Better explicating the strengths and shortcomings of these models will help refine projections and improve transparency in the years ahead.
Image credit: Witsawat.S.
Edited by David V. Goeddel, The Column Group, San Francisco, CA, and approved March 25, 2015 (received for review October 1, 2014)
Article Figures & SI
Figures
- Fig. 1.
Cross-linking of Apo2L/TRAIL augments activity against a wide range of cancer cell lines. Human cancer cell lines were treated with concentrations ranging from 0 to 5 × 102 nM of either Apo2L.0 or cross-linked Apo2L/TRAIL (Apo2L.XL). Cell viability was measured after 72 h of treatment. (A) Of the 479 cell lines tested, 146 showed at least a 50% decrease in cell viability in response to Apo2L.0 (blue circle), and 257 showed the same in response to Apo2L.XL (red circle). Representative cell lines of different populations are highlighted in detail, with fitted dose–response curves. Error bars denote SEM; n = 3 per cell line for Apo2L.0 treatments and n = 4 per cell line for Apo2L.XL treatments. (B) Cell viability in response to 2 × 102 nM of Apo2L.0 (blue points) or Apo2L.XL (red or gray points). Red points denote values for Apo2L.XL that vary from corresponding Apo2L.0 values by a statistically significant degree (P < 0.05), and gray points denote values for which P > 0.05.
- Fig. 2.
Membrane display of Apo2L/TRAIL augments proapoptotic activity. (A) Scheme illustrating generation of membrane-displayed Apo2L/TRAIL (Apo2L.M). Biotinylated Apo2L/TRAIL is linked to a supported membrane via fluorescent streptavidin. Death receptor-expressing cells are then reacted with this surface and their response is observed via live cell fluorescence microscopy. DISC, death-inducing signaling complex. (B) NB-7GFP-C8 cells were engaged with Apo2L.M. Cells were identified using bright field microscopy; the corresponding fields were imaged in the Apo2L.M (Alexa Fluor 647) and caspase-8 (GFP) channels, and the three fields were overlaid to examine colocalization between Apo2L.M and caspase-8. (C) Parental NB-7 cells were engaged with Apo2L.M then fixed and immunostained for DR5. Bright field, Apo2L.M (Alexa Fluor 647), and DR5 (Alexa Fluor 488) channels were imaged. (D) NB-7GFP-C8 cells were pretreated with the pan-caspase inhibitor zVAD-fmk and the bright-field, Apo2L.M, and caspase-8 channels were imaged. (E–G) HT-29 cells were treated with the noted reagents and analyzed for caspase-8 activity after 4 h (E), caspase-3/7 activity after 6 h (F), or cell viability after 24 h (G). Error bars denote SEM, n = 3 per treatment.
- Fig. 3.
Liposome attachment of Apo2L/TRAIL yields functional, membrane-displayed ligand that can induce apoptosis in tumors. (A) Scheme illustrating generation of liposome-displayed Apo2L/TRAIL (Apo2L.L). Liposomes are decorated with Apo2L/TRAIL via covalent cysteine–maleimide bonds. (B) SDS-HPLC chromatograms of Apo2L.0 (blue), Apo2L.L (red), and bare liposomes (green). (C) HT-29 cells were treated with various concentrations of bare liposomes (green), Apo2L.0 (blue), or Apo2L.L (red) for 24 h and viability was measured. Error bars denote SEM, n = 3 per treatment. (D) Within COLO205 tumors implanted s.c. into nude mice, fluorescent liposomes were visible 6 h after injection via tail vein i.v. (E) Tumors, livers, spleens, and kidneys were collected from nude mice bearing s.c. COLO205 xenografts, homogenized, and assessed for Apo2L/TRAIL, cleaved caspase-8, and cleaved caspase-3, by immunoblot. (F–H) Nude mice bearing COLO205 xenografts (n = 10 per group) were treated with a single 25 mg/kg dose of Apo2L.0 or Apo2L.L via i.p. for 24 or 48 h then killed. Tumors from all animals and livers, spleens, and kidneys from five animals per group were collected and homogenized. Tissues were assessed for Apo2L/TRAIL using ELISA (F), caspase-8 activity (G), and caspase-3/7 activity (H). Error bars denote SEM.
- Fig. 4.
Liposomal display of Apo2L/TRAIL improves its pharmacodynamic profile and enhances antitumor efficacy. (A and B) Nude mice bearing HT-29 xenografts (n = 10 per group) were treated with a single 50 mg/kg dose of Apo2L.0 or Apo2L.L via i.p. for 24 or 48 h then killed. All tumors were collected, homogenized, and assessed for Apo2L/TRAIL using ELISA (A) and caspase-8 activity (B). Error bars denote SEM. (C) Mice (n = 9 per group) bearing HT-29 tumor xenografts were treated with either Apo2L.0 or Apo2L.L via i.p. injection every 2 d. Tumor volumes were measured twice per week and then fit to a nonlinear mixed-effect model of tumor growth (SI Materials and Methods). Overlaid fitted curves are shown on the left and individual tumor volumes and their respective fitted curves are shown on the right. Dashed lines denote mice killed before the end of study.
Data supplements
Supporting Information
Files in this Data Supplement:
- Download Supporting Information (PDF)
- Download Dataset_S01 (XLSX)
- Download Movie_S01 (MOV) - Death receptor microclustering, caspase-8 recruitment, and apoptotic blebbing in response to membrane-displayed Apo2L/TRAIL. NB-7GFP-C8 cells were deposited on supported membranes displaying Apo2L.M. Cells were allowed to engage Apo2L.M on the surface for 2 h, at which point bright-field, Alexa Fluor 647, and GFP channels were imaged at 100× magnification every 20 min for 14 h. Samples were maintained in a live-cell imaging enclosure at 37 °C, 5% CO2 for the duration of the imaging.
Enhancing TRAIL's efficacy by membrane display
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- Biochemistry
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