Suicide HSVtk Gene Delivery by Neurotensin-Polyplex Nanoparticles via the Bloodstream and GCV Treatment Specifically Inhibit the Growth of Human MDA-MB-231 Triple Negative Breast Cancer Tumors Xenografted in Athymic Mice

<div><p>The human breast adenocarcinoma cell line MDA-MB-231 has the triple-negative breast cancer (TNBC) phenotype, which is an aggressive subtype with no specific treatment. MDA-MB-231 cells express neurotensin receptor type 1 (NTSR1), which makes these cells an attractive target of therapeutic genes that are delivered by the neurotensin (NTS)-polyplex nanocarrier via the bloodstream. We addressed the relevance of this strategy for TNBC treatment using NTS-polyplex nanoparticles harboring the herpes simplex virus thymidine kinase (HSVtk) suicide gene and its complementary prodrug ganciclovir (GCV). The reporter gene encoding green fluorescent protein (GFP) was used as a control. NTS-polyplex successfully transfected both genes in cultured MDA-MB-231 cells. The transfection was demonstrated pharmacologically to be dependent on activation of NTSR1. The expression of HSVtk gene decreased cell viability by 49% (<i>P</i><0.0001) and induced apoptosis in cultured MDA-MB-231 cells after complementary GCV treatment. In the MDA-MB-231 xenograft model, NTS-polyplex nanoparticles carrying either the HSVtk gene or GFP gene were injected into the tumors or via the bloodstream. Both routes of administration allowed the NTS-polyplex nanoparticles to reach and transfect tumorous cells. HSVtk expression and GCV led to apoptosis, as shown by the presence of cleaved caspase-3 and Apostain immunoreactivity, and significantly inhibited the tumor growth (55–60%) (<i>P</i><0.001). At the end of the experiment, the weight of tumors transfected with the HSVtk gene was 55% less than that of control tumors (<i>P</i><0.05). The intravenous transfection did not induce apoptosis in peripheral organs. Our results offer a promising gene therapy for TNBC using the NTS-polyplex nanocarrier.</p></div

Presence of apoptosis in MDA-MB-231 cell tumors after HSVtk transfection and ganciclovir treatment.

<p>NTS-polyplex nanoparticles carrying the plasmid pORF-HSVtk were administrated locally or intravenously every two days. GCV (70 mg/kg of body weight, i. p.) was administered daily. The representative micrographs show the immunofluorescence against cleaved caspase-3 (red fluorescence) in cells that were counterstained with Hoechst 33258 (blue fluorescence). Representative bright field micrographs of condensed chromatin of apoptotic bodies were stained with Apostain.</p

Cell death caused by HSVtk expression and ganciclovir treatment.

<p>MDA-MB-231 cells were transfected with NTS-polyplex harboring the plasmid pORF-HSVtk and treated with GCV (10 µg/mL) for 48 h. (<b>A</b>) Representative agarose gel showing the presence of the amplicons for HSVtk and 18S after an RT-PCR assay. (<b>B</b>) Cell viability using the MTT colorimetric assay (<i>n</i> = 5 independent experiments per group). (<b>C</b>) Cell counting of live cells using the trypan blue dye exclusion method (<i>n</i> = 6 independent experiments per group) (<b>D</b>) Representative micrographs in bright field taken 48 h after the treatments. ***<i>P</i><0.0001 when compared with untransfected and GCV untreated cells. One-way ANOVA and <i>post-hoc</i> Bonferroni test.</p

Gene delivery into breast cancer MDA-MB-231 cells via the internalization of NTSR1.

