Alteration of CD47 Expression in Human and Murine Tumor Models Changes M1/M2 Phagocytosis Effect in Vitro and In Vivo
Authors: Yang Huajun , Li Zhongliang , Tang Beibei , Wang Zegen , Wang Phillip , Li Henry , Cherry Mohamad A. , Xing Frank 
Published: October 2019

Abstract
Introduction
CD47 expression in human tumor cell represents a resistance mechanism escaping the macrophage surveillance system. To reinstate macrophage function in recognizing and engulfing tumor cells, unblocking CD47-SIRPalpha interaction and thereby re-activating macrophage activity has been an important strategy in immune-oncology therapy. To validate the strategy, we profiled a large panel of tumor models including tumor cell lines, PDX models in Crown tissue bank and clinical samples for CD47 expression. Despite of its expression in the majority of tumor cell models, the expression level of CD47 vary in flow cytometry examination. Interestingly a portion of tumor cells do not express CD47 at all.
To facilitate in vitro screening for effective macrophage therapy, we established an in vitro assay platform using CD14+ monocytes as starting material. We also engineered human and murine cell models to overexpress human CD47 at one end and delete endogenous CD47 at the other end, and observed robust phagocytosis effect in these models. Finally, the findings in the in vitro assays were validated in MC38-hCD47 homograft in vivo.
Material and Methods
Tumor cell lines were CFSE-labeled and incubated with human monocyte-derived M1 or M2 macrophages (T:M 1:1) in the presence of 20μg/ml IgG1 isotype control, or anti-CD47 blocking antibodies B6H12 and BRIC126 and non-blocking antibody 2D3, for 2hrs. Cells were then harvested and stained with a macrophage marker (CD14+). Phagocytosed tumor cells were identified by flow cytometry as CD14+CFSE+. The phagocytosis index was calculated by dividing the number of macrophages engulfing tumor cells by the total number of macrophages. CD47 overexpression and deletion clones (KARPAS299, SK-OV3, and MC38 background) were developed in house. The CD14+ cell isolation kit was from Miltenyi. M1/M2 differentiation reagents were purchased from R&D and Stem Cell. Anti-CD47 antibodies B6H12 and 2D3 and mouse IgG1 were purchased from Thermo Fisher, and BRIC126 from GeneTex. B6H12 for animal study was sourced from Bioxcell.
Results
To facilitate in vitro screening for effective macrophage therapy, we sought to establish a robust yet reproducible assay platform using CD14+ monocytes as starting material. Through macrophage differentiation we were able to achieve > 80% of M1 and M2 populations, respectively, though variation exists. Differentiated M1/M2 were then co-cultured with CFSE-labeled target tumor cells for subsequent flow cytometry analysis. Strong phagocytosis effect was observed with CD47 expression tumor cells when anti-CD47 antibody BRICK126, which blocks the CD47-SIRPalpha interaction, was introduced. In contrast, aCD274 (PD-L1) antibody didn't yield more than background phagocytosis effect in the same assay suggesting the specific activation effect of BRICK126 triggering M1/M2 functionality.
To prove the CD47-specific effect, we engineered CARPAS299 and SK-OV3 tumor cell line models and MC38 murine cell model to overexpress human CD47 at one end and delete endogenous CD47 at the other end. The recombinant and isogenic models were then subject to the CD47 biology examination and more profound changes in phagocytosis responses were observed.
To validate our findings in vivo, we introduced engineered MC38-hCD47 clone in to C57/BL6 mice to measure homograft tumor growth kinetics and pharmacological responses to B6H12, another CD47 blocking agent. Details of results will be presented.
Conclusion
Disrupting the CD47-SIRPa anti-phagocytic axis is a novel immunotherapy approach against cancer. This study reveals that CD47 expression level varies in different tumor cell models. Our in vitro screen demonstrated that M1 and M2 macrophages resulted in comparable anti-CD47 phagocytic activities, and suggests that Karpas299 with higher CD47 expression level may be a better model than SK-OV3 in cell engulfing study. We also developed Karpas299-hCD47, SK-OV-3-hCD47 and MC38-hCD47 stable clones to facilitate in vitro and in vivo screening towards macrophage therapy, and discovered strong positive correlation between CD47 expression level and phagocytic activity in in vitro and in vivo systems.

Disclosures
No relevant conflicts of interest to declare.