Chronic lymphocytic leukemia (CLL) is a common form of adult leukemia characterized by the accumulation of CD5+ B cells in the blood and lymphoid tissues. Interactions between malignant cells and endothelial cells of the vasculature influence CLL progression, though the underlying mechanisms are poorly understood. To investigate how the vasculature contributes to CLL cell dissemination, we developed a modular bioreactor called "VesselBox" by three-dimensional (3D) printing. This system enables selective perfusion of a milliscale vessel-like structure embedded within a lymphoid microenvironment designed to recapitulate selected structural features of CLL tissue niches created using 3D bioprinting and casting. We first performed numerical simulations to optimize perfusion parameters to support cell homeostasis. Upon vessel maturation, confirmed by endothelial marker expression, we demonstrated that VesselBox supports sustained perfusion for up to 7 days. By recirculating CLL cells through the system, we demonstrate its feasibility as a platform to assess CLL cell localization and immunophenotypic changes under dynamic conditions. This platform provides a framework for ex vivo analysis of CLL trafficking and offers the potential to uncover therapeutic targets by characterizing circulating and extravasated cells in the presence or absence of drug treatments.

Chronic lymphocytic leukaemia (CLL) cells circulate between the blood, bone marrow (BM) and lymphoid organs, where interactions with the lymph node (LN) microenvironment enhance their survival, proliferation and drug resistance. Most in vitro models fail to reproduce the spatial and cellular complexity of the LN niche, limiting studies of tissue-specific drug responses. To address this, we developed a 3D LN model using a gelatine scaffold and a clinorotator bioreactor previously validated for a BM system. The scaffold was seeded with human lymphatic fibroblasts and endothelial cells, which deposited extracellular matrix and supported patient-derived CLL cell viability and proliferation. Consistent with in vivo observations, CLL cells within the scaffold downregulated the chemokine receptor CXCR4, further reduced upon proliferative stimulation. Final validation involved treatment with targeted therapies: the BCL-2 antagonist venetoclax and the BTK inhibitor ibrutinib. Venetoclax treatment revealed greater CLL protection within the LN environment than in BM, whereas the mobilizing effect of ibrutinib was comparable between these two niches. This 3D LN model offers an effective ex vivo platform for studying microenvironment-tumour interactions and tissue-specific drug responses.

Breast cancer (BrCa) represents one of the most common malignancies and the leading cause of cancer-related deaths in women worldwide. Despite the advances in therapeutic treatments, de novo and/or acquired resistance still represents a major clinical challenge. Recently, a new class of therapeutic agents has been approved for the treatment of advanced/metastatic BrCa: antibody–drug conjugates (ADCs). Trastuzumab-deruxtecan (T-DXd) has recently become the prevalent treatment in different clinical settings because of its improved efficacy. Here, we identified two mechanisms of resistance: i. reduction of the payload target (Topoisomerase I) and ii. induction of sustained senescence. This phenotype correlates with increased production of reactive oxygen species (ROS), metabolic rewiring and activation of the p53/p21 axis, and is associated to the senescence-associated secretory phenotype (SASP). Furthermore, dissection of the relative contribution of the antibody (Trastuzumab) vs the payload (DXd) component of the ADC to the action of T-DXd showed that DXd alone is sufficient to promote senescence and its downstream effects. We further corroborated these conclusions exploiting another DXd-based ADC (Datopotamab-DXd) and found that DXd-based drugs promote Topoisomerase I downregulation and senescence. Altogether, these findings provide the rationale for the treatment of breast cancer patients resistant to DXd-based ADCs with senolytic or senomorphic agents.

Chronic lymphocytic leukemia (CLL) is a B cell malignancy driven by aberrant signaling and microenvironmental support. Cytoskeletal remodeling contributes to these mechanisms, where the hematopoietic protein HS1 appears to play a key role. Thus, we generated C57BL/6 HS1 knockout (HS1KO) mice to assess its impact on lymphocyte development. Young HS1KO mice showed some alterations in B cell maturation and T cell differentiation, which appeared to normalize with age. In double-transgenic Eμ-TCL1-HS1KO mice, combining HS1 loss with the standard Eμ-TCL1 CLL mouse model, we observed a trend of reduction in the CD4/CD8 ratio, and disrupted splenic architecture with loss of follicular organization. Transcriptomic and kinase profiling of the human CLL cell line MEC1, in which HS1 was knocked out (MEC1-HS1KO), revealed a coherent impairment in terms of cell mobility as highlighted by severely impaired migration in 2D and 3D environments, reduced chemotactic responses to CXCL12 and CCL19; deregulation of cytoskeletal, adhesion, and apoptotic pathways; and increased resistance to apoptosis. We posited that these defects could reduce sensitivity to BTK (Bruton's tyrosine kinase) inhibition, a standard therapeutic approach in CLL. Primary CLL cells from the spleen of Eμ-TCL1-HS1KO mice appeared to show reduced sensitivity to ibrutinib, a finding that was also observed in adoptive transfer experiments designed to equalize disease burden. Wild-type mice receiving Eμ-TCL1-HS1KO leukemic cells tend to be more resistant to ibrutinib, particularly in the peripheral blood and spleen. All together, these findings establish HS1 as a potential regulator linking B cell receptor signaling, cytoskeletal remodeling, leukemic progression, and response to therapy, supporting its potential as a biomarker for BTK inhibitor sensitivity.

