Correction: HDAC6 inhibitor WT161 downregulates growth factor receptors in breast cancer.

[This corrects the article DOI: 10.18632/oncotarget.19019.].

Effect of stent crimping on calcification of transcatheter aortic valves.

OBJECTIVES: Although many challenges related to the acute implantation of transcatheter aortic valves have been resolved, durability and early degeneration are currently the main concerns. Recent reports indicate the potential for early valve degeneration and calcification. However, only little is known about the underlying mechanisms behind the early degeneration of these valves. The goal of this study was to test whether stent crimping increases the risk for early calcification. METHODS: Stented valves that were crimped at 18-Fr and 14-Fr catheter and uncrimped controls were exposed to a standard calcifying solution for 50 million cycles in an accelerated wear test system. Subsequently, the leaflets of the valves were imaged by microcomputed tomography (micro-CT) followed by histochemical staining and microscopic analyses to quantify calcification and other changes in the leaflets' characteristics. RESULTS: Heavily calcified regions were found over the stent-crimped leaflets compared to uncrimped controls, particularly around the stent's struts. Micro-CT studies measured the total volume of calcification in the uncrimped valves as 77.31 ± 1.63 mm3 vs 95.32 ± 5.20 mm3 in 18-Fr and 110.01 ± 8.33 mm3 in 14-Fr stent-crimped valves, respectively. These results were congruent with the increase in leaflet thickness measured by CT scans (0.44 ± 0.07 mm in uncrimped valves vs 0.69 ± 0.15 mm and 0.75 ± 0.09 mm in 18-Fr and 14-Fr stent-crimped valves, respectively). Histological studies confirmed the micro-CT results, denoting that the percentage of calcification in uncrimped leaflets at the valve's posts was 5.34 ± 3.97 compared to 19.97 ± 6.18 and 27.64 ± 13.17 in the 18-Fr and 14-Fr stent-crimped leaflets, respectively. CONCLUSIONS: This study concludes that stent-crimping damage is associated with a higher level of passive leaflet calcification, which may contribute to early valve degeneration.

Multimodal imaging guides surgical management in a preclinical spinal implant infection model.

Spine implant infections portend disastrous outcomes, as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. Current imaging modalities can detect anatomical alterations and anomalies but cannot differentiate between infection and aseptic loosening, diagnose specific pathogens, or delineate the extent of an infection. Herein, a fully human monoclonal antibody 1D9, recognizing the immunodominant staphylococcal antigen A on the surface of Staphylococcus aureus, was assessed as a nuclear and fluorescent imaging probe in a preclinical model of S. aureus spinal implant infection, utilizing bioluminescently labeled bacteria to confirm the specificity and sensitivity of this targeting. Postoperative mice were administered 1D9 probe dual labeled with 89-zirconium (89Zr) and a near infrared dye (NIR680) (89Zr-NIR680-1D9), and PET-CT and in vivo fluorescence and bioluminescence imaging were performed. The 89Zr-NIR680-1D9 probe accurately diagnosed both acute and subacute implant infection and permitted fluorescent image-guided surgery for selective debridement of infected tissue. Therefore, a single probe could noninvasively diagnose an infection and facilitate image-guided surgery to improve the clinical management of implant infections.

HDAC6 inhibitor WT161 downregulates growth factor receptors in breast cancer.

We have shown that WT-161, a histone deacetylase 6 (HDAC6) inhibitor, shows remarkable anti-tumor activity in multiple myeloma (MM) in preclinical models. However, its activity in other type of cancers has not yet been shown. In this study, we further evaluated the biologic sequelae of WT161 in breast cancer cell lines. WT161 triggers apoptotic cell death in MCF7, T47D, BT474, and MDA-MB231 cells, associated with decreased expression of EGFR, HER2, and ERα and downstream signaling. However, HDAC6 knockdown shows that cytotoxicity and destabilization of these receptors triggered by WT161 are not dependent on HDAC6 inhibition. Moreover WT161 analog MAZ1793, which lacks HDAC inhibitory effect, similarly triggers cell line growth inhibition and downregulation of these receptors. We also confirm that WT161 significantly inhibits in vivo MCF7 cell growth, associated with downregulation of ERα, in a murine xenograft model. Finally, WT161 synergistically enhances bortezomib-induced cytotoxicity, even in bortezomib-resistant breast cancer cells. Our results therefore provide the rationale to develop a novel class of therapeutic agents targeting growth pathways central to the pathogenesis of breast cancer.

