Karina Christensen

CD133 identifies perivascular niches in grade II–IV astrocytomas

The aim of the present study was to investigate the localization and distribution of the putative brain tumour stem cell marker CD133 in formalin fixed paraffin embedded astrocytomas. A retrospective analysis of 114 grade II, III and IV astrocytomas was undertaken. The immunohistochemical expression of CD133 in paraffin sections was analysed using morphometry. In all grades, CD133 was expressed on tumour and endothelial cells. Tumour cells were found in perivascular niches, as dispersed single cells and in pseudopalisade formations around necrosis. There was no correlation between the mean volume fraction of CD133+ niches and all CD133+ tumour cells and tumour grade. However, the volume fraction of CD133+ blood vessels increased significantly from 0.4% in diffuse astrocytomas to 2.2% in glioblastomas. Neither of them was related to patient survival. Double immunofluorescence stainings showed that the CD133+ niches both contained CD133+ cells with and without co-expression of the intermediate filament protein marker nestin, and only few CD133+/MIB-1+ proliferating cells were found. In conclusion, a CD133+ perivascular stem cell-like entity exists in astrocytomas. CD133+ tumour vessels may play an important role in a brain tumour stem cell context, while CD133 alone does not appear to be a specific tumour stem cell marker related to patient survival.

Inconsistent Immunohistochemical Expression Patterns of Four Different CD133 Antibody Clones in Glioblastoma

The putative tumor stem cell marker CD133 is the marker of choice for identifying brain tumor stem cells in gliomas, but the use of different CD133 antibody clones possibly recognizing different CD133 splice variants with epitopes of different glycosylation status confuses the field. The aim was to investigate if current inconsistent CD133 observations could be a result of using different CD133 antibodies for immunohistochemical identification of CD133. Ten glioblastomas were immunohistochemically stained with four different CD133 antibody clones (AC133, W6B3C1, C24B9, and ab19898) and analyzed by quantitative stereology. Moreover, the CD133 staining pattern of each antibody clone was investigated in kidney, pancreas, and placenta tissue as well as in glioblastoma and retinoblastoma cultures and cell lines. All antibody clones revealed CD133+ niches and single cells in glioblastomas, but when using different clones, their distribution rarely corresponded. Morphology of identified single cells varied, and staining of various tissues, cultures, and cells lines was also inconsistent among the clones. In conclusion, the authors report inconsistent CD133 detection when using different primary CD133 antibody clones. Thus, direct comparison of studies using different antibody clones and conclusions based on CD133 immunohistochemistry should be performed with caution.

CD133+ niches and single cells in glioblastoma have different phenotypes

Putative CD133+ brain tumor stem cells have been shown to be located in niches and as single cells. This is the first study providing insight into the different phenotypes of CD133+ cells in glioblastoma according to localization. Paraffin sections were stained by double immunofluorescence with CD133 and the candidate stem cell markers Sox2, Bmi-1, EGFR, podoplanin and nestin, the proliferation marker Ki67 and the endothelial cell markers CD31, CD34, and VWF. Cell counting showed that the CD133+ cells in the niches had a significantly higher expression of Sox2, EGFR and nestin compared to CD133+ single cells, but only a 3% Ki67 labeling index versus 14% found for CD133+ single cells. Only low endothelial cell marker expression was found in the niches or the CD133− tumor areas, while 43% CD133+/CD31+ and 25% CD133+/CD34+ single cells were found. CD133+ blood vessels within CD133+ niches were less proliferative and more often Bmi-1+ than CD133+ blood vessels outside niches. In conclusion, different CD133+ cell phenotypes exist according to the in situ localization, and also the phenotype of CD133+ blood vessels vary according to the localization. CD133+ niches contain stem-like cells with a lower proliferation index than CD133+ single cells, which have an endothelial differentiation profile suggesting a role in angiogenesis.

Immunohistochemical expression of stem cell, endothelial cell, and chemosensitivity markers in primary glioma spheroids cultured in serum-containing and serum-free medium

OBJECTIVETo investigate the influence of serum-free medium (SFM) supplemented with epidermal growth factor and basic fibroblast growth factor compared with conventional serum-containing medium (SCM) on the phenotype of organotypic primary spheroids from seven gliomas. METHODSParaffin sections of the original surgical specimens, primary glioma spheroids, and U87 derived spheroids were stained immunohistochemically with the stem cell markers CD133, podoplanin, Sox2, Bmi-1, and nestin; the endothelial cell markers CD31, CD34, and Von Willebrand Factor (VWF); the chemosensitivity markers P-glycoprotein and tissue inhibitor of metalloproteinases-1 (TIMP-1); and glial fibrillary acidic protein, neural cell adhesion molecule CD56, and the proliferation marker Ki67. RESULTSScoring of the immunohistochemical stainings showed that the expression of CD133 and all other markers included was preserved in primary spheroids, confirming the in vivo-like nature of these spheroids. Spheroids in SFM better mimicked the in vivo phenotype with significantly more CD133, CD34, VWF, P-glycoprotein, TIMP-1, and Ki67 compared with SCM. CONCLUSIONIn this first study of the influence of SFM on primary glioma spheroids, the conditions favored an in vivo-like phenotype with increased expression of CD133. More vascular structures were found in SFM, suggesting that the close relationship between blood vessels and tumor stem-like cells was better preserved in this medium.

