Recombinant murine CXCL16 (cat#250-28) and CXCL12 (cat#250-20A) were from Peprotech; IL-4 (cat#12340045) and IFNγ (cat#12343536) were from Immunotools; anti-CXCL16 (cat# MAB503-100), mouse CXCR6 PE-conjugated antibody (cat# FAB2145P−025, RRID:AB_2089531), human CXCR6 PE-conjugated antibody (cat#FAB699P−025, RRID:AB_2261441) were from R&D System, APC anti mouse H-2Kb/H-2Db (cat#114613) and APC anti mouse CD1d antibody (cat#123521, RRID:AB_2715919) were from Biolegend; APC rat anti-mouse CD44 (cat# 559250), PE rat anti-mouse CD274 (PD-L1)(cat#558091) were from BD Pharmingen; IgG from rat serum antibody (cat#l4131, RRID: AB_1163627), LPS (cat#L4391), 2′,7′-Dichlorofluorescin diacetate (cat#D6883) were from Sigma-Aldrich; anti-Iba1antibody was from Wako (cat#019-19741, RRID:AB_839504); anti-GFAP (cat#NB300-141, RRID:AB_10001722), anti-5-bromo-2-deoxyuridine (cat#NB500169, RRID:AB_341913) antibodies were from Novus Biological and anti-Arg1antibody was from Santa Cruz (cat#sc-271430 RRID:AB_10648473); anti-CD68 antibody (cat#MCA1957T, RRID:AB_322219) was from AbD Serotec;. Secondary Abs were from DAKO; Microbeads CD11b⁺ were from Miltenyi Biotec; Trans-well inserts were from BD Labware (cat#353097); IPTG (Dioxane-free) was from Thermo Fisher (cat#AM9464). Hematoxylin, eosin, and BSA were from Sigma-Aldrich. All cell culture media, fetal bovine serum (FBS), goat serum, penicillin G, streptomycin, glutamine, the Thermo Script RT-PCR System, and Hoechst (cat#33342, RRID:AB_10626776) were from Invitrogen. 5-Bromo-2′-Deoxyuridine (BrdU) (cat#B5002) and lentiviral shRNA clones targeting murine CXCR6 and CXCL16 were from Sigma-Aldrich. Elisa kit for Interleukin 1 Beta (IL-1β) was from Claude-Clone Corp. (cat#SEA563Mu); Elisa kit for CXCL16was from RayBiotech (cat#ELMCXCL16); Griess reagent kit for Nitrite determination was from Molecular Probe (cat#G-7921), Red fluorescent FluoSpheres (0.03%) were from Invitrogen.

This study was approved: by the Institutional Review Board of the Policlinico Umberto I Medical Center according to the Bioethics and Safety Act and the Declaration of Helsinki. Each participant provided oral informed consent (according to the principle 22 of Ethical Principles for Medical Research Involving Human Subjects); by the Institutional Review Board of Neuromed Medical Center according to the Bioethics and Safety Act and the Declaration of Helsinki. Each participant provided written informed consent.

Tumor specimens (GBM 1, 2, 3, 11, 13, 14, 19, 28, 40, 45, 46, 51, 58) were obtained at Policlinico Umberto I (Rome) and Neuromed (Pozzilli, Isernia) from adult glioblastoma (GBM). Within half an hour from surgical resection GBM tissues were processed to obtain primary GBM cells or frozen for molecular study. Histopathological typing and tumor grading were done according to the WHO criteria resulting as grade IV. Normal cerebral tissues derived from the prefrontal cortex of patients who died from heart failure were kindly provided by Dr. Eleonora Aronica, with ethics approval of Amsterdam University.

The experiments described in the present work, were approved by the Italian Ministry of Health in accordance with the guidelines on the ethical use of animals from the European Community Council Directive of September 22, 2010 (2010/63/EU). Wild type mice C57BL/6J (cat# JAX: 000664, RRID: IMSR_JAX:000664) and Homozygous cxcr6gfp/gfp knock-in mice (cat# JAX: 005693, RRID: IMSR_JAX:00569) (23), in which the coding region of CXCR6 receptor has been substituted with the coding region of the green fluorescent protein, were from Jackson Laboratory. In the present manuscript, we refer to cxcr6gfp/gfp knock-in mice as cxcr6ko mice, and to C57BL/6J as wt mice.

The mouse GL261 glioma cell line (RRID:CVCL_Y003; kindly provided by Dr. Serena Pellegatta, Istituto Di Ricovero e Cura a Carattere Scientifico, Besta, Milan) was cultured in growth medium (DMEM with 20% heat-inactivated FBS, 100 IU/ml penicillin G, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin B, 2 mM glutamine, and 1 mM sodium pyruvate). GL261/CD133⁺ cells were obtained as previously described in Garofalo et al. (24). The cell lines were tested for mycoplasma contamination (negative). Primary GBM cells were obtained as previously described (25). Briefly tumor tissues were mechanically dissociated to cell suspensions and red blood cells were lysed with hypotonic buffer. Tumor cells were re-suspended in serum-free growth medium and cultured at 37°C in humidified atmosphere with 5% CO2. Twenty-four hours later, non-adherent cells were removed and the growth medium was supplemented with 10% heat-inactivated FBS. Cells were sub-cultured when confluent. In the current study, primary GBM cells, were used within 1–3 passages, and were named GBM13, GBM19, GBM40, and GBM45.

