Cell lines
The H1 cell line was generated from a human melanoma brain metastasis and transduced with lentiviral vectors expressing the genes for GFP and luciferase (H1_DL2) as previously described [60], or shRNAs targeting NDUFA8 (H1_shNDUFA8; TAGAAGACGCACCGGCGGTGTTTAGGGGAAGGTAAAGTTAATATTCATAGCTTTGCCTTCCTCTAAACACCGTTTTTTGGCAAGCAAAAGACGGCATACGAGATATGTACCAGTCAGTACCAGTTTCGCCGTCTTCGT). The Melmet 1 and Melmet 5 melanoma cell lines were developed from a skin and a lymph node metastasis, respectively, and were kindly provided by Ø. Fodstad (University of Oslo, Oslo, Norway). The A375 cell line was purchased from the American Type Culture Collection (ATCC). A human lung fibroblast cell line, SV-80, was purchased from CLS Cell Lines Service GmbH. The PC14-PE6 cell line was kindly provided by F. Winkler (University Hospital Heidelberg & German Cancer Research Center, Heidelberg, Germany), and a brain seeking subline PC14_PE6_Br2 was generated in our laboratory as described earlier [27]. The Melmet 1 and Melmet 5 cell lines were transduced with a lentiviral pGF1-CMV reporter vector that co-expresses copGFP and firefly luciferase linked by the self-cleaving peptide T2A (System Biosciences). The immortalized human melanocytes-hTERT (cat. no. T0463) and astrocytes (cat.no. T0281) were both purchased from Applied Biological Materials (Richmond, BC, Canada). The primary epidermal melanocytes HEMa (cat.no. PCS-200-013) were purchased from ATCC. The H1, Melmet 1, Melmet 5 and A375 cell lines have the BRAFV600E mutation. The transduced cell lines were used in all experiments, authenticated within the last six months using short tandem repeat (STR) profiling, and maintained as previously described [57].
In vivo generation of samples for RNA sequencing
Eight weeks old female NOD.CB17-Prkdcscid/NcrCrl mice were purchased from Charles River Laboratories International. Anesthesia was induced with 3% and maintained with 1.5% isofluorane in oxygen. We performed intracardiac injections in seven mice (5 × 105 H1_DL2 cells suspended in 0.1 mL PBS) as previously described [60]. Whole-body bioluminescence imaging (BLI) was used to evaluate injection failure 10 min post-injection and weekly over seven weeks to monitor metastasis formation (Additional file 2: Figure S2a) using a Xenogen Ivis 100 Small Animal Molecular Imager (Xenogen Corporation) as previously described [60]. At seven weeks, we injected 150 mg/kg D-luciferin Firefly (Gold Biotechnology), sacrificed the mice ten min later, and performed ex vivo BLI to evaluate organ involvement in detail. All animals invariably developed metastases in their brain, adrenals, ovaries, and femurs, and these organs from five animals showing the highest tumor burden on BLI, were dissociated using tailored protocols based on the Liberase TM Research Grade enzyme reagent (Roche Applied Science; Additional file 3 Table S1). Sample yields of GFP positive tumor cells were checked by fluorescence microscopy prior to cell sorting (Additional file 2: Figure S2b) using the BD Influx high-speed cell sorter (BD Biosciences). We aimed for a minimum of 150,000 cells in each sample, and picked the three tumor cell samples from each organ with the most cells for subsequent RNA sequencing: 1) brain (151,023, 150,835 and 154,797 cells); 2) adrenals (151,968, 184,506 and 276,146 cells); 3) ovaries (159,736, 171,814 and 433,703 cells); and 4) femurs (120,530, 91,055 and 73,942 cells). These 12 samples (3 samples × 4 organs) of metastatic melanoma cells were kept in 1% FBS medium, pelleted by centrifugation, transported on dry ice and stored in a − 80 °C freezer.
RNA isolation
Total cellular RNA was isolated from the cell pellets using the TRIzol reagent (Life Technologies) and a modified protocol that incorporates an additional extraction with phenol/chloroform/isoamyl alcohol (25:24:1, pH 4.3). RNA quantity and quality were assessed on a NanoDrop spectrophotometer (Thermo Scientific) and the Agilent 2100 Bioanalyzer (Agilent Technologies), respectively.
