Neurochem Res DOI 10.1007/s11064-014-1431-y

OVERVIEW

Classification of Subpopulations of Cells Within Human Primary Brain Tumors by Single Cell Gene Expression Profiling Elin Mo¨llerstro¨m • Bertil Rydenhag • Daniel Andersson • Isabell Lebkuechner Till B. Puschmann • Meng Chen • Ulrika Wilhelmsson • Anders Sta˚hlberg • Kristina Malmgren • Milos Pekny

•

Received: 27 May 2014 / Revised: 2 September 2014 / Accepted: 3 September 2014 Ó Springer Science+Business Media New York 2014

Abstract Brain tumors are heterogeneous with respect to genetic and histological properties of cells within the tumor tissue. To study subpopulations of cells, we developed a protocol for obtaining viable single cells from freshly isolated human brain tissue for single cell gene expression profiling. We evaluated this technique for characterization of cell populations within brain tumor and tumor penumbra. Fresh tumor tissue was obtained from one astrocytoma grade IV and one oligodendroglioma grade III tumor as well as the tumor penumbra of the latter tumor. The tissue was dissociated into individual cells and the expression of

36 genes was assessed by reverse transcription quantitative PCR followed by data analysis. We show that tumor cells from both the astrocytoma grade IV and oligodendroglioma grade III tumor constituted cell subpopulations defined by their gene expression profiles. Some cells from the oligodendroglioma grade III tumor proper shared molecular characteristics with the cells from the penumbra of the same tumor suggesting that a subpopulation of cells within the oligodendroglioma grade III tumor consisted of normal brain cells. We conclude that subpopulations of tumor cells can be identified by using single cell gene expression profiling.

Special Issue: In Honor of Michael Norenberg.

Keywords Astrocytoma grade IV  Oligodendroglioma grade III  Glioblastoma multiforme  Single cell gene expression profiling

E. Mo¨llerstro¨m (&)  D. Andersson  I. Lebkuechner  T. B. Puschmann  M. Chen  U. Wilhelmsson  A. Sta˚hlberg  M. Pekny (&) Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, Box 440, 405 30 Go¨teborg, Sweden e-mail: [email protected] M. Pekny e-mail: [email protected] B. Rydenhag (&)  K. Malmgren Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, Go¨teborg, Sweden e-mail: [email protected] D. Andersson  A. Sta˚hlberg Sahlgrenska Cancer Center, Department of Pathology, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Go¨teborg, Sweden M. Pekny Florey Institute of Neuroscience and Mental Health, Parkville, VIC, Australia

Introduction Quantitative measurement of mRNA isolated from nondissociated tissue reflects average mRNA expression levels within the tissue and thereby masks variation in gene expression between individual cells. The gene expression of a specific group of cells, in particular when these constitute a minority subpopulation, can be hidden in the noise of other cells within the tissue [1, 2]. In contrast, single cell gene expression profiling using reverse transcription quantitative PCR (RT-qPCR) is a method to analyze gene expression at the level of individual cells and allows to obtain their true molecular signature. Until recently, analyses of single cells have usually been performed by using imaging techniques such as immunocytochemistry, immunohistochemistry, FISH, or by flow cytometry. These techniques assess morphological

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properties, DNA copy numbers or largely qualitative expression of specific proteins [3]. By implementing single cell gene expression profiling, it is possible to detect, measure and correlate the expression of a larger number of genes within individual cells. Single cell gene expression profiling reveals molecular properties of the cells and complements as well as substantially expands histological classifications of cells and tissues. Single cell gene expression profiling starts to be implemented in the characterization of several types of malignant tumors, for example colon cancer [4], circulating melanoma cells, or breast cancer cells [5, 6]. We previously applied this technique on primary astrocyte cultures and neurosphere cells and it allowed us to identify distinct subpopulations of astrocytes and to make interaction maps for the assessed genes [7]. In 2011, 1 349 patients in Sweden were diagnosed with brain tumors or tumors in other parts of the nervous system, which corresponds to 2.5 % of total cancer incidence [8]. The majority of these were gliomas [8]. Gliomas arise from either neural stem/progenitor cells or glial cells within the nervous system and astrocytomas and oligodendrogliomas are the most common subtypes. The survival varies widely depending on the tumor grade [9]. Oligodendrogliomas are generally divided into grade II and grade III, where grade II patients have a median survival time of 11.6 years and grade III patients 3.5 years. When it comes to astrocytomas, 96 % of patients with grade I astrocytomas survive for more than 10 years from diagnosis, whereas median survival time for astrocytomas grade IV (glioblastoma multiforme) patients is only 0.4 years. Unfortunately, astrocytomas grade IV account for 69 % of all astrocytomas and oligodendrogliomas [9], and therefore most glioma patients have a low long-term survival. Individual gliomas are known to be very heterogeneous, both genetically and histologically, and with respect to the expression of specific molecules [10–12]. The existence of selfrenewing cancer stem cells, responsible for the tumor initiation and progression [13, 14] has been postulated and it is possible that many of the cells within a tumor are tumor supporting cells rather than highly proliferative tumor cells. Bonavia et al. [15] compare an astrocytoma grade IV tumor to a community where all different types of cells contribute to create a tumor-beneficial environment within the cancer tissue. In this study, we developed a protocol for obtaining molecular signature of individual cells from fresh adult human brain tissue, using single cell gene expression analysis of selected genes. This was used to assess the molecular differences and similarities between cell populations within brain tumors and the tumor penumbra tissue.

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Methods Tumor Tissue Tumor and tumor penumbra tissue was collected during surgery from a male, 49 years old, diagnosed with astrocytoma grade IV (tumor tissue) and a male, 63 years old, diagnosed with oligodendroglioma grade III (tumor tissue and tumor penumbra; Fig. 2). Ethics Statement The patients’ written informed consent was obtained and tumor tissue was collected in accordance with the ethical guidelines of the Sahlgrenska Academy University Hospital (approved by the Regional Ethics Board, registration number 179-08, 2008-06-12). Tissue Dissociation The tissue specimens were collected at the surgical theatre and kept in ice cold Hibernate A medium (Brain Bits, Springfield, Illinois, USA) for approximately 20 min before enzymatic and mechanic dissociation. To dissociate the tissue into single cells, Brain tumor dissociation kit (Trypsin) from Miltenyi Biotech (Cologne, Germany) was used according to the manufacturer’s protocol with minor modifications (plastic 1 ml tips were used instead of firepolished Pasteur-pipettes). Briefly, the tissue was cut into small pieces, incubated with enzymes at 37 °C and mechanically dissociated via titration, and then passed through a 70 lm cell strainer. To remove red blood cells and myelin debris the single cell suspension was treated with Pharmlyse (Becton–Dickinson, Franklin Lakes, New Jersey, USA) followed by magnetic associated cell sorting (MACS) using Myelin removal beads II (Miltenyi Biotech) according to the manufacturers´ protocols. Briefly, cells were washed with Pharmlyse, followed by a wash with MACS buffer (PBS containing 0,5 % BSA, autoMACS rinsing solution and MACS BSA stock solution 20:1, Miltenyi Biotech). Then, the cell suspension was incubated with magnetic beads connected to myelin antibodies, passed through a magnetic field where the myelin debris remained and the flow-through was collected, leaving a suspension of viable single cells. The cells were kept in MACS buffer on ice until sorted by FACS. Collection of RNA from Individual Cells To place single cells in separate wells of a 96-well plate (Applied Biosystems, Life Technologies, Carlsbad, California, USA), the flow-cytometric cell sorting function of a

Neurochem Res

FACS Aria II (Becton–Dickinson) was used. The cells were sorted into wells containing 5 ll Ultrapure water (Gibco, Life Technologies, Carlsbad, California, USA) with 1 lg/ll bovine serum albumin (BSA; Fermentas, Thermo Fisher Scientific, Waltham, Massachusetts, USA). This immediately lysed the cells and made the RNA available for further experiments. The plate containing lysed single cells was snap frozen on dry ice and stored at -80 °C until reverse transcription was performed. Reverse Transcription (RT) RT was carried out using SuperScriptTM III Reverse Transcriptase (Invitrogen, Life Technologies, Carlsbad, California, USA). 1.5 ll suspension containing dNTPs (Sigma-Aldrich, Munich, Germany), oligo(dT) 15 primers and random hexamers (both Eurofins MWG Operon, Ebersberg, Germany) were added to the 5 ll of RNA suspension and the mixture was incubated at 65 °C for 5 min. Next, 3.5 ll suspension containing 59 SuperScript buffer, RNaseOut, dithiothreitol (DTT) and SuperScript III enzyme (all Invitrogen) was added and the plates were incubated at 25 °C for 5 min, 50 °C for 60 min, 55 °C for 15 min and 70 °C for 15 min using CFX 96 Real Time System (Bio-Rad, Hercules, California, USA). The final concentrations in a total reaction volume of 10 ll were 0.5 mM dNTPs, 2.5 lM oligo(dT) 15 primers, 2.5 lM random hexamers, 19 SuperScript buffer, 5 mM DTT, 10 U RNaseOut and 50 U Superscript III enzyme. Preamplification The primers for the genes of interest (Tables 1, 2) were designed using the Primer 3 software (http://frodo.wi.mit. edu/primer3/input.htm), tested for primer dimers in Netprimer (Premier Biosoft International, Palo Alto, California, USA) and checked for specificity using the BLAST algorithm (NCBI, NIH, Bethesda, Maryland, USA) and obtained from Sigma-Aldrich. They were designed to span introns when possible and expected qPCR product length was confirmed using agarose gelelectrophoresis (Invitrogen). For preamplification, 20 ll IQ supermix (Bio-Rad), 1.6 ll primerpool (containing 1 lM of each primer) and 14.4 ll Ultrapure H2O (Gibco) were added to 4 ll of cDNA for each sample in a total volume of 40 ll. Each 96-well plate contained three positive controls with cDNA extracted from solid tumor tissue and three negative controls containing H2O. The mixture was incubated at 95 °C for three minutes, followed by 14 cycles of 95 °C for 20 s, 55 °C for 3 min and 72 °C for 20 s, in a LightCycler 480 (Roche Diagnostics, Basel, Switzerland). The run was

Table 1 List of the assessed genes Gene

Gene name

Function

AIF1

Allograft inflammatory factor 1

Cell M

ALDH1L1

Aldehyde dehydrogenase 1 family, member L1

Cell A

CCNE1

Cyclin E1

CC

CD63

CD63 molecule

BM

CDK4

Cyclin-dependent kinase 4

CC

CNP

2’,3’-Cyclic nucleotide 3’ phosphodiesterase

Cell O

EDNRB

Endothelin receptor type B

Cell A, BM

EGFR

Epidermal growth factor receptor

Prol

ENO2

Enolase 2 (gamma, neuronal)

Cell N

ERBB2

v-erb-b2 Avian erythroblastic leukemia viral oncogene homolog 2

BM

FN1

Fibronectin 1

Met

GFAP

Glial fibrillary acidic protein

Cell A

GFAPdelta

Glial fibrillary acidic protein delta

Cell A

GLUL

Glutamate-ammonia ligase (glutamine synthetase)

Cell A

IL8

Interleukin 8

Cell M, Angio

MAPT

Microtubule-associated protein tau

Cell N

MBP

Myelin basic protein

Cell O

MCM2

Minichromosome maintenance complex component 2

Prol

MDM4

Mdm4 p53 binding protein homolog (mouse)

