Immunocompetent 3D Follicular Lymphoma Model: A Preclinical Tool to Design Tailored Immunotherapies

Follicular Lymphoma (FL), the most common indolent non-Hodgkin Lymphoma, is a paradigm of the contribution of the immune tumor microenvironment (TME) to disease onset, progression, and therapy resistance. Moreover, macrophages, together with TFH and TREG, are fundamental players of FL immunosuppression. Patient-derived models are scarce and fail to recapitulate immune TME. Thus, we have established the first FL Patient Derived Lymphoma Spheroids (FL-PDLS) model that recapitulate the crosstalk between malignant B cells and the tumor immune microenvironment, and constitutes a pre-clinical tool to design tailored immunotherapies. Lymph node (LN) biopsies and peripheral blood (PB) samples were cultured in 96-well ultra-low-attachment plates within the context of an optimized cytokine cocktail representing the TME signals. FL-PDLS include tumor B cells and autologous T cells obtained from patient samples, together with healthy donors’ monocytes to complete the LN immune microenvironment. These populations self-organize in disc-shaped structures, as determined by light sheet microscopy, where B and T cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. Interestingly, RNA-seq analysis demonstrated that tumor cells in FL-PDLS recapitulate fundamental hallmarks of FL-LN (i.e. cell cycle, mTOR, EZH2, VEGF, IFNγ, IL2, IL10 and IL15, among others). Using public data basis, we have determined by differential expression analysis (FL-LN vs normal tonsils) an immune profile characteristic of FL-LN. Flow cytometry analysis of FL-PDLS determined that immune exhaustion profile is recapitulated in our model, including high expression of PD-1, TIM-3, LAG-3 and ICOS, and their corresponding ligands. Finally, we have validated FL-PDLS as a tool for immunotherapy drug screening. First, we have demonstrated that the combination of the standard of care rituximab (anti-CD20) with nivolumab (anti-PD1) reduce tumor load in a significant proportion of FL-PDLS, in accordance with previously published clinical results. Noteworthy, this combination leads to immune reactivation demonstrated by the increase of Granzyme B, IFNγ and TNFα secretion in FL-PDLS supernatants. Likewise, FL-PDLS has been useful to identify novel potential combinatorial immunotherapies (i.e. nivolumab + anti-ICOS, rituximab + anti-Tim3) that may be effective in specific set of patients. In summary, this novel FL-PDLS system represents a step towards personalized medicine, and contributes to reduce animal usage in biomedicine. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal