Estudio de la reconstitución inmunológica en pacientes con neoplasias hematológicas sometidos a trasplante de sangre de cordón umbilical

El trasplante de sangre de cordón umbilical (TSCU) ha demostrado ser un procedimiento alternativo a otros tipos de trasplante alogénico en pacientes adultos con neoplasias hematológicas que no disponen de un donante familiar HLA-idéntico. Una de las principales limitaciones del TSCU es el retraso en la reconstitución inmunológica que expone a los pacientes a sufrir graves complicaciones relacionadas fundamentalmente con infecciones oportunistas y enfermedad injerto contra huésped (EICH). La citometría de flujo multiparamétrica (CFM) ha propiciado el desarrollo de numerosos estudios en el área de los trasplantes alogénicos. En concreto, en el TSCU ha favorecido una compresión más profunda de la dinámica y los mecanismos de la reconstitución inmunológica. El conocimiento de su inmunobiología podría facilitar el seguimiento de los pacientes y el diseño de nuevas estrategias terapéuticas orientadas a controlar las complicaciones que surgen en el periodo postrasplante, lo que contribuiría a mejorar la supervivencia y la calidad de vida de los pacientes que reciben un TSCU como única posibilidad de curación. En esta tesis se analizaron los resultados de las poblaciones linfocitarias obtenidos mediante CFM en diferentes momentos postrasplante en una serie de 221 pacientes adultos tratados con regímenes de acondicionamiento mieloablativo y trasplantados con sangre de cordón umbilical en el Hospital Universitario y Politécnico La Fe de Valencia. Se realizó un análisis descriptivo de las poblaciones linfocitarias para establecer la dinámica de su recuperación a lo largo del periodo de estudio. Se analizaron los factores pronóstico de reconstitución inmunológica y se estudió el impacto de ésta sobre la mortalidad relacionada con el trasplante y la supervivencia global. La dinámica de reconstitución inmunológica tras el TSCU en esta serie de pacientes muestra una rápida recuperación de las cifras absolutas de células NK, un lento restablecimiento del recuento de linfocitos B y un retraso importante en la normalización de los valores de los diferentes subtipos de linfocitos T, especialmente evidente para los linfocitos T CD4+. Se observan diferencias en la recuperación de los valores absolutos de los linfocitos T CD8+, las células NK y los linfocitos B en función de la presencia o ausencia de seropositividad para citomegalovirus (CMV). La normalización del recuento absoluto de los linfocitos B experimenta un retraso en los pacientes con diagnóstico de linfoma o síndrome linfoproliferativo. El recuento absoluto de linfocitos T es significativamente menor en los pacientes que desarrollaron EICH aguda y el tratamiento empleado para la profilaxis de la EICH presenta diferencias en la recuperación de los linfocitos T y T CD4+. Por último, la recuperación del número absoluto de linfocitos T y B se asocia directamente a la supervivencia global e inversamente a la mortalidad relacionada con el trasplante. La información aportada en esta tesis permitirá mejorar nuestro conocimiento sobre el estado inmunológico de los pacientes sometidos a TSCU. Son necesarios estudios prospectivos funcionales y de poblaciones minoritarias en un mayor número de pacientes que contribuyan a ampliar la información sobre la reconstitución inmunológica en este tipo de trasplante. La aplicación de estos análisis podría favorecer el desarrollo de terapias inmunomoduladoras para el manejo de las complicaciones relacionadas con este procedimiento, acortando el retraso en la recuperación inmunológica y mejorando la supervivencia de los pacientes con TSCU

Novel microgel culture system as semi-solid three-dimensional in vitro model for the study of multiple myeloma proliferation and drug resistance

