In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy

BMC Cancer

Technical Advance

Sophie Richon¹  Louis-Bastien Weiswald¹  Virginie Dangles-Marie²  Dominique Bellet³  Bruno Saubaméa⁴  Jean-Marc Guinebretière⁵ 

[1] IFR71 Sciences du Médicament, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Paris, France;IFR71 Sciences du Médicament, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Paris, France;Preclinical Investigation Laboratory, Department of Translational Research, Institut Curie, INSERM U932, Université Paris Descartes, Paris, France;IFR71 Sciences du Médicament, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Paris, France;Service de Médecine Nucléaire, Laboratoire d'Oncobiologie, Hôpital René Huguenin, Institut Curie, Saint Cloud, France;INSERM U705, UMR CNRS 8206 and Plateau Technique d'Imagerie Cellulaire et Moléculaire, IFR71 Sciences du Médicament, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Paris, France;Service de Pathologie, Hôpital René Huguenin, Institut Curie, Saint Cloud, France;

关键词: Cancer Stem Cell;    HT29 Cell;    Colon Cancer Cell Line;    Tumour Spheroid;    Tumour Sphere;   

DOI  :  10.1186/1471-2407-10-106

 received in 2009-09-10, accepted in 2010-03-22,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundMulticellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs). CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC) after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids.MethodsProtein expression in whole spheroids (150 μm in diameter) from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC) and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied.ResultsWe first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express β-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9+ cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively). Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to the luminal cell surface of colorectal cancer acini, with CD133+ cellular debris into glandular lumina.ConclusionThe present protocol enables in situ visualization of protein expression in compact three-dimensional models by whole mount confocal imaging, allowing the accurate localization and quantification of cells expressing specific markers. It should prove useful to study rare events like CSCs within tumour spheres.

【 授权许可】

Unknown   
© Weiswald et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.