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. 2019 Jan 15;116(3):997-1006.
doi: 10.1073/pnas.1721434116. Epub 2019 Jan 2.

Microglia are effector cells of CD47-SIRPα antiphagocytic axis disruption against glioblastoma

Gregor Hutter  1   2   3   4 , Johanna Theruvath  1   2   3 , Claus Moritz Graef  1   2   3 , Michael Zhang  1 , Matthew Kenneth Schoen  1   2   3 , Eva Maria Manz  1   2   3 , Mariko L Bennett  5 , Andrew Olson  6 , Tej D Azad  1   2   3 , Rahul Sinha  2   3 , Carmel Chan  7 , Suzana Assad Kahn  1   2   3 , Sharareh Gholamin  1   2   3 , Christy Wilson  1 , Gerald Grant  1 , Joy He  1   2   3 , Irving L Weissman  8   3 , Siddhartha S Mitra  9   2   3   10 , Samuel H Cheshier  9   2   3   11
Affiliations

Microglia are effector cells of CD47-SIRPα antiphagocytic axis disruption against glioblastoma

Gregor Hutter et al. Proc Natl Acad Sci U S A. .

Abstract

Glioblastoma multiforme (GBM) is a highly aggressive malignant brain tumor with fatal outcome VSports手机版. Tumor-associated macrophages and microglia (TAMs) have been found to be major tumor-promoting immune cells in the tumor microenvironment. Hence, modulation and reeducation of tumor-associated macrophages and microglia in GBM is considered a promising antitumor strategy. Resident microglia and invading macrophages have been shown to have distinct origin and function. Whereas yolk sac-derived microglia reside in the brain, blood-derived monocytes invade the central nervous system only under pathological conditions like tumor formation. We recently showed that disruption of the SIRPα-CD47 signaling axis is efficacious against various brain tumors including GBM primarily by inducing tumor phagocytosis. However, most effects are attributed to macrophages recruited from the periphery but the role of the brain resident microglia is unknown. Here, we sought to utilize a model to distinguish resident microglia and peripheral macrophages within the GBM-TAM pool, using orthotopically xenografted, immunodeficient, and syngeneic mouse models with genetically color-coded macrophages (Ccr2RFP) and microglia (Cx3cr1GFP). We show that even in the absence of phagocytizing macrophages (Ccr2RFP/RFP), microglia are effector cells of tumor cell phagocytosis in response to anti-CD47 blockade. Additionally, macrophages and microglia show distinct morphological and transcriptional changes. Importantly, the transcriptional profile of microglia shows less of an inflammatory response which makes them a promising target for clinical applications. .

Keywords: anti-CD47; checkpoint inhibition; glioblastoma; immunotherapy; microglia. V体育安卓版.

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"V体育安卓版" Conflict of interest statement

Conflict of interest statement: S VSports最新版本. G. , S. S. M. , S. H. C. , and I. L. W. are coinventors on patents regarding the use of CD47 antibody targeting brain tumors. I. L. W. is the inventor of multiple patents regarding CD47 antibody targeting non-CNS tumors that have been licensed to Forty Seven, Inc. He serves on the board of directors and as a consultant. He has equity ownership in Forty Seven, Inc.

Figures (VSports)

