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Background/Objectives: This study evaluated a novel PET tracer, 68Ga-NOTA-CYT-200, which targets human granzyme B (GZB) as a biomarker for cytotoxic T-cell activation in a clinically relevant model of melanoma-bearing mice with a humanized immune system treated with immune checkpoint inhibitor (ICI) therapy. Methods: The binding affinity of the tracer was determined using an enzymatic colorimetric assay. Tumor-bearing humanized NSG mice underwent PET imaging before and during ICI monotherapy or combination therapy to assess 68Ga-NOTA-CYT-200 uptake within tumors and other organs. The tumor growth was carefully monitored. The treatment response was evaluated based on the percentage change in tumor size at days 5 and 15 after the treatment started. A tracer biodistribution study and immunohistochemical staining of the tumors and organs were also performed. Results: The inhibition constant (Ki) of 68Ga-NOTA-CYT-200 was estimated at 4.2 nM. PET imaging showed a significantly higher 68Ga-NOTA-CYT-200 uptake in mice receiving the combination therapy compared to those receiving monotherapy or a vehicle (p < 0.0001 or p = 0.0005, respectively), which correlated with the greatest reduction in tumor size in the combination ICI group. Regardless of treatment, the responders presented with a significantly higher 68Ga-NOTA-CYT-200 uptake at days 4 or 7 after the treatment began (p = 0.0002 and p = 0.0109, respectively). An increased uptake of 68Ga-NOTA-CYT-200, especially in the intestines and liver within the combination ICI group, suggested immune-related adverse events (IrAEs). Conclusions: Our study demonstrates that 68Ga-NOTA-CYT-200 PET imaging can predict the early treatment response in melanoma models treated with ICI and may also help in detecting IrAEs.

: Granzyme B (GZB) PET Imaging is a non-invasive tool that can determine tumoral and systemic effects in immunotherapy. We aim to evaluate Ga-NOTA-CYT-200 PET Imaging as a molecular imaging approach to determine CAR T-cell therapy response in a human melanoma mouse model. Our goal is to provide a method to monitor CAR T-cell therapy for patients with melanoma and other solid tumors. : A human melanoma mouse model was generated by implanting naïve NSG mice (n = 28) with a human melanoma cell line (A375) subcutaneously (s.c.). After tumor implantation, mice were randomly assigned to receive either the treatment (CAR T) or vehicle solution (controls). After treatment, tumor sizes were measured every other day up to 35 days after cell implantation. Ga-NOTA-CYT-200 PET Imaging was performed on days 2, 7, and 14 after CAR T-cell administration to assess T-cell activity within the tumors and organs. The PET Imaging results were correlated with IHC and immunofluorescent staining and cytokine assessment of tumor samples. : Tracer uptake within tumors of the CAR T group was significantly greater on days 2 (3.1 ± 1.2 vs. 1.1 ± 0.4, = 0.002) and 7 (2.0 ± 1.1 vs. 1.1 ± 0.1, = 0.01) after treatment, even before the CAR T group first presented with significantly lower tumor volumes on day 11 after treatment (61.8 mm ± 8.7 vs. 287.1 mm ± 157.6, = 0.05). GZB ( = 0.03) and CAR T ( = 0.001) staining were also significantly greater in tumors of CAR T-cell-treated mice. Inflammatory cytokines such as IFN gamma ( = 0.03), CXCL10 ( = 0.004), and CCL5 ( = 0.02) concentrations were also significantly greater in CAR T-cell-treated tumors. : CAR-T-treated tumors show significantly elevated Ga-NOTA-CYT-200 uptake compared with controls, consistent with enhanced effector activity.

Here, we report the effect of newly regenerated axons via scaffolds on reorganization of spinal circuitry and restoration of motor functions with epidural electrical stimulation (EES). Motor recovery was evaluated for 7 weeks after spinal transection and following implantation with scaffolds seeded with neurotrophin producing Schwann cell and with rapamycin microspheres. Combined treatment with scaffolds and EES-enabled stepping led to functional improvement compared to groups with scaffold or EES, although, the number of axons across scaffolds was not different between groups. Re-transection through the scaffold at week 6 reduced EES-enabled stepping, still demonstrating better performance compared to the other groups. Greater synaptic reorganization in the presence of regenerated axons was found in group with combined therapy. These findings suggest that newly regenerated axons through cell-containing scaffolds with EES-enabled motor training reorganize the sub-lesional circuitry improving motor recovery, demonstrating that neuroregenerative and neuromodulatory therapies cumulatively enhancing motor function after complete SCI.

