Heterogeneous expression of the atypical chemokine receptor ACKR3 in glioblastoma patient-derived tissue samples and cell cultures.

doi:10.21203/rs.3.rs-3996294/v1

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Heterogeneous expression of the atypical chemokine receptor ACKR3 in glioblastoma patient-derived tissue samples and cell cultures.

https://doi.org/10.21203/rs.3.rs-3996294/v1

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Background: Glioblastoma (GBM) is the most aggressive glial tumor of the adult brain, associated with invariably fatal outcome, and a deeper understanding of the underlying malignant mechanisms is necessary to address the current therapeutic failure. We previously demonstrated the role of the CXCL12/CXCR4 axis in GBM cell migration and resistance to ionizing radiation. The atypical receptor ACKR3, responsible for CXCL12 scavenging, was previously suggested as additional important player in the context of GBM.

Methods: Flow cytometry was used on GBM cell lines and patient-derived GBM cell cultures to quantify the level of ACKR3 expression. Moreover, we have at our disposal patient- derived formalin-fixed paraffin-embedded (FFPE) tissue, which is used for immunofluorescent analysis to characterize precisely ACKR3-positive cells. We used also an orthotopic xenograft model to study the impact of ACKR3 in vivo. Finally, qPCR analysis was also realized to study different gene expression on GBM cells.

Results: Following validation of our detection tools, we observed that ACKR3 is expressed within GBM patient tumor tissue, distributed in diverse cell types. In contrast to CXCR4, ACKR3 expression in patient-derived stem-like cells (GSCs) remains very low while ACKR3gene expression by tumor cells appears to be modulated by the in vivo environment. Using overexpression models, we also showed that in vitro ACKR3 had no significant effect on cell proliferation or invasion.

Conclusions: Altogether, these results suggest that ACKR3 plays a minor role in malignant GBM cells, although its expression is possibly regulated by in vivo influences. The subtle and multifaceted functions ACKR3 could exert in GBM should therefore only be tackled within a comprehensive tumor microenvironment considering tumoral but also non-tumoral cells.

Glioblastoma

chemokines receptors

ACKR3

Gliomas are glial primary tumors of the central nervous system (CNS) which are classified by the World Health Organization (WHO) based on their specific histological characteristics and molecular features(1). According to this classification, glioblastoma (GBM) is identified as the most common and aggressive grade 4 glioma and is distinguished by a set of genetic alterations(1, 2). These include the absence of the isocitrate dehydrogenase (IDH) mutation, designating GBM as an IDH wild-type (IDH WT) tumor, the presence of mutations in the telomerase reverse transcriptase (TERT) promoter and copy number alterations in chromosome 7 and 10 (+ 7/-10), most often associated with epithelial growth factor receptor (EGFR) amplification(1, 2). The standard-of-care therapy associating maximal safe surgery and concomitant radio-chemotherapy using temozolomide (TMZ) allows a median survival of about 16 months from diagnosis(3). Moreover, the progression of the disease is characterized by a systematic recurrence that relies on (1) GBM cell infiltration through the brain tissue, hindering total resection of the tumor(4), and (2) an extreme heterogeneity(5) of GBM cells that transit through diverse functional states(6–8), which overall lead to therapeutic resistance.

Chemokines constitute a subgroup of chemotactic cytokines secreted by various cell types in different tissues and playing an important role in inducing and guiding cell migration(9). They are important regulators of various processes such as development, immune responses and tissue repair(9). Chemokine receptors, which are G protein-coupled receptors, have been widely studied for their role in cancer development and metastasis(10, 11). Besides malignant cells, chemokines and their receptors are also expressed by a wide range of cell subtypes found within the tumor bed including tumor-associated macrophages, tumor infiltrating-lymphocytes, vascular cells, non-malignant glial cells and neurons, and play diverse roles in GBM growth, angiogenesis and resistance to treatment(12).

We have previously demonstrated the function of CXCR4, the receptor for the CXCL12 chemokine, in the migration of GBM cells towards the subventricular zone (SVZ) in orthotopic xenografts models, as well as in the CXCL12-mediated protection from radiation therapy(13, 14). ACKR3 (formerly named CXCR7), a second receptor for CXCL12, expressed in diverse cell types, including leukocytes, neurons or endothelial cells was identified(15, 16). ACKR3 was subsequently demonstrated to play a crucial role in regulating CXCL12-dependent processes including cardiovascular and neuronal development as well as in the migration and homing of hematopoietic stem/progenitor cells(17–20). Unlike CXCR4 which signals via G protein pathways to induce cell migration and proliferation, ACKR3 activity relies mainly on β-arrestin recruitment and its ability to signal through G protein-independent pathways is still controversial. ACKR3 is proposed to act as a scavenger or “sink” receptor for CXCL12, shaping its gradient and regulating its availability, thereby controlling the directional migration and homing of CXCR4-expressing cells(21–25). So far, the exact role and expression of ACKR3 in GBM and its crosstalk with CXCR4 through their shared ligand, CXCL12, remain to be elucidated.

