Repetitive Bleomycin-Based Electrochemotherapy Improves Antitumor Effectiveness in 3D Tumor Models of Conjunctival Melanoma

Background: Conjunctival melanoma (CM) is associated with a high rate of local recurrence and poor survival rate. Novel therapeutic options are needed to reduce recurrence rate. The objective of the study was to demonstrate the improved effectiveness of electrochemotherapy (ECT) on CM using repetitive application. Methods: Tumor spheroids of three CM cell lines (CRMM1, CRMM2, CM2005.1) were treated repetitively with ECT using the chemotherapeutic agent bleomycin on days 3, 5, and 7 of culture. Application of bleomycin alone and electroporation alone served as controls. The cytotoxic effect was analyzed on day 10 compared to untreated control using an independent t-test. The spheroid outgrowth rate was measured. Result: CM tumor spheroid size (median value: 78%, SD: 32%) and viability (median value: 11%, SD: 11%) were dramatically reduced after repetitive ECT treatment (p-value < 0.001). Decreased proliferation capacity (down to 8%) and an increase of apoptotic cells were observed. In most repetitive ECT-treated spheroids, no viable or proliferating cells were detected. Only 33–40% of repetitive ECT-treated spheroids exhibited single outgrowing cells with a delay of time up to 38 days. Conclusion: Repetitive ECT application effectively induces cytotoxic effects in CM spheroids by inducing apoptosis, inhibiting proliferation and decreasing the percentage of surviving tumor cells. Thus, repetitive ECT results in improved antitumor effectiveness in CM and could be an alternative therapy option.