International Journal of Molecular Sciences · 8 authors, 2 centres
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This study established a chick chorioallantoic membrane (CAM) model of uveal melanoma to compare calcium electroporation (CaEP) with electrochemotherapy (ECT) using bleomycin. Both CaEP and ECT significantly reduced tumor size, proliferation, and melanoma cell count while increasing apoptosis, with CaEP showing comparable efficacy to ECT without using a chemotherapeutic agent. These findings suggest CaEP is a promising, non-toxic alternative for local tumor control in uveal melanoma.
**Background:** Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, with an incidence of 2–8 cases per million per year. Despite local control, metastasis occurs in about 50% of patients within 10 years, and metastatic disease carries a very poor prognosis (80% mortality at 1 year). Current treatments include brachytherapy, teletherapy, and surgical excision, but novel adjuvant therapies are urgently needed. Electrochemotherapy (ECT) uses reversible electroporation (EP) to enhance the cytotoxicity of drugs like bleomycin. Calcium electroporation (CaEP) is a newer approach that induces intracellular calcium overload, leading to cell death without systemic toxicity. This study aimed to establish a CAM-based UM xenograft model and compare the efficacy of CaEP versus ECT with bleomycin.
**Methods:** Fertilized chicken eggs were incubated, and on embryonic day (ED) 7, 1×10^6 UPMD2 (primary UM, disomy 3) or UPMM3 (metastatic UM, monosomy 3) cells mixed with Matrigel were implanted onto the CAM. On ED 14, tumors were treated with: (1) no treatment (control), (2) EP alone (8 pulses, 100 µs, 5 Hz, 1000 V/cm), (3) intratumoral bleomycin (1 or 2.5 µg/mL) alone, (4) intratumoral calcium chloride (5 or 10 mM) alone, (5) EP + bleomycin (1 or 2.5 µg/mL), or (6) EP + calcium chloride (5 or 10 mM). Tumors were harvested on ED 18. Outcome measures included tumor size (length, width, cross-sectional area, perimeter), tumor density (mean gray value via ImageJ), histology (H&E), and immunofluorescence for Ki67 (proliferation), CD31 (vascularization), caspase-3 (apoptosis), and a melanoma marker cocktail (HMB45, MART-1 clones). Statistical analysis used two-way ANOVA with Tukey's multiple comparisons test.
**Key Results:** A total of 235 eggs were implanted, yielding 53 UPMD2 and 56 UPMM3 evaluable tumors. Mortality was approximately 49%. For UPMM3 tumors, ECT with 2.5 µg/mL bleomycin significantly reduced tumor length (p=0.0477), and CaEP with 5 mM and 10 mM calcium significantly reduced both length and width (p<0.05). For UPMD2 tumors, ECT with 1 µg/mL bleomycin significantly reduced length (p=0.038), and CaEP with 5 mM and 10 mM calcium significantly reduced width (p<0.01). Histological measurements confirmed a significant size reduction after CaEP with 5 mM calcium in UPMD2 (length: 0.2±0.082 cm vs. 0.36±0.055 cm control; width: 0.138±0.111 cm vs. 0.3±0.071 cm control; p=0.0445). In UPMM3, CaEP with 5 mM calcium reduced length to 0.183±0.075 cm and width to 0.15±0.055 cm (p=0.005 and p=0.01, respectively). Ki67 proliferation was significantly reduced in all EP-combination groups regardless of drug concentration (p<0.05 to p<0.001). CD31 vascularization was significantly reduced only in UPMM3 tumors treated with 5 mM Ca + EP (p=0.0023 vs. control). Caspase-3 apoptosis was significantly increased in UPMD2 tumors after 10 mM Ca + EP (p=0.0001) and in UPMM3 tumors after 5 mM Ca + EP (p=0.014). Melanoma cell count (melanoma-mix) was significantly reduced in both cell lines after EP + 10 mM Ca (p<0.0001 for UPMM3, p=0.0002 for UPMD2) and EP + 2.5 µg/mL bleomycin (p=0.001 for UPMM3, p=0.0093 for UPMD2). Image analysis showed significant reduction in cross-sectional area after EP + 2.5 µg/mL bleomycin (p=0.0023 for UPMD2, p=0.0036 for UPMM3) and after EP + 5 mM Ca (p=0.0028 for UPMD2, p=0.0049 for UPMM3) and EP + 10 mM Ca (p<0.0001 for UPMD2, p=0.024 for UPMM3). Histology revealed regressive changes, dissociation, and pigmented cell migration in treated groups.
**Clinical Implications:** This study demonstrates that CaEP is as effective as ECT with bleomycin in reducing tumor size, proliferation, and melanoma cell viability while increasing apoptosis in a CAM-based UM model. CaEP offers the advantage of avoiding chemotherapeutic agents and their systemic side effects. The CAM model proved to be a time- and cost-effective in vivo platform for preclinical screening. These results support further investigation of CaEP as a potential adjuvant treatment for UM, possibly enabling eye preservation and improved quality of life. However, the short observation period (ED 7–18) and the use of only two cell lines are limitations. Future studies should validate these findings in rodent models and explore clinical translation with customized electrodes for ocular application.