Assessment of CAR-T Cell-Mediated Cytotoxicity in 3D Microfluidic Cancer Co-Culture Models for Combination Therapy

Abstract

Chimeric antigen receptor (CAR)-T cell therapy is efficacious against many haematological malignancies, but challenges remain when using this cellular immunotherapy for treating solid tumours. Classical 2D in vitro models fail to recapitulate the complexity of the tumour microenvironment, whilst in vivo models, such as patient-derived xenografts, are costly and labour intensive. Microfluidic technologies can provide miniaturized solutions to assess CAR-T therapies in 3D complex preclinical models of solid tumours. Here, we present a novel microfluidic immunoassay for the evaluation of CAR-T cell cytotoxicity and targeting specificity on 3D spheroids containing cancer cells and stromal cells. Monitoring the interaction between CAR-T cells and spheroid co-cultures, we show that CAR-T cells home towards target-expressing cancer cells and elicit a cytotoxic effect. Testing CAR-T cells in combination therapies, we show that CAR-T cell cytotoxicity is enhanced with anti-PD-L1 therapy and carboplatin chemotherapy. We propose this proof-of-concept microfluidic immunoassay as a materialsaving, pre-clinical screening tool for quantification of cell therapy efficacy. Index Terms -Immunotherapy, Lab-on-a-chip, Solid Tumour Microenvironment, Three-dimensional in vitro complex model. Impact Statement-Microfluidic platforms and protocols canprovide powerful, cost-effective and miniaturised in vitro assays to preclinically assess CAR-T cell therapies in solid tumours.

Expanding CAR-T cell immunotherapy horizons through microfluidics

“…Droplet microfluidic chip for CAR-T cell cytotoxicity assessment 189,[190][191][192][193][194][195][196][197] Microfluidic cell trapping for cell interaction studies 54 3D microfluidic cancer spheroid co-culture models for assessment of CAR-T cell cytotoxicity 186 Made-to-order droplet ensembles for characterizing cell interactions 188 Size difference-based hierarchical loading chip 185 Droplet digital PCR for DNA copy number quantification [194][195][196][197] Lymphocyte interaction monitoring via microfluidic cell pairing 183,184 3D cell culture for tumor microenvironment characterization…”

“…11d). 186 This approach involved two open wells connected by a microfluidic channel with an array of independent culture chambers, facilitating the sedimentation of cells into microwell traps below the channel level. Assessment of CAR-T cell targeting, and cytotoxicity revealed the ability of the cells to induce disaggregation of MDA-MB-468 spheroids and subsequent cell death.…”

The challenge of making the right choice: patient avatars in the era of cancer immunotherapies

“…examined the response to trastuzumab and the impact of the tumor stroma in a breast cancer OOC ( 115 ) and Paterson et al. evaluated a CAR T construct in another breast cancer OOC ( 116 ). These studies clearly indicate the potential of OOC as patient avatar for individual therapy response prediction.…”

CAR T cell infiltration and cytotoxic killing within the core of 3D breast cancer spheroids under control of antigen sensing in microwell arrays

“…Another microfluidic study demonstrated how enhanced vascularization of cancer spheroids is critical for CAR T cell delivery and cytotoxic function 37 . Furthermore, a microfluidic platform has previously shown the generation of cancer spheroids with co-encapsulation of stromal cells to test combination immunotherapies with CAR T cells 38 . Despite these advances, the spatiotemporal patterns of CAR T cell infiltration and cytotoxicity following sensing of a target antigen, inside cancer spheroids remain poorly understood.…”