We have previously demonstrated that neuroblastoma cells increase the expression of interleukin-6 by bone marrow stromal cells and that stimulation does not require cell-cell contact. In this study we report the purification and identification of a protein secreted by neuroblastoma cells that stimulates interleukin-6 production by stromal cells. Using a series of chromatographic purification steps including heparin-affinity, ion exchange, and molecular sieve chromatography followed by trypsin digestion and liquid chromatography tandem mass spectrometry, we identified in serum-free conditioned medium of neuroblastoma cells several secreted peptides including galectin-3-binding protein, also known as 90-kDa Mac-2-binding protein. We demonstrated the presence of the galectin-3-binding protein in the conditioned medium of several neuroblastoma cell lines and in chromatographic fractions with interleukin-6 stimulatory activity. Consistently, bone marrow stromal cells express galectin-3, the receptor for galectin-3-binding protein. Supporting a role for galectin-3-binding protein in stimulating interleukin-6 expression in bone marrow stromal cells, we observed that recombinant galectin-3-binding protein stimulated interleukin-6 expression in these cells and that interleukin-6 stimulation by neuroblastoma-conditioned medium was inhibited in the presence of lactose or a neutralizing anti-galectin-3 antibody. Down-regulation of galectin-3-binding protein expression in neuroblastoma cells also decreased the interleukin-6 stimulatory activity of the conditioned medium on bone marrow stromal cells. We also provide evidence that stimulation of interleukin-6 by galectin-3-binding protein involves activation of the Erk1/2 pathway. The data, thus, identifies galectin-3-binding protein as a factor secreted by neuroblastoma cells that stimulates the expression of interleukin-6 in bone marrow stromal cells and provides a novel function for this protein in cancer progression.It has become increasingly evident that the tumor microenvironment plays an important contributory role to cancer progression (1). Tumor cells closely interact with non-malignant cells present in the tumor stoma through a series of adhesiondependent and adhesion-independent mechanisms. Among the adhesion-independent interactions, cytokines, chemokines, and growth factors play a critical role (2, 3). In this regard, the bone marrow, which is a frequent site of metastasis, provides a microenvironment that is particularly favorable to cancer progression because it is a source of hematopoietic and mesenchymal cells that produce a large number of cytokines and chemokines (4 -6).Neuroblastoma is a neural crest-derived cancer that is the second most common solid tumor in children (7). At the time it is diagnosed, it has metastasized in 56% of the cases, primarily to the bone marrow, the bone, and the liver (8). The mechanisms by which neuroblastoma cells invade the bone have been recently elucidated by several laboratories, including our group (9 -11). We have previou...