Ex vivo culture of hematopoietic stem and progenitor cells (HSPCs) is required for gene therapy applications but inadvertently triggers detrimental cellular responses, potentially threatening clinical success. In this study, we employ nichoids, biocompatible 3D culture substrates with cell-scale resolution, to provide HSPCs with mechanical support during ex vivo manipulation. This innovative 3D system improves HSPC multi-lineage differentiation and engraftment capacity by leveraging mechanobiological control over nuclear morphology, cytoskeleton organization, metabolism, and DNA integrity. Notably, 3D culture enables efficient genetic engineering across multiple platforms, including long-range gene editing, base- and prime-editing, and lentiviral-mediated gene addition. Moreover, this scaffold increases the clonal output and persistence of genetically engineered cells in xenotransplantation experiments, including a clinical protocol for lentiviral gene addition in Wiskott-Aldrich syndrome. Overall, we propose a transformative approach to enhance the efficacy and safety of emerging and established hematopoietic stem cell-based gene therapy applications.

Actin is one component of the intracellular cytoskeleton, forming filaments that are thin, flexible fibers approximately 7 nm in diameter and extending up to several micrometers in length. These filaments organize into higher-order assemblies, creating bundles of three-dimensional networks that behave like semisolid gels. Individual actin filaments (~7 nm) cannot be resolved with conventional light microscopy; however, super-resolution techniques such as STED, PALM, and STORM might increase resolution up to ~20-30 nm, enabling visualization of single filaments and fine structural details within cellular networks. For even higher resolution and ultrastructural detail, electron microscopy remains indispensable. Here, we describe STED procedures for imaging immunostained actin, including critical steps for laser alignment, a method for determining the actual spatial resolution, and criteria for selecting fluorophores and preparing samples.

