Fig. 1: Patient-derived intestinal organoids co-cultured with PBMCs recapitulate physiological target-expression patterns, enabling the ‘back-translation’ of TCB-induced on-target off-tumour toxicity.
a, Expression of CEA and EpCAM target antigens in healthy small intestine and colon primary tissue as well as in patient-derived organoids captured by chromogenic DAB (brown) staining at ×20 magnification. Scale bar, 100 µm. b, Quantification of the DAB staining by area quantification of individual colon organoids (n > 50). Scale bar, 100 µm. Red line displays mean. c, F-actin+ outlined organoids (orange) co-cultured with bright DAPI+ (blue) PBMCs displayed as maximum intensity projection of a z-stack of ~100 μm. ×20 magnification; scale bar, 200 μm. c’, A 3D reconstruction of c highlights spatial arrangement of PBMCs around the organoid (x, y, z axes). d, Schematic of low-resolution imaging assay to capture on-target off-tumour toxicity using an organoid–PBMC co-culture. Organoids (5-d expanded and 3-d differentiated) were collected and resuspended with PBMCs before assessing the TCB treatment by brightfield and IF imaging. Schematic created with BioRender.com. e, Representative single tiles of merged brightfield and caspase-3/7 IF (green) images of the co-culture treated with EpCAM, CEA(hi), CEA(lo) and non-targeting TCB (0.1–10 µg ml−1) over a time course of 72 h at ×5 magnification. Scale per tile, 500 µm. f, Heat map of quantified caspase-3/7 arbitrary fluorescence units (a.f.u.) in >20 segmented organoids per well (n = 3) for each treatment condition (0.1–10 µg ml−1) across time. All displayed experiments in this figure were replicated at least five times, yielding similar results.
