Download effects of hemodynamic shear stress on circulating tumor cells

EFFECTS OF HEMODYNAMIC SHEAR STRESS ON CIRCULATING
TUMOR CELLS
Xiaofeng Liu1, Afu Fu1, Yun Han How1, and Kathy Qian Luo1*
1
Nanyang Technological University, SINGAPORE
ABSTRACT
Metastasis is an inefficient process, but a consensus has not been reached as to which steps of
metastasis are potentially rate-limiting. In this study, a microfluidic system was developed to mimic the
physiological flow profile in the blood vessel to study how hemodynamic shear stress affects circulating
tumor cells (CTCs). Breast cancer MDA-MB-231 cells transfected with a fluorescence resonance energy
transfer (FRET)-based biosensor (231-C3) were circulated and recovered repeatedly to obtain series of
fluid force-selected cell lines. These cells were shown to acquire enhanced survival ability. They also
exhibit elongated cell morphology and greater drug resistance compared to parental 231-C3.
KEYWORDS: Metastasis, Circulating Tumor cells, Hemodynamic Shear Stress, Cell Survival
INTRODUCTION
Metastases, rather than primary tumors, are responsible for more than 90% of all cancer-related
deaths [1]. During metastasis, cancer cells leave the primary tumor, enter the circulatory system and then
encounter a hemodynamic environment. In order for the cancer cells to metastasize, CTCs must survive
the circulatory system. One of the forces that CTCs need to withstand is pulsatile shear stress, caused by
the blood flow and by collisions with the walls of the blood vessels and with other cells in the blood.
However, the effects of these hemodynamic shear stresses on CTCs are not fully understood [2].
In this project, we investigated how pulsatile shear stresses affect the properties of 231-C3 cells
during circulation in a microfluidic system (Figure 1A). An FRET-based biosensor C3 was transfected to
generate 231-C3 cells which allows a real-time detection of apoptosis as shown in Figure 1B [3,4]. We
selected 231-C3 cells by exposing them to pulsatile flow at 15 dyne/cm2 for 24 hours. The recovered cells
were designated as 231-S1 and the selection processes were repeated until the 6th generation of cells (231S6) was obtained. Recovery durations were compared between each cell line. Differences in cell length,
survival ability after circulation and resistance to doxorubicin were determined.
Figure 1: A) Schematic of investigations on effects of hemodynamic shear stress on circulating tumor
cells, and B) Principle of FRET biosensor in detecting caspase-activation mediated apoptosis.
978-0-9798064-7-6/µTAS 2014/$20©14CBMS-0001
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18th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
October 26-30, 2014, San Antonio, Texas, USA
RESULTS AND DISCUSSIONS
231-C3 cells were injected into the microfluidic system and were exposed to pulsatile flow at 15
dyne/cm2 for 24 hours, after which the cells were collected and incubated at normal cell culture condition
for recovery. After 24 hours of circulation, survived cells became highly stressed, but the cells underwent
more times of selections required a shorter duration to recover and proliferate as shown in Table 1, which
indicates that the property of flow-resistant could be maintained and inherited along with the selection
process.
Table 1. Recovery period of fluid force-selected cells.
Cell lines
231-S1
231-S2
231-S3
231-S4
231-S5
231-S6
Duration for cells to recover
after circulation (days)
16-18
10-12
7-9
6-8
5-8
3-6
We have also measured the average cell length in each batch of selected cell lines. 231-S1 cells have an
average length of 68 µm, which is similar to the parental 231-C3 cells with an average length of 64 µm.
However, after the second time of circulation, the cells elongated up to more than 70 µm in 231-S2, and
eventually reached to 87 µm in 231-S6 (Figure 2). These results indicate that hemodynamic shear stress can
contribute to regulation of cytoskeleton, which may provide evidence for how CTCs become more
mesenchymal-like after exposure to circulation.
Figure 2: Average length of 231-C3 and fluid force-selected cells. (n = 50–100 cells) (* p < 0.05)
With the help of FRET-based caspase-3 biosensor, we can directly detect how many cells could die due
to the circulation. We used the same shear force previously used to select the flow-resistant cells (pulsatile
flow at 15 dyne/cm2) to investigate the percentage of viable cells after circulation in 231-C3 and 231-S6.
Parental 231-C3 cells achieved about 50% survival rate after 12 hours and 24 hours circulation. However,
231-S6 had a higher survival rate up to around 60% after circulation (Figure 3A). These results indicate that
survived circulating tumor cells can achieve an enhanced ability to resist the hemodynamic shear force.
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Figure 3: A) Average length of 231-C3 and fluid force-selected cells, and B) Viability of 231-C3 and 231S6 cells after circulation. (* p < 0.05, ** p < 0.01)
Besides the resistance to physical forces, we further investigated whether the flow-resistant cells can
also gain resistant to chemotoxicity. An MTT assay was conducted using doxorubicin to treat various 231
sensor cell lines for 24 hours at the concentrations of 2.5 µM, 5 µM, and 10 µM. The results show that
231-S4 and 231-S5 cells have a significantly higher chemoresistance to doxorubicin at all concentrations
(Figure 3B), which may indicate that the survival ability of CTCs is closely related to their antioxidative
ability.
CONCLUSION
In this study, a pulsatile microfluidic system, combined with a FRET-based caspase-3 biosensor, was
developed to mimic the physiological flow profile in blood vessels. The FRET biosensor provided a realtime detection of CTCs apoptosis. We selected series of fluid force-resistant cells and the cells survived
from more rounds of circulation (i.e. S5 and S6) required shorter duration to recover and proliferate, and
they appeared with more elongated cell morphology. These cells also displayed enhanced survival ability
to fluid force and higher resistance to anti-cancer drug, doxorubicin. These results suggest that
hemodynamic shear stresses may directly influence the properties of CTCs, which could be maintained
and inherited through cell passages.
ACKNOWLEDGEMENTS
This work was supported in part by the Singapore National Research Foundation NRF-CRP8-201105 (Grant No. M4092018.0S4) and the Environmental and Water Industry Development Council of
Singapore (Grant No. 1102-IRIS-05-02).
REFERENCES
[1] Hanahan D, Weinberg RA, Hallmarks of cancer: the next generation. Cell. 2011;144(5):646-674.
[2] Wirtz D, Konstantopoulos K, Searson PC, The physics of cancer: the role of physical interactions
and mechanical forces in metastasis. Nature Reviews Cancer. 2011;11(7):512-22.
[3] Luo KQ, Yu VC, Pu Y, Chang DC, Application of the Fluorescence Resonance Energy Transfer
Method for Studying the Dynamics of Caspase-3 Activation during UV-Induced Apoptosis in Living
HeLa Cells. Biochemical and Biophysical Research Communications. 2001;283(5):1054-60.
[4] Liu XF, Yu JQ, Dalan R, Liu AQ, Luo KQ, Biological factors in plasma from diabetes mellitus
patients enhance hyperglycaemia and pulsatile shear stress-induced endothelial cell apoptosis.
Integrative Biology. 2014;6(5):511-522.
CONTACT
* Associate Professor Kathy Qian Luo; +65-6790 4257; [email protected]
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