Abstract A54: Isolation and characterization of cancer stem cells with peptides.
Authors: Zhu Yimin , Wang Anxin 
Published: August 2014

Background: Cancer as the most malignant disease still demands the more effective therapeutics methods although continuous efforts have been input to cancer therapies. Recent studies indicate that only a small subset of cancer cells termed cancer stem cells (CSCs) contributes to tumor progression, metastasis and drug-resistance. Drugs targeting to CSCs might become more effective approaches to cancer therapy. To date, isolation and characterization of CSCs have largely depended on surface markers which are not specific or well-recognized for individual tumor types. Our goal is to develop methods for isolating and characterizing a set of lung CSCs based on the specific binding peptides for CSCs without any recognized definitive specific surface markers.

Materials and Methods: Stem-like cells were enriched from non-small cell lung cancer (NSCLC) cell line H460 by sphere-forming assay. Characterized these stem-like cells by colony-forming assay, drug-resistance assay and soft agar assay. Screening and identification of specific binding peptides for CSCs were performed with bacteria surface display methods. The affinity between the specific binding peptides and CSCs was determined with fluorescence microscope and flow cytometric analysis. The specificity of peptides binding with CSCs was examined through comparing the binding capacity of peptides with either H460 cells or other cell lines including stem cells. The isolation of CSCs was performed using specific fluorescent peptides with FACS machine. The further characterization of selected CSCs including exploring the source and tumorigenesis mechanism of CSCs was performed through miRNA analysis.

Results: Stem-like cells divided and formed tumor spheres in a couple of weeks, while most of cancer cells died in the serum-free medium. These stem-like cells were able to grow indefinitely as tumor spheres in long-term culture (more than 20 passages). Meanwhile, sphere cells could form colonies in the soft agar plates and exhibit the drug resistance capacity, which indicated that these sphere cells behaved as CSCs. Three peptides binding specifically to those CSCs were identified with bacteria surface display method. They could seldom bind with other cell types including H460 cells, H1299 sphere cells, HLF cells, MSCs and ESCs. One selected peptides named HCBP-1 exhibited highly specific binding capacity to H460 CSCs. The apparent dissociation constant (KD) was 6.4 × 10-7 M with a flow cytometry-based assay. In the peptides binding subgroup of H460 cells, at least 10 miRNAs presented the different expression rate from the H460 cells.

Conclusions: In our study, a panel of specific binding peptides was identified with bacteria surface display method which could be used for isolation and characterization of CSCs without definitive CSCs markers. Furthermore, this new method could be potentially expanded to characterize and isolate CSCs in any types of cancer.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A54.

Citation Format: Yimin Zhu, Anxin Wang. Isolation and characterization of cancer stem cells with peptides. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A54.