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Abstract
Background Cancer is a complex and heterogeneous disease, an ecosystem architecture that involves far more than just cancer cells. We have developed a 3D ex vivo culture platform that enables the controlled delivery of media and removal of waste metabolites via perfusion in Liquid-Like Solids (LLS) medium that acts as both a 3D support medium and an open porous network mimicking a capillary-bed for fluid transport and cell motility. Using integrated fast scanning laser confocal microscopy we collect in situ spatiotemporal measurements of cytokine concentrations, track immune cells in the tumor microenvironment, and study tumor invasion dynamics in 3D.
Methods Glioblastoma patient derived tumor microexplants (200-400 µm) and PBMCs were co-cultured in 3D LLS. Spatiotemporal cytokine profiles were measured by 3D printing arrays of ELISA beads across the experiment to measure local concentrations of cytokines in situ. Modeling the bead kinetics (cytokine on-rates and off-rates), coupled with measurements of each bead’s fluorescence at a specific time and position from the tumor periphery were fit to spatiotemporal reaction-diffusion models quantifying the tumor’s production rate, concentrations, and the immuno-regulatory micro-environment (figure 1). 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 (figure 2).
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 (figure 3). Invasive fronts of the micro-tumor protruded into interstitial space and analysis of these invasive paths revealed super-diffusive behavior of these fronts. Off-lattice agent based computational simulations reveal that the interstitial space guided tumor invasion by restricting available paths resulting in super-diffusive behavior. COL1 bioconjugation reveals that glioblastoma cancer cells utilize anchorage-dependent migration to explore their surroundings, and geometrical cues guide 3D tumor invasion along the accessible paths. 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.
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, and tumor invasion dynamics.
Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
Schema for in vitro immunotherapy assays(A) Biopsied tissue sample and blood draw from properly consented donor. (B) The tissue sample is mechanically disaggregated into microexplants on the order of 1µl each. (C) Microexplant samples are collected in the BioPelle, a custom micromanipulator mounted on a microscope, for dispensing into sample wells. (D) Immune cells are separated from the blood draw, sorted for CD8+ and CD4+ T cells and stained with CFSE prior to dispersing in LLS and being dispensed into culture wells. (E) Microexplants are placed into a 3D dispersion of immune cells in LLS in each culture well and a liquid cover is added atop the LLS layer. (F) Maximum intensity projection image of glioblastoma microexplant (cyan) in a field of immune cells (green) within the LLS microgel bed (red).
Tumor invasion(A) Glioblastoma microexplant over the course of 48 hours in inert polyacrylamide LLS (top) and Collagen-1 coated polyacrylamide LLS (bottom). Where Collagen-1 context is provided, invasion outward into the surrounding regions is seen while the microexplant without Collagen context remains adherent to self. (B) Outlines of the microexplant cultured in Collagen-1 coated LLS over the course of 48 hours. (C) Microexplant outline progression over duration of the experiment (48 hours). (D) Tortuosity index over time showing monotonic increases in LLS with COL functionalization.
Cytokine GradientsSpatiotemporal measurements of cytokine concentrations using distributed arrays of ELISA beads allow for in situ measurements that can be fit to reaction-diffusion equations to give production rates and diffusivity for specific cytokines, such as IL8.