Search

Despite the success of standard front-line chemotherapy, 20% of classical Hodgkin lymphoma (cHL) patients still relapse or have refractory disease (r/r), and a subset of them die due to disease progression. There is a critical lack of predictive factors for early identification of those r/r patients who may benefit from new therapeutic strategies. This study aimed to evaluate the dynamic expression of 586 immune-related genes in a cohort of 42 cHL patients including 30 r/r cHL after first-line chemotherapy. Gene expression profiling (GEP) using NanoString technology identified a 19-gene immune signature at diagnosis predictive of cHL relapse, but dependent on histological subtypes. Genes related to tumor survival were found upregulated while genes related to B-lineage were downregulated at diagnosis in r/r nodular sclerosis cHL. In contrast to the mixed-cellularity subtype, comparative GEP analyses between paired diagnosis/relapse biopsies of nodular sclerosis cHL showed 118 differentially expressed genes, supporting an immune contexture switch at relapse with upregulation of immunosuppressive cytokines, such as LGALS1 and TGFB1, and downregulation of the T-cell co-stimulatory receptor ICOS. These results indicate that the predictive value of immune signature in cHL is strongly influenced by histological subtype which should be considered when assessing new immunotherapy target strategies.

Background: The development of single-cell technologies yields large datasets of information as diverse and multimodal as transcriptomes, immunophenotypes, and spatial position from tissue sections in the so-called ’spatial transcriptomics’. Currently however, user-friendly, powerful, and free algorithmic tools for straightforward analysis of spatial transcriptomic datasets are scarce. Results: Here, we introduce Single-Cell Spatial Explorer, an open-source software for multimodal exploration of spatial transcriptomics, examplified with 9 human and murine tissues datasets from 4 different technologies. Conclusions: Single-Cell Spatial Explorer is a very powerful, versatile, and interoperable tool for spatial transcriptomics analysis.

Follicular lymphoma (FL) is the most common indolent lymphoma. Despite the clear benefit of CD20-based therapy, a subset of FL patients still progress to aggressive lymphoma. Thus, identifying early biomarkers that incorporate positron emission tomography metrics could be helpful to identify patients with a high risk of treatment failure with rituximab. We retrospectively included a total of 132 untreated FL patients separated into training and validation cohorts. Optimal threshold of baseline whole-body maximum standardized uptake (SUVmax) was first determined in the training cohort (n=48) to predict progression-free survival (PFS). The PET results were investigated along with the tumor and immune microenvironment, which were determined by immunochemistry and transcriptome studies involving gene set enrichment analyses and immune cell deconvolution, together with the tumor mutation profile. We report that baseline SUVmax>14.5 was associated with poorer PFS than baseline SUVmax≤14.5 (hazard ratio =0.28; P=0.00046). Neither immune T-cell infiltration nor immune checkpoint expression were associated with baseline PET metrics. By contrast, FL samples with Ki-67 staining ≥10% showed enrichment of cell cycle/DNA genes (P=0.013) and significantly higher SUVmax values (P=0.007). Despite similar oncogenic pathway alterations in both SUVmax groups of FL samples, four out of five cases harboring the infrequent FOXO1 transcription factor mutation were seen in FL patients with SUVmax>14.5. Thus, high baseline SUVmax reflects FL tumor proliferation and, together with Ki-67 proliferative index, can be used to identify patients at risk of early relapse with rituximab chemotherapy.

Simple Summary Follicular lymphoma is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20–30 percent of all non-Hodgkin lymphoma (NHL) cases. Although huge efforts have been made in the last 10 years, this pathology is still considered as incurable, leaving open the discovery and testing of new therapeutic targets requiring relevant preclinical models. Here, we report a realistic 3D model of t (14;18)-positive B-NHL cell culture (ultra-low attachment (ULA)-multicellular aggregates of lymphoma cells (MALC)), which monitored by state-of-the-art 2D and 3D imaging, allows more robust drug testing. Abstract Follicular lymphoma (FL) is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20–30 percent of all non-Hodgkin lymphoma (NHL) cases. Great advances have been made to identify the most relevant targets for precision therapy. However, no relevant models for in vitro studies have been developed or characterized in depth. To this purpose, we generated a 3D cell model from t(14;18)-positive B-NHL cell lines cultured in ultra-low attachment 96-well plates. Morphological features and cell growth behavior were evaluated by classical microscopy (2D imaging) and response to treatment with different drugs was evaluated by a high-content analysis system to determine the robustness of the model. We show that the ultra-low attachment (ULA) method allows the development of regular, spherical and viable ULA-multicellular aggregates of lymphoma cells (MALC). However, discrepancies in the results obtained after 2D imaging analyses on drug-treated ULA-MALC prompted us to develop 3D imaging and specific analyses. We show by using light sheet microscopy and specifically developed 3D imaging algorithms that 3D imaging and dedicated analyses are necessary to characterize morphological properties of 3D models and drug effects. This study proposes a new method, but also imaging tools and informatic solutions, developed for FL necessary for future preclinical studies.

International audience; γδ T lymphocytes diverge from conventional T CD8 lymphocytes for ontogeny, homing, and antigen specificity, but whether their differentiation in tumors also deviates was unknown. Using innovative analyses of our original and ~150 published single-cell RNA sequencing datasets validated by phenotyping of human tumors and murine models, here we present the first high-resolution view of human γδ T cell differentiation in cancer. While γδ T lymphocytes prominently encompass TCRVγ9 cells more differentiated than T CD8 in healthy donor's blood, a different scenario is unveiled in tumors. Solid tumors and lymphomas are infiltrated by a majority of TCRVγnon9 γδ T cells which are quantitatively correlated and remarkably aligned with T CD8 for differentiation, exhaustion, gene expression profile, and response to immune checkpoint therapy. This cancer-wide association is critical for developing cancer immunotherapies.

International audience; Tumor-associated macrophage and T-cell subsets are implicated in the pathogenesis of diffuse large B-cell lymphoma, follicular lymphoma, and classical Hodgkin lymphoma. Macrophages provide essential mechanisms of tumor immune evasion through checkpoint ligand expression and secretion of suppressive cytokines. However, normal and tumor-associated macrophage phenotypes are less well characterized than those of tumor-infiltrating T-cell subsets, and it would be especially valuable to know whether the polarization state of macrophages differs across lymphoma tumor microenvironments. Here, an established mass cytometry panel designed to characterize myeloid-derived suppressor cells and known macrophage maturation and polarization states was applied to characterize B-lymphoma tumors and non-malignant human tissue. High-dimensional single-cell analyses were performed using dimensionality reduction and clustering tools. Phenotypically distinct intra-tumor macrophage subsets were identified based on abnormal marker expression profiles that were associated with lymphoma tumor types. While it had been proposed that measurement of CD163 and CD68 might be sufficient to reveal macrophage subsets in tumors, results here indicated that S100A9, CCR2, CD36, Slan, and CD32 should also be measured to effectively characterize lymphoma-specific tumor macrophages. Additionally, the presence of phenotypically distinct, abnormal macrophage populations was closely linked to the phenotype of intra-tumor T-cell populations, including PD-1 expressing T cells. These results further support the close links between macrophage polarization and T-cell functional state, as well as the rationale for targeting tumor-associated macrophages in cancer immunotherapies.

