Pulsed-laser irradiation of multifunctional gold nanoshells to overcome trastuzumab resistance in HER2-overexpressing breast cancer

Abstract

Background: HER2-overexpressing metastatic breast cancers are challenging practice in oncology when they become resistant to anti-HER2 therapies such as trastuzumab. In these clinical situations, HER2-overexpression persists in metastatic localizations, and can thus be used for active targeting using innovative therapeutic approaches. Functionalized gold nanoparticles with anti-HER2 antibody can be stimulated by near-infrared light to induce hyperthermia.

Methods: Here, hybrid anti-HER2 gold nanoshells were engineered for photothermal therapy to overcome trastuzumab resistance in HER2-overexpressing breast cancer xenografts.

Results: When gold nanoshells were administered in HER2-tumor xenografts, no toxicity was observed. A detailed pharmacokinetic study showed a time-dependent accumulation of gold nanoshells within the tumors, significantly greater with functionalized gold nanoshells at 72 h. This enabled us to optimize the treatment protocol and irradiate the mice when the anti-HER2 gold nanoshells had accumulated most in the tumors. After weekly injections of anti-HER2 gold nanoshells, and repeated irradiations with a femtosecond-pulsed laser over four weeks, tumor growth was significantly inhibited. Detailed tissue microscopic analyses showed that the tumor growth inhibition was due to an anti-angiogenic effect, coherent with a preferential distribution of the nanoshells in tumor microvessels. We also showed a direct tumor cell effect with apoptosis and inhibition of proliferation, coherent with an immune-mediated targeting of tumor cells by anti-HER2 nanoshells.

Conclusion: This preclinical study thus supports the use of anti-HER2 gold nanoshells and photothermal therapy to overcome trastuzumab resistance in HER2-overexpressing breast cancer.

Keywords: Femtosecond laser; Functionalized gold nanoparticles; HER2-overexpressing breast cancer; Photothermal therapy; Resistance reversion; Trastuzumab resistance.

Fig. 1

Illustration of the proposed multifunctional anti-HER2 gold nanoshell with HER2 receptor targeting and photothermal therapy

Fig. 2

a Diagram showing steps in anti-HER2 gold nanoshell (GN) synthesis with corresponding TEM images. b UV-Vis absorption spectra of anti-HER2 GNs. c HER2 gene copy number, mRNA expression and immunostaining (brown) for BT474-R cell line (* P < 0.05). d Immunostaining of BT474-R cells incubated with trastuzumab-FITC (green, left panel). Dark field images of BT474-R cells incubated with anti-HER2 gold nanoshells (right panel)

Fig. 3

a Kinetic bio-distribution of anti-HER2 gold nanoshells (GNs) after intravenous administration of 3.5 × 10¹³ nanoshells in mice using magnetic resonance imaging (MRI). Results are expressed as percentages of initial signal variations (ΔSI), each signal intensity being compared with a pre-contrast signal. b Absolute gold concentration in blood and tissue samples after intravenous administration of 3.5 × 10¹³ anti-HER2 GNs using ICP-MS. Data is expressed as mean ± standard deviation, and comparisons between the liver and spleen concentrations were made using Student’s t-test (*P < 0.05). c Dark field images of tissue sections obtained from the liver and spleen after injection of anti-HER2 GNs. d TEM images of anti-HER2 GNs internalized in liver and spleen cells

Fig. 4

a T2-weighted magnetic resonance imaging of one xenografted tumor (circle) before and 72 h after intravenous administration of 3.5 × 10¹³ non-functionalized gold nanoshells (GNs) or anti-HER2 GNs, with corresponding histograms for percentage of signal initial variations (ΔSI). Each signal intensity is compared with a pre-contrast signal. b Absolute gold concentration in xenografted tumor over time after intravenous administration of non-functionalized gold nanoshells (GNs) or of 3.5 × 10¹³ anti-HER2 GNs using ICP-MS. Data is expressed as mean ± standard deviation, and comparisons between mice injected with non-functionalized GNs or with anti-HER2 GNs were made using Student’s t-test (*P < 0.05). c Dark field images of tumor sections at 72 h after injection of GNs. d TEM images of tumors at 72 h showing the presence of GNs within the cytoplasm of cancer cells. e Merged dark field images with immunofluorescence staining of human xenografted tumors 72 h after injection of anti-HER2 GNs, using anti-Cytokeratin (red) or anti-CD31 (green) antibodies (T: tumor; V: microvessel)

Fig. 5

a Human xenografted tumor growth curves using mice grafted with trastuzumab-resistant HER2-overexpressing BT474-R cell lines. For each treatment group (non-irradiated tumors, irradiated tumors after intravenous administration of non-functionalized GNs, and irradiated tumors after intravenous administration of anti-HER2 GNs), 10 xenografted mice are used. Comparisons between the three treatment groups are made using ANOVA (* P < 0.05, *** P < 0.001). b Quantitative comparisons of tumor necrosis (N), cell proliferation, cell apoptosis and microvessel density between irradiated and non-irradiated tumors. Comparisons between groups are made using Student’s t-test (* P < 0.05, ** P < 0.01). c Double immunofluorescence staining of irradiated human xenografted tumor using anti-cleaved-caspase3 and anti-CD31 antibodies (T: tumor; V: microvessel)