<p>(<b>A</b>) A representative micrograph of NTS-polyplex nanoparticles that are formed at an optimal molar ratio (1 (plasmid): 24 (NTS-carrier)), which was taken using a field emission scanning electron microscope. (<b>B</b>) Representative agarose gel showing the presence of the amplicons for NTSR1 and 18S after an RT-PCR assay. N1E = N1E-115; wMDA = wild type MDA-MB-231 cells; SiMDA = NTSR1-silenced MDA-MB-231 cells. L-929 = mouse fibroblast cell line. (<b>C</b>) Representative confocal micrographs of an immunofluorescence assay showing the presence of NTSR1 in MDA-MB-231 cells that were counterstained with TRITC-phalloidin and Hoechst 33258. One-micrometer optical sections in the x–y plane (horizontal) and in the z plane (vertical). (<b>D</b>) Representative confocal micrographs of propidium iodide (PI) NTS-polyplex harboring the plasmid pEGFP-N1 in MDA-MB-231 cells in the absence or presence of different pharmacological blockers. The cells were counterstained with Hoechst 33258 and calcein. (<b>E</b>) Representative micrographs showing GFP expression in MDA-MB-231 cells after exposure to NTS-polyplex nanoparticles carrying pEGFP-N1 plasmid. The cells were counterstained with Hoechst 33258.</p

Targeted gene delivery to MDA-MB-231 cell tumors in athymic mice.

<p>(<b>A</b>) Representative confocal micrographs of cells that were counterstained with FITC-phalloidin taken after a local (6 h) or intravenous (24 h) injection of NTS-polyplex nanoparticles carrying the plasmid pEGFP-N1 labeled with propidium iodide. One-micrometer optical sections in the x–y plane (Horizontal) and in the z plane (vertical). (<b>B</b>) Representative micrographs showing the expression of GFP in cells that were counterstained with Hoechst 33258 on day 3 after a local or intravenous injection of NTS-polyplex nanoparticles carrying the plasmid pEGFP-N1.</p

Apoptosis induced by HSVtk expression and ganciclovir treatment.

<p>Apoptosis in MDA-MB-231 cells was evaluated 24 h after the exposure to NTS-polyplex harboring the plasmid pORF-HSVtk and GCV (10 µg/mL). (<b>A</b>) Representative flow-cytometry dot-plot graphics showing the proportion of cells that were stained with Annexin V-FITC (apoptosis) and propidium iodide (necrosis) whose percentage are expressed as the mean ± SEM in the corresponding quadrant. The insert shows the percentage of apoptotic cells (<i>n</i> = 5 per group) which correspond to the sum of the percentage in the inferior right quadrant (showing early apoptosis) plus the percentage in the superior right quadrant (showing late apoptosis). (<b>B</b>) Representative confocal micrograph showing the immunofluorescence against cleaved caspase-3 in cells that were counterstained with Hoechst 33258 (<i>n</i> = 3 per group). One-micrometer optical sections in the x–y plane (horizontal) and in the z plane (vertical). (<b>C</b>) Representative bright field micrographs of condensed chromatin of apoptotic bodies that were stained with Apostain. Staurosporine was used as a positive control of apoptosis. All the assays were performed 24 h after the treatments. *<i>P</i><0.05, ***<i>P</i><0.0001 when compared with untransfected and GCV treated cells. One-way ANOVA and <i>post-hoc</i> Bonferroni test.</p

NTSR1 expression in IDCs and global survival duration.

<p>Kaplan-Maier analysis for global survival duration in both groups with low (<80%) and high (≥80%) NTSR1 expression. Probability of overall death for patients with high NTSR1 expression (n = 38) versus patients with low NTSR1 expression (n = 68).</p

Prognosis factors and deaths stratified by NT-1 receptor expression in the invasive component of IDCs.

<p>Prognosis factors and deaths stratified by NT-1 receptor expression in the invasive component of IDCs.</p

Neurotensin expression in IDCs.

<p>a) NTS immunohistochemistry was performed on IDCs, ductal (1) and invasive (2) components, magnification 200× for (1) and 400× for (2). b) NTS and NTSR1 transcripts in IDCs. One µg of total RNA from 11 patients with IDCs were reverse-transcribed, and specific PCR was performed for NTS, NT-1 receptor, or GAPDH (control). The SBR grade is indicated below each line. c) Example of NTS and NTSR1 regional co-localization by immunohistochemistry for NTS (right) and NTSR1 (left) at the original magnification 400×.</p

Correlation of the subpopulation co-expressing NT and NT1 receptor with the major prognosis factors.

<p>Correlation of the subpopulation co-expressing NT and NT1 receptor with the major prognosis factors.</p