Prostate Cancer Cell Migration Is Influenced By Suspended Fiber Structural Stiffness

Introduction:  

Cancer is the second leading cause of death in the U.S.[1], and accounts for over $200 billion in health and morbidity costs [2]. The major cause of death in cancer patients is metastasis [3]. Tumors are classified in terms of grades by the World Health Organization. While grades I-II represent tumors that are mostly contained in the periphery of the primary tumor, grades III-IV represent tumors that have migrated from the primary site of occurrence to distant tissues and organs. When higher grade tumors invade other tissues, it starts compromising the functionality of the organ of origin and the organs affected via metastasis, ultimately leading to the death of the person [4, 5].

One of the most effective ways to control tumors is by administering anti-metastatic drugs [6].  Some of the drugs that compromise the ability of cancer cells to migrate include paclitaxel, nocodazole, and marimastat [7, 8]. These drugs control metastasis by primarily depolymerizing cytoskeletal components and severely compromising the ability of cells to migrate. While this is shown to be effective, another potential strategy to minimize metastasis can include the conditioning of the tumor associated extracellular matrix (ECM) such that it does not encourage cell migration. In order to design such strategies, it is important to understand how the biochemical and biophysical properties of the ECM influences single cancer cell migration.

It has been well established that bulk mechanical properties like substrate elasticity alters integrin expression in cancer cells facilitating a migratory phenotype [9, 10]. However, it has also been observed that the local and cell specific mechanical properties might have a stronger influence on cell migration behavior than bulk properties [11]. It is still not completely understood how cancer cell migration behavior is influenced by biophysical properties of their extracellular matrix. One such property is structural stiffness measured in N/m. Unlike substrate elasticity, structural stiffness accounts for local changes in the material property, and from our preliminary investigation, have been shown to influence cancer cell migration.

In this study, we utilized a previously explained Spinneret based Tunable Engineering Parameters (STEP) pseudo-dry spinning technique to manufacture aligned, parallel, and suspended nanofibers [12, 13]. This platform offers design of: a) nanofibers of diameters close to the native fibrous ECM are manufactured, b) a non-2D environment of suspended fibers on which the cells wrap around, which closely represent the in vivo conditions, and c) change in the structural stiffness (N/m) of the fibers through different  material, diameter and length of fibers. A schematic of the suspended nanofiber substrate with change in structural stiffness along its distance from the edge is shown in Fig. 1.

Materials and Methods:

Polystyrene (E= 3GPa [14]), nanofibers were manufactured using a non-electrospinning, STEP technique. Highly aligned nanofibers of diameter 500nm and length 6 mm were deposited on plastic frames to obtain suspended nanofibers (Fig 1). The fibers were sterilized with 70% ethanol, coated with fibronectin to facilitate cell attachment, and seeded with prostate cancer cells (PC-3, purchased from ATCC) in F-12K media (ATCC). After attachment, time lapse images were obtained every 10 minutes for 6 hours using a Zeiss microscope with incubating capacity. The position of cell in terms of its distance from the edge of the substrate was calculated using Axio Vision software. Also the migration (maximum displacement within the 6 hour period) of single prostate cancer cells was measured and the distance of the cell from the substrate edge was recorded.

Results and Discussion:

Using Euler beam mechanics, the structural stiffness of the nanofiber can be calculated. Using the equation for beam deflection, it can be said that the structural stiffness decreases towards the middle span length of the fiber (Fig. 1 (ii) and (iii)). The cells attached to the fibronectin coated fibers within 2-6 hours after seeding, and formed spindle morphologies as they migrated along a single nanofiber (Fig. 2). While some cells demonstrated persistent migration, cells that migrated back and forth along the fiber were also observed. Prostate cancer cells exhibited different migration speeds along the fiber span length (Fig.3). While the migration speed of PC-3 ranged from 3-195μm/hr, linear regression analysis showed that cells migrated faster when they were migrating at the center of the fiber span length. This observation suggests that as structural stiffness decreases, prostate cancer cell migration speed increases.

Structural stiffness is a measure of local mechanical property of the nanofiber. This observation shows that even when the elastic modulus of the substrate (N/m²) was constant for polystyrene, prostate cancer cells exhibited different migratory behavior due to the change in structural stiffness (N/m), a local mechanical property. This suggests that prostate cancer cells are constantly probing their local microenvironment, and are able to manipulate their migration speeds in response to mechanical changes in the microenvironment.

Conclusion:

Cell migration is a complex phenomenon that is influenced by a myriad of factors within and outside the cell. Here, we demonstrate that by changing the structural stiffness of the immediate microenvironment of the cell, its migration speed changes. Moreover, this study shows how stiffness differences within millimeter distances can impact the migration dynamics of prostate cancer cells. Hence, drugs that alter the local mechanical property of the immediate ECM could potentially be coupled with the already existing anti-metastatic drugs to achieve lower cancer metastasis rates.

 

 

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