Phagocytosis by stroma confounds coculture studies

Abstract

Signals provided by the microenvironment can modify and circumvent pathway activities that are therapeutically targeted by drugs. Bone marrow stromal cell coculture models are frequently used to study the influence of the bone marrow niche on ex vivo drug response. Here, we show that mesenchymal stromal cells from selected donors and NKTert, a stromal cell line, which is commonly used for coculture studies with primary leukemia cells, extensively phagocytose apoptotic cells. This could lead to misinterpretation of results, especially if viability readouts of the target cells (e.g. leukemic cells) in such coculture models are based on the relative proportions of dead and alive cells. Future coculture studies which aim to investigate the impact of bone marrow stromal cells on drug response should take into account that stromal cells have the capacity to phagocytose apoptotic cells.

Keywords: Biological Sciences; Cancer; Cell Biology; Stem Cells Research.

Graphical abstract

Figure 1

Dead leukemia cells disappear from cocultures of CLL with NKTert bone marrow stromal cells (A) The percentage of alive CLL cells was higher in cocultures of CLL and NKTert (N + C) than in CLL monocultures (C). This was true for cultures treated with solvent (ctrl), fludarabine (flu) or venetoclax (ven). The readout was either performed with microscopy or flow cytometry; paired t test, ∗ = p < 0.05; thin lines = samples from four patients; thick, colored bars = mean. (B) Total cell counts decreased in solvent control treated (ctrl), fludarabine treated (flu) and venetoclax treated (ven) cocultures of CLL and NKTert (N + C) in comparison to monocultures of CLL (C). Paired t test, ∗∗ = p < 0.01, ∗ = p < 0.05; thin lines = samples from four patients; thick, colored bars = mean, assessed by microscopy. (C) Dead CLL cells disappeared in cocultures with NKTert. The total number of alive cells was comparable between the culture conditions. Paired t test, ∗∗ = p < 0.01, ∗ = p < 0.05; thin lines = samples from four patients; thick, colored bars = mean, assessed by microscopy. (D) Confocal microscopy pictures of CLL cells cultured in monocultures or in coculture with NKTert or HS-5 stromal cells. CLL cells disappeared from CLL-NKTert cocultures treated with venetoclax. Green = CellTracker Green, blue = CellTracker Blue, yellow = propidium iodide. See also Figures S1 and S2.

Figure 2

Disappearance of dead leukemia cells is due to extensive phagocytosis by NKTert bone marrow stromal cells (A) Brightfield image of venetoclax treated NKTert and CLL coculture showing a NKTert cell having phagocytosed many CLL cells. Three of the many phagocytosed CLL cells are highlighted with white arrows, two nonphagocytosed CLL cells are highlighted with black arrows. (B) Large lysosomal bodies (phagosomes) appeared in cocultures of NKTert and CLL treated with venetoclax. Some of the phagosomes are highlighted with white arrows. Blue = CellTracker Blue, red = lysosomal dye NIR. (C) Magnification of two phagosomes inside NKTert (CellTracker Blue) cells. CLL cells had been previously labeled with CellTracker Green. (D) Quantification of phagosomes in microscopy pictures of HS-5 or NKTert stromal monocultures or cocultures with CLL cells. ctrl = solvent control, ven = venetoclax; Points and lines = individual patients, colored bars = mean; samples from three CLL patients. See also Figures S3, S4 and S5.

Figure 3

Phagocytosis is not restricted to NKTert but is also observed for MSCs and is not limited to dead cells of leukemic origin (A) NKTert also phagocytosed apoptotic cells of nonhaematopoietic origin. CellTracker-Green-labeled HBL-2, OCI-AML 2, HELA, or HEK-293T cells (green), treated with doxorubicine to induce apoptosis and cocultured with NKTert. Blank areas in staining of CellTracker-Blue-labeled NKTert (blue) were observed (examples indicated by arrows). These blank areas overlapped with CellTracker-Green (green)- and propidium-iodide (yellow)-stained HBL-2, OCI-AML 2, HELA, or HEK-293T cells. Some of these phagosomes were acidic, indicated by staining with lysosomal dye NIR (red). (B) CellTracker-Blue-labeled mesenchymal stromal cell (MSC; blue) phagocytosing dead CellTracker-Green-labeled CLL cells (green). White arrows indicate examples for phagosomes containing dead CLL cells. Red arrow indicates dead CLL cell in the process of being phagocytosed by NKTert. See also Video S2 for 3D view. See also Figures S6, S7, Videos S1, and S2.

Figure 4

Further characterization of phagocytic potential of bone marrow stromal cell lines (A) Flow cytometry of HS-5 and NKTert cells stained with antibodies against CD44, CD271, CD73, podoplanin (PDPN), CD26, CD90, CD105, CD31, and CD146. (B) Relative abundances of proteins in the lysosomal pathway for HS-5 and NKTert stromal cells, assessed by proteomics in four replicates. (C) Confocal microscopy of CellTracker Blue stained NKTert cocultured with 9- to 13-μM glass spheres for 16 h and stained with lysosomal dye NIR. See also Figures S8, S9, and Data S1.