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Dual complementary liposomes inhibit triple-negative breast tumor progression and metastasis

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Science Advances  20 Mar 2019:
Vol. 5, no. 3, eaav5010
DOI: 10.1126/sciadv.aav5010
  • Fig. 1 Identification of ICAM1 and EGFR as candidates for TNBC complementary targeting.

    (A) Surface protein expression of 68 cancer targets in three human TNBC cell lines and non-neoplastic MCF10A cells. Red and green bars represent maximum and minimum expression, respectively. High-magnification version is shown in fig. S1. (B) Venn diagrams summarize the cancer targets identified by flow cytometric analysis. Each circle represents the number of cancer targets up-regulated in one human TNBC cell line compared to non-neoplastic MCF10A cells. Overlapping sets show the differential expression in two or three comparison pairs, and 16 cancer targets were identified as up-regulated in all three TNBC cell lines compared to MCF10A cells. (C) Quantified surface densities of 16 target candidates. Red bars represent the five top candidates that were overexpressed in TNBC cells. (D) ICAM1 and EGFR gene expression in human TNBC and MCF10A cells as quantified by qRT-PCR. Significance was measured by one-way analysis of variance (ANOVA) with Bonferroni post hoc test. ***P < 0.001. (E) Representative microscopic images of immunofluorescent staining of ICAM1 and EGFR in three human TNBC cell lines and MCF10A cells. Scale bars, 5 μm. DAPI, 4′,6-diamidino-2-phenylindol; FITC, fluorescein isothiocyanate; PE, phycoerythrin. (F) FRET analysis of ICAM1 and EGFR colocalization. Significance was measured by one-way ANOVA with Bonferroni post hoc test. FIU, fluorescence intensity unit; NS, not significant; **P < 0.01. (G) Correlation between overall survival and ICAM1/EGFR mRNA expression levels in patients with basal-like breast cancer, as shown with Kaplan-Meier analysis (*P < 0.05, log-rank test). Ab, antibody.

  • Fig. 2 Schematic illustration of DCL structure and biomechanisms of complementary targeting strategy.

    (A) The design of the proof-of-principle binary DCL for TNBC. (B) DCL increases cellular binding using precisely matched, multivalent ligand-receptor interactions. (C) DCL enhances internalization using cooperative ICAM1 and EGFR endocytosis pathways. (D) DCL improves therapeutic efficacy using synergistic blockade of ICAM1 and EGFR signaling cascades.

  • Fig. 3 DCL increases in vitro TNBC targeting and antitumor activities.

    (A) In vitro cellular binding and uptake of DCL-FITC and controls in human TNBC and MCF10A cells were determined by flow cytometry in reference to IgG-FITC-LP. (B) Representative fluorescent images showing TNBC-specific cellular binding and uptake of DCL-FITCs in TNBC cells in comparison with IgG-FITC-LP, ICAM-FITC-LP, and EGFR-FITC-LP. Scale bars, 20 μm. (C) Internalization ratios of DCL-FITC and controls were determined by a trypan blue (TB) quenching assay. Black bars (without trypan blue quenching) represent total cellular fluorescence from both extracellular and internalized liposomes, and gray bars (with trypan blue quenching) represent cellular fluorescence from internalized liposomes only. (D) Quantified analysis of therapeutic efficacies of DCL (vehicle without Dox) and controls on TNBC cell proliferation. Representative microscope images (E) and quantitative analysis (F) of invaded TNBC cells in a transwell invasion assay. Scale bars, 50 μm. (G) In vitro cytotoxicity of DCL-Dox was evaluated for MDA-MB-231 and MDA-MB-436 cells by Dojindo cell viability assay in reference to DCL (vehicle without Dox). Significance was measured by one-way ANOVA with Bonferroni post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 4 DCL-Dox inhibits orthotopic tumor growth and metastasis.