Abstract High-grade B-cell lymphomas (HGBCL) encompass a heterogeneous group of aggressive B-cell malignancies. The most common genetically defined subtype, HGBCL with BCL2 and MYC rearrangements (HGBCL-DH-BCL2), is characterized by hallmark chromosomal translocations and poor prognosis, even in the era of improved chemo-immunotherapy, CAR-T cells, and antibody-drug conjugates1-3. Progress in this field is hindered by the lack of robust preclinical models that accurately capture the complex biology of the disease. Our recent work has reported recurrent silencing of the B cell receptor (BCR) in primary cases of HGBCL-DH-BCL2⁴. This phenotype is associated with gene expression programs characteristic of germinal center (GC) dark-zone (DZ) B cells4,5. Histological analyses have also established that HGBCL-DH-BCL2 (particularly those silencing the BCR), display an immune-cold microenvironment that closely resembles that of the GC dark zone5,6. Mutational analyses in HGBCL-DH-BCL2 have revealed recurrent gain-of-function mutations in the GC dark-zone determinant FOXO15. In addition, HGBCL-DH-BCL2 frequently inactivate the TP53 axis and share with Burkitt lymphoma expression of degron-resistant forms of CCND3 (i.e., CCND3T283A)5. HGBCL-DH-BCL2 is often preceded by follicular lymphoma (FL), characterized by BCL2 deregulation as its molecular hallmark. Finally, constitutive expression of an often mutant form of the MYC protein (i.e. MYCT58A) in a BCL2-rearranged FL or t(14;18)+ GC DZ B cell precursor represents the ultimate oncogenic event driving transformation to HGBCL-DH-BCL2. To model this transformation trajectory, we adapted a previously established retroviral transduction system7 for genetic engineering of primary human GC B cells. We constructed retroviral vectors encoding wild-type or mutant forms of lymphoma-relevant genes linked to fluorescent or surface markers for single-cell tracking. Primary GC B cells purified from human tonsils were first transduced with a four-gene cocktail (Oncomix-1) expressing BCL2, BCL6, FOXO1M1L, and dominant-negative TP53 (TP53DD). These genes cooperatively promoted survival and proliferation for > 4 weeks in vitro on CD40L/IL-21-expressing YK6 feeder cells. However, engineered cells remained dependent on exogenous stimulation and failed to form tumors in immunocompromised mice. To mimic the MYC oncogenic event driving HGBCL-DH-BCL2 onset, Oncomix-1-infected cells were superinfected with a second retroviral cocktail (Oncomix-2) expressing mutant MYCT58A and CCND3T283A. This step conferred mitogen independence, rapid proliferation in 2D culture, and tumor formation in NSG xenografts, hallmarks of transformation. In vivo tumors showed histological features of aggressive GCB-like lymphomas. Given that most HGBCL-DH-BCL2 silence their BCR, we sought to reproduce this phenotype. We engineered Oncomix-1-infected cells with an alternative Step-2 retroviral cocktail (Oncomix-3) combining MYCT58A and CCND3T283A, to wild-type KLHL6, a GC DZ-enriched BTB-domain adaptor that targets the BCR subunit CD79B for proteasomal degradation8,9. Deregulated KLHL6 expression supported the emergence of a rapidly expanding, BCR-negative GC immortalized B cell population. These cells showed a GC DZ-like immunophenotype and feeder-independent growth, closely mimicking the phenotype of BCR-silenced HGBCL-DH-BCL2. In summary, we established a stepwise viral transduction protocol to model the progressive transformation of human GC B cells into DZ–like HGBCL, driven by consecutive deregulation of the BCL2 and MYC oncoproteins. This model recapitulates key transcriptional, immunophenotypic, and oncogenic features of HGBCL-DH-BCL2 offering a versatile tool for mechanistic dissection (including immune evasion) and pharmacologic interrogation. References Melani C et al. N Engl J Med. 2024. doi:10.1056/NEJMoa2401532. Phina-Ziebin X et al. Blood Adv. 2025. doi:10.1182/bloodadvances.2024014732 Schneider M et al. Clin Lymphoma Myeloma Leuk. 2025. doi: 10.1016/j.clml.2024.08.010. Hilton LK et al. Blood. 2024. doi: 10.1182/blood.2024024251 Varano G et al. Blood Cancer Discov. 2025. doi: 10.1158/2643-3230.BCD-25-0099 Cancila V et al. J Clin Invest. 2025.doi: 10.1172/JCI187371 Caeser R et al. Nat Commun. 2019. Doi: 10.1038/s41467-019-12494-x Meriranta L et al. Blood Cancer Discov. 2024. doi: 10.1158/2643-3230.BCD-23-0182 Corcoran SR et al. Cancer Discov. 2024. doi: 10.1158/2159-8290.CD-23-0802

This work focused on generating a three-dimensional (3D) in vitro dynamic model to study chronic lymphocytic leukemia (CLL) cell dissemination, homing, and mechanisms of therapy resistance. We used a gelatin-based, hard porous biomaterial as a support matrix to develop 3D tissue-like models of the human lymph node and bone marrow, which were matured inside bioreactors under dynamic perfusion of medium. Comparing static and dynamic cultures of these 3D constructs revealed that perfusion promoted a tissue-like internal organization of cells, characterized by the expression of specific functional markers and deposition of an intricate extracellular matrix protein network. Recirculation of CLL cells within the dynamic system led to changes in leukemic cell behavior and in the expression of key markers involved in tumor progression. These findings suggest that the model is well suited for investigating the pathophysiological mechanisms of CLL and potentially other hematological malignancies.

Cells sense physical cues from their environment and convert them into biochemical responses through mechanotransduction. Unlike solid tumours, the role of such forces in haematological cancers is underexplored. In this context, immune cells experience dynamic mechanical stimuli as they migrate, extravasate and home to specific tissues. Understanding how these forces shape B-cell function and malignancy represents a groundbreaking area of research. This review examines the key mechanosensory pathways and molecules involved in lymphocyte mechanotransduction, beginning with mechanosensory proteins at the plasma membrane, followed by intracellular signal propagation through the cytoskeleton, eventually highlighting the nucleus as a 'signal actuator'. Subsequently, we cover some measurement approaches and advanced systems to investigate tumour biomechanics, highlighting their application in the context of B cells. Finally, we focus on the implications of mechanobiology in leukaemia, identifying molecules involved in B-cell malignancies that could serve as potential 'mechano-targets' for personalised therapies. This review emphasises the need to understand how lymphocytes generate, sense and respond to mechanical stimuli, which could open avenues for future biomedical innovations. Impact statement Our review is particularly valuable in highlighting the underexplored role of mechanobiology in B cell function and malignancies, while also discussing emerging techniques that can advance this research area. It bridges mechanotransduction, immunology, and cancer biology in a way that will be of interest to researchers across these three main fields.

