Novel In Vitro Experimental Platform for High Throughput Analysis of the Effect of Drugs on Multiple Myeloma Cells and the Tumour Microenvironment In a Co-Culture Setting

• Published in: Blood 2010-11, Vol.116 (21), p.982-982
• Main Authors: Ramasamy, Karthik, Khatun, Hazera, Macpherson, Lee, Schey, Stephen, Calle, Yolanda
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V116.21.982.982

Abstract 982 Mounting evidence on the role of tumour microenvironment in supporting the growth and survival of multiple myeloma (MM) and other tumour cells makes testing of potential drug treatments in vitro in this setting almost obligatory. It is becoming evident that effective therapeutic approaches against MM must target not only MM cell viability but also the pro-survival support of the tumour cellular and non-cellular stroma. Recently developed strategies recreate the myeloma tumour microenvironment in vitro allowing for detection of myeloma cell proliferation or distribution in bone marrow compartments using cell imaging. However, there is a need for high troughput self-contained co-culture technology to facilitate differentiation in the behaviour of myeloma plasma cells from the accessory cells in the tumour microenvironment. Herein, we validate a high throughput in vitro co-culture experimental platform to analyse and measure simultaneously the effect of therapeutic agents on MM cells and the tumour stroma in co-culture. We have generated eGFP-MM cell lines (eGFP-MM1.S, eGFP-MM1.R, eGFP-U266) by lentiviral infection and clonal selection by limiting dilution and validated that eGFP-expressing cells maintain the properties of the parental cell lines. The same methodology was used to generate eGFP-K562 (myeloid malignancies), eGFP- PC3, eGFP-DU145 (both prostate cancer cell lines) and eGFP-HT29 (colon cancer cell line). We show that growth of eGFP-expressing cells can be estimated using fluorimetry (lexcitation 395/475; l emission 509) or image-based analysis in the presence of other BM cells in co-culture. The use of eGFP-MM cells also allows flow cytometry analysis of cell cycle profile and apoptosis with no significant cellular contamination from co-cultured cells such as fibroblasts, osteoclasts or stromal cells derived from bone marrow aspirates of MM patients. Additionally, we have generated and validated mCherry-expressing HS5 fibroblast cell lines as HS5 cells are commonly used to study the support by fibroblasts of proliferation and viability of MM cells. Proliferation of mCherry-HS5 cells can be evaluated using fluorimetry (l excitation 584; l emission 607) or image-based analysis in the presence of various tumour cells of haematological origin and solid tumours. The disadvantage of the MTT assay is that it is impossible to distinguish the signal of specific cell types in co-culture whereas the use of fluorescent cell lines allows us to dis