[EN] Multiple myeloma (MM) is a hematological malignancy in which the patient's drug resistance is one of the main clinical problems. As 2D cultures do not recapitulate the cellular microenvironment, which has a key role in drug resistance, there is an urgent need for better biomimetic models. Here, a novel 3D platform is used to model MM. The semi-solid culture consists of a dynamic suspension of microspheres and MM cells, termed as microgel. Microspheres are synthesized with acrylic polymers of different sizes, compositions, and functionalities (fibronectin or hyaluronic acid). Optimal conditions for the platform in terms of agitation speed and microsphere size have been determined. With these parameters the system allows good proliferation of the MM cell lines RPMI8226, U226, and MM1.S. Interestingly, when used for drug resistance studies, culture of the three MM cell lines in microgels showed close agreement in revealing the role of acrylic acid in resistance to anti-MM drugs such as dexamethasone and bortezomib. This work presents a unique platform for the in vitro modeling of non-solid tumors since it allows keeping non-adherent cells in suspension conditions but in a 3D context that can be easily tuned with different functionalizations.This work was funded by the Spanish State Research Agency (AEI) through the PID2019-106099RB-C41/AEI/10.13039/501100011033 Project. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER Actions were financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was also supported by the Spanish Ministry of Science, Innovation and Universities through Grant NO FPU17/05810 awarded to Sandra Clara-Trujillo. The Microscopy Service of the UPV (UniversitatPolitecnica de València) is gratefully acknowledged for helping with FESEM characterization.Clara-Trujillo, S.; Tolosa, L.; Cordón, L.; Sempere, A.; Gallego Ferrer, G.; Gómez Ribelles, JL. (2022). Novel microgel culture system as semi-solid three-dimensional in vitro model for the study of multiple myeloma proliferation and drug resistance. Biomaterials Advances. 135:1-13. https://doi.org/10.1016/j.bioadv.2022.21274911313

Biomimetic microspheres for 3D mesenchymal stem cell culture and characterization

[EN] Stem cells reside in niches, specialized microenvironments that sustain and regulate their fate. Extracellular matrix (ECM), paracrine factors or other cells are key niche regulating elements. As the conventional 2D cell culture lacks these elements, it can alter the properties of naive stem cells. In this work we designed a novel biomimetic microenvironment for cell culture, consisting of magnetic microspheres, prepared with acrylates and acrylic acid copolymers and functionalized with fibronectin or hyaluronic acid as ECM coatings. To characterize cell proliferation and adhesion, porcine mesenchymal stem cells (MSCs) were grown with the different microspheres. The results showed that the 3D environments presented similar proliferation to the 2D culture and that fibronectin allows cell adhesion, while hyaluronic acid hinders it. In the 3D environments, cells reorganize the microspheres to grow in aggregates, highlighting the advantages of microspheres as 3D environments and allowing the cells to adapt the environment to their requirements.PROMETEO/2016/063 project is acknowledged. This work was partially financed with FEDER funds (CIBERONC (CB16/12/00284)). The CIBER-BBN initiative is funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was also supported by the Spanish Ministry of Science, Innovation and Universities trough Sandra Clara-Trujillo FPU17/05810 grant.Clara-Trujillo, S.; Marin-Paya, JC.; Cordón, L.; Sempere, A.; Gallego Ferrer, G.; Gómez Ribelles, JL. (2019). Biomimetic microspheres for 3D mesenchymal stem cell culture and characterization. Colloids and Surfaces B Biointerfaces. 177:68-76. https://doi.org/10.1016/j.colsurfb.2019.01.050S687617

CYP2C8 gene polymorphism and bisphosphonate-related osteonecrosis of the jaw in patients with multiple myeloma

Osteonecrosis of the jaw is an uncommon but potentially serious complication of bisphosphonate therapy in multiple myeloma. Previous studies showed that the presence of one or two minor alleles of the cytochrome P450, subfamily 2C polypeptide 8 gene (CYP2C8) polymorphism rs1934951 was an independent prognostic marker associated with development of osteonecrosis of the jaw in multiple myeloma patients treated with bisphosphonates. The aim of this study was to validate the frequency of SNP rs193451 in 79 patients with multiple myeloma. In 9 (22%) patients developing osteonecrosis of the jaw, a heterozygous genotype was found, in contrast with those who did not develop osteonecrosis of the jaw (n=4, 11%) or healthy individuals (n=6, 13%). We found no differences in the cumulative risk of developing osteonecrosis of the jaw between patients homozygous and heterozygous for the major allele. We were unable to confirm a significant association between this polymorphism and the risk of developing osteonecrosis of the jaw

Protein-Functionalized Microgel for Multiple Myeloma Cells’ 3D Culture

Multiple myeloma is a hematologic neoplasm caused by an uncontrolled clonal proliferation of neoplastic plasma cells (nPCs) in the bone marrow. The development and survival of this disease is tightly related to the bone marrow environment. Proliferation and viability of nPCs depend on their interaction with the stromal cells and the extracellular matrix components, which also influences the appearance of drug resistance. Recapitulating these interactions in an in vitro culture requires 3D environments that incorporate the biomolecules of interest. In this work, we studied the proliferation and viability of three multiple myeloma cell lines in a microgel consisting of biostable microspheres with fibronectin (FN) on their surfaces. We also showed that the interaction of the RPMI8226 cell line with FN induced cell arrest in the G0/G1 cell cycle phase. RPMI8226 cells developed a significant resistance to dexamethasone, which was reduced when they were treated with dexamethasone and bortezomib in combination

