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

UNLABELLED: 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 fluorescence-based 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 reporter-transduced 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.