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

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 discriminate these signals. We found that proliferation of stromal cells is differentially stimulated by different tumour cells and drug treatments. Additionally, the spatial organisation of stromal cells is also specifically altered depending on the type of tumour. These results suggest that tumour cells generate distinct signals that can affect stromal cells specifically resulting in explicit therapeutic requirements to prevent the support of the tumour microenvironment. We also found that when MM cells were seeded at low densitiy, stromal cells promote MM cell proliferation and resistance to therapeutic agents. However, when MM cells were plated at high densities and can easily enter exponential growth, tumour cells can grow independently of the presence of the stroma. However both at high and low densities, stromal cells abrogated either partially or completely the pro-apoptotic effect of therapeutic drugs. The use of eGFP-MM and mCherry HS5 cells enables application of laboratory techniques to effectively distinguish and screen the effects of drugs on the biology of myeloma cells and stromal cells in a co-culture setting that reproduces more accurately some key aspects of the tumour microenvironment. We propose the use of this experimental platform to evaluate therapeutic drugs against MM as well as other malignancies.