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Yujin Sun, Hoe Suk Kim, Phei Er Saw, Sangyong Jon,* and Woo Kyung Moon* Breast cancer stem cells (BCSCs) that exhibit a CD44+/CD24− phenotype and high aldehyde dehydrogenase (ALDH1) activity have been linked to tumor metastasis and the risk of tumor recurrence.[1] The presence of BCSC markers correlates with poor prognosis in breast cancer patients and is considered a major obstacle for curative treatments.[2] In many cases, chemotherapy or radiation therapy leaves behind increased populations of CD44+/CD24− cancer cells in the tumors of breast cancer patients, which exhibit augmented in vitro mammosphereforming ability and tumorigenicity in xenotransplantation models.[3] In that sense, it is highly demanding to develop a drug delivery system that is able to specifically target and kill BCSCs. Extra domain B of fibronectin (EDB-FN) plays an important role in tumor angiogenesis and thus it has been considered a promising target for antiangiogenic therapy.[4] Very recently, we reported, for the first time, that a BCSC line derived from a breast cancer patient expressed high levels of EDB-FN. Furthermore, using MRI, we were able to detect tumors derived from the BCSCs using EDB-FN targeting superparamagnetic iron oxide nanoparticles.[5] It suggests that EDB-FN may be a new biomarker for targeting as well as for treating BCSCs. On the other hand, we also demonstrated that EDB-FN-targeting, PEGylated liposomes encapsulating either doxorubicin or siRNA for the RhoJ gene could effectively inhibit tumor growth in murine glioma or Lewis lung carcinoma model, respectively.[6] In those studies, a peptide specific to EDB-FN, designated APTEDB, was used as a cancer-targeting ligand; the ligand was identified from phage display-based screening of a novel class of high-affinity peptides (aptides). To our knowledge, however, there have been few studies on the function of EDB-FN in BCSCs and the effect of EDB-FN gene silencing in tumors on cancer therapy. This study therefore investigated the functional significance of the EDB-FN gene in BCSCs by using BCSC targeting liposomes encapsulating EDB-FN siRNAs. Additionally, the in vivo therapeutic potential of these targeted siRNAs encapsulated within delivery liposomes was evaluated in BCSC-derived tumors. Dr. Y. Sun, Dr. H. S. Kim, Prof. W. K. Moon Department of Radiology Seoul National University Hospital 101 Daehak-ro, Jongno-gu, Seoul 110-744, South Korea E-mail: [email protected] Dr. Y. Sun Department of Radiology Yanbian University Hospital 1327 JuZi Street, Yanji City, JiLin Province 133000, China Dr. P. E. Saw, Prof. S. Jon Korea Advanced Institute of Science and Technology (KAIST) 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, South Korea E-mail: [email protected]

