Constitutive activation of STAT3 in myeloma cells

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 cancers, how 3D culture affects multiple myeloma (MM) cells 19 is not well understood. Here, we compared MM cells (U266 and RPMI8226) in a 3D culture model 20 with those in conventional culture. While the conventionally cultured cells were present in single 21 cells or small clusters, MM-3D cells grew in large spheroids. We discovered that STAT3 was the 22 pathway that was more activated in 3D in both cell lines. The active form of STAT3 (pSTAT3), being 23 absent in MM cells cultured conventionally, became detectable after 1-2 days in 3D culture. This 24 elevated pSTAT3 level was dependent on the 3D environment, since it disappeared after 25 transferring to conventional culture. STAT3 inhibition using a pharmacological agent, Stattic, 26 significantly decreased the cell viability of MM cells and sensitized them to bortezomib in 3D 27 culture. Using an oligonucleotide array, we found that 3D culture significantly increased the 28 expression of several known STAT3 downstream genes implicated in oncogenesis. Since most 29 primary MM tumors are naturally STAT3-active, studies of MM in the 3D culture can generate 30 results that are more representative of the disease. 31