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1School of Biomedical Engineering, Science and Health Systems, Drexel University
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Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.
Keywords: Biomedical Engineering, Issue 65, Bioengineering, Biophysics, Cancer Biology, Cancer, interstitial fluid flow, invasion, mechanobiology, migration, three-dimensional cell culture, tumor microenvironment
Tchafa, A. M., Shah, A. D., Wang, S., Duong, M. T., Shieh, A. C. Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion. J. Vis. Exp. (65), e4159, doi:10.3791/4159 (2012).
The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 μm s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.
1. Assay Set-up
Example gel recipe
|Components||Stock Concentration||Final Concentration||Add volume|
|10X PBS||10X||1X||0.090 ml|
|Sterile water||0.346 ml|
|1N NaOH||0.008 ml|
|Matrigel||9.90 mg/ml||1 mg/ml||0.101 ml|
|Rat tail type I collagen||3.66 mg/ml||1.3 mg/ml||0.355 ml|
2. Cell Staining and Counting
3. Data Analysis
4. Representative Results
To measure tumor cell invasion under interstitial flow, we performed our 3-D flow invasion assay using MDA-MB-435S metastatic melanoma cells. These cells have previously been shown to invade in response to interstitial fluid flow 13-14. The cells were embedded in a matrix composed of 1.3 mg/ml rat tail tendon collagen type I and 1 mg/ml Matrigel basement membrane matrix at a final cell concentration of 5 x 105 cells/ml. Two different conditions were compared: (1) average interstitial flow = 0.1 μm s-1 and (2) static condition = no measurable flow rate (Figure 1).
After 24 hr, the cells that invaded through the pores of the membrane were stained with DAPI to facilitate cell counting Figure 2 shows a representative image of the invaded cells. Under brightfield, only the pores of the membrane are visible. Using fluorescence, the DAPI-stained nuclei were used for cell counting and the phalloidin stained F-actin structures were used to visualize the cell body (optional). Using a Student's t-test assuming equal variances, we showed that interstitial flow significantly increases MDA-MB-435S cell invasion by 2.3-fold over static conditions (p = 0.003) (Figure 3). This corroborates similar findings (but with different matrix conditions and therefore flow velocities) using this cell line 13-14.
Figure 1. Schematic of the 3-D interstitial fluid flow invasion assay. First prepare gel solution using appropriate concentrations and volumes. Then add cells to gel solution and transfer to cell culture inserts. Finally add appropriate volume of media to each condition and incubate. Interstitial fluid flow is driven by a fluid pressure head.
Figure 2. Transmigrated MDA-MB-435S cells on membrane. Invaded cells were fixed after our interstitial fluid flow invasion assay and stained with DAPI and Alexa Fluor 488-conjugated phalloidin to facilitate counting of invaded cells; A) Picture of the membrane under bright field; B) DAPI stained nuclei (in blue); C) Alexa Fluor 488-phalloidin stained F-actin (in green). Scale bar represents 50 μm.
Figure 3. Increased invasion of MDA-MB-435S cells under flow. Cell invasion after 24 hr significantly enhanced by interstitial flow (p = 0.003). The results are normalized to the average static condition and values represent mean ± SEM of 6 cell culture inserts.
Here we have described a methodology for quantifying the effect of interstitial flow on tumor cell invasion, using cells embedded in a 3-D matrix within a cell culture insert. This and similar methods have been used to study the effect of interstitial flow on a variety of cell types 13-15, 17. Our approach partially mimics the matrix microenvironment of the tumor by using type I collagen and Matrigel which contain proteins found in the basement membrane of epithelial tissue and the surrounding stroma 21-22. This system is relatively easy to set-up, straightforward, and more cost effective than most microfluidic devices that are used to study interstitial fluid flow in vitro 23-24. It does not require pumps or specialized equipments and allows for multiple conditions to be tested simultaneously. Furthermore, the measurements are comparable to those of existing Boyden chamber assays commonly used to test invasion and migration of cancer cells.
By changing the cell type and the composition of the matrix, different biological systems can be modeled, such as breast cancer 13, blood vessels 15, and dendritic cell trafficking 17. Altering the matrix properties and composition and modulating the pressure head will also change the interstitial fluid flow velocity, thus allowing for flexibility in the assay parameters dependent on the biological system of interest. This system can also be used in co-culture assays.14, 17
In addition to measuring invasion, the interstitial fluid flow assay described here could be expanded to measure changes in other cell behaviors such as protein expression, cell proliferation and differences in cell signaling events. The cells can easily be isolated directly from the gel for RNA, DNA, and protein extraction and used for subsequent molecular biology assays, such as PCR and western blot This is a highly flexible assay that can be easily set up and adapted to investigate the effects of interstitial flow on a range of cellular processes in a number of cell systems.
One of the main drawbacks of this assay is the fact that flow velocities are very dependent of matrix concentration. Flow velocity increases as Matrigel concentration decreases. This means that a matrix consisting solely of collagen, which can be used in the study of stroma associated cells, will have higher flow velocities (> 1 μm s-1) than one consisting mainly of Matrigel (< 0.05 μm s-1) for a given pressure differential. If a specific flow velocity is required, one can first perform an initial test to identify the matrix composition that provides the desired flow velocity. The thickness of the gel can also be varied to adjust the flow velocity. The assay also does not allow for live cell imaging, where the behavior of individual cells and changes in their speed and directionality can be measured. Instead, this assay provides an endpoint measurement of changes in invasion across a population of cells.
No conflicts of interest declared.
|Collagen (Rat Tail)||BD Biosciences||354236||Keep sterile|
|Millicell cell culture insert||EMD Millipore||PI8P01250||8 μm pore diameter, polycarbonate membrane|
|Matrigel||BD Biosciences||354234||Keep sterile|
|PBS||Sigma-Aldrich||100M-8202||10x for preparing gel solution, 1x for washing steps|
|Sodium Hydroxide, 1.0N Solution||Sigma-Aldrich||S2770||Keep sterile|
|DMEM 1X||Cellgro||10-013-CV||Keep sterile|
|Fetal Bovine Serum||Atlanta Biologicals||511150||Keep sterile|
|Penicillin Streptomycin||Cellgro||30002CI||Keep sterile|
|Triton X-100||Sigma-Aldrich||X100-500ml||0.5% in PBS|
|Paraformaldehyde||Fisher Scientific||04042-500||4% in PBS|
|Deionized Water||Keep sterile|
|4',6-diaminido-2-phenylindole (DAPI)||MP Biomedicals||0215757401||1mg/ml stock solution|
|Mounting Solution||Thermo Fisher Scientific, Inc.||TA-030-FM|
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