ACKR3 can also heterodimerize with CXCR4 and modify its signaling properties(26). Several years ago, different studies reported high expression of ACKR3 in brain tumor cell lines and tissue samples, which correlated with bad prognosis and increased aggressiveness in preclinical models(23). ACKR3 was also shown to be expressed on cells delimiting the SVZ in the mouse brain(27–29), which suggests an important role ACKR3 could play in glioma(30). On that basis, we decided to investigate the precise function ACKR3 could exert in GBM cells, in a potential interplay with CXCL12 and CXCR4, using patient-derived models. Recently, based on large-scale patient-based transcriptomic data, we have shown that ACKR3 is one of the most abundant chemokine receptor-encoding genes in glioma tissue(12). However, how these results are correlated to detection of the corresponding protein ex vivo and in various patient-derived cell cultures remain elusive.

In this study, we assessed the expression of ACKR3 protein expression in patient-derived GBM tissue and GBM stem-like cells, in different in vitro and ex vivo settings, with the ultimate aim to better understand its function and impact on GBM and to guide novel ACKR3-targeting therapeutic strategies.

Human tissues - Human GBM samples were obtained from residual tumor tissue after surgical resection, in collaboration with the Neurosurgery department of the Liège University Hospital (CHU), and the University Hospital Biobank (BHUL, Liège, Belgium). Fresh tissue was further (1) dissociated to establish patient-derived glioblastoma stem-like cell cultures (GSCs), (2) formalin-fixed and paraffin-embedded (FFPE) to be used for immunohistofluorescence, (3) flash-frozen in liquid nitrogen for molecular biology analyses. Non-tumoral brain tissue was obtained from the BHUL, from brain donations or residual tissue following epileptic foci resections.

Cell culture – In-house patient-derived GSC cultures (T08, T013, T018 and T033) were established from resected adult GBM tumors, and cultured as 3D tumorospheres in Dulbecco's modified Eagle's medium and Nutrient Mixture F-12 (DMEM/F12 with GlutaMAX, Gibco) supplemented with 2% of B27 without vitamin A (Gibco), 100 U/mL penicillin and 100 µg/mL streptomycin (ThermoFisher Scientific), 1 µg/mL of heparin (LEO pharma), 20 ng/mL of human EGF (Peprotech) and 20 ng/mL of bFGF (Peprotech). GCS cultures informations are listed in Table 1. T033 cells were stably transduced with a lentiviral vector LV-CMV-hACKR3 or LV-CMV-mRFP for the control vector. For in vivo monitoring, T033 were stably transduced with a lentiviral vector pLV-IRES-Luciferase-mRFP. Plasmid design and related experiments were carried out with the help of the GIGA Viral Vectors platform. The U87 human GBM cell line was obtained from ATCC. U87 cells were transfected with pIRES-puro-ACKR3-WT to overexpress the human ACKR3 or with pIRES-puro-CXCR4-WT to overexpress the human CXCR4, and further selected using puromycin (1 µg/mL and 0.5 µg/mL respectively). The MCF-7 human breast cancer cell line was obtained from ATCC. These cell lines were all cultivated as adherent cell monolayers in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin and 100 µg/mL streptomycin (ThermoFisher Scientific). Mycoplasma tests were performed on a regular basis.

Immunofluorescent stainings - FFPE human brain tissue sections were heated at 60°C, dewaxed with 100% xylene (2 × 20 minutes), and rehydrated through a set of alcohol baths: 100% ethanol (2 × 5 minutes), 95% ethanol, 80% ethanol, 75% ethanol and water (1 × 2 minutes each). An antigen retrieval step was performed using Tris–EDTA buffer (10 mM Tris-base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0). Slices were heated in a pressure cooker for 3 minutes then were left to cool down at room temperature. Slices were permeabilized with PBS + 0,2% Triton-X100 for 10 minutes, incubated 30 seconds with TrueBlack® Lipofuscin Autofluorescence Quencher (Biotium), blocked with PBS + 10% normal donkey serum, then incubated with antibodies overnight at 4°C. ACKR3 (R&D systems, #MAB42273, clone 11G8) was co-stained with SOX2 (Cell Signaling Technology, #3579), PDGFRβ (R&D systems, #AF385), Iba1 (Abcam, #ab5076), GFAP (Abcam, #ab4674), EGFR (Cell Signaling Technology, #4267), Rab5 (Cell Signaling Technology, #3547) and CXCR4 (Abcam, #ab124824). After washing steps, slides were incubated for 1 hour with conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) and nuclei were counterstained with DAPI (Sigma). Cells coated on coverslips were fixed in 4% PFA for 10 minutes at room temperature, permeabilized with PBS + 0.1% Triton-X100, incubated with primary antibodies overnight at 4°C, then with fluorescently labelled secondary antibodies (Jackson ImmunoResearch Laboratories) for 1 hour at 4°C. Image acquisition was performed with an epifluorescence microscope (Zeiss Apotome) and analyzed on ZENlite and ImageJ (Fiji) softwares. Antibodies are listed in Table 2.

