PDTALL were already developed before this study after informed consent from the patients and the approval by the University Medical Center Institutional Review Board committee 18. Spleens from NRG mice harboring the different PDTALL models were harvested and proteins extracted. Western blotting was performed as before 25 with anti-DLL4 (C19035, LSBio) and anti-GAPDH (#5174, Cell Signaling technologies) primary antibodies.

PDTALL9, 13 or 19 cells were harvested from the spleen of moribund NRG mice, resuspended and cultured in DMEM+20% FBS supplemented with non-essential amino acids and pyruvate. Cells were incubated with vehicle, murine anti-DLL4 (mDLL4), human anti-DLL4 (hDLL4) antibodies, or γ-secretase inhibitor (DBZ) for 24, 48 and 72 h with different concentrations for each treatment. At indicated timepoints cell viability was assessed by FACS using DAPI (Sigma) and an anti-human CD45 antibody to discriminate murine and human cells (clone HI30, #35-0459-42 eBioscience).

For apoptosis assay PDTALLs were expanded in mice and cultured as described above. Cells incubated with vehicle, murine anti-DLL4 or human anti-DLL4 (demcizumab) antibodies (both at 20 mg/ml), or γ-secretase inhibitor (DBZ, 500 nM) for 48h were analyzed by FACS (Cytoflex, Beckman Coulter) using an anti-human CD45 antibody (clone HI30, #35-0459-42, eBioscience), AnnexinV apoptosis detection kit (#556547, BD Pharmigen) and DAPI (Sigma).