Human pancreatic cancer contains a side population expressing cancer stem cell-associated and prognostic genes

Abstract

In many types of cancers, a side population (SP) has been identified based on high efflux capacity, thereby enriching for chemoresistant cells as well as for candidate cancer stem cells (CSC). Here, we explored whether human pancreatic ductal adenocarcinoma (PDAC) contains a SP, and whether its gene expression profile is associated with chemoresistance, CSC and prognosis. After dispersion into single cells and incubation with Hoechst dye, we analyzed human PDAC resections specimens using flow cytometry (FACS). We identified a SP and main population (MP) in all human PDAC resection specimens (n = 52) analyzed, but detected immune (CD45(+)) and endothelial (CD31(+)) cells in this fraction together with tumor cells. The SP and MP cells, or more purified fractions depleted from CD31(+)/CD45(+) cells (pSP and pMP), were sorted by FACS and subjected to whole-genome expression analysis. This revealed upregulation of genes associated with therapy resistance and of markers identified before in putative pancreatic CSC. pSP gene signatures of 32 or 10 up- or downregulated genes were developed and tested for discriminatory competence between pSP and pMP in different sets of PDAC samples. The prognostic value of the pSP genes was validated in a large independent series of PDAC patients (n = 78) using nCounter analysis of expression (in tumor versus surrounding pancreatic tissue) and Cox regression for disease-free and overall survival. Of these genes, expression levels of ABCB1 and CXCR4 were correlated with worse patient survival. Thus, our study for the first time demonstrates that human PDAC contains a SP. This tumor subpopulation may represent a valuable therapeutic target given its chemoresistance- and CSC-associated gene expression characteristics with potential prognostic value.

Figure 1. Identification and molecular characterization of the human PDAC SP.

A) Dot plot of dual-wavelength FACS analysis of fresh human PDAC cells after incubation with Hoechst33342 depicting a tail of ‘Hoechst-low’ cells (SP), relative to a larger bulk of ‘Hoechst-bright’ cells, the main population (MP) (left panel). A representative example is shown and the SP proportion indicated. PI^pos (dead) cells were excluded from analysis for Hoechst33342, CD45 and CD31 labelling. The boxplot (inset) summarizes the SP proportions of the 52 PDAC samples analyzed. Verapamil blocks Hoechst efflux, thereby reducing the SP size and confirming the SP phenotype (right panel). B) FACS dot plot of the PDAC SP, immunostained for CD45 and CD31. A representative example is shown. Numbers indicate the proportions of CD45⁺, CD31⁺ and CD45^−/CD31⁻ cells within the SP. C) Principal Component Analysis (PCA) of whole genome expression profiles obtained from CD45^−/CD31⁻ SP (pSP, red) and CD45^−/CD31⁻ MP (pMP, blue) (n = 11). D) Immunohistochemical staining of ABCB1 in PDAC resection samples (n = 11). A representative example (4% SP in FACS analysis) is shown. Original magnification: x200 (left) and x400 (right).