601. An Optimized Approach to Non-Viral Transfection of Brain Tumor Initiating Cells Derived from Embryonal Tumor with Multilayered Rosettes Using Cationic Reagents

Embryonal tumor with multilayered rosettes (ETMR) is an aggressive type of primitive neuroectodermal brain tumors of the central nervous system that occur primarily in infants and young children. Despite intensive therapy, prognosis is extremely poor with typical survival outlook of <1 year after diagnosis. Resistance to conventional chemo/radiation therapy among ETMR patients has been attributed to the resilience and resurgence of a sub-population of “brain-tumor initiating cells” (BTIC) within the tumor mass. These BTICs are a type of cancer stem cell with the capacity for long-term self-renewal, multi-lineage differentiation, and tumor formation. To better understand the pathogenesis of ETMR and develop effective therapies against this disease, we have previously established and characterized a BTIC cell line (BT183) with molecular signatures and tumor-initiating properties that are hallmarks of ETMR. However, BTICs have so far proven difficult to transfect at high efficiency, making them refractory to genetic manipulation approaches. Here, we describe an optimized approach to transfect BTICs using cationic reagent. We initially tried transfecting BT183 with Lipofectamine-2000 according to manufacturer's suggested protocol, but were not able to get any GFP+ cells. Because Lipofectamine is formulated with a multivalent cationic lipid, one rate-limiting factor could be its susceptibility to serum inactivation. While BTICs are cultured in a defined, serum-free neural stem cell-promoting media, the presence of growth factors and additives (e.g. EGF, bFGF, heparin) may interfere with the maturation and stability of lipoplexes. To test this hypothesis, we incubated lipoplexes in different preparations of the NeuroCult™-XF Proliferation media, each devoid of one or more of the required additives. We then assayed for complex stability using SYBRSafe, an intercalating dye that binds to the minor grooves of free DNA more efficiently than the condensed form of complexed DNA. We noted that the fluorescent intensities of the complexes were significantly higher (1.2x) in media with EGF, bFGF, heparin, suggesting DNA may be partially dissociated in the presence of these additives. We then transfected cells in the different media preparations and saw that only transfection carried out in media devoid of heparin/growth factors had GFP+ cells. While the number of GFP+ cells were too few to quantitate at this stage, we noted that there were more GFP+ at lower seeding densities, but the GFP+ cells were mostly buried among non-transfected cells as neurospheres. We postulated that the tendency for these BTIC to aggregate in a density-dependent manner may interfere with the efficient binding and uptake of lipoplexes; hence methods to prevent them from sticking to each other during transfection might enhance efficiency. We therefore tried attaching them using a number of cell attachment methods (i.e. gelatin, PEI, CELLSTAR). Only CELLSTAR was able to facilitate uniform cell attachment without causing significant cytoxicity. When we eventually transfected BTIC attached to CELLSTAR-treated tissue culture plates in media without heparin or growth factors, we saw a remarkable increase in transfection efficiency with up to 24% of the cells being GFP+. In summary, we showed here that BTICs can be efficiently transfected using cationic reagent by temporarily remove media components that are otherwise inhibitory to transfection and by adapting cell culture format to one that is more conducive to the binding and uptake of DNA complexes. The approaches outlined here may be adapted to other hard-to-transfect suspension cultures such as lymphocytes and leukemic cells.

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