Article Figures & Data
Figures
- Figure 1.
Rapid HSPG-mediated tumor killing by AU-011. 92.1 cells were preplated at 2.5 × 104/500 μL in a 24-well plate and allowed to grow for 48 hours. A total of 7.5 μg/mL (300 pmol/L) of AU-011 was incubated for 1 hour ± 1 mg/mL heparin at 4°C and then added to the cells for 1 hour at 37°C. IR700 dye was also added in an amount equivalent to that which is conjugated to 300 pmol/L of AU-011. After 1 hour, cells were washed twice and then either kept in the dark or exposed to 25 J/cm2 of 690 nm light. Images were acquired within 15 minutes of light treatment.
- Figure 2.
HSPG specificity of AU-011 activity. HeLa (A) or 92.1MEL (B) cells were combined at a 1:1 ratio with either CHO-K1 (gray bar) or pgsA-745 cells (white bar) and coincubated with AU-011 at 300 pmol/L for 1 hour at 4°C. Cells were washed and then exposed to 25 J/cm2 or kept in the dark (0 J/cm2). Prior to combining cell lines, human cell lines were incubated with CFSE, and hamster cell lines were incubated with CellTrace Violet to distinguish the populations for flow cytometry analysis (example of the analysis provided in Supplementary Fig. S5). Data are reported as % dead as determined by viability staining. Values plotted are the mean of two experiments performed in triplicate and error bars represent the SEM.
- Figure 3.
Dose-dependent AU-011 tumor killing in vivo. AU-011 was systemically administered to nude mice with subcutaneous 92.1 tumors (40–80 mm3). After 12 hours, animals were exposed to 25 or 50 J/cm2 690 nm light. Tumors were removed after 24 hours, homogenized, and viability measured by flow cytometry. Vehicle, n = 3; AU-011, n = 5. Data are representative of two experiments. Two-tailed Student t test: *, P = 0.0091; **, P = 0.0071.
- Figure 4.
Accumulation of AU-011 in 92.1MEL ocular tumors after intravitreal injection. A, SDS-PAGE of tumor lysates from samples used in confocal microscopy (see Supplementary Fig. S6A–S6F) and analyzed on Odyssey CLx imager at 700 nm. Two examples each of rabbit 92.1MEL tumor lysates 8 (rabbits 1.2 and 1.3) or 16 hours (rabbits 3.3 and 3.4) after intravitreal injection. The negative control received vehicle only (rabbit 6.2). The arrow indicates the predominant L1 protein in AU-011. B, Measurement of AU-011 levels in tumor tissue lysates. Each dot represents AU-011 levels measured in the tumor recovered from one animal.
- Figure 5.
AU-011 anticancer activity in a rabbit orthotopic xenograft model of uveal melanoma. Panels show H&E-stained sections of tumor-bearing rabbit eyes from the vehicle (A), 50 μg (B), 20 μg (C), and 5 μg (D) treated cohorts 2 days after treatment. Higher magnifications shown on the right are demarcated by black boxes in the overview images on the left.
Tables
- Table 1.
Binding and potency EC50 values (pmol/L) of AU-011 on a panel of human tumor cell lines
Binding Killing 92.1MEL 27.37 ± 1.385 61.74 ± 2.142 NCI-H460 133.7 ± 50.94 70.32 ± 28.25 HeLa 26.17 ± 3.137 66.24 ± 2.708 HSC-3 10.99 ± 1.115 24.72 ± 0.918 MCF-7 53.28 ± 6.519 37.21 ± 4.58 SK-MEL-2 11.74 ± 0.956 25.24 ± 1.693 SK-OV-3 23.2 ± 1.414 37.86 ± 6.831 T24 41.12 ± 5.142 60.1 ± 18.53 NOTE: Values represent mean pmol/L ± SEM, obtained from three experiments performed in triplicate.
Supplementary Data
- Figures S1-S7 merged - Figure S1-video composites of real time AU-011 mediated killing of 92.1MEL cells; Figure S2-Killing and binding comparison of free IR700 dye to VLP conjugated IR700 (AU-011) on HeLa and 92.1MEL cells; Figure S3-Demonstration of both the AU-011 dose and light fluence required for AU-011 killing activity on HeLa and 92.1 cells; Figure S4-Examples of the AU-011 EC50 binding and killing curves using 8 tumor cell lines and contributing to values supplied in Table 1; Figure S5-Example of the flow cytometry gating strategy used to obtain the values plotted in the mixed cell experiment shown in Figure 2; Figure S6-Companion confocal images showing time course uptake of AU-011 in 92.1MEL tumors implanted in the eyes of rabbits and quantified in Figure 4; Figure S7-H&E staining of 92.1MEL tumors implanted in rabbit eyes showing dose response effect of AU-011 8-9 days post-treatment.
Volume 17, Issue 2
