Translocator protein (18kDA) (TSPO) marks mesenchymal glioblastoma cell populations characterized by elevated numbers of tumor-associated macrophages

TSPO is a promising novel tracer target for positron-emission tomography (PET) imaging of brain tumors. However, due to the heterogeneity of cell populations that contribute to the TSPO-PET signal, imaging interpretation may be challenging. We therefore evaluated TSPO enrichment/expression in connection with its underlying histopathological and molecular features in gliomas. We analyzed TSPO expression and its regulatory mechanisms in large in silico datasets and by performing direct bisulfite sequencing of the TSPO promotor. In glioblastoma tissue samples of our TSPO-PET imaging study cohort, we dissected the association of TSPO tracer enrichment and protein labeling with the expression of cell lineage markers by immunohistochemistry and fluorescence multiplex stains. Furthermore, we identified relevant TSPO-associated signaling pathways by RNA sequencing. We found that TSPO expression is associated with prognostically unfavorable glioma phenotypes and that TSPO promotor hypermethylation is linked to IDH mutation. Careful histological analysis revealed that TSPO immunohistochemistry correlates with the TSPO-PET signal and that TSPO is expressed by diverse cell populations. While tumor core areas are the major contributor to the overall TSPO signal, TSPO signals in the tumor rim are mainly driven by CD68-positive microglia/macrophages. Molecularly, high TSPO expression marks prognostically unfavorable glioblastoma cell subpopulations characterized by an enrichment of mesenchymal gene sets and higher amounts of tumor-associated macrophages. In conclusion, our study improves the understanding of TSPO as an imaging marker in gliomas by unveiling IDH-dependent differences in TSPO expression/regulation, regional heterogeneity of the TSPO PET signal and functional implications of TSPO in terms of tumor immune cell interactions. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-023-01651-5.


Introduction
Adult-type diffuse gliomas are the most frequent malignant brain tumors [54] and diagnosed by histological and molecular features according to the 2021 World Health Organization (WHO) classification of tumors of the central nervous system.They comprise the entities of oligodendroglioma, IDH-mutant and 1p/19q-codeleted (WHO grades 2-3), astrocytoma, IDH-mutant (WHO grade 2-4) and glioblastoma, IDH-wildtype (WHO grade 4) [45].Prognosis differs between gliomas: While IDHmutant gliomas have a more favorable prognosis, survival times for most glioblastoma (GBM) patients range between 15 and 18 months [54].
Unsupervised clustering of methylation array profiles defines distinct molecular subtypes of adult-type diffuse gliomas with IDH-mutant gliomas clustering closely together and clearly separating from IDH-wildtype GBMs [45].IDH mutations are commonly associated with a genome-wide hypermethylation phenotype [16], while molecular heterogeneity within IDH-wildtype GBMs is described by transcriptome-wide RNA sequencing profiles [69].Currently there are three clinically relevant GBM subgroups referred to as proneural (PN), mesenchymal (MES) and classical (CL) [9,12,16,34,56,72].Recently, single cell RNA sequencing revealed four main cellular states in GBMs: neural progenitor-like (NPC1/2like), oligodendrocyte progenitor-like (OPC-like), astrocyte-like (AC-like) and mesenchymal-like (MES1/2-like).MES-like cells are more abundant in the MES subgroup, are linked to tissue microenvironment interaction [52] and are promoted by macrophage-derived Oncostatin M (OSM) that interacts with its receptors (OSMR and LIFR) in complex with GP130 via STAT3 signaling pathways [30].Imaging markers to identify molecular subgroups and cellular composition are of great need, especially to support improved precision immunotherapy treatment approaches, like neoadjuvant anti-PD-1 therapy in case of high cell proportions in the tumor microenvironment (TME) or different options targeting tumor-associated macrophages (TAMs) [19,74].
TSPO is a transmembrane protein located in the outer mitochondrial barrier.The gene is located at 22q13.31 and contains four exons [24].It has been associated with a broad spectrum of functions such as steroid synthesis, regulation of proliferation, apoptosis and migration, as well as mitochondrial functions such as mitochondrial respiration and oxidative stress regulation [3].Its expression is regulated by a GC-rich promotor in breast cancer cell lines that contains binding sites for several transcription factors, including SP1 and SP3 [7].Evidence exists that the PKCε-ERK1/2-AP1-STAT3 signaling pathway can initiate TSPO transcription by upregulation of ETS and SP1/SP3 transcription factors (TFs) in MA-10 Leydig cells [6].However, TSPO transcriptional regulation in gliomas is still poorly understood.According to GTEx v8 (GTEx data release 8, dbGaP Accession phs000424.v8.p2, accessed 19.12.2021),TSPO is normally expressed at very low levels in the central nervous system (CNS) compared to other healthy tissues [1,26].However, its expression is upregulated at sites of inflammation or neurodegeneration [53] and also in gliomas [14,71,79].TSPO-PET imaging is a potential prognostic marker in patients suffering from diffuse gliomas [14,58].It has been described to mark the tumor microenvironment (TME) with its myeloid compartment and to indicate therapy-induced changes during tumor progression [22,57,79].Regarding the cellular source of TSPO-PET, tumor cells, reactive astrocytes, endothelial cells, and macrophages/ microglia have been discussed [33,53,60,79].This suggests that the presence of different cell populations and their interplay might generate a cumulative TSPO signal depending on the imaging sampling area and time point.However, further histopathological approaches to better understand TSPO imaging correlates are urgently needed.
In this study, we investigate the histological and molecular correlates of TSPO-PET tracer uptake and TSPO tissue expression.We used in silico data to get an overview of TSPO expression in low-and high-grade gliomas.Furthermore, we analyzed open-access data to assess TSPO methylation, mutation and amplification as potential regulatory mechanisms of TSPO expression.With results hinting to TSPO gene silencing by promotor hypermethylation, we performed direct bisulfite sequencing and qPCR analyses on an own tumor cohort.Most importantly, we used tissues from our unique TSPO-PET imaging study cohort that underwent TSPO-PET imaging and targeted biopsy or resection allowing for the direct correlation between tracer enrichment and histopathological/molecular features.In this cohort, we studied the regional and cellular heterogeneity of TSPO expression and used RNA sequencing (RNA-Seq) to elucidate TSPO signaling relationships.

Patient samples and tissue specimens
Fresh-frozen tumor tissues for bisulfite PCR methylation analysis and antibody validation were selected from the tumor tissue archive of the Department of Neuropathology, Regensburg University Hospital and investigated according to protocols approved by the institutional review board (ethics board approval no.18-1207-101 and 20-1799-101).Tumors were initially classified according to the WHO 2016 and re-classified according to the WHO 2021 classification [45].Parts of each tumor were snap-frozen directly after surgical resection and stored at -80 C.Only tissue samples with a tumor cell content of 70% or more were used for methylation analyses (Suppl.Table 1). 2 non-neoplastic brain samples from different individuals (NB01, NB02) served as a reference (D1234062, Biochain and X11001-1, Epigentek).As further controls, we employed commercially available hypermethylated DNA (S7821; Millipore) and unmethylated blood DNA.Antibody validation was performed on protein lysates of 4 IDH-wildtype glioblastomas (GBMs), WHO grade 4.
We used 26 IDH-wildtype GBM (biopsies/resections) from our FOR2858 (German Research Foundation, DFG) TSPO-PET imaging study (Suppl.Table 2) [58], collected in the Center for Neurosurgery/Neuropathology/ Department of Nuclear Medicine, University Hospital of Munich (LMU Munich, Munich Germany) in line with local ethics board approval (ethics board approval no.18-783).All patients had received contrast-enhanced MRI, TSPO-PET and amino acid PET within a maximum of 18 and a median of 3 days before the operation.MRI included gadolinium-enhanced T1-(1 mm slices) and T2-weighted scans (2 mm slices).For TSPO-PET, approximately 180 MBq [ 18 F]GE180 were injected intravenously.Summation scans 60 to 80 min post injection were used for image analysis.For amino acid PET, approximately 180 MBq [ 18 F]FET were injected and 40 min post injection summation images were analyzed as described previously [68].PET scans were performed on 2 subsequent days.Due to the short half-life of F18 (110 min) there was no signaling overlap.Areas of interest were defined in an interdisciplinary exchange between the attending neurosurgeon and nuclear medicine specialist.Brainlab planning software (Brainlab) was used for image fusion and either biopsy planning (Suppl.Figure 1), or intraoperative navigation in case of open tumor resection.[ 18 F]GE180 and FET uptake at the exact localization of the acquired tissue specimen were retrospectively measured by fusing the intraoperative CT or intraoperatively acquired navigation points with the PET images using a Hermes workstation (Hermes Medical Solutions).Standard histological and molecular assessment for diagnostic purposes was performed at the Center for Neuropathology and Prion Research LMU Munich according to WHO criteria as described above.Formalin-fixed and paraffin-embedded (FFPE) tissues from all 26 patients with IDH-wildtype GBM (18 newly diagnosed and 8 recurrent tumors) were used for further immunohistochemical analyses in Regensburg and assessment of tumor cell content within the framework of this study.The tumor cell content of each specimen was evaluated histologically at the Department of Neuropathology at Regensburg University Hospital using H&E stains and classified into one of four categories by the following histologic criteria: "no tumor", characterized by cortex and white matter with no visible tumor cells; "some tumor", characterized by cortex and satellitoses or only sporadic infiltrating tumor cells; "infiltration zone", characterized by a low tumor cell content intermixed with non-neoplastic tissue; "solid tumor", characterized by a tumor cell content of at least 70-80%.For whole transcriptome analyses (bulk RNA-Seq), freshfrozen tumor tissue from a subset of 18 patients with IDH-wildtype GBM was available (Suppl.Table 3).

In silico analyses
For TSPO mRNA expression analyses, open-access HTSeq count data from the TCGA database were used (TCGA Research Network, https://www.cancer.gov/tcga).We used data.table(v1.14.2, [21]) and R.utils (v2.12.2, [8]) R packages to decompress and extract counts.We linked IDs with the provided genecode v22 reference and calculated gene expression values (reads per kilobase per million, RPKM/ for dREG deconvolution: transcripts per million, TPM) for each gene and case ID.We extracted high-grade GBM (IDH-wildtype/mutant) and low-grade astrocytoma (IDH-wildtype/mutant) cases based on their histology information published in [16].GBM expression subtypes and GBM methylation subtypes were also annotated from that source.
We utilized low-grade and high-grade glioma datasets using UALCAN and cBioportal queries for analyzing ETS1 and ETS2 expression patterns.
Epitope blocking experiments for TSPO antibody validation were performed using human TSPO peptide (ab170987, abcam).Briefly, before primary antibody application on sections, the TSPO antibody was incubated with fivefold the amount of blocking peptide (30 min, room temperature).
IHC staining was scored using a Zeiss Imager M2 (200x, DL = 40-41%, 0.80 aperture, FL = on).Whole biopsy samples or four random scoring fields in resections were scored.The TSPO staining intensity was evaluated using a H Score-oriented approach [31].For area analysis, %Area positive for TSPO or CD68 were estimated in at least 5 images in the case of resections and 1-3 images in the case of biopsies (400x, DL = 59.1%, 0.90 aperture, FL = on, Axiocam 503 color camera).The images were processed with Fiji Image J [64] using background subtraction (default settings), color separation, color threshold regime (moments method), binary conversion and the analyze particles module.

RNA isolation, RNA-Seq and bioinfomatic analysis
RNA for Next Generation sequencing (NGS) was isolated using Maxwell® RSC Simply RNA Tissue kit (AS1340, Promega) according to manufacturer's instructions.NGS libraries were prepared from 10 ng total RNA with Illumina Stranded total RNA Prep kit (#20040529, Illumina).NGS was performed on a NextSeq 500 or 550Dx instrument using indexed, 75 cycles single-end read protocol and a NextSeq 500/550 High Output Kit v2.5 (#20024906, Illumina).For analysis of NGS data we used freely available, customizable tools and a workstation.Image analysis and base calling resulted in .bclfiles, which were converted into .fastqfiles by the bcl-2fastq2 tool v2.17.1.14.and were mapped to human genome assembly GRCh38.87 v102 using HiSat2 Mapper, allowing one mismatch [38].All unique hits were processed with featureCounts v2.0.1 [41].Reads were counted locus-based, i.e. for unions of exons per gene.Batch effects caused by different sequencing runs, were removed with Combat Seq of SVA package using a negative binomial regression model that retains the integer nature of count data [77].Principal component (PCA) and differential expression analyses were done within R v4.1.2(R Core Team 2021) using DeSeq2 [46].Volcano plots and heatmaps were generated with Enhanced Volcano R package [11], and Complex Heatmap R package [27,28].Functional annotation analyses were done based on differentially expressed genes (logFC ± 1, padj ≤ 0.05) with FUMA v1.4.1 [73] and Reactome release 82/Pathway browser 3.7 [25,35].Gene set enrichment analyses and single sample gene set enrichment analyses were performed with DeSeq2 normalized expression values using the GSEA module v20.3.5 [50,66] or the ssGSEA v10.0.11module [5,66] on Gene Pattern [59], respectively.Gene sets for GBM expression subtypes were used from [13,72], gene sets defining four main cellular GBM states were used from [52] and hallmark gene sets and oncogenic signature gene sets were used from [42].Genes specifying mesenchymal-like (MES-like) tumor/ immune cell interactions were used from [30].For single cell deconvolution we used dREG [20] and single cell expression values (transcripts per million/TPMs) of an open-access IDH-wildtype GBM reference (GEO access number: GSE131928).Fastq.files and raw count tables of our analyzed samples were deposited in GEO (GEO access number: GSE230453).

Elevated TSPO mRNA expression in malignant gliomas is inversely correlated with promotor methylation
Previous studies showed TSPO overexpression in gliomas compared to non-neoplastic brain tissue and suggested positive correlation of TSPO expression and glioma malignancy [14,71].However, the mechanism of upregulation of TSPO had not yet been deciphered.We therefore first retrieved TSPO expression data from open-access RNA-Seq databases to reproduce the above mentioned findings [16].We employed in silico queries from the TCGA Research Network and compared the normalized expression values (RPKMs) of TSPO among IDH-mutant/-wildtype gliomas across reported WHO grades, and across glioblastoma (GBM) transcriptional subtypes.We found that TSPO expression values increased significantly with WHO grade in IDH-wildtype gliomas (p ≤ 0.001) and were higher in IDH-wildtype than in IDH-mutant gliomas p < 0.001) (Fig. 1a/b).Within GBM transcriptional subgroups, a significantly higher TSPO expression in the prognostically unfavorable mesenchymal subgroup [9,34] compared to the classical (p = 0.004) or proneural (p = 0.002) groups was observed (Fig. 1c).
To analyze a potential epigenetic regulation mechanism of TSPO expression we analyzed in silico open-access data of Illumina K450 methylation arrays from different glioma types (TCGA-LGG and TCGA-GBM).Methylation beta values of 15 probes covering the TSPO promotor gene locus were extracted (Suppl.Figure 2a).Seven probes covering the CpG island showed lower methylation, so we focused on this area for further analyses (Fig. 1d).One out of seven probes (cg00343092) showed a significant lower methylation level in GBMs compared to anaplastic astrocytomas (intermediate beta values, p < 0.001) and to the other glioma types (high beta values, p < 0.001) (Fig. 1e).We then assessed a potential relation between the observed low methylation and high TSPO expression in GBMs by analyzing patients from which matched RNA-Seq and methylation data were available.Separated by entity, TSPO expression values (RPKMs) and beta values of cg00343092 were inversely correlated (Fig. 1f ); spearman rho analysis confirmed statistical significance (r = -0.5548,p < 0.001) (Fig. 1g).
Regarding reported GBM methylation subtypes (e.g.RTK I, RTK II, and MES), no significant TSPO expression differences between subtypes could be found (Suppl.Figure 2b).In regard to methylation of probe cg00343092 across reported GBM methylation subtypes, the MES group (n = 14) showed a significantly lower methylation (p = 0.044) than the RTK II group (n = 19) (Suppl.Figure 2c).Otherwise, no correlation of TSPO expression (RPKMs) or TSPO methylation (beta values) could be found to any of the GBM methylation subtypes (Suppl.Figure 2d).
To explore alternative regulatory mechanisms of TSPO expression we further analyzed large DNA-based glioma data sets (in total 531 samples) in cBioportal [17,23] but neither detected any relevant TSPO coding mutations nor TSPO gene amplifications (data not shown).

TSPO promotor hypermethylation depends on IDH mutation and is inversely correlated to TSPO expression
To better understand the mechanism of TSPO regulation, we extended methylation analyses beyond the 7 CpGs covered by the Illumina K450 methylation array.We performed direct bisulfite sequencing of nearly the complete TSPO promotor CpG island region (72 of 80 single CpGs covered) within 22 human glioma samples (10 IDH-mutant and 12 IDH-wildtype).We further analyzed TSPO expression by qPCR.Using JASPAR for the whole TSPO promotor CpG island region, we predicted binding sites for transcription factors (ETS1/2, SP1/2, and STAT3) known to be involved in TSPO regulation [6].We observed various possible binding sites for ETS1/2 and SP1/2 along the CpG island, and fewer sites for STAT3.Bisulfite PCR revealed areas with strong methylation in IDH-mutant gliomas compared to a weak or absent methylation in IDH-wildtype gliomas (Suppl.Table 1).Differences were especially pronounced in a TSPO CpG island subarea (chr22: 43,151,840 − 43,152,163) around the probe cg00343092 detected in the in silico dataset (Fig. 2a).Within this subarea we observed accumulated binding sites for ETS1/2.Mean methylation scores in the identified TSPO CpG island subarea were high in IDH-mutant and low or absent in IDH-wildtype gliomas (p < 0.001) (Fig. 2b).Spearman rho correlation of mean methylation scores and TSPO expression values (ΔΔCT) in the same tumors revealed a weak but significant inverse correlation (r = − 0.4404, p = 0.0403) (Fig. 2c).
Of note, non-neoplastic brain tissue also showed a low/absent methylation within the identified TSPO CpG island subarea comparable to that of IDH-wildtype tumors (GBMs).This observation leads to an enhanced understanding of the observed methylation changes: We did not recognize hypomethylation in GBMs but a denovo methylation in IDH-mutant tumors.Thus, overexpression of TSPO in gliomas is not induced by a loss of TSPO promotor methylation, but TSPO promotor subarea hypermethylation might serve as a mechanism to reduce TSPO expression levels in IDH-mutant compared to IDH-wildtype gliomas.

TSPO antibody validation showed a specific staining pattern with no unspecific binding
We thoroughly tested the monoclonal TSPO antibody with western blot experiments on transient TSPOknockdown glioma cells, on glioma cells with an antibody epitope blocking approach and on protein lysates from 4 different cryo-conserved GBM samples.Additionally, IHC was performed on TSPO-knockout microglia and an anaplastic astrocytoma with and without antibody epitope blocking.Western blots of glioma cells revealed a decrease of the antibody-binding signal in the transient TSPO-knockdown and a single band at the expected TSPO size (18 kDa).Another tested polyclonal TSPO antibody revealed unspecific binding patterns and was not used further (Suppl.Figure 3a).Epitope blocking resulted in a complete loss of the 18 kDa TSPO band in glioma cells (Suppl.Figure 3b) and a single band at the expected TSPO size was observed when glioblastoma protein lysates were analyzed (Suppl.Figure 3c).IHC staining showed TSPO expression in TSPO-wildtype microglia and no staining in TSPO-knockout microglial cells (Suppl.Figure 3d).Blocking of the epitope binding site resulted in a complete loss of IHC staining in an astrocytoma sample (Suppl.Figure 3e).In summary, all these experiments clearly demonstrate a specific staining pattern for the applied TSPO antibody with no indications for unspecific binding.

TSPO-IHC correlates with TSPO-PET signal and is highest in the tumor core of GBMs
For histopathological evaluation of TSPO as a PET imaging marker, we utilized IDH-wildtype glioblastoma (GBM) samples (n = 26) of our multidisciplinary prospective cohort [58].All study patients underwent TSPO-PET imaging and targeted biopsy or resection for correlation of imaging with histological parameters (Fig. 3).Biopsy specimens were available from 14 and resections from 13 patients.One resection sample could not be further histologically analyzed due to insufficient tissue quality.For all collected tissue samples from 26 patients (18 primary tumors) TSPO-PET imaging values were extracted, histological classification of tumor cell content (solid tumor, infiltration zone, some tumor and no tumor) was performed and TSPO protein expression was analyzed immunohistochemically.In our stereotactical approach we collected multiple tissue samples from each individual patient that could match to different tumor cell content categories (for detailed information compare Suppl.Table 2).Solid tumor was collected from 19 patients (45.0% of all samples), the tumor infiltration zone was sampled in 18 patients (28.5%), areas with even lower tumor cell content (cortex and satellitoses or only sporadic infiltrating tumor cells, "some tumor") were present in 13 patients (19.9%) and from 11 patients (6.6%) we retrieved material not containing histologically visible tumor ("no tumor").
