Tyrosine-Dependent Phenotype Switching Occurs Early in Many Primary Melanoma Cultures Limiting Their Translational Value.

Ahmad Najem,Ghanem E Ghanem,Jasper Wouters,Fabrice Journe,Ahmad Awada,Stein Aerts,Malak Sabbah,François Sales,Mohammad Krayem,Florian Rambow,Jean-Christophe Marine

doi:10.3389/fonc.2021.780654

Abstract

The use of patient-derived primary cell cultures in cancer preclinical assays, including drug screens and genotoxic studies, has increased in recent years. However, their translational value is constrained by several limitations, including variability that can be caused by the culture conditions. Here, we show that the medium composition commonly used to propagate primary melanoma cultures has limited their representability of their tumor of origin and their cellular plasticity, and modified their sensitivity to therapy. Indeed, we established and compared cultures from different melanoma patients propagated in parallel in low-tyrosine (Ham’s F10) or in high-tyrosine (Ham’s F10 supplemented with tyrosine or RPMI1640 or DMEM) media. Tyrosine is the precursor of melanin biosynthesis, a process particularly active in differentiated melanocytes and melanoma cells. Unexpectedly, we found that the high tyrosine concentrations promoted an early phenotypic drift towards either a mesenchymal-like or senescence-like phenotype, and prevented the establishment of cultures of melanoma cells harboring differentiated features, which we show are frequently present in human clinical biopsies. Moreover, the invasive phenotype emerging in these culture conditions appeared irreversible and, as expected, associated with intrinsic resistance to MAPKi. In sharp contrast, differentiated melanoma cell cultures retained their phenotypes upon propagation in low-tyrosine medium, and importantly their phenotypic plasticity, a key hallmark of melanoma cells. Altogether, our findings underline the importance of culturing melanoma cells in low-tyrosine-containing medium in order to preserve their phenotypic identity of origin and cellular plasticity.

Highlights

Phenotypic plasticity, which allows dynamic transitions between distinct cell states, enables tumor cells to survive under various sub-optimal conditions and rapidly adapt to therapeutics
We evaluated the effect of tyrosine on pigmentation and cell viability by flow cytometry in 3 representative melanoma primary cultures
Among pigmented primary cultures (n=7), we identified two mechanisms of cell adaptation following the stress induced via the continuous stimulation of melanogenesis by high concentration of tyrosine, which led us to classify primary melanoma cultures into two groups that we termed “switchers” (4/7) (LOCE #1, LOCE #2, LOCE #4 and LOCE #7) and “nonswitchers” (3/7) (LOCE #3, LOCE #5, and LOCE #6) (Table 1)

Summary

Introduction

Phenotypic plasticity, which allows dynamic transitions between distinct cell states, enables tumor cells to survive under various sub-optimal conditions and rapidly adapt to therapeutics. The hyperdifferentiated state, which has so far only be reported in drug-exposed melanoma lesions [3], and the melanocytic one (and to a lesser degree the intermediate state) are characterized by the expression of the master lineage transcription factor MITF and its downstream melanocytic markers such TYR, TYRP1 and Melan-A/MART1 implicated in cell differentiation and pigment formation [3,4,5,6,7] These cells, just like the cells-of-origin of melanoma, the melanocytes, produce melanin through a process called melanogenesis or pigmentation. The ability to switch between these different phenotypic states is strongly suspected to enable metastatic dissemination and contribute to therapy resistance (to both targeted therapy and immunotherapy) by allowing melanoma cells to adapt to various microenvironmental cues and stress conditions [1, 6, 12,13,14]

Methods

Results

Conclusion