Mouse monoclonal anti-MDK antibody (N-terminal region, MDK Ab.) and purified recombinant MDK were kindly provided by LYRAMID (Sydney, Australia). Both reagents were resuspended in PBS. Doxycycline (Dox) and puromycin were purchased from Sigma (St. Louis, MO, USA) and resuspended in DMEM and PBS, respectively. NVP-TAE 684 (TAE) was kindly provided by Sergey A. Lakatosh at the Gause Institute of New Antibiotics (Moscow, Russia) or purchased from MedChem Express (Sollentuna, Sweeden). Crizotinib and lorlatinib were kindly donated by Pfizer (New York, NY, USA). Pure THC and CBD were obtained from THC Pharm Company (Frankfurt, Germany). TMZ and lactacystin were purchased from Calbiochem-Merck (Darmstadt, Germany). E64d and pepstatin A (PA) were purchased from Enzo Life Science (Farmingdale, NY, USA).
Crizotinib
Manufactured by Pfizer
Sourced in United States
Crizotinib is a small-molecule tyrosine kinase inhibitor used in the laboratory setting. It is designed to target specific genetic mutations or alterations in proteins, which may be involved in the development and progression of certain types of cancer.
Automatically generated - may contain errors
Lab products found in correlation
Lorlatinib, by Pfizer (3 mentions)
Puromycin, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Ceritinib, by Selleck Chemicals (2 mentions)
Trametinib, by Selleck Chemicals (2 mentions)
Nvp tae684, by Axon Medchem (2 mentions)
Axitinib, by Pfizer (2 mentions)
Pf 05212384, by Pfizer (2 mentions)
Doxycycline dox, by Merck Group (1 mentions)
Lactacystin, by Merck Group (1 mentions)
Alectinib, by Selleck Chemicals (1 mentions)
Tritiated thymidine, by PerkinElmer (1 mentions)
Temsirolimus, by Pfizer (1 mentions)
Methylcellulose, by Fujifilm (1 mentions)
Midazolam, by Fujifilm (1 mentions)
Bleomycin, by Tokyo Chemical Industry (1 mentions)
Medetomidine, by Fujifilm (1 mentions)
Butorphanol, by Fujifilm (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
β actin 66009 1 ig, by Proteintech (1 mentions)
29 protocols using crizotinib
1
Reagents for Cancer Research Experiments
2
Small Molecule Compound Handling
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Crizotinib, temsirolimus and lorlatinib (PF-06463922) were provided by Pfizer Inc., Alectinib (CH5424802) was purchased from Selleck Chemicals. All compounds were dissolved in DMSO (Sigma Chemical Co., St. Louis, MO) to obtain a 10 mmol/L stock solution, aliquoted and stored at −20°C for subsequent use. Tritiated thymidine was from Perkin Elmer (Waltham, MA).
3
Mouse Models of Lung Cancer
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For the orthotopic syngeneic mouse models, the recipient C57BL/6 wild-type mouse was anesthetized with 4 mg/kg of midazolam (Wako), 0.3 mg/kg of medetomidine (Wako), and 5 mg/kg of butorphanol (Wako), and placed on a tilted platform. A 50-µL volume of bleomycin (Tokyo Chemical Industry, Tokyo, Japan) at 0.5 mg/mL was administered to the mouse via the trachea with the use of a cannula. At 2 weeks after bleomycin treatment, a single-cell suspension of KC or AC cells 1 × 105 cells in 50 µL of PBS was introduced into the lungs via the trachea as for bleomycin administration (Supplementary Figure S3, Supplementary Video S1 ). At the end of experiments, tumors were removed from mice and fixed in 4% paraformaldehyde (Wako) overnight.
For the subcutaneous injection model, a single-cell suspension of AC cells 1 × 106 cells in 100 µL of 50% growth factor–reduced was injected under the skin on the dorsal flank of a nude mouse. Tumor volume (mm3) was calculated every 2 days according to the formula: short axis2 × long axis × 0.5236. When tumor size reached ~75 mm3, mice were randomly assigned to receive vehicle (Methyl cellulose, Wako) or crizotinib (Pfizer, New York, NY, USA) at 150 mg/kg by daily oral administration.
For the subcutaneous injection model, a single-cell suspension of AC cells 1 × 106 cells in 100 µL of 50% growth factor–reduced was injected under the skin on the dorsal flank of a nude mouse. Tumor volume (mm3) was calculated every 2 days according to the formula: short axis2 × long axis × 0.5236. When tumor size reached ~75 mm3, mice were randomly assigned to receive vehicle (Methyl cellulose, Wako) or crizotinib (Pfizer, New York, NY, USA) at 150 mg/kg by daily oral administration.
