Chemotherapy sustained cancer stem cells and genomic alterations facilitate therapeutic resistance in retinoblastoma.

Abstract

Purpose : Retinoblastoma (RB) is an aggressive intraocular pediatric tumor arising from developing retina with varying genomic alterations. A subset of RB patients is resistant to the current regimen of chemotherapy. The molecular factors that facilitate chemoresistance in RB are not studied in detail. Cancer stem cells (CSC) are considered as key players of therapeutic resistance in various cancers. Hence we hypothesize that neuronal cell derived CSC may drive chemoresistance, as RB originates from neuronal cells.

Methods : In vitro models of drug resistance were established through the stepwise drug concentration increment method. Cell proliferation and apoptosis were assessed in resistant cells by MTT and TUNEL assay, respectively. Colony forming efficiency in soft-agar and invasion capability in Matrigel transwell were also evaluated in resistant cells. Expression of neuronal stem/CSC markers (SOX2, OCT4, NANOG and KLF4) and ABC transporters were analyzed in both resistant cells and non-responsive RB tumors by real-time PCR and also confirmed by Western blot. Genomic alterations of 22 genes related to cell cycle, apoptosis and DNA repair were analyzed by targeted next-generation sequencing in RB tumors.

Results : Y79 etoposide (Y79ER) and carboplatin (Y79CR) resistant cells produced larger sized colonies (Y79ER:356 ± 87.52 µm; p=0.0007 and Y79CR:4673 ± 550.4 µm; p<0.0001 vs Y79CT:24.18 ± 8.166 µm) in soft-agar, higher invasive cells (Y79ER:435 ± 70 cells; p=0.0209 and Y79CR:398 ± 40 cells; p=0.0279 vs Y79CT: 62 ± 29 cells) in transwell assay. The increased self-renewability of these cells is suggestive of sustained CSC during the treatment. Similarly, pattern with poor differentiation and intra/extra ocular metastasis was also observed in non-responsive RB tumors. Overexpression of CSC markers and ABC transporters in resistant cells and non-responsive tumors corroborated the presence of cancer stem cells in RB. Copy number analysis in non-responsive tumors showed higher genomic alterations in the genes responsible for apoptosis and DNA damage response.

Conclusions : Evidence for the existence of CSC that sustains during the treatment has been established. These CSC along with genomic alterations execute therapeutic resistance in retinoblastoma. Hence targeting CSC through overexpressed markers would improve the treatment efficacy.

This is a 2020 ARVO Annual Meeting abstract.

 

Selection of RB stem cells during treatment