Immunohistochemical evaluation of immune cell infiltration in canine gliomas

Abstract

Evasion of the immune response is an integral part of the pathogenesis of glioma. In humans, important mechanisms of immune evasion include recruitment of regulatory T cells (Tregs) and polarization of macrophages toward an M2 phenotype. Canine glioma has a robust immune cell infiltrate that has not been extensively characterized. The purpose of this study was to determine the distribution of immune cells infiltrating spontaneous intracranial canine gliomas. Seventy-three formalin-fixed, paraffin-embedded tumor samples were evaluated using immunohistochemistry for CD3, forkhead box 3 (FOXP3), CD20, Iba1, calprotectin (Mac387), CD163, and indoleamine 2,3-dioxygenase (IDO). Immune cell infiltration was present in all tumors. Low-grade and high-grade gliomas significantly differed in the numbers of FoxP3+ cells, Mac387+ cells, and CD163+ cells (P = .006, .01, and .01, respectively). Considering all tumors, there was a significant increase in tumor area fraction of CD163 compared to Mac387 (P < .0001), and this ratio was greater in high-grade tumors than in low-grade tumors (P = .005). These data warrant further exploration into the roles of macrophage repolarization or Treg interference therapy in canine glioma.

Keywords: astrocytoma; brain; cancer; dogs; immunohistochemistry; immunology; lymphocytes; macrophages; oligodendroglioma.

Figures 1–6:. High-grade oligodendroglioma, brain, dog. Immunohistochemistry.

Figure 1: Moderate numbers of CD3-immunolabeled cells are scattered through the tumor. Figure 2: CD3-immunolabeled cells form an aggregate around a focus of glomeruloid vascular proliferation. Figures 3: Moderate numbers of FoxP3-immunolabeled cells are present in the tumor. Figure 4: FoxP3-immunolabeled cells extend slightly beyond the brain-tumor interface but are rare in normal brain distant from the mass. Figure 5: Clusters of CD20-immunolabeled cells form a perivascular aggregate. Figure 6: IDO-immunolabeled cells are sporadically scattered through this tumor.

Figures 7–9:. Numbers of CD3+, Foxp3+, and CD20+ cells in canine glioma. HG = high-grade, LG = low-grade, oligo = oligodendroglioma, astro = astrocytoma, undef = undefined glioma.

Figure 7: There are no differences in CD3+ cell counts between subtypes or grades of tumors. Bar = median. Figure 8: High-grade tumors contain more FoxP3+ cells than do low-grade tumors (*, p = 0.006, Mann-Whitney test). There are no differences in FoxP3+ cell counts between tumor subtypes. Figure 9: There are no significant differences in CD20+ cell counts between tumor subtypes or grade. Bar = median.

Figures 10–15:. High-grade oligodendroglioma, brain, dog. Immunohistochemistry.

Figure 10: Iba1-immunolabeled cells have small cell bodies with long processes, consistent with a ramified morphology. Figure 11: Iba1-immunolabeled cells have medium-sized cell bodies with shortened processes, consistent with a reactive morphology. Figure 12: Iba1-immunolabeled cells have robust cell bodies with short to absent processes, consistent with an amoeboid morphology. Figure 13: Iba1-immunolabeled macrophages and microglia within the tumor exhibit an amoeboid morphology (right), which transitions towards a ramified morphology as the brain-tumor interface is approached (center). Macrophages and microglia within normal brain exhibit a ramified morphology (left). Figure 14: Mac387 immunohistochemistry. There are low numbers of Mac387-immunolabeled cells scattered through the tumor consistent with M1 like macrophages. Inset: high magnification. Figure 15: Large numbers of CD163-immunolabeled cells are scattered through the tumor consistent with M2-like macrophages. Inset: high magnification.

Figures 16–19.. Quantification of immunolabeling for immune cells based on area fraction in canine glioma. HG = high-grade, LG = low-grade, oligo = oligodendroglioma, astro = astrocytoma, undef = undefined glioma.

Figure 16: There are no significant differences in Iba1 area fraction between different tumor subtypes or grade. Bar = median. Figure 17: High-grade tumors have a higher Mac387 area fraction than low-grade tumors (*, p = 0.01, Mann-Whitney test). High-grade oligodendrogliomas have a higher Mac387 area fraction than low-grade-tumors (**, p = 0.0006, Mann-Whitney test). Bar = median. Figure 18: High-grade tumors have a higher CD163 area fraction than low-grade tumors (*, p = 0.01, Mann-Whitney test). High-grade oligodendrogliomas have a higher CD163 area fraction than low-grade oligodendrogliomas (**, p = 0.02, Mann-Whitney test). Bar = median. Figure 19: CD163 area fraction is significantly higher than Mac387 area fraction in high-grade tumors versus low-grade tumors (*, p = 0.005, Mann-Whitney test). Bar = median.