Patient-Derived Follicular Lymphoma Tumoroids: A Tool to Recapitulate Biological Features of Low and High-Grade Lymphoma

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. To recreate the cellular components, signaling cues, and the physical environment, we have optimized the generation of Patient Derived Lymphoma Tumoroids (PDLT) embedded in collagen I, highly present in the reticular network of the lymphoma lymph node (LN) where FL originates. PDLTs are composed of tumor B cells and autologous T cells. In addition, a supportive follicular dendritic cell line (FDC), or a modified variant expressing specific cytokines and factors (CD40 ligand and interleukin 21) present in FL germinal center (GC) microenvironment (FDC-GC), was integrated. These components rendered a compact tumoroid, rheologically relevant to the lymphoma LN. Our model is capable of sustaining viability for up to 10 days and promote proliferation in the presence of GC cues. Through single-cell RNA sequencing (scRNAseq), we observed that, after culture in PDLTs, both tumor cells and TME maintained a FL-LN gene expression signature. However, coculture with FDCs yielded a profile more closely related to the original biopsy sample, whereas when FDC-GC stimuli were included instead, features of a high-grade FL were recapitulated. In this regard, mass spectrometry analysis of the secretome compartment revealed that high-grade PDLTs are also able to remodel their initial extracellular matrix (ECM) and refine it with newly secreted collagens (I, IV, V and VI), fibronectin, and tenascin C (all of them present in FL-LN), together with vimentin, known to be associated with FL transformation. Finally, we used PDLTs as a preclinical tool for cell therapy. The anti-CD19 CAR-T product ARI-0001 was able to adhere to the tumoroid's surface but shows moderate infiltration and e'icacy, compared to CART activity in Patient- Derived Lymphoma Spheroids lacking ECM. This indicates that ECM may be a crucial player in cell therapy evasion in lymphoma by preventing CAR-T cells from reaching their target cells, which is a common hindrance in solid tumors.

In summary, we have developed a patient-derived model that can recapitulate both indolent and aggressive FL features, providing an in vitro model suitable to study disease biology and personalized therapy testing in the context of structural and cellular elements of the TME, while reducing animal usage.

Lopez-Guillermo:Roche: Consultancy, Research Funding; Gilead/Kite: Consultancy, Research Funding; Takeda: Consultancy; Janssen: Consultancy; Incyte: Consultancy; Abbvie: Consultancy; Genmab: Consultancy, Other: Safety Committee clinical trials. Hodson:Astra Zeneca, GSK: Research Funding.