Library preparation for RNA sequencing
RNA sequencing (RNA-Seq) libraries were prepared from 1.0 μg total RNA using the TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer’s protocol. Briefly, poly-adenylated mRNA was purified from total RNA and ribosomal RNA removed by two rounds of binding to magnetic poly-dT beads. This was followed by RNA fragmentation by incubation in the presence of divalent cations at 94 °C for 5 min. Double-stranded cDNA was then generated by random-primed first-strand synthesis with SuperScript II reverse transcriptase and subsequent second strand synthesis with RNase H and DNA Polymerase I. The cDNA was then blunt-ended with T4 and Klenow DNA polymerases to remove the 3′-overhangs and fill in 5′-overhangs, phosphorylated with T4 PNK, and then 3′-A tailed by incubation with Klenow Fragment (3′ → 5′ exo–) and dATP. Illumina paired-end (PE) adapters were then ligated, followed by purification with AMPure XP beads. The library was then enriched by high-fidelity PCR amplification (15 cycles) with Phusion DNA Polymerase (Finnzymes Oy) and adapter-specific primers. The molar concentration of the libraries was determined by measuring concentration with a Qubit fluorometer (Invitrogen), determining the insert length with an Agilent 2100 Bioanalyzer, and then qPCR-based quantification (KAPA Library Quantification Kit).
RNA sequencing
Indexed libraries were pooled, loaded on TruSeq paired-end flow cells, and paired-end sequencing (2 × 100 bp, paired-end; 4 libraries/lane) was performed with an Illumina HiSeq 2000 sequencing system (BGI@UC Davis Joint Genome Center) using standard Illumina kitted reagents (TruSeq SBS Kit v3-HS). Image processing, base calling, quality scoring (Phred), and sample demultiplexing were executed by HiSeq Control Software with Real Time Analysis (HCS 1.5/RTA 1.13) and CASAVA 1.8 software (Illumina). The Tuxedo protocol (http://compbio.mit.edu/cummeRbund) was followed using the alternate protocol of quantification of reference annotation only. We used Ensembl’s human genome build 19, and supplied TopHat with a set of known transcripts from Ensembl. The three BAM files from each individual organ were merged using Picard 1.91 (http://broadinstitute.github.io/picard), and these merged files were subsequently visualized using Integrative Genomics Viewer 2.3 (http://www.broadinstitute.org/igv). The cummeRbund package (http://compbio.mit.edu/cummeRbund) and Partek Genomics Suite 6.12 (Partek Inc.) were used to produce visualizations (Additional file 2: Figure S2c).
Gene expression analysis and brain metastasis gene signature
We developed an integrated workflow of several independent analyses to build a brain metastasis gene expression profile (Fig. 1b and Additional file 2: Figure S2d). First, there were 134 genes differentially expressed in brain metastases and metastases to any of the other organs; 122 genes were upregulated and 54 were downregulated in brain (some genes appeared in multiple lists). Second, we used Prediction Analysis for Microarrays (PAM; http://statweb.stanford.edu/~tibs/PAM) to identify which of the 134 genes consistently distinguished brain metastases from other organ metastases. We found ten upregulated genes specific to brain metastases. Third, we validated eight of these ten genes using a combination of other computational analyses: A supervised rank product analysis (RankProd; http://www.bioconductor.org/packages/release/bioc/html/RankProd.html) of brain metastases compared with all the other organs pooled together (pfp < 0.01); a meta-analysis of rank product analyses (RankProd) of brain metastases versus each of the other organs (pfp < 0.01); and significance analysis of microarrays (http://statweb.stanford.edu/~tibs/SAM) with a q-value cutoff of 0.05. Fourth, we performed hierarchical clustering using J-Express 2012 (http://jexpress.bioinfo.no/site) and identified 46 genes that clustered together with the cross-validated eight-gene list; these 54 up-regulated genes were used to build a brain metastasis gene signature. Fifth, to enable analysis with the Connectivity Map (cMap) database, we appended the 54 most downregulated genes from the supervised rank product analysis (RankProd) of brain metastases when compared with all the other organs pooled together; all of these 54 downregulated genes were also featured in the 134-gene list of differentially expressed genes, as described above. Thus, the 108-gene brain metastasis signature was comprised of 54 upregulated and 54 downregulated genes (Additional file 2: Figure S2d).
