Inhibition of GSK3ß Stimulates BMP Signaling and Decreases SOST Expression Which Results in Enhanced Osteoblast Differentiation.

Both bone morphogenetic protein (BMP) and Wnt signaling have significant roles in osteoblast differentiation and the interaction between BMP and Wnt signaling is well known. Sclerostin is an important inhibitor of bone formation, inhibiting Wnt signaling and downstream effects of BMP such as alkaline phosphatase activity and matrix mineralization in vitro. However, little is known about the effect of BMP and Wnt signaling interaction on the regulation of SOST, the gene encoding sclerostin. Possibly, uncoupling of osteoblast differentiation regulators and SOST expression could increase osteoblast differentiation. Therefore, we investigated the effect of BMP and Wnt signaling interaction on the expression of SOST and the subsequent effect on osteoblast differentiation. Human osteosarcoma cells (SaOS-2) and murine pre-osteoblast cells (KS483) were treated with different concentrations of Wnt3a, a specific GSK3ß inhibitor (GIN) and BMP4. Both Wnt3a and GIN increased BMP4-induced BMP signaling and BMP4 increased Wnt3a and GIN-induced Wnt signaling. However, the effect of GIN was much stronger. Quantitative RT-PCR analysis showed that SOST expression dose-dependently decreased with increasing Wnt signaling, while BMP4 induced SOST expression. GIN significantly decreased the BMP4-induced SOST expression. This resulted in an increased osteoblast differentiation as measured by ALP activity in the medium and matrix mineralization. We conclude that GSK3ß inhibition by GIN caused an uncoupling of BMP signaling and SOST expression, resulting in an increased BMP4-induced osteoblast differentiation. This effect can possibly be used in clinical practice to induce local bone formation, for example, fracture healing or osseointegration of implants.

Identification of receptor-type protein tyrosine phosphatase µ as a new marker for osteocytes.

Osteocytes are the predominant cells in bone, where they form a cellular network and display important functions in bone homeostasis, phosphate metabolism and mechanical transduction. Several proteins strongly expressed by osteocytes are involved in these processes, e.g., sclerostin, DMP-1, PHEX, FGF23 and MEPE, while others are upregulated during differentiation of osteoblasts into osteocytes, e.g., osteocalcin and E11. The receptor-type protein tyrosine phosphatase µ (RPTPµ) has been described to be expressed in cells which display a cellular network, e.g., endothelial and neuronal cells, and is implied in mechanotransduction. In a capillary outgrowth assay using metatarsals derived from RPTPµ-knock-out/LacZ knock-in mice, we observed that the capillary structures grown out of the metatarsals were stained blue, as expected. Surprisingly, cells within the metatarsal bone tissue were positive for LacZ activity as well, indicating that RPTPµ is also expressed by osteocytes. Subsequent histochemical analysis showed that within bone, RPTPµ is expressed exclusively in early-stage osteocytes. Analysis of bone marrow cell cultures revealed that osteocytes are present in the nodules and an enzymatic assay enabled the quantification of the amount of osteocytes. No apparent bone phenotype was observed when tibiae of RPTPµ-knock-out/LacZ knock-in mice were analyzed by µCT at several time points during aging, although a significant reduction in cortical bone was observed in RPTPµ-knock-out/LacZ knock-in mice at 20 weeks. Changes in trabecular bone were more subtle. Our data show that RPTPµ is a new marker for osteocytes.

Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone.

Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and is therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments. KS483 cells were cultured under osteogenic conditions in the presence of compounds that either stimulate or inhibit osteoblast differentiation and thereby matrix mineralization. After 21 days of differentiation, fluorescence of stained cultures was quantified with a near-infrared imager and compared to Alizarin Red S quantification. Fluorescence of both probes closely correlated to Alizarin Red S staining in both inhibiting and stimulating conditions. In addition, both compounds displayed specificity for mineralized nodules. We therefore conclude that this method of quantification of bone mineralization using fluorescent compounds is a good alternative for the Alizarin Red S staining.

