EpCAM is a putative stem marker in retinoblastoma and an effective target for T-cell-mediated immunotherapy.
OBJECTIVE
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.
L&T Department of Ocular Pathology, Vision Research Foundation, Tamil Nadu, India.
Moutushy
M
Mitra
Mallikarjuna
M
Kandalam
Anju
A
Harilal
Rama Shenkar
RS
Verma
Uma Maheswari
UM
Krishnan
Sethuraman
S
Swaminathan
Subramanian
S
Krishnakumar
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000276:immunology; D018033:Antibodies, Bispecific / Q000494:pharmacology; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000096:biosynthesis / Q000276:immunology; D051927:CD24 Antigen / Q000096:biosynthesis / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D002648:Child; D002675:Child, Preschool; D016207:Cytokines / Q000096:biosynthesis / Q000276:immunology; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis / Q000276:immunology; D007167:Immunotherapy; D007223:Infant; D008297:Male; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000276:immunology; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D061251:Primary Cell Culture; D034741:RNA, Small Interfering / Q000235:genetics; D019572:Retinal Neoplasms / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D012175:Retinoblastoma / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D018033:Antibodies, Bispecific; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D051927:CD24 Antigen; C497889:CD24 protein, human; C497483:CD44 protein, human; D015815:Cell Adhesion Molecules; D016207:Cytokines; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D018960:Hyaluronan Receptors; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering