The experimental model. The main instrument used for the fulfillment of the mentioned objectives is the co-culture system between MM cells and BMSCs.BMSCs can be separated from hematopoietic cells due to their differencial capacity to adhere to plastic surfaces: heparinised BM specimens are integrally cultured in MEM medium, 750 ml flasks, non-adherent cell fraction is removed after one week, then the medium is replenished every 7 days until confluence is reached. After at least passages, cells are detached (Thrypsin-EDTA), phenotypically analysed for confirmation of their identity (CD73, CD90, CD105), and for their adhesion molecule pattern of expression (CD44, CD45, CD49d, CD49f, CD54, CD58) before and after co-culture with MM cells (MMCLs: L363, U266, RPMI8226 and CD138+ve primary MM cells from patients). 24 well plates will be used for performing co-cultures, with 1x10e5 MM cells/BMSC-coated well.

During the second year (2009) a new element will be introduced to the co-culture system: hypoxia. For this purpose, a O2 pressure-regulatory device will be acquired from the grant’s funds.

During the last two years of the project both systems (+/- hypoxia) will be used in parallel, for comparative studies in order to validate an improved experimental model for the in vitro study of MM cells within its tumor microenvironment.

In order to evaluate signaling pathways activated in MM cells following their interaction with BMSCs, we aim to explore the three documented cascades that promote tumor survival: PI3K/Akt, Ras/MAPK and JAK/STAT. Our intention is to investigate the level of two components (phosphorylated proteins) for each enzymatic cascade involved in anti-apoptotic transduction signal: phospho- (p-) p38, p-ERK1/2, p-JNK1/2, p-AKT, p-STAT1, p-STAT3. Two alternative methods will be used, both for checking the data reproducibility and for their complementary advantages: Western blot (the enzymatic method) and flow cytometry.

Based on accumulated data, in 2010, our team will be able to select key elements involved in anti-apoptotic signal transduction and to decide on the range of specific inhibitors to be used. Other flow cytometry-related methods will be used in order to measure cell viability (7-AAD), proliferation (CFSE), and apoptosis (anexibV/PI). Statistical analysis of data will be performed.