Ovarian Cancer Cell Adhesion/Migration Dynamics on Micro-Structured Laminin Gradients Fabricated by Multiphoton Excited Photochemistry

Abstract

Haptotaxis, i.e., cell migration in response to adhesive gradients, has been previously implicated in cancer metastasis. A better understanding of cell migration dynamics and their regulation could ultimately lead to new drug targets, especially for cancers with poor prognoses, such as ovarian cancer. Haptotaxis has not been well-studied due to the lack of biomimetic, biocompatible models, where, for example, microcontact printing and microfluidics approaches are primarily limited to 2D surfaces and cannot produce the 3D submicron features to which cells respond. Here we used multiphoton excited (MPE) phototochemistry to fabricate nano/microstructured gradients of laminin (LN) as 2.5D models of the ovarian basal lamina to study the haptotaxis dynamics of a series of ovarian cancer cells. Using these models, we found that increased LN concentration increased migration speed and also alignment of the overall cell morphology and their cytoskeleton along the linear axis of the gradients. Both these metrics were enhanced on LN compared to BSA gradients of the same design, demonstrating the importance of both topographic and ECM cues on the adhesion/migration dynamics. Using two different gradient designs, we addressed the question of the roles of local concentration and slope and found that the specific haptotactic response depends on the cell phenotype and not simply the gradient design. Moreover, small changes in concentration strongly affected the migration properties. This work is a necessary step in studying haptotaxis in more complete 3D models of the tumor microenvironment for ovarian and other cancers.

Keywords: ECM; contact guidance; cytoskeleton; haptotaxis; morphology; ovarian cancer.

Figure 1

Design of the low and high slope gradients: (a) Linear ramp showing the design as a function of point density, along with the resulting phase contrast image of the fabricated gradients. (b) The resulting LN immunofluorescence and corresponding linear fit (R² > 0.95) representing the relative concentrations.

Figure 2

Representative phase contrast images of each cell line on the high slope gradient, where (a) IOSE; (b) OVCA433; (c) SKOV-3.ip1; and (d) HEY-1. The gradient increases in concentration from left to right. Scale bar = 200 microns.

Figure 3

Merged migration speed data for the four cell lines on the low and high slope gradients. The statistical analysis for each cell line is given in Table 1, Table 2, Table 3 and Table 4. The data shows the relative roles of local concentration and slope on the haptotaxis response of the cell lines. Error bars represent standard error.

Figure 4

Average migration speed of the four cell lines on LN and BSA gradients made from the same parameters for the high slope design. All cells migrated with statistically faster speeds on the LN, demonstrating the importance of both ECM and contact cues. Error bars represent standard error. Data was analyzed from 40–100 cells in each case.

Figure 5

Directed migration of the four cells lines along the fiber axis of the high slope gradient shown as polar plots. The IOSE, OVCA433, and SKOV-3.ip1 cells had similar responses of directed migration based on the Rayleigh test and the pairwise U² Watson test. The HEY-1 cells did not pass the Rayleigh test, indicating a random distribution and were different from the other cells based the pairwise U² Watson test. The slight asymmetries are a computational artifact.

Figure 6

Histograms of the alignment of OVCA433 cells on the low (a); and high (b) LN gradients relative to the fiber axis. The alignment is further broken down into low, medium, and high concentration regions of the respective gradient. The alignments were all peaked at 0°, i.e., parallel to the gradient but increased LN concentration resulted in narrower distributions until a saturated response was observed.

Figure 7

(a) Representative phase contrast images of OVCA433 cells on LN (left) and BSA (right) gradients 12 h after seeding. The cells are better spread on the LN gradient. Scale bar = 200 microns. (b) Polar plots of the orientation of the OVCA433 cells on the low, medium, and high slope ranges of the BSA gradient, showing a random distribution in each case. Polar plots are used for better visualization of the random distribution.

Figure 8

Representative cytoskeleton images (left = phalloidin; middle = anti-vinculin) for SKOV-3.ip1 cells on the LN high slope gradient for (a) low concentration; (b) medium concentration; and (c) high concentration regions. Scale bar = 40 microns. The right column is the f-actin angular distribution relative to the fiber axis, showing an increase in stress fiber alignment at higher LN concentration. The focal adhesion localization on the LN similarly increased. The statistical analysis for stress fibers and focal adhesions is given in Table 6 and Table 7, respectively.