Inter-as well as intra-tumoral heterogeneity was observed for TSPO-PET imaging and TSPO-IHC.Figure 4a/b provides visualization of one patient with low TSPO-PET signal (GBM_20) and one patient with high TSPO-PET signal (GBM_10) together with the corresponding TSPO-IHC.The case with low TSPO-PET tracer uptake showed a weak TSPO staining in IHC, whereas the case with high TSPO tracer uptake showed a strong TSPO staining.Imaging information (TSPO-PET, FET-PET and MRI) and full histology (H&E and TSPO-IHC) for both cases is supplied in Suppl.Figure 4. To further analyze TSPO-PET imaging correlation to TSPO-IHC, we compared TSPO-PET imaging SUVmax values with their corresponding TSPO-IHC %Areas.We used all specimens with solid tumor (73 biopsy/resection specimens from 17 patients) and infiltration zone (50 biopsy/ resection specimens from 15 patients).TSPO-IHC and TSPO-PET (separated by patient) showed a clear positive correlation between both parameters (Fig. 4c).Statistical significance was confirmed by a sample-to-sample spearman rho analysis within the respective tumor content cell group weighted per patients' sample numbers (solid tumor: r = 0.588, p < 0.001; infiltration zone: r = 0.300, p < 0.001).Of note, when comparing TSPO protein expression across GBM expression subtypes (Fig. 4d), we found a significantly higher TSPO expression in the solid tumor areas of patients with mesenchymal transcriptome patterns compared to patients with proneural or classical patterns (p < 0.001, both with an intensity-based H score and %Area score).
We also compared TSPO signal intensity across the different tumor cell content groups.TSPO-IHC staining intensity increased with increasing tumor cell content.Analyzing the mean distribution of TSPO-IHC semiquantified both with an intensity-based H score and %Area score in all 26 patients we found a significantly higher TSPO expression in the solid tumor areas compared to areas containing some tumor cells or no tumor at all (p < 0.05, p < 0.001) (Fig. 4e/f ).Analyzing TSPO-PET (SUVmax) in the same way, we also found a significantly higher TSPO signal in the solid tumor area compared to all other areas with lower tumor cell content (p < 0.001, p < 0.05) (Fig. 4g).Thus, the highest overall TSPO signals were observed in the solid tumor core regions with a high glial tumor cell content.

TSPO is expressed by diverse cell populations and CD68positive macrophages/microglia drive TSPO signal in the infiltration zone
To further analyze the cellular source of TSPO, immunofluorescence co-staining for TSPO with cell type differentiation and tumor-markers was performed in Fig. 3 CONSORT flow diagram of the FOR2858 TSPO study (tissue-based aspects).The FOR2858 TSPO study cohort included in total 27 patients with GBM.All patients had received contrast-enhanced MRI, TSPO-PET and amino acid PET.Areas of interest were defined in an interdisciplinary exchange between the attending neurosurgeon and nuclear medicine specialist.Imaging-coordinated biopsy/resection along trajectory resulted in 14 patients with biopsy material and 13 with resection material for further analyses.Formalin-fixed and paraffin-embedded (FFPE) material from 26 patients with IDH-wildtype GBM (18 primary and 8 recurrent tumors) was used for further histological/molecular analyses.Tumor cell content was assessed by using H&E stains of each specimen with subdivision into the following categories: "no tumor", "some tumor", "infiltration zone", "solid tumor" (see text for further explanations).In our stereotactical approach we collected multiple tissue samples from each individual patient that could match to different tumor cell content categories (for more detailed information compare Suppl.Table 2).After some dropouts, where no reliable immunohistochemical staining or no TSPO-PET value extraction was possible, 17-18 patients with solid tumor samples, 15-16 patients with infiltration zone samples, 12 patients with "some tumor" samples and 8-10 patients with no tumor were available for data evaluation (depending on the respective comparisons).For molecular analyses, fresh-frozen cryo material from 24 patients with IDH-wildtype GBM was collected in addition to the FFPE material.After exclusion of specimens with no solid tumor (5 patients) and specimens without sufficient RNA yield (1 patient) a subset of 18 patients with IDH-wildtype GBM (13 primary and 5 recurrent tumors) was available for molecular data evaluation.
a TSPO-enriched patient with IDH-wildtype GBM (GBM_11 II).As described in the literature, microglia/macrophages, endothelial cells and tumor cells can be a cellular source for TSPO [33,53,60,79].We used p53/GFAP for staining astrocytic tumor cells, CD11b/ CD68 for microglia and macrophages [36], and CD31 for endothelial cells [43] (Fig. 5a).Double staining revealed that on a single cell level in tumor core regions TSPO was expressed by all these cell populations (i.e.p53/GFAPpositive astrocytic tumor cells, CD68/CD11b-positive microglia/macrophages and CD31-positive endothelial cells).As microglia/macrophages are often described cellular sources of TSPO [22,79], we stained all 26 patients with IDH-wildtype GBM with a well-established CD68 [PG-M1] antibody.When correlating CD68-IHC with TSPO-PET in the same way as we did for TSPO-IHC, we observed clear differences (Fig. 5b).While there was almost no association between TSPO-PET imaging and CD68-IHC (Spearman correlation: r = 0.090, p = 0.015) in the solid tumor areas, there was a weak association between TSPO-PET and CD68-IHC in the infiltration zones (Spearman correlation: r = 0.559, p < 0.001).When analyzing CD68-IHC (%Area) across the tumor content groups, we also observed clear differences in CD68 expression with the lowest CD68 expression in the solid areas and higher CD68 expression in the tumor-adjacent areas (infiltration zone, p = 0.089, some tumor, p < 0.01) (Fig. 5c).