International audience; Follicular lymphoma (FL) is a common non Hodgkin's lymphoma subtype in which immune escape mechanisms are implicated in resistance to chemo-immunotherapy. Although molecular studies point to qualitative and quantitative deregulation of immune checkpoints, in depth cellular analysis of FL immune escape is lacking. Here, by functional assays and in silico analyses we show that a subset of FL patients displays a 'high' immune escape phenotype. These FL cases are characterized by abundant infiltration of PD1+ CD16+ TCRVγ9Vδ2 γδ T lymphocytes. In a 3D co-culture assay (MALC), γδ T cells mediate both direct and indirect (ADCC in the presence of anti-CD20 mAbs) cytolytic activity against FL cell aggregates. Importantly, PD-1, which is expressed by most FL-infiltrating γδ T lymphocytes with ADCC capacity, impairs these functions. In conclusion, we identify a PD1-regulated γδ T cell cytolytic immune component in FL. Our data provide a treatment rational by PD-1 blockade aimed at boosting γδ T cell anti-tumor functions in FL.

Introduction The role of the programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) axis is well established in classical Hodgkin lymphoma (HL), where PD-1 blockade demonstrated spectacular efficacy in relapsed/refractory disease. However, little is known about the frequency and cellular distribution of other immune checkpoints in HL samples. Patients and Methods Using immunohistochemistry, we investigated, along with PD-L1 and PD-1, the expression of lymphocyte-activation gene 3 (LAG-3) and T-cell immunoglobulin and mucin-domain containing 3 (TIM-3) in 57 biopsy samples of patients with classical HL. Results Hodgkin and Reed/Sternberg (HRS) cells were strongly positive for PD-L1 in nearly all cases. HRS cells were TIM-3 positive in 36% of samples, whereas LAG-3 was rarely expressed (5.2%). In the microenvironment, PD-1, LAG-3, and TIM-3 were expressed by ≥ 5% of cells in 65%, 98%, and 96% of cases, respectively. T-cell rosettes surrounding HRS cells consisted of CD4+ FoxP3− helper T cells expressing both PD-1 and LAG-3, with a variable expression of TIM-3. Conclusion This study demonstrates for the first time that LAG-3 and TIM-3 are nearly always expressed in the microenvironment of classical HL. This may constitute the basis for targeting LAG-3 or TIM-3 in combination with anti–PD-1 antibodies in the treatment of relapsed/refractory HL.

Therapeutic blockade of PD-1/PD-L1 shows promising results in Hodgkin&rsquo, s lymphoma (HL) and in some diffuse large B-cell lymphoma (DLBCL) patients, but biomarkers predicting such responses are still lacking. To this end, we recently developed a transcriptional scoring of immune escape (IE) in cancer biopsies. Using this method in DLBCL, we identified four stages of IE correlated with overall survival, but whether Hodgkin&rsquo, s lymphomas (HL) also display this partition was unknown. Thus, we explored the transcriptomic profiles of ~1000 HL and DLBCL using a comparative meta-analysis of their bulk microarrays. Relative to DLBCL, the HL co-clustered at the advanced stage of immune escape, displaying significant enrichment of both IE and T-cell activation genes. Analyses via transcriptome deconvolution and immunohistochemistry showed more CD3+ and CD4+ tumor-infiltrating lymphocytes (TILs) in HL than DLBCL. Both HL and non-GCB DLBCL shared a high abundance of infiltrating CD8+ T-cells, but HL had less CD68+CD163+ macrophages. The same cellular distribution of PD-1 and TIM-3 was observed in HL and DLBCL, though HL had more PD-L1 tumor cells and LAG-3 ME cells. This study illuminates the advanced stage of immune activation and escape in HL, consistent with the response to checkpoint blockade therapies for this type of lymphoma.

