JoVE Logo
Faculty Resource Center

Sign In





Representative Results





Cancer Research

Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle

Published: April 27th, 2021



1Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, 2Holden Comprehensive Cancer Center, University of Iowa, 3MD program, Carver College of Medicine, University of Iowa, 4Department of Pathology, Carver College of Medicine, University of Iowa, 5Department of Urology, Carver College of Medicine, University of Iowa, 6Department of Radiation Oncology, Carver College of Medicine, University of Iowa

Here we demonstrate a method to apply fluid shear stress to cancer cells in suspension to model the effects of hemodynamic stress on circulating tumor cells.

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed to mechanical stress due to hemodynamic flow. One of these stresses that circulating tumor cells (CTCs) experience is fluid shear stress (FSS). While cancer cells may experience low levels of FSS within the tumor due to interstitial flow, CTCs are exposed, without extracellular matrix attachment, to much greater levels of FSS. Physiologically, FSS ranges over 3-4 orders of magnitude, with low levels present in lymphatics (<1 dyne/cm2) and the highest levels present briefly as cells pass through the heart and around heart valves (>500 dynes/cm2). There are a few in vitro models designed to model different ranges of physiological shear stress over various time frames. This paper describes a model to investigate the consequences of brief (millisecond) pulses of high-level FSS on cancer cell biology using a simple syringe and needle system.

Metastasis, or the spread of cancer beyond the initial tumor site, is a major factor underlying cancer mortality1. During metastasis, cancer cells utilize the circulatory system as a highway to disseminate to distant sites throughout the body2,3. While en route to these sites, circulating tumor cells (CTCs) exist within a dynamic fluid microenvironment unlike that of their original primary tumor3,4,5. It has been proposed that this fluid microenvironment is one of many barriers to meta....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Cell preparation

  1. Release cells from tissue culture dish when 70-90% confluent by following the recommended guidelines for the cell line in use.
    1. For example, aspirate the growth medium for PC-3 cells, and wash the 10 cm dish of cells with 5 mL of calcium- and magnesium-free phosphate-buffered saline (PBS).
    2. Aspirate the PBS before adding 1 mL of 0.25% trypsin using manufacturer's protocol.
    3. After observing the detachment of the cells under an inverted microscope, add 5 mL.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Elevated resistance to FSS-induced mechanical destruction has been previously shown to be a conserved phenotype across multiple cancer cell lines and cancer cells freshly isolated from tumors relative to non-transformed epithelial cell comparators15,24. Here, additional cancer cell lines from a variety of tissue origins (Table 2) were tested to demonstrate that the majority of these cells display viability ≥ 20% after 10 pulses of FSS at 25.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This paper demonstrates the application of FSS to cancer cells in suspension using a syringe and needle. Using this model, cancer cells have been shown to be more resistant to brief pulses of high-level FSS relative to non-transformed epithelial cells15,22,24. Furthermore, exposure to FSS using this model results in a rapid increase in cell stiffness, activation of RhoA, and increased cortical F-actin and myosin II-based contrac.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Development of the model demonstrated here was supported by DOD grant W81XWH-12-1-0163, NIH grants R21 CA179981 and R21 CA196202, and the Sato Metastasis Research Fund.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
0.25% Trypsin Gibco 25200-056
14 mL round bottom tubes Falcon - Corning 352059
30 G 1/2" Needle BD 305106
5 mL syringe BD 309646
96-well black bottom plate Costar - Corning 3915
Bioluminescence detector AMI AMI HTX
BSA, Fraction V Sigma 10735086001
Cell Titer Blue Promega G8081
crystal violet Sigma C0775
D-luciferin GoldBio D-LUCK
DMEM Gibco 11965-092
FBS Atlanta Biologicals S11150
PBS Gibco 10010023
Plate Reader BioTek Synergy HT
Sodium Azide (NaN3) Sigma S2002
Syringe Pump Harvard Apparatus 70-3005

