Mice and men: An antibody conjugate with trans‐cyclooctene was administered to tumor‐bearing mice, and the resulting chemically tagged tumors were subsequently treated with an 111In‐labeled tetrazine ...probe in an inverse‐electron‐demand Diels–Alder reaction. The adduct was formed in a remarkable 52–57 % yield in vivo and used for non‐invasive pretargeted tumor imaging in mice (see picture).
Significance: Currently, tissue biopsies are sectioned into 3- to 5-μm-thick slices that are used for conventional pathology analysis. Previous work by confocal microscopy and light-sheet microscopy ...has shown that analyzing biopsies intact in three-dimensions (3D) is possible and may lead to a better understanding of cancer growth patterns. Although accurate, these methods require fluorescent staining of the tissue, in addition to tissue clearing. If the 3D biopsy analysis could be done sufficiently swiftly, this approach may be used for on-site assessment of the adequacy of a biopsy taken.
Aim: We aim to show that, by transmission microscopy of optically cleared tissue punches, the tissue architecture can be determined without the need for fluorescent staining.
Approach: Transmission microscopy is used by combining bright field microscopy with dark field and epifluorescent microscopy to compare samples that have also been analyzed by fluorescent confocal microscopy.
Results: With increasing distance to the focal plane, the higher-frequency part of the spatial frequency spectrum of transmitted light is attenuated increasingly. This property is exploited for tissue segmentation, detecting whether tissue is present at a certain position in the focal plane image. Using this approach, we show that a 3D rendering of the internal cavity or tubules structure of punch biopsies, which are up to 1-mm thick, can be acquired in ≈1 min scan time per imaging modality. The images of the overall tissue architecture that are obtained are similar to those from the confocal microscopy benchmark, without requiring fluorescent staining.
Conclusions: Images of the overall tissue architecture can be obtained from transmission microcopy; they are similar to those from the confocal microscopy benchmark without requiring fluorescent staining. Tissue clearing is still needed. The total scan time of the present method is significantly shorter at a fraction of the device costs.
The application of intact monoclonal antibodies (mAbs) as targeting agents in nuclear imaging and radioimmunotherapy is hampered by the slow pharmacokinetics of these molecules. Pretargeting with ...mAbs could be beneficial to reduce the radiation burden to the patient, while using the excellent targeting capacity of the mAbs. In this study, we evaluated the applicability of the Staudinger ligation as pretargeting strategy using an antibody–azide conjugate as tumor-targeting molecule in combination with a small phosphine-containing imaging/therapeutic probe. Up to 8 triazide molecules were attached to the antibody without seriously affecting its immunoreactivity, pharmacokinetics, and tumor uptake in tumor bearing nude mice. In addition, two 89Zr- and 67/68Ga-labeled desferrioxamine (DFO)-phosphines, a 177Lu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-phosphine and a 123I-cubyl phosphine probe were synthesized and characterized for their pharmacokinetic behavior in nude mice. With respect to the phosphine probes, blood levels at 30 min after injection were <5% injected dose per gram tissue, indicating rapid blood clearance. In vitro Staudinger ligation of 3.33 μM antibody–azide conjugate with 1 equiv of radiolabeled phosphine, relative to the azide, in aqueous solution resulted in 20–25% efficiency after 2 h. The presence of 37% human serum resulted in a reduced ligation efficiency (reduction max. 30% at 2 h), while the phosphines were still >80% intact. No in vivo Staudinger ligation was observed in a mouse model after injection of 500 μg antibody–azide, followed by 68 μg DFO-phosphine at t = 2 h, and evaluation in blood at t = 7 h. To explain negative results in mice, Staudinger ligation was performed in vitro in mouse serum. Under these conditions, a side product with the phosphine was formed and ligation efficiency was severely reduced. It is concluded that in vivo application of the Staudinger ligation in a pretargeting approach in mice is not feasible, since this ligation reaction is not bioorthogonal and efficient enough. Slow reaction kinetics will also severely restrict the applicability of Staudinger ligation in humans.
Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic ...device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.
Much effort has been devoted to prodrug systems that effect drug release at the tumor through enzymatic action. To widen the scope of prodrug therapy, the use of the selective Staudinger reaction as ...prodrug activator, instead of relying on enzymatic action, was investigated. Doxorubicin was conjugated to a p-azidobenzyl trigger that is cleaved after reacting with the chemical activator, triphenylphosphine. The prodrug activation was confirmed in water, cell growth medium, and serum, using HPLC and LCMS. Next, this approach was tested in a cell proliferation assay with A431 human vulvar skin squamous carcinoma cells. The doxorubicin prodrug was shown to exhibit a 176-fold higher IC50 of 15.1 µM vs 0.086 µM for the parent drug, doxorubicin. Addition of triphenylphosphine (5 × 60 µM in 72 h) to the prodrug in cell culture effected the complete recovery of the activity of the parent drug as evidenced by an IC50 value of 0.074 µM. Furthermore, high levels of triphenylphosphine were tolerated well by the cells. The demonstrated usefulness of the Staudinger reaction in cell culture and its in vivo potential opens up new avenues for prodrug therapy.
Wie sieht's aus in der Maus? Ein trans‐Cycloocten‐Antikörper‐Konjugat wurde Tumormäusen verabreicht. Die derart chemisch markierten Tumore wurden dann mit einer 111In‐markierten Tetrazin‐Sonde in ...einer Diels‐Alder‐Reaktion mit inversem Elektronenbedarf umgesetzt, die das Addukt in vivo mit einer bemerkenswerten Ausbeute von 52–57 % ergab. Mit diesem nichtinvasiven Verfahren gelang die Abbildung von Tumoren in Mäusen (siehe Bild).
Diels‐Alder‐Reaktionen mit inversem Elektronenbedarf können effizient in lebenden Tieren ausgeführt werden. In ihrer Zuschrift auf S. 3447 ff. beschreiben M. S. Robillard et al. eine Ausbeute von 52 ...% für die In‐vivo‐Umsetzung von trans‐Cycloocten‐Markierungen an einem Antikörper, der in einem Tumor lokalisiert ist, mit einer 111In‐markierten Tetrazin‐Sonde. Die schnelle Reaktion führte zu einer merklichen Ansammlung von Radioaktivität im Tumor eines Mausmodells, die durch nichtinvasive Bildgebung nachweisbar war.
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