Effects of tumor grade and dexamethasone on myeloid cells in patients with glioma

Abstract

Efforts to reduce immunosuppression in the solid tumor microenvironment by blocking the recruitment or polarization of tumor associated macrophages (TAM), or myeloid derived suppressor cells (MDSCs), have gained momentum in recent years. Expanding our knowledge of the immune cell types, cytokines, or recruitment factors that are associated with high-grade disease, both within the tumor and in circulation, is critical to identifying novel targets for immunotherapy. Furthermore, a better understanding of how therapeutic regimens, such as Dexamethasone (Dex), chemotherapy, and radiation, impact these factors will facilitate the design of therapies that can be targeted to the appropriate populations and retain efficacy when administered in combination with standard of care regimens. Here we perform quantitative analysis of tissue microarrays made of samples taken from grades I-III astrocytoma and glioblastoma (GBM, grade IV astrocytoma) to evaluate infiltration of myeloid markers CD163, CD68, CD33, and S100A9. Serum, flow cytometric, and Nanostring analysis allowed us to further elucidate the impact of Dex treatment on systemic biomarkers, circulating cells, and functional markers within tumor tissue. We found that common myeloid markers were elevated in Dex-treated grade I astrocytoma and GBM compared to non-neoplastic brain tissue and grade II-III astrocytomas. Cell frequencies in these samples differed significantly from those in Dex-naïve patients in a pattern that depended on tumor grade. In contrast, observed changes in serum chemokines or circulating monocytes were independent of disease state and were due to Dex treatment alone. Furthermore, these changes seen in blood were often not reflected within the tumor tissue. Conclusions: Our findings highlight the importance of considering perioperative treatment as well as disease grade when assessing novel therapeutic targets or biomarkers of disease.

Keywords: Dexamethasone; Glioblastoma; cancer immunology; immunotherapy; tumor associated macrophage.

Figure 1.

CD163 is elevated in grades I and IV astrocytoma. (A) Representative cores from gliosis, grade I, II, III and GBM TMAs stained for CD163. Bottom right image is an example image scored by InForm software, where blue indicates nuclei, and brown indicates CD163+ cells. (B-C) Quantitative analysis of TMAs represented as (B) % of total cells that are CD163+, or (C) number of CD163+ cells per square millimeter. P-values calculated with the Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparisons test. (D) Linear regression analysis of patient age versus CD163 positivity in grade I pilocytic astroctyomas. P-value 0.5 indicates the slope does not significantly deviate from zero. (E) Comparison CD163 infiltration in IDH wild-type versus mutant samples from grade II-III astrocytomas. (F) Comparison CD163 infiltration in grade II versus III astrocytomas among IDH mutant samples. P-values were calculated with the Mann-Whitney test. Statistical significance is denoted with an asterisk where * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Line indicates median.

Figure 2.

Impact of Dexamethasone on CD163 infiltration is tumor grade-dependent. Comparison of CD163 positivity in Dex-treated and Dex-naïve samples from (A) gliosis, (B) grade I astrocytoma, (C) grade II-III astrocytoma, and (D) GBM patients. P-values were calculated with the Mann-Whitney test. Statistical significance is denoted with an asterisk where * = p < 0.05, **** = p < 0.0001. Line indicates median.

Figure 3.

CD68, CD33, S100A9 are not associated with high grade disease. (A) Quantitative analysis of TMAs represented as % of total nuclei that are CD68 + . (B) Ratio of CD163 to CD68 positivity calculated from adjacent TMA sections. (C) Comparison of CD68 positivity in Dex-treated and Dex-naïve samples from grade I astrocytoma patients. Quantitative analysis of TMAs represented as % of total cells that are (D) CD33+ and (E) S100A9 + . (F) Comparison of S100A9 positivity in Dex-treated and Dex-naïve samples from grade I astrocytoma patients. (G) CD15 (left images) S100A9 (right images) showing vascular CD15+ neutrophils, and both vascular and tumor infiltrating S100A9+ cells. Images from left to right were taken from serial sections. Statistical significance is denoted with an asterisk where * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Line indicates median.

