ABSTRACT: IFN-g-induced gene expression profiles of UM-Chor1 cells were assessed by microarray analysis Chordoma is a rare bone tumor derived from notochord, which has shown to be resistant to conventional therapy, including chemotherapy and radiotherapy. Immunotherapy has the potential to be effective in cancers with resistance to conventional therapy. Checkpoint inhibition, including Programmed cell Death 1 (PD-1)/Programmed cell Death protein 1 (PD-L1) blockade has shown great promise in treating diverse cancers. The purpose of this study was to investigate the potential of anti-PD-L1 antibody therapy for chordoma. To this end, using 4 chordoma cell lines, we examined the expression of PD-L1 and performed in vitro NK cells killing assay for antibody-dependent cell-mediated cytotoxicity (ADCC) activity mediated by the anti-PD-L1 antibody, avelumab. Here, the expression of PD-L1 was markedly upregulated by IFN-g in all 4 chordoma cell lines, which resulted in increasing the sensitivity to ADCC. Next, to model a patient that received a tumor-antigen specific vaccine, chordoma cells were co-incubated with brachyury specific CD8+ T cells, culminating in significant upregulation of PD-L1 on the tumor cells mediated by CD8+ T cells IFN-g production. The functional consequence was the sensitivity of chordoma cells to ADCC mediated by avelumab. Finally, residential cancer stem cell subpopulations of chordoma cells were killed by avelumab-mediated ADCC to the same degree as non-cancer stem cell populations. Our findings show the potential of avelumab therapy in chordoma; a) alone, to enable endogenous NK cells to kill chordoma cells via ADCC, or b) in conjunction with T cells immunotherapy such a vaccine, to exploit enhanced NK cell killing of chordoma cells via ADCC. Overall design: UM-Chor1 cells were treated or untreated with 50 ng/mL of IFN-gamma for 24 hours. Three replicates of control (C1) or treated (G1) were analyzed.
INSTRUMENT(S): [HuGene-2_0-st] Affymetrix Human Gene 2.0 ST Array [probe set (exon) version]
Project description:PD-L1 suppresses host immunity and promotes tumor growth. We investigated how IFN-γ regulates PD-L1 in the ovarian cancer microenvironment. In clinical samples, the number of stromal CTLs in peritoneally disseminated tumors was correlated with PD-L1 expression on the tumor cells, and the lymphocyte number was significantly related to the IFN-γ signature score. In mouse models, PD-L1 was induced in peritoneal disseminated tumors, where lymphocytes were prominent, but not in subcutaneous tumors. Depleting IFNGR1 resulted in lower PD-L1 expression and longer survival in peritoneal dissemination model. Injection of IFN-γ into subcutaneous tumors increased PD-L1 expression and tumor size, and PD-L1 depletion abrogated tumor growth. These data suggest that IFN-γ works as a tumor progressor through PD-L1 induction. The source of IFN-γ in ovarian cancer microenvironment and its biological effect to the tumor cells is unclear. The immortalized human ovarian surface epithelial cell line, HOSE-E7/hTERT (HOSE) was treated with IFN-γ and expression microarray analysis was performed, and probes showing significantly higher values in IFN-γ-added group were termed “IFN-γ signature genes (295 probes)”. We then applied this signature to our ovarian cancer microarray data, which included 75 ovarian cancer clinical samples, by means of ss-GSEA. IFN-γ signature score was strongly correlated to the number of infiltrating CD4-positive or CD8-positive lymphocytes in the tumors. These data suggest that the IFN-γ in the ovarian cancer microenvironment is derived from lymphocytes, and an IFN-γ-rich microenvironment is strongly correlated to a lymphocyte-rich microenvironment. Genome-wide transcriptional changes in human ovarian cancer tissue were observed in different tumor immunological microenvironment.
Project description:TNF-α is an important inflammatory cytokine. Recent findings suggest tumor promoting inflammation could be a driving factor for chordoma progression. We used microarray to monitor the global changes in gene expression after TNF-α treatment for 1 year in two chordoma cell lines Overall design: Chordoma cell lines U-CH1 and MUG-Chor1 were treated 3 times a week with 10 ng/mL TNF-α. Cells were harvested and microarray was conducted
Project description:TNF-α is an important inflammatory cytokine. Recent findings suggest tumor promoting inflammation could be a driving factor for chordoma progression. We used microarray to monitor the global changes in gene expression after TNF-α treatment for 1 week in two chordoma cell lines Overall design: Chordoma cell lines U-CH1 and MUG-Chor1 were treated 3 times a week with 10 ng/mL TNF-α. Cells were harvested and microarray was conducted
Project description:Leukemia Inhibitory Factor is an important cytokine of the IL family. Recent findings suggest it has a crucial role in cancer progression We used microarray to monitor the global changes in gene expression after LIF treatment for 3 weeks in two chordoma cell lines Overall design: Chordoma cell lines U-CH1 and MUG-Chor1 were treated 3 times a week with 100ng/mL recombinant human Leukemia Inhibitory Factor. Cells were harvested and microarray was conducted.