Microglia cells were obtained from mixed glia cultures derived from the cerebral cortices of post-natal day 0–2 (p0–p2) wt mice. Cortices were chopped and digested in 15 U/ml papain for 20 min at 37°C. Cell suspensions were plated (5 × 10⁵ cells/cm²) on poly-L-lysine (0.1 mg/ml) coated flasks in growth medium supplemented with 10% FBS. After 9–11 days, cultures were shaken for 2 h at 37°C to detach and collect microglia cells. These procedures gave almost pure microglial cell populations as previously described (26). For microglia polarization, cells were seeded on poly-L-lysine (cat#P2636 from Sigma-Aldrich) coated six-well plate and the day after they were treated with LPS 100 ng/ml + IFNγ 20 ng/ml or glioma conditioned medium (GCM) with rat AbCXCL16 or IgG (1 μg/ml) for 24 h.

GL261 cells were transduced by lentiviral particles directing IPTG-inducible expression of CXCR6 shRNA or constitutive expression of CXCL16 shRNA constructs. Cells (1.6 × 10⁴) were plated in 96-well plates and infected for 24 h according to the manufacturer's instructions. Transduced cells were selected with 2 μg/ml puromycin for 3–12 days. IPTG (5 mM) was added for 10 days to culture medium to induce CXCR6 shRNA expression. Knockdown efficiency of CXCR6 receptor and CXCL16 was evaluated by PCR or chemotaxis assay. Silenced cell lines were named GL261shCXCR6 and GL261shCXCL16 in this study.

GL261, GL261shCXCR6 and human primary GBM cells were pre-incubated in chemotaxis medium (DMEM without glutamine, 100 IU/ml penicillin G, 100 μg/ml streptomycin, 0.1% BSA, and 25 mM HEPES, pH 7.4) with AraC (10 μM, 15 min) to block cell duplication. Cells (4 × 10⁴) were plated in the upper wells of 48-well boyden chamber (NeuroProbe) on 8 μm-pored Poly-L-Lysine coated membrane. The lower wells contained CXCL16 (0.1, 1, 10, 50, or 100 nM), CXCL12 (50 ng/ml), or vehicle (C). Cells were left migrate for 4 h (GBM cells) or 24 h (GL261). For invasion assay, GL261 and GBM19 were plated at a density of 2 × 10⁴ cells/cm² on matrigel-coated transwells (8 μm pored membrane) and left invade toward CXCL16 (1, 10 nM) or vehicle, respectively, for 48 or 24 h at 37°C. Migrated/invaded cells were fixed and stained with a solution containing 50% isopropanol, 1% formic acid, and 0.5% (w/v) brilliant blue R 250. For each membrane, stained cells were counted in at least 20 fields with a 32 × objective of a phase-contrast microscope (Zeiss).

GL261, GL261shCXCR6, and GBM19 cells were seeded into 96 well plates (5 × 10³) and treated with vehicle (C) or with CXCL16 (10 nM) for different time points (0, 24, 48, 72, or 96 h). MTT solution (500 μg/ml) was added into each well for 1.5 h. DMSO was then added to stop the reaction and the formazan produced was measured at 570 nm. Viability of cells was expressed relative to absorbance values.

For protein analysis, microglial cells (6 × 10⁵) were seeded on six-well plates and treated with vehicle, CXCL16 (200 nM), glioma conditioned medium (GCM) with or without rat AbCXCL16 for 24 h; cells were washed with PBS and lysed in hot 2 × Laemmli buffer, boiled 5 min, and sonicated. The same amount of proteins was separated on 12% SDS-polyacrylamide gel and analyzed by Western immunoblot using the following primary antibodies: ARG-1 1:200, ACTIN 1:2,000. HRP-tagged goat anti-mouse and anti-rabbit-IgG were used as secondary antibodies (1:2,000; Dako), and detection was performed by the chemiluminescent assay Immun-Star WesternC Kit (Bio-Rad, CA). Densitometric analysis has been carried out with Quantity One software (Biorad, CA).

Microglial cells were seeded on poly-L-lysine-treated 10 mm glass coverslips (7 × 10⁴ cells) and stimulated with CXCL16 (200 nM) or vehicle for 24 h and GCM with or without rat AbCXCL16 for 24 h. Medium was then removed, 0.05% (corresponding to 1.8 × 10⁷ spheres/ml) red fluorescent FluoSpheres were added for 1 h in serum-free medium (0.1% BSA), and nuclei were stained by Hoechst. Cells were washed three times with PBS to remove non-phagocytized spheres and fixed in 4% PFA for 1 min. Phagocytosis was quantified by counting the number of phagocytizing cells (scoring as positive only cells with at least five FluoSpheres to avoid possible false positives due to sphere adhesion to cell surface) in at least 20 random fields per coverslip.