  1. Dillekås, H., Rogers, M. S., Straume, O. Are 90% of deaths from cancer caused by metastases. Cancer medicine. 8 (12), 5574-5576 (2019).
  2. Hanahan, D., Weinberg, R. A. Hallmarks of cancer: the next generation. Cell. 144 (5), 646-674 (2011).
  3. Strilic, B., Offermanns, S. Intravascular survival and extravasation of tumor cells. Cancer Cell. 32 (3), 282-293 (2017).
  4. Labelle, M., Hynes, R. O. The initial hours of metastasis: the importance of cooperative host-tumor cell interactions during hematogenous dissemination. Cancer Discovery. 2 (12), 1091-1099 (2012).
  5. Krog, B. L., Henry, M. D. Biomechanics of the circulating tumor cell microenvironment. Advances in Experimental Medicine and Biology. 1092, 209-233 (2018).
  6. Weiss, L. Metastatic inefficiency. Advances in Cancer Research. 54, 159-211 (1990).
  7. Zeidman, I., Mc, C. M., Coman, D. R. Factors affecting the number of tumor metastases; experiments with a transplantable mouse tumor. Cancer Research. 10 (6), 357-359 (1950).
  8. Fidler, I. J. Metastasis: quantitative analysis of distribution and fate of tumor embolilabeled with 125 I-5-iodo-2'-deoxyuridine. Journal of the National Cancer Institute. 45 (4), 773-782 (1970).
  9. Cameron, M. D., et al. Temporal progression of metastasis in lung: cell survival, dormancy, and location dependence of metastatic inefficiency. Cancer Research. 60 (9), 2541-2546 (2000).
  10. Luzzi, K. J., et al. Multistep nature of metastatic inefficiency: dormancy of solitary cells after successful extravasation and limited survival of early micrometastases. American Journal of Pathology. 153 (3), 865-873 (1998).
  11. Kienast, Y., et al. Real-time imaging reveals the single steps of brain metastasis formation. Nature Medicine. 16 (1), 116-122 (2010).
  12. Takagi, H., et al. Analysis of the circulating tumor cell capture ability of a slit filter-based method in comparison to a selection-free method in multiple cancer types. International journal of molecular sciences. 21 (23), 9031 (2020).
  13. Scott, J., Kuhn, P., Anderson, A. R. Unifying metastasis--integrating intravasation, circulation and end-organ colonization. Nature Reviews Cancer. 12 (7), 445-446 (2012).
  14. Wirtz, D., Konstantopoulos, K., Searson, P. C. The physics of cancer: the role of physical interactions and mechanical forces in metastasis. Nature Reviews Cancer. 11 (7), 512-522 (2011).
  15. Barnes, J. M., Nauseef, J. T., Henry, M. D. Resistance to fluid shear stress is a conserved biophysical property of malignant cells. PLoS One. 7 (12), 50973 (2012).
  16. Malek, A. M., Alper, S. L., Izumo, S. Hemodynamic shear stress and its role in atherosclerosis. JAMA. 282 (21), 2035-2042 (1999).
  17. Brass, L. F., Diamond, S. L. Transport physics and biorheology in the setting of hemostasis and thrombosis. Journal of Thrombosis and Haemostasis. 14 (5), 906-917 (2016).
  18. Stein, P. D., Sabbah, H. N. Turbulent blood flow in the ascending aorta of humans with normal and diseased aortic valves. Circulation Research. 39 (1), 58-65 (1976).
  19. Strony, J., Beaudoin, A., Brands, D., Adelman, B. Analysis of shear stress and hemodynamic factors in a model of coronary artery stenosis and thrombosis. The American Journal of Physiology. 265 (5), 1787-1796 (1993).
  20. Chalmers, J. J. Mixing, aeration and cell damage, 30+ years later: what we learned, how it affected the cell culture industry and what we would like to know more about. Current Opinion in Chemical Engineering. 10, 94-102 (2015).
  21. Vennin, C., et al. Trsient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis. Science Translational Medicine. 9 (384), 126 (2017).
  22. Mitchell, M. J., et al. Lamin A/C deficiency reduces circulating tumor cell resistance to fluid shear stress. American Journal of Physiology: Cell Physiology. 309 (11), 736-746 (2015).
  23. Ortiz-Otero, N., et al. Cancer associated fibroblasts confer shear resistance to circulating tumor cells during prostate cancer metastatic progression. Oncotarget. 11 (12), 1037-1050 (2020).
  24. Moose, D. L., et al. Cancer cells resist mechanical destruction in circulation via RhoA/actomyosin-dependent mechano-adaptation. Cell Reports. 30 (11), 3864-3874 (2020).
  25. Miller, B. E., Miller, F. R., Wilburn, D. J., Heppner, G. H. Analysis of tumour cell composition in tumours composed of paired mixtures of mammary tumour cell lines. British Journal of Cancer. 56 (5), 561-569 (1987).
  26. Aslakson, C. J., Miller, F. R. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Research. 52 (6), 1399 (1992).
  27. Chivukula, V. K., Krog, B. L., Nauseef, J. T., Henry, M. D., Vigmostad, S. C. Alterations in cancer cell mechanical properties after fluid shear stress exposure: a micropipette aspiration study. Cell Health Cytoskeleton. 7, 25-35 (2015).
  28. Gensbittel, V., et al. Mechanical adaptability of tumor cells in metastasis. Developmental Cell. 56 (2), 164-179 (2021).
  29. O'Leary, B. R., et al. Pharmacological ascorbate inhibits pancreatic cancer metastases via a peroxide-mediated mechanism. Scientific Reports. 10 (1), 17649 (2020).
  30. Williams, A. R., Hughes, D. E., Nyborg, W. L. Hemolysis near a transversely oscillating wire. Science. 169 (3948), 871-873 (1970).
  31. Rooney, J. A. Hemolysis near an ultrasonically pulsating gas bubble. Science. 169 (3948), 869-871 (1970).
  32. Connolly, S., McGourty, K., Newport, D. The in vitro inertial positions and viability of cells in suspension under different in vivo flow conditions. Scientific Reports. 10 (1), 1711 (2020).
  33. Brooks, D. E. The biorheology of tumor cells. Biorheology. 21 (1-2), 85-91 (1984).
  34. Triantafillu, U. L., Park, S., Klaassen, N. L., Raddatz, A. D., Kim, Y. Fluid shear stress induces cancer stem cell-like phenotype in MCF7 breast cancer cell line without inducing epithelial to mesenchymal transition. Internation Journal of Oncology. 50 (3), 993-1001 (2017).
  35. Fan, R., et al. Circulatory shear flow alters the viability and proliferation of circulating colon cancer cells. Scientific Reports. 6, 27073 (2016).
  36. Fu, A., et al. High expression of MnSOD promotes survival of circulating breast cancer cells and increases their resistance to doxorubicin. Oncotarget. 7 (31), 50239-50257 (2016).
  37. Li, S., et al. Shear stress promotes anoikis resistance of cancer cells via caveolin-1-dependent extrinsic and intrinsic apoptotic pathways. Journal of Cellular Physiology. 234 (4), 3730-3743 (2019).
  38. Xin, Y., et al. Mechanics and actomyosin-dependent survival/chemoresistance of suspended tumor cells in shear flow. Biophysical Journal. 116 (10), 1803-1814 (2019).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved