Hermore, EGFR signaling is well-known to enhance tumor cell motility [16,17]. Still, researchers are only beginning to explore tumor cell invasion in more complex microenvironments [4,18] such as those that exist not only in the primary tumor stroma but also niche sites for disseminated cells such as bone marrow or lymph nodes [6]. Furthermore, EGF-secreting macrophages were shown to be recruited to tumor-associated blood vessels that secrete SDF-1a from pericytes in a rat breast cancer model [19,20]. Since such signaling pathways may have synergistic or antagonistic interactions, if any, it is important to build models and methods for qualitatively understanding cell response to complex environments, which is ultimately needed in future efforts aimed at building a predictive model for chemoinvasion in cancer [1]. Limitations of current models widely used to study chemotaxis or chemoinvasion, such as Boyden Gulated and 25 probesets (16 genes) were downregulated in the high CINGEC group. chambers, include (i) the lack of precise gradients that are stable in space or time [21], (ii) the lack of ability to differentiate chemotaxis from chemokinesis (i.e., enhancement of random motility but not directedness, which is less efficient for cell transport) [4,11], and (iii) endpoint quality of the assay, which does not allow imaging Title Loaded From File during migration and thus misses information on the dynamics, distribution, and cell morphology during cell migration. Microfluidic chemoinvasion models have recently been introduced to overcome these limitations and create more physiologically relevant models [11,22,23,24,25,26]. Additionally, current cancer cell chemotaxis studies using microfluidic models are largely limited to 2D, where cells are plated on a 2D substrate [27,28]. 2D tumor cell chemotaxis is fundamentally different from that of 3D. In 2D, MDA-MB-231 cells use a mesenchymal migration strategy only because it requires integrin activities (or adhesion). In 3D, mammalian cells can either squeeze through the pores of the biomatrix via amoeboid motion or climb along the collagen fibers via mesenchymal motion. In the case of leukocytes in steady state conditions, cells have been found to move within collagen fibers via amoeboid motion and independent of integrin binding [29]. MDA-MB-231 cells have been shown to undergo mesenchymalto-amoeboid transition when pericellular proteolysis is blocked [30]. In this study, we examine how tumor cell chemoinvasion behaviors can be affected by two competing chemical gradients, using a 3D microfluidic model with well-defined chemical gradients that are stable in space and time. A highly invasive and metastatic human breast cancer cell line, MDA-MB-231, was used because of the extent of characterization of this cell line [14], including its migration behavior in the presence of EGF or SDF1a gradients using conventional Boyden chamber [12,14,31]. Additionally, the methodologies presented here are readily applicable to other tumor cells or to more complex tumor microenvironments.schematics in Figure 1B. Briefly, chemokine and buffer flow through two side channels respectively, and a linear chemokine gradient is established in the center channel via diffusion of chemokine molecules though the agarose ridges. The time for the gradient establishment depends on the diffusion coefficient of the molecules. For EGF (6.2 kDa) or SDF-1a (8.0 kDa), the molecular diffusion coefficient is about 111 mm2/s [33], it takes about 30 min to establish a steady gradient. To characterize the chemical gradients in the cen.Hermore, EGFR signaling is well-known to enhance tumor cell motility [16,17]. Still, researchers are only beginning to explore tumor cell invasion in more complex microenvironments [4,18] such as those that exist not only in the primary tumor stroma but also niche sites for disseminated cells such as bone marrow or lymph nodes [6]. Furthermore, EGF-secreting macrophages were shown to be recruited to tumor-associated blood vessels that secrete SDF-1a from pericytes in a rat breast cancer model [19,20]. Since such signaling pathways may have synergistic or antagonistic interactions, if any, it is important to build models and methods for qualitatively understanding cell response to complex environments, which is ultimately needed in future efforts aimed at building a predictive model for chemoinvasion in cancer [1]. Limitations of current models widely used to study chemotaxis or chemoinvasion, such as Boyden chambers, include (i) the lack of precise gradients that are stable in space or time [21], (ii) the lack of ability to differentiate chemotaxis from chemokinesis (i.e., enhancement of random motility but not directedness, which is less efficient for cell transport) [4,11], and (iii) endpoint quality of the assay, which does not allow imaging during migration and thus misses information on the dynamics, distribution, and cell morphology during cell migration. Microfluidic chemoinvasion models have recently been introduced to overcome these limitations and create more physiologically relevant models [11,22,23,24,25,26]. Additionally, current cancer cell chemotaxis studies using microfluidic models are largely limited to 2D, where cells are plated on a 2D substrate [27,28]. 2D tumor cell chemotaxis is fundamentally different from that of 3D. In 2D, MDA-MB-231 cells use a mesenchymal migration strategy only because it requires integrin activities (or adhesion). In 3D, mammalian cells can either squeeze through the pores of the biomatrix via amoeboid motion or climb along the collagen fibers via mesenchymal motion. In the case of leukocytes in steady state conditions, cells have been found to move within collagen fibers via amoeboid motion and independent of integrin binding [29]. MDA-MB-231 cells have been shown to undergo mesenchymalto-amoeboid transition when pericellular proteolysis is blocked [30]. In this study, we examine how tumor cell chemoinvasion behaviors can be affected by two competing chemical gradients, using a 3D microfluidic model with well-defined chemical gradients that are stable in space and time. A highly invasive and metastatic human breast cancer cell line, MDA-MB-231, was used because of the extent of characterization of this cell line [14], including its migration behavior in the presence of EGF or SDF1a gradients using conventional Boyden chamber [12,14,31]. Additionally, the methodologies presented here are readily applicable to other tumor cells or to more complex tumor microenvironments.schematics in Figure 1B. Briefly, chemokine and buffer flow through two side channels respectively, and a linear chemokine gradient is established in the center channel via diffusion of chemokine molecules though the agarose ridges. The time for the gradient establishment depends on the diffusion coefficient of the molecules. For EGF (6.2 kDa) or SDF-1a (8.0 kDa), the molecular diffusion coefficient is about 111 mm2/s [33], it takes about 30 min to establish a steady gradient. To characterize the chemical gradients in the cen.

Hermore, EGFR signaling is well-known to enhance tumor cell motility [16,17]. Still

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