Abstract
Cancer stem cells (CSCs), a small subset of the tumor bulk with highly malignant properties, are deemed responsible for tumor initiation, growth, metastasis, and relapse. In order to reveal molecular markers and determinants of their tumor-initiating properties, we enriched rare stem-like pancreatic tumor-initiating cells (TICs) by harnessing their clonogenic growth capacity in three-dimensional multicellular spheroid cultures. We compared pancreatic TICs isolated from three-dimensional tumor spheroid cultures with nontumor-initiating cells (non-TICs) enriched in planar cultures. Employing differential proteomics (PTX), we identified more than 400 proteins with significantly different expression in pancreatic TICs and the non-TIC population. By combining the unbiased PTX with mRNA expression analysis and literature-based predictions of pro-malignant functions, we nominated the two calcium-binding proteins S100A8 (MRP8) and S100A9 (MRP14) as well as galactin-3-binding protein LGALS3BP (MAC-2-BP) as putative determinants of pancreatic TICs. In silico pathway analysis followed by candidate-based RNA interference mediated loss-of-function analysis revealed a critical role of S100A8, S100A9, and LGALS3BP as molecular determinants of TIC proliferation, migration, and in vivo tumor growth. Our study highlights the power of combining unbiased proteomics with focused gene expression and functional analyses for the identification of novel key regulators of TICs, an approach that warrants further application to identify proteins and pathways amenable to drug targeting.
Highlights
Cancer represents a broad set of heterogeneous malignant diseases driven by distinct genetic and epigenetic alterations, where combinations of driver mutations and driver signals determine over time and space the malignant phenotype characterized by largely unrestricted proliferative capacity.Studies have shown that tumor initiation and growth are not stochastic events but processes controlled by a hierarchical organization within the heterogeneous tumor mass: malignant development and growth are fueled by a rare subpopulation of tumor-initiating cancer cells, frequently referred to as cancer stem cells (CSCs), with the ability of self-renewal and differentiation into the diverse cells that comprise the heterogeneous tumor bulk [1,2,3,4,5,6]
In order to identify candidate proteins involved in the determination of the malignant phenotype of pancreatic tumor-initiating cells (TICs), we applied an unbiased differential proteomics approach followed by candidate-based gene-expression analysis and functional studies
We employed an unbiased enrichment method for pancreatic TICs followed by a quantitative proteomics approach comparing TICs to non-TICs in order to identify enriched proteins
Summary
Cancer represents a broad set of heterogeneous malignant diseases driven by distinct genetic and epigenetic alterations, where combinations of driver mutations and driver signals determine over time and space the malignant phenotype characterized by largely unrestricted proliferative capacity.Studies have shown that tumor initiation and growth are not stochastic events but processes controlled by a hierarchical organization within the heterogeneous tumor mass: malignant development and growth are fueled by a rare subpopulation of tumor-initiating cancer cells, frequently referred to as cancer stem cells (CSCs), with the ability of self-renewal and differentiation into the diverse cells that comprise the heterogeneous tumor bulk [1,2,3,4,5,6]. Identifying the molecular players controlling the malignant properties of CSCs is critical for the development of innovative multimodal treatments that include targeting of the tumor-initiating cancer cell population or its surrounding microenvironment [9]. In order to study molecular properties of cancer stemness, CSCs have been characterized in a multitude of cancer models and cancer cell lines employing transplantation, lineage tracing, or cell ablation assays [10,11,12]. The size of the CSC population in a tumor or a specific cell line can vary significantly [14], the low abundance of CSCs in the majority of tumors makes their characterization using established high-throughput techniques challenging and frequently requires the application of error-prone amplification and/or selective enrichment before investigations at a molecular level can be performed
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