Flow cytometry - Cells were collected by centrifugation at 300g for 5 minutes. The supernatant was removed, and cells were washed with PBS. Cells were dissociated with 1 mL Accutase (StemCell Technologies) and incubated at 37°C for 5 minutes. Twice the volume of flow buffer (PBS with 1% BSA, 1 mM EDTA, 0.1% Azide) were added to quench Accutase action. Cells were collected by centrifugation at 300g for 5 minutes. Cell pellet was resuspended in 1 mL of flow buffer and were counted to prepare 3x10⁵ cells in 100 mL. APC-conjugated ACKR3 antibody (Biolegend, #331114, clone 8F11-M16) or mouse-anti ACKR3 (R&D systems, #42273, clone 11G8) were added and incubated for 1 hour at 4°C, in the dark. Cells were washed three times by adding 1 mL of flow buffer. A centrifugation step at 300 g for 4 minutes at 4°C was performed. After the third wash, supernatant was removed, and cells were resuspended in flow buffer to a final volume of 300 µL. Samples were recorded on a flow cytometer Canto II (BD Biosciences). Percentage of positive cells as well as Mean Fluorescence Intensity values (ΔMFI = MFI of stained cells – MFI of unstained cells) were analyzed using FlowJo software. Antibodies are listed in Table 2.

CXCL11 and CXCL12 stimulation - Cells were collected by centrifugation at 300g for 5 minutes. The supernatant was removed, and cells were washed with PBS. Cells were dissociated with 1 mL Accutase (StemCell Technologies) and incubated at 37°C for 5 minutes. 3x10⁵cells were plated in a 6-well plate with 1 mL of medium and 2.5 nM or 10 nM human CXCL12 (Peprotech) or 10 nM of human CXCL11 (Peprotech) were added for 24 to 48 hours. Flow cytometry analysis was performed as described above.

Quantitative RT-PCR – Total RNA was isolated using the RNA isolation Nucleospin kit (Macherey-Nagel). RNA was reverse transcribed by using ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs) with random primers mix. For qRT-PCR reaction samples, a mix of a total volume of 5 μL was prepared. qRT-PCR Mix contained 2 μL of the diluted cDNA (10 ng per reaction), 2.5 μL of SYBRGreen (Eurogentec) and 0.25 μL of each primer (reverse and forward). Quantitative RT-PCR was performed using LightCycler 480 Roche. Primer sequences are listed in Table 3.

Cell counting – 1x10⁵ cells were seeded in 1 mL of medium and incubated at 37°C for 4 and 7 days. Then, cells were dissociated with trypsin-EDTA (Gibco) and counted with trypan blue staining by using an automatic cell counter (Countess™ II Automated Cell Counter, Thermo Fisher Scientific).

Cell Trace/CFSE – GSCs were stained with CellTrace^TM Violet Stain (Invitrogen, 5 µM /10⁶ cells) and incubated for 15 minutes at 37°C in the dark. Medium was added to quench the excess of dye and centrifuged at 300g for 3 minutes. Supernatant was removed and cells were resuspended in culture medium and incubated for 30 minutes at 37°C. Then, a cell suspension of 3x10⁵ cells was prepared for each condition. The CXCL12 condition was treated with 10 nM human recombinant CXCL12. Cells were incubated for 4 days at 37°C in the dark. Before flow cytometry analysis, cells were stained with 100 µL of a 1/1000 Zombie NIR^TM fixable viability dye (SONY) and incubated for 15 minutes at RT. Cells were analyzed with FACS Canto II (Becton Dickinson).