Despite recent therapeutic progress, plasmablastic lymphoma (PBL), a distinct entity of high grade B cell lymphoma, is still an aggressive lymphoma with adverse prognosis. PBL commonly occurs in patients with HIV infection and PBL cells frequently express Epstein Barr virus (EBV) genome with type I latency. Occasionally however, PBL may develop in patients with an immunodepressed status without EBV and HIV infection. The aim of this study was to determine which PBL patients may benefit from the emerging strategies of immune checkpoint blockade. Here, we produced and analyzed the transcriptomic profiles of such tumors to address this question. Unsupervised hierarchical clustering analysis of PBL samples revealed they segregate according to their tumor EBV-status. Moreover, EBV(+) PBL displays abundant leucocyte infiltrates and T-cell activation signatures, together with high expression levels of mRNA and protein markers of immune escape. This suggests that EBV infection induce an anti-viral cytotoxic immunity which progressively exhausts T lymphocytes and promotes the tolerogenic microenvironment of PBL. Hence, most EBV(+) PBL patients presenting an early stage of cancer immune-editing process appear as the most eligible patients for immune checkpoint blockade therapies.

International audience; Plasmablastic lymphoma is a rare and aggressive diffuse large B-cell lymphoma commonly associated with Epstein-Barr virus co-infection that most often occurs in the context of human immunodeficiency virus infection. Therefore, its immune escape strategy may involve the upregulation of immune-checkpoint proteins allowing the tumor immune evasion. However, the expression of these molecules was poorly studied in this lymphoma. We have investigated 82 plasmablastic lymphoma cases of whom half were Epstein-Barr virus positive. Although they harbored similar pathological features, Epstein-Barr virus positive plasmablastic lymphomas showed a significant increase in MYC gene rearrangement and had a better 2-year event-free survival than Epstein-Barr virus negative cases (P=0.049). Immunostains for programmed cell death-1, programmed cell death-ligand 1, indole 2,3-dioxygenase and dendritic cell specific C-type lectin showed a high or moderate expression by the microenvironment cells in 60%–72% of cases, whereas CD163 was expressed in almost all cases. Tumor cells also expressed programmed cell death-1 and its ligand in 22.5% and 5% of cases, respectively. Both Epstein-Barr virus positive and negative plasmablastic lymphomas exhibited a high immune-checkpoint score showing that it involves several pathways of immune escape. However, Epstein-Barr virus positive lymphomas exhibited a higher expression of programmed cell death-1 and its ligand in both malignant cells and microenvironment as compared to Epstein-Barr virus negative cases. In conclusion, plasmablastic lymphoma expresses immune-checkpoint proteins through both malignant cells and the tumor microenvironment. The expression of programmed cell death-1 and its ligand constitutes a strong rationale for testing monoclonal antibodies in this often chemoresistant disease.

Upregulation of T cell immunoglobulin-3 (TIM-3) has been associated with negative regulation of the immune response in chronic infection and cancer, including lymphoma. Here, we investigated the possible correlation between TIM-3 expression by ex vivo cytotoxic T cells (CTL) from follicular lymphoma (FL) biopsies and their functional unresponsiveness that could limit the favorable impact of CTL on disease progression. We report a high percentage of CD8+TIM-3+T cells in lymph nodes of FL patients. When compared to their CD8+TIM-3− counterparts, CD8+TIM-3+ T cells exhibited defective cytokine production following TCR engagement. Furthermore, CD8+TIM-3+ T cells display ex vivo markers of lytic granule release and remain unresponsive to further TCR-induced activation of the lytic machinery. Although confocal microscopy showed that TIM-3 expression on CD8+ T cells correlated with minor alterations of immunological synapse, a selective reduction of ERK signaling in CD8+TIM-3+T cells was observed by phospho-flow analysis. Finally, short relapse-free survival despite rituximab(R)-chemotherapy was observed in patients with high content of TIM-3+ cells and a poor infiltrate of granzyme B+ T cells in FL lymph nodes. Together, our data indicate that, besides selective TCR early signaling defects, TIM-3 expression correlates with unresponsiveness of ex vivo CD8+ T cells in FL. They show that scores based on the combination of exhaustion and cytolytic markers in FL microenvironment might be instrumental to identify patients at early risk of relapses following R-chemotherapy.

