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. 2010 Mar 22:10:106.
doi: 10.1186/1471-2407-10-106.

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

Louis-Bastien Weiswald  1 , Jean-Marc Guinebretière, Sophie Richon, Dominique Bellet, Bruno Saubaméa, Virginie Dangles-Marie
Affiliations

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

Louis-Bastien Weiswald et al. BMC Cancer. .

Abstract

Background: Multicellular 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.

Methods: Protein expression in whole spheroids (150 mum 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.

Results: We 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 beta-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.

Conclusion: The 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.

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Figures

Figure 1
Figure 1
Homogeneous penetration of EpCAM-FITC antibody in whole spheroids. (A) Flow cytometry analysis of CT320X6 (left histogram) and HT29 (right histogram) cells from dissociated spheroids. Cells were labelled with the same FITC-conjugated EpCAM antibody (red area) or FITC-conjugated isotype control antibody (white area). All cells were EpCAM positive. (B) CT320X6 (left panels) and HT29 (right panels) spheroids were fixed and permeabilized as described in Methods. Spheroids were then directly labelled using FITC-conjugated EpCAM antibody (yellow) and counterstained with TOPRO-3 (cyan). Acquisitions were taken at 40 μm and 70 μm depth within the same spheroid (scale bar = 50 μm).
Figure 2
Figure 2
CK20 expression in colon cancer spheroids. (A) CT320X6 (upper panel) and HT29 (lower panel) spheroids were immunostained against CK20 (yellow) and nuclei were counterstained with TOPRO-3 (cyan). Acquisitions were taken at 70 μm depth. Almost all cells were positive (scale bar = 50 μm). (B) Permeabilized cells from dissociated CT320X6 (upper panel) and HT29 (lower panel) spheroids were labelled with the same mAb against CK20. FC analysis confirmed a population with almost 100% of positive cells.
Figure 3
Figure 3
CA 19-9 and β-catenin expression in HT29 spheroids. (A) HT29 spheroids were immunostained against CA 19-9 (red, upper panel) or β-catenin (red, lower panel) and nuclei were counterstained with TOPRO-3 (blue). Acquisitions were taken at 70 μm depth. HT29 spheroids displayed a heterogeneous CA 19-9 expression with a subpopulation of 28% of CA 19-9 negative cells while cytoplasmic, nuclear and membrane associated β-catenin was found in virtually all cells. White arrowheads show nuclear β-catenin immunostain. Scale bar = 50 μm (whole spheroid) and 25 μm (zooms). (B) Permeabilized cells from dissociated HT29 spheroids were labelled against CA 19-9 (upper histogram) or β-catenin (lower histogram) and analysed by flow cytometry. All HT29 spheroid cells were positive for β-catenin whereas 26% of cells were negative for CA 19-9. (C) HT29 spheroids were embedded using the Cytoblock method and stained with the anti-CA 19-9 antibody (upper image) or the anti-β-catenin antibody (lower image) using conventional immunohistochemistry and counterstained with hematoxilin. A subset of cells did not express CA 19-9 (49%) while β-catenin staining was observed in nearly all cells, mainly associated with plasma membrane but also within cytoplasm and nuclei.
Figure 4
Figure 4
CD44 and ALDH1 expression in HT29 spheroids. (A) HT29 spheroids were immunostained against CD44 (yellow, upper panel) or ALDH1 (yellow, lower panel) and nuclei were counterstained with TOPRO-3 (cyan). Acquisitions were taken at 70 μm depth (scale bar = 50 μm). All cells expressed CD44 while a large population of cells (86%) displayed ALDH1 staining. (B) Conventional pIHC using Cytoblock embedding technique confirmed that all cells were CD44+ and 85% were ALDH1+. (C) All cells from dissociated HT29 spheroids were found CD44+ in FC analysis.
Figure 5
Figure 5
CD133 expression at the luminal surface of some epithelial tumour glands with shedding into the lumina within tumour spheroids. (A) CD133 (yellow) and EBP50 (magenta) were visualized within CT320X6 (upper panel) and HT29 (lower panel) spheroids by immunofluorescence staining. Spheroids were counterstained with TOPRO-3 (cyan). CD133 was found into the lumina and colocalized with EBP50 at the luminal surface of some acini. Acquisitions were taken at 70 μm depth (scale bar = 50 μm). (B) Dissociated cells from CT320X6 (upper histogram) and HT29 (lower histogram) spheroids were similarly labelled against CD133 and analysed by flow cytometry.
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