U87 and U87 ACKR3 cells were stained with CellTrace^TM CFSE Cell Proliferation kit (Invitrogen, 0.25 µM/2.10⁶ cells) for 15 minutes at 37°C in the dark. The excess of free dye was quenched with medium during 5 minutes at 37°C in the dark. The cells were washed with complete medium and incubated for additional 10 minutes at 37°C to allow the reagent to undergo acetate hydrolysis. Then, part of the cells (2.5x10⁵ cells per condition) were washed with PBS and stained with Zombie NIR^TM fixable viability dye (Biolegend, dilution 1/3000) during 30 minutes at 4°C. The cells were again washed with PBS and analyzed by FACS using a NovoCyte Quanteon Flow Cytometer (Agilent) (condition day 0). The remaining cells (7.5x10⁵ cells per condition) were seeded in 6 cm culture dishes either in complete medium (untreated control) or in complete medium supplemented with 10 nM of CXCL12. The cells were incubated for 5 days at 37°C in the dark. After 5 days, the cells were detached using Versene (Gibco), washed with PBS and stained with Zombie NIR^TM fixable viability dye (Biolegend, dilution 1/3000) during 30 minutes at 4°C. The cells were washed with PBS and analyzed by FACS using a NovoCyte Quanteon Flow Cytometer (Agilent).

Invasion assay – For in vitro invasion assays, transwell chambers (Boyden chambers with 8 µm pore diameter, Thincert, Greiner) were coated with a 1:1 mixture of 0.05 mg/mL collagen type I (Gibco) and 0.5 mg/mL protein of ECM gel (Sigma-Aldrich) in 1:1 PBS-DMEM for 2 hours at 37°C. U87, U87 ACKR3 and U87 CXCR4 cells were detached using Versene (Gibco), washed with PBS and seeded in the upper compartment of the previously coated inserts (3x10⁴ cells) in DMEM supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco). DMEM was added as chemoattractant in the lower compartment. After 24 hours, cells were fixed with 4% formaldehyde (VWR) in the presence of 1 µg/mL of Hoechst 33342 (ThermoFisher) for 15 minutes at room temperature. Non-invading cells were removed, and the invasion was assessed by counting the cells on the lower side of the membrane under fluorescence microscope (Axio Observer Z1, Zeiss). The cell number was determined using QuPath software.

Western blotting – Whole cell lysates were treated with RIPA (Thermo Fisher Scientific) buffer, and extracted proteins were then denatured for 5 minutes at 95°C. 20 μg of proteins were loaded on a 10% acrylamide/bis-acrylamide gel for SDS-PAGE, then transferred onto a PVDF membrane. The membrane was later incubated with blocking solution for 1 hour at room temperature, and with primary antibody overnight at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch) were then incubated for 1 hour at room temperature and signals were revealed with a chemiluminescent HRP substrate before being imaged using the ImageQuant LAS 4000. Antibodies are listed in Table 2.

In vivo orthotopic xenografts – Adult female immunodeficient mice (Crl:NU-Foxn1^nu) were obtained from Charles River Laboratories and were used for xenograft experiments. Mice were anesthetized in a cage containing isoflurane. Mice were placed in a stereotactic frame and kept under isoflurane anesthesia. After a precise bone drill, 1x10⁵ T033 (GSCs) cells suspended in 2 μL of PBS were slowly infused into the right striatum (from Bregma: -0.5 mm AP, +2 mm VL, +2.5 mm DV). Monitoring of tumor growth was performed with in vivo bioluminescence imaging (IVIS, Xenogen), mice health status was evaluated daily, and body weight was recorded every week. Mice were sacrificed when they showed first clinical signs of significant discomfort or suffering.

Mouse brain tissue processing and immunohistochemistry – Mice were euthanized with intraperitoneal injection of 400 mg/kg of Euthasol vet in NaCl 0.9% and immediately perfused intracardially with ice-cold NaCl 0.9% solution containing heparin (LEO Pharma) and then with 4% paraformaldehyde (PFA) in PBS. Brains were removed and postfixed in 4% PFA for 24 hours. Brains were cryoprotected in PBS + 30% sucrose for 24 hours before being frozen at -80°C. Coronal brain slices of 14 mm were generated with a cryostat and stored at -20°C. For the detection of T033 cells in mouse brain tissue, sections were stained with human Vimentin antibody (MAB3400, Millipore), with the Enzo PolyView IHC kit, according to the manufacturer’s instructions.