Given this finding, we further analyzed the dependency of TSPO/CD68 expression by performing direct spearman correlation analyses between TSPO-IHC (%Area) and CD68-IHC (%Area) in the different tumor cell content groups (Fig. 5d).We observed a decrease/loss of association of TSPO expression and CD68 expression with increasing tumor cell content (no tumor: r = 0.686, p < 0.001; some tumor: r = 0.414, p < 0.001; infiltration zone: r = 0.403, p < 0.001; solid tumor: r = 0.027, p = 0.472).Thus, we conclude that CD68-positive microglia/ macrophages are a relevant source of TSPO expression/enrichment, predominantly in tumor-adjacent zones and less in solid tumor core areas.
For further analysis, we split the RNA-Seq data according to the median TSPO expression (median = 92.90)into a TSPO-low and a TSPO-high group.The distribution of normalized expression counts in the TSPO-low/high group is shown in Suppl.Figure 5b.Differential expression analysis between both groups revealed that 1581 genes in total were differentially expressed (logFC ± 1, padj < 0.05), with 1213 genes upregulated and 368 genes downregulated (Fig. 6a).In subsequent functional annotation analyses (Reactome and FUMA), mostly the upregulated genes led to significant overrepresentation hits.The top 50 overrepresented pathways in the FUMA/ Reactome database using all differentially expressed genes (Reactome: FDR ≤ 0.25, FUMA: padj ≤ 0.05) were mainly related to three functional clusters: extracellular matrix (ECM) reorganization/cell migration (16 pathways), immune system interaction (13 pathways) and oncogenic pathways (2 pathways) (Fig. 6b).Furthermore, the analysis of normalized expression values (DeSeq normalized expression values) with gene set enrichment analysis (GSEA) revealed 30 significantly enriched hallmark gene sets (FDR ≤ 0.05, NES > 2.0) mainly from those functional clusters (Fig. 6c).Eight gene sets were involved in immune system interaction, 6 gene sets were indicative of higher tumor malignancy and 1 gene set was related to extracellular matrix organization.Additionally, we performed GSEA with gene sets for oncogenic signatures (Fig. 6d) and found 136 significant enriched pathways (FDR < 0.25).The vast majority of the gene sets (133) were enriched in the TSPO-high group, clearly indicating a more malignant transcriptional phenotype in the TSPO-high group.Taken together, our results suggest that tumor regions with high TSPO expression are highly malignant and exhibit a pronounced tumor-immune system interaction and extracellular matrix organization.

High TSPO expression marks mesenchymal glioblastoma cell subpopulations characterized by elevated numbers of tumor-associated macrophages
When further characterizing the GBMs from our RNA-Seq analysis in terms of their transcriptional subtypes [13,72], it was striking that all 5 GBM samples with the prognostically unfavorable mesenchymal subtype were in the TSPO-high group (Fig. 6a, Suppl.Figure 6a).Unsupervised clustering of differentially expressed genes revealed a separate cluster within the TSPO-high group consisting of these 5 GBMs with the mesenchymal subtype, and principal component analysis also showed a separated cluster with these 5 GBMs (Figs. 6a and 7a).Of note, these 5 GBM samples also showed the highest TSPO expression values (Fig. 6a, Suppl.Figure 6b).We, therefore, performed further analyses with the three clusters revealed by unsupervised clustering of differentially expressed genes (TSPO LOW I, TSPO HIGH II and TSPO HIGH III) (Figs. 6a and 7a).
It had been shown recently that single cell RNA-Seq analysis of a large GBM cohort resulted in four main cellular states: neural progenitor-like (NPC1/2-like), oligodendrocyte progenitor-like (OPC-like), astrocyte-like (AC-like) and mesenchymal-like (MES1/2-like) [52].MES-like states were linked to NF1 mutations and tissue microenvironment interaction and are more abundant in the mesenchymal expression subtype.We performed ssGSEA with all cellular state gene sets in our bulk RNA-Seq data and observed a significant enrichment of MES1/2-like genes in the TSPO HIGH III cluster (Fig. 7b).Hara and colleagues described that MES-like cellular states in GBM cells were promoted by macrophage-derived Oncostatin M (OSM) that interacts with its receptors (OSMR and LIFR) in complex with GP130 (also known as IL6ST) via STAT3 signaling pathways [30].Indeed, in the TSPO HIGH III cluster we observed a significant overexpression of the MES-like TAM tumor interaction genes OSM, OSMR, STAT3 and of CD44 (Fig. 7c).Additional direct gene-to-gene expression correlations in our IDH-wildtype GBMs (TSPO study cohort, TPMs) revealed moderate to strong associations between expression of TSPO and MES-like cellular state markers (Suppl. Figure 6c).Thus, our RNA-Seq results We then performed deconvolution analysis with a single cell RNA-Seq dataset (scRNA-Seq) as basis for specific cell type genes: tumor cells, tumor-associated macrophages (TAMs), oligodendrocyte cells (ODC) and T cells [52].Displaying TSPO expression for cell type clusters (malignant cells, macrophages, oligodendrocytes, and T cells) in this scRNA-Seq dataset demonstrated TSPO expression in all cell types (Suppl.

