All experiments were performed with the approval of the Institutional Animal Care and Use Committee at Mayo Clinic (Rochester, MN). All mice used in this study were maintained under specific pathogen-free conditions, 21 ± 2°C relative humidity of 45 ± 15%, and a 12-h light/dark cycle. IGROV1 cisplatin resistance cells were subcutaneously injected into the flanks of 6-week-old female athymic nude Ncr nu/nu (NCI/NIH) mice using 18-gauge needles. Each mouse received injections of a 0.2-ml mixture of 2 million cells with 50% growth factor-reduced MATRIGEL (BD Bioscience). Mice bearing tumors of 50 mm3 (link) were randomly assigned into the indicated groups: vehicle control (saline), PARPi (purchased from LC-lab, O-9201, 50 mg/kg), Fostamatinib (purchased from Selleck Chemicals, 80mg/kg). The treated mice were intraperitoneally injected with PARP inhibitor and gavaged with Fostamatinib 5 times/week. Tumor volume was measured every 7 days using calipers, and tumor volume was calculated using the formula length × width2 (link). Mice were sacrificed for tumor dissection on day 24 of treatment.
Fostamatinib
Manufactured by Selleck Chemicals
Sourced in United States
Fostamatinib is a chemical compound used as a laboratory reagent. It is a phosphate ester of the active pharmaceutical ingredient Tamatinib. Fostamatinib serves as a precursor for the synthesis and analysis of Tamatinib, a tyrosine kinase inhibitor.
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Lab products found in correlation
P505 15, by Selleck Chemicals (2 mentions)
Ibrutinib, by Selleck Chemicals (2 mentions)
Idelalisib, by Selleck Chemicals (2 mentions)
Dasatinib, by Selleck Chemicals (2 mentions)
Matrigel, by BD (1 mentions)
Saracatinib, by Selleck Chemicals (1 mentions)
Buparlisib, by Selleck Chemicals (1 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (1 mentions)
Bosutinib, by Selleck Chemicals (1 mentions)
Trametinib, by LC Laboratories (1 mentions)
Dasatinib, by LC Laboratories (1 mentions)
A770041, by Axon Medchem (1 mentions)
Abt 199, by Selleck Chemicals (1 mentions)
Cd40l, by R&D Systems (1 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Sorafenib, by LC Laboratories (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
F ab 2 fragment, by Jackson ImmunoResearch (1 mentions)
6 protocols using fostamatinib
1
Evaluating PARP Inhibitor and Fostamatinib in Ovarian Cancer
2
Phospho-protein Analysis of Leukemia Cells
3
In vitro cellular assay protocol
4
Evaluating Anticancer Agents in Cell Lines
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Dasatinib (S1021) and fostamatinib (S2206) were obtained from Selleckchem (Houston, TX, USA). Sorafenib (S-8502) was purchased from LC Laboratories (Woburn, MA, USA). Antibodies were purchased from either Santa Cruz Biotechnology (Dallas, TX, USA) (EGFR #sc-03; STAT3 #sc-482; β-actin #s-47778) or Cell Signaling Technology (Danvers, MA, USA) (pAkt #4060; Akt #9272; pERK #4370; ERK #9102; pEGFR #3777; pSTAT3 #9131; pPTEN #9551; PTEN #9559; pJNK #9251; JNK #9252)
5
Primary B Cell Isolation and Culture
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Primary B lymphocytes (normal and CLL) were used in all the experiments described. Cells were isolated from peripheral blood and buffy coats by centrifugation over Lymphoprep (Axis-Shield, Oslo, Norway). Normal B cells were purified by negative selection using a B cell isolation kit (Miltenyi Biotec, Bisley, U.K.) (>98% CD19+). All cultures involving primary lymphocytes employed RPMI 1640 medium containing 1% fatty acid-free BSA (Sigma-Aldrich, Poole, U.K.); HUVEC were cultured in IMDM containing 20% FCS, whereas HS-5 and CD40L-transfected fibroblasts were cultured in DMEM containing 10% FCS. All culture media were supplemented with 2 mM l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Paisley, Scotland). Ibrutinib, fostamatinib, and idelalisib were all used at 1 μM (Selleckchem). These drug concentrations were sufficient to maximally inhibit the respective target kinases following BCR ligation (data not shown); IC50 values and peak plasma concentrations are shown in Supplemental Table II . Goat anti-human IgM [F(ab)2 fragments (Jackson ImmunoResearch Laboratories)] was used at 20 μg/ml.
6
Selective SYK Inhibitor Entospletinib Protocol
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The selective SYK inhibitor entospletinib (ENTO)32 (link) was provided as a dispersible powder for in vitro studies and in rodent chow formulation in 0.03%, 0.05%, and 0.07% concentrations for in vivo animal studies by Gilead Sciences Inc. (Foster City, CA, USA). Rodent chow concentrations were selected and optimized based upon PK levels achieved in ENTO-treated adult patients with acute leukemia (clinicaltrials.gov identifiers: NCT02404220 and NCT02343939).33 Vincristine and dexamethasone were purchased from the Children’s Hospital of Philadelphia investigational pharmacy (Philadelphia, PA, USA). The MEK inhibitor selumetinib, SYK inhibitor fostamatinib, and multi-kinase inhibitor dasatinib were purchased from Selleckchem (Houston, TX, USA) or LC Labs (Woburn, MA, USA). Cell viability and phosphoflow cytom etry signaling analyses of human B-ALL cell lines and PDX model cells treated with vehicle, kinase inhibitors, or chemotherapy (in vitro or in vivo) are detailed in the Online Supplementary Methods with data shown in Online Supplementary Figures S1-S6.
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