Ibrutinib

Ibrutinib (MedChemExpress, cat. no. HY-10997) was prepared in a vehicle consisting of 0.5% methylcellulose and 0.2% Tween 80 in water and dosed orally, daily, at 50 mg/kg in a volume of 10 mL/kg. The dose was selected based on the Center for Drug Evaluation and Research Pharmacology Review for Imbruvica (Ibrutinib), in which complete BTK occupancy by Ibrutinib in splenocytes from BALB/c mice was found two hours after oral dosing at 50 mg/kg [56 ]. The present study also confirmed high brain BTK occupancy at a dose of at least 45 mg/kg Ibrutinib in naïve C57BL/6J mice (Fig. 7c).
FTY720 (fingolimod; Selleck Chemicals, cat. no. S5002) was prepared in a vehicle consisting of 2% ethanol in water and dosed orally, daily, at 3 mg/kg in a volume of 5 mL/kg.
GB7208 (preclinical drug product candidate provided by Gossamer Bio) was prepared in a vehicle consisting of 0.5% methylcellulose and 0.1% Tween 80 in water and dosed orally, daily, at 5 mg/kg in a volume of 5 mL/kg. The dose used in the present study was determined based on brain target occupancy and brain GB7208 concentration in naïve C57BL/6J mice (Figs. 7c, d).
All treatments were administered at the same time ± 1 h each day.

PBMCs from patients with CLL were treated with anti-BTLA blocking antibody (clone 3B1, Genentech) or isotype control (10 µg/mL) alone or in combination with 1 µM ibrutinib (MedChemExpress) or vehicle (DMSO) for 72 h. Then, PBMCs were stained for leukemic/T cell identification, and an equal volume of cell count reference microbeads was added to each condition (Sigma-Aldrich). 5 × 10³ reference beads were acquired in each well by flow cytometry and absolute leukemic cell count was determined.

CC-292 (Selleckchem) and ibrutinib (Medchem Express) were used at indicated concentrations dissolved in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich, D8418). DMSO at 0.1% (v/v) was used as vehicle control in all the in vitro experiments. LPS from Escherichia coli (O55:B5) was obtained from Sigma-Aldrich and used at a concentration of 1 μg/ml for in vitro experiments. The siRNA pool for BTK knockdown was obtained from Dharmacon and Silencer® negative control No. 1 siRNA was obtained from ThermoFisher.

Human cancer cell lines (obtained from ATCC, USA, or DSMZ, Germany) were cultured according to the distributors’ instructions. In brief, MCF7, SKBR3, BT20, JIMT1 and BT474 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10–15% FBS, and T47D, HCC1806 and EFM192A cells were maintained in RPMI 1640 medium supplemented with 10–20% FBS. All media were supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine. For treatment, cells were seeded at densities of 1.5-2 million cells per dish in 60 mm dishes and allowed to adhere overnight. The BTK inhibitors ibrutinib, evobrutinib, tirabrutinib, acalabrutinib and zanubrutinib were purchased from MedChemExpress, and the EGFR/HER2 inhibitor lapatinib was purchased from LC Laboratories.

Ovarian cancer cells were seeded into 6-well plates. When the cells were adherent, they were stimulated with appropriate inhibitors, such as SP600125 (MedChemExpress, America, #HY-12041), ibrutinib (MedChemExpress, America, # HY-10997), IN-8(MedChemExpress, America, #HY13319), selonsetib (MedChemExpress, America, #HY18938), T-5224 (MedChemExpress, America, #HY-12270) and cisplatin (MedChemExpress, America, # HY-17394) or a combination of both for 2-3 weeks. Then, the medium was removed and fixation solution was added into the plates at room temperature for 10 min. The colony cells were soaked in 0.5% crystal violet solution and incubated at room temperature for 2 h after removing the fixation solution.

Jump In T-Rex human embryonic kidney 293 cells (HEK293-JumpIn) with doxycycline-inducible expression of human OCT1, OCT2 or OCT3 (HEK293-JI-OCT1-3) were provided by Research Center for Molecular Medicine, Medical University of Vienna, Austria (CEMM) and constructed as reported previously [17 (link)]. HeLa cells were obtained from AddexBio (C0008001, San Diego, CA, USA). Corticosterone, 1-Ethyl-2-[(1-ethyl-2(1H)-quinolinylidene)methyl]quinolinium iodide (decynium-22), 1-Methyl-4-phenylpyridinium iodide (MPP⁺), 4-(4-diethylaminostyryl)-1-methyl-pyridinium iodide (DiASP), doxycycline hyclate, lansoprazole, ouabain, Dulbecco’s modified Eagles medium (DMEM), Minimum Essential Medium Eagle (MEM) and G418 were purchased from Sigma Aldrich (Merck, Darmstadt, Germany). Nilotinib, and ibrutinib were purchased from MedChemExpress (South Brunswick, NJ, USA). Hank’s Balanced Salt Solution (HBSS) and poly-D-lysine were obtained from ThermoFisher Scientific (Waltham, MA, USA). All other chemicals were bought from standard commercial resources and were of analytical grade.

The following cell lines were purchased from the American Type Culture Collection (ATCC): K-562R, THP-1, KASUMI-6, AML-193, SUP-B15, KU-812, U-937, CA46, SU-DHL-6, REH, NCI-H929, JVM-2, JEKO-1, MAVER-1, MINO, REC-1, Z-138, RPMI8226, SU-DHL-4, and SU-DHL-10. OCI-LY1 was purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). DOHH-2, OCI-LY2, OCI-LY-8, TMD8, HAP1, AA8, UV5, UV20, UV21, and EM9 were available at and authenticated by our contract research organizations. Human fibroblast cells GM00637 and GM05849 were purchased from Coriell Institute for Medical Research. All the cell lines were cultured in medium according to the vendors’ recommendation in cell culture incubators set at 37°C with 5% CO2. LP-284 was provided by Lantern Pharma Inc., and stored at −80°C. LP-284 was formulated from powder by dissolving in 5% EtOH and 95% saline (in vivo studies) or DMSO (in vitro studies). Bortezomib was purchased from MedChemExpress (Cat. HY-10227) and formulated with 1% DMSO and 99% Saline. Ibrutinib was purchased from MedChemExpress (Cat. HY-10997) and formulated with 10% DMSO, 40% PEG 300, 5% Tween 80, and 45% saline. Spironolactone was purchased from Selleckchem (Catalog No.S4054) and formulated with 100% DMSO.