The role of tumor cell-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) in the progression of 4T1 murine breast cancer. Teizo Yoshimura, Kaoru Nakamura, Chunning Li, Miwa Sato, Masayoshi Fujisawa and Akihiro Matsukawa. Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.
GM-CSF is widely used to enhance immune responses against cancer by promoting the production of antigen presenting cells. In the meantime, cancer cells endogenously produce GM-CSF and cancer cell production of GM-CSF is demonstrated to contribute to the progression of human breast cancer. We previously reported that in the 4T1 murine breast cancer, (1) non-tumor stromal cells, including macrophages, are the major source of MCP-1, (2) stromal cell-derived MCP-1 promotes their spontaneous lung metastasis and (3) 4T1 cells produce GM-CSF that directly up-regulates the expression of a unique set of genes, including MCP's, CCL7 and CCL17, by macrophages. In the present study, we attempt to define the role of cancer cell-derived GM-CSF in the progression of the 4T1 breast cancer. As recently shown by others, 4T1 cells are heterologous and contain multiple populations. Therefore, we first established 12 individual clones. Among them, we chose the clone E4 to generate GM-CSF-deficient cells because orthotopically injected E4 cells spontaneously metastasize to the lung. After transfection of E4 cells with a CRISPR-cas9 targeting construct, selection with puromycin and single cell cloning, we obtained several clones without GM-CSF production. The presence of deletions in the GM-CSF gene was confirmed by DNA sequencing. These cells will be transplanted into mice to examine the effects of GM-CSF-deficiency on the progression of 4T1 breast cancer. The results obtained by this study will provide important information as to the contribution of endogenous GM-CSF to the tuning of tumor microenvironments.