In vivo investigation of breast cancer progression by use of an internal control

Abstract

Optical imaging of breast cancer has been considered for detecting functional and molecular characteristics of diseases in clinical and preclinical settings. Applied to laboratory research, photonic investigations offer a highly versatile tool for preclinical imaging and drug discovery. A particular advantage of the optical method is the availability of multiple spectral bands for performing imaging. Herein, we capitalize on this feature to demonstrate how it is possible to use different wavelengths to offer internal controls and significantly improve the observation accuracy in molecular imaging applications. In particular, we show the independent in vivo detection of cysteine proteases along with tumor permeability and interstitial volume measurements using a dual-wavelength approach. To generate results with a view toward clinically geared studies, a transgenic Her2/neu mouse model that spontaneously developed mammary tumors was used. In vivo findings were validated against conventional ex vivo tests such as histology and Western blot analyses. By correcting for biodistribution parameters, the dual-wavelength method increases the accuracy of molecular observations by separating true molecular target from probe biodistribution. As such, the method is highly appropriate for molecular imaging studies where often probe delivery and target presence are not independently assessed. On the basis of these findings, we propose the dual-wavelength/normalization approach as an essential method for drug discovery and preclinical imaging studies.

Figure 1

Multispectral multimodal imaging system schematic. Configuration allowed for investigations of epi-illumination imaging at two wavelengths (672 and 748 nm).

Figure 2

In vivo NFRI signals obtained from tumor and nondiseased contralateralmammary fat pad (CMFP) tissue for each probe of interest. All signal values obtained fromadenocarcinomas are statistically higher than background; however, ProSense expression is detected at amuch higher extent compared withMMPSense. AngioSense is a nonspecific probe used in this study to account for probe delivery.

Figure 3

In vivo dual-wavelength epi-illumination images of cysteine protease expression in transgenic mammary tumor-forming mice. (A) White light image identifies tumor location and category: S indicates small, M, medium, L, large tumors. (B) AngioSense channel shows an accumulation of probe in tumor and represents vascularization and biodistribution of activatable probe. (C) Protease channel (either ProSense or MMPSense) shows accumulation of probe in tumor, but tumor size is not ascertainable by expression levels. (D) Superposition of protease signal divided by AngioSense signal with white light image shows how dual-wavelength/normalization approach can improve the observation accuracy.

Figure 4

In vivo protease signals from tumors of various sizes either plotted with or normalized to corresponding vascular signals. Raw mean ProSense (A) and MMPSense (C) along with AngioSense signals for each tumor. As seen, protease expressions remain rather constant and are statistically independent of tumor size, which is not caused by a lack of available protease probe at the cancerous region as evident by AngioSense signals. Mean ProSense (B) and MMPSense (D) expressions normalized to respective biodistribution signals (AngioSense) for tumors of different categorical areas. This internal control provides a statistically relevant way to observe disease progression.

Figure 5

In vivo fluorescence signals plotted against ex vivo signals obtained by Western blot analyses. Mean ProSense (A) and MMPSense (C) signals plotted against corresponding total protein level expressions. Mean ProSense (B) and MMPSense (D) normalized to drug delivery (i.e., divided by AngioSense) plotted against corresponding total protein level expressions. Normalizing in vivo protease signals with biodistribution signals allows for an internal correction that also provides an improved correlation to the underlying physiology.