Comparison of mouse mammary gland imaging techniques and applications: Reflectance confocal microscopy, GFP Imaging, and ultrasound

Maddalena T Tilli,Michael D Johnson,Laundette P Jones,Angela R Parrish,Priscilla A Furth,Ion Cotarla

doi:10.1186/1471-2407-8-21

Abstract

BackgroundGenetically engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue.MethodsWe evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging.ResultsIn the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice.ConclusionIn conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary.

Highlights

Engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology
A 3-D rendering of terminal end bud structures is included as Additional File 1. To determine if this technique could be used to visualize structural elements prior to whole mammary organ culture, we treated mammary glands from estradiol and mg progesterone (E&P) pellet primed fiveweek-old mice with phosphate buffered saline (PBS), 1%, 3%, or 5% acetic acid, imaged the gland with reflectance confocal microscopy (RCM), and subjected them to whole organ culture (WOC) media supplemented with IPAH for 1 week
Only the glands treated with 1% acetic acid were able to grow consistently and differentiate normally in response to IPAH in whole organ culture (Figure 2H and 2I) as compared to the non-imaged glands immediately put into culture (Figure 2B and 2C) and imaged glands treated with PBS alone (Figure 2E and 2F)

Summary

Introduction

Engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. Transgenic mouse models have been developed to recapitulate the complex effects of genes known to be involved in human breast cancer These models can help to elucidate the mechanism of action of these genes during carcinogenesis, as well as their impact on normal mammary gland development. In order to study early phenotypic effects of gene overexpression or lack of expression on mammary gland development and cancer traditional methods require that tissue is harvested from the animal and subjected to histological techniques to detect morphologically aberrant growth. These invasive procedures preclude further examination of the effects of these genetic changes on the process of carcinogenesis. No information about initiation and progression can be gathered; only at the end of the experiment [2] can information about the morphology or gene expression profile of the developing tumor be obtained

Methods

Results

Conclusion