Mantle cell lymphoma (MCL), a rare and aggressive B-cell non-Hodgkin lymphoma, mainly develops in the lymph node (LN) and creates a protective and immunosuppressive niche that facilitates tumor survival, proliferation and chemoresistance. To capture disease heterogeneity and tumor microenvironment (TME) cues, we have developed the first patient-derived MCL spheroids (MCL-PDLS) that recapitulate tumor oncogenic pathways and immune microenvironment in a multiplexed system that allows easy drug screening, including immunotherapies. MCL spheroids, integrated by tumor B cells, monocytes and autologous T-cells self-organize in disc-shaped structures, where B and T-cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. RNA-seq analysis demonstrated that tumor cells recapitulate hallmarks of MCL-LN (proliferation, NF-kB and BCR), with T cells exhibiting an exhaustion profile (PD1, TIM-3 and TIGIT). MCL-PDLS reproduces in vivo responses to ibrutinib and demonstrates that combination of ibrutinib with nivolumab (anti-PD1) may be effective in ibrutinib-resistant cases by engaging an immune response with increased interferon gamma and granzyme B release. In conclusion, MCL-PDLS recapitulates specific MCL-LN features and in vivo responses to ibrutinib, representing a robust tool to study MCL interaction with the immune TME and to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.

Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma, constitutes a paradigm of immune tumor microenvironment (TME) contribution to disease onset, progression, and heterogenous clinical outcome. Here we present the first FL-Patient Derived Lymphoma Spheroid (FL-PDLS), including fundamental immune actors and features of TME in FL lymph nodes (LNs). FL-PDLS is organized in disc-shaped 3D structures composed of proliferating B and T cells, together with macrophages with an intermediate M1/M2 phenotype. FL-PDLS recapitulates the most relevant B-cell transcriptional pathways present in FL-LN (proliferation, epigenetic regulation, mTOR, adaptive immune system, among others). The T cell compartment in the FL-PDLS preserves CD4 subsets (follicular helper, regulatory, and follicular regulatory), also encompassing the spectrum of activation/exhaustion phenotypes in CD4 and CD8 populations. Moreover, this system is suitable for chemo and immunotherapy testing, recapitulating results obtained in the clinic. FL-PDLS allowed uncovering that soluble galectin-9 limits rituximab, rituximab, plus nivolumab/TIM-3 antitumoral activities. Blocking galectin-9 improves rituximab efficacy, highlighting galectin-9 as a novel immunotherapeutic target in FL. In conclusion, FL-PDLS maintains the crosstalk between malignant B cells and the immune LN-TME and constitutes a robust and multiplexed pre-clinical tool to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.

Follicular Lymphoma (FL), the most common indolent non-Hodgkin’s B cell lymphoma, is a paradigm of the immune microenvironment’s contribution to disease onset, progression, and heterogeneity. Over the last few years, state-of-the-art technologies, including whole-exome sequencing, single-cell RNA sequencing, and mass cytometry, have precisely dissected the specific cellular phenotypes present in the FL microenvironment network and their role in the disease. In this already complex picture, the presence of recurring mutations, including KMT2D, CREBBP, EZH2, and TNFRSF14, have a prominent contributory role, with some of them finely tuning this exquisite dependence of FL on its microenvironment. This precise characterization of the enemy (FL) and its allies (microenvironment) has paved the way for the development of novel therapies aimed at dismantling this contact network, weakening tumor cell support, and reactivating the host’s immune response against the tumor. In this review, we will describe the main microenvironment actors, together with the current and future therapeutic approaches targeting them.

