A dual CEST-fluorescent reporter enables cross-validated tracking of hematopoietic engraftment in deep bone marrow

Reporter gene imaging can enable detection of engineered cells in vivo, but tracking hematopoietic engraftment within deep bone marrow (BM) remains challenging.We addressed this challenge by developing a dual CEST-MRI and fluorescent reporter system for cross-validated detection of reporter-positive donor-derived hematopoietic cells and their progeny in BM, with fluorescencebased assessment of HSPC-enriched compartments.Methods: A lentiviral dual-reporter cassette co-expressing the red fluorescent protein mScarlet-I (mScarlet) and the CEST-MRI reporter SuperCESTide (SC) via a T2A linker was evaluated in CT26 colon carcinoma cells in vitro and in a bilateral flank tumor model with contralateral reporter-transduced and non-transduced tumors in BALB/c mice (n = 6 tumors per group).In vivo optical imaging, CEST-MRI, ex vivo optical imaging, fluorescence microscopy, and histology were used for validation.The platform was then applied to primary murine hematopoietic lineage-negative (Lin⁻) cells and Lin⁻Sca-1⁺c-Kit⁺ (LSKs), with reporter expression analyzed by fluorescence microscopy and flow cytometry.Reporter-transduced Lin⁻ cells were transplanted into C57BL/6J mice after half-body irradiation, and engraftment was assessed 8 weeks later by whole-body fluorescence imaging, femoral CEST-MRI, intravital calvarial microscopy, ex vivo confocal microscopy, optical imaging, and flow cytometry.Femoral CEST-MRI was compared among non-irradiated controls, irradiated recipients transplanted with non-reporter-transduced Lin⁻ cells, and irradiated recipients transplanted with mScarlet-SC reporter-transduced Lin⁻ cells.Results: The dual mScarlet-SuperCESTide reporter showed efficient expression in CT26 cells and generated detectable red fluorescence and increased CEST-MRI contrast in reporter-positive tumors compared with contralateral non-transduced controls.Ex vivo optical imaging and histology confirmed reporter expression in tumor tissue.In murine BM-derived Lin⁻ cells, reporter expression was detected by fluorescence microscopy and flow cytometry, supporting feasibility in sensitive hematopoietic populations.Following transplantation, reporter-transduced Lin⁻ cells were detected eight weeks after engraftment using fluorescence imaging and femoral CEST-MRI.The recipients with reporter-transduced Lin⁻ cells showed increased femoral CEST contrast associated with the reporter compared with both irradiated recipients transplanted with non-transduced Lin⁻ cells and non-irradiated BM controls.Additionally, intravital microscopy visualized mScarlet⁺ cells within the BM niches of the calvarium.Ex vivo confocal microscopy and optical imaging confirmed reporter retention in BM, and flow cytometry confirmed mScarlet signal within Lin⁻ and LSK hematopoietic compartments in reporter-transduced recipients compared with non-transduced controls.Conclusion: We developed a dual CEST-fluorescent reporter platform that enables cross-validated imaging of reportertransduced hematopoietic cells in deep BM.By integrating noninvasive CEST-MRI with fluorescence-based cellular validation, this feasibility study establishes a preclinical framework for cross-validated, noninvasive assessment of engineered hematopoietic cell localization and persistence in vivo and supports future evaluation of the platform for serial imaging.