Microglia were seeded on glass coverslips, treated as necessary, fixed, permeabilized, blocked and stained with Alexa-Fluor 488 Phalloidin (Invitrogen) for 20 min together with Hoechst. Fluorescent images were processed using the MetaMorph 7.6.5.0 software (Molecular Device, Sunnyvale, CA, USA), and form factor was calculated according the formula: 4π area/perimeter² (27). Form factor is a parameter taken as 1 for round cells, and correspondingly < 1 when the morphology deviates from the spherical shape.

NO production by microglia cultures was assessed by measuring nitrite accumulation in the culture medium by Griess Reagent Kit according to manufacturer's instructions (Molecular Probes, MA, USA). The absorbance was measured at 570 nm in a spectrophotometer microplate reader (BioTek Instruments Inc., VT, USA).

Microglial cells (6 × 10⁵ cells) were seeded onto a 6-well culture plate, after 24 h cells were stimulated with LPS 100 ng/ml + IFNγ 20 ng/ml or LPS 100 ng/ml + IFNγ 20 ng/ml + CXCL16 (200 nM) for 24 h. Medium was than collected, centrifuged at 1,000 × g for 20 min, and supernatant was stored at −80°C. Control cells were stimulated only with vehicle. IL-1β present in the supernatant was measured using a specific ELISA for mouse IL-1β (Cloud-Clone Corp.) as described by the manufacturer. For each sample, cells were detached and proteins were quantified (BCA assay). For quantification of mouse CXCL16 in glioma conditioned medium (GCM) we used the mouse CXCL16 ELISA Kit (RayBiotech, Norcross, GA, USA) as described by the manufacturer. All supernatants were centrifuged (1,000 × g for 5 min) to eliminate floating cells and then samples were 10-fold concentrated with 10 KDa Microcon Centrifugal Filter devices (Merck Millipore, Darmstadt, Germany). Samples were measured in duplicate and confirmed in two independent experiments.

Primary microglia cultures (3 × 10⁵ cells) were treated for 18 h with 200 nM CXCL16 and then cells were incubated with 20 μM of 2′,7′-Dichlorodihydrofluorescein diacetate (DCF-DA, Sigma-Aldrich, #D6883) for 30 min at 37°C. Cell fluorescence was detected in FL1 channel and analyzed with a FACSCanto II (BD Biosciences). Data were elaborated using FlowJo v9.3.2 software (TreeStar, Ashland, OR, USA).

Cells were harvested in PBS with 5 mM EDTA and washed in staining buffer (PBS without Ca²+ Mg²⁺, 0.5% BSA, 2 mM EDTA, 0.025% NaN₃). mAbs directly conjugated to PE and APC fluorochromes and specific for the following antigens were used: MHC class I (APC anti-mouse H-2Kb/H-2Db, BioLegend), CD1 (APC anti-mouse CD1d, clone 1B1, BioLegend), CD44(APC rat anti-mouse CD44, clone IM7, BD Pharmingen), PD-L1 (BD Pharmingen), CXCR6 (R & D systems). Corresponding isotypes were used for negative control. Immunostaining was performed with saturating amounts of Abs for 30 min at 4°C. Samples were acquired with a flow cytometer FACSCanto II (BD Biosciences) and data were elaborated using FlowJo 9.3.2 software (TreeStar).

Samples were lysed in TRYzol reagent for isolation of total RNA. The quality and yield of RNAs were verified using NANODROP One (Thermo Scientific). For RT-PCR one microgram of total RNA was reverse transcribed using ThermoScript RT-PCR System and 150 ng of the reverse transcription products were used as a template for PCR amplification. The PCR protocol was as follows: 95°C for 5′, 30 cycles 94°C for 30″, 55°C for 30″, and 72°C for 30″. MJ Mini Thermal Cycler (Bio-Rad) was used for all reactions and amplification products were analyzed on 1.8% agarose gel stained with ethidium bromide. For RT-qPCR Reverse transcription reaction was performed in a thermocycler using IScript TM RT Supermix (Biorad) under the following conditions: incubation, 25°C, 5′; reverse transcription, 42°C, 45′; inactivation, 85°C, 5′. Real Time-PCR was carried out in a I-Cycler IQ Multicolor RT-PCR Detection System using Sso Fast Eva Green Supermix (Biorad). The PCR protocol consisted of 40 cycles at 95°C, 30″ and 60°C, 30″. For quantification analysis, the comparative Threshold Cycle (Ct) method was used. The Ct values from each gene were normalized to the Ct value of GAPDH in the same cDNA samples. Relative quantification was performed using the 2^−ΔΔCt Ct method (28) and expressed as fold increase in arbitrary values. Primers sequences are reported in Supplementary Table 1. Primers used for CXCR6 and CXCL16 were not intron spanning, and “no-RT” reactions were used as controls to rule out priming off of genomics DNA. As control for cxcl16 and cxcr6 mRNA expression, we used RNA from Human fibroblast cell line HFF-1 (ATCC® SCRC-1041™, RRID:CVCL_3285); RNAs from Mouse fibroblast NIH/3T3 cells (ATCC® CRL-1658™, RRID:CVCL_0594); RNA from primary human T lymphocytes kindly provided by Dr. Samantha Cialfi, Department of Molecular Medicine, Sapienza, Rome); RNA from mouse primary CD4⁺ T cells derived from spleen.

