Published OnlineFirst May 29, 2013; DOI: 10.1158/1078-0432.CCR-12-3725 Clinical Cancer Human Cancer Biology Research S100B Promotes Glioma Growth through Chemoattraction of Myeloid-Derived Macrophages Huaqing Wang1, Leying Zhang5, Ian Y. Zhang5, Xuebo Chen2, Anna Da Fonseca8, Shihua Wu3, Hui Ren2, Sam Badie5, Sam Sadeghi5, Mao Ouyang4, Charles D. Warden6, and Behnam Badie5,7 Abstract þ Purpose: S100B is member of a multigenic family of Ca2 -binding proteins, which is overexpressed by gliomas. Recently, we showed that low concentrations of S100B attenuated microglia activation through the induction of Stat3. We hypothesized that overexpression of S100B in gliomas could promote tumor growth by modulating the activity of tumor-associated macrophages (TAM). Experimental Design: We stably transfected GL261 glioma cell lines with constructs that overexpressed (S100Bhigh) or underexpressed (S100Blow) S100B and compared their growth characteristics to intracranial wild-type (S100Bwt) tumors. Results: Downregulation of S100B in gliomas had no impact on cell division in vitro but abrogated tumor growth in vivo. Interestingly, compared to S100Blow tumors, S100Bwt and S100Bhigh intracranial gliomas exhi- bited higher infiltration of TAMs, stronger inflammatory cytokine expression, and increased vascularity. To identify the potential mechanisms involved, the expression of the S100B receptor, receptor for advanced glycation end products (RAGE), was evaluated in gliomas. Although S100B expression induced RAGE in vivo, RAGE ablation in mice did not significantly inhibit TAM infiltration into gliomas, suggesting that other pathways were involved in this process. To evaluate other mechanisms responsible for TAM chemoattraction, we then examined chemokine pathways and found that C-C motif ligand 2 (CCL2) was upregulated in S100Bhigh tumors. Furthermore, analysis of The Cancer Genome Atlas’s glioma data bank showed a positive correlation between S100B and CCL2 expression in human proneural and neural glioma subtypes, supporting our finding. Conclusions: These observations suggest that S100B promotes glioma growth by TAM chemoattraction through upregulation of CCL2 and introduces the potential utility of S100B inhibitors for glioma therapy. Clin Cancer Res; 1–12. Ó2013 AACR. Introduction olism, motility, and proliferation (1, 2). In the nervous þ S100B is a member of the multigene family of Ca2 system, S100B is constitutively released by astrocytes into -binding proteins of the EF-hand type and has been impli- the extracellular space and at nanomolar concentrations, it cated in the regulation of cellular activities such as metab- can promote neurite outgrowth and protect neurons against oxidative stress (3). S100B expression, however, can be augmented by various stimuli such as trauma and inflam- Authors' Affiliations: 1Department of Neurosurgery, Provincial Hospital mation (4). Acting in an autocrine fashion, micromolar Affiliated to Shandong University, Shandong University, Jinan, Shandong Province; 2Department of General Surgery, The Second Hospital of Jilin concentrations of S100B activate microglia and astrocytes University, Changchun, Jilin Province; 3Research Center of Siyuan Natural through the induction of iNOS and NF-kB, and increase the Pharmacy and Biotoxicology, College of Life Sciences, Zhejiang University, 4 expression of proinflammatory cytokines such as interleu- Hangzhou; Department of Cardiology, Third Xiangya Hospital, Central b a South University, Changsha Hunan, P.R. China; 5Division of Neurosurgery; kin (IL)-1 , IL-6, and TNF- (5–7). Through these func- 6Bioinformatics Core, Department of Molecular Medicine; and 7Depart- tions, S100B has been implicated in the pathogenesis of ment of Cancer Immunotherapeutics & Tumor Immunology, Beckman neurodegenerative brain disorders such as Alzheimer’s dis- Research Institute, City of Hope, Duarte, California; and 8Laboratório de Morfogênese Celular, Instituto de Ciências Biomédicas, Universidade ease (8). Federal do Rio de Janeiro, Bolsista do CNPq, Rio de Janeiro, Brazil S100B is also overexpressed by most malignant gliomas. Note: Supplementary data for this article are available at Clinical Cancer It is proposed to contribute to tumorigenesis by inhibiting Research Online (http://clincancerres.aacrjournals.org/). the function of the tumor suppressor protein p53 (9, 10) H. Wang and L. Zhang contributed equally to this article. and regulating cell proliferation and differentiation by stimulating the activity of the mitogenic kinases Ndr (11) Corresponding Author: Behnam Badie, Division of Neurosurgery, Beck- man Research Institute, City of Hope, 1500 East Duarte Road, Duarte, CA and Akt (protein kinase B; ref. 12). Furthermore, S100B 