S100B suppression alters polarization of infiltrating myeloid-derived cells in gliomas and inhibits tumor growth

Abstract

S100B, a member of the multigene family of Ca²⁺-binding proteins, is overexpressed by most malignant gliomas but its biological role in gliomagenesis is unclear. Recently, we demonstrated that low concentrations of S100B attenuated microglia activation through the induction of STAT3. Furthermore, S100B downregulation in a murine glioma model inhibited macrophage trafficking and tumor growth. Based on these observations, we hypothesized that S100B inhibitors may have antiglioma properties through modulation of tumor microenvironment. To discover novel S100B inhibitors, we developed a high-throughput screening cell-based S100B promoter-driven luciferase reporter assay. Initial screening of 768 compounds in the NIH library identified 36 hits with >85% S100B inhibitory activity. Duloxetine (Dul, an SNRI) was selected for the initial proof-of-concept studies. At low concentrations (1-5 μM) Dul inhibited S100B and CCL2 production in mouse GL261 glioma cells, but had minimal cytotoxic activity in vitro. In vivo, however, Dul (30 mg/kg/14 days) inhibited S100B production, altered the polarization and trafficking of tumor-associated myeloid-derived cells, and inhibited the growth of intracranial GL261 gliomas. Dul therapeutic efficacy, however, was not observed in the K-Luc glioma model that expresses low levels of S100B. These findings affirm the role of S100B in gliomagenesis and justify the development of more potent S100B inhibitors for glioma therapy.

Keywords: Brain tumor; Duloxetine; Glioblastoma; Microglia; RAGE; Serotonin-norepinephrine reuptake inhibitor; Tumor-associated macrophages.

Fig. 1.. Duloxetine (Dul) inhibits S100B expression In vitro.

(A) Dul inhibition of S100B promoter activity by a luciferase reporter assay in the human U251 glioma cell line. Luciferase activity was measured 24 h after Dul treatment. n= 3 ± SD (B) Dul cytotoxicity in GL261 glioma cell line. GL261-Luc cells were incubated with Dul for 24 h before measuring luciferase activity per plate (upper panel); LD50 = 25.1 μM. Cell proliferation was not significantly inhibited at Dul at concentration < 10 μM (lower panel). n = 3 ± SD, *: p < 0.05 compared to control. (C) Inhibition of S100B expression by GL261 cells after 24 h (qPCR, top panel) and 48 h (Western analysis, ELISA and immunostains) incubations with Dul. n = 3 ± SD. (D) In vitro inhibition of CCL2 protein (left panels) and RNA expression (right panel) in GL261 cells after a 48 h incubation with Dul. n = 5 ± SD. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Experimental results are representative of at least two separate experiments.

Fig. 2.. In vivo activity of Duloxetine (Dul) in the mouse intracranial (i.c.) GL261 glioma model.

(A) Dul did not significantly inhibit total brain S100B, RAGE or CCL2 protein levels in tumor-bearing animals. One day after tumor implantation, tumor-bearing mice (n = 3) were treated daily with different doses of Dul or vehicle. Fourteen days later, tumors and peritumoral tissue was harvested for protein analysis. (B) Serum levels of S100B and soluble RAGE (sRAGE) of tumor-bearing mice with and without a two-week treatment with Dul. n = 5–6 mice/group ± SD. (C) Representative immunostains of i.c. GL261 tumors (T, left panels) following a two-week treatment with Dul demonstrating significant inhibition of S100B and RAGE-positive tumor cells (bar graphs). n = 3 mice/ group ± SD. *: p < 0.05, **: p < 0.01. (D) Effect of Dul therapy on the expression of S100B by i.c. GL261 tumors (T) and peritumoral reactive astrocytes (GFAP⁺ cells). Low (top panels) and high magnification (lowest panel) of tumor sections demonstrating effective inhibition of S100B by tumors, but not by peritumoral reactive astrocytes (arrows). GFAP: glial fibrillary acidic protein. Representative results from two separate experiments is shown.

Fig. 3.. S100B expression in gliomas is enhanced by norepinephrine (NE).

