Authors
Margherita Pauri, Riccardo Pinos, Federica Barbaglio, Giulia Maria Di Gravina, Lydia Scarfò, Emanuela Sant’antonio, Paolo Ghia, MD, Michele Conti, Cristina Scielzo.
Background
The trafficking of malignant B cells between peripheral blood, bone marrow, and lymph node is pivotal for Chronic Lymphocytic Leukemia (CLL) cell survival, proliferation, and resistance to therapies. However, the mechanisms governing intra and extravasation from lymphoid tissues remain poorly understood, one reason being the absence of models able to capture these dynamic interactions in vitro. We propose a system to study leukemic cells trafficking using an in-house designed and 3D printed bioreactor (Vesselbox) which perfuses a 3D bioprinted vascularized scaffold.
Methods
The scaffold includes an external layer, mimicking the lymphoid tissue microenvironment and composed of Human Lymphatic Fibroblasts (HLF) and an inner layer, vascularized by Human Umbilical Vein Endothelial Cells (HUVEC) and Bone Marrow – Mesenchymal Stem Cells (BM-MSC). First, the external layer is bioprinted followed by the casting the inner layer. The scaffold is allocated in the VesselBox for selective perfusion. A peristaltic pump enables controlled medium and cells recirculation. MEC1-GFP (CLL cell line) and primary CLL cells have been circulated in the system and immunophenotype analysis has been performed after 1, 3, and 7 days of circulation to assess changes in markers expression in presence or absence of the 3D bioprinted microenvironment or drugs.
Results
After biofabrication, the scaffolds are cultured in static condition for 14 days to allow cell organization and vascular lumen formation. Immunofluorescence confirmed the expression of endothelial markers (CD31, Ve-Cadherin, VWF), revealing a vascularized network. In the external layer, cells appear elongated and homogenously distributed. Considering the positive computational simulations on oxygen diffusion, pressure distribution, and shear stress, the scaffolds were perfused in the VesselBox. We used MEC1 cells to set up all the culture settings. Primary CLL cells (n=3) were added to circulating medium and their extravasation and immunophenotype were monitored through confocal microscopy and flow cytometry at 1, 3, and 7 days of recirculation. We observed that cells can extravasate in the scaffold and a CXCR4high/CD5high population emerges (24%) when cells circulate in the presence of the microenvironment. This subset is associated with tissue homing (Burger JA et al., Br J Haematol., 2007) and has been described as highly proliferative (Friedman et al., Hemasphere, 2024). Consistent with these results, CD23 expression increased when cells circulate in the presence of the microenvironment (67%) compared to the empty bioreactor (52%), potentially indicating cell activation and growth (Lampert I A et al., Hum Pathol., 1999). Moreover, CD62L+ cells increase with the microenvironment (38%) compared to its absence (2%) suggesting a tissue-homing phenotype, as CD62L has a role in the extravasation of CLL cells (Lafouresse F et al., Blood, 2015). CXCR4 and CD62L increase was also confirmed by RT-PCR, showing coherent results.
We are currently validating the system for drug testing. In this setting, CLL cells are 3D bioprinted in the external layer together with HLF. Then, treatment with BTK-inhibitors (i.e. ibrutinib) or BCL2-antagonists is performed by adding the drug to the circulating medium meaning that the drugs have to pass the endothelial lumen to reach CLL cells. So far, we confirmed that the drugs can pass the barrier and preliminary results show that ibrutinib mobilizes cells from the microenvironment as expected towards the circulation, leading to a reduction of the number of cells in the scaffold. Additionally, cells in the untreated scaffolds form aggregates and clusters, which are reduced in number and dimension by ibrutinib treatment. Finally, ibrutinib leads to a downregulation of CXCR4, mimicking what occurs in vivo (Chen SS, et al., Leukemia, 2016).
Conclusion
We demonstrated that the VesselBox device enables the investigation of CLL cells dissemination ex vivo, supporting the study CLL dynamic interactions with the microenvironment, their extravasation through the endothelial barrier and the colonization of the tissue-specific microenvironment in real-time. These features make the device suitable for elucidating the cross-talk between CLL cells and the endothelium also in comparison with healthy B cells. Additionally, the VesselBox provides a controlled platform to investigate the function and relevance of specific cell subsets, such as CXCR4high/CD5high which dynamic regulation in vivo remains unclear. We are exploiting the system to characterize CLL phenotype during the response to therapies. Moreover, this set-up is adaptable for other applications by customizing both microenvironment and the endothelial barrier, potentially enabling the study of other malignancies.
Keywords : CLL trafficking, vascularized microenvironment, ex vivo modelling
Please indicate how this research was funded.: EHA Advanced Research Grant 2020. Associazione Italiana per la Ricerca sul Cancro AIRC under IG 2018 – ID. 21332 and Special Program on Metastatic Disease – 5 per mille #2119.
Please indicate the name of the funding organization. : EHA. AIRC.