Peripheral nerve injury-induced remodeling of the tumor-associated macrophages promotes immune evasion in breast cancer

Peripheral nerve damage is intricately linked to the progression of various solid tumors and play a pivotal role in the tumor ecosystem. However, its effect on antitumor immunity and precise underlying mechanisms remain poorly understood. This study aimed to elucidate the effect of peripheral nerve damage and subsequent immune modulation on breast cancer progression. Overall design: The experimental design involves culturing purified mouse CD8? T cells in two distinct conditions: (1) control group - CD8? T cells cultured with supernatant from untreated macrophages, and (2) experimental group - CD8? T cells cultured with supernatant from NF-L (neurofilament light chain)-activated macrophages. Mouse CD8? T cells will be isolated from spleens by mechanical dissociation followed by density gradient centrifugation using Mouse Lymphocyte Separation Medium and positive selection with REAlease® CD8a MicroBead Kit (Miltenyi). Macrophages will be derived from CD11b? monocytes isolated from spleen using EasySep™ Mouse CD11b Positive Selection Kit II (Stemcell) and differentiated with M-CSF (50 ng/mL) for 7 days. For NF-L activation, macrophages will be stimulated with 5 µg/mL NF-L (prepared from sciatic nerve lysate via three freeze-thaw cycles in liquid nitrogen) for 24 hours before supernatant collection. Both control and NF-L-activated macrophage supernatants will be filtered (0.22 µm) and used at 50% concentration in fresh RPMI-1640 medium supplemented with 2% FBS for CD8? T cell culture.