Project description:Epigenetic regulators have emerged as exciting targets for cancer therapy. Additionally, restoration of antitumor immunity by blocking the PD-L1 signaling using antibodies has proven to be beneficial in cancer therapy. Here we show that BET bromodomain inhibition suppresses PD-L1 expression and restores antitumor immunity in ovarian cancer. CD274 (encoding PD-L1) is a direct target of BRD4-mediated gene transcription. In mouse models, treatment with the BET inhibitor JQ1 significantly reduced PD-L1 expression on tumor cells and tumor-associated dendritic cells and macrophages, which correlated with an increase in the activity of antitumor cytotoxic T cells. Together, these data demonstrate an epigenetic approach to block PD-L1 signaling to restore antitumor immunity. Given the fact that BET inhibitors have been proven safe with manageable reversible toxicity in clinical trials, our findings indicate that pharmacological BET inhibitors represent a novel treatment strategy for targeting PD-L1 expression. Overall design: RNA-seq for JQ1 treated and shBRD4 knockdown cells with controls
Project description:PD-L1 is a ligand for the inhibitory PD1 receptor on T cells and its expression in some cancers inhibits anti-cancer immune response. In melanoma, PD-L1 expression is induced in response to immune stimuli but in a proportion of melanomas it is constitutively expressed. Factors that drive constitutive expression of PD-L1 are unknown. Here we performed RNA-Seq analysis of six cell lines that constitutively express PD-L1 (PD-L1 positive, referred to as PD-L1CON) and six cell lines that only express PD-L1 after treatment with IFN- (PD-L1 negative, referred to as PD-L1IND) Overall design: Here we generated gene expression profile of 12 cell lines using RNA-Sequening. We have two group each consisting of 6 cell lines (inducible and constitutive)
Project description:PD-L1 is a ligand for the inhibitory PD1 receptor on T cells and its expression in some cancers inhibits anti-cancer immune response. In melanoma, PD-L1 expression is induced in response to immune stimuli but in a proportion of melanomas it is constitutively expressed. Factors that drive constitutive expression of PD-L1 are unknown. Here we performed genome-scale methylation analysis of six cell lines that constitutively express PD-L1 (PD-L1 positive, referred to as PD-L1CON) and six cell lines that only express PD-L1 after treatment with IFN- (PD-L1 negative, referred to as PD-L1IND) Overall design: Here we generated single-nucleotide resoultion DNA methylation map of a total of 12 cell lines using Reduced Representation Bisulfite Sequencing (RRBS). We have two groups, each with 6 cell lines each (inducible group and constitutive group)
Project description:Hematopoiesis generates cell diversity through evolutionarily-determined differentiation programs defined at steady-state conditions. Here, we show that peripheral innate immune activation can generate cell diversity and functional specialization during the activation process. We found that activation of steady state human plasmacytoid pre-dendritic cells (pDCs) with a single microbial stimulus induced their differentiation into three phenotypically, morphologically, and functionally distinct subsets, in the absence of cell division: P1 (PD-L1+CD80-), P2 (PD-L1+CD80+) and P3 (PD-L1-CD80+). Different stimuli induced variable proportions of the subsets, suggesting that steady state pDC are multipotent. P1-pDCs display a plasmacytoid morphology and strong specialization on IFN production, whereas P3-pDCs adopt a dendritic morphology and adaptive immune functions. P2-pDCs have an intermediate functional profile. We found that a P1-pDC phenotype is present in human autoimmune diseases associated to type I IFN production as lupus and psoriasis, supporting their pathophysiological relevance. We propose that peripheral innate activation represents a differentiation mechanism to generate subsets diversity and functional complementarity in human pDCs. Overall design: 15 samples: medium (control condition), ex-vivo, P1, P2 and P3, each from 3 healthy donors. Ex-vivo plasmacytoid dendritic cells from the blood of healthy human donors were treated for 24h with influenza virus. Treated cells were sorted as P1, P2, and P3 subsets depending on surface markers: P1 (PD-L1+CD80-), P2 (PD-L1+CD80+) and P3 (PD-L1-CD80+). Non-treated control cells correspond to pDCs kept in medium.
