P12.14.B HIGHLY INVASIVE GLIOMA CELLS MOTILITY MEASUREMENTS ON A VARIETY OF EXTRACELLULAR MATRIX PROTEINS

Abstract

Abstract BACKGROUND Glioblastoma multiforme (GBM) is a heterogeneous highly invasive malignant brain tumor. The extracellular matrix (ECM) produced by tumor microenvironment plays an important role in the regulation of cell motility and invasion into surrounding healthy tissue. In this research we determined the effect of various ECM proteins on the motility of primary tumor cells. MATERIAL AND METHODS Primary tumor cells were isolated from biopsy material obtained during GBM surgical removal. Cultivation of the piece of tumor (0.5 mm3) was carried out in the form of an 3D explant model at 37°C in 5% CO2-incubator. After several months, cells migrated from the tumor spontaneously formed spheroids. To assess the rate of cell migration, spheroids were seeded on 24-well plate coated with poly-L-lysine, fibrinogen, collagen type I, and Matrigel. After 3 days, the field of view in the well (n=9) was microphotographed every 15 min for 56 hours, after which the tracks of cells outside the spheroids were manually marked (n=50) on the obtained images. The average and maximum speeds were calculated, as well as the straightness of the track (the ratio of the Euclidean distance to the track length). The Mean square displacement (MSD) was plotted against time for each protein studied in comparison with random walk simulation calculated for the equal track parameters (number of steps, mean and sd) on correspondent protein. RESULTS Live-cell imaging of the patient-derived cells revealed the heterogeneous population of actively moving cells outside the spheroids. The following results of measuring cell motility were obtained (mean ± sd): (1) Mean speed on poly-L-lysine 4.3 ± 2.5 μm/h; on fibrinogen 11.1 ± 5.7 μm/h; on collagen type I 8.8 ± 5.1 μm/h; on Matrigel 9.7 ± 3.6 μm/h. (2) Max speed on poly-L-lysine 63.8 ± 30.1 μm/h; on fibrinogen 90.0 ± 38.9 μm/h; on collagen type I 96.1 ± 42.1 μm/h; on Matrigel 84.1 ± 34.1 μm/h. (3) Straightness on poly-L-lysine 0.24 ± 0.14 μm/h; on fibrinogen 0.18 ± 0.11 μm/h; on collagen type I 0.14 ± 0.09 μm/h; on Matrigel 0.20 ± 0.12 μm/h. MSD analysis showed the greatest contribution of random walk in the motility of cells on collagen type I, the smallest - on Matrigel. CONCLUSION Proteins of ECM affect not only the speed of cell movement, but also the tortuosity of their tracks. Further studies will clarify the mechanisms of GBM invasion depending on the composition of the ECM.