cDNA expression profiling of 4T1 breast cancer cells isolated from tumor-draining lymph node (4T1LN) or primary tumors (4T1PT)

Investigating the gene expression profile changes between 4T1LN and 4T1PT. Balb/c mice were injected with 4T1 cells at both site of 4th mammary fat pad. Three weeks later, mice were sacrificed and primary tumors and inguinal tumor-draining lymph nodes were harvested. Primary tumors from both 4th fat pads were excised, minced with scissors, and then digested in collagenase type I (150 U⁄ ml) plus hyaluronidase (50 U/ml) for 16 hr at humidified 37 °C incubator supplemented with 5% CO2. Inguinal tumor-draining LNs were excised and dissociated by mechanical disruption. Tissues were dissociated by 100 μm cell strainers from BD Biosciences (San Jose, CA, USA) and maintained in complete RPMI 1640 medium containing 60 μM 6-thioguanine (Sigma-Aldrich, St. Louis, MO, USA) and seeded into 10 cm dishes. After 10-14 days, cells were harvested and stained with APC-conjugated rat anti-mouse CD24 (#101814, Biolegend, San Diego, CA, USA) and PE-conjugated hamster anti-mouse CD29 antibodies (#102208, Biolegend, San Diego, CA, USA) for 30 mins at 4℃. The CD24+CD29+ 4T1 cell population was sorted on a FACS Aria II cell sorter. CD24+CD29+ 4T1 cell sorted from Inguinal tumor-draining LNs called 4T1LN cells, CD24+CD29+ 4T1 cell sorted from primary tumors called 4T1PT cells.