1138 Engineering 3D living systems with in situ high resolution imaging to capture the dynamics of cancer immunotherapy

<h3>Background</h3> 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. <h3>Methods</h3> 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 (75x10³) 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. <h3>Results</h3> 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 &gt; 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). <h3>Conclusions</h3> 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. <h3>References</h3> Flores EF, Sawyer WG.<i> Cancer Cell,</i> 2024. Bhattacharjee T, <i>et al. ACS Biomater. Sci. Eng</i>. 2016;<b>2</b>(10):1787–1795.