A novel luciferase fusion protein for highly sensitive optical imaging: from single-cell analysis to in vivo whole-body bioluminescence imaging.

Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 10(3) and 5 × 10(4) cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging.

Near-infrared fluorescence imaging of liver metastases in rats using indocyanine green.

BACKGROUND: Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) is a promising technique to obtain real-time assessment of the extent and number of colorectal liver metastases during surgery. The current study aims to optimize dosage and timing of ICG administration. MATERIALS AND METHODS: Liver tumors were induced in 18 male WAG/Rij rats by subcapsular inoculation of CC531 rat colorectal cancer cells into three distinct liver lobes. Rats were divided in two groups: imaging after 24 and 48 h or 72 and 96 h after intravenous ICG administration. In each time group, rats were allocated to three dose groups: 0.04, 0.08, or 0.16 mg ICG. Intraoperative imaging and ex vivo measurements were performed using the Mini-FLARE imaging system and confirmed by fluorescence microscopy. Fluorescence intensity was quantified using the Mini-FLARE software and the difference between tumor signal and liver signal (tumor-to-liver ratio; TLR) was calculated. RESULTS: In all 18 rats, all colorectal liver metastases (n = 34), some as small as 1.2 mm, were identified using ICG and the Mini-FLARE imaging system. Average tumor-to-liver ratio (TLR) over all groups was 3.0 ± 1.2. TLR was significantly higher in the 72 h time group compared with other time points. ICG dose did not significantly influence TLR, but a trend was found favoring the 0.08 mg dose group. Fluorescence microscopy demonstrated a clear fluorescent rim around the tumor. CONCLUSIONS: This study demonstrates that colorectal cancer liver metastases can be clearly identified during surgery using ICG and the Mini-FLARE imaging system, with optimal timing of 72 h post-injection and an optimal dose of 0.08 mg (0.25 mg/kg) ICG. NIR fluorescence imaging has the potential to improve intraoperative detection of micrometastases and, thus, the completeness of resection.

Optically Coupled PtOEP and DPA Molecules Encapsulated into PLGA-Nanoparticles for Cancer Bioimaging.

Triplet-triplet annihilation upconversion (TTA-UC) nanoparticles (NPs) have emerged as imaging probes and therapeutic probes in recent years due to their excellent optical properties. In contrast to lanthanide ion-doped inorganic materials, highly efficient TTA-UC can be generated by low excitation power density, which makes it suitable for clinical applications. In the present study, we used biodegradable poly(lactic-co-glycolic acid) (PLGA)-NPs as a delivery vehicle for TTA-UC based on the heavy metal porphyrin Platinum(II) octaethylporphyrin (PtOEP) and the polycyclic aromatic hydrocarbon 9,10-diphenylanthracene (DPA) as a photosensitizer/emitter pair. TTA-UC-PLGA-NPs were successfully synthesized according to an oil-in-water emulsion and solvent evaporation method. After physicochemical characterization, UC-efficacy of TTA-UC-PLGA-NPs was assessed in vitro and ex vivo. TTA-UC could be detected in the tumour area 96 h after in vivo administration of TTA-UC-PLGA-NPs, confirming the integrity and suitability of PLGA-NPs as a TTA-UC in vivo delivery system. Thus, this study provides proof-of-concept that the advantageous properties of PLGA can be combined with the unique optical properties of TTA-UC for the development of advanced nanocarriers for simultaneous in vivo molecular imaging and drug delivery.

Image-guided tumor resection using real-time near-infrared fluorescence in a syngeneic rat model of primary breast cancer.

Tumor involvement of resection margins is found in a large proportion of patients who undergo breast-conserving surgery. Near-infrared (NIR) fluorescence imaging is an experimental technique to visualize cancer cells during surgery. To determine the accuracy of real-time NIR fluorescence imaging in obtaining tumor-free resection margins, a protease-activatable NIR fluorescence probe and an intraoperative camera system were used in the EMR86 orthotopic syngeneic breast cancer rat model. Influence of concentration, timing and number of tumor cells were tested in the MCR86 rat breast cancer cell line. These variables were significantly associated with NIR fluorescence probe activation. Dosing and tumor size were also significantly associated with fluorescence intensity in the EMR86 rat model, whereas time of imaging was not. Real-time NIR fluorescence guidance of tumor resection resulted in a complete resection of 17 out of 17 tumors with minimal excision of normal healthy tissue (mean minimum and a mean maximum tumor-free margin of 0.2 ± 0.2 mm and 1.3 ± 0.6 mm, respectively). Moreover, the technique enabled identification of remnant tumor tissue in the surgical cavity. Histological analysis revealed that the NIR fluorescence signal was highest at the invasive tumor border and in the stromal compartment of the tumor. In conclusion, NIR fluorescence detection of breast tumor margins was successful in a rat model. This study suggests that clinical introduction of intraoperative NIR fluorescence imaging has the potential to increase the number of complete tumor resections in breast cancer patients undergoing breast-conserving surgery.

19F-nanoparticles: Platform for in vivo delivery of fluorinated biomaterials for 19F-MRI.

Fluorine-19 (19F) magnetic resonance imaging (MRI) features one of the most investigated and innovative techniques for quantitative and unambiguous cell tracking, providing information for both localization and number of cells. Because of the relative insensitivity of the MRI technique, a high number of magnetically equivalent fluorine atoms are required to gain detectable signals. However, an increased amount of 19F nuclei induces low solubility in aqueous solutions, making fluorine-based probes not suitable for in vivo imaging applications. In this context, nanoparticle-based platforms play a crucial role, since nanoparticles may carry a high payload of 19F-based contrast agents into the relevant cells or tissues, increase the imaging agents biocompatibility, and provide a highly versatile platform. In this review, we present an overview of the 19F-based nanoprobes for sensitive 19F-MRI, focusing on the main nanotechnologies employed to date, such as fluorine and theranostic nanovectors, including their design and applications.

Novel intraoperative near-infrared fluorescence camera system for optical image-guided cancer surgery.

Current methods of intraoperative tumor margin detection using palpation and visual inspection frequently result in incomplete resections, which is an important problem in surgical oncology. Therefore, real-time visualization of cancer cells is needed to increase the number of patients with a complete tumor resection. For this purpose, near-infrared fluorescence (NIRF) imaging is a promising technique. Here we describe a novel, handheld, intraoperative NIRF camera system equipped with a 690 nm laser; we validated its utility in detecting and guiding resection of cancer tissues in two syngeneic rat models. The camera system was calibrated using an activated cathepsin-sensing probe (ProSense, VisEn Medical, Woburn, MA). Fluorescence intensity was strongly correlated with increased activated-probe concentration (R2= .997). During the intraoperative experiments, a camera exposure time of 10 ms was used, which provided the optimal tumor to background ratio. Primary mammary tumors (n = 20 tumors) were successfully resected under direct fluorescence guidance. The tumor to background ratio was 2.34 using ProSense680 at 10 ms camera exposure time. The background fluorescence of abdominal organs, in particular liver and kidney, was high, thereby limiting the ability to detect peritoneal metastases with cathepsin-sensing probes in these regions. In conclusion, we demonstrated the technical performance of this new camera system and its intraoperative utility in guiding resection of tumors.

An in vitro model that can distinguish between effects on angiogenesis and on established vasculature: actions of TNP-470, marimastat and the tubulin-binding agent Ang-510.

In anti-cancer therapy, current investigations explore the possibility of two different strategies to target tumor vasculature; one aims at interfering with angiogenesis, the process involving the outgrowth of new blood vessels from pre-existing vessels, while the other directs at affecting the already established tumor vasculature. However, the majority of in vitro model systems currently available examine the process of angiogenesis, while the current focus in anti-vascular therapies moves towards exploring the benefit of targeting established vasculature as well. This urges the need for in vitro systems that are able to differentiate between the effects of compounds on angiogenesis as well as on established vasculature. To achieve this, we developed an in vitro model in which effects of compounds on different vascular targets can be studied specifically. Using this model, we examined the actions of the fumagillin derivate TNP-470, the MMP-inhibitor marimastat and the recently developed tubulin-binding agent Ang-510. We show that TNP-470 and marimastat solely inhibited angiogenesis, whereas Ang-510 potently inhibited angiogenesis and caused massive disruption of newly established vasculature. We show that the use of this in vitro model allows for specific and efficient screening of the effects of compounds on different vascular targets, which may facilitate the identification of agents with potential clinical benefit. The indicated differences in the mode of action between marimastat, TNP-470 and Ang-510 to target vasculature are illustrative for this approach.

