ADAM8 affects glioblastoma progression by regulating osteopontin-mediated angiogenesis

ADAM8 expression is increased in glioblastoma multiforme and is localized to tumor cells and tumor-associated macrophage/microglia

A total of 50 GBM tissues obtained from hospitalized patients were examined for ADAM8 expression by qPCR, indicating that ADAM8 expression is significantly higher in GBM compared to normal brain tissue (Figure 1a). Western Blot analyses confirmed higher expression levels of ADAM8 (Figure 1b). Furthermore, ADAM8 localization in GBM tissue was assessed by immunohistochemistry (Figure 1c). In normal brain tissue, ADAM8 expression is hardly detectable and restricted to some neuronal cells. In GBM tissue, ADAM8 expression is significantly higher (approx. 10-fold, p < 0.05) and can be detected in tumor cells, neoplastic endothelial cell with proliferations, and in tumor-associated cells. In order to characterize these cells further, we performed double staining with immune cell markers and found that ADAM8 co-localizes with CD68, a marker for macrophages/microglia in the brain (Figure 1c, image g). Thus, we conclude that ADAM8 might be functionally important for tumor cells and for tumor-associated macrophages.

Figure 1:

Levels of ADAM8 mRNA expression in GBM tissue.

(A) qPCR analysis of control access CNS tissue (“Ctrl”) and glioblastoma (“GBM”) tissue (n = 5 for access tissue, n = 50 for GBM; *, p < 0.05). (B) Western Blot analysis of representative control and GBM tissue samples (n = 2). The arrowhead indicates the inactive proform, the circle the active mature (“matA8”) form of ADAM8. Note that significantly more mature ADAM8 is detected in GBM tissue. (C) Immunohistochemistry of ADAM8 in normal brain (a) and GBM tissue sections (b-f); ADAM8⁺ cells are tumor cells (b-c), endothelial proliferates (d) which can be detected in the core tumor (e) and the infiltration zone (f). (g) Double staining of ADAM8 (brown) and macrophage marker CD68 (pink) to detect ADAM8 in macrophages/microglia, indicated by arrows. Scale bar in (a), (f): 100 μm, in (g): 30 μm.

To analyze the function of ADAM8 in GBM, we performed loss-of-function experiments in U87 MG cells. Among all human GBM cell lines, U87 MG cells showed the highest level of ADAM8 expression. Briefly, endogenous ADAM8 was knocked down by small hairpin loop RNAs (shRNA) and a number of cell clones (n = 8) were obtained with significantly reduced ADAM8 expression levels (>50-fold). The cell clone with the highest ADAM8 reduction assessed by qPCR and western blot (Figure 2a) was used for further functional analyses in vitro and in vivo (Figure 2). The proliferation behavior of cells showed no difference (Figure 2b), while cell migration was significantly reduced in U87_shA8 cells (Figure 2c).

Figure 2:

The effect of ADAM8 knockdown in a GBM model.

(A) Knockdown of ADAM8 was confirmed in U87 MG cells by qPCR and Western Blot. In the ADAM8 knockdown clones selected for analysis, no difference in ADAM8 expression compared to U87 wild-type cells was observed. (B) Proliferation of cell clones was comparable in all three U87 cell lines as confirmed by MTT assays. (C) Cell migration was analyzed by scratch assays using U87_shCtrl and U87_shA8 cells. A significant reduction of cell migration was observed in U87 MG cells with ADAM8 knockdown. (D) U87 cells (80.000 in total) were stereotactically injected into the striata of nude mice. 3 weeks after injection (tumor mass circled, injection channel marked by arrow), tumor volumes formed by U87_shA8 cells in the brain is much smaller compared to U87_shCtrl cells (p < 0.01). Results are obtained from n = 14 mice per experimental group. (E) From paraffin sections, vascular structures marked by CD31 staining in the ADAM8 knockdown group were significantly fewer than those of the control group. The scale bar corresponds to 100 μm. Data from multiple replicates are presented as mean and error bars indicate mean ± SEM.*p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test.

