Header

UZH-Logo

Maintenance Infos

Improved visualization of lung metastases at single cell resolution in mice by combined in-situ perfusion of lung tissue and X-Gal staining of lacZ-tagged tumor cells


Arlt, Matthias J E; Born, Walter; Fuchs, Bruno (2012). Improved visualization of lung metastases at single cell resolution in mice by combined in-situ perfusion of lung tissue and X-Gal staining of lacZ-tagged tumor cells. Journal of Visualized Experiments, (66):e4162.

Abstract

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.

Abstract

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.

Statistics

Altmetrics

Downloads

32 downloads since deposited on 16 Nov 2012
12 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Balgrist University Hospital, Swiss Spinal Cord Injury Center
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2012
Deposited On:16 Nov 2012 12:54
Last Modified:05 Apr 2016 16:05
Publisher:Journal of Visualized Experiments
ISSN:1940-087X
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.3791/4162
Official URL:http://www.jove.com/video/4162/improved-visualization-lung-metastases-at-single-cell-resolution-mice
PubMed ID:22929213

Download

Download PDF  'Improved visualization of lung metastases at single cell resolution in mice by combined in-situ perfusion of lung tissue and X-Gal staining of lacZ-tagged tumor cells'.
Preview
Content: Published Version
Language: English
Filetype: PDF
Size: 387kB
View at publisher