(A) Norepinephrine transporter (NET) is expressed by GL261 glioma, but not by primary astrocytes. (B) In vivo expression of NET in i.c. GL261 gliomas (T) but not peritumoral astrocytes (GFAP⁺ cells). (C) Expression of S100B by GL261 cells is induced by NE. Cells (4 × 10⁵ cells/plate) were treated with NE every 3 h. After 24 h, cells were lysed and subjected to Western analysis. (D) Inhibition of S100B promoter activity by Dul. U251 human gliomas (5000 cell/plate) that were stably transfected with S100B reporter gene were incubated with NE (added every 3 h to the medium) in presence and absence of Dul (5 μM). Luciferase activity was measured in 12 h n = 3/group ± SD. *: p < 0.05, **: p < 0.01 compared to control group. Representative results from two separate experiments is shown.

Fig. 4.. Duloxetine (Dul) inhibited the growth of GL261 gliomas.

(A) Representative histology (left) and tumor volume measurements (right) demonstrating inhibition of i.c. GL261 tumor growth with Dul. Mice were treated with Dul a day after tumor implantation. Fourteen days later, brains were harvested and the largest tumor area was used for size calculations. (n =5 mice/group ± SD). (B) Kaplan–Meier survival curve of tumor-bearing mice treated with daily vehicle or Dul for 14 days (n = 6 mice/group). Animals demonstrating signs of elevated intracranial pressure were euthanized and tumor presence confirmed by histology. *: p < 0.05, **: p < 0.01 compared to control group. MS: median survival. Results are representative of two separate experiments.

Fig. 5.. Effect of duloxetine (Dul) on tumor inflammatory responses.

(A) To assess the overall inflammatory changes in tumor and tumor microenvironment (TME), tumors and peritumoral tissue from brains of control and Dul-treated GL261-bearing mice were dissected and analyzed by Nanostring. Pathway Scores for a number of inflammatory processes were elevated in Dul-treated tumors, but not TME. (B) Representative dot plots (upper panel) and FACS quantification (lower panel) demonstrating a decrease in the proportion of macrophages (i.e. CD11b⁺ CD45^high F4/80⁺), monocytic myeloid-derived cells (CD11b⁺ Ly6G⁻ Ly6G⁺) and neutrophils (Ly6B⁺), but not microglia (i.e. CD11b⁺ CD45^low) following a two-week treatment with Dul (30 mg/kg x 14 days). n = 3 mice/group ± SD. *: p < 0.05. (C) Representative sections of tumor-bearing brains demonstrating distribution of microglia and macrophages (eGFP⁺) into GL261 tumors (T) and tumor edge. CX₃CR₁^GFP mice were implanted with GL261 cells and treated a day later with Dul (30 mg/kg) or vehicle for two weeks. Fewer eGFP⁺ cells (myeloid-derived cells) were seen within the Dul-treated tumors (D). Representative immunohistochemistry demonstrating decrease in CCL2 expression by tumors after Dul therapy. GFAP: glial fibrillary acidic protein (astrocyte marker). Lowest panel represents higher magnification of insets in the upper panel. (E) Inflammatory cells isolated from GL261 gliomas by Percoll separation mostly consisted of monocytic lineage (i.e. microglia, macrophages and monocytic myeloid-derived cells). (F) Effect of Dul on the cytokine expression profile of tumor-associate leukocytes. GL261 gliomas from vehicle or Dul-treated (30 mg/kg/14 days) mice were harvested at 14 days post tumor implantation and subjected to Percoll gradient. Leukocytes were isolated and incubated with LPS (0.5 μg/ml, 24 h). Cytokine expression was assessed by q-PCR. (n = 4 mice/group ± SD). Representative results from two separate experiments is shown.

Fig. 6.. In vitro and in vivo activity of duloxetine (Dul) on K-Luc gliomas.

(A) Dul inhibited K-Luc proliferation even at low concentrations. n = 3 ± SD. *: p < 0.05. (B) Dul did not inhibit brain S100B or RAGE protein levels in tumor-bearing animals. One day after K-Luc implantation, tumor-bearing mice were treated daily with Dul or vehicle. Fourteen days later, tumors and peritumoral tissue was harvested for protein analysis. (n = 3 mice/group ± SD). (C) Representative immunohistochemistry of i.c. K-Luc tumors (T) following a two-week treatment with Dul demonstrating persistent S100B expression by peritumoral astrocytes (GFAP⁺ cells). (D) Dul did not inhibit K-Luc tumor growth. Tumor bearing mice were treated with daily vehicle or Dul for 14 days (n =6 mice/group). Animals demonstrating signs of elevated intracranial pressure were euthanized and tumor presence confirmed by histology. MS: median survival. Results are representative of two separate experiments.