Computational prediction of candidate compounds
We queried the cMap build 2 database (http://www.broadinstitute.org/cmap) using our 108-gene signature for candidate drugs. cMap is an initiative by the Broad Institute of MIT and Harvard, where the effects of 1309 small molecules on a number of cultured human cells were examined systematically, resulting in a collection of more than 7000 expression profiles that can be freely queried using their web interface. This resource can be used to select for drugs that produce a disease-negating gene signature. In this study, cMap was hypothesized to predict drugs with the potential to induce opposite expression profiles to those observed in our brain metastasis gene signature (Additional file 2: Figure S2e). Before cMap analysis, gene symbols were mapped to HG-U133A probe IDs (microarray used in cMap). The HG-U133A probe file (Platform GPL96) was downloaded and mapping was done with a code written in our laboratory. Of the 54 upregulated genes, 37 genes had at least one matching probe and 55 probes were retrieved in total. Of the 54 downregulated genes, 29 genes had at least one matching probe, and 40 probes were retrieved in total. Mappings were subsequently verified with the GeneAnnot (http://genecards.weizmann.ac.il/geneannot/index.shtml) and GeneCards (http://www.genecards.org) databases.
Candidate drugs
Nine drugs were purchased from Santa Cruz Biotechnology, Inc.: Meticrane, tolazamide, β-sitosterol, memantine hydrochloride (herein referred to as memantine), valproic acid, letrozole, todralazine, thiostrepton, and levocabastine. Niridazole was not available. For in vitro screening, all compounds were dissolved in 100% dimethyl sulfoxide (DMSO) to a stock concentration of 250 mM. All stock solutions were subsequently diluted in complete DMEM to nine working solutions ranging from 0.2 to 1312.3 μM. This dose spectrum covered well below and above the reported dose levels for all drugs described in cMap. PLX4720 (ChemieTek) was solubilized and diluted in a similar manner to nine working solutions ranging from 0.01 to 1562.5 μM. Prior to in vitro testing, we pre-warmed (37 °C) and sonicated all working solutions.
Proliferation assays
Cells from four BRAF mutated cell lines (H1_DL2, Melmet 1, Melmet 5, A375) and three normal cell lines (SV80; fibroblasts, hTERT melanocytes and human astrocytes) were quantified using a hemocytometer and seeded into 96-well plates (5000 cells per well) in 100 μL complete DMEM. After 24 h incubation, we added 100 μL of the nine graded candidate drug dilutions, PLX4720, or 100 μL 0.1% or 1.0% DMSO in complete DMEM to each well (n = 18 per cell line per drug per drug concentration). Three days later, we added 20 μL of resazurin 0.1 mg/mL (Sigma-Aldrich Co.) per well and read the plates 4 h later (Fig. 2a and Additional file 4: Figure S3a-f) using a VICTOR X3 multilabel plate reader (PerkinElmer) with Workout 2.5 data analysis software (560 nm excitation and 590 nm emission). Wells with 200 μL complete DMEM and no cells were used for background correction (n = 60). IC50 values were calculated (Fig. 2c, and Additional file 4: Figure S3) using GraphPad Prism 6 for Mac OS X (GraphPad Software Inc.).
Cells were fixed by adding 50 μL 8% paraformaldehyde (PFA) per well for 24 h after which time the liquid contents of all wells were replaced with 50 μL PBS. Differential interference contrast (DIC) and 4′,6-diamidino-2-phenylindole (DAPI) image stacks were acquired (Fig. 2a) with 10× magnification using a Nikon Eclipse TE2000 inverted microscope (Nikon Instruments Inc.).
Tumorsphere assay
A standardized 3D tumor spheroid growth assay was carried out as reported previously [58]. Briefly, 100 μL of the graded drug dilutions and DMSO controls were added to 96-well plates with 4000 cells in soft agar per well (n = 6 per cell line per drug concentration). After a ten-day incubation period, a resazurin assay was performed, and cells were fixed as described above. DIC and GFP z-stack images were acquired with 10× magnification using a BD Pathway 855 High-Content Imager (Becton, Dickinson and Company).