DOI: 10.1002/adhm.201500190

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NDY-1 cells, a putative BCSC isolated from a patient with breast cancer, have been shown to express higher quantities of EDB-FN than other breast cancer cells, including SUM-225, MCF-7, BT-474, and MDA-MB-231 cells (Figure S1, Supporting Information).[5] To determine the function of EDB-FN in NDY-1 cells, EDB-FN was specifically downregulated by treatment with EDB-FN siRNA and the levels of expression of BCSC-associated genes assayed by RT-PCR. This specific siRNA achieved effective EDB-FN knockdown at 72 h after transfection (Figure 1A). This EDB-FN knockdown led to downregulation of expression of CD44 and ALDH1, both characteristic markers of BCSCs (Figure 1B), as well as of genes related to self-renewal capacities (KLF-4, c-Myc, Oct-4, and Nanog) (Figure 1C). In addition, genes related to the epithelial–mesenchymal transition (EMT) (N-cadherin, Slug, and Twist) and drug resistance (ABCG-2) were substantially downregulated by EDB-FN siRNA (Figure 1D). Flow cytometry analysis showed that the percentage of CD44+/CD24− cells was lower in EDB-FN knockdown than in the nontreated cells (59.6% vs 81.5%; Figure 1E). Further, immunostaining showed that transfection of EBD-FN siRNA reduced the expression levels of EDB-FN, CD44, and KLF-4 (Figure 1F). In addition, knockdown of EDB-FN led to the translocation of integrin-α5, which functions as a FN receptor, from the cytoplasm to the nucleus (Figure 1F). The effects of EDB-FN knockdown on tumor cell proliferation were assessed. EDB-FN knockdown by transfection of EDB-FN siRNA significantly reduced the proliferative activity of NDY-1 cells, up to 67.2 ± 11.7% relative to control after 4 d (p = 0.003; Figure 2A). The EDB-FN-mediated activation of ERK and AKT, which are involved in the proliferation, survival, and differentiation of cancer and normal cells, can be assessed by evaluating the levels of phosphorylation of ERK and AKT. EDB-FN knockdown induced the phosphorylation of ERK, whereas the ERK inhibitor, PD98059 (10 × 10−6 M), suppressed ERK phosphorylation completely (Figure 2B). However, AKT phosphorylation was not changed by EDB-FN knockdown but was diminished by treatment with the AKT inhibitor, LY29004 (20 × 10−6 M) (Figure 2B). We also examined the effects of EDB-FN knockdown on the mammosphere-forming ability of BCSCs, considered a typical property of these cells. Compared with control cells, EDB-FN knockdown cells showed a significant reduction in the size and number of mammospheres per cell (89.8 ± 13.8% vs 141.7 ± 32.8%, p = 0.03; Figure 2D,E). We further explored whether KLF-4 overexpression could restore the mammosphere-forming ability of EDB-FN-knockdown cells. Transfection of EDB-FN siRNA decreased KLF-4 expression, whereas cotransfection of EDB-FN siRNA and the plasmid KLF-4 upregulated KLF-4 expression (Figure 2C). As expected, cells transfected with both EDB-FN siRNA and the plasmid KLF-4 showed a considerable increase in the numbers of mammospheres per well (Figure 2E, p = 0.03).

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Figure 1. A) RT-PCR analysis on the knockdown efficacy of EDB-FN siRNA, scrambled siRNA, GAPDH siRNA, and no treatment (control) in NDY-1 cells using Lipofectamine as a transfection reagent. RT-PCR analysis on genes associated with B) BCSC markers (CD44, CD24, and ALDH1A), C) self-renewal (KLF-4, c-Myc, Oct-4, and Nanog), D) EMT (N-cadherin, Slug, and Twist) and drug resistance (ABCG-2) in NDY-1 cells after transfection with EDB-FN siRNA/Lipofectamine complexes. Specific EDB-FN siRNA strongly silenced EDB-FN expression, as well as suppressing the expression of CD44, ALDH1A1-3, KLF-4, c-Myc, Oct-4, Nanog, N-cadherin, Slug, Twist, and ABCG-2 mRNAs in BCSCs. E) Flow cytometry analysis of EDB-FN knockdown cells. F) Immunofluorescence images of EDB-FN, CD44, KLF-4 and integrin-α5 expression. EDB-FN knockdown with specific siRNA downregulated expression of EDB-FN, CD44, and KLF-4 and caused integrin-α5 to localize to the nucleus. Scale bars, 20 µm.

To address the in vivo significance of our in vitro observations of EDB-FN siRNA, we subcutaneously injected both flanks of each nude mouse (three mice per group) with equal numbers (2 × 106 per mouse) of NDY-1 or EDB-FN knockdowned NDY-1 cells. The maximal tumor volumes in the control and EDB-FN siRNA-treated groups at 27 d after injection were about 500– 550 and 250–300 mm3, respectively. Xenografts in the EDB-FN siRNA-treated group grew more slowly and exhibited significantly lower tumor volumes compared with the control group (Figure 3A,B). Immunohistochemistry was utilized to investigate why EDB-FN knockdown NDY-1 cells grew more slowly in xenografts. As expected, EDB-FN was expressed strongly in the tumor sections of the control group but its expression decreased in the tumor sections of the EDB-FN siRNA-treated group (Figure 3C). Additionally, the expression of CD44 and KLF-4 was substantially lower in the EDB-FN siRNA-treated group than in the control group. Consistent with the in vitro immunostaining of integrin-α5, integrin-α5 expression was detected in the cytoplasm of the tumor sections of the control group, whereas translocation of integrin-α5 to the nucleus was observed in tumor sections of the EDB-FN siRNA. The overall preparation process of the EDB-FN-targeting, PEGylated liposome encapsulating EDB-FN siRNA (designated as APTEDB-LS-siRNAEDB) is illustrated in Figure S2 (Supporting 1676