Ex vivo isolation of cells after tumor growth - Mice were sacrificed by cervical dislocation and brain were collected. Brains were dissected into thick coronal sections at the level of the dorsal horn of the lateral ventricles. In both hemispheres, restricted tissue areas were removed, (1) in the close proximity of the lateral wall of the lateral ventricle (“SVZ in”) and (2) in the temporal cortex, distant from the lateral ventricle (“SVZ out”). Tissue pieces were mechanically and enzymatically dissociated in a solution of Hibernate-A (Fisher Scientific) containing 10 U/mL DNase and 2.5 U/mL papain and then incubated for 10 minutes at 37°C under gentle agitation. DMEM/F12 was then added to dilute the enzymes and the solution was passed through a 100 µm strainer to remove debris. Cells were collected by centrifugation (160g for 10 minutes) and resuspended in DMEM/F12 supplemented with 2% of B27 without vitamin A (Gibco), 100 U/mL penicillin and 100 µg/mL streptomycin (ThermoFisher Scientific), 1 µg/mL of heparin (LEO pharma), 20 ng/mL of human EGF (Peprotech) and 20 ng/mL of bFGF (Peprotech). Cells were maintained in culture for 7 days, enriched in RFP-positive tumor cells, then collected before a qRT-PCR analysis.

Statistical analyses - The GraphPad Prism 8 software was used for generating graphs and for statistical analysis. The normal distribution of data was verified, and independent comparisons were further performed using unpaired t-tests, Kruskall-Wallis or parametric one-way ANOVA tests. Data were represented as mean ± SD, with the n representing the number of independent experiments. A p-value ≤ 0.05 was considered as statistically significant.

3.1 Validation of monoclonal antibodies for the detection of ACKR3 at the cell membrane

Detecting ACKR3 at the protein level is a widely acknowledged challenge in the field. While genetic models (e.g. reporter genes) can be employed for this purpose(12,21), the crucial requirement remains the recognition under native conditions, especially in order to elucidate ACKR3 role and relevance in cancer. In this study, we first aimed to assess the efficacy of two ACKR3-specific monoclonal antibodies: the 8F11-M16 antibody and the 11G8 antibody, commonly employed in our experiments for ACKR3 detection using flow cytometry and immunofluorescence, respectively. Results indicate that both antibodies yield a strong signal in U87 stably expressing ACKR3 (Figure 1, A). No signal was observed in U87 and U87 CXCR4 cells, showing a good specificity of both antibodies for ACKR3 (Figure 1, A). Additionally, we provide experimental evidence that, among several antibodies that fail to specifically detect the receptor, the 11G8 antibody reliably identifies ACKR3 in immunostaining and immunoblotting experiments using overexpression GBM cell models (Figure 1, B and Additional file 1, A-B). Based on these results, we consider that both and 8F11-M16 and 11G8 antibodies are reliable tools for ACKR3 detection using flow cytometry and immunostaining, respectively.

3.2 ACKR3 exhibits diverse expression patterns in GBM tissue

Over the last years, different studies reported ACKR3 expression in numerous cancer cell lines and tissue(23). Based on data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases (via the GEPIA® platform), ACKR3 expression appears higher in low-grade gliomas (LGGs) and GBM samples compared to normal brain (Figure 2, A), which speaks for a potential role of ACKR3 in malignant cells. In line with this, we performed immunofluorescence staining to detect ACKR3 at the protein level in patient-derived FFPE tissues. We observed that ACKR3 is expressed in both glioma tissue and non-tumoral brain tissue with high inter- and intra-tumoral heterogeneity. We identified three different expression patterns of ACKR3 expression (named here after type 1, 2 and 3). In most GBM samples, ACKR3 appeared present around cell nuclei (“type 1”). Similar pattern was found in non-GBM brain samples, such as the hippocampus (HIPPO) or the subventricular zone (SVZ). In “type 2”, ACKR3 positive regions displayed blood vessel-like shapes and were found in GBM tissue, as well as in the non-tumoral SVZ, in line with previous reports showing the expression of ACKR3 on tumor-associated vasculature(31). Finally, in several samples, ACKR3 was detectable in the whole tissue, without apparent specificity to any cell type or structure (“type 3”). No ACKR3 expression was found in non-tumoral cortical (CORTEX) tissue samples (Figure 2, B-C).

Different co-staining experiments have been performed to analyze the cell types that appeared positive for the receptor. ACKR3 was expressed in SOX2+ cells (“stem-like”) and in GFAP+ cells (astrocytes), with “type 1” features. ACKR3 did not colocalize with PDGFRb (pericytes) or Iba1 (microglia) markers. Importantly, EGFR+ cells in tumor tissue were rarely ACKR3+ (Figure 3, A-B). Blood vessel-like (“type 2”) CXCR4+ cells also appeared ACKR3+ (Figure 3, B). Altogether, these results show that ACKR3 expression within GBM tissue is rather heterogeneous, and it can be distributed among different cell types, which mostly correspond to SOX2, GFAP, and CXCR4-expressing cells. This is in line with single-cell RNAseq data showing that ACKR3 is expressed, although at a low level, in cells assigned to various categories (Figure 3, C).