4
Investigating TGFβ Pathway Modulation
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Crizotinib was kindly provided by Pfizer. Brigatinib (AP26113; Cat. #S8229) and lorlatinib (PF-6463922; Cat. #S7536) were purchased from Selleckchem (Houston, TX, USA). Silibinin (Cat. #S0417) was purchased from Sigma-Aldrich (Madrid, Spain). All reagents were dissolved in sterile dimethylsulfoxide (DMSO) to prepare 10 mmol/L stock solutions, which were stored in aliquots at −20 °C until use. Working concentrations were diluted in culture medium prior to each experiment.
Antibodies against E-cadherin (#3195), SMAD2/3 (#3102) and phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (#9510) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against GADPH (#60004-1-Ig) and β-actin (#66009-1-Ig) were purchased from Proteintech Group, Inc (Rosemont, IL, USA). Antibodies against vimentin (#V6630) and SNAIL (#MA5-14801) were purchased from Sigma-Aldrich and ThermoFisher Scientific Inc. (Waltham, MA, USA), respectively.
The Applied BiosystemsTM TaqManTM Array Human TGFβ Pathway 96-well Plate (Cat. #4414097) was purchased from Applied Biosystems (Foster City, CA, USA). RayBio® C-Series Human TGFβ Array C2 (Cat. #AAH-TGFB-2-2) was purchased from RayBiotech, Inc. (Norcross, GA, USA).
Antibodies against E-cadherin (#3195), SMAD2/3 (#3102) and phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (#9510) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against GADPH (#60004-1-Ig) and β-actin (#66009-1-Ig) were purchased from Proteintech Group, Inc (Rosemont, IL, USA). Antibodies against vimentin (#V6630) and SNAIL (#MA5-14801) were purchased from Sigma-Aldrich and ThermoFisher Scientific Inc. (Waltham, MA, USA), respectively.
The Applied BiosystemsTM TaqManTM Array Human TGFβ Pathway 96-well Plate (Cat. #4414097) was purchased from Applied Biosystems (Foster City, CA, USA). RayBio® C-Series Human TGFβ Array C2 (Cat. #AAH-TGFB-2-2) was purchased from RayBiotech, Inc. (Norcross, GA, USA).
5
Preclinical Evaluation of ALK/MYCN Tumor Models
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All experiments, including the breeding of transgenic animals, were performed in accordance with the local ethical review panel, the UK Home Office Animals (Scientific procedures) Act 1986, the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (35 (link)) and the UK NCRI guidelines (36 (link)).
For GEMM experiments, crizotinib and lorlatinib were provided by Pfizer, alectinib was provided by Hoffman-La Roche/Genentech, and ceritinib was purchased from Selleck Chemicals (Stratech Scientific Ltd).
Th-ALKF1174L/MYCN tumor-bearing animals were enrolled into therapeutic trials when their abdominal tumors reached 5 mm in diameter according to palpation. Changes in tumor volume in the TH-ALKF1174L/TH-MYCN mice were quantified using MRI on a 7T horizontal bore MicroImaging system (Bruker Instruments) using a 3 cm birdcage coil. Anatomic T2-weighted coronal images were acquired through the mouse abdomen, from which tumor volumes were determined using segmentation from regions of interest drawn on each tumor-containing slice (37 (link)).
For GEMM experiments, crizotinib and lorlatinib were provided by Pfizer, alectinib was provided by Hoffman-La Roche/Genentech, and ceritinib was purchased from Selleck Chemicals (Stratech Scientific Ltd).
Th-ALKF1174L/MYCN tumor-bearing animals were enrolled into therapeutic trials when their abdominal tumors reached 5 mm in diameter according to palpation. Changes in tumor volume in the TH-ALKF1174L/TH-MYCN mice were quantified using MRI on a 7T horizontal bore MicroImaging system (Bruker Instruments) using a 3 cm birdcage coil. Anatomic T2-weighted coronal images were acquired through the mouse abdomen, from which tumor volumes were determined using segmentation from regions of interest drawn on each tumor-containing slice (37 (link)).