Follicular lymphoma (FL) is a common non Hodgkin's lymphoma subtype in which immune escape mechanisms are implicated in resistance to chemo-immunotherapy. Although molecular studies point to qualitative and quantitative deregulation of immune checkpoints, in depth cellular analysis of FL immune escape is lacking. Here, by functional assays and in silico analyses we show that a subset of FL patients displays a 'high' immune escape phenotype. These FL cases are characterized by abundant infiltration of PD1+ CD16+ TCRVgamma9Vdelta2 gamma delta T lymphocytes and high expression of gamma delta cytolytic markers. In a 3D co-culture assay (MALC), gamma delta T cells could mediate both direct and indirect (ADCC in the presence of anti-CD20 mAbs) cytolytic activity against FL cell aggregates. Importantly, PD-1, which is expressed by most FL-infiltrating gamma delta T lymphocytes with ADCC capacity, impaired these functions. In conclusion, we identify a PD1-regulated gamma delta T cell cytolytic immune component in FL. Our data provide a treatment rational by PD-1 blockade aimed at boosting gamma delta anti-tumor function in FL. Disclosures Morin: IMACTIV-3D: Employment. Lagarde:IMACTIV-3D: Employment. Savina:Institut Roche: Employment. Klein:F. Hoffmann-La Roche Ltd.: Employment, Other: stock, Patents & Royalties.

International audience; Lymphomas grow as dense aggregates in patients, but whether this spatial organization affects lymphoma cell biology is unknown. We grew follicular lymphoma (FL) cells in vitro as multicellular aggregates of lymphoma cells to investigate this question. Gene expression analysis revealed that 612 genes were differentially expressed when cells grew in multicellular aggregates of lymphoma cells rather than in suspension. These genes correspond to several GO biological processes, such as hypoxia, activation of NF-κB pathway, and negative regulation of cell cycle, a gene signature also found in the transcriptomes from FL biopsies. Pimonidazole staining, HIF-1A accumulation, and VEGFA release confirmed that cells in multicellular aggregates of lymphoma cells actually respond to hypoxia. In adaptation to such conditions, they also displayed an activated NF-κB pathway and a quiescent status far more frequently than in suspension. When cultured in three dimensions, FL cells display resistance to doxorubicin and bendamustine, two drugs largely used in FL therapy, compared to FL cultured in suspension. Finally, multicellular aggregates of lymphoma cells were also found to be less sensitive to purified natural killer cells. To conclude, our study shows that in FL, spatial organization results in dramatic changes in FL biology, including gene expression, proliferation, drug resistance, and immune escape.

Upregulation of T cell immunoglobulin-3 (TIM-3) has been associated with negative regulation of the immune response in chronic infection and cancer, including lymphoma. Here, we investigated the possible correlation between TIM-3 expression by ex vivo cytotoxic T cells (CTL) from follicular lymphoma (FL) biopsies and their functional unresponsiveness that could limit the favorable impact of CTL on disease progression. We report a high percentage of CD8+TIM-3+T cells in lymph nodes of FL patients. When compared to their CD8+TIM-3- counterparts, CD8+TIM-3+ T cells exhibited defective cytokine production following TCR engagement. Furthermore, CD8+TIM-3+ T cells display ex vivo markers of lytic granule release and remain unresponsive to further TCR-induced activation of the lytic machinery. Although confocal microscopy showed that TIM-3 expression on CD8+ T cells correlated with minor alterations of immunological synapse, a selective reduction of ERK signaling in CD8+TIM-3+T cells was observed by phospho-flow analysis. Finally, short relapse-free survival despite rituximab(R)-chemotherapy was observed in patients with high content of TIM-3+ cells and a poor infiltrate of granzyme B+ T cells in FL lymph nodes. Together, our data indicate that, besides selective TCR early signaling defects, TIM-3 expression correlates with unresponsiveness of ex vivo CD8+ T cells in FL. They show that scores based on the combination of exhaustion and cytolytic markers in FL microenvironment might be instrumental to identify patients at early risk of relapses following R-chemotherapy.