Molecular imaging of tumor angiogenesis using alphavbeta3-integrin targeted multimodal quantum dots.

Molecular imaging of angiogenesis is urgently needed for diagnostic purposes such as early detection, monitoring of (angiostatic) therapy and individualized therapy. Multimodality molecular imaging is a promising and refined technique to study tumor angiogenesis, which has so far been largely unexplored due to the lack of suitable multimodal contrast agents. Here, we report on the application of a novel alphavbeta3-specific quantum dot-based nanoparticle, which has been optimized for both optical and magnetic resonance detection of tumor angiogenesis. Upon intravenous injection of RGD-pQDs in tumor-bearing mice, intravital microscopy allowed the detection of angiogenically activated endothelium at cellular resolution with a small scanning window and limited penetration depth, while magnetic resonance imaging was used to visualize angiogenesis at anatomical resolution throughout the entire tumor. Fluorescence imaging allowed whole-body investigation of angiogenic activity. Using these quantum dots and the aforementioned imaging modalities, the angiogenic tumor vasculature was readily detected with the highest angiogenic activity occurring in the periphery of the tumor. This nanoparticle may be employed for multimodality imaging of a variety of diseases that are accompanied by activation of endothelial cells. Furthermore, the current technology might be developed for molecular imaging of other pathophysiological processes.

Tissue Engineering: An Alternative to Repair Cartilage.

Herein we review the state-of-the-art in tissue engineering for repair of articular cartilage. First, we describe the molecular, cellular, and histologic structure and function of endogenous cartilage, focusing on chondrocytes, collagens, extracellular matrix, and proteoglycans. We then explore in vitro cell culture on scaffolds, discussing the difficulties involved in maintaining or obtaining a chondrocytic phenotype. Next, we discuss the diverse compounds and designs used for these scaffolds, including natural and synthetic biomaterials and porous, fibrous, and multilayer architectures. We then report on the mechanical properties of different cell-loaded scaffolds, and the success of these scaffolds following in vivo implantation in small animals, in terms of generating tissue that structurally and functionally resembles native tissue. Last, we highlight future trends in this field. We conclude that despite major technical advances made over the past 15 years, and continually improving results in cartilage repair experiments in animals, the development of clinically useful implants for regeneration of articular cartilage remains a challenge

A transgenic Tie2-GFP athymic mouse model; a tool for vascular biology in xenograft tumors.

We report the generation of a transgenic Tie2-GFP athymic nude mouse, carrying green fluorescent blood vessels throughout the body. This transgenic mouse is a tool for studies in vascular biology, and is especially of interest for imaging of tumor angiogenesis and the study of anti-angiogenesis strategies in (human) xenografts. Intravital microscopy identified the presence of blood conducting structures that are not lined by endothelial cells. Dedifferentiation of aggressive tumor cells can lead to acquisition of endothelial characteristics. This process of tumor cell plasticity, also referred to as vasculogenic mimicry, has been suggested to contribute to the circulatory system in a tumor. In plastic EW7 Ewing sarcoma tumors in these Tie2-GFP mice, we observed blood flow in both regular blood vessels and non-fluorescent tumor cell-lined channels, visualizing in vivo hemodynamics in vasculogenic channels. These results demonstrate that the transgenic Tie2-GFP athymic mouse model is a valuable tool for vascular biology research.

Whole-body optical imaging in animal models to assess cancer development and progression.

