6 well ultra low attachment surface dishes

Poly(DL-lactide-co-glycolide) (PLGA) with a molar ratio of lactide:glycolide of 50:50 (40–75 kDa), polyvinyl alcohol (PVA; 30–70 kDa), RA and organic reagents were all purchased from Sigma-Aldrich; EMD Millipore (Billerica, MA, USA). The lipids, including [(1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-maleimide- polyethylene glycol-2000; DSPE-PEG-Mal, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-carboxyfluorescein; PECF)], phosphatidylcholine, and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The CD133 MicroBead kit was provided by Miltenyi Biotec, Inc. (Shanghai, China). The ultra-low attachment surface 6-well dishes were purchased from Corning Life Sciences (Tewksbury, MA, USA). R&D Systems, Inc. (Minneapolis, MN, USA) provided recombinant anti-human CD133 Alexa Fluor^® 488-conjugated antibody (cat. no. FAB11331G). The synthesis of CD133 aptamers with the sequence of 5′-SH-CCCUCCUACAUAGGG-3′ was performed by Ruibo Co., Ltd. (Guangzhou, China). The Pierce BCA Protein Assay kit, FBS, B27, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-transferrin-selenium (ITS), 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI) and the cell dissociation reagent (StemPro^® Accutase^®) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

Tumorsphere formation of the lung cancer cells was assessed to evaluate the self-renewal ability of the lung cancer initiating cells when the single cells were suspended in serum-free medium. In short, lung cancer cells suspended in stem cell medium were cultured in Corning^® ultra-low attachment surface 6-well dishes. The density of the suspended cells was 5,000 cells/well, and the components of the stem cell medium included DMEM/F12, B27 (1×), ITS (1X), EGF and bFGF (both cytokines were at a concentration of 20 ng/ml). The cells were cultured in the stem cell medium for 7 days at 37°C, following which the tumorspheres were counted under an Olympus CKX41 conventional light microscope. For the second-passage tumorspheres, those of the first passage were washed with PBS and dissociated with a cell dissociation reagent (StemPro^® Accutase^®), and then propagated.