Tumor cell injections, quantification of tumor cell load in the brain and multimodal imaging of metastasis formation
Six to eight weeks old female NOD/SCID mice were purchased from the University of Bergen animal facility. Anesthesia was induced with 3% and maintained with 1.5% isoflurane in oxygen. Mice were monitored daily and sacrificed upon signs of illness. Intracardiac injections were performed using ultrasound-guidance (5 × 105 cells per 0.1 mL PBS per mouse) as reported previously [58]. H1_DL2 cells were pre-labeled with superparamagnetic iron oxide nanoparticles and MRI was carried out 24 h after injections [57]; T2*-weighted images for automated quantification of tumor cell load in the mouse brains and T2-weighted images to assess the presence of focal brain lesions. MRI equipment and sequence details have previously been described [58]. For T2*-weighted quantification we developed a cell-line specific training set for the integrated neural network analysis. Only mice with comparable tumor cell load in their brains and without ischemic lesions were included in further studies. We omitted five mice in the candidate drug study and six mice in the β-sitosterol study, based on an uneven tumor load to the brain.
We performed brain MRI to evaluate the brain metastatic burden with T2-weighted sequences and pre−/post-contrast T1-weighted sequences as previously described [58]. Images were obtained at pre-defined week numbers (Figs. 3a, b and 4a, b). The number of tumors, contrast enhancement and volume (4/3 × π × r3) were assessed using OsiriX 5.8.1 32-bit (Pixmeo SARL).
In parallel with the MRI, we did BLI to evaluate brain metastatic burden. Mice were imaged 10 min after i.p. injection of 150 mg/kg D-luciferin Firefly (Gold Biotechnology). To reduce distress, they were not depilated. Images were acquired using an Optix MX3 Small Animal Molecular Imager (ART Inc.) and analyzed with Optix OptiView 3.02 (ART Inc.). Total photon counts (ventral + dorsal) were registered from identically sized regions of interest (ROIs) placed over the head.
In vivo treatment protocols
In the drug screening study on H1_DL2 brain metastases, treatment started one week post-injection and stopped at the end of week seven (Fig. 3a and Additional file 5: Figure S4). Test groups received 0.2 mL i.p. injections of 40 mg/kg thiostrepton every second day (n = 5), 10 mg/kg memantine daily (n = 5), 5 mg/kg β-sitosterol daily (n = 4), or 0.5% DMSO (n = 5) daily. Stock solutions of the respective drugs, as described above, were diluted with saline solution, pre-warmed (37 °C) and sonicated prior to i.p. injections (the partly miscible thiostrepton solution was homogenized before administration). Fresh solutions were made every second week and stored at 4 °C.
In the validation study with β-sitosterol treatment on H1_DL2 cells and PC14_PE6_Br2 brain metastases, treatment started one week prior to intracardiac injections and was continued until sacrifice for the vehicle groups (n = 10) and for ten weeks for the β-sitosterol groups (n = 9). Test groups received daily i.p. injections of 0.1 mL olive oil or 5 mg/kg β-sitosterol in 0.1 mL olive oil. β-sitosterol was mixed with olive oil (Santa Cruz Biotechnology Inc) and solubilized over 4 h with a heated magnetic stirrer (50 °C). Fresh solutions were made every second week and stored at room temperature.
In the subcutaneous tumor model, we used the Melmet 5 melanoma cell line. We injected 1 × 106 cells in 0.1 mL PBS in the cervical skin fold of 32 mice. After two weeks, when the average tumor volume was approximately 30 mm3, the mice were randomized to daily i.p. injections of 0.1 mL vehicle (olive oil), 20 mg/kg β-sitosterol in olive oil, 25 mg/kg PLX4720 in 0.05% DMSO, or a combination of the two latter (n = 8 in each group). Caliper measurements were carried out every sixth day to evaluate tumor growth. Tumor volume was calculated using the formula (width2 × length)/2.
Functional classification of the brain metastasis gene signature
We performed a functional classification of human biological processes and signaling pathways associated with the 108-gene signature using the Protein Analysis Through Evolutionary Relationships (PANTHER) classification system (http://www.pantherdb.org/).
Protein interactions of β-sitosterol
We examined known and predicted protein interactions of β-sitosterol using the Search Tool for Interactions of Chemicals (STITCH 4.0; http://www.stitch.embl.de). We applied a high confidence level cut-off (0.700) and a maximal number of interactions (n = 50). We next investigated with which cellular processes these protein targets were associated using the Human Experimental/Functional Mapper (HEFalMp).
