Increased expression of stress inducible protein 1 in glioma-associated microglia/macrophages

Abstract

Factors released by glioma-associated microglia/macrophages (GAMs) play an important role in the growth and infiltration of tumors. We have previously demonstrated that the co-chaperone stress-inducible protein 1 (STI1) secreted by microglia promotes proliferation and migration of human glioblastoma (GBM) cell lines in vitro. In the present study, in order to investigate the role of STI1 in a physiological context, we used a glioma model to evaluate STI1 expression in vivo. Here, we demonstrate that STI1 expression in both the tumor and in the infiltrating GAMs and lymphocytes significantly increased with tumor progression. Interestingly, high expression of STI1 was observed in macrophages and lymphocytes that infiltrated brain tumors, whereas STI1 expression in the circulating blood monocytes and lymphocytes remained unchanged. Our results correlate, for the first time, the expression of STI1 and glioma progression, and suggest that STI1 expression in GAMs and infiltrating lymphocytes is modulated by the brain tumor microenvironment.

Keywords: Brain tumor microenvironment; Glioma; Glioma progression; Glioma-associated microglia/macrophages; Stress-inducible protein 1.

Figure 1. STI1 expression increases with tumor progression

(A) Representative Xenogen images of three mice 5, 12 and 19 days after tumor implantation. (B) Graph illustrating the tumor size of samples used for STI1 analysis; n=4 animals per group; * p < 0.05. (C) Western blot of normal brain (CTR) and brain tumors 1, 2 and 3 weeks after intracranial injection. All samples probed with anti-STI1 antibody showed a band at the expected molecular weight (66 kDa). (D) A two-week old GL261 glioma in a CX3CR1^GFP mouse was immunolabeled with anti-STI1 antibody (red) demonstrating the expression of STI1 by tumor and GAMs (inset, arrow); nuclei were stained with DAPI (blue). Bar, 100 μm. Data is representative of two separate experiments.

Figure 2. STI1 expression in GAMs and infiltrating lymphocytes is upregulated in tumor-bearing brain

(A) Flow cytometry was performed to identify STI1–positive cells after intracranial inoculation of GL261 gliomas. Representative dot plots demonstrating increase in STI1 expression (red events) in tumor macrophages (MP; CD11b⁺/CD45^high), microglia (MG; CD11b⁺/CD45^low), and lymphocytes (Lym; CD11b⁻/CD45⁺) after gating the leukocyte population. Quantification of the percentage of STI1–positive cells (B, D and F) and the mean fluorescence intensity (C, E and G) by cell population; n=4 animals per group; * p < 0.05, ** p < 0.01, *** p < 0.001. Data is representative of two separate experiments.

Figure 3. STI1 expression in circulating blood monocytes and lymphocytes in glioma-bearing mice

(A) Flow cytometry was performed to identify STI1–positive cells (red events) in blood after intracranial GL261 glioma inoculation. Representative dot plots demonstrating the expression of STI1 in monocytes (Mon; CD11b⁺/CD45^high) and lymphocytes (Lym; CD11b⁻/CD45⁺) after gating the CD45⁺ (leukocyte) population. Quantification of the percentage of STI1–positive cells (B and D) and the mean fluorescence intensity (C and E) by cell type; n=4 animals per group; * p < 0.05, ** p < 0.01, *** p < 0.001. Data is representative of two separate experiments.

Figure 4. STI1 expression in GAMs and infiltrating lymphocytes is modulated by the brain tumor microenvironment

(A) Flow cytometry wasperformed to identify STI1–positive inflammatory cells in GL261 tumors propagated in either the brain or subcutaneous tissue. Representative dot plots demonstrating the expression of STI1 in monocytes/macrophages (Mon/MP; CD11b⁺/CD45^high) and lymphocytes (Lym; CD11b⁻/CD45⁺) after gating the CD45⁺ (leukocyte) population. The size of the tumors is identified in each dot plot. B, D and F: brain tumors (n=15 mice in total); C, E and G: subcutaneous tumors (n=13 mice in total). Y axis: percentage of microglia (MG), monocytes/macrophages (Mon/MP) and lymphocytes (Lym) positives for STI1; X axis: tumor area (mm²) or volume (mm³). Brain, blood and subcutaneous tissue (SC) were analyzed. The thicker lines are the result of a linear regression from each of the curves.