Integrin specific hydrogels as lymphoid tumors organoids for B cell receptor signaling and drug resistance

摘要

Introduction: Non-Hodgkin lymphomas (NHL) are a heterogeneous group of lymphoproliferative disorders of B and T cell origin. Pre-clinical research in NHL has relied on testing compounds with suspension cultures of human lymphoma cell lines, without taking into account the lymphoid niche in which these tumors arise and reside. This traditional approach for drug testing risks losing potential useful compounds by considering them as ineffective due to the conditions of the assays. This is the case for compounds with a mechanism of action that relies on suppressing survival signals from microenvironmental cues, such as integrin inhibitors. Our recent findings have demonstrated that knockdown or pharmacologic inhibition of the integrin αvβ3 abrogated the proliferation of malignant T cells in vitro and in patient-derived xenograft mice models, an effect partially mediated by defective angiogenesis. Given the increasing importance of the ECM and the stromal cells to NHL biology and drug response, there is a need to develop 3D tissues that mimic the diseased lymphoid microenvironment and are adaptable to disease-specific needs. Method: Human B- and T-NHL cell lines were encapsulated in organoids of 4-arm Maleimide functionalized polyethylene glycol (PEG-MAL) and thiolated, enzymatically degradable crosslinker peptides along with human tonsil tissue derived follicular dendritic cells (FDC). Prior to organoid formation, PEGMAL was functionalized with thiolated peptides targeting αvβ3 and α4β1 on B and T-NHLs. The survival, proliferation, and drug studies were performed with confocal microscopy and biochemical assays. For drug-response analysis, organoids were cultured for 4 days before the 24 hr of exposure to Doxorubicin or Panobinostat, a histone deacetylate inhibitor and apoptosis studies were performed. Results: Our gene expression analysis shows that while αvβ3 integrin is consistently overexpressed in T-NHLs, these cells also express varying levels of α4β1. B-NHLs on the other hand, do not have a predictable pattern of integrin expression. When NHLs were presented with integrin ligands in the organoids, both B and T-NHLs formed large clusters as a function of integrin ligands (αvβ3 ligand RGD ＞ α4β1 ligand REDV). Importantly, REDV peptide significantly increased B-NHL proliferation while T-NHL proliferation was dependent on RGD signaling. Both B and T-NHLs, when cultured in organoids, demonstrated drug-resistance to Doxorubicin and the Panobinostat (Fig 1). We observed the 3D microenvironment to upregulate IgM B cell receptors BCR expression level (Fig. 2), which indicate enhanced survival and possibly explains the enhanced proliferation observed with B cell lymphomas. We predicted that activation of BCR is likely supporting the survival of B NHLs in 3D niches over 2D cultures by means of both up-regulating BCR and cross-talk with integrins. This hypothesis was supported by our findings with Syk inhibition that caused significantly higher apoptosis in the 3D group as compared to the 2D groups. Conclusions: Taken together, these results emphasize the role of biomaterials-based 3D tissues and the importance of integrin mediated signaling in malignant B and T cell tumors, as well as the role of FDCs in lymphoma proliferation. In the past, these factors have been largely ignored in drug evaluation or mechanistic studies of B and T cell lymphomas, ex vivo.