Ultrasound-guided photoacoustic imaging for the selective detection of EGFR-expressing breast cancer and lymph node metastases

Abstract

We assessed the use of ultrasound (US)-guided photoacoustic imaging (PAI) and anti-EGFR antibody-conjugated gold nanorods (anti-EGFR-GNs) to non-invasively detect EGFR-expressing primary tumor masses and regional lymph node (LN) metastases in breast tumor mice generated by injecting MCF-7 (EGFR-negative) or MDA-MB-231 (EGFR-positive) human breast cells using a preclinical Vevo 2100 LAZR Imaging system. Anti-EGFR-GNs provided a significant enhancement in the PA signal in MDA-MB-231 tumor and the axillary LN metastases relative to MCF-7 tumor and non-LN metastases. We demonstrated that US-guided PAI using anti-EGFR-GNs is highly sensitive for the selective visualization of EGFR-expressing breast primary tumors as well as LN micrometastases.

Keywords: (170.3880) Medical and biological imaging; (170.4580) Optical diagnostics for medicine; (170.5120) Photoacoustic imaging; (170.7170) Ultrasound.

Fig. 1

Analysis of US-guided photoacoustic (PA) images and histology of the axillary LN of mice injected with ICG or gold nanorods (GN). (a) Non-invasive and dynamic fusion of US and PA images of the axillary LN region before and 0.5 h, 1 h, 2 h, 4 h and 24 h after ICG injection (5 pmol/g mouse). (b) Plot of PA signal amplitude in axillary LNs versus pre- and post-injection time of ICG. (c) Non-invasive and dynamical fusion of US and PA images of axillary LN region before and 4, 12 h, 24 h, 48 h and 72 h after GN injection (0.005 pmol/g mouse). (d) Plot of PA signal amplitude in axillary LNs versus pre- and post-injection time of GN. The results represent the mean ± standard deviation in the ICG-mice group (n = 5) and the GN-mice group (n = 5), *p<0.05, **p<0.01, Arbitrary units (AU). (e, f) Photographs of isolated axillary LN before and after injection of ICG or GN. (g) H&E staining of microsectioned axillary LN isolated from mice 4 h post-injection of ICG. (h) H&E staining and silver staining of microsectioned axillary LN isolated from mice at 48 h post-injection of GN. The control (Cont) is a microsectioned axillary LN without injection of ICG or GN. Scale bar: 100 μm.

Fig. 2

Analysis of EGFR expression in human breast cancer cell lines and US-guided PAI and histology acquired in MCF-7 and MDA-MB-231 primary tumors of mice intravenously injected with anti-EGFR-GNs. (a) EGFR expression in MCF-7, BT-474, HCC-1954, HCC-1937, MDA-MB-453 and MDA-MB-231 human breast cancer cell lines (by western blot). (b) Silver staining of MCF-7 and MDA-MB-231 cells incubated with or without 120pmol/L of anti-EGFR-GNs for 24 h. Control (Cont) was incubated without 120pmol/L of anti-EGFR-GNs. (c) Non-invasive and dynamical fusion of US and PA images of MCF-7 and MDA-MB-231 tumors before and 2 h, 4 h, 8 h, 24 h, 48 h and 72 h after intravenous injection with anti-EGFR-GNs (0.5 pmol/g mouse). (d) Plot of PA signal amplitude in MCF-7 and MDA-MB-231 tumors versus pre- and post-injection of anti-EGFR-GNs. The results represent the mean ± standard deviation in the MCF-7-mice group (n = 5) and MDA-MB-231-mice group (n = 5). **p<0.01, Arbitrary units (AU). (e) H&E staining, EGFR immunostaining and silver staining of microsectioned MDA-MB-231 and MCF-7 primary tumors isolated from mice 48 h post-injection of anti-EGFR GN. Scale bar: 100 μm.

Fig. 3

Analysis of US-guided PAI and histology acquired in MCF-7 and MDA-MB-231 primary tumors of mice intravenously injected with non-targeted GNs. (a) Non-invasive and dynamical fusion of US and PA images of MCF-7 and MDA-MB-231 tumors before and 2 h, 4 h, 8 h, 24 h, 48 h and 72 h after intravenous injection with non-targeted GNs (0.5pmol/g mouse). (b) Plot of PA signal amplitude in MCF-7 and MDA-MB-231 tumors versus pre-and post-injection of non-targeted GNs. The results represented the mean ± standard deviation in the MCF-7-mice group (n = 5) and MDA-MB-231-mice group (n = 5). **p<0.01, Arbitrary units (AU). (c) H&E staining and silver staining of microsectioned MDA-MB-231 and MCF-7 primary tumor isolated from mice 48 h post-injection of non-targeted GNs. Scale bar: 100 μm.

Fig. 4

Analysis of US-guided PAI and histology acquired in LN metastases of MDA-MB-231-Luc tumor-bearing mice injected with anti-EGFR-GNs into the primary tumor. (a, b) Non-invasive and dynamical fusion of US and PA images of axillary LN metastases and non-LN metastases in MDA-MB-231-Luc tumor-bearing mice 24 h after intratumoral injection with anti-EGFR-GNs (0.5 pmol/g mouse). (c) Plot of PA signal amplitude in axillary LN at pre- and post-injection of anti-EGFR-GNs. The results represent the mean ± standard deviation in the LN metastases mice group (n = 2) and non-LN metastases mice group (n = 4), Arbitrary units (AU). (d) In vivo (left) and ex vivo (right) bioluminescence imaging of MDA-MB-231-Luc tumor-bearing mice injected intraperitoneally with D-luciferin (150 μg/g mouse). Red and white circles indicate the excised primary tumor and axillary LN from ex vivo bioluminescence imaging. (e, f) H&E staining, EGFR and cytokeratin 8/18/19 immunostaining and silver staining of microsectioned axillary LNs isolated from mice at 48 h post-injection of anti-EGFR GNs. Scale bar: 100 μm.