Mimicking Retinoblastoma Treatment With Repeated Topotecan or Melphalan Develops Cross-Resistance to Classic Agents But Not to Repurposed Drugs

Abstract

Purpose: Refractory or recurrent retinoblastoma results from acquired chemoresistance and the management of these eyes often requires surgical removal. Our objective was to develop retinoblastoma models resistant to chemotherapy by exposing cancer cells to repeated chemotherapy mimicking the clinical scenario. These newly resistant cells were used to evaluate potential novel therapies.

Methods: Chemoresistant cells were obtained by exposing two primary retinoblastoma cell cultures to three weekly doses of melphalan or topotecan. The sensitivity of these resistant cells to each chemotherapy was evaluated, and cross-resistance to topotecan, melphalan, and carboplatin was assessed. Genomic alterations and differential expression of efflux/influx transporters between chemoresistant and parental cells were analyzed. Subsequently, sensitivity of both resistant and parental cells to the repurposed agents digoxin, methylene blue, and gemcitabine was assessed.

Results: Four chemoresistant models were successfully established, showing significantly higher half-maximal inhibitory concentration (IC50) values for melphalan and topotecan compared to their corresponding parental cells (P < 0.05). Cross-resistance between melphalan and topotecan was demonstrated, with a 3-fold increase in the IC50. Chemoresistant cells also showed reduced sensitivity to carboplatin (P < 0.05) compared to parental cells, whereas sensitivity to the evaluated repurposed agents remained unchanged. Genomic analysis revealed no selective alterations in the resistant cells, although differential expression of influx/efflux transporters was observed across all chemoresistant models.

Conclusions: In vitro simulation of patient treatment was useful to establish chemoresistant retinoblastomas and to identify strategies to overcome resistance to topotecan or melphalan through drug repurposed. Our results warrant further investigation to support the clinical translation.

Figure 1.

Schematic representation of the establishment of chemoresistant retinoblastoma cells. The RBT primary cell cultures RBT-7, RBT-12L, and RBT-5 were exposed to 3 weekly doses of melphalan IC50 resulting in melphalan resistant RBT-ML cells or topotecan resistant RBT-TP cells after repeated exposure to topotecan. IC50, half maximal cell growth inhibitory concentration.

Figure 2.

Sensitivity to chemotherapy of parental and chemoresistant cells. The dose-response curves for melphalan (A) and topotecan (D) in the parental cell line are represented by the black lines, whereas dose-response curves for melphalan-resistant and topotecan-resistant cells are shown in green and blue lines, respectively. Cell viability was assessed at 72 hours using the MTT assay. The IC50 values for melphalan (B) and topotecan (E) IC50 values in RBT-7, RBT-12L, and RBT-5 cells are detailed. Fold changes in melphalan IC50 (C) or topotecan IC50 (F) are shown for the chemoresistant cells (black box) relative to the parental subtype (white box). Symbols represent the percentage (%) of cell proliferation relative to control cells and are expressed as the mean (SEM) of three independent experiments, each one performed in triplicate. *P value less than 0.05 (Mann-Whitney test).

Figure 3.

Cross-resistance in chemoresistant cells. The IC50 values for melphalan (A), topotecan (C), and carboplatin (E) in parental and chemoresistant cells to the non-exposed agent are expressed. Fold-changes in IC50 values for melphalan (B; green), topotecan (D; blue), and carboplatin (F; violet) are shown for the chemoresistant (black box) cells relative to the parental cells (white box). Symbols represent the percentage (%) of cell proliferation compared to control cells and are expressed as the mean (SEM) of three independent experiments, each performed in triplicate. *P value < 0.05 (Mann-Whitney test).

Figure 4.

Chromosomal copy number alterations of chemoresistant cells compared to parental cells. Whole genome visualization of the chromosomal profiles of (A) RBT-7-ML and RBT-7-TP cells, and (B) RBT-12-ML and RBT-12-TP cells. Each dot represents a log-ratio of the tumor signal compared to the parental cell model. Overlap in chromosomal alterations and gene mutations were detected between chemoresistant and matched parental cells.

Figure 5.

Relative expression of ABCB1, ABCG2, ABCC1, and LAT1. Relative mRNA levels of ABCB1, ABCG2, ABCC1, and LAT1 in (A) RBT-7-ML, (B) RBT-12L-ML, (C) RBT-7-TP, and (D) RBT-12L-TP compared to the parental cells. The mRNA levels of the different transporters in chemoresistant cells are expressed relative to the gene expression in the parental cell models. *P < 0.05 (ANOVA test, relative to gene expression in the parental cell model).

Figure 6.

Reversion of chemoresistance by ABC transporter inhibitors. (A) Dose-response curves for topotecan (circles), topotecan + ivermectin (triangles), and topotecan + elacridar (squares). Symbols represent the percentage (%) of cell proliferation compared to untreated control cells and performed in triplicate in two independent experiments. (B) Topotecan IC50 in the absence or presence of ivermectin or elacridar. *P value < 0.05 (Mann-Whitney test).