Protein phosphorylation assay
For the determination of the relative levels of protein phosphorylation of 43 kinases and 2 related total proteins (HSP60 and β-Catenin), we used the Human Phospho-Kinase Array (R&D Systems, Inc.) as specified by the manufacturer. H1_DL2 cells were treated with vehicle (0.05% DMSO) or β-sitosterol (50 μM) for 24 h.
Linkage analyses to mitochondrial metabolism
We obtained the mitochondrial metabolism genes from the Gene Ontology (GO) database using AmiGO (http://amigo.geneontology.org/amigo) and the search term «oxidative phosphorylation» (filter: Homo sapiens). This resulted in 121 unique gene symbols among a total of 209 gene-term associations. β-sitosterol targets were extracted from ChEMBL (Chemical Database of the European Molecular Biology Laboratory; https://www.ebi.ac.uk/chembl): 16 targets reported in humans. CHEMBL_IDs were converted to gene symbols with UnitProtKB (Universal Protein Resource Knowledgebase; http://www.uniprot.org). Network generation and visualization were implemented in Cytoscape 3.3.0 (http://www.cytoscape.org). Interactions between query and mitochondrial metabolism gene sets were extracted with ANAT (Advanced Network Analysis Tool; http://www.cs.tau.ac.il/~bnet/ANAT). β-sitosterol targets and signature genes were defined as network anchors, and the oxidative phosphorylation genes as network terminals. Anchored network analysis was applied with focus on: human interactions, protein-protein and protein-DNA interactions and default search parameters. Interaction directionality from anchors to terminals was specified.
Extracellular flux analysis
Extracellular flux analysis with concurrent detection of oxygen (mitochondrial respiration) and pH (lactate production indicating glycolysis) was employed. The measurements were performed in 96-well H1_DL2 and HEMa cell cultures using the Seahorse XF96 Analyzer system (Seahorse Bioscience), according to manufacturer’s protocol. The assay conditions were optimized with regard to cell number and concentrations of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin. Cells were seeded in quadruplicate wells (2 × 104 cells per well), and exposed to 50 μM β-sitosterol for 24 h before the analysis. Control cells received 0.05% DMSO. Following this treatment, the growth medium was replaced with assay medium consisting of phenol-free DMEM supplemented with 2 mM L-glutamine and 2 mM sodium pyruvate. 10 mM glucose was added to the medium for measurement of mitochondrial respiration. The assay medium was adjusted to a pH of 7.4. The cells were incubated at 37 °C in a CO2-free incubator (Seahorse XF Prep station) for 1 h before they were transferred to the analyzer. To investigate mitochondrial respiratory function, assessment of the initial oxygen consumption rate (OCR), indicating the basal rate, was followed by sequential additions of several modulators: 1) the ATP synthase inhibitor oligomycin (3 μM for H1_DL2 and 2 μM for HEMa cells) was used to measure phosphorylation independent respiration (Leak); 2) the uncoupler CCCP or FCCP (1.5 or 2 μM for H1_DL2 and HEMa cells, respectively) to measure the capacity of the electron transport system (respiratory capacity); 3) the Complex I (CI) inhibitor rotenone (1 μM) to determine CI independent respiration; and 4) the CIII inhibitor antimycin A (1 μM) to assess residual background OCR not related to mitochondrial respiration; this was subtracted as background from the other measurements in the statistical analysis. To analyze the glycolytic function, the extracellular acidification rate (ECAR) was measured after sequential additions of glucose (10 mM) to determine basal glycolysis and oligomycin (3 μM) to obtain glycolytic capacity. CCCP (1.5 μM) was injected to address the possible influence of uncoupling, and 2-deoxyglucose (2-DG; 100 mM) was used to obtain the non-glycolytic background. In statistical analysis, the residual ECAR was subtracted as background from the other measurements. All experiments were performed three times.