Figure 6d
).As shown in Fig. 7d, TSPO HIGH III cluster tumors showed a significant decrease in tumor cells and a significant increase in the TAM cell population patterns.Results could be confirmed when extending our previously performed double fluorescence stains for TSPO/CD68 in respect to the here defined TSPO clusters (TSPO LOW I, n = 2, TSPO HIGH II, n = 1 and TSPO HIGH III, n = 2).Representative images of all three clusters (GBM_17 = TSPO LOW I, GBM_11 II = TSPO HIGH II, GBM_25 = TSPO HIGH III) showed highest relative amount of CD68-positive macrophages/microglia in TSPO HIGH III group tumors (Fig. 7e).A quantitative analysis of cell populations in all stained samples confirmed a higher relative content of TSPO/CD68-positive macrophages/microglia in the TSPO HIGH III group tumors (Fig. 7f ).
Taken together, our RNA-Seq results showed that the TSPO-high group consists of two clusters, whereof the cluster with the highest TSPO expression shows an enrichment of mesenchymal signatures and immune system genes and contains an altered cellular composition with higher relative amounts of TSPO/CD68-positive macrophages/microglia.

Discussion
TSPO is frequently overexpressed in glioma [3,14,71,79], and a possible connection between TSPO enrichment and high malignancy has been suggested.TSPO is intensely discussed as an imaging target for prognosis [14,58], during therapy [22,57,62,63], and in CNS pathologies with neuroinflammatory components [4,18,29,78].Nevertheless, systematic approaches to link TSPO imaging to its histopathological correlates that would add informational content on TSPO as a biomarker are largely missing.Furthermore, it is still unclear how TSPO expression is regulated in CNS neoplasia.
To address these open questions we used large in silico datasets and precisely clinically annotated patient collectives from which we had matching TSPO-PET imaging data and tissue specimens for histological and molecular analyses.
Regarding TSPO regulation, initial in silico analyses and confirmation on an own cryo-conserved tissue collective established a role for TSPO promotor hypermethylation in the reduction of TSPO expression levels in the molecularly defined subgroup of IDH-mutant gliomas.Our finding of an epigenetic regulation of TSPO is in line with results reported in a Jurkat human T cell leukemia cell line where demethylation with 5-aza-2′-deoxycytidine caused a dose-dependent increase in TSPO mRNA [48].It is also known that IDH-mutant gliomas commonly exhibit a genome-wide hypermethylation phenotype [10,67] and the observed TSPO hypermethylation might be part of this.We found that the hypermethylated TSPO promotor subarea contains a number of potential transcription factor binding sites that might be blocked.One of these potentially affected transcription factors is ETS1/2, for which a transcriptional regulation of TSPO [6] and a binding inhibition through DNA methylation has already been described [32].Own in silico analyses revealed substantial ETS1 and ETS2 expression in nonneoplastic brain and tumor tissue.ETS1 was upregulated and ETS2 downregulated in GBMs compared to nonneoplastic brain tissue.Correlation analysis of TSPO and ETS revealed a weak but significant association of TSPO and ETS2 mRNA expression in GBMs in silico and in our own study cohort (Suppl.Figure 8).Of note, we found the TSPO promotor unmethylated in non-neoplastic brain tissue.Thus, the hypermethylation we report is a de novo methylation restricted to IDH-mutant gliomas.In glioblastomas/IDH-wildtype gliomas the TSPO promotor was equally unmethylated as in the non-neoplastic controls and we neither observed TSPO gene amplifications nor TSPO gain of function mutations.In this setting, transcription factor binding to an unmethylated TSPO promotor most likely explains the overexpression of TSPO in IDH-wildtype gliomas.In MA-10 Leydig cells it has been shown that PKC ε regulates TSPO gene expression through MAPK (Raf-1-MEK1/2-ERK1/2)-mediated transcriptional activation [6] and we know that these pathways besides others are commonly dysregulated in glioblastoma [37,55].In summary, TSPO transcriptional regulation in gliomas might be the result of a complex interplay between changes in TSPO promotor methylation and their effects on transcription factor binding.
The current literature on TSPO as an imaging marker (for review see [40]) clearly strengthens the need for histopathological evaluation of TSPO imaging correlates.The high-affinity TSPO ligand [ 18 F]GE180 was first used by Albert and colleagues for TSPO-PET imaging of untreated and pretreated GBM and showed remarkably high tumor-to-background contrast and TSPO-PET signal even in areas without contrast-enhancement on MRI [2].This study was then extended with more cases of IDH-wildtype/-mutant gliomas by Unterrainer and colleagues [68].We now provide a concomitant histopathological evaluation of patients with GBM that underwent the [ 18 F]GE180 TSPO imaging protocol.First, we successfully showed that the TSPO-PET signal correlates with TSPO expression as detected by immunohistochemistry with a thoroughly validated antibody.Secondly, our results revealed that the TSPO signal originates from multiple cellular sources, including tumor cells, reactive astrocytes, microglia/ macrophages and endothelial cells.Assessment of the regional heterogeneity of TSPO revealed that solid tumor-cell-rich areas are the major contributors to the overall TSPO signal.Tumor-adjacent areas show a lower TSPO enrichment/expression.Of note, in these regions the TSPO signal is mainly driven by CD68-positive microglia/macrophages.
