Nd analyzed for SP. The tumour SP proportion was larger in mice treated with gemcitabine than in the corresponding controls (median 6.6 versus 2.7 , respectively; n = 7; p = 0.028; Figure 2B-C). SP enrichment was even higher when only considering the tumours that responded to gemcitabine with tumour shrinkage (of at least 50 ) (median: 4.7 versus 1.3 , respectively; n = 4; Figure 2B). CD45+ andVan den broeck et al. BMC Cancer 2012, 12:354 http://www.biomedcentral.com/1471-2407/12/Page 5 ofABCprobes were used, thereby excluding the detection of mouse transcripts (such as from the infiltrating CD31+ and CD45+ SP cells). Comparison revealed that 145 probe sets, representing 121 genes, were differentially expressed between the SP and MP (p < 0.001); 80 genes were upregulated in the SP and 41 genes downregulated (complete list in Additional file 1: Table S1; extract of genes PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10663624 in Table 2, selected on the basis of relevance from the literature and from DAVID analysis as below). Gene-clustering analysis of all differentially expressed genes (p < 0.001) by DAVID showed 3 functionally related groups of genes enriched in the SP: one group of transcription factors, one of adhesion molecules and one of homeobox genes (Table 3). KEGG pathway analysis revealed 3 significantly upregulated pathways in the SP versus the MP, including `cancer' and `adherens junctions' (Table 3). Two KEGG pathways were significantly downregulated in the SP, including `cell adhesion molecules'. Visualization of the interaction network of SP-upregulated genes by STRING analysis (Figure 3) reveals that genes involved in chemoresistance [ETS1, KIT ligand (KITLG) or stem cell factor (SCF), SNAI2], regulation of apoptosis (FASLG, GRB10, BCL2L11, ETS1, SNAI2), epithelial-mesenchymal transition (EMT) (SNAI2, LEF1) and tumourigenesis (oncogenes like FGF7, GATA1, KITLG, ETS1) occupy a central position. Moreover, multidrug transporters, linked to chemoresistance and some considered responsible for the SP phenotype, also show a clear tendency of upregulation in the SP (ABCG2, 3.63 fold, p = 0.006; ABCA9, 3.66-fold, p = 0.003).The SP is enriched in sphere-forming PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22047565 cellsFigure 2 The SP in human PDAC is resistant to gemcitabine as analyzed in vivo. (A) SCID mice with corresponding PDAC xenograft tumours treated with vehicle (control or Co, left) or gemcitabine (GEM, right). Tumours measured 276 mm3 and 266 mm3 M617 in the control mouse and 144 mm3 and 123 mm3 in the GEM-treated mouse, respectively. (B) Overview of the data, indicating PDAC xenograft number, percent change in tumour volume after treatment with vehicle (Co) or GEM (response to GEM when >50 reduction), and proportion of SP in the tumours after treatment. (C) Boxplot of SP proportion in the PDAC xenografts of the vehicle-treated control mice (Co) and the mice treated with GEM (n = 7). *, p < 0.05.CD31+ cells did not rise in the SP in response to gemcitabine (p = 0.21 and p = 0.66, respectively) when compared to control mice.Whole-genome expression analysis of the PDAC xenograft SP reveals upregulation of genes related to therapy resistanceTumourigenic (CSC) activity was analyzed in vitro using the sphere-forming assay [22]. SP and MP from xenograft tumours were first depleted from the (murine) endothelial and immune cells by FACS and then seeded in defined culture conditions (see Methods). Viability of the sorted SP and MP cells was identical (data not shown). The CD45-/CD31- SP generated spheres in all experiments (n = 6; media.