br Monocytes macrophages promote stemness
3.5. Monocytes/macrophages promote stemness and the EMT of PDAC cells
As cancer progresses, tumor Relebactam may reprogram the surrounding cells to create a supportive microenvironment that facilitates their growth and metastasis. Monocyte and derived macrophages determine the initiation and metastatic processes of PDAC [8–13]. However, little is known about the crosstalk between monocytes/macrophages and pancreatic CSCs. Here, we wondered whether tethered monocytes/ macrophages by CD90hi PDAC cells could contribute to their stemness maintenance and metastasis. Based on the above coculture system, PDAC cells cocultured with or without THP-1 cells were sorted for multiple assays. Here, we found that the expression of core stemness genes (SOX2, NANOG, POU5F1) and EMT-related genes (ZEB2, TWIST1) in PDAC cells was highly upregulated after coculture with THP-1 cells compared with those cultured alone (Fig. 5A). Moreover, increased expression of SOX2 and ZEB2, as well as reduced E-cadherin was further confirmed at the protein level (Fig. 5B).
In addition, the sphere assay further confirmed that coculture with THP-1 cells enhanced the stemness of PDAC cells (Fig. 5C). We reasoned that direct contact of monocytes/macrophages with CD90hi cells might allow monocytes/macrophages to provide near-signals, which are cru-cial for PDAC cells to maintain their stemness. To test this hypothesis, PDAC cells were treated with conditioned medium from THP-1 mono-cytes. Here, we found that stemness-related genes, especially SOX2, were upregulated in PDAC cells (Fig. S4A). In addition, conditioned medium from THP-1 cells had a comparable ability to promote the stemness of PDAC cells according to sphere formation and ALDH ac-tivity (Fig. 5D and E). By analyzing medium from THP-1 cells, we no-ticed that IL8 was the most abundant cytokine (Fig. S4B). Moreover, PDAC cells could promote monocytes to express even more IL8, as shown in Fig. 4. Consistent with previous reports [32–34], we found that IL8 could enhance the stemness of PDAC cells, as confirmed by stemness-related gene expression, sphere formation and ALDH activity (Figs. S4C–E). To further determine whether THP-1 cell-promoted PDAC stemness was mediated by IL8, we blocked IL8 with its neu-tralizing antibody. Notably, ALDH activity enhanced by THP-1 CM in PDAC cells was largely abrogated after neutralization with the IL8 an-tibody (Fig. 5F).
As shown in Fig. 5A, the crosstalk between THP-1 monocytes and PDAC cells also enhanced the expression of EMT-related genes. Con-sistent with the qPCR assay, PDAC cells cocultured with THP-1 cells gained higher migrative and invasive capabilities (Fig. 5G). SHH, a li-gand of sonic hedgehog signaling, was dramatically induced in PDAC-primed THP-1 monocytes (THP-1 monocytes cocultured with PDAC) (Fig. 4). Consistent with previous reports [35,36], we found that SHH could promote PDAC migration (Fig. 5H). To further explore the in-volvement of SHH/Gli signaling in the crosstalk between PDAC cells and THP-1 monocytes, SW1990 cells cocultured with THP-1 monocytes were treated with DMSO or GANT61 (a specific inhibitor of Gli1/2). After coculture for 36 h, SW1990 cells from the DMSO/GANT61-treated groups were sorted for the Transwell migration assay. Notably, blocking SHH/Gli signaling with GANT61 could impair the migration of SW1990 cells, suggesting that SHH is involved in the crosstalk between r> Fig. 2. CD90hi PDAC cells harbor stemness properties. (A) GSEA showed that CD90hi cells were positively enriched for stem cells versus CD90low cells in TCGA (PAAD) database. (B) PANC1 and SW1990 cells were grown as monolayers and spheres. qPCR analysis of stemness-associated genes and CD90 expression in spheres versus adherent cells. Data were normalized to GAPDH expression and are presented as the fold change relative to adherent cells. (C) ALDEFLUOR analyses of CD90hi and CD90− cells from PANC1 cells. DEAB, a specific inhibitor of ALDH1, was used as a control. (D) CD90hi and CD90− cells were sorted with flow cytometry. The relative expression of CD90 and stemness-associated genes was determined by qPCR. Data are shown as the mean ± SEM, n = 3, *P < 0.05 (multiple unpaired Student's t-test). (E) CD90hi and CD90− cell subpopulations from PANC1 and SW1990 cells were sorted for sphere assay and colony formation. The mean number of spheres and proliferation between two groups are shown. Scale bar: 50 μm. (F) CD90hi and CD90− cells (103 each) sorted from PANC1 cells were injected sub-cutaneously into two sides of BALB/c nude mice. After 2 months, CD90hi cells, but not CD90− cells, formed tumor nodules. Arrows indicate the sites of injection (n = 10 per group). All data are shown as the mean ± SEM from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test).