Currently, there is one other similar human study investigating TSPO imaging and histopathological features in combination [79].However, this study used the [ 18 F]DPA-714 imaging tracer and a different spectrum of tumors (smaller total number: 9 vs. 26 patients in our study, focus on IDH-mutant gliomas, only one GBM vs. 26 in our study) so that the results may not be entirely comparable.Zinnhardt and colleagues found a strong relationship between [ 18 F]DPA-714 uptake and activation of glioma-associated myeloid cells.TSPO expression was mainly restricted to tumor-infiltrating HLA-DR + myeloid-derived suppressor cells (MDSCs) and TAMs.These findings match our observations in the tumor-infiltration zone.However, we additionally describe a relevant degree of intratumoral heterogeneity with higher TSPO expression in the solid tumor core that is characterized by the highest tumor cell content.It appears indeed very likely that in our patient cohort there is a stronger contribution of tumor cells to the overall TSPO signal, as GBM/IDH-wildtype gliomas (as outlined above) have an unmethylated TSPO promotor and overall higher TSPO expression levels than the IDH-mutant gliomas studied in the Zinnhardt paper [79].
Our observation of divergent cell populations contributing to the TSPO signal is also reinforced by investigations on human or murine GBM implantation-based mouse models [14].There, comparable to our results, TSPO expression was observed in tumor cells, microglia, tumor-associated macrophages and endothelial cells and the authors proposed a combination of TSPO-PET and FET-PET as a promising way to visualize tumor-associated myeloid cells.
Interestingly, another study on GBM mouse models described an increase of tracer uptake during temozolomide (TMZ) chemotherapy [22].Our study contained a limited number of 8 recurrent GBM only, but we also observed higher TSPO uptake/expression in those tumors compared to primary, therapy-naïve GBMs.It is indeed very likely that the cellular composition might change under therapy (decrease in tumor cells, increase in reactive and myeloid cells).Therefore we plan to extend our human study to a longitudinal setting in order to increase the number of matched pairs of primary and recurrent tumors and to investigate changes in TSPO enrichment/expression in the course of the disease.
Important findings were revealed by the molecular characterization of our current study.By RNA-Seq we linked high TSPO expression to the three functional clusters "oncogenic signaling, immune system interaction and extracellular matrix organization".We further found that high TSPO expression indicated the mesenchymal transcription subtype and MES-like cell populations, which are associated with a worse prognosis [9,34] and a pronounced interaction of tumor cells with the immune system [30].In line with our findings, various signaling pathways related to inflammation are upregulated in tumors with high TSPO expression [3].TSPO can be either involved in an anti-tumor/pro-inflammatory setting with M1 type microglia/macrophages or a pro-tumor/anti-inflammatory setting with M2 type microglia/macrophages [76].Our transcriptional analysis suggests a complex role of TSPO in tumor-associated inflammation.In tumors with high TSPO expression, on the one hand, we see an enrichment in IFN/TNF-signaling typical for M1 type microglia/macrophages.On the other hand, we observe an overrepresentation in IL-10/-4/-13 signaling genes reactivating the M2 type microglia/ macrophages.Interestingly, M2 macrophages are significantly associated with the mesenchymal phenotype [72] where we see the highest TSPO expression.
By analyzing in silico samples from patients with GBM of the TCGA database, Cai et al. first described that TSPO is highly expressed in the prognostically unfavorable mesenchymal GBM transcriptional subtype [14].We now could confirm this finding in our own patient cohort of the TSPO imaging study.In addition, we further linked high TSPO expression to recently described, MES-like cellular states [52] with their described induction patterns by macrophages [30] and high numbers of TAMs.Indeed, mesenchymal GBMs display the highest percentage of microglia, macrophage, and lymphocyte infiltration from all transcriptional GBM subtypes [47].Interestingly, TSPO as a marker for MES-like cellular states might be of therapeutical interest.In view of their high interaction with immune cells, MES-like cells are potential emerging targets for immune checkpoint inhibition [74].Moreover, TSPO could qualify as a predictive biomarker for TAM-targeting immunotherapy [19].
In conclusion, our study improves the understanding of TSPO as an imaging marker in gliomas.We describe a novel mechanism of TSPO silencing in IDH-mutant astrocytomas.The histological and molecular evaluation of 26 patients with GBM that underwent a defined TSPO imaging procedure provides novel insights into the intratumoral heterogeneity of the TSPO signal.While high signal intensities are observed in the tumor-cell-rich solid core regions, lower TSPO signals in the tumor rim are mostly driven by CD68-positive microglia/macrophages.Finally, we identify TSPO expression as an indicator for the presence of a prognostically unfavorable mesenchymal GBM cell subpopulation characterized by a higher amount of TAMs and pronounced immune system interactions.