Resistance to fluid shear stress is a conserved biophysical property of malignant cells

Abstract

During metastasis, cancer cells enter the circulation in order to gain access to distant tissues, but how this fluid microenvironment influences cancer cell biology is poorly understood. A longstanding view is that circulating cancer cells derived from solid tissues may be susceptible to damage from hemodynamic shear forces, contributing to metastatic inefficiency. Here we report that compared to non-transformed epithelial cells, transformed cells are remarkably resistant to fluid shear stress (FSS) in a microfluidic protocol, exhibiting a biphasic decrease in viability when subjected to a series of millisecond pulses of high FSS. We show that magnitude of FSS resistance is influenced by several oncogenes, is an adaptive and transient response triggered by plasma membrane damage and requires extracellular calcium and actin cytoskeletal dynamics. This novel property of malignant cancer cells may facilitate hematogenous metastasis and indicates, contrary to expectations, that cancer cells are quite resistant to destruction by hemodynamic shear forces.

Figure 1. Fluid shear stress induces cell death in a magnitude-dependent manner.

A. Scale illustration of a PC-3 cell subjected to FSS in this model. Note the gradient of increasing stress from the axis of flow to the wall of the needle. Suspensions of PC-3 cells were subjected to FSS at increasing flow rates and monitored for changes in viability. Survival is represented as percent viability of non-FSS treated cells which are held in suspension for the duration of the assay. B. Viability after ten passages at the indicated flow rate. (**, p<0.01; ***, p<0.001 vs. 0 control. One-way ANOVA, Bonferroni’s multiple comparison test; for each flow rate, n = 5 using syringe pump.) C. Survival over repeated passages at 20 and 250 µL/s. (**p<0.01, ***p<0.001 vs. 20 µL/s. Repeated measures ANOVA, Bonferroni’s multiple comparison test; for each flow rate n = 10 using syringe pump). The time taken to perform ten passages at each flow rate is indicated.

Figure 2. Loss of cell viability is due to exposure to fluid shear stress.

A. The viability of PC-3 cells exposed to 10 passages of the FSS assay at 250 µL/s was not significantly different when assessed by three independent techniques: BLI, WST-1 assay, and clonogenic plating (p>0.05 for each pair, Bonferroni’s mulitiple comparison test, minimum of n = 3 for each method). B. Variation in FSS exposure but not cell suspension/preparation conditions significantly affected endpoint viability of PC-3 cells. Conditions tested include altered suspension cell concentration (p>0.05), degree of confluency prior to collection (p>0.05), suspension media (p>0.05), collection technique (p>0.05), and needle length. Only under this final condition, where the time of exposure to FSS was effectively doubled, was a significant difference in endpoint survival noted. (*p<0.01 vs standard 0.5″ needle, Bonferroni’s multiple comparison test. All experiments n = 4 using pump method.) C. FSS survival of PC-3 cells is not affected by changes in pH. PC-3 cells suspended in DMEM/F12, 10% FBS in the presence or absence of 20 mM HEPES (avg. pH at room temperature: 7.3 vs. 7.7, respectively) (p>0.05, Bonferroni’s multiple comparison tests). D. FSS survival of primary cells was not affected by changes in temperature. (p>0.05, Bonferroni’s multiple comparison test, HMEC, n = 2 and PrEC, n = 5 experiments using pump method). E. Response to FSS does not depend on the time cells are held in suspension. Survival is represented as percent viability of non-FSS-treated cells held in suspension for the duration of the assay (p>0.05, one-way ANOVA). F. Loss of viability of PC-3 and primary cells due to detachment-induced cell death during the protocol were not significantly different for up to one hour. Loss of viability due to detachment over the first 30 minutes is insignificant (p>0.05 one-way ANOVA, Bonferroni’s multiple comparison test, n = 5 for each cell line). All error bars = ±SEM. Details on preparations for controls can be found in Methods under Control conditions.

Figure 3. Transformed cells of various histological origins exhibit resistance to fluid shear stress.