Tumor-intrinsic immuno-resistance is a prerequisite for emergence of follicular lymphomas. Here we show that in vitro, such cells are more resistant to immune cytolysis when grown as follicle-mimicking tridimensional aggregates than when grown as cell suspensions. So in patients, this innate adaptation to tumor immunity might precede its selective pressure.

Dynamic interactions between cells and extracellular matrix (ECM) through integrins influence most cellular functions. Normal cells, but even more, tumor cells are subjected to different forms of stress, including ischemia, radical oxygen species production, starvation, mechanical stress or genotoxic insults due to anti-cancer drugs or irradiation. In these situations, an adaptative cellular response occurs, integrating a complex network of intracellular signaling modules, which, depending on stress intensity, may result to either damage repair followed by complete restitution of cellular functions, or programmed cell death. Because of its implication in oncogenesis and anti-cancer therapy, cellular stress response has been thoroughly investigated. However, most of these studies have been performed in the context of isolated cells without taking into consideration that most cells are part of the tissue within which they interact with ECM through integrin. Few studies have described the influence of stress on cell-to-ECM interaction. However, one can speculate that, in these conditions, cells could functionally interact with protein microenvironment either to create positive interactions to survive (for example by facilitating protective pathways) or negative interaction to die (for example by facilitating detachment). In this review, we summarize the knowledge relative to the influence of different stress modalities on ECM remodeling, integrin expression and/or function modifications, and possible functional consequences, independently from the cellular model as these findings came from a large variety of cells (mesenchymal, endothelial, muscular, epithelial and glandular) and fields of application (cancer, vascular biology and tissue engineering). Most studies support the general notion that non-lethal stress favors ECM stiffness, integrin activation and enhanced survival. This field opens large perspectives not only in tumor biology but also in anti-cancer therapy by targeting one or several steps of the integrin-mediated signaling pathway, including integrin ligation, or activation of integrin-linked enzymes or integrin adaptors.

