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 ( CRMM 1, CRMM 2) and one normal conjunctival epithelial cell line ( HC jE‐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. HC jE‐Gi cells showed higher sensitivity to electric pulses over 750 Volts/cm. MMC and 5‐ FU demonstrated a higher cytotoxicity for the HC jE‐Gi cell line. The CM cell lines were resistant to MMC and 5‐ FU . Bleomycin (1 μ g/ml) alone had no significant effect on the HC jE‐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.

Characterization of Uveal Melanoma Cell Lines and Primary Tumor Samples in 3D Culture

“…39 Methods used to increase drug penetrance into spheroids have also been described, including electrochemotherapy. 40 With respect to drug efficacy, studies have shown that in 2D cell culture systems potency is enhanced when compared with 3D models, 21 such that the predicted clinical efficacy in patients is often overestimated. 1 Interestingly, in our study, doxorubicin had similar effects on 92.1 and MM66 cells grown in 2D or 3D culture irrespective of spheroid parameters such as compactness, growth rate, or cross-sectional area.…”

Recent Advances in Electrochemotherapy

“…The reasons for difference in response are multifaceted and include physical features pertinent to electrochemotherapy, such as unavailability of the drug and insufficient coverage with electric field and biological, pertinent to patient, such as previous treatments, difference in tumor microenvironment, mutational status, and so on. 2,14,18 Thus, electrochemotherapy is still evaluated in different types of cancer, either in vitro or in vivo, including colon carcinoma CT26 (in vitro) 19 ; bladder cancer SW780 (in vitro and in vivo) 20 ; fibroblasts, human umbilical vein endothelial cells (HUVEC; in vitro) and two squamous cell carcinomas, CAL-27 and SCC-4 (in vitro) 21 ; ovarian cell lines OvBH-1 and SKOV-3 (in vitro) 22 ; human neuroblastoma SH-SY5Y cells (in vitro) 23 ; BRAF-mutated melanoma cells (SK-MEL-28) and their counterpart without mutation, CHL-1 cells (in vitro) 24 ; chemoresistant uveal melanoma cells (Mel 270, 92-1, OMM-1, OMM-2) (in vitro) 25 ; primary cells from metastatic pancreatic tumors (in vitro) 26 ; human papillomavirus (HPV)-positive head and neck squamous cell carcinoma 2A3 (in vitro and in vivo) 27 ; Lewis lung carcinoma and CT 25 colorectal carcinoma (in vivo) 28 ; conjunctival melanoma CRMM1 and CRMM2 and normal conjunctival epithelial cells HCjE-Gi (in vitro) 29 ; radioresistant head and neck squamous cell carcinoma Fa-DuRR (in vitro and in vivo) 30 ; and small cell lung cancer H69AR cells (in vitro). 31 In general, it was demonstrated that electrochemotherapy with bleomycin or cisplatin causes immunogenic cell death in colon carcinoma 19,28 ; that human endothelial cells are more susceptible to electrochemotherapy than tumor cells, 21 supporting the antivascular effect in vivo; that electrochemotherapy is effective in ovarian carcinoma cells resistant to standard therapy 22 ; and that electrochemotherapy is also effective in neuroblastoma and primary pancreatic tumor cells, which was not demonstrated before.…”

Image Analysis of 3D Conjunctival Melanoma Cell Cultures Following Electrochemotherapy

“…An image analysis and the integrated algorithms offer a quantitative characterization and a more precise calculation of various morphological features of the tested spheroids. Moreover, an image analysis complements the established assays for the spheroid monitoring, while it provides a comprehensive platform with a variety of techniques, such as statistical analysis, graph representation and computational topology and, therefore, 3D visualization [ 17 ]. In previous studies of our group, various EP parameters and differing concentrations of 5-FU, Mitomycin C and bleomycin were tested to identify the structural changes of the spheroids histologically and chemically, and to assess the toxicity of the tested drugs on conjunctival 2D and 3D cell models [ 17 ].…”

“…Moreover, an image analysis complements the established assays for the spheroid monitoring, while it provides a comprehensive platform with a variety of techniques, such as statistical analysis, graph representation and computational topology and, therefore, 3D visualization [ 17 ]. In previous studies of our group, various EP parameters and differing concentrations of 5-FU, Mitomycin C and bleomycin were tested to identify the structural changes of the spheroids histologically and chemically, and to assess the toxicity of the tested drugs on conjunctival 2D and 3D cell models [ 17 ]. The CRMM1 cell lines were most sensitive to 5-FU and the CRMM2 cell lines to MMC but the cytotoxic effect of the drugs was not further enhanced by EP [ 17 ].…”

“…The drawbacks of its application are localized pain, muscle contraction, erythema and edema [ 14 ]. The effect of ECT in ocular melanomas remains unclear, since the research and acquired knowledge in this field is very limited [ 17 , 18 , 19 ].…”