Protection in the form of an immunosuppressive barrier created by the tumor microenvironment is a significant obstacle to many therapies. Concentrations of secreted cytokines and signaling molecules involved in immunoregulation in this interphase are particularly difficult to measure because of the exquisitely small volume. This region may only comprise a volume of microliters if considering its thickness to be that of a cell, or about 10 micrometers. However, studies of tumor explants and tumoroids have shown interphase regions on the order of 1-10 nanoliters, further exacerbating the difficulty of measurements in this space. As well, the 3D nature of novel tumor models introduces complication in terms of sampling and imaging. Accurate measurements within such volumes, in an ongoing experiment, pose non-trivial challenges. To overcome these obstacles, ELISA beads are suspended within the Liquid-Like Solid (LLS™) culture medium, where they participate in normal capture dynamics and are imaged via confocal microscopy to enable quantifiable detection of signaling molecules in the tumor vicinity.
ELISA beads are widely used in traditional cell culture protocols to measure cytokine concentrations and often then to estimate cellular production or consumption rates. The surfaces of ELISA beads are coated with capture antibodies for specific cytokines and when analyzed after being bathed in the media solution, their fluorescence intensity corresponds to the cytokine concentration. Because LLS™ is a yield-stress solid, ELISA beads (6-8 micrometers in diameter) can be dispersed in a given experiment and remain stationary for the duration. LLS™ microgels suppresses convection, but permits diffusion, so secreted cytokines and the capture antibodies needed for the ELISA principal are free to diffuse throughout the system. Consequently, the ELISA principal remains perfectly valid for beads in LLS™.
Contrary to the traditional ELISA bead protocol, this method requires the fluorescently labelled detection antibodies to be continuously present throughout the experiment. Without this, the bead will not fluoresce and thus would not be detectable in real time. Careful determination of the on-and off-rates associated with the capture sites on the beads was performed in order to understand the minimum required concentration of detection antibodies that would not otherwise affect the dynamics of the system.