ADAM8 reduced angiogenesis and tumor volume in a GBM model

Using U87 control (scramble, U87_shCtrl) and U87 ADAM8 knockdown (U87_shA8) cell clones, intracranial tumor cell injections into striatum of nude mice were carried out to assess tumorigenesis. To detect tumors, MRI scanning was started three weeks after implantation in weekly intervals and tumor volumes were calculated (n = 14 for each group, Figure 2d). All mice injected with U87_shA8 cells survived over an observation time of nine weeks. In contrast, all mice injected with U87_shCtrl cells reached the endpoint criteria or died spontaneously within the observation time (Supplementary Figure 1). Compared to the U87 control group, tumor volumes of U87_A8kd cells were significantly smaller and, in some cases (6 out of 14), tumor cells were completely absent three weeks after implantation (Figure 2a). To assess a possible tumor graft rejection, injection sites were analyzed one week after injection (Figure 2e). Whereas tumors derived from U87_shCtrl cells grew as a compact cell mass after one week, U87_A8kd cells showed a patchy growth characteristic. After staining for vascular structures using CD31 staining, we found significantly less (reduced to 46%, p < 0.01) vascular staining in U87_shA8 (8.7 × 10⁴ ± 8) compared to the U87_shCtrl group (Figure 2e).

ADAM8 regulates GBM cell angiogenesis via osteopontin

Given the results of the in vivo experiments, we postulated that U87_shA8 cells could be deficient in angiogenesis induction. To test this hypothesis in vitro, we utilized conditioned medium from U87_shCtrl and U87_shA8 cells for angiogenesis induction of human umbilical vein endothelial cells (HUVEC) and quantified formation of tubes. After 6 h incubation with the respective cell culture supernatants, tube formation was quantified by counting the number of meshes formed (Figure 3a, b). ADAM8 containing supernatants from U87_shCtrl cells induced significantly higher numbers of tubes as compared to supernatants from ADAM8-deficient U87_shA8 cells (p < 0.05).

Figure 3:

ADAM8 regulates angiogenesis by GBM cells.

(A) HUVEC cells were stimulated with conditioned medium from U87_shCtrl and U87_shA8 cells and tube formation was quantified after 6 h using Calcein-AM dye. (B) Compared to U87_shCtrl supernatants, HUVEC cell tube formation ability was significantly decreased when U87_shA8 supernatants were applied. (C) Protein Profiler Array to analyze differentially secreted proteins; the expression of osteopontin (OPN), a cytokine related to GBM angiogenesis, was by at least 4-fold decreased. (D) In supernatants of U87_shA8 cells, the mRNA expression of OPN decreased accordingly. (E) Addition of 40, 80, and 200 ng OPN to angiogenesis assay performed with U87_shCtrl and U87_shA8 supernatants. After addition OPN, the number of meshes formed in the ADAM8 knockdown group is similar to those ones observed in the ADAM8 wildtype group, but significantly increased compared to ADAM8 knockdown U87 without OPN (p < 0.05). The scale bar corresponds to 500 μm. Data from multiple replicates are presented as mean and error bars indicate mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test and two-way ANOVA.

To identify the molecular mechanism by which ADAM8 affects angiogenesis, a differential analysis of cell supernatants from U87_shCtrl and U87_shA8 cells was conducted in an angiogenesis protein profiler array. Array membranes with spotted antibodies against proteins involved in angiogenesis were incubated with conditioned supernatants of U87_shCtrl and U87_shA8 cells and the proteins captured by antibodies on the array membrane were detected by chemiluminescence and quantified (Figure 3c). Several candidates were identified, most prominently IL-8 and Osteopontin (OPN, Figure 3c).

Differences in expression levels of OPN between U87_shCtrl and U87_shA8 cells were confirmed by qPCR, suggesting that OPN expression levels are dependent on the ADAM8 gene dosage (Figure 3d).

To provide a direct proof for our hypothesis, we analyzed whether exogenous OPN can be used to recover the deficiency of U87_shA8 cells in angiogenesis assays (Figure 3e, f). Different concentrations of OPN were added to U87 supernatants in angiogenesis assays. When adding IL-8, the reduced angiogenesis capacity of U87_shA8 cells was not restored (data not shown). In contrast, addition of recombinant OPN at different concentrations caused an increase in the number of meshes formed in the U87_shA group and reached similar values as in the U87_shCtrl group (Figure 3f). As the OPN concentration increases, the number of meshes increases in both groups, although to a smaller extent in U87_shCtrl cells due to saturation effects (Figure 3e).

