Students

Sim Jingwei, Quek Li Ling
Raffles Girls’ School (Secondary), Singapore
National University of Singapore-Science Mentorship Programmes (NUS-SMP) (4 Months)

Presentation and Awards

Best Poster Award, 10th Youth Science Conference, 2004, Singapore

Supervisor

Charles A Gullo, Research Scientist, Department of Clinical Research (DCR), SGH

Sponsor

Multiple Myeloma Research Laboratory, SingHealth

Summary

Interleukin-6 (IL-6) is a major and potent inducer of multiple myeloma (MM) cell proliferation. Increased IL-6 levels in MM patient sera is known to parallel disease progression.¹ In vitro, MM cell IL-6 production may be increased by CD40L activation.² The enzyme-linked immunosorbent assay (ELISA) assay can be used to quantify IL-6 production for MM research.^3,4 Previous attempts to define IL-6 production lacked specificity and sensitivity of cytokine detection. Furthermore, the in vivo bone marrow microenvironment where the MM cells thrive is recreated in vitro by co-culture conditions with bone marrow stromal cell line, AA101. Experiments were undertaken using two human-myeloma-derived cell lines (RPMI and SGH-MM5), varied cytokine dose responses, anti-cytokine receptors and a stromal-myeloma co-culture system. In order to improve the lower limits of detection and to optimise the kinetics of IL-6 detection, a 60ng/ml CD40 ligand (CD40L) dose at an 18h-stimulation time was found to be optimal. Furthermore, the addition of the anti-IL-6 receptor alpha (that blocks the autocrine absorption of IL-6) and a 2h co-culture condition with bone marrow stromal cells was ideal for detecting maximum analyte from supernatants. These conditions resulted in increased sensitivity of ELISA. This work will contribute to the development of potential clinical diagnostic tools using IL-6 as a surrogate marker. Of particular interest is the fact that this is the first time IL-6 detection directly from the MM cell is achieved.

Figure Legend

With supernatants obtained from the MM cell-AA101 co-culture system (fixed cell concentration of 2´ 10⁶  cells/ml and CD40L stimulation time of 18h), the following data was obtained. Following CD40 stimulation, both RPMI and MM5 showed a dose dependent increase in IL-6 production. Addition of an anti-IL-6 receptor antibody, which blocks the binding site of the IL-6 receptor and prevents re-absorption, enhanced the detection of IL-6 by ELISA. Thus, increasing cell numbers, time of stimulation, and finally blocking the IL-6R resulted in maximal detection of this very important MM growth promoting analyte.

Fig. 1. ELISA graph of CD40L concentration versus concentration of IL-6 production. RPMI and SGH-MM5 are both long term MM cell lines. The later was derived from a MM patient at the MMRL in SGH. AA101 is a bone marrow stromal cell line (kind gift from Dr Ken Anderson, Harvard Medical). The co-culture experiments mimic the in vivo bone marrow microenvironment by allowing for interaction of paracrine and autocrine growth factors as well as adhesion molecules in vitro.

Acknowledgements

We thank Dr Charles Gullo, Dr Gerrard Teoh and the staff of Singhealth, MMRL for their invaluable guidance. This work was supported by the Science Mentorship Program Grant (Ministry of Education) and DCR Research Grant #DCR/P07/2004.

References