Participation of Tumor-Associated Myeloid Cells in Progression of Amelanotic Melanoma (RMM Tumor Line) in F344 Rats, with Particular Reference to MHC Class II- and CD163-Expressing Cells.

Tumor progression is often influenced by infiltration of myeloid cells; depending on the M1- or M2-like activation status, these cells may have either inhibitory or promoting effects on tumor growth. We investigated the properties of tumor-associated myeloid cells in a previously established homotransplantable amelanotic melanoma (RMM tumor line) in F344 rats. RMM tumor nodules were allowed to reach the sizes of 0.5, 1, 2 and 3 cm, respectively. Immunohistochemistry and flow cytometry was performed for macrophage markers CD68 and CD163, and for the antigen-presenting cell marker, MHC class II. Although no significant change was observed in the number of CD68⁺ and CD163⁺ macrophages during RMM progression, the number of MHC class II⁺ antigen-presenting cells was reduced in 3 cm nodules. Real-time RT-PCR of laser microdissection samples obtained from RMM regions rich in MHC class II⁺ cells demonstrated high expressions of M1-like factors: IFN-γ, GM-CSF and IL-12a. Furthermore, fluorescence-activated cell sorting, followed by real-time RT-PCR for CD11b⁺ MHC class II⁺ (myeloid antigen-presenting cells), CD11b⁺ CD163⁺ (M2 type myeloid cells), CD11b⁺ CD80⁺ (M1 type myeloid cells) and CD11b⁺ CD11c⁺ (dendritic cells) cells was performed. Based on the levels of inflammation- and tumor progression-related factors, MHC class II⁺ antigen-presenting cells showed polarization towards M1, while CD163⁺ macrophages, towards M2. CD80⁺ and CD11c⁺ myeloid cells did not show clear functional polarization. Our results provide novel information on tumor-associated myeloid cells in amelanotic melanoma, and may become useful in further research on melanoma immunity.