Background Immune Checkpoint Inhibitors (ICI) have revolutionized cancer treatment. Unfortunately, existing FDA approved biomarkers like PD-L1, MSI and dMMR do not accurately predict which patients will respond. The Elephas Cybrid™ platform aims to improve ICI response prediction using multiple biological assays and imaging of the patient’s live tumor fragments (LTF)™ treated with ICI ex vivo.

Multiphoton microscopy (MPM) is a powerful imaging technique and one of the primary modalities Elephas uses to interrogate the biological changes in LTF following ICI treatment. MPM allows rapid visualization of biological processes in 3D living tissues without interference and can be used to characterize T cell responses induced by ICI. We recently described an MPM assay to detect and quantify cell viability without the use of dyes and are building on these methods to enable the Cybrid platform to distinguish responding from non-responding T cells.

Methods Activated and resting T cells isolated from both mice and humans were characterized using MPM to observe differences in metabolic state via fluorescence lifetime imaging (FLIM). Murine cytotoxic T cells were generated, isolated, and co-cultured for 48-hours with antigen-expressing tumor cells. Anti-CD8 nanobodies and cleaved caspase 3/7 dye were used to observe and characterize antigen-specific T cell cytotoxicity. Cytotoxic T cell phenotype was confirmed with flow cytometry and secreted cytokine assays.

Tumors were cut into LTF, sorted, and cultured in multi-well plates using the Cybrid platform. LTF structure and metabolic status based on the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavin adenine dinucleotide (FAD) were assessed. Multi-channel fluorescence intensity and lifetime were obtained over 48-hours using a custom four-channel, multiphoton microscope.

Results Using the lifetime metabolic ratio (LMR, LMR=NAD(P)H α₂/FAD α₁), pockets of cell death surrounding CD8 T cells were detected in LTF that were co-cultured with cytotoxic T cells, but not in fragments cultured in the absence of cytotoxic T cells.

Conclusions The methods presented herein will advance the field of cancer diagnostics by progressing from fixed tissue imaging and genetic characterization to assessment of clinically relevant ICI-treated LTF over days as an alternative to months of waiting for a clinical response. Understanding the response of LTF to immunotherapies will allow clinicians to make more informed treatment decisions and improve the lives of those with cancer.