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Information).[6] The corresponding phospholipid components were mixed at fixed molar ratios, dried to form a lipid film, and rehydrated to form APTEDB-LS-siRNAEDB. Protamine, an arginine-rich cationic protein, was used to form a complex with EDB-FN siRNA because it has been shown that it forms stable complexes with siRNAs or plasmids and exhibits relatively high gene knockdown or expression efficiency.[7] Fluorescencebased analysis using Oligreen revealed that the encapsulation of EDB-FN siRNA/protamine complexes into APTEDB-LS was optimal at the molar ratio of siRNA to protamine of 1:4. The mean sizes of the liposomes, as determined by dynamic light scattering, were 103 ± 15 nm for LS-siRNAEDB and 118 ± 4 nm for APTEDB-LS-siRNAEDB, respectively. A schematic diagram of APTEDB-LS-siRNAEDB is illustrated in Figure 4A. For facile tracking or imaging, all liposomes used herein were labeled with a fluorescent dye by incorporating rhodamine-phospholipid (Rh-DSPE) as a component during the liposome preparation. Cellular uptake and EDB-FN knockdown were examined after treatment of NDY-1 cells with Rh-APTEDB-LS-siRNAEDB and Rh-LS-siRNAEDB. The former system showed much higher cellular uptake than did the latter one (Figure 4B). Furthermore, RT-PCR analysis showed significant EDB-FN knockdown in cells treated with Rh-APTEDB-LS-siRNAEDB but not with the nontargeting, Rh-LS-siRNAEDB (Figure 4C).

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COMMUNICATION Figure 2. A) MTT assay on cell proliferation. The proliferative activity of NDY-1 cells was significantly suppressed 4 d after transfection with EDB-FN siRNA compared to the control, nontreated cells. B) Western blot analysis of EDB-FN, p-ERK/ERK, and p-AKT/AKT. EDB-FN knockdown induced marked phosphorylation of ERK but not of AKT. C) RT-PCR analysis of KLF-4. Cotransfection of the plasmid KLF-4 upregulated KLF-4 expression in EDB-FN-knockdown NDY-1 cells. D,E) Mammosphere forming assay. Mammosphere forming activity of NDY-1 cells was significantly inhibited by EDB-FN knockdown with siRNA but was partially reversed by KLF-4 overexpression, increasing mammosphere size, and number per well. Scale bars, 100 µm, * p < 0.05.

To explore the in vivo therapeutic efficacy of APTEDB-LSsiRNA, we established NDY-1 xenograft models in NOG (NOD/ Shi-scid/IL-2Rγnull) mice. Two weeks after injection of 2 × 106 NDY-1 cells, the tumors reached a volume of 200 mm3. Mice were intravenously injected with saline, EDB-FN siRNA, LSsiRNAEDB, or APTEDB-LS-siRNAEDB through the tail vein and tumor volumes were measured. Following treatment with EDB-FN siRNA and LS-siRNAEDB, the tumors grew as rapidly as those in the saline group; however, the tumor volumes of the APTEDB-LS-siRNAEDB-treated group did not increase for 5 d after treatment (Figure 5A). At 21 d after treatment, tumors were removed from mice treated with saline, EDB-FN siRNA, LS-siRNAEDB, and APTEDB-LS-siRNAEDB. The mean ± SD tumor volumes in these four groups were 853 ± 137, 763 ± 192, 718 ± 165, and 344 ± 55 mm3, respectively. The tumor volumes in the EDB FN siRNA- and LS-siRNA-treated groups were slightly lower than those of saline-treated mice, whereas those of the APTEDB-LS-siRNAEDB-treated group were much lower (Figure 5B, p < 0.001), suggesting that injection of APTEDBLS-siRNAEDB effectively knocked down EDB-FN, whereas injection of EDB-FN siRNA or LS-siRNAEDB did not suppress expression of EDB-FN. Histological analysis further showed that treatment with APTEDB-LS-siRNAEDB markedly reduced the expression of CD44 and KLF-4 and induced the translocation of integrin-α5 into the nucleus of the tumor, while the total level of immunostained integrin-α5 was similar in all groups (Figure 5C,D).

