Novel immunofluorescence protocol for multimarker assessment of putative disseminating breast cancer stem cells.
OBJECTIVE
The phenotypical and functional variety of breast cancer cells is well recognized. This variety is evident in primary tumors and in disseminated tumor cells (DTCs) and solid metastases as shown for recognized prognostic factors, such as estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2/neu and also for cancer stem cell markers such as CD44, CD24, or aldehyde dehydrogenase (ALDH). For the development of new therapeutic strategies, the identification and characterization of disseminated breast cancer cells are needed. This requires the use of multiple antibodies (ie, cytokeratin, Her2/neu, ALDH1, CD44, and CD24) labeled with fluorochromes of different colors and spectral image analysis to separate different color spectra.


METHODS
We have focused here on putative breast cancer stem cell markers and evaluated the feasibility of triple and quadruple labeling of breast cancer cells. Using breast cancer cell lines we have developed a method optimized for multimarker analysis by employing novel DyLight Technology. Single marker immunofluorescence was performed in 6 replicates, and reproducible results had to be obtained before proceeding to multimarker immunofluorescence.


RESULTS
Three of the markers, CD44, ALDH1, and cytokeratin have been directly conjugated with DyLight dyes. CD24 could not be conjugated directly to the fluorescent dye. A labeled secondary antibody was used for visualization. Single and multimarker immunofluorescence gave consistent results throughout the replicates.


CONCLUSIONS
This novel protocol will facilitate detection and phenotypical characterization of disseminated tumor cells. In addition, by adding additional markers, distinct subpopulations could be evaluated for the expression of particular therapeutic targets.
Balic
Marija
M
Department of Internal Medicine, Medical University of Graz, Austria. marija.balic@medunigraz.at
Rapp
Nadine
N
Stanzer
Stefanie
S
Lin
Henry
H
Strutz
Jasmin
J
Szkandera
Joanna
J
Daidone
Maria Grazia
MG
Samonigg
Hellmut
H
Cote
Richard James
RJ
Dandachi
Nadia
N
D000912:Antibodies, Neoplasm / Q000737:chemistry* / Q000276:immunology; D014408:Biomarkers, Tumor / Q000276:immunology / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods* / Q000592:standards; D056945:Hep G2 Cells; D006801:Humans; D014411:Neoplastic Stem Cells / Q000276:immunology / Q000378:metabolism*
D000912:Antibodies, Neoplasm; D014408:Biomarkers, Tumor