Mice: female BALB/c background mice were bought from Tongji Medical College and maintained in animal experiment center of Tongji Hospital. All procedures were approved by the animal ethetic committee of Tongji Hospital. In our study, 8 weeks-old mice were chosen for vaccination. The detailed vaccine injection time and sequence was detailed as follow:
Cell: 4T1 cell line was also bought from Tongji Medical College and maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco). For orthotopically transplanted in mammary, 1 × 106 4T1 cells were dissolved in 100 ul Phosphate Buffered Saline (PBS), and then injected to the #4 mammary fat pad of BALB/c female mice.
Vaccine: 4 separate epitope peptides derived from TPX2 protein were combined as our vaccine; their peptides sequences were RGTRGCTI, PVPHFDTI, GAYKAEMW, RTPNRYHL, respectively; which were synthesized by Ontoresinc Cor. (http://www.ontoresinc.cn/). Each 50ug of 4 epitopes were combined and dissolved in 50 ul PBS, and then 50 ul adjuvant were added for vaccination (complete Freund’s adjuvant at first time and incomplete Freund’s adjuvant for the following 3 times). The vaccination place was subcutaneously near the scapula. For mice in control group, 50 ul PBS plus 50 ul adjuvant were combined and then injected in the same place.
IFN-γ ELISPOT: TIL was extracted by tumor dissociation kit (Nwbiotec, Beijing), following the company’s guide. Spleen was crushed and then filtered through a 70um cell strainer (Absin) and lysed by ACK lysis buffer for eliminating red blood cell, then supernant was collected as splenocyte. 2.0 × 105 cells (TIL or splenocyte, seperately) were plated into each well of a 96 well plate coated with anti IFN-γ antibody (MabTech, OH) and vaccine(all 4 peptides) or control. Then after 72 h, plates were washed and a secondary biotinylated anti-mouse antibody (MabTech, OH) was added and incubated over night at 4℃. Then plate was washed by PBS and HRP streptavidin was added to each well and incubated for 1 h. Then AEC substrate was added, incubated until the color change. Then the plate was washed and dry, and read by automated plate reader system (CTL Technologies, Shaker Heights, OH).
Flow cytometry: single TIL or splenocyte was detected using flow cytometry for expression of surface markers such as CD4, CD8, CD25 (eBioscience), and intracellular marker of FoxP3 by the instrument of company (BD). Data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Statistic: tumor was measured by vernier caliper when nodule palpable and parameter was recorded by maximum diameter (A) and minimum diameter (B) and calculated by the formula volume = 1/2AB2 (8). Data were analyzed by t-test.