Abstract
Gliomas are invasive cancers that resist all forms of attempted therapy. Immunotherapy using Ag-pulsed dendritic cells has improved survival in some patients. We present evidence that another level of complexity may also contribute to lack of responses by the lymphocytes toward gliomas. Atomic force microscopy of four different glioma types-human U251 and rat T9 and F98 glioma cells, including freshly isolated human glioblastoma multiforme neurosphere cultures (containing "stem cell-like cells")-revealed a complex surface topography with numerous microvilli and filopodia. These structures were not found on other cell types. Electron microscopy and immunofluorescence microscopy of glioma cells confirmed that microvilli are present. U251 cells with microvilli resisted the cytolytic actions of different human effector cells, (lymphokine-activated killer cells, γδ T cells, conventional CTLs, and chimeric Ag-receptor-redirected T cells) better than their nonmicrovilli-expressing counterparts. Killer lymphocytes released perforin, which was detected within the glioma's microvilli/filopodia, indicating these structures can receive the cytolytic effector molecules, but cytotoxicity is suboptimal. Air-dried gliomas revealed nodes within the microvilli/filopodia. The microvilli that penetrated 0.4-μm transwell chamber's pores resisted the actions of CTLs and physical damage. Those nodelike structures may represent a compartmentalization that resists physical damage. These microvilli may play multiple roles in glioma biology, such as invasion and resistance to lymphocyte-mediated killing.
Highlights
atomic force microscopy (AFM) utilizes a sharp imaging silicon nitride tip attached to a mechanical cantilever that physically moves over the surface of the cells
AFM allows the cell surface topography of cells to be visualized with minimal postfixative manipulation
Some artifacts are seen with this technology, because the cell surface is still flexible when the cantilever runs over the membranes
Summary
We performed experiments where Ag nonspecific effector lymphocytes, such as lymphokine activated killer (LAK) cells and gd T cells [7], together with Ag-specific CTL and T cells engineered to possess a CAR toward the her2/neu receptor [8], could kill microvilli expressing U251 cells (Fig. 7). Because the her2/neu receptor is present on the microvilli (Fig. 9), we used the CAR-T cells as the effector lymphocytes for an additional experiment.
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