Time of Day Bias for Biological Sampling in Studies of Mammary Cancer

Despite its demonstrated biological significance, time of day is a broadly overlooked biological variable in preclinical and clinical studies. How time of day affects the influence of peripheral tumors on central (brain) function remains unspecified. Thus, we tested the hypothesis that peripheral mammary cancer tumors alter the transcriptome of immune responses in the brain and that these responses vary based on time of day; we predicted that time of day sampling bias would alter the interpretation of the results. Brain tissues collected at mid dark and mid light from mammary tumor-bearing and vehicle injected mice were analyzed using the Nanostring nCounter Mouse Neuroinflammation Panel V1. Peripheral mammary tumors significantly affected expression within the brain of over 100 unique genes of the 770 represented in the panel, and fewer than 25% of these genes were affected similarly across the day. Indeed, between 65-75% of GO biological processes represented by the differentially expressed genes were dependent upon time of day of sampling. The implications of time-of-day sampling bias in interpretation of research studies cannot be understated. We encourage considering time of day as a significant biological variable in studies and to appropriately control for it and clearly report time of day in findings. Mammary tumor or vehicle injected female mice; brains collected at two times of day (mid day, mid dark); n=5 for each tumor condition and time of day; 3 brain regions dissected; total RNA extracted from each region resulting in 60 samples for simultaneous gene expression analysis of of 770 neuroinflammation-related genes.