Figure 6: IR mechanism sustained in in vivo xenograft model, ex vivo primary MCL specimens and recapitulated in drug screening assay.

(a) Xenograft experiments showed that HK cells enhanced tumour formation of both parental (Sen) and IR MCL cells (HBL-2). IR MCL cells had more robust tumour formation in vivo compared with parental cells. *P<0.05, n=5 per cohort (Student's t-test). (b) Immunoblot and flow cytometry conducted on MCL samples from NOD/SCID mice injected with HBL-2 cells and IR HBL-2 with and without co-injection of HK cells of cohorts as described in a. 1–8 represent eight xenografts, with relative expression of pAKT and p-4EBP were quantitated by quantitative densitometry and shown in coloured bar graph. (c) Immunohistochemistry stains showing elevated AKT activation (c) and β1 expression (d) in IR MCL patient samples when compared with ibrutinib-sensitive samples or samples on the progression after ibrutinib treatments. Scale bar, 10 μm. (d) A cell-based drug screening assay was used to measure cell growth and chemosensitivity to indicated chemical agents in a reconstructed TME. Parental and IR cells (SP49-Sen and SP49-IR) were seeded in 384-well plates of reconstructed bone marrow, including high physiological cell densities (1–10 × 10⁶ cells per ml), extracellular matrix (collagen 1) and human bone marrow derived stromal cells (BMSC). A panel of drugs at five different concentrations was added to the media, and plates were continuously imaged for 96 h using a digital image analysis algorithm to identify viable cells based on membrane motion (pseudo-coloured in green). Changes in viability are quantified by area under curve (AUC). Left, cell growth and viability of sensitive (Sen) and IR MCL cells was measured with no treatment, treatment with ibrutinib, doxorubicin, MK2206 or INK128; right, images at 48 h in these conditions. Negative controls (no drug) were included, as well as positive controls for each drug (cell line MM1.S at highest drug concentration). Scale bar, 30 μm. (e) Heat map showing chemosensitivity of parental (Sen) and IR cells (Resis) to 31 agents including protein kinase inhibitors, inhibitors of enzymatic processes and chemotherapeutic agents. See also Supplementary Figs 6, 8 for full gel scan of the WB.