Next-generation sequencing to define the impact of metastatic prostate cancer on neutrophils

Purpose: Prostate cancer most frequently metastasizes to bone and is incurable. Metastatic prostate cancer cells thrive in bone through molecular regulation of surrounding bone stroma; however, it is unclear how non-metastatic vs. metastatic cancer differentially alter the bone cells. Since neutrophils are the most abundant stromal cell in bone, the goal of this study was to identify prostate cancer-induced transcriptomic changes in bone marrow neutrophils for comparison to non-metastatic prostate cancer cells. Overall design: For comparison of non-metastatic and metastatic prostate cancer, we utilized LNCaP (originally derived from prostate cancer patient lymph node; non-metastatic) and C42B (bone metastatic cells derived from LNCaP) cells. For cancer conditioned media, LNCaP and C42B cancer cells were cultured overnight in serum-free media to collect soluble factors. The supernatant was collected and overall protein content measured. Human neutrophils were isolated from bone marrow using ficoll density centrifugation and were incubated in cancer conditioned media for 3 hours total. After incubation, media was washed off of the neutrophils and RNA was collected using Trizol reagent, for downstream bulk RNA-seq analyses.