Periodic Reporting for period 1 - SMART-AML (SMART-AML: Shaping Marrow Adiposity to Redefine Treatment in Acute Myeloid Leukemia)
Período documentado: 2025-01-01 hasta 2026-12-31
Resumen del contexto y de los objetivos generales del proyecto
Acute Myeloid Leukemia (AML) is an aggressive hematologic malignancy characterized by the uncontrolled proliferation of myeloid cells, which hijack the bone marrow (BM) and compromise its physiological functions. AML prognosis remains poor, with <10% of patients above 60 years old surviving after 5 years. Recent discoveries have revealed the key role of the BM microenvironment in harboring and protecting leukemic stem cells from chemotherapy, contributing to AML relapse. Therefore, targeting the hijacked BM niche and transforming it into a “tumor-inhospitable” microenvironment represents a promising therapeutic strategy. In adults, bone marrow adipose tissue (BMAT) is the predominant component of the BM microenvironment, making it a potential target to render the niche inadequate for leukemic cells survival. Nevertheless, the role of BMAT in AML development, drug resistance, and survival are still controversial. The overall aim of SMART-AML was to identify AML cell-related changes within the BMAT/BM microenvironment and evaluate the potential of BMAT manipulation as a therapeutic strategy to sensitize leukemic cells to chemotherapy. To achieve this goal, the first focus was to exploit state-of-the-art bioprinting technologies to engineer a perfusable, humanized bone/BMAT 3D biomimetic microenvironment. Specifically, volumetric bioprinting was selected as the biofabrication technique because it allows the fabrication of cm3-scale constructs with complex geometries within seconds without compromising cell viability. To ensure the physiological relevance of the engineered BM environment model, it was initially evaluated whether the bioprinting process introduced any additional cellular stress or alteration in the cellular DNA, as such alterations could compromise the model’s baseline integrity, introducing artefactual changes and thereby limit the biological relevance of the system. Next, the biomaterials and bioprinting conditions for the engineering of the two different components of the BM, i.e. the bone and the BMAT, were selected. Finally, the model was completed by evaluating the attachment and survival of a blood cancer cell line in both compartments of the engineered BM niche. Future studies will focus on culturing patient-derived AML cells within the engineered platform to characterize the AML-induced BMAT changes in a controlled environment, with limited variables. By manipulating the BMAT it will be possible to determine the potential of this therapeutic approach to enhance the efficacy of chemotherapy on leukemic stem cells and reduce the chance of relapse. In addition to the value of the developed model for cancer research, the developed two-compartment platform could be used as a starting point for studying other pathological conditions where the relationship between bone and BMAT is key, such as osteoporosis.
Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha
Three main achievements can be identified in this project:
1) Established the biological impact of tomographic VBP on bioprinted cells.
Advances in biofabrication have driven a shift from largely homogeneous 3D constructs toward engineered tissues that more closely recapitulate the macro- and micro- organization of native tissues. Volumetric bioprinting was selected in this project because it allows the fabrication of generates centimeter-scale, cell-laden hydrogel constructs with complex architectures in a layerless manner within seconds. While it is a suitable technique to reach the architectural complexity and biomimicry planned for the humanized bone/BMAT 3D platform of SMART-AML, cytocompatibility is still commonly inferred from post-printing viability alone, which can overlook sublethal oxidative stress or genotoxicity relevant for the long-term function of 3D constructs. The presence of such effects would represent a confounding factor for subsequent biological interpretation. Thus, in this project an in-depth evaluation of the biological impact of tomographic VBP on mesenchymal stromal cells (MSCs), the multipotent cells that will be differentiated to produce the osteoblasts (bone cells) and adipocytes (fat cells) used in this model was performed. Here, I demonstrated that VBP is not only cytocompatible but also allow the preservation of key cellular functions beyond simple survival. Furthermore, by establishing a multiparametric framework that captures oxidative, genotoxic, and functional endpoints, this work supports more confident adoption of VBP for engineering tissue substitutes and predictive 3D models.
2) Selection of biomaterials and printing condition to engineer the bone and BMAT compartments.
The BM niche model developed in SMART-AML comprises two compartments: a bone compartment and a BMAT compartment. Mesenchymal stromal cells (MSCs) were selected as multipotent progenitors capable of giving rise to both tissues. However, their differentiation is strongly influenced by the physicochemical properties of the 3D hydrogels where they are embedded and cultured. Specifically, hydrogel porosity, which can be tune adjusting the biomaterials properties, and stiffness, which can be tuned by adjusting printing parameters such as light dose, are known to affect MSC differentiation. At the same time, printing parameters also influence the shape fidelity of the 3D constructs. For example, excessive light exposure may lead to overpolymerization and the formation of structures that do not accurately reproduce the original design. Therefore, the second major achievement of this project was the optimization of both biomaterial properties and VBP printing parameters to promote MSC differentiation toward osteoblasts and adipocytes while preserving the structural fidelity of the construct. This was essential to ensure proper integration of the model with the perfusion system required for the dynamic seeding of leukemic cells.
3) Proof of concept of BM niche model-blood cancer cell line interactions.
