Sartorius eBook - 42

THE FUTURE OF DRUG DISCOVERY: LIVE 3D CELL ANALYSIS

BT-474-CyG

MCF7-NR

+ PBMCs

+ Herceptin (1 μg mL)

+ Herceptin (1 μg mL)

SKOV3-NR

Figure 7: Impact of Herceptin-induced PBMCs on multi-spheroid proliferation. Tumor cells either stably expressing nuclear restricted
RFP (SKOV3-NR, MCF7-NR) or cytoplasmically restricted GFP (BT-474-CyG) were seeded in flat bottom 96-well plates (1,000 cells/well
on a bed of Matrigel). Multi-spheroids were allowed to form (3 d) prior to addition of freshly isolated PBMCs (E:T, 5:1) and Herceptin.
Incucyte® S3 brightfield and fluorescence images (7 d; SKOV3-NR, MCF-NR or 10 d; BT-474-CyG) compare the effect of Herceptin on
spheroid proliferation in absence (top panel) and presence (bottom panel) of PBMCs (Brightfield outline mask shown in yellow). Note
the loss of fluorescence intensity in HER2-postive (SKOV3 and BT474) multi-spheroids in the presence of PBMCs.

Tumor multi-spheroids, either stably expressing
nuclear restricted RFP (NR) or cytoplasmic

a marked loss of MS viability.
Herceptin induced a concentration-

GFP (CyG), were co-cultured with freshly

dependent, cytotoxic effect in HER2-positive

isolated peripheral blood mononuclear cells

multi-spheroids but not HER2-negative multi-

(PBMCs) (E:T, 5:1) and treated with Herceptin.

spheroids. BT-474 multi-spheroids appeared

Multi-spheroid proliferation and immune cell-

more sensitive to Herceptin cytotoxicity, with the

mediated cytotoxicity were quantified kinetically

highest test concentration of Herceptin (1 μg/

using fluorescence intensity within brightfield

mL) producing approximately 80% inhibition

boundary, which does not require masking of

compared with approximately 50% in SKOV3

fluorescent tumor multi-spheroid.

multi-spheroids (Figure 8, concentration response

PBMCs alone (data not shown) or Herceptin

curve). Activation of the PBMCs T cell population

alone had little or no effect on multi-spheroid

with an AntiCD3 antibody and IL-2 cocktail (10

size or viability (fluorescence intensity, Figure 7).

ng/mL of each) resulted in maximal killing across

However, Herceptin activated PBMCs, resulting in

all cell types (Figure 8, time-courses).

42 |

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