3.3 Patient-derived GBM stem-like cell cultures express low levels of ACKR3 at the membrane

Fresh tumor tissue obtained after surgical resection were processed in order to obtain patient-derived GBM stem-like cells (GSCs) cultured as floating, 3D tumorospheres (Figure 4, A). The expression of ACKR3 was assessed at mRNA level by qRT-PCR as well as at the cell membrane by flow cytometry. ACKR3 mRNA was detected in patient-derived GSCs (Figure 4, B), although weakly expressed, e.g. compared to the SOX2 gene (Additional file 2, A). Using flow cytometry, we aimed at detecting ACKR3 at the cell surface of GSCs. The signal intensity for ACKR3 appeared rather close to the negative control values (Figure 4, C). The percentage of ACKR3-positive cells in each GSC culture was as follows: T08 =1.22±0.76%; T013=3.91±1.78%; T018=2.90±0.66%; T033=0.78±0.18% (vs U87 ACKR3=81.40±2.31%) (Figure 4D). We selected T018 and T033 GSCs to assess whether ACKR3 expression at the cell membrane could be upregulated upon stimulation with the ACKR3-binding chemokines CXCL12 and CXCL11. T018 and T033 cells were incubated for 24 or 48 hours with different concentrations of chemokines and flow cytometry analysis was performed to detect ACKR3 as well as CXCR4, which also binds CXCL12 but not CXCL11. When T018 cells were stimulated for 24 hours with 10 nM of CXCL12, we discovered a statistically significant decrease of the percentage of CXCR4-positive cells (82.6±4.8%), compared to untreated cells (92.4±2.2%), which suggested CXCL12-mediated CXCR4 downregulation (N=4; p=0.03) (Figure 4, E). No differences of CXCR4 expression were observed when T018 cells were stimulated with a lower concentration of CXCL12 (2.5 nM) or with CXCL11 (10 nM) ) (Figure 4, E). After 48 hours of stimulation, we observed again a reduced expression of the CXCR4 receptor at the membrane (Figure 4, F). Importantly, no difference was observed for ACKR3 expression upon stimulation with CXCL12 or CXCL11, for 24 or 48 hours (Figure 4, E-F). Whereas reduction in CXCR4-positive cells was suggested in T033 cells as well after 24h of stimulation by CXCL12, no significant differences were recorded. Again, the expression of ACKR3 remained very low across all conditions (Additional file 2, B-C). We observed a strong signal for ACKR3 detection in U87 genetically modified for expressing stably ACKR3 (positive control), but also in MCF-7 breast cancer cells, which were previously described to endogenously expressing ACKR3(23,24). Then, we tested a 5-minute incubation of CXCL12, which revealed a significant decrease in ACKR3 signal, revealing receptor internalization in the MCF-7 cells endogenously expressing ACKR3 (N=4 or 5; p=0.014), but the signal in GSCs remained close to zero due to scare surface expression of ACKR3 (Additional file 2, D-E). In conclusion, these results indicate that ACKR3 is not detected at the membrane of patient-derived GSCs, irrespective of chemokine presence in the culture medium. To make sure ACKR3 is not localized exclusively inside the cells and therefore not detected by surface staining, we used immunofluorescence and high-magnification microscopy. This revealed a mild ACKR3 staining, without specific colocalization with Rab5, ruling out a restricted localization of ACKR3 in endosomes in GSCs (Figure 4, G).

3.4 ACKR3 overexpression does not directly influence GBM cell proliferation and invasion

To investigate the role ACKR3 could play in GBM cells that initially express low levels of this receptor, we took advantages of U87 cells and U87 ACKR3 cells and carried out various assays to determine whether the expression of ACKR3 in GBM cells influenced cell proliferation (Figure 5, A). Cell counting revealed no changes in proliferation between U87 ACKR3 and U87 parental cells. CFSE labeling experiments confirmed these results (Figure 5, B-C). We further modified patient-derived GSC T033 via lentiviral transduction to overexpress the ACKR3 receptor (Figure 5, D). Again, no difference in cell proliferation was observed in T033 hACKR3 cells compared to T033 mRFP in the presence or absence of CXCL12 (Figure 5, E-F). We also checked whether ACKR3 expression modified the invasive properties of U87 cells using Boyden chamber assays. The invasive capacities of U87 ACKR3 cells were not modified compared to U87 wild-type cells, even upon stimulation with CXCL12 or CXCL11. Conversely, the presence of CXCL12 increased the invasiveness of U87 CXCR4 cells, which was not observed upon CXCL11 stimulation (p=0,010) (Figure 5, G).

