Phenotypic switch in blood: effects of pro-inflammatory cytokines on breast cancer cell aggregation and adhesion.

Hematogeneous metastasis can occur via a cascade of circulating tumor cell adhesion events to the endothelial lining of the vasculature, i.e. the metastatic cascade. Interestingly, the pro-inflammatory cytokines IL-6 and TNF-α, which play an important role in potentiating the inflammatory cascade, are significantly elevated in metastatic breast cancer (BCa) patients. Despite their high metastatic potential, human breast carcinoma cells MDA-MB-231 lack interactions with E-selectin functionalized surfaces under physiological shear stresses. We hypothesized that human plasma, 3-D tumor spheroid culture, and cytokine-supplemented culture media could induce a phenotypic switch that allows BCa cells to interact with E-selectin coated surfaces under physiological flow. Flow cytometry, immunofluorescence imaging, and flow-based cell adhesion assay were utilized to investigate the phenotypic changes of MDA-MB-231 cells with various treatments. Our results indicate that plasma, IL-6, and TNF-α promote breast cancer cell growth as aggregates and induce adhesive recruitment of BCa cells on E-selectin coated surfaces under flow. 3-D tumor spheroid culture exhibits the most significant increases in the interactions between BCa and E-selectin coated surfaces by upregulating CD44V4 and sLe(x) expression. Furthermore, we show that IL-6 and TNF-α concentrations in blood may regulate the recruitment of BCa cells to the inflamed endothelium. Finally, we propose a mechanism that could explain the invasiveness of 'triple-negative' breast cancer cell line MDA-MB-231 via a positive feedback loop of IL-6 secretion and maintenance. Taken together, our results suggest that therapeutic approaches targeting cytokine receptors and adhesion molecules on cancer cells may potentially reduce metastatic load and improve current cancer treatments

Plasma, IL-6, and TNF-α promote breast cancer cell growth as aggregates.

<p>Morphology and viability of MDA-MB-231 cells cultured in (a) regular medium, (b) IL-6 (5 ng/mL) spiked medium, (c) IL-6 (5 ng/mL) and 0.1 mmol Metformin added medium, (d) TNF-α (5 ng/mL) spiked medium, (e) IL-6 and TNF-α (5 ng/mL each) spiked medium, and (f) healthy donor plasma. A blue fluorescent cell-permeable nucleic acid dye (for live cell staining) and a red fluorescent cell-impermeable nucleic dye (for dead cell staining) were used to visualize the live and dead populations. (g) Cell proliferation results via MTT assay of control media, IL-6, TNF-α, and Metformin treated MDA-MB-231 cells.</p

A positive feedback loop: activation and maintenance of the adhesive phenotypic switch.

<p>Step A: Tumor cells encounter proinflammatory cytokines such as IL-6 and TNF-α secreted by tumor promoting immune cells in the microenvironment. Step B: Cytokine conditioned tumor cells form aggregates. Step C: Aggregates/spheroids of tumor cells release more IL-6 and TNF-α, turning on a phenotypic switch for more tumor cells nearby by upregulating E-selectin ligand and binding moiety expressions, and promote heterotypic interactions between tumor cells and the inflamed endothelium. Step D: Tumor cells ready to invade the endothelium.</p

IL-6 and TNF-α concentrations in blood can regulate the recruitment of BCa cells to the inflamed endothelium.

<p>(a) Calculated concentrations of IL-6 (left) and TNF-α (right) of healthy female donor plasma and tumor spheroid conditioned media on ELISA standard curves. IL-6 concentration in the tumor spheroid conditioned media was diluted with equal volume of culture media to remain in the detectable range (0–200 pg/mL) of the ELISA kit. (b) Average rolling velocities (left) and average numbers of MDA cells found interacting with E-selectin coated surfaces (right) from untreated conditions and culture media spiked with a range of IL-6 concentration including 1, 5, 10, and 25 ng/mL. For the cell number analysis, all conditions are significantly different (p<0.0001) except for the 5 and 10 ng/mL treatments. Bars represent mean ± SEM. Two-tailed unpaired student t-test was used.</p

Plasma triggers an adhesive phenotypic switch of breast cancer (BCa) cells on E-selectin coated surface under flow.

<p>(a) Left: Untreated MDA-MB-231 cells (2-D culture with regular medium) show no interactions with the E-selectin coated surfaces under flow. Right: Cells establish stable rolling on E-selectin coated surface after 48 h plasma treatment. (b) Flow cytometry results of CD44V4 (left) and sLe^x (right) expression on untreated and plasma treated MDA-MB-231 cell surfaces. Mean fluorescence intensity ratio of sample over isotype has been included. (c) Left: Average rolling velocity of untreated (control) and plasma treated MDA-MB-231 cells (n = 35 cells). Shear stress of 1 dyn/cm² was used. Right: Average numbers of cells interacting with the E-selectin coated surface per 5×10⁵ μm² of untreated (n = 30 frames) and plasma treated (n = 36 frames) MDA-MB-231 cells. Bars represent mean ± SEM. P<0.0001, two-tailed unpaired student t-test.</p

Pro-inflammatory cytokines IL-6 and TNF-α also induce adhesive recruitment of BCa cells and is blocked by the anti-inflammatory drug Metformin.

<p>(a) Average rolling velocity (left) and average number of MDA-MB-231 cells interacting with E-selectin coated surfaces (right) after treatments including 5 ng/mL of IL-6 (n = 71), 5 ng/mL TNF-α (n = 48), IL-6+ TNF-α (5 ng/mL each), 5 ng/mL of IL-6 with 0.1 mmol Metformin, 5 ng/mL of IL-6 with 1 μg/mL anti-IL-6R MAb, 5 ng/mL of IL-6 with neuraminidase treatment prior to rolling experiment. For conditions where MDA-MB-231 were treated with IL-6 with MAb, Metformin, or neuraminidase (after IL-6 treatment), there were insufficient numbers of cells found on the E-selectin coated surface for velocity analysis. Bars represent mean ± SEM. P<0.0001, two-tailed unpaired student t-test. Average number of MDA-MB-231 cells from control experiment was found to be significantly less than all other conditions (P<0.0001). (b) Flow cytometry measurements of CD44V4 (left) and sLe^x (right) expression on untreated, IL-6 (5 ng/mL), and TNF-α (5 ng/mL) treated MDA-MB-231 cell surfaces. Mean fluorescence intensity ratio of sample over isotype has been included.</p