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This study has shown that EDB-FN plays important roles in the survival and growth of BCSCs, as well as in the expression of genes associated with self-renewal and those encoding surface markers. APTEDB-LS-siRNAEDB was able to selectively target EDB-FN present in the BCSC-derived tumors and further effectively knockdown expression of EDB-FN in the cancer cells. To our knowledge, this study is the first demonstration for successful treatment of BCSC tumors in vivo by targeting as well as controlling EDB-FN function. EDB-FN, which is associated with tumor angiogenesis, may be a molecular target for tumor imaging and treatment.[8] Our recent study suggested that EDB-FN might be a new biomarker for the identification of BCSCs, because EDB-FN is highly expressed in NDY-1 cells, more than in other breast cancer cell lines.[5] These earlier results prompted us to consider that EDB-FN might play a critical role in maintaining the properties of BCSCs. Cellular EDB-FN serves to poise the ligand for productive integrin-α5β1 adhesive interactions and promotes efficient capillary morphogenesis in endothelial cells.[9] siRNAmediated EDB-FN knockdown resulted in decreased VEGF expression in endothelial cells, leading to antitumor activity.[4] The present study has shown that selective EDB-FN knockdown using siRNA markedly suppressed the mammosphereforming ability of these cells, as well as their expression of BCSC markers (CD44, ALDH1), self-renewal-related genes (KLF-4, c-Myc, Oct-4, Nanog), a drug-resistance-related gene (ABCG-2), and EMT markers (N-cadherin, Slug, Twist). In

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Figure 3. A) Tumor volume in BALB/c nude mice after subcutaneous injection of NDY-1 cells and EDB-FN knockdowned NDY-1 cells. Tumors injected with EDB-FN knockdowned NDY-1 cells grew slowly and were significantly smaller in number than control tumors. B) Image of tumors isolated from BALB/c nude mice 27 d after subcutaneous injection with NDY-1 cells and EDB-FN knockdowned NDY-1 cells. C) Representative immunohistochemical staining of EDB-FN, integrin-α5, CD44 and KLF-4 in microsectioned tumors. Expression of all four proteins was lower in the EDB-FN knockdowned tumors than in the control tumors. Scale bars, 20 µm, * p < 0.05.

addition, transfection of EDB-FN siRNA into BCSCs suppressed primary tumor growth, as well as decreasing the expression of CD44 and KLF-4 and inducing the translocation of integrin-α5 into the nucleus of BCSCs. These results indicate that EDB-FN knockdown can alter the gene expression profiles involved in maintaining the specific characteristics of BCSCs, such as self-renewal and tumorigenicity. In the sense, EDB-FN knockdown by siRNA can be used as a new therapeutic approach in the treatment of EDB-FNpositive BCSC-derived tumors by abrogating the self-renewal capacity of BCSCs. Despite progress in siRNA-associated therapeutic strategies, systemic siRNA therapy remains hampered by barriers that prevent siRNAs from reaching their intended targets in the cytoplasm and exerting their gene silencing activity. Liposomes modified with peptide targeting ligands and polyethylene glycol may be a good carrier for siRNA delivery due to their selective targeting and long circulation time, thus improving the clinical outcomes of nanomedicine.[10] Our previous study demonstrated that the uptake of

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Figure 4. A) A schematic depiction of rhodamine-labeled APTEDB-LS-siRNAEDB (Rh-APTEDB-LSsiRNAEDB). B) Fluorescence images of the cellular uptake of Rh-LS-siRNAEDB or Rh-APTEDBLS-siRNAEDB. Effective uptake of Rh-APTEDB-LS-siRNAEDB by NDY-1 cells was observed at 4 h after treatment. C) RT-PCR analysis of EDB-FN mRNA in NDY-1 cells treated with Rh-APTEDBLS-siRNAEDB or Rh-LS-siRNAEDB. Rh-APTEDB-LS-siRNAEDB strongly suppressed EDB-FN mRNA expression, whereas Rh-LS-siRNAEDB did not. Scale bars, 20 µm.