Figure 4.

Chemokine levels are influenced by Dex-treatment. (A) Serum samples from healthy donors (HD) or Dex-treated and naïve GBM patients were analysed using the Biorad Human Chemokine Panel 40-plex. Dex-dependent differences were found in 6Ckine (CCL21), CTACK (CCL27), IP-10 (CXCL10), MCP-1 (CCL2), MDC (CCL22), MPIF-1 (CCL23), and SDF-1 (CXCL12). P-values calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. Line indicates mean. (B) Representative cores from gliosis, grade I, III and GBM TMAs stained for MCP-1. (C-D) Quantitative analysis of TMAs represented as (C) % of total cells that are MCP-1+, or (D) number of MCP-1+ cells per square millimeter. P-values calculated with the Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparisons test. Line indicates median. Linear regression analysis of MCP-1 versus CD163 positivity in (E) grade I pilocytic astroctyoma and (F) GBM. P-values indicate the slope does not significantly deviate from zero. Comparison of MCP-1 positivity in Dex treated and Dex naïve samples from (G) gliosis (H) grade I, and (I) grade II-III TMAs.

Figure 4.

(Continued).

Figure 4.

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Figure 5.

MHC-II and CD86 protein levels are unchanged by pre-operative Dex-treatment. Quantitative image analysis of FFPE samples used in Nanostring analysis (Table 1) reveal no difference in (A) HLA-DR, DP, DQ or (B) CD86 protein expression. Line indicates median. (C) Immunofluorescent staining for HLA-DR, DP, DQ (green) and CD163 (red, top image) or GFAP (red, bottom image) show that MHC-II is restricted to CD163+ cells and is not expressed by GFAP+ tumor cells or astrocytes.

Figure 6.

Dex-treatment, but not tumor grade, influences circulating monocyte phenotype. Fresh, RBC-lysed blood from healthy donors (HD), and Dex-treated and naïve grade II-III glioma, and GBM patients was immunostained for myeloid markers (A) HLA-DR, (B) CD163, (C) CD33, and (D) PD-L1 and analyzed by flow cytometry. All frequencies are represented as the percent of CD45+, CD14+ cells. Line and error bars indicate mean and SEM.

Figure 7.

Dex-treatment suppresses expression of myeloid markers in vitro. Monocyte derived macrophages were treated with 0.1, 1, 10 μM Dex for 24 hours, then detached and immunostained for (A) HLA-DR, DP, DQ (MHC-II), (B) CD86, (C) CD80, (D) CD40, (E) HLA-ABC (MHC-I), (F) CCR2 (G) PD-L1, and (H) CD163 and analyzed by flow cytometry. The background mean fluorescence intensity (MFI) of unstained samples was subtracted from experimental samples. Data was then expressed as the fold of treated MFI over untreated (control) MFI. P-values calculated with a one-way ANOVA followed by Dunnett’s multiple comparisons test comparing treated samples to control. Line indicates mean, error bars indicate SEM.

Figure 8.

GM-CSF-differentiated macrophages were treated for 24 hours with dexamethasone, then allowed to recover in growth media for six days. Cells were then detached and immunostained for (A) HLA-DR, DP, DQ (MHC-II), (B) CD86, (C) CD80, (D) CD40, (E) HLA-ABC (MHC-I), (F) CCR2 (G) PD-L1, and (H) CD163 and analyzed by flow cytometry. The background mean fluorescence intensity (MFI) of unstained samples was subtracted from experimental samples. Data was then expressed as the fold of treated MFI over untreated (control) MFI. P-values calculated with a one-way ANOVA followed by Dunnett’s multiple comparisons test comparing treated samples to control. Line and error bars indicate mean and SEM.