Crizotinib

Formalin test was performed as originally described (33 (link)) and as routinely performed in our lab (55 (link)). Briefly, mice were acclimatized in the laboratory for at least 60 minutes before experiments. Animals received 20 μl of formalin solution (1.25 %) prepared in PBS and injected in the plantar surface of the right hind paw (i.pl.). Following i.pl. injections of formalin, mice were immediately placed individually into observation chambers and the time spent licking or biting the injected paw was recorded and considered as a nocifensive response. We observed animals individually and measured nocifensive responses from 0 to 5 minutes (acute nociceptive phase) and 15 to 30 minutes (inflammatory phase). Crizotinib (Sigma-Aldrich) was delivered by i.t. injection 20 minutes prior to testing. Lorlatinib (Sigma-Aldrich) was delivered systemically via intragastric gavage (i.g.) 30 minutes prior to testing.

Crizotinib, sunitinib malate, cisplatin, carboplatin, doxorubicin and paclitaxel were purchased from Sigma-Aldrich^® (St. Louis, MO, USA). Imatinib and gefitinib were purchased from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions for the TKIs, doxorubicin, and paclitaxel were prepared in dimethyl sulfoxide (DMSO) with maximal final concentrations of 1%. The stock solutions for cisplatin and carboplatin were prepared in culture medium, since DMSO interacts with the platinum complexes resulting in inactivation (Hall et al. 2014 (link)).

Crizotinib (PF-2341066), was purchased from Sigma-Aldrich (Oakville, Ontario, Canada). The HSP90 inhibitor (NVP-AUY922) and Ceritinib (LDK378) were purchased from Selleck Chemicals. Doxorubicin was purchased from LC Laboratories (Woburn, MA, USA). Each compound was dissolved in DMSO for cell culture experiments. The pcDNA3-flag-ALK wild-type and ALK^F1174L were kindly provided by Dr. Junko Takita (The University of Tokyo, Tokyo, Japan)61 (link). The EML4-ALK expression vector was a kind gift from Dr. James Dalton (University of Tennessee Health Science Center)62 (link). The NPM-ALK expression vector was a kind gift from Dr. S. Morris (St. Jude Children’s Research Hospital)63 (link). For the siRNA knockdown experiments, ALK and β-catenin specific ON-Target Plus SMARTpool small interfering RNA (siRNA) and scramble control were purchased from Thermo Scientific (Chicago, USA).

Erlotinib and osimertinib were purchased from Selleck Chemicals (Houston, TX, USA). Crizotinib was from Sigma-Aldrich (St. Louis, MO, USA). Chloroquine was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibodies used for immunoblot analyses were the products of Cell Signaling Technology (Danvers, MA, USA); anti-eIF3c (#2068), EGFR (#4267), phospho-EGFR (Tyr1068) (#3777), Akt (#9272), phospho-Akt (Ser473) (#9271), ERK1/2 (#9102), phospho-ERK1/2 (Thr202/Tyr204) (#9101), Met (#8198), phospho-Met (Tyr1234/1235) (#3077), LC3B (#3868), and β-actin (#4970).

We purchased the drugs from the commercial sources: paclitaxel, doxorubicin/adriamycin, 5-fluorouracil, epirubicin, cyclophosphamide, crizotinib, salinomycin, thioridazine, phenethyl isothiocyanate (PEITC), sodium valproate, sodium butyrate, 5-azacytidine, and 6-mercaptopurine (Sigma-Aldrich, St. Louis, MO), NVP-BEZ235 (Cayman Chemical, Ann Arbor, MI), and itraconazole (Selleckchem, Houston, TX). Naoto Ueno kindly provided AZD6244 (AstraZeneca, Wilmington, DE) and erlotinib (ChemieTek, Indianapolis, IN). We dissolved BEZ235, PEITC, itraconazole, crizotinib, salinomycin, doxorubicin, paclitaxel, AZD6244, and erlotinib in dimethyl sulfoxide (DMSO), thioridazine, sodium valproate, and sodium butyrate in water, 6-mercaptopurine in 0.1 M NaOH, and 5-azacytidine in dulbecco’s phosphate buffered saline. To prepare FAC (5-fluorouracil, adriamycin, cyclophosphamide) and FEC (5-fluorouracil, epirubicin, cyclophosphamide) chemotherapy combinations, drugs were measured individually and dissolved into DMSO and combined. Final concentrations of drugs were 250 nM 5-fluorouracil, 25 nM adriamycin, and 250 nM cyclophosphamide for FAC and 250 nM 5-fluorouracil, 50 nM epirubicin, and 250 nM cyclophosphamide for FEC. We added equal volume of the solvent in all dishes including the control dishes without drugs. DMSO volume was ≤0.04% of the volume of the culture medium.

Crizotinib, diltiazem, erythromycin, imatinib, nilotinib, pazopanib, roxithromycin, and verapamil were obtained from Sigma-Aldrich (St. Louis, MO). Nefazodone and azithromycin were purchased from Toronto Research Chemicals (Toronto, ON) and telithromycin from Cayman Chemical Company (Ann Arbor, MI). Midazolam was obtained from Cerilliant Corporation (Round Rock, TX), 1′-hydroxyMidazolam from Ultrafine Chemical Co. (Manchester, England) and nicotinamide adenine dinucleotide phosphate (NADPH) from AppliChem (Darmstadt, Germany). HLM from 50 individual mixed-sex donors were purchased from Xenotech (Lenexa, KS). Human hepatocytes were isolated in house from liver tissue obtained from a female donor undergoing surgical resection at the Department of Surgery, Uppsala University Hospital (Sweden) and cryopreserved as described previously^{34 (link),35 (link)}. Ethical approval was granted by the Uppsala Regional Ethics Committee (ethical Approvals Nos 2009/028 and 2011/037). Donors gave informed consent and all studies were performed in accordance with the current national regulations and ethical guidelines.