Extended Data Fig. 4: Unique targeting features of CD4⁺ and CD8⁺ TEGs and behavioral signatures in relation to NCAM1 expression.

(a) Representative FACS plots showing CD4, CD8, αβ TCR and Vψ9/V82 TCR expression for cultured CD4⁺ and CD8⁺ LM1s or TEGs. (b) Representative image of long-term tracking of TEGs in co-culture with 13 T organoids (grey surface rendering at t = 0) showing full tracks (up to 20 hrs; rainbow-colored). Scale bar, 50 µm. (c) Time series color plot showing long-term tracks of TEGs (co-cultured with 13 T) and how they change their behavioral signature overtime for each time interval. (0- 3.3 hrs; 3.3-6.6 hrs; 6.6-10 hrs; 10-13.3 hrs). Colors indicate cluster identity for each TEG (see j). Tracks were classified into 6 different groups (named according to their most distinct behavior) and the proportion of CD4⁺TEG and CD8⁺TEG is indicated per group. TEGs were pooled from 3 independent experiments. (d) CD4⁺ TEG moving away from a 13 T organoid without killing. Scale bars, 20 µm. (e) Images showing 13 T organoids and CD8⁺ TEGs with defined anchor points. Scale bars, 10 µm. (f) Quantification of fold increase in cell length. Individual cells pooled from 6 independent experiments. Boxplot depicts the median, first and third quartiles, whiskers extend 1.5 times from the interquartile range. (g) UMAP plot shows distinct TEGs subsets unexposed to PDOs, pooled from three independent experiments. (h) Gene-expression dot plot of a curated set of differentially expressed genes in each cell subpopulation. Rows depict cell subpopulations as in g, while columns depict genes. (i) Quantification of breast cancer PDO targeting using a CellTiter-Glo® viability assay upon overnight co-culture with sorted NCAM1⁻CD8⁺TEGs or NCAM1⁺CD8⁺TEGs. Data corrected for organoid only responses. Unpaired T test: 34 T p = 0,0263; 27 T p = 0,0198; 10 T p = 0,0289. (n = 3 individual wells, representative data of 3 independent experiments; mean ± s.d.). (j) Relative behavioral cluster distribution of NCAM1⁻CD8⁺ TEGs or NCAM1⁺CD8⁺ TEGs co-cultured with 13 T PDOs. (k) FACS histogram plots showing NCAM1 expression in TEGs that were cultured in the absence (grey) or presence of IL-15 (black) for 10 days. (Representative data of 3 (b,d,e,k) or 4 (a) independent experiments).