Eight week old male mice (wt or cxcr6ko) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic head frame. Animals were injected with 1 × 10⁵ GL261, GL261shCXCR6, or GL261shCXCL16 cells at 2 mm lateral and 1 mm anterior to the bregma in the right striatum. Cell suspensions, in sterile phosphate-buffered saline (PBS) (5 μl) were injected with a Hamilton syringe at a rate of 1 μl/min at 3 mm depth. For GL261 shCXCR6, after 10 days of IPTG treatment, cells were injected in mice and shRNA expression was maintained by adding IPTG (10 mM) in drinking water. For survival analysis glioma injected mice were daily monitored. The end points were determined by lack of physical activity or 20% weight loss in glioma-bearing mice. The mean survival time was calculated using the Kaplan–Meier method and statistical analysis was performed using a log-rank test.

Seventeen days after tumor cell injection glioma-bearing mice were killed and brains were isolated and fixed in 4% buffered paraformaldehyde. Brains were snap frozen and cut in 20 μm coronal brain cryosections. Tumor volume was evaluated with hematoxylin–eosin staining as previously described. Briefly, after staining, brain slices (20 μm of thickness) were analyzed by the Image Tool 3.0 software (University of Texas, Health Science Center, San Antonio, TX, USA). Tumor volume was calculated according to the formula (volume = t × ΣA), where A = tumor area/slice and t = thickness (29). For tumor cell proliferation in vivo, 17 days after tumor cells injection glioma-bearing mice were injected intraperitoneally with bromo-2-deoxyuridine (50 mg/kg). Two hours later, mice were killed and brains processed for BrdU immunostaining. For immunostaing analysis cryosections were washed in PBS and blocked with blocking solution (3% goat serum, 0.3% Triton X-100 in PBS) for 1 h at room temperature. Sections were then incubated with specific antibodies (anti-Iba1 1:500, anti-CD68 1:200, anti-GFAP 1:750, anti-BrdU 1:200) in 1% goat serum and 0.1% Triton X-100/PBS solution overnight at 4°C. After several washes, sections were stained with the respective secondary fluorophore-conjugated antibody and Hoechst for nuclei visualization. For Iba1 staining, citrate buffer antigen retrieval protocol was used. For BrdU immunostaining, sections were pretreated with HCl 1N for 15 min, HCl 2N for 25 min at 37°C, and neutralized with 0.1 M borate buffer. Digitized fluorescent cell images were collected using an inverted fluorescence microscope (Nikon Ti Eclipse) and analyzed with MetaMorph analysis software (Molecular Devices, USA).

GL261 cells were grown on glass coverslips at a density of 5 × 10⁴ cells/cm² and treated for 4 h with CXCL16 10 nM or vehicle. Cells were then incubated with 10 μg/ml BrdU for 30 min, washed with PBS and fixed in 4% paraformaldehyde for 20 min. Fixed cells underwent immunostaining protocols as described for brain sections. Hoechst was used for nuclear staining. BrdU positive cells were counted out of 800 cells for condition.

Seventeen days after GL261 injection, mice brains were isolated and fixed in 4% buffered formaldehyde for morphological evaluation. Coronal brain sections (20 μm), prepared using the standard procedures, were stained with hematoxylin and eosin. For analysis of tumor invasiveness, glioma cells protruding more than 150 μm from the main tumor mass were counted in at least 20 fields, obtained from six slices per mice.

Glioma-bearing wt or cxcr6ko mice after 17 days from inoculation were deeply anesthetized and intracardially perfused with ice cold PBS. Brains were removed, cut into small pieces and single-cell suspensions were achieved by enzymatic digestion in trypsin (0.25 mg/ml) solution in Hank's balanced salt solution (HBSS). Cell suspensions were labeled with CD11b⁺ Microbeads, loaded onto a MACS Column (MiltenyiBiotec) and placed in the magnetic field of a MACS Separator. After removing the magnetic field, CD11b⁺ cells were eluted and used for RNA extraction. CD11b⁺ cells were also isolated from human GBM tissues surgically removed from patients as described above.

Data are expressed as the mean ± SEM. Statistical significance was assessed by Student's t-test, Student's paired t-test or one-way ANOVA, as indicated; Holm–Sidak, Turkey post-hoc test or Student-Newman-Keuls Method were used as a post-hoc test. For Kaplan–Meier analysis of survival, the log-rank test was used. All statistical analyses were done using Sigma Plot 11.0 software.

