Research ArticleCancer Therapy

FolamiRs: Ligand-targeted, vehicle-free delivery of microRNAs for the treatment of cancer

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Science Translational Medicine  02 Aug 2017:
Vol. 9, Issue 401, eaam9327
DOI: 10.1126/scitranslmed.aam9327
  • Fig. 1. Specificity of FolamiR uptake in cancer cells in culture.

    (A) Proposed mechanism of action of FolamiRs. (B) Identification of FRα in FR+ MB-231 breast cancer cells and in FR A549 lung cancer cells. Histograms represent overlaid flow cytometry data as a percentage of unstained, FRα+, and isotype control–stained cells. (C) NIR-FolamiR-34a uptake in FR+ MB-231 cells compared to FR A549 cells. Histograms represent overlaid flow cytometry data as a percentage of unstained and NIR-FolamiR-34a (50 nM)–stained cells. (D) Fol-FITC uptake in FR+ MB-231 cells compared to FR A549 cells. Scale bars, 50 μm. (E) Targeted silencing of miR-34a Renilla sensor using FolamiR in MB-231 and A549 cells in vitro. Data points were normalized to FolamiR-NC (negative control: scrambled miRNA) for each time point. Error bars: means ± SD. Each experiment corresponds to n = 3 with at least four technical replicates per treatment. **P < 0.01, *P < 0.05, one-way analysis of variance (ANOVA) and Bonferroni correction. ns, not significant.

  • Fig. 2. Cellular response to FolamiRs in vitro.

    (A) Targeted silencing of miR-34a Renilla sensor using FolamiRs in MB-231 breast cancer cells. Data points were normalized to FolamiR-NC (negative control: scrambled miRNA) for each time point. (B) Proliferation of MB-231 cancer cells as a function of FolamiR treatment (50 nM). Data points were normalized to FolamiR-NC for each time point. Error bars: means ± SD. Each experiment corresponds to n = 3 with at least three technical replicates per treatment. (C) Dose response of MB-231 cells to FolamiR-34a. Renilla values were measured 96 hours after treatment. Data points were normalized to FolamiR-NC. Duplex miRNA represents unconjugated miR-34a. Error bars: means ± SD. Each experiment corresponds to n = 3 with at least four technical replicates per treatment. (D) Displacement of NIR-FolamiR-34a binding from MB-231 cells (50 nM, 4°C) with increasing concentrations of folate-glucosamine conjugate. Histograms represent overlaid flow cytometry data as a percentage of unstained and NIR-FolamiR-34a–stained cells. (E) FolamiR-34a competition assay in MB-231 breast cancer cells in vitro. MiR-34a Renilla sensor response to FolamiR-34a (50 nM, 96 hours) in the presence of increasing concentrations of folate-glucosamine conjugate. Data points were normalized to FolamiR-NC (negative control: scrambled miRNA) for each experimental condition. Experiment corresponds to n = 3 with at least three technical replicates per treatment. *P < 0.05, **P < 0.01, one-way ANOVA with post hoc Bonferroni correction.

  • Fig. 3. FolamiR-34a inhibits the growth of MB-231 tumors in mice.

    (A) Representative live imaging of female Nu/Nu congenic mice implanted with MB-231 sensor xenografts after intravenous injection of NIR-FolamiR-NC, NIR-FolamiR-SS-34a, or NIR-FolamiR-34a (4 mg/kg, 5 nmol). Left: NIR fluorescence distribution. Right: MiR-34a Renilla luciferase sensor signal. (B) Gross images of excised MB-231 tumors (T) and whole organs (Int, intestines; S, spleen; K, kidneys; Lv, liver; HLu, heart and lungs) visualized for fluorescence. (C) Effects of NIR-FolamiR-34a delivery on miR-34a Renilla sensor activity over time; data normalized to Renilla signal at day 0 (error bars: means ± SEM, n = 3). (D) miR-34a expression from excised MB-231 tumors measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) 72 hours after injection with NIR-FolamiR conjugates (n = 3; error bars: means ± SD). (E) NIR epifluorescence quantification from live animals. Nude mice were implanted with A549 cells on the left shoulder and MB-231 cells on the right shoulder, and live imaging was conducted after intravenous injection of NIR-FolamiR-34a (4 mg/kg, 5 nmol) in the presence (right graph) or absence (left graph) of ≥100-fold molar excess of folate-glucosamine (n = 3, error bars: means ± SD). (F) Fluorescence distribution of excised organs and tumors from (E). A549: FR tumor; MB-231: FR+ tumor. (G) Tumor size after FolamiR-34a treatment (n = 5; error bars: means ± SEM). Arrows represent treatment times (0.8 mg/kg, 1 nmol intravenous injection). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way (D and E) or two-way (B and G) ANOVA with Bonferroni correction.

  • Fig. 4. Murine KrasLSL-G12D/+;p53flx/flx lung adenocarcinomas express FR.

