AACR 2017 - American Association for Cancer Research

In this study, we characterised anti-CD47 antibody-mediated engulfment of living tumour cells (CCRF-CEM) by mouse bone-marrow derived macrophages (BMDMs) or immortalised mouse macrophages (J774A.1). Phagocytosis was quantified using a pH-sensitive cell-labelling fluorescent probe, pHrodo, and automated kinetic live-cell analysis (IncuCyte®). CCRF-CEM cells were first labelled using pHrodo (250ng ml-1 for 1h), washed and then treated with antibody for 1 h. Target cells were then added to BMDMs or J774A.1 that had been seeded overnight on 96-well plates. Phase- and fluorescence images were captured and quantified every 15 min. Anti-CD47 antibody (B6H12.2, 0.04-5μg ml-1), but not IgG-control, produced time- and concentration-dependent engulfment of CCRF-CEMs by BMDMs (30’-4h), as evidenced by an increase in intracellular fluorescence as the label accumulates in the acidic phagosome. After 4h the red fluorescence area was increased by 25-fold (1975 ± 391 μm2 vs 80 ± 41 μm2). From close inspection of the time-lapse images cellular engulfment could be clearly observed, coincident with the appearance of the fluorescent signal. Similar observations were made with J774A.1 as the effector cell. Interestingly, the rate and degree of engulfment appeared effector cell-dependent. The mechanism of engulfment was not via induction of target cell apoptosis since anti-CD47 did not induce PS externalisation (Annexin V) or activate caspase 3/7. Anti-CD47 had no direct effect on CCRF-CEM proliferation for the first 4 h but upon longer exposures (>8 h) cell growth was attenuated. Our experimental findings substantiate the known pro-phagocytic effects of anti-CD47 antibodies, and provide a model system and method for quantitative functional analysis and mechanistic insight of CD47 modulators as cancer therapeutics.

Here, we describe novel high-throughput live-cell image-based assays for immune cell activation and killing of target cells that are geared toward screening for new treatments for these malignancies. Myeloid and lymphoid cells (Jurkats, Raji, Ramos, WIL2-NS, THP-1, PBMCs) were plated on poly-L-ornithine (PLO) or fibronectin coated 96-well flat plates and monitored over time (h to days) using non-invasive live-cell analysis (IncuCyte®). The dynamics of proliferation were quantified via phase-contrast image analysis (% confluence), and validated as a robust measure of cell number by correlating to direct cell counts (Scepter, Millipore) and ATP assays (PerkinElmer). Anti-CD3/IL-2 (0.1-100/10 ng/mL) or anti-CD28 activation (1-100 ng/mL) of PBMCs evoked time-dependent proliferation (0-5d) that was sensitive to the initial cell density and concentration of stimulus. L-Kynurenine (4.69 - 300 µM), a metabolite of the amino acid L-tryptophan caused concentration and time-dependent inhibition of proliferation of PBMCs. To quantify immune cell killing in co-cultures, WIL2Ns and Ramos B-cell myelomas were first transduced with nuclear-targeted RFP (NucLight Red) to enable direct cell counting. PBMCs, either pre-activated or activated in situ (IL-2/CD3), were then added and the time-course of killing quantified through live (RFP) and dead/apoptotic (annexin-V) cell counting. Together, these protocol developments and validation data illustrate non-invasive continuous measurement of proliferation, activation, clustering and immune-cell killing of non-adherent tumour cells at industrial scale. Unlike flow cytometry, this approach follows the full time-course of the biology without perturbing the cells and allows cell-cell interactions to be visualised. These assays are amenable to testing new therapeutic antibodies, small molecules and genetic T-cell modulation such as CAR-T.