In the last decade, molecular ultrasound imaging has been rapidly progressing. It has proven promising to diagnose angiogenesis, inflammation, and thrombosis, and many intravascular targets, such as VEGFR2, integrins, and selectins, have been successfully visualized in vivo. Furthermore, pre-clinical studies demonstrated that molecular ultrasound increased sensitivity and specificity in disease detection, classification, and therapy response monitoring compared to current clinically applied ultrasound technologies. Several techniques were developed to detect target-bound microbubbles comprising sensitive particle acoustic quantification (SPAQ), destruction-replenishment analysis, and dwelling time assessment. Moreover, some groups tried to assess microbubble binding by a change in their echogenicity after target binding. These techniques can be complemented by radiation force ultrasound improving target binding by pushing microbubbles to vessel walls. Two targeted microbubble formulations are already in clinical trials for tumor detection and liver lesion characterization, and further clinical scale targeted microbubbles are prepared for clinical translation. The recent enormous progress in the field of molecular ultrasound imaging is summarized in this review article by introducing the most relevant detection technologies, concepts for targeted nano- and micro-bubbles, as well as their applications to characterize various diseases. Finally, progress in clinical translation is highlighted, and roadblocks are discussed that currently slow the clinical translation.

Active targeting can improve the retention of drugs and drug delivery systems in tumors, thereby enhancing their therapeutic efficacy. In this context, vitamin receptors that are overexpressed in many cancers are promising targets. In the last decade, attention and research were mainly centered on vitamin B9 (folate) targeting; however, the focus is slowly shifting towards vitamin B2 (riboflavin). Interestingly, while the riboflavin carrier protein was discovered in the 1960s, the three riboflavin transporters (RFVT 1-3) were only identified recently. It has been shown that riboflavin transporters and the riboflavin carrier protein are overexpressed in many tumor types, tumor stem cells, and the tumor neovasculature. Furthermore, a clinical study has demonstrated that tumor cells exhibit increased riboflavin metabolism as compared to normal cells. Moreover, riboflavin and its derivatives have been conjugated to ultrasmall iron oxide nanoparticles, polyethylene glycol polymers, dendrimers, and liposomes. These conjugates have shown a high affinity towards tumors in preclinical studies. This review article summarizes knowledge on RFVT expression in healthy and pathological tissues, discusses riboflavin internalization pathways, and provides an overview of RF-targeted diagnostics and therapeutics.

Automation of medical data analysis is an important topic in modern cancer diagnostics, aiming at robust and reproducible workflows. Therefore, we used a dataset of breast US images (252 malignant and 253 benign cases) to realize and compare different strategies for CAD support in lesion detection and classification. Eight different datasets (including pre-processed and spatially augmented images) were prepared, and machine learning algorithms (i.e., Viola–Jones; YOLOv3) were trained for lesion detection. The radiomics signature (RS) was derived from detection boxes and compared with RS derived from manually obtained segments. Finally, the classification model was established and evaluated concerning accuracy, sensitivity, specificity, and area under the Receiver Operating Characteristic curve. After training on a dataset including logarithmic derivatives of US images, we found that YOLOv3 obtains better results in breast lesion detection (IoU: 0.544 ± 0.081; LE: 0.171 ± 0.009) than the Viola–Jones framework (IoU: 0.399 ± 0.054; LE: 0.096 ± 0.016). Interestingly, our findings show that the classification model trained with RS derived from detection boxes and the model based on the RS derived from a gold standard manual segmentation are comparable (p-value = 0.071). Thus, deriving radiomics signatures from the detection box is a promising technique for building a breast lesion classification model, and may reduce the need for the lesion segmentation step in the future design of CAD systems.

Background: Humanized mice transplanted with CD34⁺ hematopoietic cells (HPCs) are broadly used to study human immune responses and infections in vivo and for testing therapies pre-clinically. However, until now, it was not clear whether interactions between the mouse major histocompatibility complexes (MHCs) and/or the human leukocyte antigens (HLAs) were necessary for human T-cell development and immune reactivity. Methods: We evaluated the long-term (20-week) human hematopoiesis and human T-cell development in NOD Scid Gamma (NSG) mice lacking the expression of MHC class I and II (NSG-DKO). Triplicate experiments were performed with HPCs obtained from three donors, and humanization was confirmed in the reference strain NOD Rag Gamma (NRG). Further, we tested whether humanized NSG-DKO mice would respond to a lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN-α, and the human cytomegalovirus gB antigen. Results: Human immune reconstitution was detectable in peripheral blood from 8 to 20 weeks after the transplantation of NSG-DKO. Human single positive CD4⁺ and CD8⁺ T-cells were detectable in lymphatic tissues (thymus, bone marrow, and spleen). LV delivery harnessed the detection of lymphocyte subsets in bone marrow (αβ and γδ T-cells and NK cells) and the expression of HLA-DR. Furthermore, RNA sequencing showed that LV delivery increased the expression of different human reactome pathways, such as defense responses to other organisms and viruses. Conclusions: Human T-cell development and reactivity are independent of the expression of murine MHCs in humanized mice. Therefore, humanized NSG-DKO is a promising new model for studying human immune responses, as it abrogates the xenograft mouse MHC interference.

Humanized mice transplanted with CD34 ⁺ hematopoietic progenitor cells (HPCs) are used to study human immune responses in vivo . However, the mismatch between the mouse major histocompatibility complexes (MHCs) and the human leukocyte antigens (HLAs) is not optimal for T-cell development and can trigger xenograft reactivity. We evaluated human T-cell development in NOD.Scid.Gamma mice lacking expression of MHC class I and II (NSG-DKO). Human leukocyte engraftment was detectable at 8 weeks post-transplantation. Human CD4 ⁺ and CD8 ⁺ T-cells were detectable in blood, thymus, bone marrow and spleen of humanized NSG-DKO mice for up to 20 weeks post-transplantation. Further, we evaluated the effects of lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN-α and the human cytomegalovirus gB antigen. LV delivery promoted development and activation of human central memory, αβ and γδ T-cells with amplifications of the T-cell repertoire. LV administration unleashed multiple reactome pathways such as type-I interferon responses, cell cycle and metabolic processes. In summary, development of human T-cells in humanized mice does not rely on mouse MHCs and can be boosted systemically via LV administration.