Abstract 468: 70kDa fibronectin splice variant induced human mammary fibroblast migration

Abstract

Abstract A soluble factor hypothesized to play an important role in metastasis is migration stimulating factor (MSF). MSF is a truncated splice variant of fibronectin, with a structure nearly identical to the long-studied 70 kDa N-terminal catheptic-D fragment. MSF is secreted by fetal fibroblasts, some tumor cells, tumor-associated macrophages (TAM), cancer associated fibroblasts (CAF), and tumor-associated vascular endothelial cells. MSF is a potent bioactivator of normal adult fibroblast, and endothelial cell migration, but previous studies have failed to regulate and characterize the MSF gradient and the gradient effect on cell migration. In the current study, we compared the motogenic activity of the 70 kDa fibronectin N-terminal fragment (70kDA-FN) at different concentrations to baseline media containing 2% fetal calf serum (FCS) using microfluidic channels with well-characterized chemoattractant gradients. Microfluidic channels with 5um x 1um x 500 um dimensions were developed from PDMS. They comprise of two half-Y-shaped channels connected by a 1.35 um long gradient controlling migration channel. In this design, cells were introduced into the right half channel. To distinguish gradient dependent migration from random migration, MSF was introduced into the non-cell-containing half channel for chemotaxis, or placed directly into the cell containing half channel for chemokinesis. GFP-labeled human mammary fibroblast (HMF; originally termed RMF/EG) cells were provided by Dr. Kuperwasser, and maintained in high glucose DMEM supplemented with 10% FCS. Prior to the experiments, HMFs were cultured overnight in DMEM supplemented with 2% FCS. Microfluidic channels were imaged after 36 hours by an Olympus IX71 microscope, and migrating cells were quantified using the custom image analysis program JeX. Prior to the migration experiments, time-lapse fluorescent imaging of FITC-labeled 70kDa-FN was used to characterize and verify the presence of the chemoattractant gradient for 36 hours. The numbers of cells present in different experimental conditions were compared using two-sided Mann-Whitney Wilcoxon test. There was a statistically significant difference between the chemotactic experimental conditions containing 70kDa-FN concentrations of 14pMol/mL (p<0.01) and 1.4pMol/mL (p<0.05). Normalizing to migrating cells present in the control channel, the 14pMol/mL and 1.4pMol/mL 70kDa-FN chemotaxis channels on average contained 70% and 53% more cells, respectively. Furthermore, the experimental setups testing chemokinesis on average contained more cells compared to the control, supporting a possible role for 70kDa-FN in HMF chemokinesis. MSF and 70kDa-FN are potent activators of fibroblast migration. In the current experiment we utilized microfluidic channels to test and confirm that 70kDa-FN induced fibroblast migration is both chemokinetic and chemotactic, and dose dependent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 468. doi:1538-7445.AM2012-468