Conjunctival melanoma and electrochemotherapy: preliminary results using 2D and 3D cell culture models in vitro

Abstract

Purpose: To investigate the cytotoxic effect of bleomycin, mitomycin C (MMC) and Fluorouracil (5-FU) in combination with electroporation (EP) on human conjunctival melanoma (CM) and normal conjunctival cell lines using 2D and 3D cell culture systems in vitro.

Methods: Two CM (CRMM1, CRMM2) and one normal conjunctival epithelial cell line (HCjE-Gi) were treated with various EP conditions and increasing concentrations of 5-FU, MMC and bleomycin. Cell survival was assessed by MTT viability assay. All cell lines were seeded to create spheroids and were treated with bleomycin on day 3 and day 8 combined with EP. Spheroids were collected, fixed in buffered formalin and subsequently paraffin embedded for histological assessment of the effects of the treatment on cell viability.

Results: CM cell lines were resistant to electroporation alone and showed a reduction in cell number only when treated with 1000 Volts/cm and 8 pulses. HCjE-Gi cells showed higher sensitivity to electric pulses over 750 Volts/cm. MMC and 5-FU demonstrated a higher cytotoxicity for the HCjE-Gi cell line. The CM cell lines were resistant to MMC and 5-FU. Bleomycin (1 μg/ml) alone had no significant effect on the HCjE-Gi even when combined with EP conditions ≥750 Volts/cm. In contrast, it significantly (p -, paired t-test) reduced cell viability in the CM cell lines. Spheroids treated with bleomycin and EP showed a reduction in tumour mass and proliferation rates after treatment.

Conclusion: Our in vitro study using 2D and 3D models indicates that the application of EP may effectively enhance chemotherapy with bleomycin in CM. This may offer new viable perspectives for CM treatment.

Keywords: bleomycin; conjunctival melanoma; electrochemotherapy; electroporation.