91010. Phone: 626-471-7100; Fax: 626-471-7344; E-mail: stimulates glioma proliferation at low concentrations (13) [email protected] and modulates astrocyte function through an interaction doi: 10.1158/1078-0432.CCR-12-3725 with Src kinase (14). These reports suggest that S100B may Ó2013 American Association for Cancer Research. contribute to astrocyte differentiation and could promote www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst May 29, 2013; DOI: 10.1158/1078-0432.CCR-12-3725 Wang et al. murine s100b cDNA or shRNA vectors to respectively Translational Relevance increase (S100Bhigh) or knockdown (S100Blow) S100B Tumor-associated macrophages are heterogeneous expression in GL261 gliomas (OriGene). The control cell populations that constitute a major component vector for shRNA was used to generate the control GL261 of inflammatory cells in tumor microenvironment. cells (S100Bwt). After transfection, cells were selected by These cells, when found in gliomas, are derived in Puromycin to generate monoclonal cell lines. To validate part from brain microglia and circulating monocytes, our findings in a second glioma model, K-luc cells (which and have been implicated in glioma angiogenesis, express very low levels of S100B at baseline) were elec- invasion, local tumor recurrence, and immunosup- troporated with s100B cDNA or control vectors, and cells pression. Here, we show that S100B overexpression were cultured in presence of Puromycin to generated by tumor cells promotes the growth of gliomas and monoclonal lines. S100B expression in each cell line enhances the infiltration of macrophages through was confirmed by Western blotting every month and CCL2 upregulation. To our knowledge, this is the first remained stable throughout the study. report that shows the role of S100B in gliomagenesis and introduces the potential utility of S100B inhibitors In vitro cell-proliferation assay for glioma therapy. S100Blow, S100Bhigh, and S100Bwt GL261 cells were placed in 6-well plates (1.5 Â 105 cells/well). Cell prolifer- ation was measured by counting the trypsinized cells at different time intervals. For proliferation rate measure- their activation and motility in the central nervous system ments, cells were incubated with BrdU (10 mmol/L) for (CNS). 30 minutes before quantifying the labeled cells by flow The role of S100B in gliomagenesis has not been exten- cytometry. sively studied. We recently showed that even at low levels, S100B modulated the activity of tumor-associated macro- Tumor implantation and sample collection phages (TAM) in a glioma model by activating Stat3 (15). Mice were housed and handled in accordance to the We hypothesized that expression of S100B could also guidelines of City of Hope Institutional Animal Care promote the growth of gliomas through modulation of and Use Committee under pathogen-free conditions. All GFP TAM activity in vivo. Here, we show that S100B overexpres- mice were on C57BL/6J background. CX3CR1 Knock- sion in gliomas enhanced TAM infiltration and promoted in mice that express enhanced green fluorescent protein tumor growth in vivo. Furthermore, S100B upregulation under control of the endogenous Cx3cr1 locus were resulted in increased angiogenesis and C-C motif ligand purchased from The Jackson Laboratory. Receptor for 2 (CCL2) overexpression. These findings were confirmed advanced glycation end products (RAGE) knockout using publicly available data showing S100B directly cor- mice, a generous gift from Dr. Y. Yamamoto (Kanazawa relates with CCL2 expression in proneural and neural University, Japan), were bred at our institution and glioma subtypes. To our knowledge, this is the first report PCR genotyped using tail DNA (17). Intracranial tumor that shows the role of S100B in gliomagenesis and intro- implantation was conducted stereotactically at a depth duces the potential utility of S100B inhibitors for glioma of 3 mm through a bur hole placed 2 mm lateral and therapy. 0.5 mm anterior to the bregma as described earlier (15). Briefly, GL261 glioma cells were harvested by trypsini- zation, counted, and resuspended in culture medium. Materials and Methods Female C57BL/6 mice (The Jackson Laboratory) weigh- Reagents and cell lines ing15to25gwereanesthetized by intraperitoneal GL261 glioma cells were obtained from Dr. K. Aboody’s (i.p.) administration of ketamine (132 mg/kg) and laboratory in 2006 and stably transfected with firefly xylazine (8.8 mg/kg) and implanted with 105 tumor luciferase expression vector. Positive clones (GL261-luc) cells using a stereotactic head frame at a depth of 3 mm were selected using zeocin (1 mg/mL) and G418. Lucif- through a bur hole placed 2 mm lateral and 0.5 mm erase-expressing KR158B cells (or K-luc),