Acoustic tweezers for studying intracellular calcium signaling in SKBR-3 human breast cancer cells

Abstract

Extracellular matrix proteins such as fibronectin (FNT) play crucial roles in cell proliferation, adhesion, and migration. For better understanding of these associated cellular activities, various microscopic manipulation tools have been used to study their intracellular signaling pathways. Recently, it has appeared that acoustic tweezers may possess similar capabilities in the study. Therefore, we here demonstrate that our newly developed acoustic tweezers with a high-frequency lithium niobate ultrasonic transducer have potentials to study intracellular calcium signaling by FNT-binding to human breast cancer cells (SKBR-3). It is found that intracellular calcium elevations in SKBR-3 cells, initially occurring on the microbead-contacted spot and then eventually spreading over the entire cell, are elicited by attaching an acoustically trapped FNT-coated microbead. Interestingly, they are suppressed by either extracellular calcium elimination or phospholipase C (PLC) inhibition. Hence, this suggests that our acoustic tweezers may serve as an alternative tool in the study of intracellular signaling by FNT-binding activities.

Keywords: Acoustic tweezers; Fibronectin; High-frequency ultrasound transducer; Intracellular calcium elevation; SKBR-3 breast cancer cells.

Fig. 1

Illustration of the acoustic trapping mechanism.

Fig. 2

(a) System configuration for microbead trapping experiment. (b) Pulse-echo characteristics of a 47 MHz LiNbO₃ transducer. (Upper: a photographic image of transducer, lower-left: pulse-echo signals, and lower-right: frequency characteristics of the pulse-echo signals.) The red line indicates −6 dB bandwidth of 74% (c) Illustration of the experimental procedure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Calcium elevation in SKBR-3 cells due to FNT-coated microbeads: (a) comparison of calcium elevation between FNT-coated- (upper) and non-FNT-coated microbead attachment (lower). Each arrow indicates a trapped microbead location (b) Temporal profiles of calcium elevation arising from FNT-coated- (curved line) and non-FNT-coated microbead binding (flat line). (c) Quantitative analysis of calcium elevation due to FNT-coated- (right) and non-FNT-coated microbeads (left). (n = 9, p-value = ~0.009 < 0.05).

Fig. 4

Calcium elevations in SKBR-3 cells induced by the 5 μm (n = 10) and 15 μm (n = 12) FNT-coated microbead binding (p-value = ~0.00001 < 0.05).

Fig. 5

Calcium elevation in SKBR-3 cells by free FNT binding (a) Calcium fluorescence images of SKBR-3 cells at the indicated time-points (50 μg/ml: upper and 100 μg/ml: lower). (b) Temporal calcium changes in SKBR-3 cells due to free FNT binding at 50 (blue-dotted line) and 100 μg/ml (red-solid line) (representative ones). The black-solid arrow indicates the time point when free FNTs are added into the cell. (c) Quantitative analysis of calcium elevations due to free FNT binding at 50 (left) and 100 μg/ml (right) (n = 13, p-value = ~0.045 < 0.05). The scar bar indicates ~20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Calcium propagation over a SKBR-3 cell when the FNT microbead is attached to the cell: (a) Sequential fluorescence images obtained at the indicated time points after the microbead attachment (b) Temporal fluorescence changes at the selected areas [1′: contact spot, 2′: middle area, and 3′: far area) of the cell. The scar bar indicates ~10 μm.

Fig. 7

Quantitative analysis of calcium elevation in SKBR-3 cells under calcium free HBSS (No calcium) and treated with PLC inhibitors (U73122) (n = 10).

Fig. 8

Quantitative analysis of SKBR-3 cell viability influenced by an acoustically trapped microbead: (a) Plot of mean Calcien florescence intensity as a function of time after FNT-microbead binding (n = 12). The arrow indicates the moment when a trapped FNT-coated microbead is attached to a cell. (b) Cell viability in 24 h after the attachment of a 5 μm (left) and a 15 μm (right) FNT-microbead to cells. The arrows indicate the cells undergoing the FNT-coated microbead binding activity.