Fluorescence lifetime imaging of unstained, fixed FaDu tumor slides

Fluorescence lifetime imaging microscopy (FLIM) of metabolic coenzymes NAD(P)H and FAD monitors cancer treatment response and heterogeneity in in vitro and in vivo models. FLIM offers the potential to inform cancer patient treatment in a clinical setting, but requires further preclinical validation as an accurate predictor of patient response. Tissue banks offer readily available tumor samples with follow-up patient data that could be used for FLIM validation, but the effects of fixation and processing on autofluorescence have not been well characterized. This study aims to determine if trends seen with treatment in live tumor samples are conserved in formalin-fixed, paraffin-embedded tumors. Previously, fluorescence lifetime images of FaDu xenografts were acquired in vivo in mice before fixation and embedding. Here, corresponding lifetime images of the fixed FaDu tumors were acquired and compared with the in vivo data. The results demonstrate that while NAD(P)H lifetime values are generally conserved between in vivo and fixed tumors, FAD lifetime values are not. Additionally, the cancer response trends seen in vivo are positively correlated for most FLIM components in fixed tumors, but they vary in magnitude. Further investigation is required to determine the cause of the discrepancies.