Robey (NIH, MD) for providing the SW620, SW620/Ad300, S1, and S1-M1-80 cells. anti-tumor Oleandomycin effects of paclitaxel in P-gp-overexpressing MDR SW620/Ad300 xenograft tumors. With medical trials beginning to add newly approved immune checkpoint-based immunotherapy into standard-of-care immunogenic chemotherapy to improve patient outcomes, our findings support the rationale of adding IPI-549 to both the chemotherapeutic and immunotherapeutic aspects of cancer combination treatment strategies. gene, and also known as ABCB1 , is composed of two homologous nucleotide binding domains and two transmembrane domains joined by a linker region . Each transmembrane domain name is made of six transmembrane helices which make up a twelve transmembrane helix efflux pump that binds hydrophobic Oleandomycin drug substrates . Its hydrophilic region contains the ATP binding site which binds two molecules of ATP. Efflux of a drug substrate leads to hydrolysis of ATP into ADP and inorganic phosphate, allowing the transmembrane domain name to bind another substrate to be effluxed. This continuous cycle leads to low intracellular concentrations of substrate drugs Oleandomycin and thus survival of MDR cancer cells exposed to drugs of chemotherapy . Anticancer drug substrates of P-gp include the taxanes (paclitaxel, docetaxel), anthracyclines (doxorubicin, daunorubicin), vinca alkaloids (vincristine, vinblastine), epipodophyllotoxins (etoposide, teniposide) and tyrosine kinase inhibitors of EGFR, VEGFR, and Bcr-Abl such as lapatinib, nilotinib, and sunitinib, respectively . In addition to cancer cells, P-gp is usually highly expressed at the apical surface of epithelial cells, such as in the colon, hepatic bile duct, renal proximal convoluted tubule, pancreatic ductules, adrenal gland, placenta (blood-placenta barrier), testis (blood-testis barrier), and brain capillaries (blood-brain barrier) . Anatomically, P-gp functions as an efflux transporter that limits cellular uptake of drugs from the blood into the brain, and from intestinal lumen into enterocytes. On the other hand, P-gp enhances the elimination of drugs out of the hepatocytes and renal epithelial cells into the bile and urine, respectively . Overexpression of P-gp has been associated with various cancers, including hematological malignancies, breast cancers, acute myeloid leukemia, and solid tumors [16C19]. In order to counteract P-gp-mediated MDR, strategies to develop small molecule drugs which inhibit or block the efflux function of P-gp, referred to as P-gp inhibitors or modulators, or chemosensitizers/reversal brokers have been undertaken and have gone through three generations of development . IPI-549 is an investigational first-in-class, small molecule, gamma isoform selective phosphoinositide 3-kinase (PI3K) inhibitor [21,22]. In preclinical studies, inhibition of PI3K by IPI-549 reprogrammed macrophages from an immune-suppressive M2 phenotype to an immune-activating M1 phenotype . The shift of macrophages to the proinflammatory antitumor M1 phenotype enhanced the recruitment, infiltration, and activation of cytotoxic T cells at the tumor site . IPI-549 in combination with anti-CTLA4 or anti-PD-1 immune checkpoint blockers showed synergistic effects in a mouse model . In NEU a phase 1 clinical trial, IPI-549 in combination with nivolumab (anti-PD-1) showed favorable tolerability and signs of clinical activity with immune modulation, and recruiting is currently underway for phase 2 clinical trials . In our own studies, we chose to test whether IPI-549 could act as a chemosensitizing agent to the P-gp-overexpressing MDR phenotype of cancer cells. Most immuno-oncology brokers are biological-based therapy in the form of monoclonal antibodies . As a small molecule kinase inhibitor, IPI-549 is an ideal candidate for combination therapy with conventional chemotherapy targeting P-gp-mediated MDR. 2.?Materials and Methods 2.1. Reagents [3H]-paclitaxel (37.9 Ci/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, CA). Dulbeccos Modified Eagles Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and Trypsin/EDTA were purchased from Hyclone, GE Healthcare Life Science (Pittsburgh, PA). Secondary horseradish peroxidase-labeled rabbit anti-mouse IgG was purchased from Cell Signaling Technology (Danvers, MA). 3-(4,5-dimethylthiazol-yl)-2,5- diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), Triton X-100, propidium iodide and paraformaldehyde were purchased from Sigma-Aldrich (St. Louis, MO). P7965 Monoclonal Anti-P-Glycoprotein (MDR) antibody produced in mouse, monoclonal antibodies BXP-21 to ABCG2, GAPDH, and the secondary horseradish peroxidase-labeled rabbit antimouse.