OG

MGMT

O-6-Methylguanine-DNA methyltransferase

TS

MKI67

Marker of proliferation Ki-67

Prol

NES

Nestin

Cell NS

NOTCH1

Notch 1

TS

OLIG2

Oligodendrocyte lineage transcription factor 2

BM

PCNA

Proliferating cell nuclear antigen

Prol

PECAM1

Platelet/endothelial cell adhesion molecule 1

Cell E, Angio

PROM1

Prominin 1 (CD133)

Cell CS, OG

RB1

Retinoblastoma 1

TS, CC

SEC61G

Sec61 gamma subunit

BM, OG

SOX2

SRY (sex determining region Y)-box 2

Cell NS

SUZ12

SUZ12 polycomb repressive complex 2 subunit

BM

SYNM

Synemin, intermediate filament protein

Cell A, Cell SM, BM

TP53

Tumor protein p53

TS

TUBB3

Tubulin, beta 3 class III

Cell N, BM

VEGFA

Vascular endothelial growth factor A

Angio

VIM

Vimentin

Cell A

The genes were selected as cell type markers (Cell A astrocyte marker; Cell CS cancer stem cell marker; Cell E endothelial cell marker; Cell M microglia marker; Cell N neuronal marker; Cell NS neural progenitor marker; Cell O oligodendrocyte marker; Cell SM smooth muscle marker) Prol proliferation markers, Angio angiogenesis markers, CC involved in cell cycle control, BM cancer biomarkers, OG proto-oncogenes, TS tumor suppressor genes, Met genes involved in tumor metastasis

ended with a final step of 72 °C, during which the run was interrupted and the plate put on dry ice. The preamplified cDNA were kept cold while diluted 1:20 in TE-buffer (Invitrogen), before qPCR analysis.

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Quantitative Real Time PCR (qPCR)

Results

qPCR analysis was performed in 10 ll reactions, containing 2 ll diluted preamplified cDNA, 19 TATAA SYBR GrandMaster Mix (TATAA Biocenter, Gothenburg, Sweden) and a final primer concentration of 0.4 lM. Same primers were used as for preamplification (Table 2). The temperature was set to 95 °C for three minutes followed by 45 cycles of 95 °C for 5 s, 60 °C for 10 s and 72 °C for 15 s, followed by a melting curve step ranging from 60 °C to 95 °C. The qPCR experiments were performed in 384-well plates (4titude, Wotton, Surrey, UK) in a Viia7 instrument (Applied Biosystems).

Development of a Protocol for Obtaining Viable Single Cells from Freshly Isolated Human Brain Tissue for Single Cell Gene Expression Profiling

Data Analysis and Statistics The qPCR data was pre-processed using the GenEx Professional version 5.4.4 software (MultiD, Gothenburg, Sweden) according to the single cell data handling steps described by Sta˚hlberg et al. [16], with few modifications. In brief, elimination of false positives (based on melt curves and gel electrophoresis) was followed by interplate calibration using the mean of the three positive controls, gene expression above Cq 40 was set to Cq 40 and all negatives were set to Cq 41. The relative gene expression for each cell and gene was calculated by assigning one molecule to Cq 41, and thereafter log 2 transforming all the data. Cells expressing fewer than two of the selected genes were excluded from further analysis. The binary data were analysed by using Fisher’s exact test in PASW Statistics 18 (SPSS Inc., IBM, Armonk, New York, USA). Spearman correlations were calculated in the PASW software (IBM) using log scaled quantitative data including zeros for non-expressing cells and the genes with the highest number of correlations were compiled in correlation tables. Excel (Microsoft, Albuquerque, New Mexico, USA) was used to prepare diagrams showing percentages of cells expressing genes. Multivariate analyses and calculation of differences in gene expression were all performed in GenEx. Principal component analyses (PCA) [17], Kohonen Self Organizing Maps (SOMs) [18] were performed using autoscaled data, while hierarchical clusterings [19] with heat maps were performed using mean-centred data. For the SOMs, the following settings were used; 0.40 learning rate, 2 neighbours and 5,000 iterations and 2 groups. The cells separated into identical groups in repeated analyses. To calculate differences in gene expression levels between two groups, non-scaled log 2 transformed data was used and non-parametric Mann–Whitney tests [20] with Bonferroni correction for multiple testing [21] were performed.

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We developed a protocol for dissociating human brain tumor tissue as well as the tissue of the adjacent tumor penumbra into single viable cells and analyzed the gene expression of these cells using reverse transcription quantitative real time PCR (RT-qPCR, Fig. 1). The tissue was resected in the surgical theatre, where a neuronavigation system was used to determine the precise location of the resected tissue (Fig. 2). The tumor/tumor penumbra tissue dissociation steps included mechanical dissociation, enzymatic digestion and removal of myelin debris and lysis of erythrocytes. The complete tissue dissociation process and collection of single cells took approximately 3 h and the cells remained viable until lysis and, if placed in cell cultures, they survived and propagated (data not shown). RNA from individual cells in 96-well plates was snap frozen and stored at -80 °C. This was followed by reverse transcription, pre-amplification using the primers for selected genes (Tables 1, 2), qPCR, statistical analysis and classification of individual cells with the use of unsupervised algorithms, specifically Self organizing map (SOM), Principal component analysis (PCA) and hierarchical clustering. It was previously demonstrated that SOM constitutes a useful bioinformatics approach to identify cell subpopulations [7]. The protocol was applied to tumor resections from two patients; one with an oligodendroglioma grade III tumor, the other with an astrocytoma grade IV (glioblastoma multiforme) tumor. From the first patient, we assessed the gene expression in cells from the tumor proper as well as from the tumor penumbra, and for the second patient we only had access to the tumor proper. Our panel of genes included cell type markers, genes expressed by proliferating cells (non-tumor and tumor cells), angiogenesis-related genes, proto-oncogenes and tumor suppressor genes, and other tumor cell markers (Table 1). Oligodendroglioma Grade III Cells Clustered into Two Distinct Subpopulations We assessed gene expression in 79 cells from the oligodendroglioma grade III tumor (Table 3) and classified them into subpopulations according to their molecular signature (Fig. 3). SOM analysis was used for separation of the cells into subpopulations. The SOM analysis repeatedly identified two cell clusters, which were also detectable in the PCA plot (Fig. 3a) and further confirmed in the hierarchical clustering (Fig. 3e).

