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Introduction: The cellular microenvironment plays a key role in the pathogenesis of B-cell malignancies (Amé-Thomas, Blood 2007; Podar, Leukemia 2009). Bone marrow stromal cell interactions can provide survival signals to CLL cells (Kurtova, Blood 2009), and stromal cell gene signatures and markers can predict survival outcomes and add prognostic value, respectively, for patients with DLBCL (Lenz, NEJM 2008; Meyer, Am J Clin Pathol 2011). Ibrutinib (ibr), a first-in-class, oral, once-daily inhibitor of Bruton’s tyrosine kinase, has demonstrated single-agent activity in R/R DLBCL (Wilson, Nat Med 2015). In this study, we established an in vitro coculture system and report the effects of stromal cells on ABC-DLBCL cells and their sensitivity to ibr. Methods: The effect of ibr on cell growth was evaluated by luminescent cell viability assay. Flow cytometry was used to evaluate the effect of ibr on cell death in different cell populations. Live-cell imaging detected drug effects on apoptosis at different time points. Gene expression levels in B lymphoma cells were examined by quantitative PCR and NanoString panels. Human cytokines/chemokines were quantified using MILLIPLEX® MAP Kit. Results: Reduced ibr sensitivity was observed in TMD8 DLBCL cells cocultured with HS-5 bone marrow-derived stromal cells. HS-5 cells protected TMD8 cells from ibr-induced apoptosis. Treatment with ibr induced TNFSF10 expression only in TMD8 cells without HS-5 coculture, providing a survival mechanism for escape of ibr-induced cell death. In addition, coculturing with HS-5 decreased surface expression of CD20 but not CD19. Several cytokines/chemokines showed differential expression in the coculture system (increased CCL22 and IL-10; decreased CCL4 and TNFβ). Reduced expression of BCR signaling-related miR-155 was observed in ABC-DLBCL and CLL cell lines with HS-5 coculture, suggesting downregulation of BCR signaling in these cells. The adhesion molecule, SELL, showed decreased expression in the ABC-DLBCL cells cultured with HS-5 as well as in ibr-resistant TMD8 cells, consistent with our observation that ABC-DLBCL patients with poor responses (PD+SD) to ibr have lower SELL expression in their tumor samples. We further assessed expression levels of amino acid metabolism-related genes and demonstrated significant increases of ASS1 and TDO2 in the TMD8 cells cocultured with HS-5. Expression levels of amino acid metabolism-related genes varied across patients with different responses to ibr, further suggesting the importance of amino acid levels in the tumor microenvironment and ibr response. Conclusions: The changes in ABC-DLBCL cells cocultured with stromal cells identified here may provide new insights into cellular cross-talk in the microenvironment. Further investigation into cell-cell interactions and ibr may help stratify patient populations and provide new therapeutic strategies. Citation Format: Hsu-Ping Kuo, Sidney Hsieh, Mint Sirisawad, Chun-Te Chen, Leo W. Cheung, Karl J. Schweighofer, Chia-Lin Hsu, Chi-Ling Fu, Jing Liu, Shiquan Wu, Karl Eckert, Hugh Wang, Mutiah Apatira, Kamaldeep Dhami, Kevin Kwei, Jeff Hsu, Betty Y. Chang. Interaction of B lymphoma cells with the microenvironment affects ibrutinib sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4197. doi:10.1158/1538-7445.AM2017-4197