Fascin-1 knock-down of human glioma cells reduces their microvilli/filopodia while improving their susceptibility to lymphocyte-mediated cytotoxicity

Abstract

Cancer cells derived from Glioblastoma multiforme possess membranous protrusions allowing these cells to infiltrate surrounding tissue, while resisting lymphocyte cytotoxicity. Microvilli and filopodia are supported by actin filaments cross-linked by fascin. Fascin-1 was genetically silenced within human U251 glioma cells; these knock-down glioma cells lost their microvilli/filopodia. The doubling time of these fascin-1 knock-down cells was doubled that of shRNA control U251 cells. Fascin-1 knock-down cells lost their transmigratory ability responding to interleukin-6 or insulin-like growth factor-1. Fascin-1 silenced U251 cells were more easily killed by cytolytic lymphocytes. Fascin-1 knock-down provides unique opportunities to augment glioma immunotherapy by simultaneously targeting several key glioma functions: like cell transmigration, cell division and resisting immune responses.

Keywords: CTL; Fascin-1; filopodia; glioblastoma; microvilli; shRNA.

Figure 1

Intracellular flow cytometry of fascin-1 protein level knocked down by siRNA and shRNA. (A) The U251 cells, siRNA control (shaded area) or siRNA fascin-1 knockdown (light line) and (B), shRNA control (shaded area) or initial non-cloned shRNA fascin-1 knockdown U251 cell (light line) were examined by intracellular flow cytometry using the monoclonal antibody towards fascin-1. Ten thousand cells were analyzed. By the Kolmogorov-Smirnov statistics’ test, the fluorescent readings were determined to be statistically different (P<0.001) from each other.

Figure 2

The morphology of U251 cells before and two days after fascin-1 siRNA knock-down. U251 cells (untreated Top Row) and siRNA-fascin-1 knock-down (2 days after transfection, Second Row), shRNA control (third row) or shRNA fascin-1 (bottom row) were allowed to adhere overnight onto a slide. The cells were fixed, permeabilized and then stained with the anti-fascin-1 antibody (red) and anti-F-actin antibody (green). Magnification is 100 X.

Figure 3

Atomic force microscopy reveals that fascin-1 knock-down results in loss of microvilli/microspikes. The U251, siRNA control, siRNA fascin and shRNA fascin, cells were allowed to adhere overnight on sterile cover-slips. The cells were fixed in 2% paraformaldehyde and imaged by atomic force microscopy. Left Panel: the siRNA controls U251 cells with multiple microspikes/microvilli. While Middle Panels (siRNA fascin treated) and Right Panel (shRNA fascin treated) displays a smoothed cell-surface topography.

Figure 4

Fascin-1 knock-down cells loss of transmigration ability by the U251. Ten thousand cells (U251 untreated, shRNA-control U251 and fascin-1 knockdown U251 cells) were placed in the top chamber of the NeuroProbe Invasive unit. The chemo-attractants (20% FBS, 100 ng/ml rh-IL-6 or rhIGF-1) were placed in their respective bottom chambers. After 5 hours, the number of invading cells that penetrated through the 8 µm pore was measured and then calculated for the number of cells. Data is expressed as the mean of sextuplicate cultures ± standard deviation (SD). The asterisk denotes a significant difference by the student’s t test (P<0.05) of the fascin-1 knock cells compared to both the untreated U251 or shRNA control U251 cells.

Figure 5

LAK cells kill siRNA knock-down cells better than control siRNA treated cells. Human lymphokine activated killer cells were tested against adherent U251 non-treated, shRNA control U251 and U251 fascin-1 knock-down cells. The non-adherent U251 cells which lack microvilli served as the maximum killing. Data is expressed as the mean of specific killing of quadruplicate cultures ± SD. The asterisk denotes a significant difference by the student’s t test (P<0.05) from the shRNA-fascin-1 knock-down cells compared to the adherent U251 or shRNA-control U251 cells.

Figure 6

shRNA knock-down of fascin-1 does not alter expression of HLA-A2 or gBK antigens. A-C. The U251 untreated, shRNA control or fascin-1 shRNA treated cells were stained for the presence of membrane HLA-A2 (Top Row) or the cells were fixed, permeabilized and stained for gBK (Bottom Row). D-F. Ten thousand were analyzed by flow cytometry.

Figure 7

CTL-mediated cytotoxicity is enhanced on fascin-1 knocked-down U251 cells. HLA-A*0201+ human CTLs sensitized to gBK1 or gBK2 peptides were tested against adherent U251 shRNA control U251, U251 fascin-1 knock-down and gBK knock-down cells. The top panels show the representative results of one experiment done out of three different assays. The Bottom Panel shows the results when adherent siRNA control or siRNA fascin-1 U251 cells were used. A, C. The gBK1 specific CTLs are shown in the left panels while the gBK2 CTLs are displayed on the right panels B, D. Data is expressed as the mean of specific killing of quadruplicate cultures ± SD. The asterisk denotes a significant difference by the student’s t test (P<0.05) from the fascin-1 knock-down cells compared to the adherent control U251 cells. In the Top Panel, the asterisk also indicates that the gBK shRNA knock-down U251 cells were significantly killed less than the shRNA control U251 cells.