STUDY OF THE EFFECT OF TUMOUR EDUCATED MACROPHAGES ON NEURAL CELLS

Background: Traumatic injuries to the brain and spinal cord, including spinal cord injury (SCI), represent devastating conditions that lead to permanent loss of function, mainly due to the poor intrinsic regenerative capacity of the central nervous system (CNS). At the lesion site, the hostile microenvironment which develops within the CNS prevents regeneration. Current therapeutic strategies for traumatic CNS injury aim at patient stabilization and symptoms management, however without promoting neural tissue regeneration. Ongoing studies focus on the development of both acellular and cell-based therapies to allow neuroprotection, improve cell survival, and modulate the pro-inflammatory microenvironment. However, they proved very poor functional improvement in clinical trials. Given the complexity of the lesion formed following a loss of neural tissue, effective treatments would require a multi-targeted strategy capable to both reprogram the hostile injured microenvironment and enhance CNS regeneration. Hypothesis: We hypothesised that Tumour-associated macrophages (TAMs), a distinctive cell type within the tumour microenvironment, could represent an innovative therapeutic approach for neuro-regenerative medicine. Indeed, TAMs possess a unique set of properties, including immune suppression, extracellular matrix remodelling, and neoangiogenesis, that, while detrimental in cancer, could be harnessed for promoting tissue regeneration following neurotrauma. Moreover, studies conducted in our laboratory further identified that TAMs are endowed with a strong nerve growth property both in vitro and in vivo in a mouse model of severe contusive compressive SCI (scSCI). In the light of that, my PhD project aimed to characterize in vitro generated TAMs’ biological and functional properties as well as to study the key molecular mechanisms underlying such neuro-regenerative activities. Methods: To achieve these goals, I performed a multi-level in vitro characterization, including transcriptomic, phenotypical and secretome analysis of in vitro generated TAMs, generated both from mouse (mTAMs) and humans (hTAMs). Moreover, functional assays were conducted to test TAMs chemotactic, immune modulatory, neuroprotective and neural regenerative activities. Furthermore, zebrafish model of axonal regeneration in combination with mechanistic studies using pharmacological and genetic modulations of TAMs were employed to validate the translational potential of our findings and to elucidate the molecular mechanisms of TAMs action. Results: Our findings revealed that in vitro generated hTAMs exhibit a TAM-like phenotype, with the upregulation of genes involved in immune suppression, extracellular matrix remodelling and angiogenesis. Moreover, we confirmed that hTAMs display the peculiar neurogenic signature already observed in mTAMs. From in vitro characterization, we demonstrated that TAMs are endowed with chemotactic, immune modulatory, neuroprotective and neurite outgrowth properties. TAMs effectively promote axonal regeneration in a zebrafish model, suggesting its pro-regenerative capacity conserved throughout species. This PhD thesis work also revealed that secreted phosphoprotein 1 (SPP1) is as a required key mediator for TAMs neurogenic activities. TAM-derived SPP1 promote nerve growth by upregulating mTORC2/Rictor pathway in neural cells. Conclusions: Our findings revealed that in vitro generated hTAMs can be exploited as a novel, multifaceted cell-based therapy for CNS injuries. TAMs integrate chemotactic, immune modulatory, metabolic and neurotrophic activities, allowing them to function in hostile environments where many other cellular therapies lose their efficacy. The identification of the SPP1-Rictor axis as a central component of TAM-induced neurotrophic effects provides the molecular framework for further investigations. Overall, these findings support the development of TAM-based autologous cell therapies as a promising strategy for neural repair and may pave the way for future translational applications.