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EpCAM is a putative stem marker in retinoblastoma and an effective target for T-cell-mediated immunotherapy.

Mitra M, Kandalam M, Harilal A, Verma RS, Krishnan UM, Swaminathan S, Krishnakumar S - Mol. Vis. (2012)

Bottom Line: EpCAM+ cells showed significantly higher proliferative invasive potential and neurosphere formation in vitro compared to EpCAM⁻ Y79 cells.EpCAM+ cells showed higher β-catenin expression compared to EpCAM- cells.EpCAM×CD3 significantly retarded proliferation of RB primary tumor cells.

View Article: PubMed Central - PubMed

Affiliation: L&T Department of Ocular Pathology, Vision Research Foundation, Tamil Nadu, India.

ABSTRACT

Purpose: The molecular markers cluster of differentiation (CD)24, CD44, adenosine tri-phosphate (ATP) binding cassette protein G2 (ABCG2), and epithelial cell adhesion molecule (EpCAM) are widely used, individually or in combination, to characterize some types of cancer stem cells. In this study we characterized the EpCAM+ retinoblastoma (RB) cells for their cancer stem-like properties in vitro. Additionally, we targeted RB tumor cells via redirecting T cells using bispecific EpCAM×CD3 antibody.

Methods: Flow cytometry was used to study the co-expression of EpCAM with putative cancer stem cell markers, such as CD44, CD24, and ABCG2, in RB primary tumors. In vitro methyl thiazol tetrazolium (MTT) assay, invasion assay, and neurosphere formation assay were performed to characterize EpCAM+ cells for their cancer stem/progenitor cell-like properties. We assessed the in vitro efficacy of bispecific EpCAM×CD3 antibody on RB tumor cell proliferation and validated the results by evaluating effector cytokine production in the culture medium with the ELISA method.

Results: EpCAM was co-expressed with all cancer stem cell markers (CD44, CD24, and ABCG2) in primary RB tumors. EpCAM+ cells showed significantly higher proliferative invasive potential and neurosphere formation in vitro compared to EpCAM⁻ Y79 cells. EpCAM+ cells showed higher β-catenin expression compared to EpCAM- cells. EpCAM×CD3 significantly retarded proliferation of RB primary tumor cells. EpCAM×CD3 effectively induced the secretion of effector cytokines, such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-2, and transforming growth factor (TGF)-β1, and also perforin levels by pre-activated lymphocytes.

Conclusions: EpCAM might be a novel cancer stem cell marker in RB. EpCAM×CD3 antibody redirecting T cells to attack RB tumor cells may prove effective in RB management. Further preclinical studies are needed to confirm the initial findings of our study.

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Investigation of the co-expression of epithelial cell adhesion molecule (EpCAM) with cluster determinant (CD)44, CD24 and ATP binding cassette protein G2 (ABCG2) markers in retinoblastoma (RB) tumor 4. A: Scatter plot shows the co-expression of EpCAM with CD44. B: Scatter plot shows the co-expression of EpCAM with CD24. C: Scatter plot shows the co-expression of EpCAM with ABCG2. EpCAM-fluorescein isothiocyanate (FITC) represents EpCAM positive cells identified using FITC labeled antibody. CD44-PE represents CD44 positive cells identified using phycoerythrin labeled antibody. CD24-APC represents CD24 positive cells identified using allophycocyanin labeled antibody. ABCG2-APC represents ABCG2 positive cells identified using allophycocyanin labeled antibody..
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f6: Investigation of the co-expression of epithelial cell adhesion molecule (EpCAM) with cluster determinant (CD)44, CD24 and ATP binding cassette protein G2 (ABCG2) markers in retinoblastoma (RB) tumor 4. A: Scatter plot shows the co-expression of EpCAM with CD44. B: Scatter plot shows the co-expression of EpCAM with CD24. C: Scatter plot shows the co-expression of EpCAM with ABCG2. EpCAM-fluorescein isothiocyanate (FITC) represents EpCAM positive cells identified using FITC labeled antibody. CD44-PE represents CD44 positive cells identified using phycoerythrin labeled antibody. CD24-APC represents CD24 positive cells identified using allophycocyanin labeled antibody. ABCG2-APC represents ABCG2 positive cells identified using allophycocyanin labeled antibody..

Mentions: We investigated the expression of four markers that have previously been described for isolation of several types of human CSCs in human cancer cell lines: CD44, CD24, ABCG2, and EpCAM [25-32]. In this study CD44, CD24, ABCG2, and EpCAM could be detected by flow cytometry in all the RB patient tumor samples. The individual expression of CD44, CD24, ABCG2, and EpCAM in primary tumor samples ranged from 19% to 64%, 15% to 77%, 2% to 7%, 4% to 65%, respectively (Figure 2). The co-expression of EpCAM+CD44+, EpCAM+CD24+, and EpCAM+ABCG2+ ranged from 0.83% to 42.6%, 0.65% to 41.4%, and 1.6% to 6.5%, respectively, in the eight RB tumor samples (Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, and Figure 10). The percentage of cancer cells expressing a single surface marker in individual tumors was significantly different. Except tumor No.1, all other tumors showed a significantly higher percentage of cells positive for the combination markers. The percentage of ABCG2 was less in all eight tumors, which is consistent with a previous study [31]. Although the expression of AGCB2 was less in tumors, we could still see that a small subpopulation of ABCG2-positive cells co-expressed EpCAM.


