Journal: Nature Biomedical Engineering

Article Title: Analysis of off-tumour toxicities of T-cell-engaging bispecific antibodies via donor-matched intestinal organoids and tumouroids

doi: 10.1038/s41551-023-01156-5

Figure Lengend Snippet: 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.

Article Snippet: HE and IHC stained slides were imaged with a brightfield whole-slide scanner at ×40 (Hamamatsu, NanoZoomer S360).

Techniques: Expressing, Derivative Assay, Staining, Cell Culture, Imaging, Co-Culture Assay, Fluorescence

Journal: Nature Biomedical Engineering

Article Title: Analysis of off-tumour toxicities of T-cell-engaging bispecific antibodies via donor-matched intestinal organoids and tumouroids

doi: 10.1038/s41551-023-01156-5

Figure Lengend Snippet: a , Representative single tiles of merged brightfield and IF images of the 14-organoid donor lines co-cultured with PBMCs treated with EpCAM, CEA(hi), CEA(lo) and NT TCB (0.5 µg ml −1 ) at 48 h post TCB administration. ×5 magnification; scale bar per tile, 1 mm. Organoid donor number is random, following no particular order. b , Heat map of quantified caspase-3/7 fluorescence signal in dozens of segmented organoids per well ( n = 3) for each TCB treatment across time. Mean fluorescence signal for each TCB condition normalized to the mean a.f.u. detected in the NT TCB control at each timepoint. The 14 different patient-derived organoid lines are displayed on the x axis and ordered by extent of apoptosis experienced. c , Representative IHC of CEA (top) and EpCAM (bottom) expression of the 14 embedded organoid lines at ×40 magnification. Organoid donors ordered by expression levels. Scale bar, 50 µm. d , Quantified target expression levels of cross sections in c for CEA (top) and EpCAM (bottom) indicated as positive area for each individual organoid. Donors are ordered by increasing target expression. e , Data in d as boxplots, with whiskers showing all points (minimum to maximum) of the mean target expression across all donors distinguished between the indicated intestinal regions for each organoid line ( n = 7). Unpaired t -test (two-tailed) was performed. f , Correlation plots of the target expression between CEA or EpCAM and normalized caspase-3/7 signal of the respective TCB. R 2 is provided per plot. g , Data as boxplots, with whiskers showing all points (min. to max.) of the mean of normalized caspase-3/7 across all donors distinguished between the indicated intestinal regions for each organoid line at 48 h post administration ( n = 7). Unpaired t -test (two-tailed) was performed. All displayed experiments in this figure were replicated at least three times, yielding similar results.

Article Snippet: HE and IHC stained slides were imaged with a brightfield whole-slide scanner at ×40 (Hamamatsu, NanoZoomer S360).

Techniques: Cell Culture, Fluorescence, Derivative Assay, Expressing, Two Tailed Test