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  • br An immunosuppressive microenvironment is a

    2020-08-30


    An immunosuppressive microenvironment is a common feature in PDAC. Tumor cells, especially CSCs, redirect immune Mitoquinone to evade immune surveillance and even coopt immune cells to support their
    growth and metastasis [7,8]. Monocytes and derived macrophages, which are abundant in both clinical and experimental PDAC tissue, have been proved to be indispensable for the initiation and metastasis of pancreatic cancer [9–13]. However, the crosstalk Mitoquinone between pancreatic CSCs and adjacent immune cells is still elusive. Investigation of the crosstalk between CSCs and immune cells would reveal how CSCs re-spond to cues from their surroundings for disease progression.
    CD90, also named Thy-1, is a 25–37 kDa glycosylpho-sphatidylinositol (GPI)-anchored glycoprotein expressed in various cell types, including hematopoietic stem cells, activated microvasculature endothelial cells and fibroblasts, and is mainly responsible for cell ad-hesion, migration and fibrosis [14,15]. Moreover, CD90 has been re-ported to be expressed on tumor-initiating cells in several solid tumors [16–20]. However, except for reports about CD90 expression in PDAC
    ∗ Corresponding author. State Key Laboratory of Oncogenes and Related Genes, Stem Cell Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Rd, Shanghai, 200127, China. ∗∗ Corresponding author. Stem Cell Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Rd, Shanghai, 200127, China. ∗∗∗ Corresponding author. Department of Biliary-Pancreatic Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Rd, Shanghai, 200127, China.
    stroma [21,22], little is known about the expression pattern of CD90 in pancreatic cancer cells and its potential role in PDAC tumorigenesis.
    These considerations prompted us to identify “stem-like” cells in PDAC and to explore the mechanism by which tumors survive and evade immune surveillance. Here, we show that CD90 is expressed on both stromal and tumor cells in PDAC tissues, and its high expression is linked to a poor prognosis. PDAC cells with high expression of CD90 harbor stemness characteristics. Notably, CD90 acts as an anchor for monocyte/macrophage adhesion in PDAC cells, providing crosstalk between CD90hi PDAC cells and monocytes/macrophages in a near-secretory manner. Moreover, CD90hi PDAC cells express high levels of PD-L1 and suppress the T cell response. Thus, CD90hi PDAC cells harbor stemness properties and create an immunosuppressive niche, which enhances the stemness and epithelial-mesenchymal transition (EMT) state of PDAC. These results provide invaluable insights and new therapeutic strategies for the diagnosis and treatment of PDAC.
    2. Materials and methods
    Human pancreatic cancer cell lines (SW1990, PANC1, MiaPaCa2, AsPC-1 and HPAC) were purchased from the American Type Culture Collection (ATCC). SW1990, PANC1 and MiaPaCa2 cells were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM), and AsPC-1 and HPAC cells were cultured in RPMI 1640 medium and a 1:1 mixture of DMEM and F12, respectively, containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Pancreatic patient-derived tumor cells (PDCs), including PDC0034 and PDC0049, were isolated from pancreatic PDX (patient-derived xenograft) tumors and cultured in complete RPMI 1640 medium plus 10 ng/ml EGF (Epidermal growth factor) and 1% insulin-transferrin-selenium (ITS) as previously de-scribed [23]. Human pancreatic tissue specimens were derived from patients with confirmed diagnoses of pancreatic ductal adenocarcinoma (PDAC) at Renji Hospital. Primary tumor cells from PDAC patient samples were directly isolated by collagenase digestion (collagenase IV, 2 mg/ml, 30–60 min). THP-1 cells were obtained from the ATCC and grown in RPMI 1640 medium supplemented with 10% FBS, 1% peni-cillin-streptomycin and 5 mM β-mercaptoethanol. All cells were cul-tured in a humidified incubator at 37 °C with 5% CO2.
    For the coculture experiment, tumor cells and THP-1 cells (2:3) were cultured together for 36 h in DMEM supplemented with 10% FBS. Primed PDAC cells or THP-1 cells were sorted for further use. Control cells underwent the same sorting process.
    Cells were prepared as a single-cell suspension for FACS staining. For CD90 staining, cells were stained with APC-CD90 antibody (Clone #5E10, Biolegend) for 30 min at 4 °C. CD90+ and CD90− cells were identified based on isotype gating. ALDH activity was determined by the ALDEFLUOR Kit following the manufacturer's instructions (STEM CELL). The stained cells were acquired for analysis or sorted on an LSRFortessa or a FACS Aria II flow cytometer (BD). Flow cytometry data were analyzed with FlowJo software (Tree Star Inc.).
    2.3. Quantitative RT-PCR
    Total RNA was extracted from the indicated cells using Trizol (Invitrogen) according to the manufacturer's protocol. cDNA was syn-thesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Specific primers are included in the supplemen-tary information (Supplement Table 1). Quantitative RT-PCR was per-formed with SYBR Green Master Mix (Roche) on a Step One Plus system (Applied Biosystems). The relative expression of target genes was cal-culated according to the Ct value with normalization to GADPH.