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. 2015 Jun 1;75(11):2316-2325.
doi: 10.1158/0008-5472.CAN-14-3485. Epub 2015 Apr 9.

Silencing β3 Integrin by Targeted ECO/siRNA Nanoparticles Inhibits EMT and Metastasis of Triple-Negative Breast Cancer

Jenny G Parvani #  1   2 , Maneesh D Gujrati #  1 , Margaret A Mack  1 , William P Schiemann  2 , Zheng-Rong Lu  1
Affiliations

Silencing β3 Integrin by Targeted ECO/siRNA Nanoparticles Inhibits EMT and Metastasis of Triple-Negative Breast Cancer

Jenny G Parvani et al. Cancer Res. .

Abstract

Metastatic breast cancer is the second leading cause of cancer-related deaths among women. Triple-negative breast cancer (TNBC) is a highly aggressive subcategory of breast cancer and currently lacks well-defined molecular targets for effective targeted therapies. Disease relapse, metastasis, and drug resistance render standard chemotherapy ineffective in the treatment of TNBC. Because previous studies coupled β3 integrin (ITGB3) to epithelial-mesenchymal transition (EMT) and metastasis, we exploited β3 integrin as a therapeutic target to treat TNBC by delivering β3 integrin siRNA via lipid ECO-based nanoparticles (ECO/siβ3). Treatment of TNBC cells with ECO/siβ3 was sufficient to effectively silence β3 integrin expression, attenuate TGFβ-mediated EMT and invasion, restore TGFβ-mediated cytostasis, and inhibit three-dimensional organoid growth. Modification of ECO/siβ3 nanoparticles with an RGD peptide via a PEG spacer enhanced siRNA uptake by post-EMT cells. Intravenous injections of RGD-targeted ECO/siβ3 nanoparticles in vivo alleviated primary tumor burden and, more importantly, significantly inhibited metastasis. In the span of 16 weeks of the experiments and observations, including primary tumor resection at week 9 and release from the treatment for 4 weeks, the mice bearing orthotopic, TGFβ-prestimulated MDA-MB-231 tumors that were treated with RGD-targeted ECO/siβ3 nanoparticles were free of metastases and relapse, in comparison with untreated mice. Collectively, these results highlight ECO/siβ3 nanoparticles as a promising therapeutic regimen to combat TNBC.

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Figures

Figure 1
Figure 1
ECO/siβ3 nanoparticles induced sustained gene silencing of β3 integrin. A, ECO forms nanoparticles with siRNA through electrostatic interactions, disulfide cross-linking and hydrophobic interactions. B, β3 integrin mRNA expression in quiescent or TGF-β stimulated (5 ng/mL, 72 hours) NME and MDA-MB-231 cells with the indicated treatment groups at 100 nM siRNA by semi-quantitative real-time PCR (n=3, mean ± SE, p≤0.01 for all time points beyond 8 hours). Western blot analysis of β3 integrin expression in quiescent or TGF-β stimulated (5 ng/mL, 72 hours) NME C, and MDA-MB-231 d) cells at the indicated time points post-nanoparticle treatment with the indicated treatment groups.
Figure 2
Figure 2
ECO/siβ3 nanoparticles attenuated TGF-β-mediated EMT, invasion and proliferation. A, Immunofluorescence images of actin cytoskeleton visualized with rhodamine-conjugated phalloidin in mouse NME cells with different treatments (scale bar, 100 μm; inset scale bar, 50 μm). B, Semi-quantitative real-time PCR analysis (n=3) of EMT markers in NME cells (**p≤0.01). C, Western blot analysis of E-cadherin and N-cadherin in NME cells. D, Invasion assay of quiescent (white bars) or TGF-β stimulated (gray bars) NME cells (n=3, *p≤0.05, **p≤0.01). E, Proliferation as measured by [3H]thymidine incorporation of either quiescent (white bars) or TGF-β stimulated (gray bars) NME cells (n=3, *p≤0.05, **p≤0.01). For all experimental groups, NME cells were pre-treated with TGF-β (5ng/mL; 72 hours) followed by ECO/siRNA nanoparticle treatment using 100nM siRNA. For panels B, D-E, data represent mean ± SE. Results for panels D-E are representative of three independent experiments.
Figure 3
Figure 3
ECO/siβ3 nanoparticles attenuated 3D organoid outgrowth. NME and MDA-MB-231 cells were grown in a compliant 3D-organotypic microenvironment and treated with ECO nanoparticles containing Alexa Fluor 488-labeled siRNA. Cellular uptake of ECO/siRNA nanoparticles monitored by fluorescence confocal microscopy (scale bar, 100 μm). A, Bright-field microscopic image of a single organoid and fluorescence confocal microscopic images of ECO/siRNA nanoparticle uptake in the organoid over the course of 24 hours. B, NME and C, MDA-MB-231 cells were grown in a compliant 3D-organotypic microenvironment for up to 10 days with or without prior TGF-β stimulation (5ng/mL) for 72 h. On day 4, 6 and 8, cells were treated with ECO/siNS or ECO/siβ3 nanoparticles at 100 nM siRNA. Organoid growth at day 10 was monitored via longitudinal bioluminescence (n=4, *p≤0.05, **p≤0.01). For panels C-D, data represent mean ± SE.
Figure 4
Figure 4
RGD modification of ECO/siRNA nanoparticles enhances uptake in post-EMT breast cancer cells. A, RGD-ECO/siRNA nanoparticles were prepared by modifying the ECO/siRNA nanoparticles with RGD-targeted PEG ligand via thiol-maleimide chemistry. B, Conjugation of RGD-PEG-maleimide to ECO was confirmed from MALDI-TOF mass spectrometry. The center of the bell was observed at m/z 5200 confirming the conjugation of RGD-PEG-maleimide (m/z, 4100) to ECO [m/z, 1046 (M+Na+)]. C, The size of RGD-ECO/siRNA nanoparticles was determined by dynamic light scattering (DLS). NME cells, with or without TGF-β stimulation (5ng/mL; 72 hours), were treated with ECO nanoparticles with Alexa Fluor 488-labeled siRNA at 40 nM siRNA for 4 hours. NME cells that were stimulated with TGF-β exhibited a higher cellular uptake of RGD-targeted ECO/siRNA nanoparticles compared to the non-targeted ECO/siRNA nanoparticles as confirmed by D, confocal microscopy (scale bar, 50 μm) and E, quantified by flow cytometry (n=3, **p≤0.01). Quantitative analysis of β3 integrin mRNA levels following treatment with siβ3 nanoparticles by real-time PCR (n=3, **p≤0.01) in F, NME and G, MDA-MB-231 cells revealed RGD-targeted ECO/siRNA nanoparticles maintain gene silencing. H, Cellular uptake in NME cells, both with and without TGF-β stimulation (5ng/mL; 72 hours), was quantified by flow cytometry for RGD-ECO/siRNA nanoparticles containing Alexa Fluor 488-labelled siRNA 4 hours after treatment. One group of TGF-β stimulated NME cells (TGF-β + ECO/siβ3) was treated with ECO/siβ3 nanoparticles at 100 nM siRNA for 48 hours prior to cellular uptake with the RGD-targeted nanoparticles to quantify the effect of β3 integrin silencing on targeted uptake (n=3, ±SE, *p≤0.05, **p≤0.01). For panels C-F, data represent mean ± SE.
Figure 5
Figure 5. Pulmonary outgrowth of NME cells treated with the ECO/siRNA treatment regimen
TGF-β-pre-treated NME cells (1 × 106) were injected into the lateral tail vein of nude mice. Tail vein administration of the ECO/siRNA treatment regimen (siRNA dose of 1.5 mg/kg, ECO dose of 18.6 mg/kg) was conducted every 5 d, starting at day 18 after the cancer cell inoculation (n = 4, mean ± SE, *p ≤ 0.05).
Figure 6
Figure 6
RGD-targeted ECO/siβ3 nanoparticles inhibited primary tumor growth and EMT in mice after systemic administration. A, Schematic of targeted ECO/siRNA nanoparticle treatment (siRNA dose of 1.5 mg/kg, ECO dose of 18.6 mg/kg) schedule in vivo. Tumor growth was monitored at the indicated time points B, and quantified by C, BLI (data represents mean ± SE, n=5, *p≤0.05, **p≤0.01), and D, caliper measurements (data represents mean ± SE, n=5, *p≤0.05, **p≤0.01). E, Primary tumors were resected at week 9, and final tumor weights of the indicated treatment groups were obtained (data represents mean ± SE, n=5, **p≤0.01). F, Semi-quantitative real-time quantification of β3 integrin mRNA expression from resected primary tumors of the indicated groups (data represents mean ± SE, n=5, **p≤0.01). G, H&E staining at 200X of primary tumors. H, H&E, DAPI and fibronectin immunostaining of the indicated primary tumors (scale bar, 300 μm).
Figure 7
Figure 7
RGD-ECO/siβ3 nanoparticles inhibited breast cancer metastasis and primary tumor recurrence. A, BLI images of mice at week 12 revealed differences in metastasis and primary tumor recurrence for the different treatment groups after primary tumor resection on week 9. B, Quantification of primary tumor recurrence (data represents mean ± SE, n=5, *p≤0.05). C, Quantification of thoracic metastasis by BLI (data represents mean ± SE, n=5, *p≤0.05, **p≤0.01). Mice were released from ECO/siRNA therapeutic regimen at week 12. D, Representative BLI of mice on week 16. E, Quantification of whole body tumors from D. (data represents mean ± SE, n=5, *p≤0.05). F, Change in the body weight of mice bearing MDA-MB-231 primary tumors across various treatment groups over the course of 16 weeks. The body weight was measured weekly and reported as mean ± S.E. (n = 5) for each group. No significant difference was observed between the various treatment groups at any time point.
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