NM_005228.3

NM_001975.2

NM_004448.2

NM_212482.1 NM_002055.4

NM_001131019.2

NM_002065.5

NM_000584.3

NM_016835.4

NM_001025081.1

NM_004526.3

NM_002393.4

NM_002412.3

NM_002417.4

NM_006617.1

NM_017617.3

NM_005806.3

NM_002592.2

NM_000442.4

NM_006017.2 NM_000321.2

NM_014302.3

NM_003106.3

NM_015355.2

NM_145728.2

NM_000546.5

EDNRB

EGFR

ENO2

ERBB2

FN1 GFAP

GFAPdelta

GLUL

IL8

MAPT

MBP

MCM2

MDM4

MGMT

MKI67

NES

NOTCH1

OLIG2

PCNA

PECAM1

PROM1 RB1

SEC61G

SOX2

SUZ12

SYN

TP53

NM_003380.3

NM_000115.3

CNP

NM_001025366.2

NM_033133.4

CDK4

VIM

NM_000075.3

CD63

VEGFA

NM_001780.5

CCNE1

NM_006086.3

NM_001238.2

ALDH1L1

TUBB3

NM_001623.3

NM_001270364.1

AIF1

NCBI accession number

Gene

Table 2 Primer sequences

yes

yes

yes

yes

no

yes

no

yes

yes yes

yes

yes

no

yes

no

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

Intron spanning

CAGATGCGTGAAATGGAAGA

CAGAAGGAGGAGGGCAGAA

GTGCGGAAGGAGTGTGAAAA

CCCTTCCCAGAAAACCTACC

TTCCCAACCCTCCATTCAT

AGCCATCACCAAACTCAGAAA

ACACCAATCCCATCCACACT

GGGATTCATTGGCTTCTTTG

CACTTACGGCACTCTTCACC AAAGGACCGAGAAGGACCA

TCAGAAGGACAAGGCGATT

GTGGAGAACTTGGAAATGGAA

CCGTTCCTCCCTGTCTCTC

GCCAGACCAACATCAACGAG

GCTGGAGTGGAAAATGAGGA

GAGAGGAGGGAGAAAGAGTGG

CTGAATGCCTATTTCCACCA

CCAGAAAAAGAACTACAGAAGACGA

CGGCGTGACAACAATGAG

GATGAAAACCCCGTAGTCCA

CCTGGCGGAGGAAATAAAA

CCTGATTTCTGCAGCTCT

AGATTGCGGGGACTAATGC

CCGTGCAGACCTTCTCCAA

GGAGACACCTGGAGCAAGAA GCAGACCTTCTCCAACCTG

ACCTGGAACTCACCTACCTG

AGGCTGGCTACACGGAAAA

CCTTCACACATACTCCTCCTCTG

AGCAAAAGATTGGTGGCTATTC

CGCTCTACTTCGGCTGGTTC

CAGATGGCACTTACACCCGT

ATCATCGCAGTGGGTGTCT

CCGGTATATGGCGACACAAG

CCTTCCAACCCTCCTGCTAC

CTGTCTCCCCACCTCTACCA

Forward primer

TGGAAGAGGCAGAGAAATCC

TCAGGGGCACACAGGATG

CGGAAGCAGATGTCGTAGAG

GCCTCACAACCTCCGTCA

GTTCCTTCCCCAAAACATCC

GCTTTTTACCTGTGGGAACTTG

CCTCCCCAGGTTTTCTCTGT

GAGTTTCTCACACCCTCACACTT

TCTATTCCACAAGCAGCAAAA AAGGCTGAGGTTGCTTGTGT

GGATGGAGCAGGACAGGTT

ACCGTTGAAGAGAGTGGAGTG

TCGGCAGTTTTGGGTTATTC

GGGGCAGACACAGGAGAAG

ACCTGTTGTGATTGCCCTTC

TTGGTTGGAAATGAAGTTGTTG

ATTGCTCCTCCCACTGCTC

CTACATCCCACTCCTCAAATCC

CCCAAACCAGAATCCCAAG

TCCCTTGAATCCCTTGTGAG

GCTGAGATGCCGTGGAGA

TTTGGGGTGGAAAGGTTT

TGGTGCTGAAGTTGGTATGG

CGTATTGTGAGGCTTTTGAGATATCT

GCATCCCCACAGAGTAGACC TGCCTCACATCACATCCTTG

TCACTTGGTTGTGAGCGATG

ACATTGGCTGTGAACTTGGA

TCACATCTCCATCACTTATCTCCTT

CAGAGGGCAAAGACAAGGAC

GCCTGGGGGTCTCTTTCC

CAGCCCAATCAGGTCAAAGA

CGAAGCAGTGTGGTTGTTTT

TACGCAAACTGGTGCAACTT

CGGCACTCCATCCTTCTC

AAGTTTCTCCAGCATTCGTTTC

Reverse primer

222

153

281

233

184

126

116

133

177 180

233

160

142

212

236

388

206

150

287

185

138

102

210

100

235 161

99

235

246

192

185

195

234

123

177

265

Length (bp)

