CD47 antibody-induced engulfment of human leukaemic T-cells by bone-marrow derived macrophagesG. Lovell, C. Szybut, K. Patel, H. Campwala, N. Bevan, D. Appledorn, T. Dale & D. Trezise

Essen BioScience, Welwyn Garden City, AL7 3AX UK or Ann Arbor, MI, 48108, USA

www.essenbioscience.com

Anti-CD47 engulfment does not involve target cell apoptosis

T-lymphoblast CCRF-CEM (1.5 x104

cells/well) were incubated with 5µg/ml anti-CD47 for 1 hour. Thesetreated cells were then added into a96-well micro-titre plate containingbone marrow derived macrophages(BMDM) seeded at 1 x104 cells/well.The plate was placed in an IncuCyte®live-cell analysis system within astandard cell incubator and phaseand fluorescence images werecaptured every 15-30 minutes at 20xmagnification.

Labelled CCRF-CEM cells were treated with increasing concentrations of anti-CD47 antibody (B6H12.2, Abcam; ab3283)and added to human bone marrow-derived macrophages (BMDM; values shown are mean ± SEM, n=4).

• Target cells are labelled using the IncuCyte pHrodo Red Cell Labelling Kit which covalently attaches a pH-sensitivedye to proteins on the exterior of the cell.

• Labelled target cells are added to a standard 96-well microtitre plate containing effector phagocytes (e.g.macrophages, dendritic cells), then placed in an IncuCyte live cell analysis system within an incubator.

• Phase contrast and fluorescence images are taken at regular intervals.

• Fluorescence (area or intensity) increases as the target cells are engulfed into the effector cell lysosome (low pH).

• A kinetic profile showing real-time cell engulfment is generated using the IncuCyte software.

• Inhibitors with varying mechanisms of action (actin polymerisation inhibitor, microtubule disruptor, PKC inhibitor)were applied to the effector cells (J774A.1 mouse macrophages).

• Four concentration-response curves generated (triplicate data, 7 point curves) from a single 96-well plate.

Fluorescence intensity

Object count

• Engulfed cells are observed inside the macrophages and have high fluorescence intensity.• The fluorescence (total fluorescent area or intensity) is proportional to the amount of cell phagocytosis.• Rapid imaging (2 minute intervals) can be performed to create movies showing individual engulfment events.

Phagocytosis ApoptosisAnnexin V

ApoptosisCaspase 3/7

Continuous Live-Cell Analysis: Methodology

• CD47 is a cell-surface marker of self, or “don’t-eat-me” signal. Expression of CD47 enables tumour cellsto evade clearance by host macrophages.

• Blocking CD47 holds great promise as a therapeuticstrategy for both solid and hematologic cancers.

• Here, we have developed and validated anautomated image-based 96-well assay for anti-CD47antibody-mediated cellular phagocytosis.

• Tumour cells are labelled with pHrodo, a pH-sensitivedye, and added to phagocytes (e.g. BMDMs).

• When the labelled cell is engulfed into the acidiclysosome; the change in pH causes the labelled cellsto become fluorescent.

• The approach is amenable to many cell types andenables researchers to screen for small molecules orantibodies that enhance or inhibit phagocytosis.

Overview

Phagocytosis time-course is effector-cell dependent

Automated 96-well assay and analysis

Imaging and quantification

Anti-CD47 Ab induces phagocytosis of CCRF cells

Anti-CD47 directly inhibits proliferation at later time points

Jurkat CCRF-CEM Neutrophils

J774A.1

RAW264.7 NT NT

THP-1 NT

BMDM NT

Effe

cto

r

Target

Confluence at 3h Confluence at 24h

• Anti-CD47 antibody induces phagocytosis of CCRF-CEM cells by J774A.1 mouse macrophages without inducingapoptosis. Data shown are for 5 µg ml-1 anti-CD47- and camptothecin-treated (apoptotic) CCRF-CEM cells.

Cellular phagocytosis assay principle

Validation of labelling step:

• pHrodo-labelled Jurkat cells display red fluorescence in low pH buffer (pH4).

• Fluorescence intensity and the number of cells (objects) exceeding threshold brightness were determined using an IncuCyte live-cell analysis system.

• Intensity increase is proportional to the label concentration.

• The number of labelled cells exceeding the intensity analysis threshold plateaus.

• Anti-CD47 antibody exerts a direct anti-proliferative effect on CCRF-CEM cells in mono-culture; no effect observedafter 3 h, whereas a marked reduction seen at 24 h. Data shown are for 5 µg ml-1 anti-CD47- and camptothecin-treated (apoptotic) CCRF-CEM cells.

Proliferation

Differential time-courses observed for anti-CD47-induced phagocytosis in different effector cells.

• Exemplified target:effector combinations.

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