Fig. 1.
Fig. 1.
TAM composition of T387 grafted NSG-Ccr2RFP/wtCx3cr1GFP/wt and Ccr2RFP/RFPCx3cr1GFP/wt mice is dominated by microglia. (A) Representative FACS plots of the TAM pool in T387-EBFP2-luc grafted NSG-Ccr2RFP/wtCx3cr1GFP/wt and NSG-Ccr2RFP/RFPCx3cr1GFP/wt mice, gated on CD45 positive cells. (B and C) Composition of the TAM pool in T387-EBFP2-luc grafted (B) NSG-Ccr2RFP/wtCx3cr1GFP/wt (*P = 0.018 and **P < 0.0001) and (C) NSG-Ccr2RFP/RFPCx3cr1GFP/wt mice as assessed by flow cytometry analyses on RFPnegativeGFPbright (microglia) and GFPlowRFP+ (macrophages) signal gated on CD45 positive cells. *P = 0.036; **P = 0.030. Results are pooled from three independent experiments (NSG-Ccr2RFP/wtCx3cr1GFP/wt control group n = 11, anti-CD47 group n = 15) (NSG-Ccr2RFP/RFPCx3cr1GFP/wt control group n = 11, anti-CD47 group n = 10). Mean ± SEM.
Fig. 2.
Fig. 2.
Anti-CD47 treatment enhances microglial tumor phagocytosis independent of macrophage recruitment. (A and B) Representative FACS plots of dissociated tumors from EBFP2+T387-grafted (A) NSG-Ccr2RFP/wtCx3cr1GFP/wt and (B) NSG-Ccr2RFP/RFPCx3cr1GFP/wt mice treated with anti-CD47 or control until they reached morbidity. Microglia were defined as GFPhighRFPnegative and macrophages as RFPhighGFPlow. Anti-CD47 led to a significant increase of the double positive EBFP2+GFP+ microglial and EBFP2+RFP+ macrophage population in the Ccr2RFP/wt mouse model. In the Ccr2RFP/RFP model anti-CD47 treatment led to a significant increase of the EBFP2+GFP+ microglial population only. (C and D) Statistical analysis of the percentage of EBFP2+GFP+ microglia and EBFP2+RFP+ macrophages ± anti-CD47 treatment in (C) NSG-Ccr2RFP/wtCx3cr1GFP/wt and (D) NSG-Ccr2RFP/RFPCx3cr1GFP/wt mice after 3 wk of treatment. ***P = 0.0003; ****P < 0.0001; **P = 0.0019; Welch’s t test. Results are pooled from three independent experiments. (NSG-Ccr2RFP/wtCx3cr1GFP/wt control group n = 12, anti-CD47 group n = 17) (NSG-Ccr2RFP/RFPCx3cr1GFP/wt control group n = 7, anti-CD47 group n = 7). Mean ± SEM. ns, not significant.
Fig. 3.
Fig. 3.
Anti-CD47 treatment leads to significant survival benefit in T387 grafted mice even in absence of phagocytizing macrophages. (A and B) Kaplan–Meier curves of (A) NSG-Ccr2RFP/wtCx3cr1GFP/wt (*P = 0.01) and (B) Ccr2RFP/RFPCx3cr1GFP/wt mice (*P = 0.049) grafted with T387-EBFP2-luc treated with anti-CD47 (H5F9-G4) or human IgG control; purple, anti-CD47 treated; blue, control (human IgG). Arrows indicate beginning of treatment on day 7 after tumor injections. (C) Images of luminescence measurements detected with an IVIS spectrum instrument on day 30 after tumor injections. Minimum and maximum values were adjusted to the same level for the NSG-Ccr2RFP/wtCx3cr1GFP/wt and Ccr2RFP/RFPCx3cr1GFP/wt model. Representative survival experiment is shown. (NSG-Ccr2RFP/wtCx3cr1GFP/wt control group n = 5, anti-CD47 group n = 5) (Ccr2RFP/RFPCx3cr1GFP/wt control group n = 7, anti-CD47 group n = 6). Mean ± SEM.
Fig. 4.
Fig. 4.
Anti-CD47 treatment leads to macrophage-independent survival benefit in a syngeneic glioma model. (A) Representative FACS plots of the TAM pool in anti-CD47 treated and control condition in CT-2A-luc grafted B6-Ccr2RFP/RFPCx3cr1GFP/wt mice, gated on CD45 positive cells. (B) Composition of the TAM pool in CT-2A-luc grafted B6-Ccr2RFP/RFPCx3cr1GFP/wt mice as assessed by flow cytometry analyses on GFPlowRFP+ (macrophages) and RFPnegativeGFPbright (microglia) signal. Expressed as a percent of CD45 positive cells. *P = 0.041; **P = 0.003. (control group n = 5, anti-CD47 group n = 5) (C) Luminescence measurements (photon flux in p/s) for CT-2A-luc grafted B6-Ccr2RFP/RFPCx3cr1GFP/wt mice treated with anti-CD47 (MIAP401) or mouse IgG control. *P = 0.041, (control group n = 6, anti-CD47 group n = 6) Mann–Whitney U test. (D) Representative images of luminescence measurements detected with an IVIS spectrum instrument on day 23 after tumor injections. Mean ± SEM.
Fig. 5.
Fig. 5.
In vivo imaging shows real-time phagocytosis of living glioma cells by microglia and morphologic changes upon anti-CD47 treatment. (A and B) Two-photon in vivo laser scanning image of T387-EBFP2+ grafted NSG-Ccr2RFP/wtCx3cr1GFP/wt mice after 2 wk of anti-CD47 treatment. Tumors were implanted at a depth of 2 mm below the right frontal cortex and cranial windows were implanted; microglia, green; tumor cells, blue; and macrophages, red. (C) Violin plots of microglial filament tracking parameters using binomial logistic regression for anti-CD47 treated or control condition (based on corresponding Movies S6 and S7). Multivariate logistic regression identified an increase of number of processes (*P = 0.046) and process straightness (**P = 0.0057) in anti-CD47 treated cells, whereas process speed decreased significantly (***P = 0.005). Process length was not significantly different between conditions [P = not significant (ns)].
Fig. 6.
Fig. 6.
RNA-seq analysis reveals distinct transcriptional profiles of tumor-associated macrophages and microglia. (A) Experimental scheme for the RNA-seq experiment. (B) Venn diagram of significantly differentially expressed genes (q value <0.1) in TA-MAC (red) (control group n = 3, anti-CD47 group n = 3) and TA-MG (green) (control group n = 3, anti-CD47 group n = 3) of mice treated with anti-CD47 or control. (C and D) Volcano plots of differentially expressed genes between (C) TA-MAC control versus treated and (D) TA-MG control versus treated. The P values are Benjamini–Hochberg corrected. (E) Heatmap depicting row z scores of log2-transformed gene expression of TA-MAC control (light red), TA-MAC treated (red), TA-MG control (light green), and TA-MG treated (green).
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