Pulmonary Delivery of Engineered Exosomes to Suppress Postoperative Melanoma Lung Metastasis through Preventing Premetastatic Niche Formation

16 Premetastatic niche (PMN) is a prerequisite for initiation of tumor metastasis. Targeting prevention 17 of PMN formation in distant organs is becoming a promising strategy to suppress metastasis of primary 18 tumor. Based on “organotropic metastasis”, melanoma tends to metastasize to lungs, where 19 granulocytic myeloid-derived suppressor cells (G-MDSCs) recruitment in lungs significantly 20 contributes to the PMN formation. Herein, functional exosomes ( G Exo I ) were designed to present 21 pulmonary targeting peptide GFE1 on the membrane and load PI3Kγ inhibitor (IPI549) inside, aiming 22 at suppressing postoperative lung metastasis of melanoma. In postoperative mice model, intravenously 23 injected G Exo I could significantly accumulate in lungs and release IPI549 to block G-MDSCs 24 recruitment through interfering with CXCLs/CXCR2/PI3Kγ signaling. The increased percentages of CD4 + T cells and CD8 + T cells in lungs could transform microenvironment from immunosuppression to immunostimulation, leading to metastasis inhibition. This study suggests an effective anti-metastasis strategy of targeting prevention of PMN formation through specifically blocking G-MDSCs recruitment.


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Dynamic light scattering (DLS) analysis showed that BExo, GExo, BExo I and GExo I had similar 131 hydrodynamic sizes of 116±3.3, 109±4.9, 110.4±5.6 and 114.3±4.1 nm respectively (Fig. 2a). Western 132 blot analysis revealed that BExo, GExo, BExo I and GExo I contained the abundant exosome marker 133 proteins, such as TSG101, CD9 and CD63 (Fig. 2b), suggesting that the successful isolation of 134 exosomes from MSCs. To confirm GFE-1 was located on the surface of exosomes, the binding ability 135 of BExo I and GExo I to His-tagged recombinant DPEP1 that is the receptor for the lung-targeting peptide 136 GFE1 was detected by assessing the levels of His-tag in BExo I and GExo I . Western blot analysis showed 137 that GExo I could strongly bind to DPEP1, but no detectable binding was observed in BExo I group (  Fig. 3 and 4). GFE1 peptide has been reported to efficiently target pulmonary 156 vascular [44]. In order to explore the ability of GExo I targeting to pulmonary vascular, pulmonary the highest one in the liver ( Fig. 4a and b). This result reveals the excellent pulmonary targeting ability secretions was intravenously injected into normal C57BL/6 mice, and free IPI549, BExo I or GExo I was 234 injected intravenously at the same time, followed by intravenous injection of B16/F10 cells (Fig. 5a).

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The PMN characteristic gene expression in lungs, including Bv8, S100a8, S100a9, and MMP9, which increased the expression of Bv8, S100a8, S100a9, and MMP9 in the lung than that of normal mice 239 ( Supplementary Fig. 9). However, additional treatment with free IPI549, BExo I or GExo I decreased 240 their expression, where GExo I exhibited the most potent performance, suggesting GExo I can effectively 241 inhibit lung PMN establishment. Furthermore, the metastatic nodules in the lung of mice were counted.

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Only TCM-treated mice showed the most metastatic nodules, accelerating the metastasis, but the 243 further treatment with IPI549, BExo I or GExo I induced the reduced metastatic nodules, showing 244 inhibited metastasis (Fig. 5b and 5c). Importantly, GExo I triggered the least metastatic nodules, 245 exhibiting the best inhibitory activity against metastasis. All above results indicate that GExo I can 246 significantly inhibit tumor metastasis by blocking PMN formation in the lung.  (Fig. 5d). Compared to the mice treated with free IPI549, the number of macroscopic 270 metastatic nodules of both exosomes-treated mice was dramatically decreased, where GExo I still 271 showed more significant metastases inhibition than BExo I (Fig. 5e and 5f). From the hematoxylin-eosin 272 (H&E) staining of the lung metastatic lesions (Fig. 5g) Fig. 6a and Supplementary Fig. 10 show that IPI549 and BExo I could reduce the 287 percentage of G-MDSCs in premetastatic lungs, but GExo I even decreased the percentage close to the 288 normal level of healthy mice, which is because on the one hand GExo I has a longer circulation time than 289 IPI549, capable of more effectively blocking the chemotactic migration of G-MDSCs, and on the other 290 hand, GExo I has a higher drug delivery efficiency than BExo I , thus releasing the stronger 291 anti-recruitment ability. Moreover, the expression of PI3Kγ in G-MDSCs from premetastatic lungs of 292 mice with different treatments was observed to show the same trend as the percentage of G-MDSCs did 293 (Fig. 6b). were increased, compared with that of PBS-treated mice ( Fig. 6d and Supplementary Fig. 12),

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indicating that the reduction of G-MDSCs can achieve the remodeling of acquired immunity. Moreover, 306 the percentage of CD8 + T cells in the lungs of the GExo I -treated mice increased most significantly (Fig.   307 6d and Supplementary Fig. 13). Based on the above results, the composition ratio of various immune

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The loading capacity and encapsulation efficiency of IPI549 were calculated using the following 407 formula, where W1 was the weight of the IPI549 enveloped in the GExo I , W was the weight of the 408 exosomes, and W0 was the initial amount of IPI549 added to the culture medium.