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose-response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.

In vitro cell cultures are fundamental and necessary tools in cancer research and personalized drug discovery. Currently, most cells are cultured using two-dimensional (2D) methods, and drug testing is mainly performed in animal models. However, new and improved methods that implement three-dimensional (3D) cell-culturing techniques provide compelling evidence that more advanced experiments can be performed, yielding valuable new insights. In 3D cell-culture experiments, the cell environment can be manipulated to mimic the complexity and dynamicity of the human tissue microenvironment, possibly leading to more accurate representations of cell-to-cell interactions, tumor biology, and predictions of drug response. The 3D cell cultures can also potentially provide alternative ways to study hematological cancers and are expected to eventually bridge the gap between 2D cell culture and animal models. The present review provides an overview of the complexity of the lymphoid microenvironment and a summary of the currently used 3D models that aim at recreating it for hematological cancer research. We here dissect the differences and challenges between, and potential advantages of, different culture methods and present our vision of the most promising future strategies in the hematological field.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. It is known that the ability of lymphocytes to recirculate strongly depends on their capability to rapidly rearrange their cytoskeleton and adapt to external cues; however, little is known about the differences occurring between CLL and healthy B cells during these processes. To investigate this point, we applied a single-cell optical (super resolution microscopy) and nanomechanical approaches (atomic force microscopy, real-time deformability cytometry) to both CLL and healthy B lymphocytes and compared their behavior. We demonstrated that CLL cells have a specific actomyosin complex organization and altered mechanical properties in comparison to their healthy counterpart. To evaluate the clinical relevance of our findings, we treated the cells in vitro with the Bruton's tyrosine kinase inhibitors and we found for the first time that the drug restores the CLL cells mechanical properties to a healthy phenotype and activates the actomyosin complex. We further validated these results in vivo on CLL cells isolated from patients undergoing ibrutinib treatment. Our results suggest that CLL cells' mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance, thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells.

In this protocol, we describe how to generate 3D culture surrogates of chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) bone marrow microenvironments. We detail the use of culturing scaffolds populated with BM stromal cells and tumor cells in the RCCS™ bioreactor. This 3D culture can efficiently recapitulate tumor-stroma crosstalk and allows the testing of drugs such as ibrutinib and bortezomib. Moreover, this protocol can be used for the generation of other and more complex tumor microenvironments. For complete details on the use and execution of this protocol, please refer to Belloni et al. (2018) and Barbaglio et al. (2021).

Introduction: For decades, in vitro 2D cell culture techniques have been employed in research, but they fail to recapitulate the complexity of natural tissues. 3D bioprinting could potentially overcome this drawback due to the possibility to control the spatial disposition of living cells and the geometry of the 3D scaffold.Materials and methods: This study reports the design and characterization of a novel bioink for extrusion bioprinting, analyzing different blend formulations composed of alginate, gelatin, and methylcellulose, suitable as cell-laden bioink for lymphoid cells, in particular those isolated from patients with Chronic Lymphocytic Leukemia (CLL). The rheological properties as a function of temperature and the printability of the formulations were investigated to define the optimal printing parameters. In vitro stability of the printed scaffolds was investigated under culture conditions and compression tests were performed on printed and bioprinted scaffolds to compare their mechanical properties with those of fresh lymphoid tissue. Finally, MEC1, a CLL cell line, was bioprinted to investigate cell viability, cell density, and cell capability to be released from the scaffold over time.Results and discussion: Results showed that, for the selected blends, good shape fidelity and printing accuracy were achieved with a limitation on the number of printed layers. Scaffolds withstood culture conditions showing stability for up to 3 weeks and their mechanical properties were similar to those of lymphoid tissues already reported in the literature. High cell viability after 21 days was observed for both MEC1 and primary peripheral mononuclear cells, confirming the possibility to use the selected formulation to successfully bioprint lymphoid cells by possibly mimicking their native lymphoid microenvironment.

Experimental models and scientific questions require a choice of specific read-outs to allow the analysis of the results. Recently, the establishment of the three-dimensional (3D) bioprinting strategy in current laboratory practice has opened the issue to find effective and reproducible way to analyze tissue engineering constructs from the manufacturing point of view, as well as from the perspective of cells and tissues, in this context. We here report a summary of optimized read-out strategies that can be applied to analyze 3D bioprinted constructs.