Diffuse Large B-Cell Lymphoma Cells Promote Paracrine Activation of NF-kB Pathway in Tumor-Associated Stromal Cells
Vasiliki Leventaki, Estelle Bourbon, Changju Qu, Yadong Liu, Kranthi Kunkalla, Martin Nguyen, Carlos Bueso-Ramos, Rajesh R Singh, Nitin K Agarwal, Francisco Vega. UT MD Anderson Cancer Center, Houston, TX
Background: Interactions between tumor cells and the stromal microenvironment are increasingly implicated as relevant components of tumor biology. Previously, we found that co-culturing diffuse large B-cell lymphoma (DLBCL) cell lines with human bone marrow stromal cells (HS-5) resulted in decreased chemosensitivity to doxorubicin and methotrexate that was associated to marked increased expression levels of the drug transporters ABCG2, MDR1 and ABCC1 as well as of the expression levels of BCL2, BCL2A1 and BCL-xL and activation of hedgehog (Hh) and NF-kB pathways in lymphoma cells. Here, we explore the activation status of NF-kB pathway in tumor associated stromal cells in DLBCL in vitro and in tumor samples.
Design: 5 DLBCL cell lines, activated cell type (LP & HBL1) and germinal center (GC) type (BJAB, DOHH2 & SUDHL4) and the human stroma cell line HS-5 were used. Co-culture experiments were performed for 48h in trans-well experiments (not direct cell-to-cell contact was allowed). The activation status of the NF-kB pathway in HS-5 cells was evaluated by western blot measuring phosphorylation levels of ser536p-P65 and total levels of IkBa and confirmed using protein nuclear extracts and DNA binding ELISA assays (P65, Rel, c-Rel, P52 and P50). Single and/or double immunohistochemical studies using antibodies P52, p-P65 and CD68 were performed in a series of 20 DLBCL tumors.
Results: Co-culturing HS-5 cells with DLBCL cell lines resulted in an increased activation of NF-kB pathway in HS-5 cells as indicated by increased levels of ser536p-p65 and downregulation of IkB alpha. The increased activation status of the canonical and non-canonical NF-kB pathway in the co-cultured stromal cells was confirmed by the statistically significant increase of nuclear binding of P65, P52 and P50 when compared with HS-5 cells alone. Immunohistochemical studies also showed activation of canonical and non-canonical NF-kB pathway in tumor associated accessory cells (macrophages and in a subset of stromal spindle cells) in tumor samples. This was confirmed with double immunostains for p-P65 and CD68.
Conclusions: DLBCL cells initiate a reciprocal paracrine crosstalk with the surrounding accessory cells. This crosstalk results in the activation of canonical and non-canonical NF-kB pathway not only in the tumor cells but also in the stromal cells. To understand mechanisms involved in this double lymphoma-stroma cross-talk and their consequences will help to further understand the pathobiology of this neoplasm.
Tuesday, March 20, 2012 1:00 PM
Poster Session IV # 201, Tuesday Afternoon