Figure 4.

ADSL expression levels predict response to 6-mercaptopurine in in vitro, in vivo and PDOs. (A) Schematic diagram of the role of ADSL as well as 6-MP inhibitory effect in the de novo purine biosynthetic process. (B) From the left: pictures of Caco-2 control cells pre-treated with DMSO, Caco-2 control cells pre-treated with 6-MP, ADSL-overexpressed Caco-2 cells pre-treated with DMSO and ADSL-overexpressed Caco-2 cells pre-treated with 6-MP implanted in CAMs and grown for 4 days post-implantation. (C) Quantification of tumor growth derived from the CAM experiment (n = at least 4 tumors from 2 independent experiments); mean ± SD. Values are normalized to the mean of CTR DMSO. (D) Representative micrographs of Caco-2 tumors extracted 4 days post-implantation. Tissue sections were stained with Hematoxylin-eosin (H&E) and immunostained with the apoptotic marker cleaved Caspase 3 in the different treatment conditions. (E) Quantification of cleaved caspase 3 positive cells in the tumor from (C). (F) Immunoblot showing ADSL protein expression in the different CRC-PDOs. Quantification is relative to the loading control (Actin). (G) Representative pictures of matched tissue-organoids pairs. Both tissues and organoids sections were stained with Hematoxylin-eosin (H&E) (upper panel) and ADSL antibody (lower panel). (H) Percentage of viable cells relative to DMSO in CRC-PDOs treated with 2.5 µM of 6-MP. (I) Representative pictures of the organoid growth after 5 days treatment with 2.5 µM of 6-MP. Data are mean ± SD (B, E, H) n ≥ 3 replicates. For all experiments, statistical significance was assessed by unpaired t-test. Scale bars are 50 and 100 µm for (D, G) and 200 µm for (I).