Abstract Positively charged oligo-polyethylene glycol fumarate (OPF+) hydrogel scaffolds, implanted into a complete transection spinal cord injury (SCI), facilitate a permissive regenerative environment and provide a platform for controlled observation of repair mechanisms. Axonal regeneration after SCI is critically dependent upon the availability of nutrients and oxygen from a newly formed blood supply. In this study, the objective was to investigate fundamental characteristics of revascularization in association with the ingrowth of axons into hydrogel scaffolds, and to define the spatial relationships between axons and the neovasculature. A novel combination of stereologic estimates and precision image analysis techniques are described to quantitate neurovascular regeneration in rats. Multichannel hydrogel scaffolds containing Matrigel-only (MG), Schwann cells (SCs), or SCs with rapamycin-eluting poly(lactic co-glycolic acid) (PLGA) microspheres (RAPA) were implanted for 6 weeks following complete spinal cord transection. Image analysis of 72 scaffold channels identified a total of 2,494 myelinated and 4,173 unmyelinated axons at 10 micron circumferential intervals centered around 708 individual blood vessel profiles. Blood vessel number, density, volume, diameter, inter-vessel distances, total vessel surface and cross-sectional areas, and radial diffusion distances in each group were measured. Axon number and density, blood vessel surface area, and vessel cross-sectional areas in the SC group exceeded that in the MG and RAPA groups. Axons were concentrated within a concentric radius of 200-250 microns from the blood vessel wall in Gaussian distributions which identified a peak axonal number (mean peak amplitude) corresponding to defined distances (mean peak distance) from each vessel. Axons were largely excluded from a 25 micron zone immediately adjacent to the vessel. Higher axonal densities correlated with smaller vessel cross-sectional areas. A statistical spatial algorithm was used to generate cumulative distribution F- and G-functions of axonal distribution in the reference channel space. Axons located around blood vessels were definitively organized as clusters and were not randomly distributed. By providing methods to quantify the axonal-vessel relationships, these results may refine spinal cord tissue engineering strategies to optimize the regeneration of complete neurovascular bundles in their relevant spatial relationships after SCI. Impact Statement Vascular disruption and impaired neovascularization contribute critically to the poor regenerative capacity of the spinal cord after injury. In this study, hydrogel scaffolds provide a detailed model system to investigate the regeneration of spinal cord axons as they directly associate with individual blood vessels, using novel methods to define their spatial relationships and the physiologic implications of that organization. These results refine future tissue-engineering strategies for spinal cord repair to optimize the re-development of complete neurovascular bundles in their relevant spatial architectures. Competing Interest Statement The authors have declared no competing interest. Footnotes * This version includes new data, updated figures, and additional interpretations.

Abstract We report the effect of newly regenerated neural fibers via bioengineered scaffold on reorganization of spinal circuitry and restoration of motor functions with electrical epidural stimulation (EES) after spinal transection (ST). Restoration across multiple modalities was evaluated for 7 weeks after ST with implanted scaffold seeded with Schwann cells, producing neurotrophic factors and with rapamycin microspheres. Gradual improvement in EES-facilitated stepping was observed in animals with scaffolds, although, no significant difference in stepping ability was found between groups without EES. Similar number of regenerated axons through the scaffolds was found in rats with and without EES-enabled training. Re-transection through the scaffold at week 6, reduced EES-enabled motor function, remaining higher compared to rats without scaffolds. The combination of scaffolds and EES-enabled training demonstrated synaptic changes below the injury. These findings indicate that sub-functional connectivity with regenerated across injury fibers can reorganize of sub-lesional circuitry, facilitating motor functions recovery with EES. Competing Interest Statement The authors have declared no competing interest.

An understanding of spinal cord functional neuroanatomy is essential for diagnosis and treatment of multiple disorders including, chronic pain, movement disorders, and spinal cord injury. Till now, no information is available on segment-specific spinal roots orientation in humans. In this study we collected neuroanatomical measurements of the dorsal and ventral roots from C2-L5, as well as spinal cord and vertebral bone measurements from adult cadavers. Spatial orientation of dorsal and ventral roots were measured and correlated to the anatomical landmarks of the spinal cord and vertebral column. The results show less variability in rostral root angles compared to the caudal angles across all segments. Dorsal and ventral rootlets were oriented mostly perpendicular to the spinal cord at the cervical level and demonstrate more parallel orientation at the thoracic and lumbar segments. The number of rootlets was the highest in dorsal cervical and lumbar segments. Spinal cord transverse diameter and size of the dorsal columns were largest at cervical and lumbar segments. The strongest correlation was found between the length of intervertebral foramen to rostral rootlet and vertebral bone length. These results could be used to locate spinal roots and spinal cord landmarks based on bone marks on CT or X-rays. These results also provide background for future correlations between anatomy of spinal cord and spinal column structures that could improve stereotactic surgical procedures and electrode positioning for spinal cord neuromodulation.