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Neurochem Res Fig. 1 Gene expression profiling of individual cells in human brain tissue—a schematic presentation of the experimental procedure

The first cell cluster consisted of 39 % of all oligodendroglioma grade III tumor cells (Fig. 3, red). Genes that influenced separation of cells in Fig. 3a in the PCA were CD63, CDK4, CNP, EDNRB, EGFR, ENO2, GFAP, MAPT, NOTCH1, OLIG2, SEC61G, SUZ12 and TUBB3 (Fig. 3b, PCA loadings). These genes were also expressed in a higher proportion of cells in the red group compared to the green group, together with SOX2 and RB1 (Fig. 3c). Furthermore, CD63, CDK4, CNP, EGFR, ENO2, MAPT, OLIG2, SEC61G and SUZ12 showed a higher quantitative expression level in the red group compared to the green group (Fig. 3d). Although GFAP is usually expressed in astrocytes [22, 23], CNP in oligodendrocytes [24], ENO2 and TUBB3 [25] in neurons and SOX2 in neural progenitor cells [26] and astrocytes [27], and MAPT (TAU) more known for its involvement in Alzheimer’s disease [28], all

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these genes have also been implicated in the context of malignant tumors (CD63 [29], CDK4 [30–32], CNP [33], EDNRB [34], EGFR [10, 35], ENO2 [36], MAPT [28, 37, 38], NOTCH1 [39], OLIG2 [40, 41], RB1 [42–44], SEC61G [45], SOX2 [46], SUZ12 [47], TUBB3 [48] ). Hence, this subpopulation showed gene expression profiles that corresponded to a more cancer-like phenotype than the cells in the green group and we thereby classified them as tumor cells. In addition, the gene expression of CD63, CDK4, CNP, EGFR, ENO2 NOTCH1, OLIG2, SEC61G, SUZ12 and TUBB3 correlated individually in single cells from this tumor (Table 4), which indicates that those genes are coregulated in these tumor cells. The second cell cluster consisted of 61 % of all oligodendroglioma grade III cells (Fig. 3, green). Those cells localized to the right in the PCA, a consequence of the

Neurochem Res Table 3 Percentage of cells expressing the selected genes in cells of oligodendroglioma grade III tumor (OT), the penumbra of oligodendroglioma grade III tumor (OP) and astrocytoma grade IV tumor (A)

Fig. 2 Patient data and the location of the resected tissue. a Preoperative MRI of the astrocytoma grade IV patient with the area of resected tumor tissue indicated in blue. b Pre-operative MRI of the oligodendroglioma grade III patient with neuronavigation coordinates (red dots) at surgery, pointing to the tumor (left) and tumor penumbra (right) tissue that are to be resected. Bottom panel shows photographs of temporal lobe surface at surgery with the area encircled in blue of the tumor proper (left) and the tumor penumbra (right) that are to be resected

expression of the microglia marker AIF1 [49] expressed by 29 %, an inflammation marker IL8 [50] expressed by 33 %, and an endothelial cell marker PECAM1 [51] expressed by 6 % of cells in this cluster (Fig. 3b). Expression of AIF1 and IL8 correlated in the cells of the oligodendroglioma tumor (cc. 0.53, p value\0.001) and it has previously been

Gene symbol

OT n = 79 (%)

OP n = 38 (%)

A n = 86 (%)

P value positive cells OT versus OP

AIF1

18

50

6

\0.001***

ALDH1L1

4

0

1

0.304

CCNE1

–

–

28

–

CD63

44

45

86

0.560

CDK4

32

13

91

0.024*

CNP

22

8

73

0.053

EDNRB

9

0

6

0.058

EGFR

35

0

57

\0.001***

ENO2

33

3

73

\0.001***

ERBB2 FN1

3 1

0 0

16 6

0.454 0.675

GFAP

22

5

8

0.020*

GFAPdelta

4

0

–

0.304

GLUL

66

76

83

0.175

IL8

22

26

2

0.361

MAPT

42

3

70

\0.001***

MBP

–

–

3

–

MCM2

3

0

56

0.454

MDM4

38

39

83

0.516

MGMT

1

3

6

0.546

MKI67

–

–

49

–

NES

5

3

10

0.475

NOTCH1

25

5

73

0.006**

OLIG2

49

8

74

\0.001***

PCNA PECAM1

38 4

39 34

81 2

0.516 \0.001***

PROM1

–

–

15

–

RB1

28

21

66

0.290

SEC61G

41

24

91

0.055

SOX2

16

5

47

0.075

SUZ12

51

42

88

0.253

SYNM

14

5

9

0.138

TP53

8

3

33

0.271

TUBB3

16

5

77

0.075

VEGFA

10

18

42

0.168

VIM

11

13

13

0.500

N number of cells; P values for binary comparisons were obtained by Fisher’s exact test *** P \ 0.001; ** P \ 0.01; * P \ 0.05

shown that microglia cells produce IL8 [52]. Furthermore, the cells in this cell cluster expressed fewer genes related to tumor properties than the cells of the first cell cluster (Fig. 3c, d). Thus, these cells show expression profiles corresponding to normal cells present within the tumor, and

