4t1 murine breast carcinoma cells

4T1 murine breast carcinoma cells were purchased from American Type Culture Collection, cultured in the recommended media, and implanted (5x10⁴ cells) into the mammary fat pad of 10-14-week old wild-type Balb/c mice obtained from the NCI Frederick Cancer Research Facility. Tumor-inoculated mice were subsequently treated with OX40L-FP (10 mg/kg, IP) and MVA-Twist-TRICOM (1x10⁸ PFU, sc), which were obtained from MedImmune LLC and Bavarian Nordic, respectively, under separate Cooperative Research and Development Agreements (CRADAs). MVA-Twist-TRICOM was administered on days 7, 14, and 21 after tumor implant, while OX40-FP was administered on days 10, 13, 17, and 20. Mice were sacrificed on day 28 when primary tumor sizes from untreated mice reached their ethical limits (2000 mm³). Pulmonary metastases were evaluated by enumerating the number of clonogenic 4T1 tumor colonies cultured from each lung, as previously described [18 (link)].

MDA-MB-231 human breast carcinoma cells, 4T1 murine breast carcinoma cells, and HEK293T human embryonic kidney cells were purchased from the American Type Culture Collection (ATCC). The LM2-4 cell line, a lung metastatic variant of the MDA-MB-231 cell line, was kindly provided by Prof. Robert Kerbel (Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada). 67NR, a low metastatic variant of the 4T1 cell line, was kindly provided by Prof. Jonathan Sleeman (Medical Faculty Mannheim, Centre for Biomedicine and Medical Technology Mannheim (CBTM), University of Heidelberg, Mannheim 68167, Germany). In some cases, cells were fluorescently tagged with green fluorescent protein (GFP). All cells were used within 6 months of resuscitation. Human cell lines were cultured in RPMI 1640 (Sigma-Aldrich, Rehovot, Israel) and murine cell lines in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich). In all cases, medium was supplemented with 5% fetal bovine serum (FBS), 1% l-glutamine, 1% sodium pyruvate, and 1% streptomycin, penicillin, and neomycin in solution (10 mg/mL, Biological Industries, Beit, Ha’Emek, Israel). Cells were tested routinely to be mycoplasma-free. Human cell lines were tested to be authentic or they were used within 6 months from resuscitation.

4T1 murine breast carcinoma cells were purchased from American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA). To produce breast cancer models, 4-week-old female BALB/c nude mice were purchased from the Shanghai Laboratory Animal Center at the Chinese Academy of Sciences (Shanghai, China) and subcutaneously injected in the left lateral thigh with 1 × 10⁶ tumor cells suspended in 100 μL of PBS. All animal experiments were performed according to protocols established by the Ethics Committee of Shanghai General Hospital.