Follicular lymphoma (FL) is the second most common type of B non-Hodgkin’s lymphomas (NHL) and makes up to 40% of all adult lymphomas. Like most carcinomas, NHL grow as spherical tumors. Based on carcinoma 3D models (often described as spheroids) it is acknowledged that spatial organization may profoundly affect tumor cell behavior since important functions are dictated by the collective properties of a cell population rather than those of a single cell. These include growth, metastasis, cell-to-matrix and cell-to-cell interaction, as well as intracellular signaling and resistance to anti-tumor agents or even immune escape. Spatial organization can exert these effects in cancer cells by affecting gene expression profiles or influencing major signaling pathways such as those driven by MAPK, Akt and oncogenic products such as HER-2. More importantly, spatial organization is known to influence the response to antibodies such as Trastuzumab or Pertuzumab. Conventional 2D NHL cell culture models do not reflect the true effects of antibodies as they occur in vivo and may be of limited use. Thus, we recently created a NHL 3D culture system, the so-called Multicellular Aggregates of Lymphoma Cells (MALC) model, using a modification of the “hanging drop” method. This model, which displays similar transcriptomic profiles compared to biopsies from FL patients, is not only useful for studying FL biology, but also for evaluating tumor sensitivity to antibodies since diffusion/distribution is different within a solid 3D tumor. Thus, we recently showed that 3D organization influences direct response to Rituximab (RTX) and GA101 (Obinutuzumab), two monoclonal anti-CD20 antibodies, as we observed a more potent efficiency in MALC model compared to conventional 2D cell culture in term of cell death induction (apoptosis, senescence, lysosomal cell death) and intracellular signaling targeting. Here, we evaluate antibody-dependent cell cytotoxicity in MALC in presence of immune effectors cells such as NK cells and Tgd lymphocytes. We showed that RTX and GA101 induce immune cells activation as attested by CD69 and IFNg increase and immune cells cytotoxic activity as attested by CD107 increase as well as granzyme B and perforine decrease, reflecting granules release. GA101 appears to be more efficient than RTX in effectors cells activation. We correlate this phenomenon with a more potent effect on target cells as attested by MALC volume decrease and cell death induction observed by flow cytometry and confocal microscopy. By investigating intracellular signaling pathways induced during ADCC on both effector (NK) and target cells (MALC), we observed on NK cells the activation of CD16 downstream signaling molecules such as Syk, PLCg2 and Akt, with a more potent effect in presence of GA101 compared to RTX for proximal proteins activation. On the other hand, we showed that RTX and GA101 induced inhibition of classical overactivated signaling pathways such as Syk, Akt, ERK and p38 on target cells. Finally, we asked the question of the presence of immune cells in lymph node isolated from FL patients. By flow cytometry, we were able to determine the percent of NK and gdT cells and present evidences that immune cells number is higher in FL lymph node compared to normal tonsil. Altogether, this study completes our previous works on the identification of MAbs mechanisms of action in a relevant 3D FL model.A better understanding of these mechanisms is necessary to improve therapy by identifying mechanisms of resistance and new therapeutic targets. Moreover, we believe that MALC is a powerful and relevant model for further investigation on FL biology, and also useful for the development/screening of new genotoxic drugs and therapeutic monoclonal antibodies in lymphoma. Disclosures Bezombes: Roche: Research Funding. Savina:roche: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Follicular lymphoma (FL) is the second most common type of B non-Hodgkin’s lymphomas (NHL) and makes up 40% of all adult lymphomas. Like most carcinomas, NHL grow as spherical tumors. Based on carcinoma 3D models (often described as spheroids) it is acknowledged that spatial organization may profoundly affect tumor cell behavior since important functions are dictated by the collective properties of a cell population rather than those of a single cell. These include growth, metastasis, cell-to-matrix and cell-to-cell interaction, as well as intracellular signaling and resistance to anti-tumor agents or even immune escape. Spatial organization can exert these effects in cancer cells by affecting gene expression profiles or influencing major signaling pathways such as those driven by MAPK, Akt and oncogenic products such as HER-2. More importantly, spatial organization is known to influence the response to antibodies such as Trastuzumab or Pertuzumab. Conventional 2D NHL cell culture models do not reflect the true effects of antibodies as they occur in vivo and may be of limited use. Thus, we recently created a NHL 3D culture system, the so-called Multicellular Aggregates of Lymphoma Cells (MALC) model, using a modification of the “hanging drop” method. This model is not only useful for studying FL biology, but also for evaluating tumor sensitivity to antibodies since diffusion/distribution is different within a solid 3D tumor. In this study, we present transcriptomic and extracellular matrix proteins expression profiles in 2D, 3D FL cell cultures and patients biopsies, and observed that MALC, but not 2D cultures, display similarities to patients samples. Indeed, we observe an upregulation of genes involved in the response to hypoxia, activation of NFκB pathway and negative regulation of cell cycle promoting the emergence of chemoresistant quiescent cells. Moreover, MALC, in contrast to 2D cultures but as observed in biopsies, exhibit higher expression of fibronectin, laminin, vitronectin and collagen. Recently, we showed that spatial organization also influence response to anti-CD20 monoclonal antibodies such as Rituximab and Obinutuzumab. Here, we investigate the antibody-dependant cell cytotoxicity induced by these two monoclonal anti-CD20 antibodies in the MALC model: cell cytotoxicity, effector and target cells signaling, and spatial localization are presented. Thus, we present an invaluable model for further investigation of FL biology, and also for the development/screening of new genotoxic drugs and therapeutic monoclonal antibodies in lymphoma. Disclosures: Bezombes: Roche: Research Funding. Decaup:Institut de Recherche Roche: Employment. Klein:Roche Glycart AG: Employment. Varoqueaux:F. Hoffman-La Roche AG: Employment. Savina:Roche S.A.S: Employment.

