Metastasis is the main cause of death in the majority of
cancer types and consequently a main focus in
cancer research. However, the detection of
micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited. While animal models have proven to be invaluable tools for
cancer research, the monitoring/visualization of
micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of
metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of
tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging. We are currently optimizing these techniques for in vivo monitoring of primary
tumor growth and
metastasis in different
osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of
metastasis beside classical methods like qPCR, FACS or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of
tumor cells with the lacZ gene encoding the bacterial
enzyme β-
galactosidase that metabolizes the
chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (
X-Gal) to an insoluble
indigo blue dye and allows highly sensitive and selective histochemical blue staining of
tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of
metastasis in studies assessing new anticancer
therapies. A limiting factor of
X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al. who perfused the lungs of mice under
anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of
X-gal staining of lacZ-expressing
tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung
micrometastases in a mouse model, which was originally described to be non-metastatic.