Integrin α_vβ₃ is overexpressed on neoendothelial cells and frequently on tumor cells. We have developed a peptide-like scaffold (regioselectively addressable functionalized template, RAFT), which holds four cyclo(-RGDfK-) (cRGD) motifs and proved that this molecule (called regioselectively addressable functionalized template-arginine-glycine-aspartic acid, RAFT-RGD) targets integrin α_vβ₃ in vitro and in vivo. Using fluorescence correlation spectroscopy (FCS), we measured the constant of affinity (K_D) of the RAFT-RGD for purified integrins. K_D values rose from 3.87 nmol/l for RAFT-RGD to 41.70 nmol/l for cyclo(-RGDfK-). In addition, RAFT-RGD inhibited α_vβ₃ lateral mobility in the cell membrane, probably due to the formation of integrin clusters as demonstrated by fluorescence recovery after photobleaching (FRAP). This was confirmed by electronic microscopy data, which established the formation of molecular complexes containing two integrins in the presence of RAFT-RGD but not cRGD or regioselectively addressable functionalized template-arginine-alanine- aspartic acid (RAFT-RAD). Using an enzyme-linked immunosorbent assay (ELISA), we proved that 1 µmol/l RAFT-RGD increased by 79% α_vβ₃ internalization via clathrin-coated vesicles. Conversely, cRGD was internalized without modifying α_vβ₃ internalization. Although RGD has been known for >20 years, this is the first study to formerly establish the relationships among multimeric presentation, increased affinity, and subsequent integrin-mediated cointernalization. These results strongly support the rationale for using multimeric RGD-peptides as targeting vectors for imaging, diagnosis, or therapy of cancers.

Advanced ovarian cancer is the most lethal gynecological cancer, with a high rate of chemoresistance and relapse. Photodynamic therapy offers new prospects for ovarian cancer treatment, but current photosensitizers lack tumor specificity, resulting in low efficacy and significant side-effects. In the present work, the clinically approved photosensitizer verteporfin was encapsulated within nanostructured lipid carriers (NLC) for targeted photodynamic therapy of ovarian cancer. Cellular uptake and phototoxicity of free verteporfin and NLC-verteporfin were studied in vitro in human ovarian cancer cell lines cultured in 2D and 3D-spheroids, and biodistribution and photodynamic therapy were evaluated in vivo in mice. Both molecules were internalized in ovarian cancer cells and strongly inhibited tumor cells viability when exposed to laser light only. In vivo biodistribution and pharmacokinetic studies evidenced a long circulation time of NLC associated with efficient tumor uptake. Administration of 2 mg·kg⁻¹ free verteporfin induced severe phototoxic adverse effects leading to the death of 5 out of 8 mice. In contrast, laser light exposure of tumors after intravenous administration of NLC-verteporfin (8 mg·kg⁻¹) significantly inhibited tumor growth without visible toxicity. NLC-verteporfin thus led to efficient verteporfin vectorization to the tumor site and protection from side-effects, providing promising therapeutic prospects for photodynamic therapy of cancer.

Several abnormalities in the insulin-like growth factor-1 (IGF1) and erbB receptors pathways stimulate the growth and survival of lung cancer cells, but their mechanisms of action and cooperation are poorly understood. In this report, we have identified a new mechanism of apoptosis inhibition by amphiregulin through an IGF1-dependent survival pathway in non-small cell lung cancer (NSCLC) cells: amphiregulin activates the IGF1 receptor that in turn induces the secretion of amphiregulin and IGF1. In the absence of serum, the NSCLC cell line H358 resists apoptosis and secretes factors protecting the NSCLC cell line H322 from serum deprivation apoptosis. IGF1 receptor inhibitor AG1024 as well as epidermal growth factor receptor inhibitors AG556 and ZD1839 restore apoptosis in H322 cells cultured in H358-conditioned medium. Accordingly, the anti-apoptotic activity of H358-conditioned medium is completely abolished after incubation with anti-amphiregulin neutralizing antibody and only partially with anti-IGF1 neutralizing antibody. H358-conditioned medium and amphiregulin induce IGF1 receptor phosphorylation in H322 cells, which is prevented by anti-amphiregulin neutralizing antibody but not by AG556 or ZD1839. H358 cells secrete a high level of amphiregulin that, in combination with IGF1, prevents serum deprivation apoptosis. Finally, IGF1 receptor inhibitor blocks amphiregulin and IGF1 release by H358 cells.