Project description:The programmed cell death protein 1 (PD-1) limits effector T-cell functions in peripheral tissues and its inhibition leads to clinical benefit in different cancers. To better understand how PD-1 blockade therapy modulates the tumor-host interactions, we evaluated three syngeneic murine tumor models, the BRAFV600E-driven YUMM1.1 and YUMM2.1 melanomas, and the carcinogen-induced murine colon adenocarcinoma MC38. The YUMM cell lines were established from mice with melanocyte-specific BRAFV600E mutation and PTEN loss (BRAFV600E/PTEN-/-). Administration of anti-PD-1 or anti-PD-L1 antibody therapy had strong antitumor activity against MC38 and YUMM2.1, but not YUMM1.1. There was no difference in PD-L1 expression between the three models at baseline or upon interferon stimulation. While mutational load was high in MC38, it was lower in both YUMM models. In YUMM2.1, the antitumor activity of PD-1 blockade had a critical requirement for both CD4 and CD8 T-cells, as well as CD28 and CD80/86 co-stimulation, with an increase in CD11c+CD11b+MHC-IIhigh dendritic cells and tumor associated macrophages in the tumors after PD-1 blockade. Compared to YUMM1.1, YUMM2.1 exhibited a more inflammatory profile by RNA sequencing analysis, with an increase in chemokine-trafficking gene expression levels related to immune cell recruitment and T-cell priming. In conclusion, response to PD-1 blockade therapy in tumor models requires CD4 and CD8 T cells, and co-stimulation mediated by dendritic cells and macrophages. Overall design: mRNA profiles of YUMM murine melanoma cell lines
Project description:Therapeutic antibodies trigger antibody-dependent cellular cytotoxicity (ADCC) of cancer cells and also their antibody-dependent cellular phagocytosis (ADCP) by tumor-associated macrophages (TAMs). We report here our unexpected finding that TAMs that have undergone ADCP of tumor cells induced by therapeutic antibodies display immunosuppressive characteristics and inhibit the proliferation and tumoricidal effects of NK and tumor-specific CD8+ T cells in breast cancers and lymphomas. Mechanistically, we show that DNA released from the phagocytosed tumor cells after ADCP activates caspase 1 inflammasome, leading to IL-1β-mediated PD-L1 and IDO upregulation in these cells. Combined treatment with anti- HER2 antibody and inhibitors of PD-L1 and IDO increased the infiltration of NK and CD8+ T cells and enhanced anti- HER2 therapeutic efficacy in mouse models of HER2+ breast cancers. Furthermore, neo-adjuvant Trastuzumab therapy significantly increased PD-L1 and IDO expression in the TAMs of HER2+ breast cancer patients, which correlate with poor Trastuzumab response and reduced NK and CD8+ T cells in the tumors. Collectively, our findings unveil an unexpected role of ADCP induced by therapeutic antibodies in cancer immunosuppression, and suggest that antibody plus immune checkpoint blockade may provide synergistic therapeutic effects in cancer patients. Overall design: HER2+ breast cancer cells pre-dyed by CellTtracker Deep Red Dye (Cat# C34565, Thermo Fisher) were co-cultured with macrophages pre-dyed by CellTracker CMFDA Dye (Cat# C7025, Thermo Fisher) with or without Trastuzumab (Roche). The ratio between tumor cells and macrophages was 1:5. After 24 hr, the cells were rigorously washed by PBS, digested by 10×TrypLE Selected Enzyme (Cat# A1217701, Gibco) diluted 5× in PBS with 1mM EDTA and sorted by CD14 Isolation Kit (Cat# 130-050-201, Miltenyi Biotec) to collect macrophages followed the instructions. we named untreated macrophages as Mɸ and macrophages pre-incubated with HER2+ tumor cells in the presence or absence of Trastuzumab as ADCP-Mɸs or TC-Mɸs respectively. Total RNA was extracted from macrophages with indicated treatments (5 x 106 cells) using the TRIzol Reagent (Cat#A33251, Thermo Fisher). The quality of extracted RNA was assessed using the NanoDrop ND-2000 spectrophotometer.