Fig 2: The experimental design of the 3D-RDL assay and CEA target expression validation: (A) In the preparation of the 3D-RDL assay, single cells from cell lines (MKN-45 and DLD-1) and organoids (Rectum and Small intestine_2) are seeded in 96-well microwell plates. At day 2, hPBMCs are rested overnight in cRPMI medium. At day 3, hPBMCs (E:T, 5:1) and Cibisatamab (six doses; 0.001–100 µM) are added to the aggregated spheroids and organoids and incubated for 72 h. (B) CEA protein and RNA expression levels in healthy human organs. CEA is expressed in a variety of epithelial tissues such as the urogenital, respiratory, and gastrointestinal tracts. In the healthy human colon, the CEA protein is restricted to the apical side of the differentiated epithelial cells forming the luminal surface. Source: Human Protein Atlas. (C) CEACAM5 bulk RNA expression levels in healthy colon, rectal, and intestinal organoids (Doppl SA Biobank). (D) Relative CEA protein expression levels of control cell lines MKN-45 (CEAhigh) and DLD-1 (CEAlow) and 2 different healthy organoid lines (Rectum and Small intestine_2) analyzed by flow cytometry.

Fig 3: Granzyme B quantification after 72 h of co-culture of rectal/intestinal healthy organoids and 3D grown MKN-45/DLD-1 spheroids in the presence of hPBMCs treated at different doses of CEA TCB. (A,B) The quantification of Granzyme B released in the supernatant after 72 h RDL assay by flow cytometry. Data represent non-linear regression analysis of the mean ± SD from 3 different hPBMCs donors tested in duplicate. Ordinary 1-way ANOVA with Dunnett’s multiple-comparisons test was performed, and the mean of each column compared to the mean of MKN-45 treated at 100 nM. ** p < 0.01 and **** p < 0.0001. Only significant values are displayed (p < 0.05).