Single-cell sequencing reveals cancer cell heterogeneity in a murine breast cancer lymph node metastasis model

Using a mouse model of breast cancer that develops spontaneous lymph node metastasis, we performed high-resolution single-cell RNA sequencing (scRNA-Seq) of the primary tumor and TDLN to measure how cancer cells adapt to the dynamic lymph node microenvironment. To understand the dynamic change of lymph node microenvironment after cancer cell invasion, we also compared the gene-expression alteration between naive lymph node and TDLN at single-cell level. 4T1 cells were implanted into the left-side (supine position) fourth mammary fat pad of adult (6-10 weeks) female Balb/c mice. The primary tumors were collected at day 14 or when tumor size reached 250 mm3. After resection, we sutured the wound and allowed these mice to grow spontaneous lymph node metastases. The primary tumors were cut into 1-2 mm pieces and dissociated into single cells. To decrease bias from individual mice, we collected primary tumors from 4 mice and pooled them together for single-cell library preparation. On day 28, we sacrificed the mice and collected the inguinal lymph nodes (n = 4) to create the sample for the TDLN datasets. All analyzed lymph nodes were dissociated into single cells with digestion buffer (200 ug/ml collagenase I, 800 ug/mL Dispase, 100ug / ml DNase I). All samples used ACK buffer (Thermo Fisher) to lyse red blood cells and had the cell concentration and viability tested using a Nadia instrument (Dolomite Bio). The loading quantity of viable cells for 10x Genomics platform was estimated to capture around 5000 cells.