Co-culture of functionally enriched cancer stem-like cells and cancer-associated fibroblasts for single-cell whole transcriptome analysis

Integr Biol (Camb). 2019 Dec 31;11(9):353-361. doi: 10.1093/intbio/zyz029.


Considerable evidence suggests that breast cancer development and metastasis are driven by cancer stem-like cells (CSCs). Due to their unique role in tumor initiation, the interaction between CSCs and stromal cells is especially critical. In this work, we developed a platform to reliably isolate single cells in suspension and grow single-cell-derived spheres for functional enrichment of CSCs. The platform also allows adherent culture of stromal cells for cancer-stromal interaction. As a proof of concept, we grew SUM149 breast cancer cells and successfully formed single-cell-derived spheres. Cancer-associated fibroblasts (CAFs) as stromal cells were found to significantly enhance the formation and growth of cancer spheres, indicating elevated tumor-initiation potential. After on-chip culture for 14 days, we retrieved single-cell derived spheres with and without CAF co-culture for single-cell transcriptome sequencing. Whole transcriptome analysis highlights that CAF co-culture can boost cancer stemness especially ALDHhigh CSCs and alter epithelial/mesenchymal status. Single-cell resolution allows identification of individual CSCs and investigation of cancer cellular heterogeneity. Incorporating whole transcriptome sequencing data with public patient database, we discovered novel genes associated with cancer-CAF interaction and critical to patient survival. The preliminary works demonstrated a reliable platform for enrichment of CSCs and studies of cancer-stromal interaction.

Keywords: RNA-Seq; cancer-associated fibroblast; cell co-culture; single cell; tumor sphere.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Breast Neoplasms / pathology*
  • Cancer-Associated Fibroblasts / cytology*
  • Cell Line, Tumor
  • Coculture Techniques / methods*
  • Dimethylpolysiloxanes / chemistry
  • Epithelial-Mesenchymal Transition
  • Female
  • Humans
  • Lab-On-A-Chip Devices
  • Neoplastic Stem Cells / cytology*
  • RNA-Seq
  • Transcriptome*


  • Dimethylpolysiloxanes
  • baysilon