A549 lung adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC) and cultured in standard Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 IU penicillin, and 100 μg/ml streptomycin. To generate 3D spheroids, 2D-cultured A549 cells (50-75% confluent) were harvested by trypsinization, counted using a hemocytometer, and seeded into ultra-low-attachment round-bottom 96-well plates (Spheroid Microplates, Corning) at a density of 5,000 or 10,000 cells per well.
Immortalized human umbilical vein endothelial cells (HUVECs) expressing green fluorescent protein (GFP) were provided from the laboratory of the late Dr. J. Folkman (Children’s Hospital, Boston). HUVECs were cultured in endothelial basal medium (EBM-2, Lonza, Walkersville, MD) containing supplements from the EGM-2 kit, 10 % fetal bovine serum (FBS), and 1 % penicillin-streptomycin glutamine (PSG). The cells were cultured in an incubator supplied with 5 % CO2 at 37°C. The culture medium was changed every 3 days.
Preparation of Gelatin Methacrylate (GelMA)
The culture matrix, GelMA, was prepared using a previous protocol . Briefly, a 10 % (w/v) gelatin solution was prepared by stirring 10 g of gelatin type-A powder in 100 ml of ultra-pure distilled water and reacted at 50 °C for an hour. Then, 7.5 % (v/v) methacrylic anhydride (MA) was added to the gelatin solution dropwise, and the reaction continued at 50 °C for 6 hours. The mixture was dialyzed in distilled water using dialysis tubing (molecular weight cut off: 13,000-14,000 Da) for 6 days. After dialysis, the obtained solution was filtered and freeze-dried for 2-3 days and then stored at -20 °C until use.
Co-culture of A549 and HUVEC cells
The GelMA hydrogel solution was prepared by mixing 10 % (w/v) GelMA and 0.1 % (w/v) of lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP) in sterile PBS. The solution was sterile-filtered before mixing with cells. Cancer spheroids (day-3 after seeding 10,000 cells/spheroid or 5,000 cells/spheroid) were mixed with 400,000 HUVECs in 100 µl of GelMA solution. The GelMA prepolymer solution containing cells was photo-crosslinked by visible light exposure for 1.5 minutes to create a 200 µm-thin hydrogel coating layer. The thickness of the cell culture was verified using a confocal z-scan (Zeiss, LSM880). The cell containing GelMA was transferred into a 24-well plate and cultured for 4 days. Morphological cell changes were observed using a Zeiss Axiovert 200 microscope and Zeiss Axiovision software. Fluorescent images of the cells were taken on day 0 and day 3.
FDG labeling and sample preparation
For imaging of glucose metabolism using RLM, the hydrogel-cell mixture was cast on a CdWO4 scintillator plate (0.5 mm thick) to form a 200 µm-thin layer. After 5 days, the co-culture (and mono-cultures) were incubated in glucose-free DMEM medium for 1 h before the addition of FDG (1 mCi/ml). FDG was produced at the Stanford radiochemistry facility using an on-site cyclotron. The culture was then incubated with FDG for 1-2 h followed by washing with phosphate buffer saline (PBS) thrice with gentle rocking. The CdWO4 scintillator plates were taken out from the 6-well plates and mounted onto a standard glass coverslip (0.1 mm thick, Fisher Scientific). The specimens were then placed on the RLM stage for imaging. For imaging with a high NA objective, the scintillator plates should be placed upside-down to reduce the working distance.
Instrumentation and multimodal imaging
The set-up was mounted on a custom-built wide-field microscope equipped with a short focal tube lens (50 mm, 4X/0.2 NA; Nikon, CFI Plan Apochromat λ), 20X/0.75 NA air objective (Nikon, CFI Plan Apochromat λ), and deep-cooled electron-multiplying charge-coupled device (EM-CCD; Hamamatsu Photonics, ImagEM C9100-13). Brightfield images were acquired with no EM gain and used as a reference focus. A 469/35 nm filter set (Thorlabs, filter ref: MDF-GFP1) was used for GFP (green) imaging of HUVEC cells. The imaging field of view was 1.5 mm with an image pixel size of 3.2 µm. For RLM imaging, images were taken with a 10X or 20X objective, an exposure time of 10-300s, an EM gain of 600/1200, and 1×1 pixel binning. Although high-quality digital imaging can be achieved for 2D cell cultures by constructing an image from individual detected events , the high count-rate from 3D tissue constructs creates enough scintillation signal for fast and direct analog measurements of the whole sample using a single short camera exposure. Additionally, tumor cells labeled with NucRed™ Live 647 Reagent were imaged using a 685/40 nm emission filter in a commercial fluorescence microscope (EVOS imaging system).
Quantification of FDG concentration
To estimate the relative FDG concentration within A549 spheroids (tumor) and the HUVEC microvascular network (stroma), the EMCCD radioluminescence signal was calibrated by imaging a drop of FDG containing a known amount of FDG. Timelapse RLM imaging was performed to record decreasing EMCCD counts, while the radioactivity corresponding to those time points was calculated using the standard 18F decay curve. The recorded EMCCD signal was found to be linearly correlated to the computed amount of FDG. The slope of this calibration curve was used to convert EMCCD pixel counts to radioactivity in Bq/pixel. Line and radial distribution profiles of the radioluminescence signal were analyzed using NIH ImageJ software. For kinetic measurement, FDG-treated cells were washed thrice and collected from the standard cell-culture dish by trypsinization. Cellular FDG uptake was calculated by measuring the total radioactivity by gamma counting (Hidex) and dividing by the number of cells.
Fourier ring correlation (FRC) analysis
To evaluate the spatial resolution of RLM for imaging thick tissue-engineered specimens, we applied FRC analysis to images collected during this study. The FRC is computed from two images of the same sample and provides an estimate of the spatial resolution of the imager. The FRC computation was performed using a publically available MATLAB implementation of the method . Spatial resolution was determined according to the standard 1/7 threshold.