A New Serum Macrophage Checkpoint Biomarker for Innate Immunotherapy: Soluble Signal-Regulatory Protein Alpha (sSIRPα)

Yoanna V Vladimirova, Marie K Mølmer, Kristian W Antonsen, Niels Møller, Nikolaj Rittig, Marlene C Nielsen, Holger J Møller, Yoanna V Vladimirova, Marie K Mølmer, Kristian W Antonsen, Niels Møller, Nikolaj Rittig, Marlene C Nielsen, Holger J Møller

Abstract

Background and Aims: The macrophage “don’t eat me” pathway CD47/SIRPα is a target for promising new immunotherapy. We hypothesized that a soluble variant of SIRPα is present in the blood and may function as a biomarker. Methods: Monocyte derived macrophages (MDMs) from human buffy-coats were stimulated into macrophage subtypes by LPS and IFN-γ (M1), IL-4 and IL-13 (M2a), IL-10 (M2c) and investigated using flow cytometry. Soluble SIRPα (sSIRPα) was measured in cell cultures and serum by Western blotting and an optimized ELISA. Serum samples were obtained from 120 healthy individuals and from 8 individuals challenged by an LPS injection. Results: All macrophage phenotypes expressed SIRPα by flowcytometry, and sSIRPα was present in all culture supernatants including unstimulated cells. M1 macrophages expressed the lowest level of SIRPαand released the highest level of sSIRPα (p < 0.05). In vivo, the serum level of sSIRPα increased significantly (p < 0.0001) after an LPS challenge in humans. The median concentration in healthy individuals was 28.7 µg/L (19.8−41.1, 95% reference interval), and 20.5 µg/L in an IFCC certified serum reference material. The protein was stable in serum for prolonged storage and repeated freeze/thawing. Conclusions: We demonstrate that sSIRPα is produced constitutively and the concentration increases upon macrophage activation both in vitro and in vivo. It is present in human serum where it may function as a biomarker for the activity of tumor-associated macrophages (TAMs), and for monitoring the effect of immunotherapy.

Trial registration: ClinicalTrials.gov NCT01705782.

Keywords: CD47; SIRPα; biomarker; cancer; checkpoint; immunotherapy; macrophage.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
CD47 on tumor cells protects against phagocytosis. It interacts with SIRPα on the surface of TAMs resulting in decreased macrophage phagocytic capacity. Activation of macrophages leads to shedding by metalloproteases of soluble receptors (sCD163, sCD206) that function as biomarkers of macrophage activation in the blood. Soluble SIRPα (sSIRPα) is thought to be the product of extracellular domain shedding by the action of ADAM10 that, upon activation, cleaves SIRPα at a juxta-membrane position (15). Created with BioRender.com.
Figure 2
Figure 2
Expression and shedding of SIRPα. (A) Monocyte derived macrophages (M0) were polarized into macrophage subtypes by LPS and IFN-γ (M1), IL-4 and IL13 (M2a), IL-10 (M2c) and the expression of SIRPα was investigated using flowcytometry. (B) Soluble SIRPα (sSIRPα) was measured in the cell cultures by ELISA. (* p < 0.05, ** p < 0.01). (C) Western blotting of serum-samples from four individuals (Lane 2–5: samples, Lane 6: Recombinant extracellular SIRPα).
Figure 3
Figure 3
Shedding of sSIRPα in vivo. The concentration of sSIRPα (A), sCD206 (B), and sCD163 (C) were measured at t = 0, t = 180 and t = 360 min in blood samples from 8 healthy individuals. Participants were injected with a bolus of LPS at t = 0. Repeated measures ANOVA.
Figure 4
Figure 4
(A) Whole blood samples stored at room temperature or 4 °C were stable for up to 24 h before centrifugation. (B) Pipetted serum samples were stable at 4 °C for up to 8 days and underwent a small reduction in concentration between days 8 and 14 (p = 0.0313), * p < 0.05. (C) Pipetted serum samples were stable at −20 °C for up to 29 days (p = 0.3125). (D) Concentration of sSIRPα in pipetted serum was unaffected for at least five freeze–thaw cycles at −80 °C (p = 0.5347).

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