Different optical-based imaging models were used to investigate tumor progression and metastasis with particular emphasis on metastasis to bone and bone marrow. We describe how optical imaging can be used to follow important processes in tumor development and treatment response, including angiogenesis, apoptosis, and proteolysis. Finally, we discuss the translation of one optical imaging modality, near-IR fluorescence, from animal validation studies to applications in the clinic related to cancer management.

Prolonged in vivo gene silencing by electroporation-mediated plasmid delivery of small interfering RNA.

For the successful application of RNA interference in vivo, it is desired to achieve (local) delivery of small interfering RNAs (siRNAs) and long-term gene silencing. Nonviral electrodelivery is suitable to obtain local and prolonged expression of transgenes. By intramuscular electrodelivery of a plasmid in which two opposing human polymerase III promoters (H1 and U6) drive the expression of siRNA constructs that form functional double-stranded siRNAs, in combination with in vivo bioluminescence imaging, we were able to knock down exogenous delivered luciferase for at least 100 days in murine calf muscles. This effect was sequence specific, because scrambled siRNA had no effect. Moreover, we were able to demonstrate in vivo reduction of endogenous TLR4 expression for at least 1 week, using a similar vector expressing an siRNA for TLR4 in the muscle. In this study, we demonstrate that in vivo suppression of both endogenous (for at least 1 week) and introduced genes (>100 days) is feasible via plasmid-driven siRNA expression after electroporation-mediated intramuscular gene transfer. With this approach the short-term effect of oligonucleotides and the drawbacks of viral gene delivery, like immunological responses, could be circumvented. Therefore, this application of RNA interference is a useful tool with which to investigate gene function and might be promising as a therapeutic tool for locally acting diseases such as restenosis or tumors.

Advances in optical imaging and novel model systems for cancer metastasis research.

Research into the genetic and physiological interactions of tumours with their host environment requires in vivo assays to address molecular expression patterns and function. In recent years much of this work has been performed using bioluminescent and fluorescent imaging techniques that allow real-time and non-invasive imaging of gene expression and (tumour) tissue development. Luminescence imaging has until now been more or less the only tool that allows the imaging of intra-osseous breast cancer cells and indeed this technique has been pioneered in our laboratory. Here we summarise some recent innovations and developments using cancer cells and some of the first imaging models of multimodal dual luminescence and luminescence combined with fluorescence of intra-osseous tumours. We further engineered our models to incorporate a specific insertion site in the genome and will discuss some of the possible applications. These include the insertion of signalling pathway-specific reporters and studying the fate of multiple injected populations in a single mouse. We conclude that recent improvements in luminescence- and fluorescence-detection platforms now clearly allow multimodal imaging which will greatly enhance our ability to assess gene function and for the first time to visualise multiple gene- and cellular interactions in real time and in vivo.

Traumatic Brain Injury: Preclinical Imaging Diagnostic(s) and Therapeutic Approaches.

BACKGROUND: Traumatic brain injury (TBI) is the result of an external physical force to the head that harms the brain. TBI is a major public health problem worldwide and mainly results from falls, vehicle accidents and violence. Clinical problem: The management of TBI, causing a wide spectrum of possible health outcomes, has barely changed over the years as encouraging outcomes from many pre-clinical therapeutic and pharmacological studies have only rarely been translated to the clinical situation. New management options: In the last decades management of TBI is rapidly advancing and new innovative imaging modalities with sophisticated treatment options by using nanomedicine based drug delivery systems are under investigation. Nano formulations such as PLGA, exosomes and liposomes have the advantage of a targeted and controlled delivery of their cargo, such as diagnostic probes and/or therapeutic drugs. SUMMARY: Here we provide an overview of new promising pre-clinical developments in TBI management that may find their way to the clinic in the near future. Nanotechnology and nanomedicine in TBI intervention may establish new platforms for targeted drug delivery to the traumatized brain to improve the quality of life and survival of TBI patients.

Development of a Multicolor Bioluminescence Imaging Platform to Simultaneously Investigate Transcription Factor NF-κB Signaling and Apoptosis.

