Ultrasound-guided photoacoustic imaging for diagnosis and near infrared photothermal therapy of triple negative breast cancer using EGFR antibody-conjugated gold nanorods

Abstract

Introduction: Triple negative breast cancer (TNBC), lacking human epidermal growth factor receptor 2 (HER2), and estrogen receptor (ER) and progesterone receptor (PR) expression, are associated with an aggressive natural history compared with other disease subtypes. Approximately 70 to 80% of TNBCs more frequently overexpress the epidermal growth factor receptor (EGFR), which is emerging as a therapeutic target. Photoacoustic imaging (PAI), which can create multi-contrast images of living biological structures ranging from organelles to organs, has shown great translational potential from bench to bedside due to it being relatively inexpensive and convenient for combination with clinical ultrasound (US). Gold nanorods (GNs) are extensively used for PAI and NIR-PTT. The first aim of this study is to assess the use of US-guided PAI and anti-EGFR antibody-conjugated GNs (anti-EGFR-GNs) to detect the EGFR-expressing primary tumor mass and regional LN metastases in a mouse model of human TNBC. Secondary aim of this study is to prove that anti-EGFR-GNs combined with NIR-PTT is an encouraging therapeutic strategy for highly selective cancer cell targeting and synergistic anti-cancer activity in aggressive TNBCs.

Methods: The human breast cancer cell lines (ER+) subtype

MCF-7, HER2+ subtype

BT-474, and TN subtype

HCC-578T, HCC-38, HCC-1937, MDA-MB-453 and MDA-MB-231) were used. The intracellular signaling events were evaluated by Western blot. Cell proliferation activity was evaluated by MTT assay. For analysis of apoptotic cell damage, fluorescence of annexin V and propidium iodide was measured using flow cytometry. MDA-MB-231 and MCF-7 cells were fat pad injected into the back right flank of female BALB/c nude mice (4-5 week old) for xenograft tumor model. GNs and anti-EGFR-GNs, 10 nm in diameter and 41 nm in length used as PAI contrast agents. In vivo real-time US-guided PAI and bioluminescence imaging of primary tumor and LN were performed using a preclinical Vevo 2100 LAZR Imaging system and IVIS Lumina II Imaging system, respectively. For the study of anti-EGFR-GNs-combined NIR-PTT heat (up to 43°C ) of the culture medium and tumor skin was produced by irradiation of a water-filtered infrared A lamp (at 1.5 W/cm2) for 3 min at 48 h post-treatment with anti-EGFR-GNs. Hematoxylin and eosin staining, silver enhancement staining, and immunostaining were performed on tissue sections.

Results: Representative western blots showed that TNBC cell lines (Hs578T, HCC-38, HCC-1937, MDA-MB-468 and MDA-MB-231) highly expressed EGFR whereas the other ER+ (MCF-7) and HER2+ cell lines (BT474) did not. A large amount of nanoparticles in the intracellular region was apparent in MDA-MB-231 cells treated with anti-EGFR-GNs for 24 h, whereas very few nanoparticles in the cytoplasm were observed in the MDA-MB-231 cells treated with GNs. In the EGFR-targeting study in cultured MCF-7 and MDA-MB-231 cells, selective binding of anti-EGFR-GNs on MDA-MB-231 cells which highly expressed EGFR was evident, but no binding observed in MCF-7 cells. Intratumor injection of anti-EGFR-GNs provided a significant enhancement in PA signal in axillary LN of mice after 24h, which correlated histologically with LN metastases with small number of cancer cells and anti-EGFR-GNs accumulation. The treatment of anti-EGFR-GNs combined with NIR-PTT augmented the induction of HSP70 and cleaved caspase-3 and decreased Ki-67 and EGFR and EGFR accompanying the strong inhibition of intracellular signaling molecules (mTOR, AKT, ERK1/2, and FAK), eventually exerting synergic anti-proliferative and apoptotic effects on the MDA-MB-231 cells. With the help of longitudinal real-time US-guided PAI and bioluminescence imaging, the combination of NIR-PTT, after three intravenous injections of anti-EGFR-GNs in tumor models, resulted in the greatest therapeutic effectiveness of almost complete tumor regression without virtually normal tissue damage compared with the treatment responses with free anti-EGFR antibodies or anti-EGFR-GNs alone.

Conclusions: In conclusion, US-guided PAI technology using anti-EGFR-GNs is feasible and highly sensitive for the selective visualization of EGFR-targeted primary tumors as well as LN micrometastases in a murine model of human TNBC. Anti-EGFR-GNs combined with NIR-PTT is an effective therapeutic strategy for TNBC, which enables selective tumor cell targeting through the synergistic mechanism of anti-proliferation and apoptosis without side effects and complications compared with previous conventional therapy strategy for EGFR-overexpressing TNBCs.