Cancer Letters br Sphere formation assay br Sphere culture was

 Cancer Letters 453 (2019) 158–169
2.4. Sphere formation assay
Sphere culture was performed as previously described [24,25]. Briefly, Amiloride HCL were plated in 24-well Ultra-Low Attachment Plates (Corning) with DMEM: F12 medium plus 2% B27 (Gibco) and 20 ng/ml bFGF (PeproTech). The culture medium was replenished every 3 days. After 7 days, the sphere diameter and number (> 50 μm) were eval-uated and quantified.
2.5. Monocyte adhesion assay
For the monocyte adhesion experiment, pancreatic cancer cells were seeded at 70–80% confluency before adding THP-1 monocytes (THP-1:PDAC cells = 3:2). The non-adherent monocytes were gently rinsed away with PBS after 1 h of coculture, and then the remaining cells in the culture dish were trypsinized and stained with the indicated antibody for flow cytometry.
2.6. PBMC isolation and in vitro culture
PBMCs (peripheral blood mononuclear cells) were isolated by density gradient centrifugation from healthy donors using Ficoll-Hypaque. Prepared PBMCs were seeded in 96-well round bottom plates and then treated with PDAC conditioned medium for 6 days before staining and flow cytometry analysis. DAPI (Life Technology) was used to exclude dead cells.
2.7. Immunohistochemistry (IHC) and immunofluorescence (IF)
Pancreas tissues were fixed in 10% buffered formalin or frozen in Tissue-Tek OCT compound. Fixed and frozen tissues were sectioned for IHC and IF, respectively, using CD90 (Abcam, 133350), CD68 (Abcam, ab955), CD68 (Biolegend, #Y1/82A) and CD8 (Abcam, ab93278). Cells were mounted with DAPI and prolonged gold antifade reagent (Thermo Fisher Scientific) before analysis with a confocal microscope.
2.8. Lentiviral transduction
The CD90 gRNA expression oligos and control oligos were in-troduced into LentiCRISPR V2 (Addgene). The sequences of these oligos are shown in Supplementary Table 1. A lentivirus containing the CD90 gRNA sequence and the control gRNA sequence was transfected into PANC1 cells. After 2 weeks of selection with puromycin (2 μg/ml), multiple colonies were picked and passaged for sequencing. Knockout efficiency was determined by qPCR and flow cytometry.
2.9. Cell viability analysis
Cell proliferation and gemcitabine sensitivity assays were per-formed with CellTiter-Glo® 2.0 reagent (Promega) according to the manufacturer's instructions.
2.10. Migration and invasion assays
Transwell-based cell migration and invasion assays were performed in polyethylene terephthalate-based migration chambers with an 8 μm pore size according to the manufacturer's instructions. The 1:8 mixture of Matrigel and serum-free DMEM was plated onto filters in the upper chambers one hour in advance when the invasion assay was performed. Then, tumor cells (5 × 103) in serum-free DMEM were seeded onto filters in the upper chambers, while DMEM containing 10% FBS was added to the lower chambers. After 36 h of incubation for the migration assay and 48 h of incubation for the invasion assay, cells on the upper surface of the filters were removed, and filters were fixed with 4% PFA and stained with crystal violet. After imaging, the stained cells were stained with acetic acid to detect the absorbance at OD595.
The indicated numbers (103 each) of CD90− and CD90hi cells sorted from PANC1 cells were subcutaneously transplanted into nude mice (from Shanghai SLAC Laboratory Animal Co., Ltd.). Tumor incidence was monitored for 8 weeks. All animals were maintained and used in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Renji Hospital.
2.12. T cell proliferation assay
CD4+ and CD8+ T cells were isolated and sorted from the fresh human peripheral blood of healthy volunteers. Then, enriched CD4+ and CD8+ T cells were labeled with 1.0 μM CFSE (Invitrogen) in PBS and incubated at 37 °C for 30 min as previously described [26]. The cells were cultured at 5*104 cells per well in 96-well round bottom plates and then activated with anti-CD3 and anti-CD28 antibodies (2 μg/ml each) for 5 days under different conditions.
The Pearson correlation together with the P value was computed to measure the correlation between the indicated genes. The survival curve was estimated by the Kaplan-Meier method. Two-way ANOVA or one-way ANOVA together with Bonferroni's post hoc test was used for multiple group analysis. An unpaired Student's t-test was used to de-termine statistical significance in the rest of the experiments, and a P value of less than 0.05 was considered significant. Values are expressed as the mean ± SEM or the mean ± SD (Prism 6; GraphPad Software). Unless indicated, the results were obtained from at least two or three independent experiments.
3. Results
3.1. CD90 is upregulated in pancreatic cancer and expressed on both stromal and tumor cells
To determine the expression level of CD90 in PDAC (pancreatic ductal adenocarcinoma), we analyzed gene expression from 4 published databases, TCGA (PAAD), GSE15471 [27], GSE28375 [28] and GSE102238 (Renji cohort) [29]. Notably, the expression of CD90 was significantly higher in PDAC than in adjacent normal tissues (Fig. 1A). Previous studies have shown that CD90 is expressed on PDAC stroma cells, such as fibroblasts and vascular endothelial cells [21,22]. To further elucidate the distribution of CD90 in PDAC, we performed im-munohistochemical staining for CD90 using a tissue array. In line with previous reports, we observed that CD90 was dominantly expressed on PDAC stromal cells (34.74%), whereas CD90 could also be detected on tumor cells in a small portion of PDAC patients (10%) (Fig. 1B). Moreover, the expression of CD90 gradually increased from normal tissues to intraepithelial neoplasia (PanINs) and PDAC in the GSE43288 familial pancreatic cancer database [30] (Fig. 1C).