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Fig. 3. CD90 acts as an anchor for monocyte/macrophage adhesion in PDAC Exendin 4 (A) GSEA revealed that CD90hi cells (versus control CD90low cells) were positively enriched for integrin binding and integrin cell surface interaction. Data were collected from TCGA (PAAD) database. (B) Plots show significant correlations between monocyte/macrophage-associated genes (CD14, CD68, MRC1 and CD163) and CD90 expression in TCGA (PAAD) database. (C) THP-1 monocytes were cultured with PDAC cells. Monocytes that did not adhere to the PDAC monolayer were washed away after 1 h. Attached monocytes were quantified by flow cytometry based on CD45 expression. Representative images and flow cytometry profiles are shown on the left. The ratios of attached monocytes are shown on the right. (D) CD90 expression (log10 transformed MFI) was positively associated with the adhesion of THP-1 monocytes. (E) CD90 knockout eﬃciency in PANC1 cells was examined by flow cytometry. (F) Representative flow cytometry profiles of THP-1 cells adhered to PANC1 sgCD90 and sgCON cells are shown on the left. The ratios of attached THP-1 cells were calculated and are shown on the right. Data are shown as the mean ± SEM, **P < 0.01, ***P < 0.001.
3.6. CD90hi PDAC cells exhibit high PD-L1 expression and suppress T cell proliferation
As shown above, CD90hi PDAC cells with high stemness properties tethered and reprogrammed monocytes/macrophages into an
immunosuppressive state. In turn, these monocytes/macrophages fur-ther promoted the stemness and EMT of PDAC cells. Creating an im-munosuppressive niche is a strategy for tumor cells, especially CSCs, to evade immune surveillance [13,37]. It is well known that the PD-L1/ PD-1 axis drives T cell dysfunction and exhaustion, thus enabling tumor cells to escape immune surveillance. Surprisingly, we found that CD90hi PDAC cells exhibited higher expression of PD-L1 compared with CD90−
Fig. 4. PDAC cells promote immunosuppressive features of monocytes/macrophages. (A) Overview of the procedure to coculture PDAC cells with THP-1 monocytes.
(B) THP-1 monocytes were cultured with or without PDAC cells for 36 h, and THP-1 monocytes were isolated for qPCR analysis. Bar graphs show the upregulation of
SHH, IL6, and IL8 in THP-1 cells after coculture with PDAC cells. (C) PBMCs isolated from healthy donors were cocultured with PDAC conditioned medium in 96-well round bottom plates for 6 days. Representative flow cytometry plot shows that CD14+ cells were upregulated after coculture with conditioned medium from PDAC cells. (D) Downregulation of HLA-DR expression upon PDAC_CM exposure. Flow cytometry was performed to examine HLA-DR expression on CD14+ cells. (E) PBMCs from healthy donors were treated with PDAC conditioned medium in 6-well plates for 6 days. Then, CD14+ cells were isolated by magnetic beads to examine the expression of the indicated genes by qPCR. (F) THP-1 monocytes were cultured with or without PDAC cells for 36 h. Then, THP-1 and primed THP-1 cells were isolated for coculture with CFSE-labeled CD4+ and CD8+ cells for 5 days. (G) Immunostaining of CD90, CD68 (macrophage marker) and CD8 (CD8+ T cell marker) in PDAC tissues. All data are shown as the mean ± SD, Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 5. Monocytes/Macrophages promote the stemness and EMT of PDAC cells (A, B) THP-1 monocytes were cultured with PDAC cells (SW1990 or PANC1) for 36 h. Then, PDAC cells were sorted for qPCR and western blot analyses. (C) A sphere assay was performed with sorted cells described in (A). Representative images and the number of spheres is shown (n ≥ 3, mean ± SEM). (D) A sphere assay was performed in SW1990 and PANC1 cells upon VE or THP-1 conditioned medium treatment. Representative images and the number of spheres is shown (n ≥3, mean ± SEM). (E) SW1990 cells were treated with VE or THP-1 CM for 36 h before ALDEFLUOR analyses. (F) ALDEFLUOR analyses of SW1990 cells after CM (top) or IL8 (bottom) treatment in the presence of αIgG or αIL8. Data represent the mean ± SEM. (G) THP-1 monocytes were cultured together with PDAC cells for 36 h. Then, cells were sorted for Transwell-based migration and invasion assays. (H) Migration rates of SW1990 cells upon SHH or VE treatment were evaluated with the migration assay. SW1990 cells were cocultured with THP-1 cells in the presence of GANT61 or DMSO treatment. SW1990 cells from 2 groups were sorted to perform the Transwell migration assay. Bar graphs depict the OD595 value of migrated cells (mean ± SEM of three independent experiments).
Fig. 6. CD90hi PDAC cells exhibit high PD-L1 expression and suppress T cell proliferation (A, B) Representative flow cytometry plot showing the expression of PD-L1 in CD90hi and CD90− cells from PANC1 (A) and SW1990 (B) cells. (C) Bar graphs depict the high expression of PD-L1 in CD90hi cells in both PANC1 and SW1990 cells. (D) CFSE-labeled CD4+ and CD8+ cells isolated from PBMCs were cocultured with CD90hi or CD90− cells for 5 days. A T cell suppression assay showed that CD90hi cells could inhibit the proliferation of T cells. (E) Schematic graph depicting the crosstalk between CD90hi PDAC cells and monocytes/mac-rophages and T cells.