Glioblastoma Phagocytic Cell Death: Balancing the Opportunities for Therapeutic Manipulation

Abstract

Macrophages and microglia are professional phagocytes that sense and migrate toward "eat-me" signals. The role of phagocytic cells is to maintain homeostasis by engulfing senescent or apoptotic cells, debris, and abnormally aggregated macromolecules. Usually, dying cells send out "find-me" signals, facilitating the recruitment of phagocytes. Healthy cells can also promote or inhibit the phagocytosis phenomenon of macrophages and microglia by tuning the balance between "eat-me" and "don't-eat-me" signals at different stages in their lifespan, while the "don't-eat-me" signals are often hijacked by tumor cells as a mechanism of immune evasion. Using a combination of bioinformatic analysis and spatial profiling, we delineate the balance of the "don't-eat-me" CD47/SIRPα and "eat-me" CALR/STC1 ligand-receptor interactions to guide therapeutic strategies that are being developed for glioblastoma sequestered in the central nervous system (CNS).

Keywords: gliomas; immune checkpoint blockade; myeloid cells.

Figure 1

The expression of key molecules regulating phagocytosis in human glioblastoma. (a) Correlation of mRNA expression with overall survival in newly diagnosed IDHwt glioblastoma patients as analyzed from TCGA database using Gliovis. Kaplan–Meier curve is calculated based on high versus low expression of markers dichotomized based on the median expression in glioma patients. CD47: n = 142; median survival for high–CD47 (n = 70) and low–CD47 (n = 72) are 11.0 and 14.5 months, respectively (p = 0.24). SIRPα: n = 145; median survival for high–SIRPα (n = 74) and low–SIRPα (n = 71) are 13.3 and 13.0 months, respectively (p = 0.82). CALR: n = 142; median survival for high–CALR (n = 71) and low–CALR (n = 71) are 13.1 and 13.0 months, respectively (p = 0.88). STC1: n = 142; median survival for high–STC1 (n = 70) and low–STC1 (n = 72) are 10.8 and 14.9 months, respectively (p = 0.03). Mantel–Cox test was used to compare groups: ns denotes p > 0.05, * denotes p ≤ 0.05. (b) Expression of markers at the mRNA level as a function of glioma grade as analyzed from TCGA database using Gliovis. Ordinary one–way ANOVA with Tukey’s multiple comparison test was used to compare each grade: * denotes p ≤ 0.05, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001, **** denotes p ≤ 0.0001. (c) Expression of markers based on anatomical location as analyzed from the Ivy Atlas using Gliovis. Ordinary one–way ANOVA with Tukey’s multiple comparison test was used to compare locations: **** denotes p ≤ 0.0001. LE, leading edge; IT, infiltrating tumor; CT, cellular tumor; PZ, perinecrotic zone; PS, pseudopalisading cells around necrosis; HPV, hyperplastic blood vessels in cellular tumor; MP, microvascular proliferation. The markers are annotated with the axes they are part of, respectively.

Figure 2

Original representative images of the spatial and cellular localization of CD47/SIRPα expression. (a) A newly diagnosed glioblastoma specimen resected as a lobectomy in continuity with the adjacent infiltrating brain was analyzed using automated sequential multiplex immunofluorescence (n = 2) as previously described [21,22,23]. SIRPα (yellow; Cell Signaling, clone D613M, dilution 1/100) is expressed at the leading edge. CD47 (red; Novus Bio, clone B6H12.2, dilution 1/100) is expressed at minimal levels. Scale bar = 200 µm. (b) Dot plot displaying marker expression within gliomas analyzed with the scRNA seq dataset from Abdelfattah et al. [24]. Bubble size corresponds to the percentage of cells expressing gene marker; colors indicate average expression. Glioma, glioma tumor cells and astrocytes; oligo, oligodendrocytes; endo, endothelial cells. (c) Adjacent infiltrating region of the brain analyzed using automated sequential multiplex immunofluorescence as previously described with magnified insert [21,22,23]. SIRPα (yellow) is seen in association with microglia (P2RY12; Atlas Antibodies, polyclonal, dilution 1/1000). Scale bar = 100 µm (main panel) and 50 µm (insert). (d) Newly diagnosed glioblastoma sample with adjacent peritumoral brain (lobectomy resection) analyzed using automated sequential multiplex immunofluorescence as previously described [21,22,23]. SIRPα (yellow) expression is confined to a focal area at the edge. Dotted line represents the edge between the tumor and the adjacent brain. Scale bar = 2 mm. (e) Positive control multiplex immunofluorescence images of a glioblastoma expressing CD47 (red) within the tumor region with panel (f) representing the magnified window in (e) as highlighted with the dotted box. Red arrows denote rare cells expressing CD47. The other markers were turned off to see the CD47 expression more clearly. Scale bars = 500 µm (e) and 100 µm (f). Nuclei are denoted with DAPI (dark blue, ThermoFisher Scientific); glioblastoma tumor cells and astrocytes are marked with GFAP (light blue; Sigma, clone GA5, dilution 1/2000); vasculature is marked with CD31 (cyan blue; Abcam, clone EPR17259, dilution 1/1500); microglia are marked with P2RY12 (pink).

Figure 3

Original representative images of the spatial localization of CALR/STC1 expression. (a) Representative images showing the colocalization of CALR (purple; Abcam, clone FMC75, dilution 1/1000) and CD163 (green; Abcam, clone EPR19518, dilution 1/600) within the same cell. Multiple regions were analyzed and show concordant expression of both CD163 (green) and CALR (purple). Location 2 is highlighted in panel (c) with boxed window “(a)”. Scale bar = 50 µm (location 1) and 20 µm (location 2). (b) CALR (purple) colocalized with CD163 (green) in the perinecrotic zone within the glioblastoma (top panels). This area is highlighted in panel (c) within boxed window “(b)”. In the region of the adjacent brain, CD163 (green) expression is present in the blood vessels, but CALR (purple) is absent (bottom panels). Panels on the right are magnified views of the highlighted area on the left. Scale bar = 200 µm (top left), 50 µm (top right), 100 µm (bottom left), and 50 µm (bottom right). (c) Newly diagnosed glioblastoma sample with adjacent peritumoral brain (lobectomy resections, n = 2) analyzed using automated sequential multiplex immunofluorescence as previously described [21,22,23]. CALR (purple) is expressed throughout the tumor region. Area shown in boxed window “(a)” is location 2 of panel (a). Area shown in boxed window “(b)” is tumor in panel (b). Scale bar = 2 mm. Nuclei are denoted with DAPI (dark blue; ThermoFisher Scientific); glioblastoma tumor cells and astrocytes are marked with GFAP (light blue; Sigma, clone GA5, dilution 1/2000); vasculature is marked with CD31 (cyan blue; Abcam, clone EPR17259, dilution 1/1500); macrophages are marked with CD163 (green).