Abstract Fluorescence molecular imaging is an emerging field with potential to aid in optically guided surgery for cancer treatment. To overcome the drawbacks of always-on fluorescent probes, such as indocyanine green, activatable fluorescent probes are being developed that allow high signal-to-background imaging for better discrimination of cancerous cells from normal cells. The activatable fluorescent probes designed in our research group rely on the presence of two-electron reductase hNQO1 (human NAD(P)H:quinone oxidoreductase isoenzyme 1), which is highly upregulated in tumor cells. Two-dimensional monolayer cell culture systems lack the features of real tissues, such as complex microenvironment and cellular heterogeneity. To overcome the limitations of two-dimensional (2-D) systems, we expanded our research to three-dimensional (3-D) cell culture systems. Our goal is to understand the dependence of probe activation on the availability and activity of hNQO1 in the heterogeneous regions of solid tumors. Hence, 3-D cell cultures were incorporated in our study using the traditional liquid overlay method for the HT-29 cancer cell line. Following the original approach for monolayer cell imaging, probe activation is observed in the outer proliferating cell regions of 3-D HT-29 colon cancer cell assemblies (multicellular spheroids, MCSs). We are utilizing the in vitro cultured MCSs as mimics of solid tumors to investigate the activity of hNQO1 in the avascular regions of solid tumors with the help of hNQO1 activatable fluorophores, such as previously reported QMeNN. Paraffin-embedded sectioning is used to determine hNQO1 presence in the avascular, or necrotic regions, of these tumor mimics. Immunofluorescence data obtained from more than 20-day-old HT-29 MCSs exhibited a distribution of hNQO1 throughout the peripheral cellular region and in the fragmented cells and debris of the necrotic center of the tumor mimics. Preliminary results, from wide-field fluorescence microscopy of MCSs that were trypsinized for periods of time to yield released cells that were then incubated with the QMeNN probe, showed minimal probe activation in the inner regions of MCSs, implying diminished hNQO1 activity in the core region. These results counter increased hNQO1 activity, based on literature and our work, that has been observed in the later stages of the monolayer growth cycle of HT-29 cells. Currently, experiments are being performed to determine if hNQO1 expression and its activity are dependent on the age of MCSs, through the use of immunofluorescence, and UV/vis and fluorescence spectrometry methods, with the aid of fluorescent probes. hNQO1 is a potential tumor target, not just for contrast agents, but for bioactivatable prodrugs as well. Understanding the bioavailability of the hNQO1 biomarker in the avascular regions of solid tumors and their activity may answer some aspects of the reasons for chemoresistance. Citation Format: Bijeta Prasai, Robin L. McCarley. Fluorescent probe activation and hNQO1 activity in solid tumor mimics. [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 3095.