High-resolution respirometry
Oxygen consumption was analyzed using the Oxygraph O2K instrument and DatLab software (Oroboros Instruments). The H1_DL2 cells were harvested and washed in PBS before they were suspended in the assay medium (20 mM K-hepes, 83 mM KCl, 4 mM KH2PO4, 1 mM EGTA and 1 mM MgCl2) and transferred to the assay chamber (37 °C) at 1.25 × 106 cells per 2 mL assay medium. Digitonin (8.1 μM) was added to permabilize the cell membrane. The combined CI + CII-driven respiration was obtained in the presence of malate (2 mM), pyruvate (1 mM), succinate (10 mM), and FCCP (titrated to 0.18 μM). Following injection of β-sitosterol (50 μM) or DMSO (0.05%) and assessment of the resulting OCRs, rotenone (0.5 μM) was injected to determine if the effect was linked to CI and/or CII. Antimycin A (2.3 μM) was added to provide the non-mitochondrial background activity, which was subtracted during data analysis. This experiment was performed three times.
Measurement of reactive oxygen species and apoptosis assay
H1_DL2 cells were treated with 0.05% DMSO or 50 μM β-sitosterol for 24 h, and apoptosis was detected and quantified using the disodium salt of 3, 4, 5, 6,-tetrachloro-2′, 4′, 5′, 7′-tetraiodofluorescein (TCTF) as previously described (n = 3) [34]. The cellular content of reactive oxygen species (ROS) was measured using the CM-H2DCFDA probe according to the manufacturer’s instructions (Life Technologies). Briefly, naïve H1 cells were treated with 0.05% DMSO or 50 μM β-sitosterol for 2 h before exposure to 5 μM CM-H2DCFDA for 15 min (two experiments with triplicate measurements). Data were analyzed using a BD Accuri C6 flow cytometer (BD Biosciences).
Western blot
Protein extraction and western blotting was carried out as described previously [58]. Antibodies were diluted in blocking buffer and the following antibodies were used: Caspase-3 (1:500) and PGC1α (1:500) (Cell Signaling Technology); and NDUFA8 (ab74126; 1:500), GAPDH (1:20,000) and β-actin (1:20,000) (Abcam).
MitoTracker red fluorescence
MitoTracker Red (Invitrogen) mean fluorescence intensity was measured in H1 cells treated with DMSO (0.05%) or PLX4720 (1.5 μM) for 72 h (n = 3). Data were analyzed using a BD Accuri C6 flow cytometer (BD Biosciences).
Colony formation assay
5 × 103 H1 cells were cultured in 6-well plates and treated with DMSO (0.05%), PLX4720 (1.5 μM), β-sitosterol (50 μM), or a combination of the latter two for one (controls) or three weeks (treatment groups) (n = 3). The same protocol was used for H1_shNDUFA8 cells, but these were not treated with β-sitosterol.
Immunohistochemistry of human brain metastases
We stained 197 formalin-fixed and paraffin-embedded human brain metastases using NDUFB8 (ab110242; Abcam). Samples were derived from melanoma (n = 78), non-small cell lung cancer (n = 52), breast cancer (n = 25), renal cell cancer (n = 9), colon cancer (n = 9), small cell lung cancer (n = 7), carcinomas not otherwise specified (n = 9) and other rare cases like ovarian and esophageal cancer (n = 8). All staining procedures were performed using an automated immunostainer (Leica Bond III) and analyzed by ≥ two observers using a semi-quantitative score (H-Score) ranging from 0 to 300. H-Score is defined by the staining intensity (1 = weak, 2 = moderate, 3 = strong) multiplied with the frequency of positive cells (%).
Statistics
Statistical analyses were conducted with SPSS 21 for Mac (SPSS Inc.) and Prism 7 for Mac, Version 7.0b (La Jolla, CA, USA). An independent samples Student’s t-test was used to compare two normally distributed groups. The Kruskal-Wallis test was used to analyze nonparametric data and Dunn’s or Sidak’s test was used to correct for multiple testing. Levene’s test was used to assess the variance. Kaplan-Meier survival analysis and Mantel-Cox log-rank test were used to assess survival differences between groups. Values are presented as means ± standard error of the mean (s.e.m.) unless otherwise specified. A two-tailed P ≤ 0.05 was considered significant.
Study approval
We obtained written informed consent before human tumor material was collected. The Regional Ethical Committee and the Norwegian Directorate of Health approved the collection and storage of human tissue. The Institutional Animal Care and Use Committee at the University of California Davis and at the University of Bergen approved the mouse experiments. At both institutions, mice were maintained in animal facilities certified by the Association for Assessment and Accreditation of Laboratory Animal Care International. The local ethical committee at the University Cancer Center Frankfurt approved the immunohistochemical analyses of human brain metastases.