Abstract 5549: Multicellular spheroid structural and metabolic characterization through radial line profiling
Authors: Bess Shelby N. , Igoe Matthew J. , Smart Gaven K. , Muldoon Timothy J. 
Published: March 2024

Three-dimensional multicellular tumor spheroids have emerged as a step between in vitro and in vivo models that can offer biological insight in fields such as tumor biology. Spheroids closely resemble in vivo solid tumors regarding their heterogeneous architecture and gradients of nutrients and oxygenation. These characteristics give the spheroid model great potential for studying basic tumor biology. However, traditional characterization methods investigate changes in the discrete regions within the spheroid, limiting the amount of spatial information regarding cellular metabolism. In this study, multicellular spheroids were created through a combination of hanging drops and gentle agitation using murine RAW 264.7 macrophages and colorectal adenocarcinoma CT26 cells. Hanging drops were maintained for three days then transferred to 6-well plates and placed on an orbital shaker set to 70 RPM for seven days. Prior to metabolic imaging, spheroids were moved to a microincubator with controllable temperature and humidified gas delivery (5% CO2). A custom inverted multiphoton imaging system (Bruker) equipped with an Ultrafast Ti:Sapphire via a 60x/1.2NA water immersion objective (Olympus) and four close-proximity, high-efficiency GaAsP detectors. NADH fluorescence was captured with a 460 (± 20) nm band pass filter at 755 nm excitation and FAD fluorescence with a 525 (± 25) nm band pass filter at 855 nm excitation. NADH and FAD fluorescence were normalized by PMT gain and laser power. An integrated fluorescence lifetime imaging microscopy (FLIM) module was used to measure mitochondrial function regarding the different components contributing to NADH autofluorescence. Immunofluorescence was used to characterize macrophage populations (M1 vs M2) and cellular proliferation (Ki67) and apoptosis (CC3). CD206, a M2 marker, showed a lower normalized fluorescent intensity at the spheroid core (0.429) compared to the proliferative edge (0.570). Similar trends are also observed for CD80, a M1 marker and Ki67. CC3 showed a higher normalized fluorescent intensity at the spheroid core (0.823) compared to the proliferative edge (0.004). The optical redox ratio displays a slightly lower value at the core (0.429) compared to the proliferative edge (0.570). Overall, these results indicate that the proliferative edge shows higher numbers of immune cells and more oxidative metabolism compared to the core. Future work includes exposing this spheroid model to low levels of oxygen to investigate how environmental changes in oxygen can affect spheroid structure and metabolism.

Citation Format: Shelby N. Bess, Matthew J. Igoe, Gaven K. Smart, Timothy J. Muldoon. Multicellular spheroid structural and metabolic characterization through radial line profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5549.