Protocol for generation of 3D bone marrow surrogate microenvironments in a rotary cell culture system

Belloni, Daniela; Ferrarini, Marina; Ferrero, Elisabetta; Guzzeloni, Virginia; Barbaglio, Federica; Ghia, Paolo; Scielzo, Cristina

doi:10.1016/j.xpro.2022.101601

Cited by 7 publications

(6 citation statements)

References 10 publications

(16 reference statements)

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“… 5 To evaluate the effect of defactinib in the presence of leukaemia‐stroma interactions, we exploited the traditional 2D culture, thus seeding CLL cells in the presence of HS5 cells (4 cases), treating with 4 μM defactinib and collecting them after 48 h for flow cytometry analysis, where we used the Annexin V‐PI Kit to evaluate cell death and we could not detect any significant protective effect at this concentration and time point (Figure S1 A). To test the ability of defactinib to mobilize CLL cells we took advantage of our established 3D in vitro model with a RCCS™ bioreactor, 25 , 32 which allowed us to study the mobilization and retention effects of defactinib in a scaffold in dynamic conditions ensured by the rotating vessels. The Spongostan™ scaffolds, made of gelatine, have a structure similar to the trabecular bone in the BM and they were populated with HS5 and CLL cells ( n = 4).…”

Section: Resultsmentioning

confidence: 99%

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Sbrana

Fiordi

Bordini

et al. 2023

J Cellular Molecular Medi

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Chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in the western world within adults. It is a lymphoproliferative disorder involving progressive accumulation of mature CD19 + CD5 + B lymphocytes in the Peripheral Blood (PB), Bone Marrow (BM) and other lymphoid organs such as the Spleen (SP) and Lymph Nodes (LN). 1 The clinical features of CLL show high heterogeneity among patients; this variability could be explained by both intrinsic factors (e.g. genetics and epigenetics) and external stimuli. Despite the introduction of new effective targeted therapies such as BTK (e.g. ibrutinib, acalabrutinib) and BCL-2 (e.g. venetoclax) inhibitors, a majority of patients eventually relapse, so CLL still remains an incurable disease. 2,3 A major role in the development of the disease and the onset of drug resistance is played by the crosstalk between the malignant clone and the Tumour Microenvironment (TME). Malignant B cells recirculate between lymphoid organs and the bloodstream but while

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Section: Resultsmentioning

confidence: 99%

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Sbrana

Fiordi

Bordini

et al. 2023

J Cellular Molecular Medi

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“…The 3D scaffolds were populated according to protocol described by Belloni et al [ 19 ] and adapted to MM cells growth in static conditions. Briefly, scaffold discs were cut from SpongostanTM sheets (Cod.…”

Section: Methodsmentioning

confidence: 99%

Euchromatic Histone Lysine Methyltransferase 2 Inhibition Enhances Carfilzomib Sensitivity and Overcomes Drug Resistance in Multiple Myeloma Cell Lines

Mereu

Abbo

Paradžik

et al. 2023

Cancers

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Proteasome inhibitors (PIs) are extensively used for the therapy of multiple myeloma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. In addition, adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. Here, to identify compounds that can increase the efficacy of PIs, we performed a functional screening using a library of small-molecule inhibitors covering key signaling pathways. Among the best synthetic lethal interactions, the euchromatic histone-lysine N-methyltransferase 2 (EHMT2) inhibitor UNC0642 displayed a cooperative effect with carfilzomib (CFZ) in numerous multiple myeloma (MM) cell lines, including drug-resistant models. In MM patients, EHMT2 expression correlated to worse overall and progression-free survival. Moreover, EHMT2 levels were significantly increased in bortezomib-resistant patients. We demonstrated that CFZ/UNC0642 combination exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone-marrow-derived stromal cells. To exclude off-target effects, we proved that UNC0642 treatment reduces EHMT2-related molecular markers and that an alternative EHMT2 inhibitor recapitulated the synergistic activity with CFZ. Finally, we showed that the combinatorial treatment significantly perturbs autophagy and the DNA damage repair pathways, suggesting a multi-layered mechanism of action. Overall, the present study demonstrates that EHMT2 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients.

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“…66 We recently demonstrated the feasibility of recreating a surrogate 3D MM and CLL microenvironment able to reproduce the functional interactions of the native BM. 20,50,67 We developed a robust technology based on the integrated use of cell-repopulated scaffolds (BM stromal cells) and the microgravity RCCS bioreactor. This system provides a balance between increased mass transfer and reduced shear stress, and this dynamic bioreactor generates optimal conditions for long-term ex vivo maintenance of tissue explants.…”

Section: Advantages Of Dynamic Culturesmentioning

confidence: 99%

“…We recently demonstrated the feasibility of recreating a surrogate 3D MM and CLL microenvironment able to reproduce the functional interactions of the native BM 20 , 50 , 67 . We developed a robust technology based on the integrated use of cell‐repopulated scaffolds (BM stromal cells) and the microgravity RCCS bioreactor.…”

Section: Advantages Of Dynamic Culturesmentioning

confidence: 99%

Emerging Strategies in 3D Culture Models for Hematological Cancers

Barozzi

Scielzo

2023

HemaSphere

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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.

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Protocol for generation of 3D bone marrow surrogate microenvironments in a rotary cell culture system

Cited by 7 publications

References 10 publications

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Euchromatic Histone Lysine Methyltransferase 2 Inhibition Enhances Carfilzomib Sensitivity and Overcomes Drug Resistance in Multiple Myeloma Cell Lines

Emerging Strategies in 3D Culture Models for Hematological Cancers

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