Abstract 4305: Invasion of primary glioma- and cell line-derived spheroids implanted into corticostriatal slice cultures

Gliomas are highly invasive tumors and the pronounced invasive features of gliomas prevent radical surgical resection. In the search for new therapeutics targeting invasive glioma cells, in vivo-like in vitro models are of great interest. We developed and evaluated an in vivo-like in vitro model preserving the invasive features and stem cell features of glioma cells. Fluorescently labelled primary glioma spheroids and U87MG cell line-derived spheroids were implanted into organotypic rat corticostriatal slice cultures and the invasion was followed over time by confocal microscopy. The invasion was validated immunohistochemically with paraffin sections using a human-specific vimentin antibody. Moreover, the preservation of immature stem cell features was evaluated immunohistochemically using the stem cell markers CD133, Sox2, Bmi-1 and nestin. The confocal and immunohistochemical results showed that the primary glioma spheroid area was constant or decreasing after implantation, with a clear increase in the number of invading cells over time. In contrast, the U87MG spheroid area increased after implantation, with no convincing tumor cell invasion. High levels of Bmi-1 and nestin were found in all spheroids, whereas high levels of Sox2 and low to moderate levels of CD133 were only found in the primary spheroids. In conclusion, the invasion of gliomas is preserved using primary glioma spheroids. Some stem cell features are preserved as well, making this model useful in drug development elucidating both invasion and cancer stemness at the early in vitro level.

Primary Glioma Spheroids: Advantage of Serum-Free Medium

In the field of cancer research, in vitro models are widely used to investigate tumor biology as well as new experimental treatments. There is a need to improve these models, making them more in vivo-like, in order to meet the new challenges arising together with emerging knowledge such as the discovery of cancer stem-like cells. When investigating glioblastomas in vitro – and especially the supposed cancer stem cells – three dimensional spheroid models recently came into focus. Spheroids have earlier been derived from commercial glioblastoma cell lines, but in the 1990s the spheroid model was further improved by the introduction of organotypic primary spheroids derived from small tumor fragments preserving tumor tissue characteristics including cell-to-cell interactions, extracellular matrix, macrophages and blood vessels. Most recently – in the cancer stem cell context – spheroids have been derived from single cells from dissociated primary glioblastoma tissue and cultured in serum-free conditions similar to neuronal stem cells. Using this methodology a much better preservation of cancer stem cell features was obtained compared to the use of serum-containing medium. Furthermore, in a study recently performed in our lab we demonstrated similar advantages of culturing primary organotypic spheroids in serum-free medium favoring an in-vivo-like cancer cell phenotype with better preservation of cancer stem cell features. In addition to this, more vascular structures were seen under serum-free conditions suggesting that the close relationship between blood vessels and cancer stem-like cells was better preserved in this medium. As it will be reviewed below, culturing primary spheroids in a serum-free medium adds a new level to the spheroid model suggesting that this model should be included in studies of tumor biology and drug development when possible.

Abstract 3369: Discordant immunohistochemical expression patterns of four different CD133 antibody clones in glioblastoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The putative tumor stem cell marker CD133 is the marker of choice for identifying brain tumor stem cells in gliomas, but the use of different antibody clones recognizing different epitopes with different glycosylation status, confuses the field. In this study, we sat out to highlight if current discordant CD133 observations could be a result of using different CD133 antibodies for immunohistochemical identification of CD133. Paraffin embedded sections of glioblastoma, kidney, pancreas and placenta tissue as well as glioblastoma and retinoblastoma cell lines were stained with four different CD133 antibody clones and analyzed using light microscopy. Ten consecutive sections of glioblastomas were analyzed with each of the four CD133 clones using quantitative stereology. Results revealed presence of CD133+ niches in glioblastomas, often in close relation to blood vessels, using all four antibody clones. The distribution of identified niches did, however, rarely correspond among each antibody clone. Staining of glioblastoma single and niche cells was predominantly cytoplasmatic, which is opposed to the membranous staining observed in epithelial cells in kidney, pancreas and placenta tissues. Quantitative stereology revealed vast dissimilarities regarding fractions of CD133+ niches and single cells among the CD133 antibody clones. Generally, the fraction of CD133 positivity identified by clone W6B3C1 was significantly higher than CD133 fractions identified by clones AC133, ab19898 and C24B9. Furthermore, clone W6B3C1 was the only clone to stain tumor blood vessels. In conclusion, we report that using different CD133 antibodies for immunohistochemical identification of CD133+ cells on paraffin sections will most likely cause dissimilar results. Thus, direct comparison of CD133 studies using different primary CD133 antibodies should be performed with caution. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3369.