    (A) Schematic design of orthotopic tumor biodistribution imaging. (B) In vivo NIR fluorescent images of nude mice at 4, 24, and 48 hours after the administration of IgG-DiR-LP, ICAM-DiR-LP, EGFR-DiR-LP, and DCL-DiR_4.2/1. n = 8 per group. (C) Quantitative analysis of in vivo tumor accumulation of DCL-DiR_4.2/1 and control liposomes. (D) Representative ex vivo NIR fluorescent images of organs (liver, spleen, kidney, lung, heart, and brain) and excised tumors. (E) Biodistribution of administrated agents in different organs and tumors after 48 hours. (F) TNBC tumor specificity of DCLs with three distinct ratios (4.2/1, 1/1, and 1/4.2, mol/mol) and a mixture of two single-targeting liposomes (ICAM-DiR-LP and EGFR-DiR-LP, 1/1, mol/mol) in the orthotopic TNBC model (MDA-MB-231) (n = 5 per group). (G) Tumor accumulation of liposomes was determined at 48 hours after injection by NIR fluorescent intensity (n = 5 per group). (H) Schematic design of orthotopic tumor therapy model. (I) Image of excised orthotopic TNBC tumors from mice treated with PBS (sham), free Dox, IgG-Dox-LP, ICAM-Dox-LP, EGFR-Dox-LP, or DCL-Dox_4.2/1 under a 21-day treatment regimen (n = 7 to 10 per group). (J) Tumor progression was closely monitored by weekly tumor volume measurement using caliper. (K) Tumor mass at end point (day 24) was quantified in weight. Significance was measured by one-way ANOVA (C, E, G, and K) or two-way ANOVA (J) with Bonferroni post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 5 DCL-Dox inhibits TNBC lung metastasis and improves survival.

    (A) Schematic design of TNBC lung metastasis therapy. (B) Representative bioluminescence images of lung metastasis at different time points in mice treated with the following agents: PBS (sham), free Dox, IgG-Dox-LP, ICAM-Dox-LP, EGFR-Dox-LP, or DCL-Dox_4.2/1 (n = 8 per group). (C) Representative tumor progression curves as depicted from in vivo bioluminescence signal intensity (n = 3 per group). (D) Size and morphology of lungs excised from mice in different treatment groups. (E) Quantification of metastasis node numbers on excised lungs from mice in different treatment groups. (F) Metastasis-free survival of mice in DCL-Dox and control groups as displayed by Kaplan-Meier curves (log-rank test). (G) Schematic design for dosage-dependent therapy. iv, intravenous. (H) In vivo bioluminescence images of mice in the dosage-dependent study. Tumor-bearing mice were treated with DCL-Dox_4.2/1 at different dosages and imaged at day 74 or an earlier sacrifice date (n = 5 per group). “*” indicates the mouse sacrificed at day 22 due to blindness caused by retro-orbital injection). (I) Quantification of metastasis node numbers on excised lungs in the dosage-dependent study. (J) Metastasis-free survival of mice in the dosage-dependent study as displayed by Kaplan-Meier curves (log-rank test). (K) Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and blood urea nitrogen (BUN) (n = 4 to 5 per group). Significance was measured by one-way ANOVA with Bonferroni post hoc test (E, I, and K). *P < 0.05; **P < 0.01; ***P < 0.001.

  • Table 1 Summary of metastasis formation in TNBC orthotopic and lung metastasis models.

    Metastatic sitePBS (sham)Free DoxIgG-Dox-LPICAM-Dox-LPEGFR-Dox-LPDCL-Dox_4.2/1
    Orthotopic breast tumor model
    Brain0/80/80/70/90/80/10
    Lung1/80/80/70/91/80/10
    Heart0/80/80/70/90/80/10
    Liver3/80/82/70/91/80/10
    Spleen3/80/82/71/91/81/10
    Kidney0/80/80/70/90/80/10
    Right hind limb (tumor bearing)6/88/85/74/95/81/10
    Left hind (normal)1/80/80/72/91/80/10
    Total7/88/86/75/95/81/10
    Lung metastasis model
    Brain0/80/80/80/80/80/8
    Lung8/85/86/82/86/80/8
    Heart0/80/80/80/80/80/8
    Liver2/80/81/80/80/80/8
    Spleen1/80/80/80/80/80/8
    Kidney0/80/80/80/80/80/8
    Hind limbs (normal)0/80/80/80/80/80/8
    Total8/85/86/82/86/80/8

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/3/eaav5010/DC1

    Fig. S1. Surface protein expression of 68 cancer targets in three human TNBC cell lines and non-neoplastic MCF10A cells.

    Fig. S2. Morphological characterization of DCL.

    Table S1. List of cell membrane proteins.

    Table S2. ICAM1 and EGFR surface density and ratio on human TNBC cells.

    Table S3. Dynamic light scattering characterization of DCL-Dox and controls.

    Table S4. Theoretical and experimental densities of ICAM1 and EGFR antibodies on DCL surfaces.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Surface protein expression of 68 cancer targets in three human TNBC cell lines and non-neoplastic MCF10A cells.
    • Fig. S2. Morphological characterization of DCL.
    • Table S1. List of cell membrane proteins.
    • Table S2. ICAM1 and EGFR surface density and ratio on human TNBC cells.
    • Table S3. Dynamic light scattering characterization of DCL-Dox and controls.
    • Table S4. Theoretical and experimental densities of ICAM1 and EGFR antibodies on DCL surfaces.

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