ABSTRACT
Title
Melanoma Heterogenity and its Role in Tumour-Associated Macrophages Differentiation
Authors
E. Assi
Dottorato in Farmacologia, Chemioterapia e Tossicologia Mediche
Dipartimento di Scienze Cliniche-Osp. L. Sacco Università degli Studi di Milano
Abstract
The pathogenesis and progression of melanoma depend on interactions between tumour cells and stromal cells in the host. Tumour-associated macrophages (TAMs) derived from the host are major components of the tumour microenvironment and have been shown to be associated with tumour progression, angiogenesis and metastasis.
As far as they have been studied, TAMs show mostly an M2-likephenotype. This preferential polarization is due to theabsence in the tumor of M1-orienting signals such as IFN-g orbacterial components as well as to the expression of M2 stimuli.Indeed, M2-Mf (or alternatively activated Mf) differentiate frommonocytes due to specific growth factors (e.g., M-CSF) and post-stimulationwith IL-4 and IL-13 (M2a), immune complexes/TLRligands (M2b), or IL-10 and glucocorticoids (M2c).
An intriguing feature of melanoma is its heterogeneity with subpopulation of cells expressing a wide range of phenotypic variations, especially in terms of pigment synthesis. This clonal heterogeneity accounts for the existence of tumour cell populations showing different tumour markers expression, drug resistance, immunogenic invasive and metastatic capacities. It has been demonstrated that pigmented cell lines are more tumorigenic, invasive and metastatic then the non pigmented ones.
Taking these data, our first purpose is to investigate the ability of ipo- and iper-pigmented melanoma clones to attract macrophages and influence their polarization.
To this end we isolated two clones of B16 murine melanoma cells that differ for their melanin content: the ipo-pigmented, W6, clone and the iper-pigmented, B9, one.
Here we demonstrated that both clones are able to promote macrophages proliferation and differentiation towards M1 and M2 phenotype, but W6 shows a major ability to differentiate Mфin M2-like TAMs.
Another interesting features of melanoma is that a number of macrophage-like traits are expressed by metastatic melanoma proposing that metastatic melanoma cells might be macrophage-melanoma hybrids.
Melanoma-macrophage hybrids isolated from patients or created in vitro, are hypermelanotic, produce “coarse melanin” (phagolysosomal-like vescicles with multiple melanosomes) and show higher tumorigenicity and/or metastatic potential.
The increase in malignancy could be due to the expression of genes characteristic of monocytes/macrophages, such as GnT-V, SPARC, c-MET and integrins, whose expression in neoplastic cells increase their aggressiveness.
Starting fromthese data we hypnotized the possibility that the highly-metastatic an iper-pigmented melanoma clones, B9, isolated in our laboratory, is a fusion hybrid with macrophage while the low metastatic and ipo-pigmented ones, W6, is not.
Thus, W6 seems to be represent melanoma cells before fusion, while B9 seems to be melanoma-macrophages fusion hybrid. Coloring W6 and B9 clones in green and M2 macrophages in red, we demonstrate that W6 cells have a major ability to fuse , as a flow citometry analysis showed more double positive cells in the co-colture between W6 and M2 then in the one between B9 and M2.
As far as they have been studied, TAMs show mostly an M2-likephenotype. This preferential polarization is due to theabsence in the tumor of M1-orienting signals such as IFN-g orbacterial components as well as to the expression of M2 stimuli.Indeed, M2-Mf (or alternatively activated Mf) differentiate frommonocytes due to specific growth factors (e.g., M-CSF) and post-stimulationwith IL-4 and IL-13 (M2a), immune complexes/TLRligands (M2b), or IL-10 and glucocorticoids (M2c).
An intriguing feature of melanoma is its heterogeneity with subpopulation of cells expressing a wide range of phenotypic variations, especially in terms of pigment synthesis. This clonal heterogeneity accounts for the existence of tumour cell populations showing different tumour markers expression, drug resistance, immunogenic invasive and metastatic capacities. It has been demonstrated that pigmented cell lines are more tumorigenic, invasive and metastatic then the non pigmented ones.
Taking these data, our first purpose is to investigate the ability of ipo- and iper-pigmented melanoma clones to attract macrophages and influence their polarization.
To this end we isolated two clones of B16 murine melanoma cells that differ for their melanin content: the ipo-pigmented, W6, clone and the iper-pigmented, B9, one.
Here we demonstrated that both clones are able to promote macrophages proliferation and differentiation towards M1 and M2 phenotype, but W6 shows a major ability to differentiate Mфin M2-like TAMs.
Another interesting features of melanoma is that a number of macrophage-like traits are expressed by metastatic melanoma proposing that metastatic melanoma cells might be macrophage-melanoma hybrids.
Melanoma-macrophage hybrids isolated from patients or created in vitro, are hypermelanotic, produce “coarse melanin” (phagolysosomal-like vescicles with multiple melanosomes) and show higher tumorigenicity and/or metastatic potential.
The increase in malignancy could be due to the expression of genes characteristic of monocytes/macrophages, such as GnT-V, SPARC, c-MET and integrins, whose expression in neoplastic cells increase their aggressiveness.
Starting fromthese data we hypnotized the possibility that the highly-metastatic an iper-pigmented melanoma clones, B9, isolated in our laboratory, is a fusion hybrid with macrophage while the low metastatic and ipo-pigmented ones, W6, is not.
Thus, W6 seems to be represent melanoma cells before fusion, while B9 seems to be melanoma-macrophages fusion hybrid. Coloring W6 and B9 clones in green and M2 macrophages in red, we demonstrate that W6 cells have a major ability to fuse , as a flow citometry analysis showed more double positive cells in the co-colture between W6 and M2 then in the one between B9 and M2.