3.5 ACKR3 protein expression is not detected in orthotopic tumors, but gene expression is upregulated ex vivo

T033 GSCs expressing low level of ACKR3 in vitro conditions, were transduced with a lentivirus to stably express RFP and Luciferase. T033-RFP-LUC were grafted in the right striatum of immunodeficient mice and tumor growth was recorded by in vivo bioluminescence imaging at different time points. T033 cells were specifically chosen because they facilitate the formation of a tumor mass within a reasonably acceptable timeframe. So, these cells were selected over others due to technical considerations. (Figure 6, A). At tumor endpoint (7 weeks post-graft), mice brains were collected and immunohistochemistry against human vimentin was performed to detect T033 tumor cells. T033 cells formed highly infiltrative tumors that invaded the whole right hemisphere and reached the contralateral hemisphere via white matter tracts (Figure 6, B). The tumor core was enriched in CXCR4-positive cells, but ACKR3 expression was not detected (Figure 6, C). In a second experiment, in vivo engrafted T033-RFP-Luc GSCs were harvested from different brain regions at 6 weeks post-implantation (before clinical endpoint), dissociated and put back in culture. We were particularly interested in the impact of the subventricular zone on GBM cell behavior(32). Interestingly, RFP-positive T033 cells were detected in the tissue harvested from the right SVZ (“SVZ in”) as well as in the right temporal cortex, away from the SVZ (“SVZ out”). We let RFP-positive T033 cells “SVZ in” and “SVZ out” form new tumorospheres in serum-free culture for seven days, then collected them to analyze their ACKR3 gene expression. Of note, only few RFP-positive cells could be harvested from the contralateral SVZ and cortex which did not regrow. Surprisingly, quantitative RT-PCR analysis showed a significant increase of ACKR3 expression of T033 GSCs ex vivo, especially from “SVZ in”, compared to T033 in vitro (Figure 6, D-E). Given that, endothelial cells present on the lateral wall of ventricle is a major source of CXCL12, we stimulated T033 GSC with CXCL12 in vitro. However, no differences were observed in terms of ACKR3 gene expression when GSCs were stimulated with CXCL12 (Figure 6, F). These results suggest that ACKR3 gene expression is modulated by specific brain microenvironments which does not only rely on CXCL12.

The chemokine receptor ACKR3 has gained increased therapeutic interest in the last years, along with the in-depth elucidation of its atypical mechanism of action, implication in different physiopathological processes and the development of new pharmacological modulators(33–41). Together with its CXCL12/CXCR4 signaling partners, it has been suggested as an important player in cancer, including in brain tumors(23). Additionally, our recent transcriptomic analysis of large patient cohorts showed a high expression of ACKR3 in glioma tissues(12), which several reports have previously associated with disease prognosis. ACKR3 has therefore been endowed with significant therapeutic potential.

Accordingly, several studies described ACKR3 expression in GBM patient tissue and in GBM cells in vitro(25, 42, 43). Here, after validation of the reliability of our detection tools(44), we analyse the expression of ACKR3 in glioblastoma patient-derived tissue samples and cell cultures. Indeed, ACKR3 high expression in RNAseq data from bulk tissue is not sufficient to obtain the full picture of its function in a tumor. On top of that, although single cell RNAseq data indicate ACKR3 is expressed in malignant cells, we firmly demonstrated here that in spite of a sustained expression at the transcript level, ACKR3 protein is merely undetected in patient-derived GBM cells in vitro, under various conditions. In a different tumor model, Antonello et al. show that ACKR3 surface expression is detected only in a fraction of B-cell lymphoma cells, yet both ACKR3 + and ACKR3- cells have similar levels of mRNA(45). Such results can partially be explained by the preferred localization of ACKR3 in intracellular compartments, e.g. endosomes(46, 47). It has also been shown that ACKR3 surface expression in the same lymphoma cells is increased upon in vivo “conditioning” in subcutaneous grafts and further ex vivo isolation, without any modification in ACKR3 gene expression, again suggesting a particular trafficking of the receptor from intracellular compartments to the plasma membrane(48). Here however, we do not detect the receptor inside patient-derived GBM cells using immunofluorescence or western-blotting on whole cell lysates. It remains puzzling to observe that in vivo “conditioning” in orthotopic brain xenografts strikingly increases ACKR3 expression in ex vivo isolated cells although the protein was not detected in tumor tissue.