Since we have shown that CXCL16 is neuroprotective in ischemia (11, 12), and neuroinflammation plays a role in brain damage following ischemic insult (30, 31), we considered the possibility that CXCL16, acting on CXCR6 expressed by microglia cells (11), might provide protective effects also modulating microglia phenotype.

We performed in vitro experiments treating primary mouse microglia for 24 h with CXCL16 (200 nM) and analyzing the expression of pro- (nos2, il1b, cd86, tnfa) and anti- (arg1, chil3, retnla, cd163) inflammatory genes (32) by RT-qPCR: as reported in Figure 1A, CXCL16 increases the expression of anti-inflammatory genes (right panel; n = 5 p < 0.05; Student's t-test), while no significant modulation of pro-inflammatory genes is observed, with the exception of nos2 (left panel; n = 5 p < 0.05; Student's t-test).The ability of CXCL16 to induce anti-inflammatory polarization was further supported by an increase in ARG-1 protein expression in microglia treated with CXCL16 (200 nM, 24 h) vs. not treated cells (n = 4, p < 0.05; Student's t-test), Figure 1B. Moreover, CXCL16 increases: the number of phagocytizing microglia (measured as number of cells that phagocytized five or more fluorescence beads) (n = 3, p < 0.001; Student's t-test) vs. control, Figure 1C; the production of reactive oxygen species (measured as generated DCF fluorescence) vs. vehicle (n = 3 experiments in duplicates, p < 0.05; Student's t-test), Figure 1D.

We then wanted to verify the hypothesis that CXCL16 could also modulate microglia polarization in the context of pro-inflammatory conditions (LPS, 100 ng/ml + IFNγ, 20 ng/ml, 24 h): as reported in Figure 1E, the presence of CXCL16 (200 nM) significantly reduced the expression of nos2, il1b, and tnfa genes (n = 4–5, p < 0.05; Student's t-test). Moreover, we measured the release of nitric oxide (NO) and IL-1β by microglia cells treated with vehicle or LPS + IFNγ, in the presence or not of CXCL16: as shown in Figures 1F,G, the release of NO (n = 7, p < 0.05; One-way ANOVA followed by Holm–Sidak post-hoc test) and IL-1β (n = 4, p < 0.05; One-way ANOVA followed by Holm–Sidak post-hoc test) induced by LPS + IFNγ was significantly reduced by treatment with CXCL16.

The activation state of microglia cells has been often correlated with their shape, although it is not possible to strictly associate a morphology to a specific phenotype (33). We measured the ramification grade of microglia calculating the “form factor,” a parameter taken as 1 for round cells, and correspondingly < 1 when the morphology deviates from the spherical shape. As shown in Figure 1H, in analogy with what previously reported (29), the form factor of cells polarized toward an anti-inflammatory ramified phenotype (IL-4 20 ng/ml, 24 h) was 0.26 ± 0.03, while in cells with an inflammatory phenotype (LPS+IFNy) was 0.66 ± 0.04. The form factor of cells stimulated with CXCL16 (0.37 ± 0.03) or treated with LPS + IFNy + CXCL16 (0.37 ± 0.04) were similar to cells treated with IL-4, and statistically different from those treated with LPS + IFNy (n = 30 cells in three different experiments, p < 0.05; One-way ANOVA followed by Tukey post-hoc test), further confirming that CXCL16 polarizes cells toward an anti-inflammatory phenotype.

We analyzed the expression of CXCL16 and CXCR6 in human GBM tissues acutely (< 2 h) removed from patients, and in normal cerebral tissues (controls) derived from the temporal and frontal cortex of patients who died for heart failure: RT-qPCR analysis revealed a significant higher expression for cxcl16 and cxcr6 mRNAs in GBM, compared to controls (Figure 2A left panel) (p < 0.001 and p < 0.05, respectively; Student's t-test). We also analyzed the expression of cxcr6 in human CD11b⁺ cells (microglia/macrophages) isolated from GBM tissues, and found a considerable expression of cxcr6 also in these cells (Figure 2A, right panel). To study the role of CXCL16/CXCR6 in glioma development in a mouse model, we analyzed cxcl16 and cxcr6 mRNAs expression in GL261, and in the more aggressive derived glioma stem cells (GL261/cd133⁺ cells): as shown in Figure 2B, both chemokine and its receptor are expressed by GL261 cells, with higher expression of cxcr6 in GL261/cd133⁺ cells (both by RT- PCR and RT-qPCR analysis). Mouse T-cells and fibroblasts were analyzed as positive controls for cxcr6 and cxcl16 mRNAs expression, respectively. Furthermore, we analyzed the expression of CXCR6 membrane protein by flow cytometry, using a mouse CXCR6 PE-conjugated antibody and confirmed the expression of CXCR6 on GL261 cells (Figure 2B, right panel).

It is known that glioma cells secrete soluble factors that contribute to the establishment of a pro-tumor microenvironment switching GAMs toward an anti-inflammatory phenotype (2, 32, 34); thus, considering that microglia cells do express CXCR6, we speculated that CXCL16 released by tumor cells might act as an effector in driving such microglia polarization.

Primary microglia cells were incubated for 24 h with glioma conditioned medium (GCM) in the presence of neutralizing anti-CXCL16 antibody (AbCXCL16) (GCM + AbCXCL16), or control IgG (GCM + IgG), and analyzed for the expression of pro- or anti-inflammatory genes. In the presence of AbCXCL16, microglia increases the expression of nos2, il1b, cd86, tnfa (pro-inflammatory genes, Figure 2C, left panel), and decreases the expression of arg1, chil3, retnla, cd163 (anti-inflammatory genes, Figure 2C, right panel) compared to cells treated with GCM+IgG (n = 14, p < 0.05; Student's t-test). As control, in each experiment we checked the expression of pro- and anti-inflammatory genes of GCM-incubated microglia (data not shown). These results suggest that CXCL16 in GCM is determinant to promote microglia polarization to establish a pro-tumor/anti-inflammatory microenvironment. To further support these data we analyzed the phagocytizing activity of microglia cells incubated with GCM or GCM + AbCXCL16 (Figure 2D), and the expression of ARG-1 in these cells (Figure 2E): we found a significant reduction in the number of phagocytizing cells (n = 4, p < 0.001; Student's paired t-test) and in the expression of ARG-1 protein (n = 3, p < 0.05; Student's t-test) in cells treated with GCM + AbCXCL16 vs. cells treated with GCM. The presence of soluble CXCL16 in GL261 conditioned medium was also confirmed by ELISA measurement (0.47 ± 0.03 pg/ml).

Since tumor micro-environment plays an important role in glioma progression, and considering the ability of CXCL16 to promote microglia anti-inflammatory phenotype in vitro, we decided to investigate the effect of CXCL16/CXCR6 signaling on tumor micro-environment in vivo: we therefore orthotopically implanted GL261 cells into the brain of wt and cxcr6ko mice. Some animals were used for a survival-analysis, others were sacrificed 17 days after implantation for tumor volume analysis (Figure 3A). As reported in Figure 3B, tumor volume was strongly reduced (62%) in cxcr6ko mice compared to wt mice (cxcr6ko: 3.83 ± 0.67 mm³; wt: 10.07 ± 0.55 mm³; n = 7–12, p < 0.001; Student's t-test). Moreover, survival studies (Kaplan–Meier analysis) revealed that cxcr6ko mice survive longer than wt mice (cxcr6ko: 45 ± 2.9 days; wt: 22.8 ± 2.3 days, n = 6–10; p < 0.001 Log rank test, Figure 3C). These data suggest that the CXCL16/CXCR6 axis plays a key role in establishing a pro-tumoral microenvironment in the brain of glioma-bearing mice. Due to the importance of GAMs in glioma progression, we also investigated Iba1 and CD68 cell immuno-reactivity. As shown in Figure 3D there was no difference in Iba1⁺cells (measured as % of Iba⁺ staining per tumor area) in wt and cxcr6ko mice (cxcr6ko: 0.69 ± 0.02%; wt: 0.72 ± 0.04%; n = 4, p = 0.56; Student's t-test), but there was a strong reduction in CD68⁺cells (measured as % of CD68⁺ staining per tumor area) in cxcr6ko mice compared to wt mice (53% reduction, cxcr6ko: 0.26 ± 0.02%; wt: 0.55 ± 0.02%; n = 4, p < 0.05; Student's t-test), indicating that although there was no difference in the recruitment of total GAMs in tumor mass, they were differently activated in cxcr6ko mice.

To confirm a role of CXCL16 in driving GAMs toward a pro-tumor phenotype in vivo, we implanted GL261 cells into the brain of wt and cxcr6ko mice and, after 17 days, CD11b⁺ cells were isolated from the ipsi- and contra-lateral brain hemispheres of each mice and analyzed by RT-qPCR. Data reported in Figure 3E show no significant differences in the expression levels of pro-inflammatory genes (n = 4–5, p > 0.05; Student's t-test); instead we found a significant reduction in the expression of anti-inflammatory genes such as arg1, chil3, retnla, cd163 in CD11b⁺ cells from cxcr6ko mice (Figure 3F; n = 4–5, p < 0.05; Student's t-test). In order to look at differences in the brain tumor microenvironment in the two genotypes, we also analyzed astrocytic activation and tumor cells invasion in the surrounding brain tissue.

As reported in Figure 3G (left), the brain of glioma-bearing cxcr6ko mice showed reduced astrogliosis (measured as % of GFAP⁺ area in brain slice) compared to wt mice (46% reduction, 0.53 ± 0.03% in wt, 0.29 ± 0.03% in cxcr6ko; n = 4, p < 0.001; Student's t-test). In addition, as revealed by the analysis of the number of glioma cells protruding more than 150 μm from the main tumor mass, cxcr6ko mice presented a reduction in the number of glioma cells invading the brain parenchyma (10.1 ± 1.0 cells for brain slice) compared to wt mice (24.8 ± 1.5 cells for brain slice) (n = 3, p < 0.05; Student's t-test), Figure 3G (right).

We investigated the direct effects of CXCR6 stimulation on GL261 cells: at this aim cells were stimulated with CXCL16 and analyzed for migration in the Boyden chamber assay. Data reported in Figure 4A (left panel) demonstrate that the chemotactic index of GL261 increased with CXCL16 dose, starting at 0.1 nM CXCL16, with maximal effect at 10 nM (n = 3 experiments in triplicate, p < 0.05; One-way ANOVA followed by Holm–Sidak post-hoc test). In vitro matrigel invasion assay with GL261 shows significant increase of cell invasion upon CXCL16 stimulation (Figure 4A, central panel; n = 3, p < 0.05; One-way ANOVA followed by Holm–Sidak post-hoc test). In accordance with this effect, we also found that CXCL16 stimulation increased the expression level of the matrix metalloproteinases mmp9 and mmp2 (Figure 4A right panel; n = 6, p < 0.05; Student's t-test), whose activity is reported to be involved with the invasion ability of glioma cells (35). To investigate whether CXCL16 might directly promote glioma cell proliferation, in analogy with CXCL12 (20), we analyzed GL261 proliferation upon stimulation with CXCL16. Figure 4B shows that CXCL16 (10 nM) significantly increased GL261 cell number after 24 and 48 h, as revealed by MTT analysis (left panel; n = 3 experiments in six-replicates, p < 0.05; One way ANOVA followed by Student-Newman-Keuls Method). Similar results were obtained measuring BrdU incorporation in GL261 cells: CXCL16 administration (10 nM, 4 h) increased the number of proliferating BrdU⁺ cells compared to vehicle (C) stimulated cells (middle and right panels; n = 3, p < 0.05; Student's t-test).

To confirm the role of CXCR6 activation on glioma cells, GL261 cells were engineered for CXCR6 silencing, using an IPTG-inducible shCXCR6 construct. As shown in Figure 4C, we selected a GL261shCXCR6-inducible cell clone that, after 10 days of treatment with IPTG, presented a strong reduction in CXCR6 mRNA expression compared to control cells (not treated with IPTG). To further confirm CXCR6 silencing in the selected clone, we performed chemotaxis experiments toward CXCL16: Figure 4D shows that shCXCR6 cells (+IPTG) did not respond to CXCL16 (n = 4 experiments in duplicate, p < 0.05; Two- way ANOVA followed by Holm–Sidak post-hoc test). Since CXCL16 is present in GCM and CXCL16 stimulation increases GL261 proliferation (Figure 4B), we speculated that basal cell proliferation might be altered in shCXCR6 cells: as reported in Figure 4E, cell proliferation measured at 24, 48, 72, 96 h was reduced in IPTG induced GL261shCXCR6 cells (+IPTG) compared to GL261shCXCR6 not treated cells (-IPTG) (n = 4 experiments in quadruplicate, p < 0.05;One way ANOVA followed by Student-Newman-Keuls Method).

To confirm a role for CXCR6 in glioma development also in vivo, we orthotopically implanted GL261shCXCR6 cells, silenced or not with IPTG (+/-IPTG), into the brain of wt mice. Tumor-bearing mice were supplied with drinking water with or without IPTG, respectively, and, 17 days after implantation, were sacrificed for tumor volume analysis (Figure 4F). Mice injected with tumor cells silenced for CXCR6 revealed a significant reduction (67%) in tumor volume compared to mice injected with GL261 expressing CXCR6 (2.19 ± 0.47 mm³ shCXCR6 + IPTG mice vs. 6.54 ± 1.26 mm³ shCXCR6–IPTG) (n = 5, p < 0.05; Student's t-test). These mice were also i.p. injected with BrdU 4 h before sacrifice, in order to analyze tumor cell proliferation in vivo: as shown in Figure 4G, shCXCR6+ IPTG cells revealed a significant reduction in BrdU incorporation (0.52 ± 0.08% of tumor area) compared to control cells (0.81 ± 0.05% of tumor area) (n = 3, p < 0.05; Student's t-test). Moreover, as reported in Figure 4H, in mice injected with tumor cells silenced for CXCR6 there was a reduced number of glioma cells that migrate and invade the surrounding brain tissue (15.00 ± 1.8 cells for brain slice) compared to mice injected with cells not silenced (28.6 ± 3.9 cells for brain slice) (n = 3–4, p < 0.05; Student's t-test).