Protein-Functionalized Microgel for Multiple Myeloma Cells’ 3D Culture

Multiple myeloma is a hematologic neoplasm caused by an uncontrolled clonal proliferation of neoplastic plasma cells (nPCs) in the bone marrow. The development and survival of this disease is tightly related to the bone marrow environment. Proliferation and viability of nPCs depend on their interaction with the stromal cells and the extracellular matrix components, which also influences the appearance of drug resistance. Recapitulating these interactions in an in vitro culture requires 3D environments that incorporate the biomolecules of interest. In this work, we studied the proliferation and viability of three multiple myeloma cell lines in a microgel consisting of biostable microspheres with fibronectin (FN) on their surfaces. We also showed that the interaction of the RPMI8226 cell line with FN induced cell arrest in the G0/G1 cell cycle phase. RPMI8226 cells developed a significant resistance to dexamethasone, which was reduced when they were treated with dexamethasone and bortezomib in combination.CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, and Consolider Program. CIBER Actions were financed by the Instituto de Salud Carlos III, with assistance from the European Regional Development Fund. The kind supplying of RPMI 8226 cells by Beatriz Martin (Josep Carreras Leukaemia Research Institute) is greatly acknowledged. The Microscopy Service of the UPV (Universitat Politècnica de València) is gratefully acknowledged.Marín Pallá, JC.; Clara Trujillo, S.; Cordón, L.; Gallego Ferrer, G.; Sempere, A.; Gómez Ribelles, JL. (2022). Protein-Functionalized Microgel for Multiple Myeloma Cells’ 3D Culture. MDPI. https://doi.org/10.4995/Dataset/10251/18995

Biomimetic 3D Environment Based on Microgels as a Model for the Generation of Drug Resistance in Multiple Myeloma

The development of three-dimensional environments to mimic the in vivo cellular response is a problem in the building of disease models. This study aimed to synthesize and validate three-dimensional support for culturing monoclonal plasma cells (mPCs) as a disease model for multiple myeloma. The three-dimensional environment is a biomimetic microgel formed by alginate microspheres and produced on a microfluidic device whose surface has been functionalized by a layer-by-layer process with components of the bone marrow’s extracellular matrix, which will interact with mPC. As a proof of concept, RPMI 8226 cell line cells were cultured in our 3D culture platform. We proved that hyaluronic acid significantly increased cell proliferation and corroborated its role in inducing resistance to dexamethasone. Despite collagen type I having no effect on proliferation, it generated significant resistance to dexamethasone. Additionally, it was evidenced that both biomolecules were unable to induce resistance to bortezomib. These results validate the functionalized microgels as a 3D culture system that emulates the interaction between tumoral cells and the bone marrow extracellular matrix. This 3D environment could be a valuable culture system to test antitumoral drugs efficiency in multiple myeloma.Marin Paya, JC.; Díaz Benito, B.; Amaro Martins, L.; Clara Trujillo, S.; Cordón, L.; Lanceros-Mendez, S.; Gallego Ferrer, G.... (2021). Biomimetic 3D Environment Based on Microgels as a Model for the Generation of Drug Resistance in Multiple Myeloma. Universitat Politècnica de València. https://doi.org/10.4995/Dataset/10251/17831

Biomimetic 3d environment based on microgels as a model for the generation of drug resistance in multiple myeloma

The development of three-dimensional environments to mimic the in vivo cellular response is a problem in the building of disease models. This study aimed to synthesize and validate three-dimensional support for culturing monoclonal plasma cells (mPCs) as a disease model for multiple myeloma. The three-dimensional environment is a biomimetic microgel formed by alginate microspheres and produced on a microfluidic device whose surface has been functionalized by a layer-by-layer process with components of the bone marrow’s extracellular matrix, which will interact with mPC. As a proof of concept, RPMI 8226 cell line cells were cultured in our 3D culture platform. We proved that hyaluronic acid significantly increased cell proliferation and corroborated its role in inducing resistance to dexamethasone. Despite collagen type I having no effect on proliferation, it generated significant resistance to dexamethasone. Additionally, it was evidenced that both biomolecules were unable to induce resistance to bortezomib. These results validate the functionalized microgels as a 3D culture system that emulates the interaction between tumoral cells and the bone marrow extracellular matrix. This 3D environment could be a valuable culture system to test antitumoral drugs efficiency in multiple myelomaFunding by PROMETEO/2016/063 project is acknowledged. This work was partially financed with FEDER funds (CIBERONC (CB16/12/00284)). The CIBER-BBN initiative is funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fun