PI3Kγ Activates Integrin α 4 and Promotes Immune Suppressive Myeloid Cell Polarization during Tumor Progression
Philippe Foubert, Megan M. Kaneda, Judith A. Varner
Cancer immunology research
Immunosuppressive myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) accumulate in tumors where they inhibit T cell-mediated antitumor immune responses and promote tumor progression. Myeloid cell PI3K plays a role in regulating tumor immune suppression by promoting integrin 4 -dependent MDSC recruitment to tumors and by stimulating the immunosuppressive polarization of MDSCs and TAMs. Here we show that integrin 4 promotes immunosuppressive polarization of
... and TAMs downstream of PI3K thereby inhibiting antitumor immunity. Genetic or pharmacological suppression of either PI3K or integrin 4 blocked MDSC recruitment to tumors and also inhibited immune suppressive myeloid cell polarization, thereby reducing expression of IL10 and increasing expression of IL12 and IFNγ within tumors. Inhibition of PI3K or integrin 4 within tumors stimulated dendritic cell and CD8 + T-cell recruitment and maturation, as well as tumor cell cytotoxicity in vivo, thereby inhibiting tumor growth. As blockade of PI3Kγ or integrin 4 prevents accumulation of MDSC and reduces myeloid cell expression of immunosuppressive factors that stimulate tumor immune escape, these results highlight PI3Kγ and integrin 4 as targets for the design of cancer therapeutics. MATERIALS AND METHODS Tumor cell lines Lewis Lung Carcinoma cells (LLC) were obtained from the American Type Culture collection (ATCC) and cultured in Dulbecco's Modified Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin. Pancreatic adenocarcinoma cells (Panc02) were obtained from the David Cheresh laboratory, University of California, San Diego and were cultured in RPMI (GIBCO) supplemented with Lglutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin and 10% fetal bovine serum. Cultures were maintained in a humidified incubator at 37°C in an atmosphere containing 5% CO 2. Both cell lines were verified by RT-PCR or RNA sequencing, in vitro morphological and biochemical criteria and in vivo tumor assays in syngeneic mice. Murine macrophage differentiation and culture Bone marrow derived cells (BMDC) were aseptically harvested from 6-8 week-old female mice by flushing leg bones of euthanized mice with phosphate buffered saline (PBS), 0.5% BSA, 2 mM EDTA, incubating in red cell lysis buffer (155 mM NH4Cl, 1 mM NaHCO3 and 0.1 mM EDTA) and centrifuging over Histopaque 1083. Purified mononuclear cells were cultured in RPMI + 20% serum + 50 ng/ml M-CSF (PeproTech). Bone marrow derived macrophages were polarized with either IFNγ (20 ng/ml, Peprotech) plus LPS (100 ng/ml, Sigma) or LPS alone for 24 h or IL4 (20 ng/ml, Peprotech) for 24-48h. Total RNA was harvested from macrophages using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.