Fluorescence imaging of bombesin and transferrin receptor expression is comparable to 18F-FDG PET in early detection of sorafenib-induced changes in tumor metabolism.

Physical measurement of tumor volume reduction is the most commonly used approach to assess tumor progression and treatment efficacy in mouse tumor models. However, it is relatively insensitive, and often requires long treatment courses to achieve gross physical tumor destruction. As alternatives, several non-invasive imaging methods such as bioluminescence imaging (BLI), fluorescence imaging (FLI) and positron emission tomography (PET) have been developed for more accurate measurement. As tumors have elevated glucose metabolism, 18F-fludeoxyglucose (18F-FDG) has become a sensitive PET imaging tracer for cancer detection, diagnosis, and efficacy assessment by measuring alterations in glucose metabolism. In particular, the ability of 18F-FDG imaging to detect drug-induced effects on tumor metabolism at a very early phase has dramatically improved the speed of decision-making regarding treatment efficacy. Here we demonstrated an approach with FLI that offers not only comparable performance to PET imaging, but also provides additional benefits, including ease of use, imaging throughput, probe stability, and the potential for multiplex imaging. In this report, we used sorafenib, a tyrosine kinase inhibitor clinically approved for cancer therapy, for treatment of a mouse tumor xenograft model. The drug is known to block several key signaling pathways involved in tumor metabolism. We first identified an appropriate sorafenib dose, 40 mg/kg (daily on days 0-4 and 7-10), that retained ultimate therapeutic efficacy yet provided a 2-3 day window post-treatment for imaging early, subtle metabolic changes prior to gross tumor regression. We then used 18F-FDG PET as the gold standard for assessing the effects of sorafenib treatment on tumor metabolism and compared this to results obtained by measurement of tumor size, tumor BLI, and tumor FLI changes. PET imaging showed ~55-60% inhibition of tumor uptake of 18F-FDG as early as days 2 and 3 post-treatment, without noticeable changes in tumor size. For comparison, two FLI probes, BombesinRSense™ 680 (BRS-680) and Transferrin-Vivo™ 750 (TfV-750), were assessed for their potential in metabolic imaging. Metabolically active cancer cells are known to have elevated bombesin and transferrin receptor levels on the surface. In excellent agreement with PET imaging, the BRS-680 imaging showed 40% and 79% inhibition on days 2 and 3, respectively, and the TfV-750 imaging showed 65% inhibition on day 3. In both cases, no significant reduction in tumor volume or BLI signal was observed during the first 3 days of treatment. These results suggest that metabolic FLI has potential preclinical application as an additional method for detecting drug-induced metabolic changes in tumors.

In vivo imaging of endogenous enzyme activities using luminescent 1,2-dioxetane compounds.

BACKGROUND: Here we present a non-invasive imaging method for visualizing endogenous enzyme activities in living animals. This optical imaging method is based on an energy transfer principle termed chemically initiated electron exchange luminescence (CIEEL). The light energy is provided by enzymatic activation of metastable 1,2-dioxetane substrates, whose protective groups are removed by hydrolytic enzymes such as ß-galactosidase and alkaline phosphatase. In the presence of a nearby fluorescent recipient, the chemical energy within the activated substrate is then transferred via formation of a charge-transfer complex with the fluorophore, a mechanism closely related to glow stick chemistry. RESULTS: Efficient CIEEL energy transfer requires close proximity between the trigger enzyme and the fluorescent recipient. Using cells stained with fluorescent dialkylcarbocyanines as the energy recipients, we demonstrated CIEEL imaging of cellular ß-galactosidase or alkaline phosphatase activity. In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity. CONCLUSIONS: In this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity. In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

A twist tale of cancer metastasis and tumor angiogenesis.