To prove a role of CXCL16 released from glioma cells in tumor progression, GL261 cells were engineered for constitutive CXCL16 silencing, using shCXCL16 construct. As shown in Figure 5A, we selected a shCXCL16 cell clone with 80% reduction in CXCL16 mRNA expression compared to GL261 cells. GL261 or shCXCL16 cells were implanted into the brain of adult wt mice and 17 days after implantation mice were sacrificed for tumor volume analysis (Figure 5B). As reported in Figure 5C, mice injected with shCXCL16 cells revealed a significant reduction (67%) in tumor volume compared to mice injected with GL261 (3.3 ± 0.40 mm³ shCXCL16 vs. 9.6 ± 0.67 mm³ GL261) (n = 4; p < 0.001; Student's t-test). Glioma-bearing mice were also injected with BrdU 4h before animal sacrifice: analysis of BrdU incorporation in tumor cells (Figure 5D) revealed a significant reduction in BrdU⁺ cells in mice implanted with shCXCL16 cells (0.45 ± 0.02% of tumor area), compared to mice implanted with GL261 cells (0.8 ± 0.15% of tumor area) (n = 3; p < 0.05; Student's t-test). In addition (Figure 5E) we found that mice injected with tumor cells silenced for CXCL16 presented a reduced number of glioma cells that migrate and invade the surrounding brain tissue (12.16 ± 0.82 cells for brain slice) compared to mice injected with GL261 (24.85 ± 1.49 cells for brain slice) (n = 3, p < 0.05; Student's t-test). To exclude that differences observed in vivo between tumor volume in GL261 and shCXCL16-GL261 bearing mice might be due to clonal differences between cells rather than to the CXCL16 silencing, we checked for the expression of markers involved in tumor immune recognition (specifically MHC class I, CD1 and PD-L1) as well as tumor cell migration and invasion (CD44). Flow cytometry analysis revealed no differences in the expression of all these markers (Supplementary Figure 1). All these data confirm that CXCL16 released by glioma cells concurs to tumor progression, and promotes tumor cell proliferation.

To investigate whether CXCL16/CXCR6 also modulates cell migration, invasion and proliferation in human GBM, we isolated tumor cells from patient's derived biopsies (GBM 13, 19, 40, 45) and analyzed their expression of CXCL16 and CXCR6 by RT-qPCR. We first compared the expression level of cxcl16 and cxcr6 mRNAs in whole patient's tissue, and in the corresponding isolated tumor cells. RNAs from primary human T-cells and a fibroblasts were used as positive controls for cxcr6 and cxcl16 expression, respectively. All the examined tissues express high levels of cxcl16 and cxcr6, compared to normal brain tissues (see Figure 2A); however, the corresponding primary cells, even if cultured for only few passages (from 1 to 3), showed a strong reduction in their expression level (Figure 6A). This reduction could be due to a culture-dependent variation in the expression level in tumor cells, but we cannot exclude that the differences observed are due to the selection of specific cell subpopulation (or the elimination of infiltrating cells in the tumor tissue) during the cell culture procedures. By flow cytometry analysis, using specific human CXCR6 PE-conjugated antibody, we confirmed the expression of membrane CXCR6 protein in primary GBM19 and GBM45 cells (Figure 6B). As reported in Figure 6C, primary GBM19 and GBM45, cells responded to CXCL16 stimulation (10 nM, 4 h) increasing their chemotactic index compared to unstimulated cells (control), thus suggesting the expression of functional CXCR6 (n = 4, p < 0.05; One way ANOVA followed by Holm–Sidak post-hoc test). Since it is known that CXCL12 is able to induce migration of GBM cells (36) and GBM19 and 45 cells do express CXCR4 (data not shown), CXCL12 stimulation was used as positive control of migratory activity.

GBM19 cells were also used to investigate CXCL16-induced cell invasion trough matrigel substrate (CXCL16 10 nM, 24 h) and cell proliferation (CXCL16 10 nM, 24, 48, 72 h). As shown in Figure 6D, upon stimulation with CXCL16, there was a significant increase in cell invasion (left panel; n = 3, p < 0.05; Student's t-test), and in cell proliferation (right panel; n = 3 experiments in five replicates, p < 0.05; Student's t-test) compared to un-stimulated cells (control). All these data indicate that activation of CXCL16/CXCR6 axis in human primary GBM cells is able to promote tumor cell proliferation, migration and invasion. To further support our data, we used cBioportal Database to look at a possible correlation between patient survival and CXCR6 expression in glioma tumor. We first explored the alteration in cxcr6 gene in a merged cohort of low grade glioma (LGG) and GBM (TCGA, Cell 2016) and found a significant increase in patient months survival associated with cxcr6 deletion (median months survival 130.7 vs. 20.6 in normal cases, Log rank Test P-value 0.0339); we than looked at cxcr6 mRNA expression data in a cohort of Glioblastoma (TCGA, Cell 2013) and found a significant decrease in months survival associated with cxcr6 mRNA overexpression (median months survival 5.2 vs. 14 in normal cases, Log rank Test P-value 0.00417), Supplementary Figure 2.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.