    (A) NIR fluorescence imaging showing that ligand OTL38 (On Target Laboratories), an FRα-targeting ligand conjugated to a NIR dye, is preferentially retained in lung tumors and cleared from normal healthy lungs. KrasLSL-G12D/+;p53Flox/Flox mice were injected with 5 nmol of OTL38 8 weeks after tumor induction and sacrificed 24 hours after injection. Whole lungs were excised and imaged using LI-COR Odyssey CLx. A noninduced healthy mouse was used as a control. RC, right caudal lobe; RM, right medial lobe; RA, right accessory lobe; RCr, right cranial lobe; L, left lobe. (B) Histological and NIR images of right lung lobes from mice treated with OTL38. Left: Whole-organ low-magnification hematoxylin and eosin (H&E) and corresponding NIR images. Right: High-magnification H&E, bright-field, and NIR images of tumorous and healthy tissue corresponding to insets in (B). Scale bars, 50 μm and 20 μm (inset). (C) Whole-organ NIR fluorescence images of excised lung lobes from mice bearing lung tumors treated with OTL38 (5 nmol) in the presence or absence of ≥100-fold molar excess of folate-glucosamine (n = 3 per group). (D) Representative H&E and fluorescence images of tissues from (C). Insets on low-magnification H&E images correspond to adjacent high-magnification images of tumor tissue. Insets on whole-organ NIR fluorescence images correspond to adjacent high-magnification NIR images. H&E images represent the types of tissue shown in NIR images. Scale bars, 20 μm.

  • Fig. 5. Targeted replacement of miR-34a via FolamiR reduces tumor size in a murine model of lung adenocarcinoma.

    (A) MRI-measured tumor burden after FolamiR-34a treatment (FolamiR-NC, n = 3; FolamiR-34a, n = 5). Error bars represent means ± SEM. **P < 0.01, two-way ANOVA and Bonferroni post hoc tests. Arrows represent treatment times (0.8 mg/kg, 1 nmol intravenous injection; total 10). (B) Representative three-dimensional volume renderings of the lungs and MRI images of the thoracic region of mice treated with FolamiRs during (day 8) and at the end of treatment (day 29). (C) Tumor/whole lung volume ratios from (B) at the indicated times showing the percentage of lung volume occupied by tumors. Error bars represent means ± SD. *P < 0.05, one-way ANOVA. (D) Representative H&E-stained tissue of the left lobe of the lung from animals from each treatment group (FolamiR-NC, FolamiR-34a). Scale bars, 1 mm. (E) Overall tumor burden calculated from total tumor area averaged from three histological sections obtained from each treated animal relative to the total area of the lung. Bars indicate the median. FolamiR-NC: n = 3, FolamiR-34a: n = 5; unpaired t test. (F) miR-34a target genes (Met, Myc, and Bcl-2) evaluated by qRT-PCR of tumor tissue normalized to Actin and graphed relative to FolamiR-NC–treated tumors (FolamiR-NC: n = 3, FolamiR-34a: n = 5). Error bars represent means ± SD. *P < 0.05, unpaired t test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/401/eaam9327/DC1

    Materials and Methods

    Fig. S1. Fol-DBCO ligand synthesis and LC-MS spectral analysis.

    Fig. S2. Evaluation of folate-miRNA conjugation measured by 15% native TAE PAGE.

    Fig. S3. MALDI spectra of FolamiRs.

    Fig. S4. NIR-folate ligand synthesis and LC-MS spectral analysis.

    Fig. S5. Evaluation of NIR-FolamiR conjugation measured by 15% native TAE PAGE.

    Fig. S6. MALDI spectra of NIR-FolamiR conjugates.

    Fig. S7. miR-34a Renilla luciferase sensor response to miR-34a transfection.

    Fig. S8. Evaluation of MB-231 miR-34a sensor cells.

    Fig. S9. Folate-mediated delivery of functional siLuc2.

    Fig. S10. Serum stability of FolamiRs and duplex RNA oligos.

    Fig. S11. In vivo tumor growth response to dose titration of FolamiR-34a.

    Fig. S12. miR-34a copy number in tumors treated with FolamiR-34a in mice.

    Fig. S13. Serum cytokines and maximum tolerated FolamiR dose study in mice.

    Table S1. Individual-level data.

    References (5964)

  • Supplementary Material for:

    FolamiRs: Ligand-targeted, vehicle-free delivery of microRNAs for the treatment of cancer

    Esteban A. Orellana, Srinivasarao Tenneti, Loganathan Rangasamy, L. Tiffany Lyle, Philip S. Low, Andrea L. Kasinski*

    *Corresponding author. Email: akasinski{at}purdue.edu

    Published 2 August 2017, Sci. Transl. Med. 9, eaam9327 (2017)
    DOI: 10.1126/scitranslmed.aam9327

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Fol-DBCO ligand synthesis and LC-MS spectral analysis.
    • Fig. S2. Evaluation of folate-miRNA conjugation measured by 15% native TAE PAGE.
    • Fig. S3. MALDI spectra of FolamiRs.
    • Fig. S4. NIR-folate ligand synthesis and LC-MS spectral analysis.
    • Fig. S5. Evaluation of NIR-FolamiR conjugation measured by 15% native TAE PAGE.
    • Fig. S6. MALDI spectra of NIR-FolamiR conjugates.
    • Fig. S7. miR-34a Renilla luciferase sensor response to miR-34a transfection.
    • Fig. S8. Evaluation of MB-231 miR-34a sensor cells.
    • Fig. S9. Folate-mediated delivery of functional siLuc2.
    • Fig. S10. Serum stability of FolamiRs and duplex RNA oligos.
    • Fig. S11. In vivo tumor growth response to dose titration of FolamiR-34a.
    • Fig. S12. miR-34a copy number in tumors treated with FolamiR-34a in mice.
    • Fig. S13. Serum cytokines and maximum tolerated FolamiR dose study in mice.
    • References (5964)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Individual-level data.

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