Single cell RNA sequencing of advanced gastric cancer

To define the cellular characteristics of malignant ascites of advanced gastric cancer patients and search for therapeutic strategies, we applied single cell RNA sequencing to cancer cells and tumor associated macrophages (TAM), present in large numbers in malignant ascites. We analyzed 180 cells from 4 malignant ascites and 1 cerebrospinal fluid metastasis. The results indicate that the anti-inflammatory characteristics of the tumor associated macrophages are concocted by the tumor cells. By constructing reference transcriptomes for M1 and M2 type macrophages, we found a strong non-inflammatory property of macrophages recovered from the malignant ascites of gastric cancer. Total five samples were collected from four patients diagnosed as advanced gastric cancer. We obtained four peritoneal ascites and one cerebrospinal fluid (AGC04CSF). To construct reference M1 or M2 type macrophages, PBMC-derived CD14+ cells were collected from two healty donors. Isolated CD14+ cells were cultured 7 days with M-CSF. At day 7, M-CSF containing media were removed then LPS and IFN-γ (for M1) or IL-4 and IL-10 (for M2) were supplied. After additional 48 hour of culture, cells were collected by smooth scrapping then applied to single-cell RNA sequencing. By removing low quality cells, total 162 cells from AGC patients, 97 cells from M1, and 45 cells from M2 were analyzed.