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

Abstract

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.

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Abbreviations

CCL:

chemokine (C-C motif) ligand

CD:

cluster of differentiation

CXCL:

chemokine (C-X-C motif) ligand

FACS:

fluorescence-activated cell sorting

FBS:

fetal bovine serum

Flt:

FMS-related tyrosine kinase

GM-CSF:

granulocyte-macrophage colony-stimulating factor

HE:

hematoxylin and eosin

HIF:

hypoxia-inducible factor

IFN:

interferon

IL:

interleukin

LMD:

laser microdissection

MDSC:

myeloid-derived suppressor cell

MHC:

major histocompatibility complex

MMP:

matrix metalloproteinase

NBF:

neutral buffered formalin

PBS:

phosphate buffered saline

PLP:

periodate-lysine-paraformaldehyde

RT-PCR:

reverse transcriptase polymerase chain reaction

TAM:

tumor-associated macrophage

TGF:

transforming growth factor

TIMP:

tissue inhibitor of metalloproteinase

TNF:

tumor necrosis factor

VEGF:

vascular endothelial growth factor

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Acknowledgements

This work was supported in part by JSPS KAKENHI grant number 22380173 to Yamate.

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Correspondence to J. Yamate.

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Bondoc, A., Golbar, H.M., Pervin, M. et al. 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. Cancer Microenvironment 10, 9–24 (2017). https://doi.org/10.1007/s12307-017-0193-x

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Keywords

  • Amelanotic melanoma
  • Tumor-associated myeloid cell
  • Macrophage
  • Antigen-presenting cell
  • Cytokine