EpCAM is a putative stem marker in retinoblastoma and an effective target for T-cell-mediated immunotherapy.

Mitra M, Kandalam M, Harilal A, Verma RS, Krishnan UM, Swaminathan S, Krishnakumar S - Mol. Vis. (2012)

Investigation of the co-expression of epithelial cell adhesion molecule (EpCAM) with cluster determinant (CD)44, CD24 and ATP binding cassette protein G2 (ABCG2) markers in retinoblastoma (RB) tumor 4. A: Scatter plot shows the co-expression of EpCAM with CD44. B: Scatter plot shows the co-expression of EpCAM with CD24. C: Scatter plot shows the co-expression of EpCAM with ABCG2. EpCAM-fluorescein isothiocyanate (FITC) represents EpCAM positive cells identified using FITC labeled antibody. CD44-PE represents CD44 positive cells identified using phycoerythrin labeled antibody. CD24-APC represents CD24 positive cells identified using allophycocyanin labeled antibody. ABCG2-APC represents ABCG2 positive cells identified using allophycocyanin labeled antibody..
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275636&req=5

f6: Investigation of the co-expression of epithelial cell adhesion molecule (EpCAM) with cluster determinant (CD)44, CD24 and ATP binding cassette protein G2 (ABCG2) markers in retinoblastoma (RB) tumor 4. A: Scatter plot shows the co-expression of EpCAM with CD44. B: Scatter plot shows the co-expression of EpCAM with CD24. C: Scatter plot shows the co-expression of EpCAM with ABCG2. EpCAM-fluorescein isothiocyanate (FITC) represents EpCAM positive cells identified using FITC labeled antibody. CD44-PE represents CD44 positive cells identified using phycoerythrin labeled antibody. CD24-APC represents CD24 positive cells identified using allophycocyanin labeled antibody. ABCG2-APC represents ABCG2 positive cells identified using allophycocyanin labeled antibody..
Mentions: We investigated the expression of four markers that have previously been described for isolation of several types of human CSCs in human cancer cell lines: CD44, CD24, ABCG2, and EpCAM [25-32]. In this study CD44, CD24, ABCG2, and EpCAM could be detected by flow cytometry in all the RB patient tumor samples. The individual expression of CD44, CD24, ABCG2, and EpCAM in primary tumor samples ranged from 19% to 64%, 15% to 77%, 2% to 7%, 4% to 65%, respectively (Figure 2). The co-expression of EpCAM+CD44+, EpCAM+CD24+, and EpCAM+ABCG2+ ranged from 0.83% to 42.6%, 0.65% to 41.4%, and 1.6% to 6.5%, respectively, in the eight RB tumor samples (Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, and Figure 10). The percentage of cancer cells expressing a single surface marker in individual tumors was significantly different. Except tumor No.1, all other tumors showed a significantly higher percentage of cells positive for the combination markers. The percentage of ABCG2 was less in all eight tumors, which is consistent with a previous study [31]. Although the expression of AGCB2 was less in tumors, we could still see that a small subpopulation of ABCG2-positive cells co-expressed EpCAM.

Bottom Line: EpCAM+ cells showed significantly higher proliferative invasive potential and neurosphere formation in vitro compared to EpCAM⁻ Y79 cells.EpCAM+ cells showed higher β-catenin expression compared to EpCAM- cells.EpCAM×CD3 significantly retarded proliferation of RB primary tumor cells.

View Article: PubMed Central - PubMed

Affiliation: L&T Department of Ocular Pathology, Vision Research Foundation, Tamil Nadu, India.

ABSTRACT

Purpose: The molecular markers cluster of differentiation (CD)24, CD44, adenosine tri-phosphate (ATP) binding cassette protein G2 (ABCG2), and epithelial cell adhesion molecule (EpCAM) are widely used, individually or in combination, to characterize some types of cancer stem cells. In this study we characterized the EpCAM+ retinoblastoma (RB) cells for their cancer stem-like properties in vitro. Additionally, we targeted RB tumor cells via redirecting T cells using bispecific EpCAM×CD3 antibody.

Methods: Flow cytometry was used to study the co-expression of EpCAM with putative cancer stem cell markers, such as CD44, CD24, and ABCG2, in RB primary tumors. In vitro methyl thiazol tetrazolium (MTT) assay, invasion assay, and neurosphere formation assay were performed to characterize EpCAM+ cells for their cancer stem/progenitor cell-like properties. We assessed the in vitro efficacy of bispecific EpCAM×CD3 antibody on RB tumor cell proliferation and validated the results by evaluating effector cytokine production in the culture medium with the ELISA method.

Results: EpCAM was co-expressed with all cancer stem cell markers (CD44, CD24, and ABCG2) in primary RB tumors. EpCAM+ cells showed significantly higher proliferative invasive potential and neurosphere formation in vitro compared to EpCAM⁻ Y79 cells. EpCAM+ cells showed higher β-catenin expression compared to EpCAM- cells. EpCAM×CD3 significantly retarded proliferation of RB primary tumor cells. EpCAM×CD3 effectively induced the secretion of effector cytokines, such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-2, and transforming growth factor (TGF)-β1, and also perforin levels by pre-activated lymphocytes.

Conclusions: EpCAM might be a novel cancer stem cell marker in RB. EpCAM×CD3 antibody redirecting T cells to attack RB tumor cells may prove effective in RB management. Further preclinical studies are needed to confirm the initial findings of our study.

Show MeSH
Related in: MedlinePlus