Cells were cultured in DMEM or RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics. to IGF-1R was hampered by cixutumumab, leading to Akt activation and cixutumumab resistance. Targeting Tafenoquine Succinate integrin 3 or Src enhanced antitumor activity of cixutumumab in multiple cixutumumab-resistant cell lines and patient-derived tumors in vitro and in vivo. Mean tumor volume of mice cotreated with cixutumumab and integrin 3 siRNA was 133.7mm3 (95% confidence interval [CI] = 57.6 to 209.8mm3) compared with those treated with cixutumumab (1472.5mm3; 95% CI = 1150.7 to 1794.3mm3; .001) or integrin 3 siRNA (903.2mm3; 95% CI = 636.1 to 1170.3mm3; .001) alone. Conclusions Increased Src activation through integrin 3 confers considerable resistance against antiCIGF-1R mAb-based therapies in HNSCC and NSCLC cells. Dual targeting of the IGF-1R pathway and collateral integrin 3CSrc signaling module may override this resistance. The insulin-like growth factor (IGF) axis, regulated by receptors (IGF-1R and IGF-2R), ligands (IGF-1, IGF-2, and insulin), and IGF-binding proteins, is critically important for numerous hallmarks of neoplasia (1,2), and thus is recognized as an attractive target for anticancer therapies. A number of clinical trials are under way to test two major antiCIGF-1R strategies, including monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) (3,4). Although a small subset of patients enrolled in phase I and II clinical trials demonstrated sporadic tumor responses to antiCIGF-1R mAbs (5C9), the anticancer effects have been very modest and unsustained when used alone (10C12). However, the mechanisms mediating resistance to antiCIGF-1R strategies are poorly Tafenoquine Succinate understood. Integrins, a family of adhesive receptors composed of 8 and 18 ATP1B3 subunits (13) activated by ligand occupancy, induce focal adhesion kinase (FAK) autophosphorylation at tyrosine 397 (Y397), which is required for p85 binding and PI3K activation (14), the recruitment of Src, and Src-dependent phosphorylation of FAK and epidermal growth factor receptor (EGFR) (13,15). Several reports have demonstrated the implications of integrin v3 in key aspects of neoplasia and antineoplastic drug resistance (16,17). Of note, a recent report showed that IGF-1 directly binds to integrin 3, but not integrin 1 (18), suggesting a direct regulatory link between the IGF system and specific integrin signals. In this study, we sought to determine the mechanisms mediating resistance to cixutumumab (IMC-A12), a fully humanized antiCIGF-1R mAb that has been evaluated in several clinical trials (19), and to discover alternative strategies for targeting of IGF-1R and other signaling molecules involved in antiCIGF-1R mAb resistance. Methods Further details for some experimental procedures are described in the Supplementary Methods (available online). Reagents, preparation of poly-(HEMA [poly-2-hydroxyethyl methacrylate])-coated plates (PCPs), cell proliferation/viability and anchorage-independent colony formation assays, Western blot Tafenoquine Succinate and enzyme-linked immunosorbent assay (ELISA), preparation of paraffin-embedded cell blocks and immunofluorescence, extracellular matrix adhesion and immunofluorescence, mouse studies, and liposomal preparation are only described online. Cell Culture, In Vivo Experiments, and Analyses of Proliferation/Viability All cell lines were authenticated/validated. Cells were cultured in DMEM or RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells were maintained at 37C in a humidified atmosphere with 5% CO2 and subcultured twice a week. Athymic nude mice were purchased from Harlan Sprague Dawley (Indianapolis, IN). The use of tissue specimens of primary head and neck squamous cell carcinoma (HNSCC) obtained from patients who had surgical resection at MD Anderson Cancer Center was approved by the Institutional Review Board, which waived the need for written informed consent. Human HNSCC and nonCsmall cell lung cancer (NSCLC) cell culture and analyses of cell proliferation/viability under the 3D-mimic and 3D culture conditions were performed as described previously (20). Further details are described in the Supplementary Methods (available online). Mouse Studies All mouse study procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Seoul National University or MD Anderson Cancer Center. Mice were cared for in accordance with guidelines set by the Association for Assessment and Accreditation of Laboratory Animal Care and the US Public Health Service Tafenoquine Succinate Policy on Human Care and Use Tafenoquine Succinate of Laboratory Animals. For 686LN, UMNSCC38, H226B, or A549m xenograft tumors, cancer cells (1106 cells/mouse in 100 L of phosphate-buffered saline) were subcutaneously injected into nude.