Antitumor effects of NGR-peptide targeted liposomal doxorubicin against human solid tumors

A21 Purpose: NGR-targeted doxorubicin (DOX)-entrapping liposomes (TVT-DOX) displayed specific binding to a CD13 isoform expressed on the vasculature of solid tumors, leading in turn to its regression and to a prolonged survival of tumor xenografts. In this work, we validated the in vivo anti-tumor activity of TVT-DOX on lung, ovarian and neuroblastoma (NB) human cancers. Experimental Design: Athymic mice were injected either intrapulmonary with the lung carcinoma cell line Colo-699N, or intraperitoneally with the ovarian carcinoma cell line OVCAR-3, or orthotopically in the capsule of the left adrenal gland with the NB cell line GI-LI-N, and treated, at different days post cell inoculation, with 5mg/kg (MTD) or with 2/3 or 1/3 of MTD of DOX, free or encapsulated in non targeted (Caelyx) or TVT-DOX, once a week for 5 weeks. Survival time was used as the main criterion for determining treatment efficacy. The visualization of the minimal residual disease (MRD) after NB surgical removal and the response to TVT-DOX therapy have been also quantified by bioluminescence (BLI) and with a magnetic resonance scanner at 1.5T (MR) before and after gadolinium injection. Histological (IHC) evaluation of cryopreserved neoplastic tissues have been carried out to assess the impact of TVT-DOX on tumor cell proliferation, viability and apoptosis, while its anti-angiogenesis-specific responses have been evaluated by both IHC analysis and the use of the chick embryo chorioallantoic membrane (CAM) assay. Results: Statistically significant improvement in anti-tumor effects was seen in all three tumor-bearing animal models treated with TVT-DOX, when compared to control mice or mice treated with free DOX or Caelyx at the MTD. Significant differences, in term of increased life span, between mice treated with TVT-DOX and Caelyx were seen also in NB- and ovarian-bearing mice when treated with 1/3 of MTD of DOX, while, at this dose, anti-tumor effects against lung carcinoma-bearing mice were similar for both the formulations used. Long term survivors were obtained only in NB-bearing animals treated with TVT-DOX and confirmed by BLI and MR evaluations. TVT-DOX treatment resulted in a dramatic inhibition of tumor endothelial cell density for all the three animal models examined. IHC evaluation of cryopreserved tumor mass, performed at the end of treatments, clearly indicated inhibition of cell proliferation with an enhanced induction of cell apoptosis in mice treated with TVT-DOX with respect to untreated mice or those treated with Caelyx. Moreover, a statistically different suppression of blood vessel density was seen in mice treated with the NGR-targeted therapy compared to Caelyx. CAMs implanted with xenografts derived from the three solid tumors showed numerous allantoic vessels radiating in a “spoked wheel” pattern towards the specimens. Incubation with TVT-DOX led to a significant reduction of microvessel area with respect to both untreated and Caelyx-treated specimens. Conclusions: In lung cancer animal model, the anti-tumor efficacy of TVT-DOX is only slightly significantly higher, with respect to Caelyx, at the MTD. TVT-DOX works better than Caelyx in NB model, independently of the dose used and of the beginning of treatment. Same conclusion is valid for ovarian cancer, but only when treatment starts after 7 days from cell inoculation. F. P. and G. P. supported by Italian Neuroblastoma Foundation.