Constitutive activation of STAT 3 in myeloma cells 2 cultured in a three-dimensional , reconstructed bone 3 marrow model 4

Malignant cells cultured in three-dimensional (3D) models have been found to be 17 phenotypically and biochemically different from their counterparts cultured conventionally. Since 18 most of these studies employed solid tumor types, how 3D culture affects multiple myeloma (MM) 19 cells is not well understood. Here, we compared MM cells (U266 and RPMI8226) in a 3D culture 20 model with those in conventional culture. While the conventionally cultured cells were present in 21 single cells or small clusters, MM-3D cells grew in large spheroids. We discovered that STAT3 was 22 the pathway that was more activated in 3D in both cell lines. The active form of STAT3 (phospho23 STAT3 or pSTAT3), which was absent in MM cells cultured conventionally, became detectable after 24 1-2 days in 3D culture. This elevated pSTAT3 level was dependent on the 3D environment, since it 25 disappeared after transferring to conventional culture. STAT3 inhibition using a pharmacological 26 agent, Stattic, significantly decreased the cell viability of MM cells and sensitized them to 27 bortezomib in 3D culture. Using an oligonucleotide array, we found that 3D culture significantly 28 increased the expression of several known STAT3 downstream genes implicated in oncogenesis. 29 Since most primary MM tumors are naturally STAT3-active, studies of MM in 3D culture can 30 generate results that are more representative of the disease. 31