Twist1 is an evolutionally conserved transcription factor. Originally identified in Drosophila as a key regulator for mesoderm development, it was later implicated in many human diseases, including Saethre-Chotzen syndrome and cancer. Twist1's involvement in cancer has been well recognized. Driven by hypoxia-induced factor-1 (HIF-1), Twist1 has been considered as a proto-oncogene and its overexpression has been observed in a wide variety of human cancers. High expression level of Twist1 is closely related to tumor aggressiveness and metastatic potential. In cancer cells, Twist1 has been shown to function as a key regulator of epithelial-mesenchymal transition (EMT), a critical process for metastasis initiation. Twist1 has also been implicated in maintaining cancer stemness for self-renewal and chemoresistance. This review first summarizes the roles of Twist1 in embryo development and Saethre-Chotzen syndrome followed by a discussion of Twist1's critical functions in cancer. In particular, the review focuses on the recent discovery of Twist1's capability to promote endothelial transdifferentiation of cancer cells beyond EMT.

Noninvasive imaging of tumor burden and molecular pathways in mouse models of cancer.

Imaging plays a central role in the diagnosis of cancer and the evaluation of therapeutic efficacy in patients with cancer. Because macroscopic imaging is noninvasive and quantitative, the development of specialized instruments for small animals has spurred increasing utilization in preclinical cancer studies. Some small-animal imaging devices are miniaturized derivatives of clinical imaging modalities, including computed tomography, magnetic resonance imaging, positron-emission tomography, single-photon emission computed tomography, and ultrasonography. Optical imaging, including bioluminescence imaging and fluorescence imaging, has evolved from microscopic cellular imaging technologies. Here, we review how current imaging modalities are enabling high-resolution structural imaging with micrometer-scale spatial resolution, thus allowing for the quantification of tumor burden in genetically engineered and orthotopic models of cancer, where tumors develop within organs not typically accessible to measurements with calipers. Beyond measuring tumor size, imaging is increasingly being used to assess the activity of molecular pathways within tumors and to reveal the pharmacodynamic efficacy of targeted therapies. Each imaging technology has particular strengths and limitations, and we discuss how studies should be carefully designed to match the imaging approach to the primary experimental question.

Quantitative bioluminescence imaging of mouse tumor models.

Bioluminescence imaging (BLI) has become an essential technique for preclinical evaluation of anticancer therapeutics and provides sensitive and quantitative measurements of tumor burden in experimental cancer models. For light generation, a vector encoding firefly luciferase is introduced into human cancer cells that are grown as tumor xenografts in immunocompromised hosts, and the enzyme substrate luciferin is injected into the host. Alternatively, the reporter gene can be expressed in genetically engineered mouse models to determine the onset and progression of disease. In addition to expression of an ectopic luciferase enzyme, bioluminescence requires oxygen and ATP, thus only viable luciferase-expressing cells or tissues are capable of producing bioluminescence signals. Here, we summarize a BLI protocol that takes advantage of advances in hardware, especially the cooled charge-coupled device camera, to enable detection of bioluminescence in living animals with high sensitivity and a large dynamic range.

Targeting oncogenic interleukin-7 receptor signalling with N-acetylcysteine in T cell acute lymphoblastic leukaemia.

Activating mutations of the interleukin-7 receptor (IL7R) occur in approximately 10% of patients with T cell acute lymphoblastic leukaemia (T-ALL). Most mutations generate a cysteine at the transmembrane domain leading to receptor homodimerization through disulfide bond formation and ligand-independent activation of STAT5. We hypothesized that the reducing agent N-acetylcysteine (NAC), a well-tolerated drug used widely in clinical practice to treat acetaminophen overdose, would reduce disulfide bond formation, and inhibit mutant IL7R-mediated oncogenic signalling. We found that treatment with NAC disrupted IL7R homodimerization in IL7R-mutant DND-41 cells as assessed by non-reducing Western blot, as well as in a luciferase complementation assay. NAC led to STAT5 dephosphorylation and cell apoptosis at clinically achievable concentrations in DND-41 cells, and Ba/F3 cells transformed by an IL7R-mutant construct containing a cysteine insertion. The apoptotic effects of NAC could be rescued in part by a constitutively active allele of STAT5. Despite using doses lower than those tolerated in humans, NAC treatment significantly inhibited the progression of human DND-41 cells engrafted in immunodeficient mice. Thus, targeting leukaemogenic IL7R homodimerization with NAC offers a potentially effective and feasible therapeutic strategy that warrants testing in patients with T-ALL.