A. A panel of transformed and normal epithelial and blood cells was compared for survival after 10 passages of FSS at 250 µL/s. ***, p<0.001 vs. all cancer cell lines; #, p<0.001 vs. RWPE-1; ††, p<0.001 vs. all non-blood cells (one way ANOVA, Bonferroni’s multiple comparison tests n = 3 for blood cells by syringe pump method, n = 6 for all other lines using manual method). B. The viability of all cells in A at every second passage. (#, p<.001 vs. all other cell types, repeated measures ANOVA, Bonferroni’s mutliple comparison test). C. The rate of cell death as a function of repeated exposure to FSS decreases as the number of exposures increases ***, p<0.001 vs. passages 1 and 2 of cancer cells; # p<0.05 vs. passages 1 to 2 of primary cells (one way ANOVA, Bonferroni’s multiple comparison test tests). D. Viability at every other passage of PrEC and HMEC suspensions subjected to ten passages of FSS at 20 µL/s.

Figure 4. Transforming oncogenes promote fluid shear stress resistance.

A. The effect of FSS (at 250 µL/s) was compared between wild type primary human prostate epithelial cells (PrEC), immortalized PrEC (LH), and Myc/PI3K (LHMK) or Ras (LHSR) transformed PrEC (***, p<0.0001 vs. WT). B. R545 melanoma cells (derived from Tyr/Tet-Ras INK4a^−/− mice) express H-Ras^G12V in a doxycycline-dependent manner. These cells were cultured for two passages in the presence or absence of 2 µg/mL doxycycline before shearing at 250 µL/s. n = 4 for all cell lines and conditions using syringe pump (***, p<0.0001). C. PrEC LHSR cells were treated with 10 µM U0126 for one hour prior to FSS exposure. Drug treatment lead to a reduction in FSS resistance (***, p<0.0001). Statistical analysis is by repeated measures ANOVA, Bonferroni’s multiple comparison test.

Figure 5. Fluid shear stress resistance is induced by membrane damage.

A. Flow cytometry analysis was performed on cells exposed to PI throughout the FSS protocol or introduced prior to passage 10 (P.10 grey box). Cell debris can be seen in the “D” gate (long, narrow polygon on the left) whereas viable cells are represented by the “V” gate, which is established on forward- and side-scatter parameters that typically defines a viable cell population. For these studies, an equal number of events in the V-gate were counted at each passage, even though overall viability of the population was decreasing as documented in Fig. 1. Green data points represent viable, PI⁻ cells, whereas fuchsia data points represent viable, PI⁺ cells. B. Histogram of the number of PI⁺ viable cells in the conditions displayed in panel A. The grey peak represents FSS-naive cells in suspension in the presence of PI (P.0), which is defined as PI-negative by gating parameters. P.1 (green) and P.10 (blue) in the constant presence of PI. The red peak represents cells passaged ten times, but with PI added prior to P.10. C. Graphical representation of accumulation of PI in viable cells over repeated passages (***, p<0.001). D. When PI was added prior to passage six, eight, or ten, less of the viable population of cells accumulated dye. (*p<0.05 vs. P.1 in the constant presence of PI; for each condition, n = 8 using syringe pump). Statistical analysis for C and D is one-way ANOVA, Bonferroni’s multiple comparisontest. All error bars±SEM.

Figure 6. Fluid shear stress resistance requires extracellular calcium and actin dynamics.

A. PC-3 cells were suspended in complete medium, calcium-free PBS, or PBS plus calcium chloride (1.16 mM final concentration) and subjected to shear stress at 250 µL/s. B. PC-3 cells were suspended in complete medium and EGTA was added to a final concentration of 10 mM prior to FSS-treatment. For A and B, *p<0.05, **p<0.01, ***p<0.001 vs. complete media (for each condition, (A) n = 6 and (B) n = 4 using syringe pump). C. PC-3 and MDA.MB.231 cells were treated with 20 µM CCD for one hour before exposure to the FSS protocol. *p<0.05, **p<0.01, ***p<0.001 vs. corresponding DMSO control (for each condition, n = 4 using manual method). D. PC-3 cells were treated with 100 µM Y27632 for 20 hours before exposure to the FSS protocol. *p<0.05, **p<0.01, ***p<0.001 vs. complete media (for each condition, n = 2). Statistical analysis is by repeated measures ANOVA, Bonferroni’s multiple comparison test. All error bars ±SEM.