ADAM8 affects secretion of OPN via the JAK/STAT3 pathway in GBM cells

From previous studies it is known that ADAM8 can directly affect intracellular signaling by either shedding of membrane proteins, binding to integrin β1, or by direct interaction with intracellular proteins interacting with the ADAM8 cytoplasmic domain. To explore the signaling pathway involved in OPN regulation by ADAM8, we used a panel of pathway inhibitors to target NF-kB, JAK/STAT3, p38, ERK1/2 and AKT/PI3K (Figure 4). U87_shCtrl and U87_shA8 cells were incubated with the respective inhibitors for 24 h and levels of secreted OPN in cell supernatants were determined by ELISA. While none of the inhibitors affects OPN secretion in U87_shA8 cells, OPN secretion was significantly decreased in U87_shCtrl cells treated with the JAK/STAT3 inhibitor only, whereas other inhibitors such as NF-κB and p38 inhibitor caused even slightly enhancing effects on OPN secretion (Figure 4). To clarify the role of the ADAM8 protease function in OPN regulation, U87_shA8 cells were transfected with either active or inactive (E to Q mutation at amino acid position 335) ADAM8. Both, on the mRNA and the protein level, ADAM8 upregulated OPN expression and secretion, however, independent of the protease activity (Supplementary Figure 2).

Figure 4:

In GBM cells, ADAM8 affects OPN secretion via the JAK/STAT3 pathway.

(A) Treatment of U87_shCtrl and U87_shA8 cells with inhibitors of NF-κB, JAK/STAT3, p38, ERK1/2, and PI3K/AKT, followed by quantification of OPN release using a specific ELISA. OPN expression of U87_shCtrl and U87_shA8 cells are different even with inhibitor treatment. When inhibiting NF-κB, p38, ERK1/2, OPN expression is not affected or even slightly higher in U87_shCtrl cells compared to non-treated controls. (B) In U87_shCtrl cells treated with JAK/STAT3 inhibitor, OPN expression relative to control conditions (set to 1). Data from multiple replicates are presented as mean and error bars indicate mean ± SD.*p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA and multiple comparison.

In macrophages, ADAM8 is involved in the regulation of angiogenesis via OPN

As shown above (Figure 1c), ADAM8 is additionally expressed in GBM associated macrophages/microglia (GAMMs) [9] which could contribute to tumor angiogenesis in GBM. To analyze if ADAM8 affects OPN expression in these cells similar to tumor cells, we used primary macrophages isolated from wild-type (WT) and ADAM8-deficient mice (Kelly et al. 2005) and no difference in macrophage development was observed (Bartsch et al., unpublished). Primary macrophages were isolated from bone marrow with a purity of >90% (Figure 5a, b). Supernatants from macrophages were collected and angiogenesis assays using HUVEC cells were performed. After 6 h incubation with supernatants from WT and ADAM8-deficient macrophages, HUVEC tube formation was significantly lower in supernatants from ADAM8-deficient macrophages compared to wild-type macrophages (Figure 5c–e). Similar to U87 cells, we analyzed differential proteins secreted from macrophages using a cytokine array. The strongest difference between WT and ADAM8-deficient macrophages was observed for osteopontin (Figure 5f).

Figure 5:

ADAM8 regulates the angiogenic potential of macrophages.

(A, B) Enrichment of bone marrow derived macrophages (BMDM) from WT and ADAM8-deficient (A8KO) mice according to the protocol described in the MandM section. CD11b was used as macrophage marker to quantify purity of macrophage preparation. In WT bone marrow derived macrophages, ADAM8 (m-A8, green) and CD11b (red) stainings are positive. In bone marrow macrophages derived from ADAM8-deficient mice, only CD11b is positive. (C, D) With WT macrophage supernatants, HUVECs form tube-like structure after 6 h, whereas A8KO macrophage supernatants lead to smaller numbers of tubes after 6 h. (E) Number of meshes quantified in 20 viewing fields (n = 5). Angiogenesis induced from ADAM8-deficient macrophages is significantly decreased. (F, G) Differential analysis of cytokines and angiogenesis factors in supernatants of WT and A8KO macrophages. Note that osteopontin (OPN, blue box for WT, red box for A8ko) shows the biggest difference between the two experimental groups. White boxes, negative controls, asterisks mark the positive controls. Scale bar in (A) and (B) corresponds to 100 μm, (C) and (D) corresponds to 500 μm. Data from multiple replicates are presented as mean values and error bars indicate mean ± SEM.*p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test.

