Tumor-associated macrofages (tams) drive neural regeneration via secreted phosphoprotein 1 (spp1)

Spinal Cord Injury (SCI) is a devastating condition that disrupts the communication between the brain and the rest of the body. This interruption affects all the regions located caudally to the lesion site, resulting in the loss of motor-sensory functions and leading to paraplegia or tetraplegia. SCI pathophysiology evolves through two phases: primary and secondary injury. Primary injury is the initial mechanical damage that leads to cell death, axonal and vascular impairment. Secondary injury, characterized by hypoxia, demyelination, and inflammation that increase neuronal loss. These processes form a hostile microenvironment that impedes axonal regeneration and functional recovery, posing a major challenge for the development of effective therapeutic strategies for SCI. Within the tumor microenvironment, a subtype of macrophages called Tumor Associated Macrophages (TAMs) plays a pivotal role in promoting tumor invasion and metastasis by contributing to angiogenesis, cell proliferation, and immunosuppression. These properties make TAMs a promising and innovative tool for the treatment of neurodegenerative conditions, including SCI. Bioinformatic analyses revealed that TAMs express a distinct neural growth gene signature and identified secreted phosphoprotein 1 (SPP1) as possible mediator of TAM-driven neural growth. In addition, repeated intraparenchymal TAMs transplantation promotes regeneration of the neural tissue, and microenvironment remodeling following SCI. This work aims to validate the neurotrophic and regenerative potential of TAMs and to elucidate the underlying molecular mechanisms, with a specific focus on the role of SPP1, employing multiple in vitro and in vivo models. Experimental validation of the neurotrophic and regenerative functions of TAMs To investigate if TAMs have a direct role in neuronal outgrowth, we tested in vitro-generated TAMs’ ability to promote neural sprouting in different neuronal cell types, in both Central and Peripheral Nervous System models. In all in vitro models, TAMs increased neural differentiation and neurite outgrowth. Strikingly, pharmacological inhibition of action potentials with Tetrodotoxin (TTX) attenuated the TAM-induced neurotrophic effects, suggesting a possible contribution of cell-cell interactions to the mechanism of TAM-mediated neuronal support. Based on the in vitro evidence, we then tested if these effects could be recapitulated in vivo using relevant pathological models, such as a sarcoma model and a severe contusive-compressive SCI (scSCI) model. In sarcoma, TAMs injection did not affect the primary tumor mass, but increased lung metastases, tumor innervation, and overall tumor progression. Repeated TAMs intrathecal transplantation in a scSCI model showed improved motor performance compared to vehicle. Ex vivo analysis of the spinal cord confirmed enhanced tissue regeneration, confirming the therapeutic efficacy of TAMs with an alternative route of administration. SPP1 as required mediator of TAM-induced neural regeneration To functionally validate SPP1 role in TAM neurotrophic effect we used both in vitro and in vivo models. SPP1 inhibition, with pharmacological blockade (Parecoxib) or with lentiviral knockdown in TAMs (TAMLV Spp1-KD), in in vitro co-culture significantly reduced TAM-promoted neuronal differentiation to levels comparable to control neurons. Consistently, in the sarcoma model, TAMLV Spp1-KD failed to promote axonal and nerve fiber growth. Likewise, in the scSCI model, repeated transplantation of TAMLV Spp1-KD did not induce locomotor recovery and was associated with impaired neural tissue regeneration. Collectively, these findings underscore the neurotrophic and regenerative effect of TAMs, identify SPP1 as a required mediator of their action, and advance their translational relevance as a promising cell-based therapeutic strategy to treat SCI.