A custom inverted multiphoton imaging system (Bruker custom system) equipped with an Ultrafast Ti:Sapphire (Mai Tai HP, Spectra Physics, Inc.) 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. An integrated fluorescence lifetime imaging microscopy (FLIM) module was used to measure mitochondrial function regarding the components contributing to NADH autofluorescence (Figure 2).
Shelby N. Bess1, Matthew J. Igoe1, Gaven K. Smart1, Timothy J. Muldoon1
1Dept. of Biomedical Engineering, University of Arkansas, 1 University of Arkansas, Fayetteville, AR 72701
INTRODUCTION
RADIAL STRUCTURAL CHARACTERIZATION
The tumor microenvironment (TME) is a dynamic, intricate system. More specifically, tumors contain micro-regions that consists of the tumor cells as well as a variety of ancillary cell types, including activated fibroblasts, blood vessels, immune cells that have infiltrated, and extracellular matrix1. Traditionally, early characterization of simulation tumor microenvironments have been performed in 2D in vitro monolayers2. However, these monolayers do not accurately represent the pathophysiology of tumor architecture due to the restriction of cell-cell interactions and improper modeling of nutrient, oxygen and metabolic waste gradients2. Therefore, there is a need to develop a 3D-simulated tumor model that can display the traditional characteristics of an in vivo tumor. However, traditional characterization methods of 3D-simulated tumor models only investigates changes in the discrete micro-regions, which limits the amount of spatial information that can be obtained regarding tumor structure and changes in metabolism3. In this study, we have developed a 3D-simulated tumor model using cancer cells and macrophages along with a custom MATLAB script used to characterize the radial changes in spheroid structure and metabolism.
MULTICELLULAR SPHEROID MODEL
IMMUNOFLUORESCENCE
Table 1. Immunofluorescent antibodies used and their respective targets to quantify individual cell populations or specific micro-regions within the multicellular spheroid model.
Multicellular Spheroid Structure and Metabolic Characterization Through Radial Profiling
Radial profiling reveals that M1 and M2 macrophages are of equal intensity at the core with M1s having a higher intensity at the edge. In line with the literature, cellular apoptosis (CC3) is highest at the core while cellular proliferation (Ki67) is highest at the edge. Within the necrotic core of a multicellular spheroid, the level of hypoxia is expected to be elevated compared to the other micro-regions. Radial profiling reveals that HIF-2𝛼 (a chronic hypoxia marker) intensity is higher than that of HIF-1𝛼 (an acute hypoxia marker) (Figure 4).
CONCLUSIONS
Overall, we have created a multicellular spheroid model that captures the heterogeneous architecture and nutrient gradients that is observed in in vivo tumors. We have also created an analysis technique that can capture acute changes in multicellular spheroid structure and metabolism within the micro-regions. Future work includes exposing the spheroid model to low oxygen levels to investigate how environmental hypoxia can change structure and metabolism gradients.
REFERENCES
ACKNOWLEDGEMENTS
This work was supported by the National Science Foundation (CBET 1751554), the National Institutes of Health, Arkansas Integrative Metabolic Research Center (5P20GM139768-02), and the Arkansas Biosciences Institute. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the acknowledged funding agencies.
Figure 1. Schematic of RAW 264.7/CT26 multicellular spheroid model.
TWO-PHOTON MICROSCOPY
Figure 2. Inverted two-photon microscopy system. Inverted two-photon microscope (Left) with close-up view of microincubator equipped with temperature and humidified gas delivery (Right).
RADIAL METABOLIC CHARACTERIZATION
Radial profiling reveals acute changes in the optical redox ratio across the multicellular spheroid radius (Figure 5, Top Row). The core shows an optical redox value of approximately 0.5 while the proliferative edge shows an optical redox value of approximately 0.6. Very little changes were observed for the mean NADH lifetime across the multicellular spheroid radius (Figure 5, Bottom Row). The core of the spheroid has a mean NADH lifetime value of approximately 1 ns, while the proliferative edge shows a mean NADH lifetime value of approximately 1.14 ns.
RADIAL PROFILING
Antibody | Target |
CD80 | M1 Macrophages |
CD206 | M2 Macrophages |
Ki67 | Proliferation |
CC3 | Apoptosis |
HIF-1𝛼 | Acute Hypoxia |
HIF-2𝛼 | Chronic Hypoxia |
Figure 3. Schematic of the generation of radial intensity profiles.
Figure 4. Radial profiling reveals changes in intensity for spheroid structural markers. Top Row: Immunofluorescent cross-section images of multicellular spheroids. Bottom Row: Left: Comparison of M1 vs M2 macrophage populations. Middle: Comparison of cellular proliferation vs apoptosis. Right: Comparisons of hypoxia marker Scale bars are 20 µm (n = 20).
Figure 5. Radial profiling reveals changes metabolic parameters. Top Row: Optical Redox Ratio. Bottom Row: Left: Mean NADH Lifetime. Scale bars are 20 µm (n = 20).
Mean NADH Lifetime (ns)
ns
ns