We also report in situ staining, paying particular attention to ACKR3 expression pattern and to the type of cells that express it. ACKR3 appears associated with blood vessels in tumor tissue (“Type 2”), and present in GFAP+/SOX2 + cells, however not present in EGFR + cells (“Type 1”). Importantly, GFAP+/SOX2 + cells may include intra-tumoral reactive astrocytes, which were previously described as ACKR3 + in different models of brain pathologies(49). In a very similar manner, Birner et al.(50) described ACKR3-positive cells as “vascular ACKR3 cells (vasACKR3 + cells detected in 58.1% of 320 cases) and much rarer “tumor cell ACKR3” (tcACKR3 cells in 11.6% of 320 cases). They described the number of vasACKR3 + cells as a bad prognosis marker in IDH mutant gliomas, with the number of tcACKR3 + cells having no prognosis value(50). Elaborating an ACKR3-targeted therapeutic strategy in cancer requires to precisely identify the cell types of interest and it is now of significant importance to orient futures research endeavors considering ACKR3 in non-malignant cells from the TME (e.g. vascular cells, glial cells, putatively immune cells).

Whether ACKR3 plays a key role in GBM tumors is still a matter of debate. Here, we concluded that patient-derived GSCs, widely used as in vitro models of GBM tumors in mechanistic studies or drug testing, do not express detectable levels of ACKR3 protein. However, we do not rule out that ACKR3 expression may be tightly regulated upon variable conditions. Given the fine-tuned chemokine-dependent cell-cell interactions, we now consider imperative to study ACKR3 as well as other chemokine receptor function in an exhaustive, well vascularized, immunocompetent tumor microenvironment.

ACKR3: atypical chemokine receptor 3

bFGF: basic fibroblast growth factor

BSA: bovine serum albumin

CNS: central nervous system

CXCL11: C-X-C motif chemokine ligand 11

CXCL12: C-X-C motif chemokine ligand 12

CXCR7: C-X-C chemokine receptor type 7

DMEM: Dulbecco's Modified Eagle’s Medium

ECM: extracellular matrix

EDTA: ethylenediaminetetraacetic acid

EGF: epidermal growth factor

EGFR: epidermal growth factor receptor

FBS: fetal bovine serum

FFPE: formalin-fixed paraffin-embedded

GAPDH: glyceraldehyde 3-phosphate dehydrogenase

GBM: glioblastoma

GFAP: glial fibrillary acidic protein

GSCs: glioblastoma stem-like cells

GTEx: genotype-tissue expression

HIPPO: hippocampus

HKG: housekeeping gene

HRP: horseradish peroxidase

IDH: isocitrate dehydrogenase

IHC: immunohistochemistry

LGG: low-grade glioma

MFI: mean fluorescence intensity

PBS: phosphate buffered saline

PDGFR: platelet-derived growth factor receptor

PFA: paraformaldehyde

PVDF: polyvinylidene difluoride

RFP: red fluorescent protein

RIPA: radioimmunoprecipitation assay

RNA: ribonucleic acid

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

SVZ: subventricular zone

TCGA: The Cancer Genome Atlas

TERT: telomerase reverse transcriptase

TMZ: Temozolomide

WB: western blotting

WHO: World Health Organization

ΔMFI: delta mean of fluorescence

Ethics approval and consent to participate

All animal experiments were approved by the ethical committee of the University of Liège (#2290).

Consent for publication

Not applicable

Availability of data and materials

All data are available when request to the corresponding author.

Competing interest

The authors declare that they have no competing interests.

Funding

This work was supported by the National Fund for Scientific Research (FNRS, Télévie) (grants 7.4593.19, 7.4529.19 and 7.8504.20) and the Leon Frédéricq Foundation (Liège, Belgium), the Luxembourg Institute of Health (LIH) through the NanoLux platform, Luxembourg National Research Fund (INTER/FNRS grant 20/15084569 and CORE C23/BM/18068832).

Authors’ contributions

DI and VN designed the study. Entire experiments and data analysis were mainly performed by DI and partially by other authors. The manuscript was written by DI and revised by VN, BR, AC and MS. All authors read and approved the final manuscript.

Acknowledgments

The authors would like to thank the patients and the Neurosurgery department of the Liège University Hospital Belgium. We also would like to thank the GIGA-ULiège Flow cytometry and Cell Imaging (S. Ormenese and colleagues), the NCP platform from LIH (Fanny Hedin, Giovanna Radicchio and Dominique Revets) and Viral Vector (E. Di Valentin and colleagues) platforms for their expert advice and technical support.

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Tables 1 to 3 are available in the Supplementary Files section.

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Heterogeneous expression of the atypical chemokine receptor ACKR3 in glioblastoma patient-derived tissue samples and cell cultures.

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