Follicular lymphoma (FL) represents a heterogeneous group of B-cell neoplasms with distinct genetic, epigenetic, microenvironmental, and clinical features. It is the most prevalent indolent non-Hodgkin lymphoma, characterized by a relapsing course and risk of transformation to aggressive diffuse large B-cell lymphoma. Recent advances in high-throughput sequencing, spatial transcriptomics, and imaging technologies uncovered genetic, epigenetic, and immunogenetic features underpinning FL, offering insights into its biology and potential therapeutic vulnerabilities. Although FL is primarily driven by the hallmark t(14;18) translocation involving BCL2, its pathogenesis requires additional oncogenic mutations, particularly in genes regulating chromatin and histone modifications. These early genetic and epigenetic alterations promote the persistence and evolution of cancer precursor cells, setting the stage for lymphomagenesis. The tumor microenvironment is also crucial in FL progression and patient prognosis, with T cells, stromal cells, and macrophages playing pivotal roles in facilitating tumor immune escape. Targeted therapies, including B-cell lymphoma 2 (BCL2) inhibitors, epigenetic modulators, and immunotherapies, have emerged from this deeper understanding of FL biology. Achieving a cure for FL will require targeted therapies that selectively eliminate cancer precursor cells with minimal impact on normal cells, thus preventing relapse and avoiding harmful side effects. Eradicating minimal residual disease should be a primary objective rather than waiting for clinical relapse. Future research must prioritize the development of accurate experimental models, the elucidation of FL precursors, and a deeper understanding of its heterogeneity, dependencies, progression, and mechanisms driving transformation. Implementing targeted therapies at FL early stages, instead of the current “watch and wait” approach, will be essential to improve patient outcomes.

Mantle cell lymphoma (MCL), a rare and aggressive B cell non-Hodgkin lymphoma, mainly develops in the lymph node (LN) and creates a protective and immunosuppressive niche that facilitates tumor survival, proliferation and chemoresistance. To capture disease heterogeneity and tumor microenvironment (TME) cues, we have developed the first patient-derived MCL spheroids (MCL-PDLS) that recapitulate tumor oncogenic pathways and immune microenvironment in a multiplexed system that allows easy drug screening, including immunotherapies. MCL spheroids, integrated by tumor B cells, monocytes and autologous T cells self-organize in disc-shaped structures, where B and T cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. RNA-seq analysis demonstrated that tumor cells recapitulate hallmarks of MCL-LN (proliferation, NF-kB and BCR), with T cells exhibiting an exhaustion profile (PD1, TIM-3 and TIGIT). MCL-PDLS reproducesin vivoresponses to ibrutinib and demonstrates that combination of ibrutinib with nivolumab (anti-PD1) may be effective in ibrutinib-resistant cases by engaging an immune response with increased interferon gamma and granzyme B release. In conclusion, MCL-PDLS recapitulates specific MCL-LN features andin vivoresponses to ibrutinib, representing a robust tool to study MCL interaction with the immune TME and to perform drug screening in a patient-derived system, advancing towards personalized therapeutic approaches.

Although first-line immunotherapy achieves remission in most patients with follicular lymphoma (FL), improved biomarkers and therapeutic strategies are required to identify and manage those who relapse. We investigated the immune microenvironment at diagnosis in uniformly treated FL patients with extended follow-up (>11 years), comparing relapsed and relapse-free cases. Transcriptomic profiling revealed enrichment of inflammatory response pathways in relapsed patients, including cytokine overexpression and upregulation of CD70, a molecule implicated in immune activation and inflammation. Multiplex immunofluorescence confirmed CD70 overexpression at diagnosis in tumor cells as well as in CD8⁺ and CD4⁺ T follicular helper (TFH) cells, correlating with inferior progression-free survival. Functional studies demonstrated that CD70⁺ tumor cells were more proliferative and induced CD70 expression in T cells, suggesting a feed-forward loop sustaining immune activation. To target these cells and enhance current CAR-T approaches, we engineered dual CD19-CD70 chimeric antigen receptor (CAR) T cells by co-transducing the CD19-CAR-T ARI-0001 with a CD27-based anti-CD70 CAR. Dual CAR-T cells exhibited enhanced in vitro cytotoxicity against patient-derived spheroids of FL, diffuse large B-cell lymphoma, and mantle cell lymphoma. In FL xenograft models, dual CAR-T treatment achieved superior disease control compared to monotargeted CAR-T cells, inducing complete tumor clearance in spleen and bone marrow. Collectively, these findings provide strong preclinical rationale for the clinical development of CD19-CD70 dual CAR-T therapy to improve outcomes in high-risk FL and potentially other B-cell non-Hodgkin lymphomas.