Anti-myeloma activity and molecular logic operation by Natural Killer cells in microfluidic droplets

Abstract

Immune-targeted therapies that activate effector lymphocytes such as Natural Killer (NK) cells are currently being investigated for the treatment of Multiple myeloma (MM), the second most common form of hematological cancer. However, individual NK cells are highly heterogeneous in their cytolytic potential, making it difficult to detect, quantify and correlate the outcome of dynamic effector-target cell interactions at single cell resolution. Here, we present a microfluidic bioassay platform capable of activity-based screening of cellular and molecular immunotherapies. We identified distinct functional signatures associated with NK-MM cell interaction. The addition of immunomodulatory drug lenalidomide altered responses of NK-susceptible MM cells but not that of NK-tolerant MM cells. Antitumor cytotoxicity was significantly increased by the blockade of PD1/PDL1 axis as well as the clinically relevant cell line NK92, which were used to construct molecular logic functions (AND and NOT gates). A predictive agent-based mathematical model was developed to simulate progressive disease states and drug efficacy. The findings of the current study validate the applicability of this microfluidic cytotoxicity assay for immunotherapy screening, biocomputation and for future employment in detection of patient-specific cell response for precision medicine.

Droplet-Based Cytotoxicity Assay: Implementation of Time-Efficient Screening of Antitumor Activity of Natural Killer Cells

“… 30 In addition to the close proximity, some effector cells require enough space to engage in “on–off conjugation” or “transient scanning” interactions with their targets. 21 , 29 , 31 , 32 Notably, in previously published studies, 21 , 29 , 32 it was reported that, instead of forming a stable immunological synapse, some of the effector cells transiently perform a “scansion” on target cells by engaging in multiple (i.e., 2–10) contacts and dissociations before releasing the cytolytic hit. 21 Additionally, we considered that activated effector cells undergo morphological changes and alternate high and low phases of motility, as shown in the studies of Sarkar and Khorshidi.…”

“… 21 , 29 , 31 , 32 Notably, in previously published studies, 21 , 29 , 32 it was reported that, instead of forming a stable immunological synapse, some of the effector cells transiently perform a “scansion” on target cells by engaging in multiple (i.e., 2–10) contacts and dissociations before releasing the cytolytic hit. 21 Additionally, we considered that activated effector cells undergo morphological changes and alternate high and low phases of motility, as shown in the studies of Sarkar and Khorshidi. 21 , 29 , 31 Therefore, if either proximity, motility, or “screening” (i.e., on–off conjugation) is restricted, the cytotoxic behavior of the effector cells could be potentially compromised.…”

“… 21 Additionally, we considered that activated effector cells undergo morphological changes and alternate high and low phases of motility, as shown in the studies of Sarkar and Khorshidi. 21 , 29 , 31 Therefore, if either proximity, motility, or “screening” (i.e., on–off conjugation) is restricted, the cytotoxic behavior of the effector cells could be potentially compromised. Finally, sufficient supply with nutrients to maintain cell viability and functionality during the entire assay time is another important factor.…”

“…Upon sedimentation, NK-92 cells showed normal motility behavior around target cells as described previously. 21 , 29 , 31 Briefly, even though no quantitative assessment has been made, NK-92 cells showed behavior such as migration toward the target cells and alternation of high, low, and no motility while changing their morphology and transiently scanning the target cells (see the Supporting Information, Figure S4 ).…”

“…Droplet-based microfluidic technology has emerged as a powerful tool for handling and investigating single-cell genetics and behavior in a high-throughput manner . Surfactant-stabilized, microsized droplets of water-in-oil emulsion generated in microfluidic devices have been tested and recently used as picoliter biocompatible three-dimensional (3D) confinements for short- (i.e., few hours) and long-time (i.e., few days) spatiotemporal screening of mammalian cells. − Besides its high-throughput screening capabilities, this technology permits the integration of multiple functional modules into one single microfabricated chip, including picoinjection, droplet content manipulation, and sorting . Moreover, droplet-based microfluidics requires minimal manual user intervention and minimal sample consumption.…”