Abstract 5812: Development of αVβ5 + exosomes for liver-targeted therapy

Abstract

Abstract Liver metastases are common in pancreatic and colorectal cancer. Their development is promoted by tumor-secreted exosomes that, thanks to the presence of the integrin αVβ5 on their membrane, home to the liver where they increase TGF-ß production by Kupffer cells, liver-resident macrophages, initiating the formation of the pre-metastatic niche. Considering that anti-TGF-β therapies have not been efficient in clinical trials or cause side-effects, our aim is to develop a liver-targeted anti-TGF-ß treatment using functionalized αVβ5 + exosomes. We selected 293T cells as a human, non-cancerous cell line to obtain exosomes. 293T have an endogenous expression of αVβ5, and exosomes were isolated using differential centrifugation of conditioned media. The vesicles obtained had a diameter of 20 to 120nm according to dynamic light scattering and electron microscopy, as expected for exosomes, and Western blot confirmed the presence of the exosome marker CD81. When fluorescently labeled exosomes (SP-DiIC18) were inoculated in the tail vein of Balb/C mice, they were detected in the liver 24 hours later. To increase the homing of 293T cell exosomes to the liver, we decided to overexpress αVβ5. 293T cells were co-transfected with plasmids coding the αV and β5 subunits. After antibiotic and clonal selections, we obtained 293T cells stably overexpressing αVβ5 (8.9x vs. parental), and isolated exosomes contained the exogenous integrin. In vitro, the internalization of αVβ5 + exosomes was increased in RAW264.7 macrophages compared to exosomes from parental 293T cells. In mice, we confirmed that the overexpression of αVβ5 increases the homing of the exosomes to the liver (4.9x vs. parental) and that αVβ5 + exosomes preferentially home to the liver when compared to lungs, kidneys, and brain. To functionalize the exosomes, we chose to express, in 293T cells, anti-TGF-ß factors that could be directly loaded into their exosomes before their isolation. First, we transduced 293T cells to express the mRNA of a soluble form of betaglycan (sBG) that can neutralize TGF-ß. sBG mRNA was detected in 293T cells, their exosomes, and in RAW264.7 cells treated with these exosomes. Low levels of sBG protein could be detected in the conditioned media of treated RAW264.7, but it did not prevent SMAD2/3 phosphorylation in HepG2 hepatocarcinoma cells, which could be due to the low levels of sBG mRNA in the exosomes. Since larger RNAs have been reported to be less transferred into exosomes, we used 2 shRNA to knock down Tgfb1. Although both shTgfb1 could efficiently knock down Tgfb1 in transduced cells, the exosomes of 293T cells expressing these shTgfb1 did not decrease Tgfb1 levels in treated RAW264.7 cells. Overall, although the functionalization of exosomes to target TGF-ß remains to be achieved, our results show that αVβ5 + exosomes are nanocarriers that could be used for liver-targeted therapies. Citation Format: Paloma Acosta Montaño, Eréndira Olvera Félix, Verónica Castro Flores, Patricia Juárez, Pierrick G.J. Fournier. Development of αVβ5 + exosomes for liver-targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5812.