Rationale: Nanobodies (Nbs) have emerged as an elegant alternative to the use of conventional monoclonal antibodies in cancer therapy, but a detailed microscopic insight into the in vivo... Show moreRationale: Nanobodies (Nbs) have emerged as an elegant alternative to the use of conventional monoclonal antibodies in cancer therapy, but a detailed microscopic insight into the in vivo pharmacokinetics of different Nb formats in tumor-bearers is lacking. This is especially relevant for the recognition and targeting of pro-tumoral tumor-associated macrophages (TAMs), which may be located in less penetrable tumor regions.Methods: We employed anti-Macrophage Mannose Receptor (MMR) Nbs, in a monovalent (m) or bivalent (biv) format, to assess in vivo TAM targeting. Intravital and confocal microscopy were used to analyse the blood clearance rate and targeting kinetics of anti-MMR Nbs in tumor tissue, healthy muscle tissue and liver. Fluorescence Molecular Tomography was applied to confirm anti-MMR Nb accumulation in the primary tumor and in metastatic lesions.Results: Intravital microscopy demonstrated significant differences in the blood clearance rate and macrophage targeting kinetics of (m) and (biv)anti-MMR Nbs, both in tumoral and extra-tumoral tissue. Importantly, (m)anti-MMR Nbs are superior in reaching tissue macrophages, an advantage that is especially prominent in tumor tissue. The administration of a molar excess of unlabelled (biv)anti-MMR Nbs increased the (m)anti-MMR Nb bioavailability and impacted on its macrophage targeting kinetics, preventing their accumulation in extra-tumoral tissue (especially in the liver) but only partially influencing their interaction with TAMs. Finally, anti-MMR Nb administration not only allowed the visualization of TAMs in primary tumors, but also at a distant metastatic site.Conclusions: These data describe, for the first time, a microscopic analysis of (m) and (biv)anti-MMR Nb pharmacokinetics in tumor and healthy tissues. The concepts proposed in this study provide important knowledge for the future use of Nbs as diagnostic and therapeutic agents, especially for the targeting of tumor-infiltrating immune cells. Show less
Berg, C.W. van den; Koudijs, A.; Ritsma, L.; Rabelink, T.J. 2020
Significance StatementThe ability to differentiate human induced pluripotent stem cells to kidney organoids in vitro holds promise for disease modeling, drug discovery, and clinical application.... Show moreSignificance StatementThe ability to differentiate human induced pluripotent stem cells to kidney organoids in vitro holds promise for disease modeling, drug discovery, and clinical application. The authors differentiated such cells to kidney tissue comprising glomerular, proximal, and distal tubular structures. Earlier research demonstrated that these structures become vascularized upon transplantation in mice and show advanced maturation. To investigate whether human induced pluripotent stem cell?derived kidney organoids can also become functional in vivo, they applied high-resolution intravital multiphoton imaging through a titanium imaging window. They demonstrated in vivo glomerular filtration and size-selective glomerular barrier function in the transplanted organoids. This technique can be instrumental for further developing stem cell?derived organoids toward clinical applications. BackgroundThe utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the vascularization and subsequent maturation of human pluripotent stem cell?derived kidney organoids after renal subcapsular transplantation. This raises the question of whether the glomeruli also become functional upon transplantation.MethodsWe transplanted kidney organoids under the renal capsule of the left kidney in immunodeficient mice followed by the implantation of a titanium imaging window on top of the kidney organoid. To assess glomerular function in the transplanted human pluripotent stem cell?derived kidney tissue 1, 2, and 3 weeks after transplantation, we applied high-resolution intravital multiphoton imaging through the imaging window during intravenous infusion of fluorescently labeled low and high molecular mass dextran molecules or albumin.ResultsAfter vascularization, glomerular structures in the organoid displayed dextran and albumin size selectivity across their glomerular filtration barrier. We also observed evidence of proximal tubular dextran reuptake.ConclusionsOur results demonstrate that human pluripotent stem cell?derived glomeruli can develop an appropriate barrier function and discriminate between molecules of varying size. These characteristics together with tubular presence of low molecular mass dextran provide clear evidence of functional filtration. This approach to visualizing glomerular filtration function will be instrumental for translation of organoid technology for clinical applications as well as for disease modeling. Show less
