SILAC Analysis of Two Bone Marrow Stromal Cell Lines

Within the bone marrow, stromal cells help support hematopoietic cells and inflammatory processes. Two major types of stromal cells related to early cancer development, cell lines HS5 and HS27a, are helpful in studying tumor cell growth and stromal interactions by deciphering crosstalk between stromal cells and clonal cells. According to Li et al., HS5 secretes multiple cytokines that support proliferation of committed progenitor cells, and HS27a supports “cobblestone area” formation by early hematopoietic progenitor cells.¹

Earlier investigations have led to the characterization of over 17,000 genes in these cell lines; however, researchers have not yet performed whole-proteome analyses. To meet this need, Li et al. used stable isotope labeling with amino acids in cell culture (SILAC) to analyze proteomic differences in HS5 and HS27a cells.

The researchers grew cells in culture, using two stable isotopes each of arginine and lysine to label proteins in the two cell lines. They then analyzed the labeled proteins using 2-D ultra-high-resolution liquid chromatography and an LTQ Orbitrap mass spectrometer (Thermo Scientific). They used MaxQuant to analyze the raw data, with a false discovery rate of 0.01 for proteins and peptides with a minimum peptide length of six amino acids. They used Swiss-Prot and the Database for Annotation, Visualization and Integrated Discovery (DAVID) to classify proteins according to their biological function.

The researchers identified a total of 4,213 unique proteins in the cell lines. Of these proteins, they detected 1,462 proteins in two independent experiments. Li and colleagues also found that 69 proteins had significant upregulation, and 48 showed significant downregulation (> 95% confidence) in HS27a relative to HS5.

Turning to gene ontology term and pathway analysis, the team determined that the differentially regulated proteins affected cellular movement, cell-to-cell signaling and interaction, and hematological system development and function. They identified a total of 55 items in both genomic and proteomic databases.

For further confirmation, the researchers used quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting on seven proteins randomly selected from 28 differentially expressed proteins. These proteins are also in open access databases, and confirmed in the top networks/pathways. The proteins included HCLS1, COL1A2, ALCAM, MAP4, HSP90 and CSE1L. The team noted higher mRNA and protein levels of HCLS1, COL1A2 and ALCAM in HS27a, whereas they saw lower mRNA and protein levels of MAP4, HSP90 and CSE1L in HS27a relative to HS5. They concluded that these findings were consistent with proteomic and genomic expression data.

In the case of another protein, integrin alpha-V (ITGAV), they saw lower gene expression in HS27a relative to HS5. However, in the proteomic database, they found increased protein levels of HS27a. Validation by RT-PCR and Western blotting also confirmed higher ITGAV levels in HS27a at both mRNA and protein levels. 

The team then inhibited the expression of ITGAV in HS27a cells by specific siRNAs. They observed a decrease in apoptosis in KG1a cells after co-cultured with modified HS27a cells under TNFɑ induction.The team further explained that these results indicate ITGAV plays a role in the co-culture system. Since this role is not understood, the team plans to specifically investigate ITGAV in connection with bone marrow stromal cells.

Li et al. maintain that this proteomic approach is a good starting place for future studies involving molecular mechanisms in the bone marrow microenvironment and crosstalk between stromal cells and myeloma cells in co-culture systems.

Reference
1. Li, X., et al. (2016) “Quantitative proteomic analysis and comparison of two bone marrow stromal cell lines using the SILAC method,” Experimental Hematology, 44(11) (pp. 1059–1071), doi: 10.1016/j.exphem.2016.08.002.