EGF as a New Therapeutic Target for Medulloblastoma Metastasis

Abstract

Medulloblastoma (MB) is a malignant pediatric brain tumor known for its aggressive metastatic potential. Despite the well-documented migration of MB cells to other parts of the brain and spinal column, MB chemotaxis is poorly understood. Herein, we examined the in vitro migratory and cellular responses of MB-derived cells to external signaling of Epidermal Growth Factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF-BB), and the stromal cell-derived factors 1-alpha (SDF-1). Experiments utilized transwell assays and immunocytochemistry to identify receptor activation in MB migration, and used a microfluidic platform to examine directionality, trajectory, and gradient-dependence of motile cells. Data illustrates that MB-derived cells respond strongly to EGF in a dosage and gradient-dependent manner with increased EGF-R activation, and show that high EGF gradient fields cause an increased number of cells to migrate longer directed distances. Our results provide evidence that EGF and its receptor play an important role than previously documented in MB chemotactic migration than previously documented and should be considered for developing migration-target therapies against MB metastasis.

Keywords: Pediatric cancer; chemotaxis; gradients; microfluidics.

Figure 1. Migration of MB-derived cells to different concentrations of selective chemotactic ligands

(A1) Schematic of transwell assay with motile cells attached to the underside of the porous membrane. (A2) Stained nuclei and cytoplasm of motile MB-derived cells toward different concentrations of (B1) EGF, (B2) HGF, (B3) PDGF, and (B4) SDF-1. The control groups indicate number of cells that migrated towards serum-free medium. An asterisk (*) indicates statistically significant data with p-values <0.05 against control group.

Figure 2. Receptor activation within motile MB-derived cells

Immunocytochemistry of basal receptor expression of MB cells without ligand stimulation to (A1) EGF, (B1) SDF-1, (C1) HGF, and (D1) PDGF-BB. Receptor activation post-stimulation with (A2) EGF, (B2) SDF-1, (C2) HGF, and (D2) PDGF-BB. The expression level of receptor following ligand stimulation of (A3) EGFR, (B3) CXCR4, (C3) c-Met, and (D3) PDGFR-BB normalized to basal control levels. Scale bars are 100μm. An asterisk (*) indicates statistically significant data with p-values <0.05.

Figure 3. The μLane system and MB migratory responses

(A) Schematic of the bridged μLane system, showing cells inserted within the sink (SKR) and source (SRR) reservoir, and adhered along the microchannel. Chemotactic agents (e.g. EGF, SDF-1) are loaded into the source chamber (SRC), and transported to SKR to reach steady-state concentration distribution. (B) Top view image of the first layer PDMS bonded onto a glass slide. Two 9-nL reservoirs are connected by a microchannel of 13mm in length and 100μm in diameter. (C) Top view image of second layer PDMS bonded to the first layer. The source (SRC) and sink (SKC) chambers are connected by a bridge channel. (D) Raw data image of motile MB cells within μLane system at (D1) source reservoir, (D2) mid channel, and (D3) sink reservoir.

Figure 4. Concentration distribution along μLane and number of motile cells

(A) Concentration profile of EGF and SDF-1 along 13-mm microchannel length of μLane system. Concentration gradients are identified by five orders of magnitude in the microchannel: 10⁺¹<G₁<10⁰ ng/(mL.mm), 10⁰<G₂<10⁻¹ ng/(mL.mm), 10⁻¹<G₃<10⁻² ng/(mL.mm), 10⁻²<G₄<10⁻³ ng/(mL.mm), and 10⁻³<G₅<0 ng/(mL.mm). (B) Fraction of MB-derived cells observed to respond via migration to the different concentration gradient fields (G₁ through G₅) of EGF and SDF-1.

Figure 5. Motility of MB-derived cells in the μLane system

(A) Representative trajectories of cells that migrated in response to 100ng/mL of EGF and 100 ng/mL of SDF-1 stimulation. Three cell paths are shown in dashed for EGF and three in solid for SDF-1, 24 hours post steady-state. Note that concentration gradients decrease from left to right within the μLane. (B) Maximum accumulated distance of motile cells stimulated by concentration profiles generated by using 100ng/mL of EGF and 100 ng/mL of SDF-1, respectively, in the SRR of the μLane.