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

• Published in: Molecular vision Vol. 18; pp. 290 - 308
• Main Authors: Mitra, Moutushy, Kandalam, Mallikarjuna, Harilal, Anju, Verma, Rama Shenkar, Krishnan, Uma Maheswari, Swaminathan, Sethuraman, Krishnakumar, Subramanian
• Format: Journal Article
• Language: English
• Published: United States Molecular Vision 2012
• Subjects: Adenosine; Antibodies; Antibodies, Bispecific - pharmacology; Antigens, Neoplasm - genetics; Antigens, Neoplasm - metabolism; ATP; ATP Binding Cassette Transporter, Sub-Family G, Member 2; ATP-Binding Cassette Transporters - biosynthesis; ATP-Binding Cassette Transporters - immunology; Biomarkers, Tumor - biosynthesis; Biomarkers, Tumor - immunology; Bispecific antibodies; Cancer; catenin; CD24 Antigen - biosynthesis; CD24 Antigen - immunology; CD44 antigen; Cell adhesion molecules; Cell Adhesion Molecules - antagonists & inhibitors; Cell Adhesion Molecules - genetics; Cell Adhesion Molecules - metabolism; Cell culture; Cell Proliferation; Child; Child, Preschool; Cytokines; Cytokines - biosynthesis; Cytokines - immunology; Differentiation; Enzyme-linked immunosorbent assay; Epithelial Cell Adhesion Molecule; Epithelial cells; Female; Flow Cytometry; Humans; Hyaluronan Receptors - biosynthesis; Hyaluronan Receptors - immunology; Immunotherapy; Infant; Interferon; Interleukin 2; Invasiveness; Lymphocytes T; Male; Neoplasm Proteins - biosynthesis; Neoplasm Proteins - immunology; Neoplastic Stem Cells - metabolism; Neoplastic Stem Cells - pathology; neurospheres; Perforin; Primary Cell Culture; Retinal Neoplasms - immunology; Retinal Neoplasms - pathology; Retinal Neoplasms - therapy; retinoblastoma; Retinoblastoma - immunology; Retinoblastoma - pathology; Retinoblastoma - therapy; Retinoblastoma protein; RNA, Small Interfering - genetics; Stem cells; T-Lymphocytes - drug effects; T-Lymphocytes - immunology; Transforming growth factor; Tumor cells; Tumor necrosis factor; Vision
• ISSN: 1090-0535; 1090-0535

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. 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. 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. 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.