosuppressive characteristics. These cytokines appear to play an important role in tumor proliferation by promoting angiogenesis, growth, and invasiveness (43, 44). Therefore, at least some of the induced inflammatory response seen in our study within the irradiated tumor tissue could have had a protective role for the tumor.

We also investigated whether any significant differences were evident between the patients receiving the 2 Gy standard dose and those receiving 3 or 3.4 Gy. Although we did not find a difference, we could identify a significant statistical correlation between baseline IL-8 and IL- 6 microdialysis levels and survival (r2 = 0.28 and r2 = 0.13, respectively). There was no correlation between the increase in IL-6, IL-8, and MCP-1 and survival following treatment.

IMMUNOHISTOCHEMISTRY (STUDY II) The immunohistochemical analysis of stereotactic biopsies could verify the histopathological diagnosis of high-grade glioma with high levels of proliferation markers such as Ki-67 and PHH3. The presence of CD68 and CD163 demonstrates a high infiltration of immunocompetent cells such as monocytes and macrophages within the tumor tissue. These findings correlate well with the literature, where it is suggested that up to 30-40% of viable cells within a glioblastoma are immunocompetent (42, 43).

The cytokine IL-6 was detected both intra- and extra-cellular in viable and peri-necrotic tissue. IL-8 and MCP-1 could be observed mainly in necrotic and peri-necrotic tissue. IL-8 was observed in five of ten patients although only in less than 25% of the cells. MCP-1 could be observed in all patients, primarily extracellularly and in macrophages. Together, these findings indicate the fact that there is prevailing inflammatory activity within glioma tissue. Immunological cells, cytokines, and chemokines are common features within the microenvironment of malignant tumors. Clearly, a better understanding of the role of these cells and molecules are important in the future development of more effective therapeutic strategies.

RETROGRADE MICRODIALYSIS WITH CISPLATIN (STUDY III AND IV) PHARMACOKINETICS Cisplatin was successfully administered to most of the patients by retrograde microdialysis in the doses according to our protocol. The drug penetrated the tumor/brain tissue up to 37 mm from the supplying catheter after 12 hours of dialysis. For the entire treatment period, the total cisplatin dose delivered to tumor tissue was 1.2-36.8 mg. The daily dose delivered ranged between 0.3 and 3.9 mg/day.

There were detectable, although low, concentrations of cisplatin in the BAT catheter. The BAT catheters were up to 30 mm from the supplying catheter (nine patients). This concentration increased during the therapy; the highest concentrations were measured on days 7, 8, and 9. Throughout the treatment, one patient had higher cisplatin concentrations in BAT than in tumor tissue.

Furthermore, in most patients, cisplatin was below or close to the level of detection in subcutaneous tissue after 12 hours and after five to six days. Blood levels of cisplatin were below the level of detection after 12 hours of treatment and very low concentrations were detected after four to seven days of treatment. Blood levels around 5 µg/mL can give rise to

56 systemic toxicity (87). In study III, the concentrations in the blood never reached one-tenth of that concentration.

The pharmacokinetic results of this study demonstrate that retrograde microdialysis is a useful method for delivering active substances to any specific tissue.

CYTOTOXICITY ASSESSED BY GLUTAMATE AND GLYCEROL A significant increase in glutamate and glycerol was observed in tumor tissue compared to BAT. The increase was gradual and peaked after three days of treatment. Thereafter, the elevated levels of the metabolites were fairly consistent. In BAT, glutamate was unchanged and glycerol levels decreased. However, this decrease was not statistically significant. These results show that cisplatin exerted a distinct restricted cytotoxic effect within the desired tumor area without any cytotoxicity in BAT, serum, or subcutaneous tissue. Furthermore, the treated patient did not show any clinical signs of systemic toxicity. This, of course, is very important as the goal of the study was to deliver clinically significant doses of cisplatin to the targeted tumor tissue without any toxicity outside the tumor.

Figure 12. Glutamate and glycerol levels in microdialysis from tumor tissue and BAT are presented before and during treatment with intertumoral cisplatin. Both these molecules are regarded as markers of cell damage and increase abruptly in tumor tissue as the treatment is started 24 hours after implantation of the catheters. (* Statistically significant value with P<0,05, ** P <0,01)

QUALITY OF LIFE, FUNCTIONAL STATUS, AND SURVIVAL Eight patients in the study answered the life quality questionnaires at the first follow-up. One patient died ten days post treatment and one patient was unable to answer the questionnaires. Of the eight patients who answered the questionnaires, three experienced a pronounced decline in their functional status and QoL, the remaining five had only a minor decline. We found large differences in QoL from the start of the treatment, during therapy, and after one and three months. This might reflect the experimental pilot character of this study: a study of patients with a very poor prognosis. The five patients who had only a minor decline, slightly improved shortly after treatment. This improvement was more evident at the one-month follow-up. Finally, five patients were regarded as long time survivals–i.e., survival of 153-492 days. Four of these patients completed their evaluation at three months.

