Figure 1. Intracranial GBM tumors are densely infiltrated with immune cells.

(A) GL26 tumor volume 7, 14 and 21 days after implantion into the brain striatum of C57BL/6 mice was determined. Reactive astrocytic staining (GFAP+) was used to define the border of the tumor with non-malignant brain tissue. Representative sections of mouse brains bearing tumors implanted 7, 14 and 21 days previously and stained with GFAP are shown on the left. Tumor growth kinetics were essentially exponential over the time period analyzed with a doubling time of approximately 1.8 days. 5 mice were used to determine tumor volume at each time point. (B) Infiltration of immune cells in intracranial GL26 tumors. Brain sections from tumor bearing mice were stained with antibodies against CD45 (leukocytes) or F4/80 (macrophages/activated microglia). Representative Confocal images show immune cells (green) within the tumor mass and in the tumor borders. DAPI (blue) was used to stain nuclei. Yellow arrows indicate immunoreactive cells. T: tumor. (C) Tumor infiltrating immune cells were isolated from tumors 24 days after tumor implantation and analyzed using flow cytometry. (i) Dot plot of CD11c against I-Ab that was first gated for live CD45+ leukocytes. DC (CD11c+ CD45+ I-Ab+) are shown in the red box. (ii) Macrophages (MΦ) were assessed by gating live leukocytes with CD45, then plotting CD11b against I-Ab. The red box outlines the population of tumor infiltrating macrophages (CD11b+ CD45+ I-Ab+). (iii) T cells were stained with CD3ε-PE, CD4-PerCP and CD8a-FITC. The plot displays CD4 against CD8a when cells were first gated for CD3ε+ live leukocytes. CD4+ T cells and CD8a+ T cells are shown in red boxes. (iv) Tumor infiltrating Tregs were observed by staining with CD3ε-PE, CD4-PerCP and Foxp3-FITC. CD3ε+ live leukocytes were gated and dot plots display Foxp3 against CD4 staining. The population of Tregs (CD4+ Foxp3+ CD3ε+) are shown in the red box. (v) NK and NK-T cells were visualized by gating live CD45+ leukocytes, then displaying NK1.1 against CD3ε. The population of tumor infiltrating NK cells (CD3ε- CD45+ NK1.1+) and NK-T cells (CD3ε+ CD45+ NK1.1+) are shown in red boxes. (vi) Tumor infiltrating B cells are visualized by gating for live leukocytes, then plotting CD45 against CD19. The population of B cells (CD19+ CD45+) is shown in a red box. The percentages of each immune cell population infiltrating the tumor with respect to the total number of tumor CD45+ cells is indicated in representative dot plots. (D) Nissl staining was used to visualize tumors in the brain. Tumors are dense in Nissl substance, so stain darker than normal brain tissue. Representative Confocal images show tumor infiltrating CD4+ T cells, CD8+ T cells, CD205+ mDCs, CD19+ B cells, and CD25+ immune cells (seen in green) and DAPI (blue) was used to visualize the nuclei. Yellow arrows indicate immunoreactive cells. (E) Flow cytometric analysis of the percentage of T cells that express CD25 in spleens, dLN and tumors in mice bearing intracranial GL26 brain tumors 24 days after tumor implantation. (F) Representative dot plots of CD25 vs CD3ε in immune cells isolated from the spleen, LN and tumor. The percentages of CD25+ cells population with respect to the total number of CD3ε+ cells in the tumor, draining lymph nodes or spleen are indicated in representative dot plots.