Abstract
Purpose :
Retinoblastoma (RB) is the most common intraocular eye cancer in children. Although intra-arterial chemotherapy is effective, there is a strong propensity for therapeutic escape in retinoblastoma and limited second-line treatment options. Characterizing RB cancer progression is essential for discovering novel targeted therapies. Here, we developed different techniques for characterizing RB in vitro and generated a new chemoresistant retinoblastoma cell line.
Methods :
A naïve retinoblastoma cell line (RB028) and a treated retinoblastoma cell line (RB006) were transduced with a constitutively expressing GFP system as a surrogate readout of viability using the Incucyte Imaging System. The sensitivity of the GFP signal in retinoblastoma cell lines was validated using DMSO. This system was then used to generate kill curves with clinically relevant chemotherapy agents (carboplatin, melphalan, and topotecan). RB028 was treated with repeated sublethal dosages of 1uM melphalan and 1uM carboplatin and was monitored for the development of chemoresistance after recovery. We characterized the recovered cell lines’ invasiveness and clonogenicity in Cultrex and in HyStem matrices.
Results :
GFP signals in both RB006 and RB028 were quenched within 4 hours of 50% DMSO treatment and were sufficiently sensitive for generating a cost-effective viability assay. Baseline corrected AUCs of kill curve assays for RB006 is higher than RB028 for carboplatin (1-10uM) and for melphalan (5-20uM), highlighting the preservation of clinically-relevant chemoresistance mechanisms in RB cells using our culturing system. Treated naïve RB028 recovered within two weeks for carboplatin and within three weeks for melphalan. RB028 was treated up to four times with carboplatin (RB028-4XC). Compared with Naïve RB028, RB028-4XC had higher baseline-corrected AUCs for carboplatin (10-40uM) and demonstrated a greater propensity for remaining viable as small clusters in preliminary characterization assays.
Conclusions :
We generated a new carboplatin chemoresistant retinoblastoma cell line useful for downstream molecular analysis of chemotherapy resistance mechanisms. Though further in vitro characterization is still necessary, preliminary findings reveal traits associated with more clinically aggressive phenotype.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.