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Neurochem Res b Fig. 3 Oligodendroglioma grade III tumor cells separated into two

groups using SOM analysis. a The groups from the SOM analysis visualized by PCA scatter plot, one rectangle represents one cell. b PCA loading plot, indicating the weight of each gene along the PC1 and PC2 separation axes. c Diagram showing percentages of cells in the two subpopulations expressing the selected genes. P values for binary comparisons were obtained by Fisher’s exact test. *** P \ 0.001; ** P \ 0.01; * P \ 0.05. d Box plots of log 2 transformed expression levels of genes which expression differed between the red and green subpopulations as determined by Mann–Whitney test and Bonferroni correction for multiple testing. The boxes indicate the 25th and 75th percentiles, red circles indicate outliers and asterisks extreme outliers. e Heat map with hierarchical clustering of individual cells with the grouping based on SOM analysis visualized by green and red at the bottom. Two groups of cells, similar to those seen in the SOM analysis, appeared in the hierarchical clustering

at least some of these cells are likely to be microglia and cells of the vascular system. Interestingly, none of the cells from the oligodendroglioma grade III tumor expressed proliferation marker MKI67 [53], and only two cells expressed proliferation marker MCM2 [54] (Table 3), which indicates that relatively low proportion of cells of this tumor were proliferating. Cells of the Second Cell Cluster of the Oligodendroglioma Tumor Shared Molecular Signature with Cells from the Oligodendroglioma Penumbra PCA plot of the cells from the oligodendroglioma grade III tumor and its penumbra showed that the penumbra cells (Fig. 4a, black) clustered together with the normal cells of the oligodendroglioma grade III (green). This was also seen

in the hierarchical clustering where the penumbra cells were within the second oligodendroglioma tumor cell cluster of normal cells, with only a single penumbra cell clustering with tumor cells (Fig. 4d). There were no differences in gene expression between the cluster of normal cells within oligodendroglioma tumor cells and oligodendroglioma penumbra cells (Mann–Whitney tests with Bonferroni correction for multiple testing). PECAM1 were expressed in a higher proportion of penumbra cells compared to ‘‘normal cells’’ of the tumor (Fig. 4c, black vs. green), but except for this gene, the groups did not differ when it comes to proportion of cells expressing specific genes. This further implies that the cells of the oligodendroglioma grade III second cell cluster were normal cells. Astrocytoma Grade IV Cells Clustered into Three Subpopulations We assessed gene expression in 86 cells (Table 3) from the astrocytoma grade IV tumor and classified them into subpopulations based on their molecular signature (Fig. 5). The PCA pointed to three groups of cells (Fig. 5a), these groups were also present in the hierarchical clustering (Fig. 5g). The group consisting of only three cells (Fig. 5a, grey) was excluded since this allowed a better separation of the remaining cells (Fig. 5c–f). These three cells were defined by the expression of GFAP, VIM, ALDH1L1, EDNRB and FN1 (Fig. 5b). VIM (vimentin) and GFAP encode intermediate filament proteins often co-expressed in astrocytes [55]. These cells expressed FN1 (fibronectin), and two of them EDNRB (endothelin receptor B). Both FN1 and EDNRB have been reported to be upregulated in

Table 4 Spearman correlation coefficients for oligodendroglioma grade III tumor cells, selected genes n = 79

AIF1 n = 14

CD63 n = 35

CDK4 n = 25

CNP n = 17

AIF1 CD63

1 -0.11

1

CDK4

-0.31**

0.43***

1

CNP

-0.21

0.48***

0.50***

1

EGFR n = 28

ENO2 n = 26

MAPT n = 33

NOTCH1 n = 20

OLIG2 n = 39

SEC61G n = 32

SUZ12 n = 40

EGFR

-0.33**

0.50***

0.41***

0.47***

1

ENO2

-0.32**

0.32**

0.52***

0.41***

0.57***

MAPT

-0.37***

0.47***

0.50***

0.61***

0.69***

0.63***

1

NOTCH1

-0.11

0.38***

0.36***

0.54***

0.38***

0.33**

0.41***

OLIG2

-0.38***

0.48***

0.46***

0.52***

0.69***

0.54***

0.72***

0.30**

1

SEC61G

-0.14

0.20

0.46***

0.41***

0.35***

0.24*

0.43***

0.28*

0.36***

1

SUZ12

-0.21

0.24*

0.22

0.26*

0.56***

0.38***

0.45***

0.06

0.51***

0.27*

1

TUBB3

-0.09

0.46***

0.35**

0.47***

0.29**

0.15

0.33**

0.39***

0.44***

0.36***

0.15

TUBB3 n = 13

1 1

1

N number of cells expressing the respective gene *** P \ 0.001; ** P \ 0.01; * P \ 0.05

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Fig. 4 Oligodendroglioma grade III tumor and tumor penumbra cells. The two groups of tumor cells separated by SOM analysis and described in Fig. 3 (red and green cells) are compared with tumor penumbra cells (black). a PCA scatter plot of the three groups of cells, one rectangle represents one cell. The green population of the tumor cells clusters with the penumbra cells. b PCA loading plot, indicating the weight of each gene along the PC1 and PC2 separation

123

axes. c Diagram showing percentages of cells in the tumor penumbra cells (black) and the two subpopulations of tumor cells (red and green) expressing the selected genes. d Heat map with hierarchical clustering of individual cells of green and red tumor cells together with black tumor penumbra cells shows that the green tumor cell population clusters with the penumbra cells

Neurochem Res

reactive astrocytes [56, 57], and therefore, these cells are likely to be reactive astrocytes. The second cell cluster consisted of 86 % of the astrocytoma grade IV tumor cells (Fig. 5, yellow). This group showed high proportion of cells expressing cancer-related genes: CD63, CDK4, CNP, EGFR, ENO2, MAPT, MCM2, MDM4 [58, 59], MKI67, NOTCH1, OLIG2, PCNA [60], RB1, SEC61G, SUZ12, TP53 [9] and TUBB3 (Fig. 5f). The expression of CD63, CDK4, CNP, ENO2, MAPT, MCM2, MKI67, NOTCH1, OLIG2, PCNA, RB1, SEC61G, SUZ12 and TUBB3 correlated in individual cells of the astrocytoma grade IV tumor (Table 5), which suggests that these genes are co-regulated. The expression of many of these genes also correlated in the cells of the oligodendroglioma (CD63, CDK4, CNP, ENO2, NOTCH1, OLIG2, SEC61G, SUZ12 and TUBB3; Table 4). The third cell cluster consisted of nine astrocytoma grade IV tumor cells (10 %, Fig. 5, blue). This cell cluster can be defined by expression of microglial marker AIF1 (55 % of the cells in the blue cluster expressed AIF1, Fig. 5d). AIF1, IL8 and VIM were all expressed in a higher proportion of cells in the third cluster compared to the second cluster of tumor cells (Fig. 5f). AIF1 expression in individual cells negatively correlated with the expression of oligodendrocyte marker CNP [24], neuronal markers ENO2 and TUBB3, and genes related to tumor cell proliferation/behavior CDK4, MCM2, MKI67, NOTCH1, OLIG2, PCNA, SEC61G, SUZ12 (Table 5). Thus, the third cell cluster consisted of cells that did not express the tumor related genes, some of them being microglia.

Subpopulations of Astrocytoma Grade IV and Oligodendroglioma Grade III Tumor Cells Shared Molecular Signature with Cells from the Oligodendroglioma Penumbra A PCA plot of all the cells from oligodendroglioma grade III tumor and penumbra, and astrocytoma grade IV tumor (Fig. 6a) showed that subpopulations of these cells (second cell cluster) of oligodendroglioma grade III (green), and third cell cluster of astrocytoma grade IV (blue) grouped together with cells of the oligodendroglioma penumbra (black). This was mainly influenced by the expression of AIF1, PECAM1 and IL8 (Fig. 6b). These three groups of cells showed comparable gene expression. Also the proportions of cells within each group expressing the selected genes were similar (Fig. 6c). This implies that also the cells of the third cell cluster of astrocytoma grade IV (blue) were nonmalignant cells present within the tumor, and that these cells did not differ between these two tumor types.

Tumor Cells of the Astrocytoma Grade IV and Oligodendroglioma Tumor Samples Have Distinct Signatures The first cell cluster of the oligodendroglioma grade III (tumor cells; red) were separated in PCA (Fig. 6a) from the second cell cluster of astrocytoma grade IV (tumor cells; yellow). These two groups differed in the expression of several genes and the three proliferation markers (MCM2, MKI67 and PCNA) showed higher expression in the astrocytoma tumor cells than in the oligodendroglioma tumor cells. Also CDK4, CNP, and NOTCH1 showed a higher expression in the astrocytoma tumor cells. GFAP, MAPT and OLIG2 had a higher expression in the oligodendroglioma tumor cells. This demonstrates that the cells of these two tumor samples have distinct molecular signatures (Fig. 6a, d).

Discussion In this study, a protocol for single cell gene expression profiling of freshly isolated brain cells was developed and evaluated on individual cells from one oligodendroglioma grade III and one astrocytoma grade IV tumor. We show that cells of the tumor proper from both tumors cluster into subpopulations with different molecular signatures. Both tumors contained subpopulations of cells which expression profile matched the profile of the tumor penumbra, i.e. cells of relatively normal brain tissue (Fig. 6). The expression of proliferation markers MCM2, MKI67, and PCNA was higher in the tumor cells of the astrocytoma grade IV tumor compared to the oligodendroglioma grade III tumor (Fig. 6d). Furthermore, MKI67 and MCM2 were expressed in half of the cells of the astrocytoma (49 and 56 %, respectively), but no oligodendroglioma tumor cells expressed MKI67, and only 3 % of these cells expressed MCM2. PCNA was expressed by 81 % of the astrocytoma tumor cells, but only 38 % of the oligodendroglioma tumor cells (Table 3). This indicated that the proliferation was substantially higher in the astrocytoma tumor, and this was also compatible with the clinical history of the patients. The oligodendroglioma patient was diagnosed 29 years ago and received a radiation therapy 10 years ago as the only treatment for his slowly growing tumor. The astrocytoma patient had clinical symptoms of the tumor for only 1 month before surgery, indicating a fast growing tumor. This was also compatible with the histopathological evaluation of the tumor tissue that showed high mitotic activity in the astrocytoma grade IV tumor, but only a limited one in the oligodendroglioma grade III tumor. Cell subpopulations in the oligodendroglioma grade III tumor and astrocytoma grade IV tumor showed similar

123

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123

*** P \ 0.001; ** P \ 0.01; * P \ 0.05

N number of cells expressing the respective gene

1

0.40*** 0.26* 0.32** 0.34** 0.42* 0.26* 0.37*** 0.26* 0.42*** 0.40*** 0.36*** 0.46*** 0.32**

0.47*** -0.32** SUZ12

-0.22*

-0.38*** SEC61G

TUBB3

1 0.32**

1

0.47*** 0.68*** 0.43*** 0.37*** 0.46*** 0.60*** 0.54*** 0.48*** 0.45***

0.40*** 0.47*** 0.34*** 0.31** 0.26* 0.35*** 0.38*** 0.51*** 0.43***

-0.23* RB1

0.48***

0.59***

1 0.47***

0.34*** 0.22* 0.42*** 0.47*** 0.23* 0.32** 0.42*** 0.53***

-0.36*** PCNA

0.41***

1 0.37***

0.29** 0.67*** 0.79*** 0.29** 0.50*** 0.47*** 0.64***

-0.32** OLIG2

0.45***

1 0.27**

0.44*** 0.37*** 0.16 0.29** 0.36*** 0.36***

-0.31** NOTCH1

0.34***

1 0.46***

0.18 0.34*** 0.30** 0.41*** 0.41***

-0.21 MKI67

0.39***

1 0.18

0.17 0.32** 0.32** 0.36***

-0.25* MCM2

0.28**

1 0.32**

0.31** 0.42*** 0.52***

-0.30**

0.35***

1 0.32**

0.25* 0.29**

1

MAPT

0.38***

0.35***

0.61***

-0.31**

0.39***

1

ENO2

0.50***

0.38***

-0.31** CNP

1

-0.39*** CDK4

1

-0.32** CD63

AIF1

0.63***

SEC61G n = 78 RB1 n = 57 PCNA n = 70 OLIG2 n = 64 NOTCH1 n = 63 MKI67 n = 42 MCM2 n = 71 MAPT n = 60 ENO2 n = 63 CNP n = 63 CDK4 n = 78 CD63 n = 74 AIF1 n=5 n = 86

