Effects of Cryo-thermal therapy  on gene expression in neutrophils and monocytes
The characteristics of the tumor immunosuppressive microenvironment represent a major challenge limiting the efficacy of immunotherapy. Our previous results suggested that cryo-thermal therapy (CTT), a tumor ablation system developed in our laboratory, could promote macrophage M1-type polarization and full maturation of DCs to remodel the immunosuppressive environment. However, it is not clear which cells respond promptly to CTT. CTT can cause extensive cell death and the release of danger-associated molecular patterns (DAMPs) and antigens. Neutrophils are the first white blood cells recruited to sites of damage and acute inflammation. We therefore hypothesized that neutrophils were the initial cells to respond to CTT and were involved in the subsequent establishment of antitumor immunity. We found that CTT led to a rapid and strong proinflammatory neutrophil response, which was essential for the long-term survival of mice. In vivo neutrophil depletion and in vitro coculture experiments demonstrated that CTT-induced neutrophils promoted the differentiation of monocytes toward to mature antigen presenting cells and further upregulated the expression of IFN-? and cytotoxic molecules in T and NK cells in a CD86-dependent manner. These results reveal the important role of neutrophil-monocyte interactions for the development of anti-tumor immunity and highlight that CTT could be used as an immunotherapy for targeting neutrophils and monocytes to enhance antitumor immunity. Overall design: Neutrophils in the spleen from tumor-bearing mice and mice at 12 h after CTT were labeled with PE anti-Ly6G and isolated  using EasySep Mouse PE Positive Selection Kit II. Monocytes in the spleen from mice 5 days after CTT  with or without anti-Ly6G treatment were labeld with  PE anti-CCR2 and isolated using EasySep Mouse PE Positive Selection Kit II. The RNA-seq analysis on neutrophils and monocyes was performed.