ADAM8 regulates OPN via JAK/STAT3 and NF-κB in macrophages

Expression levels of OPN in WT and A8KO macrophages were confirmed by qPCR (Figure 6a) and ELISA assays (Figure 6b). We further analyzed if OPN added to A8KO macrophages restores angiogenesis; 100 ngl OPN restores the decrease of angiogenic ability caused by ADAM8 deletion but had little effect on WT macrophage supernatant (Figure 6c). Signaling pathway inhibitors were used in macrophages. Protein levels of OPN in cell supernatants were determined by ELISA. Inhibition of JAK/STAT3 and NF-κB caused a significant reduction of OPN expression in WT and A8KO macrophages (Figure 6d, e), suggesting that ADAM8 is one of the upstream proteins to regulate OPN expression through JAK/STAT3 and NF-κB.

Figure 6:

In macrophages, ADAM8 regulates OPN secretion via JAK/STAT3 and NF-κB. (A,B) Relative OPN expression was determined by qPCR and OPN protein levels in supernatants by ELISA. A8KO macrophages express less OPN than WT macrophages. (C) By adding 100 ng/µl OPN, defective angiogenesis of HUVEC cells caused by ADAM8 deficiency can be restored. (D) Treatment of primary macrophages with signaling pathway inhibitors for p38, ERK1/2, PI3K/AKT, NF-κB, JAK/STAT3 followed by quantification of OPN using an ELISA. OPN expression of A8WT and A8KO is decreased after addition NF-κB and JAK/STAT3 inhibitors. (E) macrophages treated with NF-κB inhibitor and JAK/STAT3 inhibitor. Note that OPN expression is significantly lower than under control conditions (set to 1). Data from multiple replicates are presented as mean and error bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test and two-way ANOVA.

Patient specimens

Approval from the local ethics committee (file number 185/11) was obtained to collect tumor tissue samples from patients who underwent surgical resection of GBM after providing written consent. Tumor tissue was either shock-frozen in liquid nitrogen (biochemical analyses) or embedded in paraffine for immunohistochemistry.

Cells

U87 MG cells were obtained from ATCC and their authenticity was checked by STR profiling and karyotype analysis with X- and Y-specific FISH probes. Cells were grown in DMEM with 10% FCS. HUVEC cells were purchased from Promo Cell (Heidelberg, Germany) and maintained according to the manufacturer’s instructions.

To generate U87 MG cells with an ADAM8 knockdown, cells were transfected with four shADAM8 constructs (TF314948, Origene, Rockville, MD, USA with the target sequence 5′- GCGGCACCTGCATGACAACGTACAGCTCA-3′) cloned into pRFP-C-RS vector using Lipofectamine LTX (Invitrogen, UK). Twenty-four hours after transfection, cells were treated with the respective selection antibiotic (1 mg/ml puromycin). Around 15 cell clones were selected and successful knockdown of ADAM8 was confirmed. For generation of control cell clones, a construct with a scramble shRNA (target sequence 5′-GCACTACCAGAGCTAACT CAGATAGTACT-3′) sequence was used. ADAM8 expression in single U87 MG cell clones was analyzed by qRT-PCR, western blotting and ELISA.

Mice

ADAM8-deficient mice are kept on a C57B1/6 background for at least 10 generations. For genotyping, diagnostic PCR assays were performed as described (Schlomann et al. 2015). For in vivo experiments, six-week-old athymic mice were obtained from Harlan (Indianapolis, USA). All animal experiments were performed in accordance with the German laws on the protection of animals (“Tierschutzgesetz”) and with permission by the local authorities (Regierungspräsidium Gießen V54-19c2015(1) MR20/18, and G60_2016).

Mouse bone marrow-derived macrophage isolation and culture

Mouse macrophages were isolated from mouse bone marrow. Mice were sacrificed by cervical dislocation, and femur and tibia were removed to flush bone marrow out with completed culture medium (10% FCS, 20 ng/ml MCSF, DMEM) using a 21G needle. After separating the cells using a cell strainer (pore size 100 µm), cells were seeded in a petri dish in 10 ml complete medium. At day 4, 5 ml of the complete medium were replaced. After 7 days of incubation, macrophages were used for functional experiments.

Proliferation assays

Cells were plated at 5000 cells/well in 96-well plates and cultured for 1, 2, 3, 4 and 5 days. On the indicated days, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (AMRESCO, Solon, OH, USA) was added and incubated for 4 h at 37 °C, 5% CO₂. The supernatant was discarded and replaced with 200 μl DMSO (Sigma-Aldrich Co., St. Louis, MO, USA) to dissolve the formazan product. Absorbance at 570 nm was determined in a 96-well-plate reader (BMG Labtech, Offenburg, Germany).

Scratch assays

Cell migration was determined by scratch assays in vitro in culture-insert microdishes (Ibidi GmbH, Planegg, Germany) according to the manufacturer’s instructions. Cells were seeded into each chamber at densities of 1 × 10⁵ cells/ml in 100 μl DMEM medium with 10% FCS. After incubation at 37 °C, 5% CO₂ for 24 h and serum starving for another 6 h, inserts were removed and 1 ml DMEM medium with 2% FCS was added into each dish. Images were taken immediately after insert removal and 18 h later. Rates of cell migration were determined by counting cells in the gaps (n = 3 viewing fields per plate in three independent experiments) using Image J software.

Immunofluorescence microscopy

1000 cells were seeded per well in an eight well chamber on glass (Lab-Tek,4808), fixed and stained with antibodies (CD11b: Thermo Fisher, MA1-80091; mouse ADAM8: Biorbyt, orb4376; human ADAM8: R&D Systems, AF1031).

Angiogenesis tube formation assay

1 × 10⁶ macrophages/U87 cells (ADAM8 deficient compared to respective control) were seeded in a six well in 1 ml of a 1:1 mix serum free DMEM and ECM MEDIUM (Promo Cell, C-22010). After 24 h, supernatant was collected and cleared by centrifugation at 1000 rpm for 5 min. 4 × 10⁵ HUVECs per well were seeded on Angiogenesis Plate (IBIDI, 81506). After particular time points (Macrophages: 6 h; U87: 15 h), three pictures (center, upper left and lower right) were taken from each well and meshes formed were counted. For the OPN "rescue" assay, 2.5 ng/ml OPN (PeproTech, 120-35) was added to macrophage and 50 ng/ml OPN to U87 supernatants.

Cytokine array

A membrane-based antibody array [Proteome Profiler® Mouse XL Cytokine Array ARY028, RandD Systems, Abingdon, UK] was used to identify cytokine expression levels under different conditions, according to the manufacturer’s protocol. Briefly, conditioned supernatants of U87_shA8 and U87_shCtrl cells were incubated with a biotinylated antibody mix for detection on the nitrocellulose membrane by chemiluminescence. Quantification of dot optical intensities was performed using Image J software.

Real-time quantitative PCR

To determine gene expression, real-time quantitative PCR with SYBR Green (Precision® FAST master mix) on StepOnePlus Real-Time PCR System was performed using commercially available primers for m-ADAM8 (Qiagen, Hilden, Germany), m-SPP-1 (Qiagen) h-ADAM8 (Qiagen), h-SPP-1 (Apara Bioscience, Freiburg, Germany), RPLPO (Qiagen) was used as reference gene for normalization. Relative gene expression was calculated using comparative 2^−∆∆Ct method.

Statistical analysis

Unpaired two-tailed Student’s t-test was used to compare values from two independent groups. For multiple comparisons, Two-way ANOVA and multiple comparisons were used. Data from multiple replicates are presented as mean and error bars indicate mean ± SEM from at least three independent samples. Based on the obtained results, the data were considered not significant (p > 0.05), significant (*p < 0.05), highly significant (**p < 0.01) or very highly significant (***p < 0.001). Calculations were performed using the GraphPrism statistical analysis software.