Terapias inovadoras com RNA não-codificante para controlar o microambiente em osteossarcoma

The InOSTEO project strives to pioneer a cutting-edge molecular and cellular bioengineering approach, aiming to unravel the intricate role of ncRNAs in the OS microenvironment. With a specific focus on OC, the project seeks to identify, characterize and modulate the miRNA profile within the OS microenvironment, intending to leverage these findings as therapeutic tools to reshape cell behavior in the tumor environment. A key objective of the project is to establish, for the first time, a proof of concept for the use of miRNAs as effective modulators of OS behavior by regulating the profiles of non-malignant and non-immune cells, the OCs. This ambitious yet realistic proposal encompasses three scientific tasks (Tasks 1-3), each tailored to address specific aims, along with one dissemination task (Task 4) and one management task (Task 5). InOSTEO integrates diverse concepts from molecular biology, cell engineering, bioinformatics, oncology, orthopedics, and regenerative medicine. The specific objectives (SO) were stablish to address the challenges described in the previous section and include: SO1. Comprehensive analysis: Identify and characterize the miRNA transcriptional landscape at the single-cell resolution, creating a comprehensive mapping of the OC plasticity in the OS microenvironment. SO2. Functional exploration: Investigate the potential of miRNA-engineered OC to suppress OS behaviour and to overcome chemoresistance, in a co-culture system within a bone biomimetic platform. SO3. Bioengineered strategy: Develop a bone co-culture system to mimic the dynamic interactions between OC and OS, and the cell populations within the tumor microenvironment, including immune-cells, OB, endothelial cells. This will enable the pre-clinical validation the proposed miRNA-OC technology. To investigate SO1, the project will employ single-cell RNA sequencing (scRNA-seq) together with spatial transcriptomic technology. This innovative approach represents the first bioinformatics analysis conducted on OCs to evaluate their plasticity and miRNA profiles within clinical samples of OS. For SO2, building upon our previous findings, cultures of miRNA-engineered-OC and OS (chemoresistant versus chemosensitive) will be established on human bone substrates, and their real-time impact on OS progression and the structural effect extracellular matrix (ECM) will be assessed. Investigations will extend to the proteins released by the cells and the ECM, shedding light on their role in the tumor microenvironment. Finally, SO3 represent an in vitro preclinical proof-of-concept. A dynamic fluorescent multilabel system will be developed to replicate the intricate interplay between miRNA-engineered-OC, OS and surrounding bone microenvironment, including immune cells, MSC, OB, and endothelial cells. Both the cell behaviors and the molecular profile of each individual population will be assessed, uncovering additional potential targets for the after-phase project.