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  • br Collectively these results indicate

    2020-08-18


    Collectively, these results indicate that the AmiC combines GL21.T and miR-137 functions, inducing an impaired cell motility (mediated by the aptamer moiety) and viability (mediated by the miR moiety).
    GL21.T-137-Mediated Activity in Patient-Derived NSCLC Cells and In Vivo
    In order to extend the AmiC functional characterization, we deter-mined the effects of the GL21.T-137 in Axl+ primary Phenylacetic Acid (NSCLC #17 and #18) obtained from surgical specimens from lung cancer patients (Figure S4). Cells were treated with 400 nmol/L GL21.T aptamer or GL21.T-137 complex and the effects on cell viability and migration were analyzed. As shown in Figure 5, the aptamer and the complex treatments efficiently hamper cell migration (Figures 5A and 5B), reaching about 40% of inhibition in both cell lines as compared to a control aptamer or a control complex. Further, the GL21.T-137 complex significantly reduces cell viability in both cell lines (Figures 5C and 5D), confirming GL21.T-137 functional activity on NSCLC. 
    Next, to assess the effect of the GL21.T complex on tumor growth in vivo, A549 tumor-bearing mice were treated with the GL21.T or GL21.T-
    137 AmiC (1,600 pmol/injection) by intraperitoneal administration. As shown (Figure 5E), after 1 week of treatment, the GL21.T treatment induces a significant reduction of tumor growth that is further inhibited by the GL21.T-137 complex. In addition, histopathological analyses revealed that, as compared to the control Phenylacetic Acid group, the GL21.T-137-treated tumors showed a reduction of the cellularity (Figure 5F).
    DISCUSSION
    The use of aptamers for targeted delivery of interfering RNA mole-cules (siRNAs, miRs, and miR inhibitors) is emerging as a promising therapeutic strategy in cancer, aimed to enhance the therapeutic effi-cacy, reducing the occurrence of unwanted off-target effects on healthy tissues.23 In a previous study, we developed the GL21.T aptamer, demonstrating its ability to bind and inhibit Axl receptor activity, reducing cell migration and tumor growth.21 Recently, we have described that an AmiC (named GL21.T-137) containing the GL21.T aptamer and miR-137 is able to effectively reach and inhibit glioma stem cells phenotype in a receptor-dependent manner. The AmiC was shown to remain stable up to 8 h in 80% serum.18
    Here, we address the therapeutic applicability of the GL21.T-137 complex to NSCLC, demonstrating its ability to selectively bind to Axl-expressing NSCLC cells (Figure 1) and produces within target cells a functional miR-137 (Figure 2), thus leading to impairment of cell migration and viability in vitro and altering of tumor growth in vivo (Figures 3, 4, and 5). Notably, compared to miR mimic trans-fection, the treatment permits the same extent of inhibition with a more physiological level of miR, thus greatly reducing the potential occurrence of off-target effects.
    Molecular Therapy: Nucleic Acids
    A
    B
    measured by MTT assay and expressed as percentage of
    viable cells relative to untreated (“ ,” for treatment) or ctrl-
    miR (for transfection). Error bars depict mean ± SD. Sta-
    immunoblotted with anti-cycD1, anti-phospho-Rb (pRb),
    and anti-vinculin (Vinc) antibodies. Values below the blots
    indicate normalized quantization of signal levels relative to
    untreated (“ ,” for treatment) or ctrl-miR (for transfection).
    Molecular weights of indicated proteins are reported.
    Our data reveal the therapeutic potential of the GL21.T-137 in NSCLC, showing its ability to act as a multifunctional molecule affecting both cell survival and migration through the combination of the Axl-signaling inhibition, mediated by the GL21.T aptamer, and the anti-tumor growth properties of the miR-137. The used AmiC thus gives the possibility to have different functional effects in a unique molecule, well-fitting with the increased interest in the use of combination therapies in cancer.
    The two moieties of the GL21.T-137 complex have attractive potential for NSCLC therapeutic targeting. The GL21.T aptamer targets the re-ceptor tyrosine kinase Axl, whose expression has been correlated to ma-lignant progression and metastasis in lung cancer.24–26 Further, it has been demonstrated to be a fundamental role of Axl in regulating lung cancer cell invasion and metastasis27,28 and its involvement in the resistance to tyrosine kinase inhibitors.29,30 These studies highlight the therapeutic potential of the Axl-targeting therapies for NSCLC. On the other hand, it has been demonstrated that miR-137 acts as a tu-mor suppressor in NSCLC, being downregulated in NSCLC tissue sam-ples and inhibiting cell proliferation of NSCLC cells when increased.7,8