Background
We have engineered1 a 3D system with perfusion compatible with high resolution microscopy. Perfusion-based culture prevents the diffusion limited growth and reduces stress associated with the accumulation of toxic waste enabling high resolution long duration in situ microscopy. This 3D platform uses Liquid-Like Solids (LLS)2 medium that acts as both a 3D support medium and a perfusion network mimicking a capillary-bed. Integrated fast scanning laser confocal microscopy allows in situ spatiotemporal measurements of cytokine concentrations, tracking immune cells in the microenvironment, recording immune-cell killing dynamics, and studies of solid tumor invasion.
Methods
Isolated Human CD3+ T cells were activated for 24 hours with anti-CD3/anti-CD28 dynabeads and transduced with retrovirus encoded with a CD19-directed chimeric antigen receptor with CD28 and CD3z signaling domains. Transduced T cells (75×103) into custom Darcy™ inserts in RPMI media supplemented with antibiotics, 10% human AB serum, and 100 U/mL of IL–2 with a perfusion flow of 15 µL/h. Imaging was performed on a Nikon AXR with NSPARC. Spatiotemporal cytokine profiles were measured by 3D printing arrays of ELISA beads and modeling the bead kinetics coupled with measurements of fluorescence at a specific time and position from the tumor periphery. 3D imaging of immune and cancer cells created movies that were analyzed frame-by-frame to track 3D positions, motion, proliferation, action, cell death, and elimination as well as quantify tumor evolution-dynamics, and T cell killing. Surface conjugation of the LLS microgels with type 1 collagen (COL1-LLS) enabled cell adhesion to the LLS and cancer invasion studies. Cell tracking used novel AI algorithms from astrophysics data processing.
Results
Fitting spatiotemporal data of cytokine concentrations revealed production rates of 2 IL8 molecules per cell per second giving tumor margin concentrations of over 2ng/ml after 10 hours. Invasive fronts of the micro-tumor protruded into interstitial space and analysis of these invasive paths revealed super-diffusive behavior of these fronts. Tracking revealed both chemotaxis and chemokinetics of CD8+ cells: average migration speed of > 2.8 µm/min, and average killing rates ~3 cancer cells/h decreasing monotonically to ~1 cancer cell/h over 12 hours. Isolated killing events were also captured, with some CAR T cells showing killing times in under 30 minutes (figure 1).
Conclusions
The in vitro immuno-oncology platform with in situ fast scanning fluorescence microscopy was able to quantify spatiotemporal concentrations of cytokines, T Cell motions and activity, T Cell killing, and tumor invasion dynamics.
References
Flores EF, Sawyer WG. Cancer Cell, 2024.
Bhattacharjee T, et al. ACS Biomater. Sci. Eng. 2016;2(10):1787–1795.
Abstract 1138 Figure 1
CAR T killing dynamics. Early (top) and late (bottom) timepoint of a 1928z CAR T cell (red) killing of 3 burkitt’s lymphoma cells (green)