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 T_FH and T_REG, 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.