br To further verify the expression of CD on
To further verify the expression of CD90 on PDAC cells, we ex-amined its surface level with flow cytometry on cell lines, PDCs (PDX-derived cancer cells, generated from patient-derived xenografts) and primary Y 27632 (freshly isolated from patients’ tumors). Flow cytometry analysis revealed that CD90 expression was limited to a small subset of PDAC cells, which are referred to as CD90hi PDAC cells (Fig. 1D, Figure S1 A-B). The above data confirmed the expression of CD90 in pancreatic tumor cells.
Next, we sought to explore which signaling pathway CD90 might be involved in, and we performed GSEA using TCGA (PAAD) database. We grouped patients according to the median mRNA expression of CD90, in which the top 80% was defined as CD90hi and the bottom 20% was defined as CD90low. The results revealed that the pathways associated with PDAC malignance/aggressiveness were positively enriched in Cancer Letters 453 (2019) 158–169
CD90hi cells (versus control CD90low cells), including Kras signaling, epithelial mesenchymal transition (EMT), IL-6/JAK/STAT3 signaling and TNFα signaling via NFκB (Fig. 1E). Moreover, the high expression of CD90 was significantly associated with poor survival in TCGA (PAAD) database (Fig. 1F). The above data suggest that increased CD90 expression may be an important predictor of poor survival in pancreatic cancer.
To further elucidate which signaling pathway might participate in the regulation of CD90 levels, we treated PANC1 cells with inhibitors specific to Wnt, Notch, Hedgehog (Gli), Hippo (YAP1), STAT3 and mTOR signaling. Here, we found that only Gli1 and YAP1 inhibitors could partially reduce CD90 expression at both the protein and mRNA levels (Figs. S1C–D). Furthermore, CD90 expression was positively correlated with the expression of Gli1 and YAP1 in TCGA (PAAD) da-tabase (Fig. S1E), suggesting that the Sonic Hedgehog and Hippo-YAP pathways might participate in the regulation of CD90 expression in PDAC.
3.2. CD90hi PDAC cells harbor stemness properties
Previous studies have shown that CD90 is a surface marker that can be used to define cancer stem cells in various tumors [16–20]. How-ever, the role of CD90 in PDAC is still not well defined. To annotate the function of CD90 in PDAC, we first performed gene set enrichment analysis (GSEA) with the PDAC database by dividing PDAC patients into 2 groups according to the median expression of CD90 (CD90hi vs CD90low). Compared with CD90low, CD90hi PDAC showed a significant enrichment of genes related to mammary stem cells (Fig. 2A). More-over, to determine whether high expression of CD90 was associated with stemness in PDAC, pancreatic cancer stem cells (CSCs) were en-riched in nonadherent, serum-free, growth factor-supplemented con-ditions in vitro as previously reported [24,25]. We observed that CD90, along with other multiple stemness-related genes (NANOG, SOX2 and POU5F1), especially SOX2, was significantly higher in spheres com-pared to adherent monolayer cells (Fig. 2B, Fig. S2A), suggesting that CD90 is associated with cancer stemness in PDAC. In addition, the ex-pression of CD90 was positively correlated with SOX2 expression in the Renji PDAC cohort (Fig. S2B).
Aldehyde dehydrogenase A1 (ALDHA1) is an operative and well-recognized stemness marker. We found that CD90hi cells exhibited higher ALDH activity than CD90− cells in multiple PDAC cell lines (Fig. 2C and Fig. S2C). To further discern these two populations in PDAC, we used flow cytometry to purify PDAC cells based on CD90 expression. Quantitative RT-PCR analysis revealed that the expression levels of core stemness genes (NANOG, SOX2 and POU5F1) were higher in CD90hi cells than in CD90− cells (Fig. 2D, Fig. S2D). Moreover, CD90hi cells had higher clonogenic capability than the CD90− popu-lation according to the sphere assay (Fig. 2E, Fig. S2E). However, the growth rate of CD90hi cells was comparable to that of CD90− cells, indicating that the observed diﬀerences were not related to the changes in proliferation rate (Fig. 2E). In addition, CD90hi cells possessed stronger resistance to gemcitabine in vitro (Fig. S2F). To determine the in vivo tumorigenic potential of the two subpopulations, 103 CD90hi and CD90− cells sorted from PANC1 cells were subcutaneously transplanted into immunodeficient mice. Eight weeks after injection, we found that CD90hi cells possessed higher tumorigenicity (Fig. 2F). Thus, the CD90hi population widely existed in PDAC cells and displayed stemness prop-erties.
3.3. CD90 acts as an anchor for monocyte/macrophage cell adhesion in PDAC cells
CD90 is a GPI-anchored glycoprotein that has been shown to in-teract with integrins displayed by adjacent cells, such as the monocyte/ macrophage surface marker CD11b/MAC-1 (integrin alpha M/beta) . CD90, expressed on breast cancer stem cells, was shown to