Immunotherapy Bridge 2020 and Melanoma Bridge 2020: meeting abstracts The clonal heterogenicity of circulating tumor cells (CTCs) drives their metastatic potential: a new NOD‐SCID melanoma model

Background: Innovative therapies have improved the overall survival in melanoma although a high number of patients still experience disease progression or recurrence. The clonal heterogenicity of melanoma cells is a critical issue concurring to drug-resistance development and metastatic spreading that, however, could be efficiently forecasted by CTC measure- ment. A number of studies attempted to validate a method for capturing viable CTCs in metastatic melanoma and a peculiar phenotype and biolog- ical properties have been recently correlated with outcome. Materials and methods: CTCs were obtained from 15 patients with BRAFV600-mutated metastatic melanoma by DEPArray technology and cultured in vitro. Cells established in vitro were definitely trans- duced with luciferase. The mutational state was assessed by NGS. The CTC phenotype and molecular profile were investigated by cytometry and qPCR. Stemness was investigated by measuring sphere formation and ALDH activity, while limit dilution discriminated the existence of clonal subpopulations. Viability, proliferation and cell cycle were investigated. The tumorigenic potential of subclones was evaluated by their injection in 8-weeks old NOD/SCID mice. Mice were sacrificed while site of metastasis and tumor burden were evaluated by Lumina- SIII. Metastatic tissues were analysed by immunohistochemistry. Results: A single cell line was established in vitro from a patient with the highest number (n=102) of CTCs (CTC-AT). Missense mutations J Transl Med 2021, 19(Suppl 1):110 Page 9 of 12 of BRAF, TP53 and PIK3CA occurred in CTC-AT and primary tissue. An intermediate EMT phenotype was demonstrated as result of the SNAI1, TWIST1, ZEB1 and ZEB2 or both PDL-1 and CD155 levels (Fig. 1). The expression of CD44, CD90, CD10, CD73 or Oct3/4, Nanog and Sox2 confirmed the presence of stem cells, whereas the ALDH activity and sphere formation suggested the stemness (Fig. 2). Three clonal sub- populations showing different proliferative rate were obtained (Fig. 3). In vivo studies revealed the ability of two clones to develop the tumor, although they showed different metastatic potential (Fig. 4). Conclusions: Herein we described a new NOD-SCID model of metastatic melanoma induced by CTCs. The heterogeneity of subclones as well as their stemness support the role of CTCs for investigating the variability of mela- noma behaviour and offers the opportunity of pharmacogenomic studies to guide future therapeutic strategies in advanced disease.