One of the main challenges biologists currently face is overcoming the problem of tissue heterogeneity to further understand organ function. It is crucial to distinguish which cell populations produce specific molecules or to get relevant expression profiles reflecting in vivo status.
Milk is synthesized in mammary gland during lactation and though this process has been thoroughly studied, we still do not know precisely what mechanisms are involved in the intracellular transport and secretion of milk components, including supra-molecular structures, such as casein micelles [1
] which are assembled during their transit within the mammary epithelial cell (MEC).
Mammary parenchyma consists of secretory alveoli organized into lobules and interconnected by a system of branching ducts separated from adipocytes by multiple layers of fibroblastic connective tissue. In the duct and alveoli, the mammary epithelium is organized into two layers, a basal layer of myoepithelial cells (MMC) and a luminal layer of MEC that secretes milk [3
]. The extra cellular matrix comprises non-epithelial cells: fibroblast, endothelial cells, lymphocytes, adipocytes, neurons, myocytes, etc. Thus, the adult mammary gland during lactation is a complex tissue consisting of several cell types. During lactation, epithelial cells are predominant relative to adipocytes which are conversely more abundant in the nulliparous gland [3
]. Since both cell types are involved in lipid metabolism using the same metabolic pathways and enzymes, it becomes difficult to sort out the function of each cell type [4
Advances in molecular biology have provided new tools, including gene expression profiling, to analyze mechanisms controlling mammary gland development and differentiation [6
] and regulating milk synthesis and secretion. However, most of the studies performed to date on healthy mammary gland have been done without taking into account the complexity of this tissue with the exception of Grigoriadis et al
]. On the other hand, a number of integrated approaches combining advanced molecular technologies have been applied to analyze human breast cancer [9
], but few studies were carried out on healthy breast tissue compared to carcinoma [12
]. Analysis of bulk mammary tissue homogenates leads inevitably to an average measurement of biomolecules (RNA and proteins) from the various cell types it is made of. Therefore, there is a high risk that changes in the expression of genes involved in MEC functions could be masked by their expression in surrounding cells. For example, genes involved in lipid biosynthesis are expressed in MEC and adipocytes but not regulated in the same way during lactation [14
Therefore, to accurately and reliably follow molecular changes occurring in MEC for comparison purposes between physiologically different stages and genetically or environmentally perturbed systems, it is necessary to isolate MEC preserving biomolecule (RNA and proteins) integrity.
Different techniques, such as immunomagnetic separation [16
], cell sorting [20
] and tissue-depletion [15
] have been used to isolate more or less homogeneous populations of MEC from milk or mammary tissue. MECs isolated from milk are easy to collect non-invasively and constitute a valuable source of material for analyzing mammary transcript profile during lactation. Although it has been claimed that milk MECs reliably reflect the activity of the mammary epithelium in goats and cattle [21
], one can expect that cells out of their physiological context and faced with stressful purification protocols very likely induce adaptive changes modifying their expression profile. Differentially expressed membrane antigens have been used to flow-sort viable luminal epithelial and MMC from freshly disaggregated adult virgin rat mammary parenchyma [23
Another means to obtain MEC homogeneous populations is from cell culture. However, one major obstacle to molecular biological studies of MEC is the lack of established cell lines that secrete, or can be induced to secrete, fat globules and milk proteins [24
]. While culture systems have helped to identify some of the factors controlling growth [25
], morphogenesis [27
], functional differentiation [29
] and tumorigenesis [30
] of the rodent mammary gland, the heterogeneous cellular composition of primary cultures derived from the intact mammary parenchyma [32
] complicates the interpretation of responses in vitro
. In addition, it is well-established that MEC in culture are subjected to dedifferentiation [34
Laser Capture Microdissection (LCM), first described by Emmert-Buck et al
] is now well established as a powerful tool for isolating cells of interest under morphological control from heterogeneous tissues. Major issues that should be addressed when using such a sorting approach are the amount and integrity of biological material extracted for reliable subsequent analyses of biomolecules (DNA, RNA and proteins). Amplification of nucleic acids is still possible as well, provided integrity is preserved. RNA degradation remains one of the main concerns since it can extend dramatically, depending upon the tissue. Also, it may significantly impact gene expression profiling. Frozen tissues are recommended for RNA recovery.
Nevertheless, LCM is an appealing technique, but it introduces additional methodological hurdles, including tissue handling (fixation, storage and staining) and maintenance of molecular integrity. The success of a microdissection experiment first depends upon the ability to distinguish cell types of interest from their morphological features. Immunological labelling may be required and used to assist in the identification of cells. In other words, if gene expression experiments are targeted, the challenge is to design a global protocol ensuring acceptable tissue morphology to facilitate isolation of cells while preserving accessibility and integrity of RNA, keeping in mind that this is critically tissue-dependent.
Successful application of LCM in transcriptomic analyses relies upon three critical factors: good tissue morphology, capture efficiency, and maintenance of RNA molecular integrity. Effective balancing of these three factors is required to recognize regions and obtain reliable transcriptomic results. Since ruminant mammary gland is one of the richest tissues in RNAse activity, classical protocols require accommodation to preserve RNA from RNase (endogenous and exogenous) and keep it intact in captured cells. This study was carried out to address these issues with the aim of developing a convenient and reproducible protocol to isolate MECs from ruminants (goat, sheep and cow) lactating mammary gland, preserving tissue morphology and RNA integrity, to develop a comprehensive overview of the genome expressed in MECs in their physiological environment.
Given that recent studies [36
] reported the effects of tissue manipulation on RNA quality and gene expression, and that each tissue requires specific protocol for reliable results, we have evaluated the impact of the main critical steps (sampling, freezing, cryosectioning, staining, dehydration, and microdissection) during slide preparation and capture of MEC. In addition, we examined selectivity of this technique in evaluating enrichment in MEC as well as contamination by other surrounding cell types such as MMC, and immune cells (macrophages and lymphocytes) using qPCR.