Abstract Cancer chemotherapy can produce severe side effects such as suppression of immune function and damage to heart muscle, gastrointestinal tract, and liver. If serious enough, tissue injury can be a major reason for late stage termination of drug discovery research projects, so it is becoming more important to integrate safety/toxicology assessments earlier in the drug development process. There are a variety of traditional serum markers, tailored mechanistically to specific tissues, however there are no current non-invasive assessment tools that are capable of looking broadly at in situ biological changes in target and non-target tissue induced by chemical insult. We used non-invasive near infrared (NIR) fluorescence tomography for whole-body imaging of luciferase-expressing HT-29 human colon adenocarcinoma tumors implanted in nude mice. Both tumor and host tissue responses were imaged using a cocktail of near infrared fluorescent imaging agents, specific for cell death (Annexin-Vivo 750™ [AV750]), inflammatory matrix metalloproteases (MMPSense™ 750 FAST [MMP750]), and metabolic changes in transferrin receptor expression (Transferrin-Vivo™ 750 [TfV750]). Vascular changes were imaged using AngioSense® 680 (AS680). As a means of validating this efficacy/tox screening approach, HT-29-bearing nude mice were dosed with 5-Fluorouracil (5-FU). 5-FU has been a mainstay in the treatment of many cancers, including colorectal, but is associated with several peripheral toxicities, including gastrointestinal, hepatic, renal, vascular, and (less frequently) cardiac. Dosing was performed as a single IP bolus administration, using doses (50 and 100 mg/kg) known to have minimal overt effects on body weight or tumor mass using this acute dosing regimen. At 2h and 24h post-5-FU, independent cohorts of mice were injected IV with the AV750/MMP750/TfV 750 cocktail (AMT 750) combined with AS680. Imaging was performed 24h later. No apparent effects were seen on tumor mass with this very short treatment regimen, although there was a trend for decreased bioluminescence 2h following 5-FU. Interestingly, there was a measureable decrease in tumor vascular leak and increased AMT750 signal, in a dose- and time-dependent manner. A similar, but more dramatic, response profile was seen in the heart, suggesting rapid tissue damage and vascular changes that resolved by the later imaging timepoint. Kidneys showed enhanced AMT750/AS680 at the early timepoints; liver showed only AMT750 signal increases at the early timepoint and high dose; and lungs showed a trend of both AMT750 and AS680 increases that peaked at the early time and high dose. No obvious effects were seen in the intestines or stomach. These results agree well with observations in the literature and in patients and suggests that NIR fluorescence tomography of the reported imaging agents provides a sensitive approach to simultaneously detect both chemotherapeutic tumor efficacy and suggestions of adverse effects on tissue. Citation Format: Kristine O. Vasquez, Jeffrey D. Peterson. Simultaneous fluorescence tomographic imaging of efficacy and toxicity following acute 5-FU treatment of HT-29 tumor xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-189.