Mimicking white matter tract topography using core-shell electrospun nanofibers to examine migration of malignant brain tumors

Abstract

Glioblastoma multiforme (GBM), one of the deadliest forms of human cancer, is characterized by its high infiltration capacity, partially regulated by the neural extracellular matrix (ECM). A major limitation in developing effective treatments is the lack of in vitro models that mimic features of GBM migration highways. Ideally, these models would permit tunable control of mechanics and chemistry to allow the unique role of each of these components to be examined. To address this need, we developed aligned nanofiber biomaterials via core-shell electrospinning that permit systematic study of mechanical and chemical influences on cell adhesion and migration. These models mimic the topography of white matter tracts, a major GBM migration 'highway'. To independently investigate the influence of chemistry and mechanics on GBM behaviors, nanofiber mechanics were modulated by using different polymers (i.e., gelatin, poly(ethersulfone), poly(dimethylsiloxane)) in the 'core' while employing a common poly(ε-caprolactone) (PCL) 'shell' to conserve surface chemistry. These materials revealed GBM sensitivity to nanofiber mechanics, with single cell morphology (Feret diameter), migration speed, focal adhesion kinase (FAK) and myosin light chain 2 (MLC2) expression all showing a strong dependence on nanofiber modulus. Similarly, modulating nanofiber chemistry using extracellular matrix molecules (i.e., hyaluronic acid (HA), collagen, and Matrigel) in the 'shell' material with a common PCL 'core' to conserve mechanical properties revealed GBM sensitivity to HA; specifically, a negative effect on migration. This system, which mimics the topographical features of white matter tracts, should allow further examination of the complex interplay of mechanics, chemistry, and topography in regulating brain tumor behaviors.

Fig. 1

Micro-structural features of PCL and core–shell nanofibers observed via scanning electron microscopy (SEM). (A) Gelatin-PCL (B) PCL (C) PDMS-PCL (D) PES-PCL (E) PCL-Collagen (F) PCL-HA (G) PCL-Matrigel. Scale bar indicates 20 µm. Fast Fourier Transform (FFT) analyses of the associated images are shown as insets. (H) FFT analysis via radial summation of pixels normalized for all samples and plotted versus degree.

Fig. 2

Representative PDMS-PCL core–shell nanofiber. (A) Schematic and (B) transmission electron microscopy (TEM) image of the PDMS-PCL core–shell nanofiber showing the PDMS ‘core’ and PCL ‘shell’ surrounding the core indicated by white bidirectional arrows. Scale bar = 0.2 µm.

Fig. 3

Initial attachment of OSU-2 cells to nanofiber scaffolds. (A) Adhesion as a function of nanofiber mechanics and (B) Adhesion as a function of surface chemistry. * indicates statistically significant difference compared to PCL nanofiber control (p < 0.05).

Fig. 4

OSU-2 cell morphology on nanofiber scaffolds. (A) Gelatin-PCL (B) PCL (C) PDMS-PCL (D) PES-PCL (E) PCL-Collagen (F) PCL-HA (G) PCL-Matrigel. Scale bar in (A) indicates 100 µm. Bidirectional arrow indicates direction of fiber alignment.

Fig. 5

Feret diameter analysis of OSU-2 cells on various nanofibers. (A) Feret diameter as a function of mechanics and (B) Feret diameter as a function of biomimetic chemistries. N ≥ 142 individual cells analyzed for each nanofiber. * and ** indicates statistically significant difference compared to PCL nanofiber (p < 0.05). Levels marked by identical number of * are not significantly different from each other as determined by Tukey-HSD test.

Fig. 6

Single cell migration speeds on electrospun nanofiber scaffolds. (A) Migration as a function of nanofiber mechanics and (B) migration as a function of surface chemistry shown in box and whisker plots. N ≥ 95 individual cells analyzed for each nanofiber. * and ** indicate statistically significant difference compared to PCL nanofibers (p < 0.05). Levels marked by identical numbers of * are not significantly different from each other as determined by the Tukey-HSD test.

Fig. 7

Analysis of FAK/MLC2 expression via western blotting. (A) pFAK, FAK, pMLC2, MLC2 expression as a function of mechanics. (B) pFAK, FAK, pMLC2, MLC2 expression for PCL versus PCL-HA electrospun nanofibers. In both cases, tubulin served as a loading control.