Targeting Effector T Cells Against Cancer Stem Cells in Retinoblastoma

Abstract

Purpose: : Within retinoblastoma (Rb) cell lines there is a small subpopulation of cells that express the stem cell marker ABCG2 and possess increased tumorigenicity and metastatic potential. We hypothesize that activated T cells specific for this cancer stem cell subpopulation will achieve maximum tumor destruction with minimal damage to surrounding normal retina. In order to test this hypothesis, we transfected ABCG2+ Rb stem cells with a series of genes that allows them to express endogenous tumor antigens and activate specific T cells.

Methods: : Rb gene mutations in newly established Rb cell lines (Rb143 and Rb107) were identified by sequencing the 27 exons in the Rb gene. Cells expressing the ABCG2 stem cell marker were identified by flow cytometry and immunohistochemistry. Rb ABCG2 positive and negative cells were transfected (AMAXA) with vectors containing the cDNA for: (i) Class II (DRA*0101 and DRB1*0101), (ii) CD80 and (iii) Class II eGFP DR1 (eGFP attached to the beta chain) to track Class II expressing Rb cells. Two-color flow cytometry and cell sorting were used to identify ABCG2 positive and negative Rb cells that expressed the transgenes. Class II and invariant chain were detected in Rb cells via Western blot and flow cytometry.

Results: : Rb143 and Rb107 cell lines possessed a small subpopulation of ABCG2+ cells (4-6%). Only ABCG2+ Rb cells grew progressively two weeks after injection into the subretinal space of NSG immunodeficient mice. Rb143 and Rb107 cells were successfully transfected to express Class II DR1 and CD80. Moreover, we demonstrated by two-color staining and cell sorting that Rb ABCG2+ cancer stem cells were transfected and expressed Class II.

Conclusions: : Rb cancer stem cells were genetically modified to express CD80 and Class II, in order to transform the tumor cells into antigen presenting cells that stimulate CD4+ T helper cells. We predict that Rb cancer stem cells will express a unique gene profile and unique tumor antigens that are recognized by specific T cells.

Keywords: retinoblastoma • immunomodulation/immunoregulation • gene transfer/gene therapy