METABOLIC DIFFERENCES IN TUMOR VERSUS BAT AT BASELINE (STUDY IV) In total, 45 different metabolites showed a significant difference in expression in tumor tissue versus BAT. Most evident was the elevated levels of proteinogenic and non-proteinogenic amino acid and their degradation products. The most evident increase in tumor was seen for glycine, alanine, cyanoalanine, proline, and branched-chain amino acids (e.g., leucine, isoleucine, and valine). In addition, elevated levels of metabolic end products (e.g., uric acid and lactic acid) were identified in tumor. Compared to BAT, the tumor had more five-carbon monosaccharides and related five-carbon sugar alcohols, but fewer antioxidative molecules such as ascorbic acid, myo-inositol, and erythritol. In addition, the tumor had fewer purine nucleosides and metabolites used for their synthesis than BAT.

These observed differences in tumor versus BAT could be explained by the higher rate of metabolism and anabolism in tumor compared to normal tissue. Moreover, malignant tumors exhibit a higher oxidative stress when metabolizing molecules with antioxidative properties (47, 49). Patients 3 and 10 had a confirmed IDH-1 mutation and had high concentrations of the oncometabolite D-2-hydroxyglutarate, associated with the mutation of isocitrate dehydrogenase I and II.

TREATMENT EFFECTS ON METABOLITES Metabolite concentrations before treatment were compared with concentrations after three and six days of treatment. These comparisons revealed changes in both tumor and BAT. Following cisplatin treatment, the metabolic events in the tumor tissue were extensive, but changes in BAT were much less. This difference demonstrates that the desired cytotoxic effect of the treatment could be localized to the tumor. There were no correlations found between the doses of cisplatin, side effects, and survival time.

Glutamic Phosphate Spermidine Amino Cysteine Monosaccharides Citric Ascorbic Urea acid acids acid acid Tumor ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ →

Serum ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↑

BAT → → → → → → → → →

Table 3. Metabolic treatment effect in tumor, serum, and BAT. The effect in serum was observed with a delay compared to tumor.

Cisplatin is activated through hydrolysis as it enters cancerous cells. The hydrolyzed molecule is labile and reacts readily with DNA nucleobases to form cisplatin-DNA adducts, causing various cellular responses, culminating in apoptosis (88). Although this is the main mechanism of cisplatin cytotoxicity, the complete picture of cisplatin’s effects within the cell is more complex. Hydrolyzed cisplatin reacts non-discriminately with many biologically important N- donor ligands or carriers of nucleophilic groups including proteins, amino acids, and nucleic acids. Furthermore, cisplatin effectively inhibits mitochondrial respiratory chain complexes and depletes the antioxidant defense system within the mitochondria, resulting in elevated oxidative stress in the cells. In addition, cisplatin easily reacts with acetate, phosphate, and pyrophosphate groups, which in these cases replaces the chloro-ligand. As this molecule reacts with DNA nucleobases, the acetate, phosphate, and pyrophosphate groups are released (88, 89).

In tumor, the levels of glutamic acid were elevated as a sign of cytotoxicity due to the effect of cisplatin and a wide range of amino acids were elevated in tumor as a result of protein catabolism, serving as cellular maintenance and energetic currency. Spermidine, an indicator of disruption of cellular integrity, was also elevated in tumor. Phosphate increased in tumor and showed a clear treatment enhanced pattern (89, 90). This enhancement can be explained by the reaction between DNA nucleobases and cisplatin-phosphate chelates, resulting in the release of the free phosphate. Interestingly, the phosphate levels were higher in the long-survivor group, possibly indicating chemosensitivity with higher turnover and reactivity rate of the phosphate bound cisplatin intermediate in these patients.