Follicular lymphomas (FLs) account for 35–40% of all adult lymphomas. Treatment typically involves chemotherapy combined with the anti-CD20 monoclonal antibody (MAb) rituximab (RTX). The development of the type II anti-CD20 MAb obinutuzumab (GA101) aims to further improve treatment. Here, using FL cells we show that RTX and GA101 display a similar activity on RL cells cultured in 2D. However, 2D culture cannot mimic tumor spatial organization and conventional 2D models may not reflect the effects of antibodies as they occur in vivo. Thus, we created a non-Hodgkin’s lymphoma (NHL) 3D culture system, termed multicellular aggregates of lymphoma cells (MALC), and used it to compare RTX and GA101 activity. Our results show that both antibodies display greater activity towards FL cells in 3D culture compared with 2D culture. Moreover, we observed that in the 3D model GA101 was more effective than RTX both in inhibiting MALC growth through induction of (lysosomal) cell death and senescence and in inhibiting intracellular signaling pathways, such as mammalian target of rapamycin, Akt, PLCgamma (Phospholipase C gamma) and Syk. Altogether, our study demonstrates that spatial organization strongly influences the response to antibody treatment, supporting the use of 3D models for the testing of therapeutic agents in NHL.

Follicular lymphomas (FL) account for 35-40% of all adult lymphomas. Treatment typically involves chemotherapy combined with the anti-CD20 monoclonal antibody rituximab (RTX). The development of type II anti-CD20 MAb obinutuzumab (GA101) aims to further improve treatment. Here we show using FL cells that RTX and GA101 displayed similar activity on RL cells cultured in 2D. As 2D culture cannot mimic tumor spatial organization, conventional 2D models may not reflect the effects of antibodies as they occur in vivo. Thus, we created a NHL 3D culture system, termed multicellular aggregates of lymphoma cells (MALC), and compared RTX and GA101 activity. Our results show that both antibodies display greater activity towards FL cells in 3D culture compared to 2D culture. Moreover, we observed that in the MALC model GA101 was more effective than RTX in inhibiting tumor growth through induction of (lysosomal) cell death and senescence; and in inhibiting intracellular signaling pathways such as mTOR, Akt, PLCgamma and Syk. Altogether, our study demonstrates that spatial organization strongly influences response to antibody treatment supporting the use of 3D models for the testing of therapeutic agents in NHL.

Abstract 4868 Follicular lymphomas (FL) account for 35–40% of all adult lymphomas. Treatment typically involves chemotherapy combined with the anti-CD20 monoclonal antibody (MAb) rituximab (RTX). The development of type II anti-CD20 MAb obinutuzumab (GA101) aims to improve treatment further. Using FL cells, here we showed that RTX and GA101 displayed similar efficiency towards 2D culture. However, 2D culture does not mimic tumor spatial organization and conventional 2D models do not reflect the effects of MAbs seen from preclinical and in vivo studies. Thus, we created a NHL 3D culture system, the so-called multicellular aggregates of lymphoma cells (MALC), and compared RTX with GA101 activity. Our result showed that both MAbs displayed greater bioactivity towards FL cells in 3D culture compared to 2D culture. Moreover, we observed that GA101 was more effective than RTX in inhibiting tumor growth through induction of apoptosis, lysosomal cell death and senescence. GA101 was also more effective than RTX in inhibiting intracellular signaling pathways such as mTOR, Akt, PLCgamma and Syk. Altogether, our study demonstrates that spatial organization strongly influences response to MAbs treatment. Additionally, we provide evidences that GA101 is more effective than RTX in inhibiting FL development in MALC model, which suggests the possibility for better patient treatment with GA101. Disclosures: Decaup: roche: Employment, Research Funding. Klein:Roche Glycart AG: Employment. Varoqueaux:Roche S.A.S: Employment. Bezombes:Roche: Research Funding.