Article Figures & Data
Figures
- Figure 1.
iRGD inhibts spontaneous metastasis in a prostate cancer mouse model. A, neuropilin and αv integrin expression in GFP-PC-3 human prostate cancer cells analyzed by flow cytometry. The profiles represent the values of cells incubated with isotype control (red) or appropriate neuropilin (NRP) or integrin antibodies (blue) as primary antibodies. B–D, mice bearing orthotopic GFP-PC-3 tumors implanted 2 weeks earlier received intravenous injections of 4 μmol/kg of iRGD, a scrambled iRGD with a disrupted CendR motif (iRGDD: CRGDDGPKC), or a conventional non-CendR RGD peptide (RGDfV), or PBS, every other day for 21 days. The mice were necropsied after the treatment and viewed under a fluorescence imager (B). PT, primary tumor; L, liver. Metastatic burden was analyzed by quantifying fluorescence intensity with ImageJ (C). Weight of primary tumors (D). n = 5 per group. One of three experiments that gave similar results is shown. Error bars, mean ± SEM. Statistical analyses were performed with ANOVA: n.s., not significant; *, P < 0.05; **, P < 0.01.
- Figure 2.
iRGD inhibits spontaneous metastasis in a pancreatic cancer mouse model. Mice bearing orthotopic LM-PmC mouse pancreatic tumors implanted 1 week earlier received intravenous injections of 4 μmol/kg of iRGD, a conventional non-CendR RGD peptide (CRGDC), iRGDD, or iNGR (CRNGRGPDC), or PBS, every other day for 14 days. n = 10 per group. One of two experiments that gave similar results is shown. A, the mice were necropsied after the treatment and viewed under a fluorescence imager. PT, primary tumor; S, stomach. B, metastatic burden analyzed by quantification of fluorescence intensity with ImageJ. C, weight of primary tumors. Error bars, mean ± SEM. Statistical analyses were performed with ANOVA: n.s., not significant; *, P < 0.05; **, P < 0.01.
- Figure 3.
iRGD inhibits tumor cell migration in Transwell assays. LM-PmC (A and C) or GFP-PC-3 (B and C) cells were seeded on the upper side of a Transwell filter, and the number of cells that migrated to the other side of the filter was quantified. A and B, iRGD, non-CendR RGD peptides (CRGDC and RGDfV), or non-RGD CendR peptides (iNGR: CRNGRGPDC or RPARPAR) at a final concentration of 10 μmol/L or PBS was added to both upper and lower wells. C, iRGD or CRGDC at a final concentration of 10 μmol/L or PBS was added only to lower wells. Anti-NRP-1 b1b2 or control IgG was added to some of the wells. n = 3 per experiment. Nontreated columns were considered as 100%. Error bars, mean ± SEM; statistical analyses, ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistics against the nontreated columns are shown unless otherwise noted.
- Figure 4.
iRGD repels tumor cells. A, iRGD-coated silver nanoparticles (iRGD-AgNPs) or CRGDC-AgNPs were blotted on glass surfaces, and cells were seeded in the center in close proximity with the AgNPs. Cells were imaged for 48 hours. Representative images at 0, 24, and 48 hours are shown. The dark areas are coated with AgNPs. The dotted lines show the edge of the migrating cell populations. Note that the cells are repelled by iRGD-AgNPs, while they progressively migrate over CRGDC-AgNPs and completely saturate the field in 48 hours. n = 5. B, cells were seeded on glass surfaces densely coated with the indicated AgNPs and imaged for 48 hours. The speed of cell migration was quantified by measuring percentage of AgNP area covered by the cells. Error bars, mean ± SEM; statistical analyses, ANOVA; *, P < 0.05; ***, P < 0.001.
- Figure 5.
β1 integrin-dependent tumor cell attachment to fibronectin. A, LM-PmC (closed bars) or GFP-PC-3 (open bars) cells were seeded into 96-well plates coated with fibronectin, and allowed to attach for 30 minutes at 37°C in the presence of an anti-β1 integrin subunit antibody or a control IgG. The number of cells that remained attached to the wells were quantified. Cell attachment with no antibody was considered as 100%. n = 3. Error bars denote mean ± SEM. Statistical analyses were performed with the Student t test: **, P < 0.01; ***, P < 0.001. B, expression of total β1 integrins, β1 integrins in active conformation, and α5β1 integrin in LM-PmC and GFP-PC-3 cells analyzed by flow cytometry. The profiles represent the values of cells incubated with isotype control (red) or appropriate anti-β1, anti–active β1, or anti–α5β1 primary antibodies (blue).
- Figure 6.
iRGD inhibits LM-PmC cell attachment to fibronectin in a NRP-1–dependent manner. A and B, cell attachment assays. The number of LM-PmC cells that attached to fibronectin-coated wells in the presence of various peptides was quantified. A, iRGD, cleaved iRGD with an inactive RGD motif (CRGDK), non-RGD CendR peptides (iNGR: CRNGRGPDC or RPARPAR), and a RPARPAR variant that lacks affinity to NRPs (RPARPAR-NH2) were used. B, a conventional non-CendR RGD peptide (CRGDC) and its non–integrin binding variant (CRGEC) were used. Some cells were also treated with anti-NRP-1 b1b2 or control IgG. n = 3 per experiment. Nontreated columns were considered as 100%. Error bars, mean ± SEM; statistical analyses, ANOVA; n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistics against nontreated columns are shown unless otherwise noted. C, cell retraction assays. LM-PmC cells (red) cultured on fibronectin-coated coverslips were treated with 10 μmol/L of the indicated peptides for 1 hour, fixed, and stained for phospho-paxillin (green) and nuclei (blue). Representative confocal micrographs from three independent experiments are shown. Scale bars, 20 μm.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figures 1-7 - Supplementary Figures 1-7. Supplemental Figure 1: Metastatic burden and primary tumor weight after long term in vivo peptide treatment Supplemental Figure 2: In vitro and in vivo targeting of pancreatic tumor cells by iRGD and iNGR Supplemental Figure 3: Inhibition of tumor cell attachment to vitronectin by RGD peptides Supplemental Figure 4: In vitro cell toxicity of CendR peptides Supplemental Figure 5: Chemorepulsion assay design Supplemental Figure 6: Inhibition of NRP-1-dependent GFP-PC-3 cell attachment to fibronectin Supplemental Figure 7: Effects of CendR peptides on tumor cell attachment to collagen type-1 Legends of Supplementary Movies SM1 through SM5
- Supplemental Table 1 - Supplemental Table 1. Summary of statistical analysis
- Supplementary Movie SM5 - Supplementary Movie SM5. Tumor cell migration on plain silver nanoparticles
- Supplementary Movie SM4 - Supplementary Movie SM4. Tumor cell migration on CRGDC silver nanoparticles
- Supplementary Movie SM3 - Supplementary Movie SM3. Tumor cell repulsion from iRGD silver nanoparticles
- Suuplementary Movie SM2 - Suuplementary Movie SM2. Collapse of cellular protrusions upon interaction with iRGD silver nanoparticles
- Supplementary Movie SM1 - Supplementary Movie SM1. Tumor cell migration on iRGD silver nanoparticles
Volume 14, Issue 1