Cysteine, monosaccharides, citric acid, and ascorbic acids were significantly reduced during the treatment. Because cysteine binds to metals (cisplatin is a platinum-based antineoplastic medication), the decreased levels of cysteine could be explained due to cysteine-cisplatin binding. As cysteine is needed for the synthesis of the antioxidant glutathione, the accumulation of cysteine-cisplatin complexes will lead to the depletion of cysteine, resulting in elevated levels intracellular ROS and enhanced cell death (91, 92). The reduced cysteine levels could also be due to the high levels of glutamic acid seen in the samples as glutamic acid inhibits cysteine synthesis, which will result in elevated levels of intracellular ROS as discussed above. This increase of intracellular ROS could also explain the decrease of other molecules with antioxidative properties such as citric acid and ascorbic acid (88).

After three days of treatment, BAT had an overall altered metabolite level. However, these changes were small in comparison to the changes observed in tumor and serum samples, and no individual metabolite reached the predefined significance level. The picture seen in the analysis of the serum samples was similar to the one from the tumor although the changes were delayed compared to changes in the samples from tumors.

TREATMENT RESPONSE MARKERS AND SURVIVAL Five patients in the study had a notably longer survival than the other five and their survival was longer than expected from a clinical point of view. With a median survival of 188 days (153–492 days), these patients were classified as long-time survivors following treatment. The rest of the patients were classified as short-time survivors, with a median survival of 34 days (10–72 days). The difference between these two groups could not be linked to age, gender, or total dose of cisplatin.

Figure 13. Kaplan-Meier survival plot of long-time survivors versus short-time survivors.

Using OPLS-DA, simultaneous modeling using survival (long-time vs. short-time survivors) and time point (before vs. after three days) based on all identified metabolites showed significant response models for both tumor and serum. Analysis of the model estimates for both responses illustrates clearly the metabolic differences between timepoints in both tumor and serum. In serum, the differences between short-time and long-time survivors were visible at both timepoints. In tumor, metabolic differences between long-time and short-time survivors were weaker, especially before treatment.

Multivariate OPLS-DA analysis shows significantly different metabolic profiles in tumor and serum for long-time vs. short-time survivors after three days of cisplatin treatment. Interestingly, the largest difference between long-time vs. short-time survivors was mainly in metabolic serum profile before treatment. Several individual metabolites were mainly responsible for the multivariate model separation. In serum of long-time survivors, the metabolites that were in lower concentrations were ketohexoses, mainly fructose, fatty acid methyl esters, glycerol-3-phosphate, and the related 1-palmitoyl-sn-glycero-3-phosphocholin and ⍺-tocopherol. Several metabolites had elevated levels in patients with longer survival. Of these, cis-11-eisenoic acid, erythronic/threonic acid, and phosphate showed enhanced treatment responsive patterns. The distribution of metabolites in microdialysis fluids from tumor were more one-sided in distribution, as almost all altered metabolites were lower in long-time survivors. Levels of lactic acid, glyceric acid, ketohexoses, and the related deoxy sugar fucose/rhamnose were the ones most significantly changed during the treatment period and were also significantly higher in short-time survivors. The metabolites myo-inositol, N-acetyl mannoamine, glutamine, erythronic/threonic acid, and its related alcohol, erythriol, showed clear response to treatment and was significantly reduced in concentration in long-time survivors. Cysteine concentration was lower in long-time survivors. After treatment, all patients had reduced cysteine levels. Finally, we identified phosphate as the sole metabolite in tumor,

60 which was found to be higher in long-time survivors, and found a clear elevation in concentrations in both in tumor and serum after treatment.

In summary, we can state that there are extensive metabolic changes both in tumor and serum due to the cisplatin treatment administered using retrograde microdialysis. An increase in amino acids and a decrease in carbohydrates were evident due to increased energy metabolism and protein catabolism. The cytotoxicity of cisplatin is highly complex and uses several cellular mechanisms. Metabolic patterns and single metabolites, such as phosphate, in serum may be used in the future as potential markers for treatment response.

CONCLUSIONS

This thesis provides proof of the feasibility and safety of microdialysis as a method for real- time assessment of various metabolites and cytokines involved in several metabolic and immunologic processes present in a high-grade glioma tissue. We have shown the usefulness of this technique for assessment of glucose metabolites, glutamate, and glycerol (Study I and II). In study II, we assessed the immune microenvironment of malignant gliomas with the same technique, analyzing variations in cytokine and chemokine concentrations in ECM. Finally, in study IV, we investigated metabolic response patterns in malignant gliomas analyzing microdialysis samples in tumor and BAT together with samples from serum for many different metabolites involved in several cellular processes.

In study I and II, we described the glycolytic properties of malignant glioma metabolism, a finding that corresponds well with earlier studies performed with other modalities. Furthermore, we concluded that fractionated radiotherapy with 2-3.4 Gy daily doses for five days does not induce any changes in glucose metabolism or cell damage to such extent that it could be measured in elevated levels of glutamate or glycerol.