To date, most research has focused on the interactions between blood cancer cells and the endosteal and vascular BM niches. As a result, relatively few in vitro studies have investigated the culture of leukemic cells within a biomimetic BM model that includes a BMAT compartment. Therefore, the third major achievement of this project was the generation of preliminary data on cancer cell attachment and survival within the different compartments of the SMART-AML platform, together with the optimization of the culture conditions. This represented an essential preparatory step before progressing to experiments with patient-derived leukemic blasts.
1) Established the biological impact of tomographic VBP on bioprinted cells.
Advances in biofabrication have driven a shift from largely homogeneous 3D constructs toward engineered tissues that more closely recapitulate the macro- and micro- organization of native tissues. Volumetric bioprinting was selected in this project because it allows the fabrication of generates centimeter-scale, cell-laden hydrogel constructs with complex architectures in a layerless manner within seconds. While it is a suitable technique to reach the architectural complexity and biomimicry planned for the humanized bone/BMAT 3D platform of SMART-AML, cytocompatibility is still commonly inferred from post-printing viability alone, which can overlook sublethal oxidative stress or genotoxicity relevant for the long-term function of 3D constructs. The presence of such effects would represent a confounding factor for subsequent biological interpretation. Thus, in this project an in-depth evaluation of the biological impact of tomographic VBP on mesenchymal stromal cells (MSCs), the multipotent cells that will be differentiated to produce the osteoblasts (bone cells) and adipocytes (fat cells) used in this model was performed. Here, I demonstrated that VBP is not only cytocompatible but also allow the preservation of key cellular functions beyond simple survival. Furthermore, by establishing a multiparametric framework that captures oxidative, genotoxic, and functional endpoints, this work supports more confident adoption of VBP for engineering tissue substitutes and predictive 3D models.
2) Selection of biomaterials and printing condition to engineer the bone and BMAT compartments.
The BM niche model developed in SMART-AML comprises two compartments: a bone compartment and a BMAT compartment. Mesenchymal stromal cells (MSCs) were selected as multipotent progenitors capable of giving rise to both tissues. However, their differentiation is strongly influenced by the physicochemical properties of the 3D hydrogels where they are embedded and cultured. Specifically, hydrogel porosity, which can be tune adjusting the biomaterials properties, and stiffness, which can be tuned by adjusting printing parameters such as light dose, are known to affect MSC differentiation. At the same time, printing parameters also influence the shape fidelity of the 3D constructs. For example, excessive light exposure may lead to overpolymerization and the formation of structures that do not accurately reproduce the original design. Therefore, the second major achievement of this project was the optimization of both biomaterial properties and VBP printing parameters to promote MSC differentiation toward osteoblasts and adipocytes while preserving the structural fidelity of the construct. This was essential to ensure proper integration of the model with the perfusion system required for the dynamic seeding of leukemic cells.
3) Proof of concept of BM niche model-blood cancer cell line interactions.
To date, most research has focused on the interactions between blood cancer cells and the endosteal and vascular BM niches. As a result, relatively few in vitro studies have investigated the culture of leukemic cells within a biomimetic BM model that includes a BMAT compartment. Therefore, the third major achievement of this project was the generation of preliminary data on cancer cell attachment and survival within the different compartments of the SMART-AML platform, together with the optimization of the culture conditions. This represented an essential preparatory step before progressing to experiments with patient-derived leukemic blasts.
Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)
The main results of the project and structured and presented around the three key achievements outlined above.
1) Established the biological impact of tomographic VBP on bioprinted cells.
I evaluated the short-term (1 day post-printing) and long-term (7 days post-printing) expression of antioxidant genes (i.e. NRF2/SOD1/HMOX1 transcription), the presence of intracellular reactive oxygen species (ROS), and extracellular H2O2. Furthermore, I evaluated changes in the expression of genes involved in DNA repair signaling pathways and the formation of γH2AX and 53BP1 foci, which are markers for DNA double strand break and repair. Bioprinted samples were compared to 3D cast constructs and MSC grown in 2D on tissue plastic, to benchmark printing outcomes against standard tissue-engineering practice and routine culture handling. Post printing and over the course of the experiment, cells retained a high cell viability (>90%) and displayed no activation of apoptotic pathways (programmed cell death) compared to the cast control. No additional activation of oxidative stress pathways or ROS production was detected in the VBP samples compared to the 3D and 2D controls. Similarly, no variation the expression of genes involved in the DNA repair signaling pathways or in foci formation was observed compared to the baseline levels expected in cultured MSCs. Together, these data demonstrate that tomographic VBP is not only cytocompatible but also allows the preservation of cellular homeostasis beyond simple survival.
2) Selection of biomaterials and printing condition to engineer the bone and BMAT compartments.
For the BMAT compartment, the use of a porous microresin composed of jammed microgels of methacrylated gelatin (GelMA) was investigated. First, the protocol for the formation of the microresin was optimized by changing the milling time of thermally gelated (solid) GelMA to achieve microparticles of different size. A milling time of 65 seconds was selected as it allowed the formation of a porous gel, while not significantly altering the physical properties of the hydrogels. Next, MSCs adipogenic differentiation was assessed, and was enhanced in the hydrogels composed by jammed microgels compared to bulk constructs. For the bone compartment, bulk GelMA was selected. Multiple VBP printing parameters (i.e. light doses) were compared in order to the define the one required to achieve high shape fidelity while not hampering MSCs’ osteogenic differentiation. Finally, osteogenic differentiation was confirmed with multiple MSC donors, and the one displaying the highest mineralization (bone matrix) was selected for further analysis.
3) Proof of concept of BM niche model-blood cancer cell line interactions.
A cell line was initially used to evaluate the survival and attachment of the cancer cell in the different compartments of the model. Cancer cells had a low viability (<10%) when embedded directly in the hydrogel mix prior to the crosslinking that was necessary to form a solid 3D construct. Higher viability was observed when cells were seeded on top of the bulk or microgel-based hydrogels (>50%), which resembles the dynamic seeding that would occur in the bioreactor system. After assessing cell survival, their capability of interacting with the bone and BMAT compartment was evaluated. Cancer cells were able to attach to both compartments and their short-term viability was confirmed. Interestingly, the presence of cells, either adipocytes or osteoblasts, was necessary to enhance cancer cell-construct interaction, as limited attachment was observed to the empty GelMA hydrogels.
Overall, the findings of this project validate the high cytocompatibility of volumetric bioprinting and highlight its potential for regenerative medicine applications as well as for the development of biomimetic in vitro models. More specifically, this work established the feasibility of using VBP to engineer a humanized bone marrow niche model comprising both bone and BMAT compartments. These results lay the groundwork for future studies involving patient-derived AML cells and provide a 3D platform to investigate the contribution of BMAT to leukemic cell survival and resistance to chemotherapeutic treatment.
1) Established the biological impact of tomographic VBP on bioprinted cells.
I evaluated the short-term (1 day post-printing) and long-term (7 days post-printing) expression of antioxidant genes (i.e. NRF2/SOD1/HMOX1 transcription), the presence of intracellular reactive oxygen species (ROS), and extracellular H2O2. Furthermore, I evaluated changes in the expression of genes involved in DNA repair signaling pathways and the formation of γH2AX and 53BP1 foci, which are markers for DNA double strand break and repair. Bioprinted samples were compared to 3D cast constructs and MSC grown in 2D on tissue plastic, to benchmark printing outcomes against standard tissue-engineering practice and routine culture handling. Post printing and over the course of the experiment, cells retained a high cell viability (>90%) and displayed no activation of apoptotic pathways (programmed cell death) compared to the cast control. No additional activation of oxidative stress pathways or ROS production was detected in the VBP samples compared to the 3D and 2D controls. Similarly, no variation the expression of genes involved in the DNA repair signaling pathways or in foci formation was observed compared to the baseline levels expected in cultured MSCs. Together, these data demonstrate that tomographic VBP is not only cytocompatible but also allows the preservation of cellular homeostasis beyond simple survival.
2) Selection of biomaterials and printing condition to engineer the bone and BMAT compartments.
For the BMAT compartment, the use of a porous microresin composed of jammed microgels of methacrylated gelatin (GelMA) was investigated. First, the protocol for the formation of the microresin was optimized by changing the milling time of thermally gelated (solid) GelMA to achieve microparticles of different size. A milling time of 65 seconds was selected as it allowed the formation of a porous gel, while not significantly altering the physical properties of the hydrogels. Next, MSCs adipogenic differentiation was assessed, and was enhanced in the hydrogels composed by jammed microgels compared to bulk constructs. For the bone compartment, bulk GelMA was selected. Multiple VBP printing parameters (i.e. light doses) were compared in order to the define the one required to achieve high shape fidelity while not hampering MSCs’ osteogenic differentiation. Finally, osteogenic differentiation was confirmed with multiple MSC donors, and the one displaying the highest mineralization (bone matrix) was selected for further analysis.
3) Proof of concept of BM niche model-blood cancer cell line interactions.
A cell line was initially used to evaluate the survival and attachment of the cancer cell in the different compartments of the model. Cancer cells had a low viability (<10%) when embedded directly in the hydrogel mix prior to the crosslinking that was necessary to form a solid 3D construct. Higher viability was observed when cells were seeded on top of the bulk or microgel-based hydrogels (>50%), which resembles the dynamic seeding that would occur in the bioreactor system. After assessing cell survival, their capability of interacting with the bone and BMAT compartment was evaluated. Cancer cells were able to attach to both compartments and their short-term viability was confirmed. Interestingly, the presence of cells, either adipocytes or osteoblasts, was necessary to enhance cancer cell-construct interaction, as limited attachment was observed to the empty GelMA hydrogels.
Overall, the findings of this project validate the high cytocompatibility of volumetric bioprinting and highlight its potential for regenerative medicine applications as well as for the development of biomimetic in vitro models. More specifically, this work established the feasibility of using VBP to engineer a humanized bone marrow niche model comprising both bone and BMAT compartments. These results lay the groundwork for future studies involving patient-derived AML cells and provide a 3D platform to investigate the contribution of BMAT to leukemic cell survival and resistance to chemotherapeutic treatment.