6
Characterization of ALK+ ALCL Cell Lines
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Human ALK+ ALCL cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2% penicillin and streptomycin and 1% glutamine. Cell lines were grown at 37°C in a humidified atmosphere with 5% CO2. ALK+ cell lines (TS, SU-DHL1, JB6, KARPAS-299) and ALK- cell lines (CEM, JURKAT and NAMALWA) were obtained from DSMZ cell bank (German Collection of Microorganisms and Cell Cultures). ALK+ ALCL cell line, COST was kindly provided by Dr. Laurence Lamant (Institut Universitaire du Cancer Toulouse Oncopole, Toulouse, France). All cell lines were routinely tested for Mycoplasma contamination via PCR and always tested negative
ALK tyrosine kinase inhibitor, crizotinib, was obtained from Pfizer (New York, NY, USA) and dissolved in DMSO for in vitro experiments. ALK degrader TL13-112 (cat# 6745/5) was purchased from Tocris Bioscience and STAT-3 degrader SD36 (cat# HY-129602) was purchased by MedChemExpress. They were dissolved according to manufacturer’s instructions.
Inducible ALK short hairpin (shRNA) SU-DHL-1 and TS (SU-DHL-1 TTA A5 and TS TTA A5, respectively) cells were obtained by co-transduction with pLV-tTRKRAB (TTA) vector and pLVTHM vector containing the H1 promoter ALK-shRNA (A5) cassette. These cells undergo NPM-ALK silencing when 1 μg/mL doxycycline is added to the medium as previously described (19 (link)).
ALK tyrosine kinase inhibitor, crizotinib, was obtained from Pfizer (New York, NY, USA) and dissolved in DMSO for in vitro experiments. ALK degrader TL13-112 (cat# 6745/5) was purchased from Tocris Bioscience and STAT-3 degrader SD36 (cat# HY-129602) was purchased by MedChemExpress. They were dissolved according to manufacturer’s instructions.
Inducible ALK short hairpin (shRNA) SU-DHL-1 and TS (SU-DHL-1 TTA A5 and TS TTA A5, respectively) cells were obtained by co-transduction with pLV-tTRKRAB (TTA) vector and pLVTHM vector containing the H1 promoter ALK-shRNA (A5) cassette. These cells undergo NPM-ALK silencing when 1 μg/mL doxycycline is added to the medium as previously described (19 (link)).
7
Inhibition of STAT3 and JAK Signaling
8
Antibody Panel for Signaling Proteins
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Antibodies to detect MET (D1C2), mTOR (7C10), LC3B, PARP, Caspase 3, Cyclin D1, phospho-ALK (Tyr1604), phospho-MET (Tyr1234/1235), AKT, phospho-AKT (Ser473), phospho-MAPK (Tyr202/204), 4EBP1, phospho-4EBP1 (Thr37/46), S6K, phospho-S6K (Ser371), RPS6, phospho-RPS6 (Ser235/236), STAT3, phospho-STAT3 (Tyr705) were from Cell Signaling (Danvers, MA, USA). Anti-RIP3 was from Santa Cruz Biotechnology (Dallas, TX, USA), Anti-Ki-67 from Cell Marque (Rocklin, CA, USA), and Anti-β-Actin from Sigma-Aldrich (München, Germany). ALK-1A4 (immunoblotting) was from Origene (Rockville, MD, USA), ALK-D5F3 (immunohistochemistry) was from Ventana Medical Systems (Tucson, AZ, USA). Crizotinib and rapamycin were purchased from Pfizer (Berlin, Germany) and ChemieTek (Indianapolis, IN, USA), respectively.
9
Murine IL-3 and Tocopherol Signaling Pathway
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Recombinant murine IL-3 was purchased from PEPROTECH (Rocky Hill, NJ, USA). Puromycin was purchased from InVivoGen (San Diego, CA, USA). Crizotinib (PF-02341066; Xalkori) was presented by Pfizer (San Diego, CA, USA). Mitomycin C (MMC) were purchased from Kirin Brewery Co. (Tokyo, Japan). α-Tocopherol, δ-tocopherol and anti-Flag (M2) antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). β-Tocopherol and γ-tocopherol were purchased from Abcam (Cambridge, MA, USA). α-Tocotrienol and Trolox were purchased from Cayman Chemical (Ann Arbor, MI). Anti-phospho-STAT3 antibody (Tyr705), anti-phospho-STAT5 antibody (Tyr694) and anti-STAT5 antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin antibody and anti-STAT3 antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Peroxidase-conjugated rabbit anti-mouse and goat anti-rabbit secondary antibodies were from Dako (Glostrup, Denmark).
10
JAK2V617F Mouse Model Treatment
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