In study II, we demonstrated that malignant gliomas are highly infiltrated with immunocompetent cells such as monocytes, macrophages, and lymphocytes. We observed that fractionated radiotherapy with 2-3.4 Gy daily doses for five days clearly induces a rapid enhancement of the prevailing inflammation in the tumor tissue.

In study III and IV, we provided proof that the microdialysis method can be used to administer cisplatin intratumorally and, at the same time, obtain microdialysates for metabolic monitoring. Metabolomic analysis demonstrated a strong cytotoxic reaction to cisplatin in glioma tissue and important metabolic differences between glioma tissue and BAT. The metabolic response pattern displayed a complex picture regarding cisplatin’s mechanism of action. Metabolic patterns as well as single metabolites in serum could be used as biomarkers for treatment response. These two studies describe the feasibility and safety of retrograde microdialysis. We believe that retrograde microdialysis can be a useful method for interstitial drug delivery to the brain. However, this conclusion needs to be confirmed using controlled clinical trials.

ACKNOWLEDGEMENTS

The studies in this thesis were carried out between 2007-2020 at the neuroscience unit at the institution of clinical science at Umeå University. As all four studies are clinical, much of the work has been carried out at the department of Neurosurgery at Norrland University Hospital. I wish to express my sincere gratitude to all those who helped, guided, and encouraged me during these years.

I wish to especially thank professor Tommy Bergenheim, my supervisor, mentor, and friend, for everything he has taught me during these years. He has been a big inspiration and support not only in the scientific setting but also in the clinical setting during our time together at the neurosurgical department. In addition to the science and neurosurgery, professor Bergenheim have also taught me how important it is to live life and be content, drink good wine, and always visit sky bars if there are any in the vicinity.

I am also in debt to docent Mikael Johansson from the department of Oncology. As my second scientific mentor and supervisor, he has patiently followed me through the years, encouraging and guiding me. His depth of knowledge in the oncological field has been a source of wisdom and inspiration.

I wish to thank docent Thomas Asklund for his invaluable contribution both clinically and scientifically during study I and study II. He has been a source of knowledge and introduced me to the field of radiation therapy.

I wish to express my sincere gratitude to Benny Björkblom, Erik Björn, Henrik Antti, Edward Visse, Per Bergström, Thomas Brännström, Pär Jonsson, Roger Henriksson and Peter Siesjö, who all have contributed and been an important part of the different studies in this thesis. Without them this thesis would not have been possible.

I would like to thank Kristin Nyman at the department of Neurosurgery for her technical skills in managing the microdialysis bedside and monitoring our studies. She has been an invaluable asset for our institution and our research team.

I wish to thank docent Peter Lindvall, our head of department, for having understanding with me during the last year and enabled time for research while doing my clinical work at the department. I wish also to direct a big thank you to all my great colleagues at our department for their support and understanding during the last year. I would also like to express my gratitude to professor Lars Ove Koskinen for his support both as our professor enabling financial support for my research work and as a colleague.

I have a great number of friends who both have guided me and supported me during these years. I would like to direct a special thanks to Björn, Greg and Hani, who have supported me during the last year. I am also grateful to Björn, Greg and Amar for their excellent job with proofreading this thesis.

I wish to thank my family, especially my mother, sister and my uncle Reza, for all their support and love. My mother has always encouraged me to pursue scientific studies and supported me

63 since medical school with everything from her wisdom and experience to her fantastic Persian food boxes when I am on call. I wish to thank my sister, Termeh, for being the best loving sister one can wish for. She has always supported me in all my endeavors. Reza has always been so much more than just an uncle. He has always been more like a wise elder brother, always supporting me and being by my side at every big decision in my life.

My dear Elsa is my motivation and what gives me my energy and strength every day. I am so thankful for having her in my life as my best friend and biggest love. I am thankful for every day together with her and al here support and love. She is always kind, patient, and loving, making me to feel special and happy every day.

I also wish to send my love and thank to my two pugs, Walter and Gösta, for always keeping me company, even when I am extremely boring while working on my computer. They have been extremely patient with me during the last year, snoring by my side as this thesis has taken form.

This study was financially supported by the Swedish Cancer Society, Umeå University Hospital, the Swedish Research Council, the Cancer Research Foundation in Northern Sweden, the Research Foundation of Clinical Neuroscience, Umeå University, and by the regional agreement between Umeå University and Västerbotten County Council on the cooperation in the field of Medicine, Odontology, and Health.

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