Here we describe a novel multicolor bioluminescent imaging platform that enables us to simultaneously investigate transcription factor nuclear factor-κB (NF-κB) signalling and apoptosis. We genetically modified the human breast cancer cell line MDA-MB-231 to express green, red, and blue light-emitting luciferases to monitor cell number and viability, NF-κB promoter activity, and to enable specific cell sorting and detection, respectively. Z-DEVD-animoluciferin, the pro-luciferin substrate, was used to determine apoptotic caspase 3/7 activity. We used this multicolored cell line for the in vitro evaluation of natural compounds and in vivo optical imaging of tumor necrosis factor (TNFα)-induced NF-κB activation (Mezzanotte et al., PLoS One 9:e85550, 2014).

Effect of PLGA NP size on efficiency to target traumatic brain injury.

Necrotic cell death occurs exclusively under pathological conditions, such as ischemic diseases. Necrosis imaging is of diagnostic value and enables early measurement of treatment efficiency in ischemic patients. Here we explored the targeted delivery of particles, with diameters of approximately 100nm, 200nm and 800nm, consisting of a poly(lactic-co-glycolic acid) (PLGA) nanoparticle (NP) core coated with a polyethylene glycol-lipid (PEG) layer. Targeted delivery was facilitated by coupling the amino end group of the polyethylene glycol-layer to 800CW imaging agent, which specifically binds to intracellular proteins of cells that have lost membrane integrity, thus revealing the extent of the damaged area. We found that smaller NPs (100nm), with an appropriate coating, diffuse throughout the traumatic brain injury (TBI) in mice. Optical imaging revealed that smaller (100-nm) PEG-coated NPs carrying 800CW penetrated deeper into the mouse brain than large 800CW containing NPs (800nm). The importance of the 800CW as a ligand to target the necrotic tissue was further confirmed in living mice. The ability to achieve brain penetration with smaller NPs is expected to allow more uniform, longer-lasting, and effective delivery of drugs within the brain, and may find application in the treatment of stroke, brain tumors, neuroinflammation, and other brain diseases where the blood-brain barrier is compromised or where local delivery strategies are feasible.

Pre-clinical Evaluation of a Cyanine-Based SPECT Probe for Multimodal Tumor Necrosis Imaging.

PURPOSE: Recently we showed that a number of carboxylated near-infrared fluorescent (NIRF) cyanine dyes possess strong necrosis avid properties in vitro as well as in different mouse models of spontaneous and therapy-induced tumor necrosis, indicating their potential use for cancer diagnostic- and prognostic purposes. In the previous study, the detection of the cyanines was achieved by whole body optical imaging, a technique that, due to the limited penetration of near-infrared light, is not suitable for investigations deeper than 1 cm within the human body. Therefore, in order to facilitate clinical translation, the purpose of the present study was to generate a necrosis avid cyanine-based NIRF probe that could also be used for single photon emission computed tomography (SPECT). For this, the necrosis avid NIRF cyanine HQ4 was radiolabeled with 111indium, via the chelate diethylene triamine pentaacetic acid (DTPA). PROCEDURES: The necrosis avid properties of the radiotracer [111In]DTPA-HQ4 were examined in vitro and in vivo in different breast tumor models in mice using SPECT and optical imaging. Moreover, biodistribution studies were performed to examine the pharmacokinetics of the probe in vivo. RESULTS: Using optical imaging and radioactivity measurements, in vitro, we showed selective accumulation of [111In]DTPA-HQ4 in dead cells. Using SPECT and in biodistribution studies, the necrosis avidity of the radiotracer was confirmed in a 4T1 mouse breast cancer model of spontaneous tumor necrosis and in a MCF-7 human breast cancer model of chemotherapy-induced tumor necrosis. CONCLUSIONS: The radiotracer [111In]DTPA-HQ4 possessed strong and selective necrosis avidity in vitro and in various mouse models of tumor necrosis in vivo, indicating its potential to be clinically applied for diagnostic purposes and to monitor anti-cancer treatment efficacy.