Table 5 Spearman correlation coefficients for astrocytoma grade IV tumor cells, selected genes

expression profiles as cells of the oligodendroglioma penumbra. Subpopulations of cells within the oligodendroglioma and astrocytoma tumors, and tumor penumbra cells of the oligodendroglioma did not differ in the expression of any of the assessed genes, and they clustered together in the PCA of all the examined cells (Fig. 6a). A high proportion of these cells expressed microglia marker AIF1, and PECAM1, a marker of endothelial cells, indicating that some of them are likely to be microglia cells or cells of the vascular system (Fig. 6c). The cells of oligodendroglioma grade III and astrocytoma grade IV differed in the expression of several genes. A number of genes were expressed in cells in both tumors: CD63, CDK4, CNP, EGFR, ENO2, GFAP, MAPT, NOTCH1, OLIG2, SEC61G, SUZ12 and TUBB3. In the astrocytoma grade IV, additional genes were expressed: MCM2, MDM4 MKI67, PCNA, RB1 and TP53. Likewise, the oligodendroglioma tumor cells showed a higher expression level of GFAP, MAPT and OLIG2 compared to astrocytoma cells (Fig. 6d). Ligon et al. showed that oligodendrogliomas had higher expression of OLIG2 than high grade astrocytomas, although both tumor types expressed this gene [40]. It was reported that oligodendroglioma cells express GFAP [61, 62], and that the number of GFAP expressing cells decrease with increasing grade of astrocytoma [63]. This is in concordance with our results: 90 % of the second cell cluster within the oligodendroglioma tumor expressed GFAP, whereas only three cells within the astrocytoma tumor expressed GFAP. The expression of MAPT (TAU) was not previously reported in astrocytomas or oligodendrogliomas, although it was associated to several other tumor types [37, 38, 64]. NOTCH1 were more highly expressed in the cells of astrocytoma grade IV compared to the oligodendroglioma grade III. This is compatible with the finding that NOTCH1 expression increases with increasing grade of astrocytomas [65].

SUZ12 n = 76

suggested the existence of three cell populations (indicated in grey, yellow and blue). b PCA loading plot, indicating the weight of each gene along the PC1 and PC2 separation axes. c PCA scatter plot with the grey cell population removed from the analysis to improve separation of the yellow and blue groups along the PC2 axis. d PCA loading plot for (c), indicating the weight of each gene along the PC1 and PC2 separation axes. e Box plots of log 2 transformed expression levels of genes which expression differed between the yellow and blue subpopulations as determined by Mann–Whitney test and Bonferroni correction for multiple testing. The boxes indicate the 25th and 75th percentiles, red circles indicate outliers and asterisks extreme outliers. f Diagram showing percentages of cells in the yellow and red subpopulations expressing the selected genes. P values for binary comparisons were obtained by Fisher’s exact test. *** P \ 0.001; ** P \ 0.01; * P \ 0.05. g Heat map with hierarchical clustering of individual cells with the grouping based on SOM analysis visualized by grey, yellow and blue at the bottom. Three groups of cells, similar to those seen in the SOM analysis, appeared in the hierarchical clustering

0.34***

b Fig. 5 Astrocytoma grade IV tumor cells. a PCA scatter plot

1

TUBB3 n = 66

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123

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Fig. 6 Comparison of cell populations in oligodendroglioma grade III tumor and tumor penumbra with astrocytoma grade IV tumor cells. The cell populations from previous SOM analyses were used with the color-coding of individuals cells as in Figs. 3, 4 and 5. a All populations visualized by PCA scatter plot, one rectangle represents one cell. b PCA loading plot, indicating the weight of each gene along the PC1 and PC2 separation axes. c Diagram showing percentages of

cells expressing the selected genes within each cell population. d Box plots of log 2 transformed expression levels of genes which expression differed between the yellow and red subpopulations as determined by Mann–Whitney test and Bonferroni correction for multiple testing. The boxes indicate the 25th and 75th percentiles, red circles indicate outliers and asterisks extreme outliers

In conclusion, we have made the initial evaluation of a method for classification of populations of cells based on their molecular characteristics. Single cell gene expression

profiling seems to be a highly useful method allowing identification of molecular signatures of individual subpopulations of cells within a given tumor and can be used

123

Neurochem Res

to compare molecular signatures among individual tumors and between tumor types and grades. Acknowledgments We would like to thank Dr. Yolanda de Pablo for providing mouse brain tissue for development of the cell dissociation protocols. This work was supported by Swedish Medical Research Council (11548 to MP), ALF Gothenburg (11267 to MP, 11392 to BR, 137241 to AS), So¨derbergs Foundations, Hja¨rnfonden, Amlo¨v’s Foundation, E. Jacobson’s Donation Fund, NanoNet COST Action (BM1002), EU FP 7 Program EduGlia (237956) to MP, EU FP 7 Program TargetBraIn (279017), AFA Research Foundation, Gothenburg Foundation for Neurological Research, FoU (Va¨stra Go¨talandsregionen), the Swedish Cancer Society, and Wilhelm and Martina Lundgren foundation.

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