Abcb5 transporter as a biomarker to study melanoma stem cells in the TyrCre

: Pten^(-/-) Braf^(V600E): Abcb5-IRESeGFP mouse model

Abstract

BACKGROUND – Tumor heterogeneity is one of the main causes of resistance to cancer therapy and cancer relapse. It turns out that a subpopulation of cells, called cancer stem cells (CSCs), is a key determining factor that contribute to this tumor heterogeneity. This subpopulation has characteristics similar to normal stem cells such as self-renewal, differentiation and infinite proliferation capacity. They would be involved in tumor progression, angiogenesis, metastasis, and resistance to chemotherapy. Researchers discovered that CSCs were also present in melanoma, though their existence remains somewhat controversial. These melanoma CSCs are identified by cellular and intracellular markers, including ABCB5, shown to confer low level multidrug resistance and to play a role in melanoma development.
AIMS – The objective of this work was to characterize the potential association of Abcb5 with melanoma stem cells, and to better understand the biology of melanoma stem cells.
METHODS – We used a mouse model that spontaneously develops melanoma, in which cells expressing Abcb5 also express the enhanced Green Fluorescent Protein (eGFP). Mice with the expected genotype were induced with Tamoxifen/4-hydroxytamoxifen to develop melanoma. The resected tumor
was transformed into a cell suspension that was labeled and analyzed by flow cytometry. In vitro, MelJuSo cells were cultured in non-adherent plates to form spheroids enriched in stem cells. These spheroids were characterized through the study of biomarkers of melanoma stem cells by RT qPCR, western blot, and immunofluorescence.
RESULTS – A small eGFP-positive cell population was observed in the tumors taken from TyrCre:Pten(-/-) Braf(V600E): Abcb5-IRESeGFP mouse model. In vitro, we did generate spheroids enriched in stem cells, which overexpress ABCB5 full length isoform and OCT 3/4, a marker associated with stemness properties.
CONCLUSION – We did show that we could induce melanoma in mice and we did demonstrate the presence of a subpopulation of eGFP-positive cells in the tumors developed from TyrCre:Pten(-/-) Braf(V600E):Abcb5-IRESeGFP mouse model. Further characterization of the eGFP-positive cells is required to validate the expression of eGFP in this cell population and to determine the stemness
properties of these cells. In vitro, we have developed a spheroid model enriched in stem cells that will be compared with the stem cell population isolated from tumors developed in mice. This in vitro model might replace the use of mice.

Date of Award	19 Jan 2022
Original language	English
Awarding Institution	University of Namur
Supervisor	Jean-Pierre Gillet (Supervisor)