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COMMUNICATION Figure 5. A) Volumes of subcutaneous xenograft tumors in NOG mice after systemic treatment with EDB-FN siRNA, LS-siRNAEDB, or APTEDB-LSsiRNAEDB. Three intravenous injections (arrows) of APTEDB-LS-siRNAEDB (3 mg kg−1) effectively suppressed tumor growth when compared with injection of EDB-FN siRNA, LS-siRNAEDB, or saline. B) Images of tumors isolated from NOG mice at 21 d after systemic treatment with EDB-FN siRNA, LS-siRNAEDB, or APTEDB-LS-siRNAEDB. The tumors isolated from APTEDB-LS-siRNAEDB-treated mice were significantly smaller than those of the other groups. Scale bars, 1 cm. C) Representative immunohistochemical staining of EDB-FN, integrin-α5, CD44, and KLF-4 in tumors treated with EDB-FN siRNA, LS-siRNAEDB, or APTEDB-LS-siRNAEDB. Scale bars, 20 µm. D) Quantitative analysis of immunohistochemical staining proportion of tumor sections. Intravenous injection of APTEDB-LS-siRNAEDB effectively knockdowned EDB-FN expression in tumor tissues. Significant decreases in CD44 and KLF-4 expression and integrin-α5 translocation were observed in APTEDB-LS-siRNAEDB-treated tumors compared with the other groups. *p < 0.05.

APTEDB-LS was clearly detected in only the allograft tumor which overexpressed EDB-FN after intravenous injection, indicating the highly specific and selective targeted version of APTEDB-LS in vivo.[11] This study describes the development of APTEDB-LSsiRNAEDB, which specifically targets EDB-FN-positive BCSCs and enhances cellular uptake and delivery of EDB-FN siRNA. Compared with nontargeted liposomes, three intravenous injections of APTEDB-LS-siRNAEDB showed enhanced cell and tissue penetration and achieved effective EDB-FN knockdown in vitro and in vivo, resulting in effective targeted therapy for BCSC tumors. Therefore, it is anticipated that therapeutic efficacy may be further enhanced by simultaneously delivering siRNAs and chemical drugs effective to BCSCs using the present EDB-FN targeting liposome. In summary, our findings suggest that APTEDB-LS-siRNAEDB is a promising therapeutic system, enabling simultaneous EDB-FN targeting and siRNA therapy for BCSC tumors with high EDB-FN expression. The liposomal system targeting EDB-FN presented herein may have warrant applications to treat other types of aggressive tumors that express EDB-FN. Further studies using an active EDB-FN targeted liposome system are needed to improve the therapeutic efficacy, by synergistically delivering higher concentrations of siRNAs and anticancer drugs, and with long-duration circulation properties and maximal tumor distribution.

Experimental Section Experimental details are included in the Supporting Information.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

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Acknowledgements Y.S., H.S.K., and P.E.S. contributed equally to this work. Y.S., H.S.K., P.E.S., S.J., and W.K.M. conceived and designed the experiments. Y.S., H.S.K., and P.E.S. performed the experiments and analyzed the data. P.E.S. and S.J. contributed reagents/materials/analysis tools. Y.S., H.S.K., P.E.S., S.J., and W.K.M. contributed to the writing of the manuscript. This study was supported by the National R & D Program for Cancer Control, Ministry of Health & Welfare (0920030). Received: March 17, 2015 Revised: May 20, 2015 Published online: June 11, 2015

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Targeted Therapy for Breast Cancer Stem Cells by Liposomal Delivery of siRNA against Fibronectin EDB.

Targeted therapy for breast cancer stem cell (BCSC): A novel liposomal system (APTEDB -LS-siRNA(EDB) ) that enables simultaneous targeting and knockdo...
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