Article Figures & Data
Figures
- Figure 1.
A, patient sample no 13 (right) and JIMT-1 pleural effusion explant (left) were sorted for CD24-negative and CD44-positive cells and seeded at 25,000 cells per well on 24-well plates. Twenty-four hours after infection with 5 VP per cell of TSP-driven luciferase-expressing viruses, cells were lysed and luciferase activity was determined. Results are presented as relative values of TSP-driven adenoviruses compared with CMV-driven virus. For comparison, CD24+ JIMT-1 cells were analyzed for TSP expression (center). Insert, RT-PCR analysis of CD44+CD24low/− JIMT-1 cells for β-actin (350 bp), hTERT (462 bp), mdr (373 bp), cox-2 (482 bp), and ala (393 bp). All were positive except the negative control (neg ctrl). B, CD44+CD24low/− and CD24+ JIMT-1 cells were infected with 500 VP per cell of Ad5, a virus featuring serotype 5/3 chimeric capsid or a RGD-4C modified capsid. Twenty-four hours after infection, cells were lysed and luciferase activity was determined and normalized to the cellular protein content. Columns, mean; bars, SE. C, oncolytic adenoviruses featuring the TSPs and 5/3 modification of the capsid were constructed. All viruses have intact E3 except for Ad5/3-hTERT-Δgp, which is deleted for gp19K. For additional tumor selectivity, Δ24 viruses have a 24-bp deletion in the constant region 2 of E1A, which renders them unable to bind Rb, and therefore, these viruses replicate selectively in p16-Rb pathway–deficient cells.
- Figure 2.
Primary breast cancer pleural effusion samples from 3 different patients (A–C) were sorted for CD24-negative and CD44-positive cells and seeded at 20,000 cells per well on 96-well plates. After infection with promoter-driven oncolytic viruses, cells were checked daily by light microscopy, and MTS cell viability assay was performed when the most potent virus had lysed virtually all cells with the second lowest viral concentration. Results are presented as relative survival compared with mock-infected cells. Points, mean; bars, SE. ***, P < 0.001 versus Ad5wt.
- Figure 3.
JIMT-1 pleural metastasis explant cells were sorted for CD24-negative and CD44-positive cells and seeded at 20,000 cells per well on 96-well plates. After infection with promoter-driven oncolytic viruses in triplicates, daily MTS (A) or trypan blue cell viability assays (B) were performed. Results are presented as relative survival compared with mock-infected cells. Points, mean; bars, SE. ***, P < 0.001 versus mock.
- Figure 4.
To assess antitumor efficacy, CD44+CD24−/low cells were injected into mammary fat pads of nude mice and tumors were allowed to grow for 14 d, followed by intratumoral injection of viruses or mock every second day until all mock mice died (A). Data points represent relative tumor volume of eight tumors per group compared with mean tumor volume at first day of treatment. Points, mean; bars, SE. ***, P < 0.001 versus mock. For evaluation of the effect of the viruses on CD44+CD24−/low cells, tumors were allowed to grow for 14 d, followed by intratumoral injection of virus or mock thrice weekly (B). Mock or Ad5/3-mdr-Δ24 (active in CD44+CD24−/low cells) and mock or Ad5/3-ala-Δ24– treated tumors (not active in CD44+CD24−/low cells; n = 7 per group) were collected for FACS analysis after 17 d of treatment (C). Numbers, percentage.
Volume 68, Issue 14
