Quantitative secretomics analysis of stem and non-stem cells of two different stages of colon cancer cell lines


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Jeyalakshmi Kandhavelu1,Kumar Subramanian1,Previn Naicker2,Stoyan Stochev2,Paul Ruff1,Clement Penny1
1University of the Witwatersrand,2Council for Scientific and Industrial Research

Abstract

Background

The cancer stem cell secretome is considered to be a source of many proteins including, cytokines, chemokines, and growth factors. Such factors may influence cell division and cell behavior both within the CSC niche and externally (to the niche), to promote angiogenesis and enhance cell migration, leading to metastasis.  Secretome analysis of CSCs may disclose unique mechanisms whereby these cells contribute to tumour relapse and lead to the development of novel approaches for targeting CSCs. Thus, here, the secretory protein profile associated with both CD133+ stem cells (CSCs) and CD133- (non- stem cells (nCSCs) of two human colon cancer cell lines was investigated.

Method

The CSC population was isolated from the HT29 and DLD1 colorectal adenocarcinoma cells using magnetically labeled CD133 antibody microbeads. Both the CSC and nCSCs population from each cell line was cultured and secretome being harvested after 48 hrs. Mass spectrometry was used to compare secretome profiling in both cell lines, and SWATH-MS was used to perform quantitative analysis. The functional enrichment analysis of significantly changed proteins was accomplished using GO, Reactome and STRING analysis.

Results

A total of 2679 and 480 proteins was identified from the secretome of HT29-CSCs and DLD1-CSCs, respectively. From the nCSCs, 1775 and 2758 proteins were identified from HT29-nCSCs and DLD1-nCSCs, respectively. Quantitative proteomic analysis revealed that 339 and 180 proteins were upregulated in HT29 CSCs and DLD1CSCs, respectively, when compared to the respective nCSCs. Target P analysis confirmed that most of the upregulated proteins were associated with classical secretory pathways. Further, a REACTOME analysis identified several pathways linked to metabolism and Immune system in CSCs. In particular, the neutrophil degranulation and nonsense-mediated decay (NMD) surveillance pathway were significantly enriched in the CSCs populations.

Conclusion

These results provide novel insight into the metastatic mechanisms of CSCs population of HT29 and DLD1 cells