Acalabrutinib

Ibrutinib and acalabrutinib were purchased from Selleck Chemicals. Stock solutions were made in DMSO 5% and cyclodextrin 30% (vehicle).

The 3D cultures were stimulated and treated as indicated. Acalabrutinib and ibrutinib were purchased from Selleck Chemicals (Houston, Texas). Culture plates were placed in an IncuCyte live-cell imager (Essen Biosciences, Royston, United Kingdom) in an incubator at 37°C and 5% CO₂. Scans were taken every 12 hours using the single spheroid assay for live-cell analysis application using ×4 magnification. Spheroid area was quantified using IncuCyte software as a proxy for spheroid growth.

In vitro experiments were performed in this study using HD or treatment naive CLL patient PBMC treated with vehicle or BTKi during the experiment, whereas ex vivo experiments were performed using PBMC from patients collected at baseline or after long-term treatment on BTKi to examine the effect of targeted therapies on immune cell profiles and function. For in vitro analyses, PBMC were thawed and cultured at 1.0 × 10⁶ /mL in T cell media (TCM) (Gibco RPMI media 1640, 10% v/v heat inactivated foetal calf serum (FCS), 1% v/v 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 ×  Gibco GlutaMAX, 1 ×  Gibco MEM non-essential amino acids, 1 mM Gibco sodium pyruvate, 100 U/mL Penicillin/Streptomycin (all from Thermo Fisher Scientific, Watham, MA) and 50 mM 2-Mercaptoethanol (2ME) (Sigma Aldrich, St Louis, MO)) supplemented with 1 µM ibrutinib (Selleck Chemicals), 1 µM zanubrutinib (BGB-3111, Obtained under a Materials Transfer Agreement with BeiGene Co. Ltd.) or 1 µM acalabrutinib (Selleck Chemicals), or 0.5% DMSO (Sigma Aldrich) vehicle control at 37 °C, 5% CO₂ for 18 h prior to use in experiments. For ex vivo analysis, PBMC were thawed and plated at 1.0 × 10⁶ /mL in TCM, and used immediately. For all flow cytometry based assays, samples were acquired on a BD LSRFortessa Flow Cytometer (BD Biosciences). Analysis was performed using FlowJo software v10.7.1 (BD Biosciences).

For treatment for AFM-FS measurement, Cytochalasin D (Sigma, C8273, Albuquerque, New Mexico) was added directly to PBS, incubated for 10 minutes, and then maintained in the plate during the measurement. Similar protocol was used for treatment with IgM (Southern biotech, 9023-01, Birmingham); 10 µL/mL of IgM was added directly to PBS and maintained in the medium during all measurements. The experiment lasted no longer than 2 hours to avoid saturation of the BCR signal. Ibrutinib (Selleckchem, S2680, Planegg, Germany) and acalabrutinib (Selleckchem, S8116, Planegg, Germany) were administered at concentrations of 1 μM or 10 μM^{40 (link)} to cells in suspension. After 4 hours incubation, the unbound drugs were washed out by centrifuging the cell suspension. Cells were then seeded on polyornithine precoated dishes (1:10) for 2 hours to obtain stable adhesion, as described above for AFM-FS experiments in the absence of the drug.

Ibrutinib, acalabrutinib, entospletinib, idelalisib and dyngo-4A (SelleckChem), and cyctochalasin D (Merck) were dissolved in dimethyl sulfoxide (DMSO). The antibodies used were goat F(ab’)₂ anti-human IgM, control goat F(ab’)₂ and mouse F(ab’)₂ κ anti-IgM (all Cambridge Biosciences). These were used as soluble antibodies or after covalent binding to 2.8 μm epoxy-coated M-280 Dynabeads (ThermoFisher) or 3 μm carboxylate-coated latex beads (Polybead^® Carboxylate Microspheres; PolySciences) using the manufacturers’ instructions.