Design and In Vitro Evaluation of Layer by Layer siRNA Nanovectors Targeting Breast Tumor Initiating Cells

<div><p>Efficient therapeutics and early detection has helped to increase breast cancer survival rates over the years. However, the recurrence of breast cancer remains to be a problem and this may be due to the presence of a small population of cells, called tumor initiating cells (TICs). Breast TICs are resistant to drugs, difficult to detect, and exhibit high self-renewal capabilities. In this study, layer by layer (LBL) small interfering RNA (siRNA) nanovectors (SNVs) were designed to target breast TICs. SNVs were fabricated using alternating layers of poly-L-lysine and siRNA molecules on gold (Au) nanoparticle (NP) surfaces. The stability, cell uptake, and release profile for SNVs were examined. In addition, SNVs reduced TIC-related STAT3 expression levels, CD44⁺/CD24⁻/EpCAM⁺ surface marker levels and the number of mammospheres formed compared to the standard transfection agent. The data from this study show, for the first time, that SNVs in LBL assembly effectively delivers STAT3 siRNA and inhibit the growth of breast TICs <i>in vitro</i>.</p></div

Figure 4

<p>(<b>A</b>) Protein expression of p-STAT3 pathway in triple negative breast cancer cell lines, SUM159 and MDA-MB-231, after treatment with AuNPs, non-targeting siRNA in siPORT, STAT3 targeting siRNA in siPORT, non-targeting SNVs, and STAT3 targeting siRNA in SNVs. (<b>B</b>) Primary mammosphere forming efficiency from breast cancer cells after 3 days treatment with AuNPs, STAT3 siRNA in siPORT, in PLL and STAT3 targeting siRNA in SNVs. (<b>C</b>) Forming efficiency of secondary mammospheres was plotted after primary mammospheres were collected, disassociated, and re-seeded in fresh mammosphere media for 3 days. (* = statistical significance, n = 6, p<0.05).)</p

Figure 3

<p>(<b>A</b>) Number of cells emitting FITC after uptake of FITC tagged PLL layer (Layer 3-green) from SNVs after 4, 24 and 48 h treatment. (<b>B</b>) Plot of the FITC intensity measured at each time point, 4, 24, and 48 h (n = 9, n is number of regions of interest (ROI) from fluorescent images) Images correspond to fluorescent image of SUM159 cells (nuclei stained with DAPI – blue color) displaying the release of FITC tagged PLL layer (Layer 1-green) from SNVs, scale bar = 10 µm.</p

Figure 2

<p>(<b>A</b>) Absorption spectra of SNVs in FBS and in water. (<b>B</b>) Release profile of the first layer (FITC tagged poly-L-Lysine) released from LBL SNVs in pH 5.5 and 7.2 at 37°C. (<b>C</b>) Release profile of the STAT3 siRNA layer (Cy3-tagged siRNA molecules) released from the SNVs in pH 5.5 and pH 7.2 at 37°C.</p

Schematic representation on the fabrication method for SNVs using AuNPs.

<p>Plot of (<b>B</b>) zeta potential, (<b>C</b>) hydrodynamic diameter, (<b>D</b>) the amount of siRNA loaded onto SNVs and (<b>E</b>) absorbance for bare AuNPs stabilized by citrate and every subsequent layer added on to the SNVs. (<b>F</b>) AFM height image of SNVs.</p

Kaplan Meier recurrence free survival with the combination with high dose docetaxel and C188 vs. chemotherapy alone.

<p>Female mice were transplanted in one mammary fat pad with chemoresistant breast cancer xenograft 2665A (ER/PgR/HER2-negative and p-Stat3-positive). After 6 weeks, mice with ∼200 mm³ tumors were randomized and treated with either 2 cycles of docetaxel (60 mg/kg) combined with daily dose of C188 (12.5 mg/kg; solid line) at the start of treatment vs. docetaxel alone (– line). Mice were observed daily after the end of all treatment (all the tumors had receded at that time). Time to tumor recurrence as denoted by tumors being measured to be larger than 50mm³ was significantly improved with the combination vs. chemotherapy alone (p = 0.030).</p

Chemoresistant BCM2665 transplanted in the fat pad of SCID Beige mice responds to combination of Stat3 inhibitor+docetaxel treatment, with decrease in tumor volume and TIC markers by FACS and MSFE.

<p>(A) Tumor volume fold change over time + SEM in 4 treatment arms of the chemoresistant BCM2665 model for the treatment period of 14 days. Significant decrease in mean tumor volume was noted between vehicle and combination of C188+docetaxel, and between chemotherapy and combination treatment (p<0.05). (B) FACS analysis of 10,000 cells BCM2665 tumor cells for ALDF+. Statistically significant decreases were observed with C188 vs. control (p<0.05), C188 vs. chemotherapy (p<0.05), and combination of C188+chemotherapy vs. chemotherapy alone (p<0.05). Data depicted as Mean + SEM. (C) Mammosphere forming efficiency of tumor cells harvested and seeded at 20,000 cells/well. Statistically significant decrease with C188 treatment vs. control (p<0.05), and C188 treatment vs. chemotherapy was observed (p<0.05). (D) The epithelial compartment of the BCM2665 tumors treated with C188 or vehicle control was isolated using laser capture microscopy. pStat3 levels were determined using Western blot analysis (representative picture shown). (E) Immunohistochemical analysis of pStat3Stat expression in Vehicle vs C188Stat treated BCM2665 tumor tissue (representative picture shown).</p

Chemosensitive MC1 transplanted in fat pad of SCID Beige mice, with non-significant decrease in TIC markers (ALDF, CD44+/CD24−, and MSFE) with Stat3 inhibitor compared to chemotherapy alone.

<p>(A) Tumor volume fold change over time (14 days). Decrease in mean tumor volume + SEM was observed with chemotherapy alone (chemosenstive tumor line). (B) FACS analysis of 100,000 cells MC1 tumor cells for CD44+/CD24−, ALDF. Data were depicted as Mean + SEM. Non-significant decrease in TIC markers (ALDF, CD44+/CD24) with Stat3 inhibitor compared to chemotherapy alone was observed. (C) Mammosphere forming efficiency of tumor cells seeded at 10,000 cells/ml. Data depicted as Mean + SEM. Non-significant decrease in MSFE with C188 compared to chemotherapy alone was observed.</p

Ingenuity Analysis identifies Stat3 as an important target for TIC self renewal.

<p>(A) Ingenuity Analysis of 493-gene tumorigenic stem cell signature, looking for direct and indirect interactions of these genes among themselves identified Stat3 as one of the important targets of TIC self renewal. (B) Mammosphere forming efficiency in SUM159 treated with Stat3 shRNA vs. empty vector (EV) showed a significant decrease (p<0.05). Data depicted as Mean+ SEM. Western analysis of empty vector vs. Stat3 shRNAs treated cells depicts reduction in pStat3 levels upon treatment. (C)Mammosphere forming efficiency for SUM 159 and BT 549 cells treated with C188 at 10 µM concentration. Significant decrease in mammosphere formation upon treatment with C188 (p<0.05). Data depicted as Mean+SEM.</p