Enhanced efficacy of CKD-516 in combination with doxorubicin: pre-clinical evaluation using a hepatocellular carcinoma xenograft model.

AIM: To evaluate the anticancer efficacy of CKD-516, a novel vascular-disrupting agent, alone and in combination with doxorubicin in the treatment of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: In mice bearing luciferized HCC cells, therapeutic efficacy was assessed for seven days after single administration of CKD-516, doxorubicin, or combination of CKD-516 and doxorubicin. RESULTS: Bioluminescence-imaging (BLI) signals in the CKD-516 group abruptly decreased initially, but recovered at seven days after treatment. BLI signals in the doxorubicin group gradually decreased over the 7-day period. In the combination group, BLI signals were abruptly reduced and remained suppressed for the 7-day period. On histopathological examination, CKD-516-treated tumors showed extensive central necrosis, whereas the peripheral layers remained viable. Doxorubicin-treated tumors showed mild and scattered necrosis. Tumors from the combination group showed more extensive central and peripheral necrosis, with smaller viable peripheral layers than the CKD-516 group. CONCLUSION: Combination therapy can have additive effects for treatment of HCC compared with CKD-516 or doxorubicin monotherapy.

Using glow stick chemistry for biological imaging.

PURPOSE: This study describes an imaging strategy based on glow stick chemistry to non-invasively image oxidative stress and reactive oxygen species (ROS) production in living animals. PROCEDURES: Upon stimulation, phagocytes produce toxic levels of ROS to kill engulfed microorganisms. The mitochondrial respiratory chain continually generates low levels of superoxide (O2·(-)) that serve as a source for generation of downstream ROS, which function as regulatory signaling intermediaries. A ROS-reacting substrate, 2-methyl-6-[4-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one hydrochloride, was used as the chemical energy donor for generating energy transfer luminescence in phagosomes and mitochondria. RESULTS: Using targeted photoluminescent dyes with specific subcellular localization that serve as chemical energy recipients, our imaging data demonstrate proof-of-concept for using glow stick chemistry to visualize ROS production associated with phagocytosis and mitochondrial respiration in living mice. CONCLUSIONS: Glow stick imaging is a complementary hybrid of chemiluminescence and photoluminescence imaging, capable of generating red or far-red emission for deep tissue imaging.

In vivo imaging method to distinguish acute and chronic inflammation.

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

Tumor-specific targeting with modified Sindbis viral vectors: evaluation with optical imaging and positron emission tomography in vivo.

PURPOSE: Sindbis virus (SINV) infect tumor cells specifically and systemically throughout the body. Sindbis vectors are capable of expressing high levels of transduced suicide genes and thus efficiently produce enzymes for prodrug conversion in infected tumor cells. The ability to monitor suicide gene expression levels and viral load in patients, after administration of the vectors, would significantly enhance this tumor-specific therapeutic option. PROCEDURES: The tumor specificity of SINV is mediated by the 67-kDa laminin receptor (LR). We probed different cancer cell lines for their LR expression and, to determine the specific role of LR-expression in the infection cycle, used different molecular imaging strategies, such as bioluminescence, fluorescence molecular tomography, and positron emission tomography, to evaluate SINV-mediated infection in vitro and in vivo. RESULTS: All cancer cell lines showed a marked expression of LR. The infection rates of the SINV particles, however, differed significantly among the cell lines. CONCLUSION: We used novel molecular imaging techniques to visualize vector delivery to different neoplatic cells. SINV infection rates proofed to be not solely dependent on cellular LR expression. Further studies need to evaluate the herein discussed ways of cellular infection and viral replication.

In Vivo Fluorescent Labeling of Tumor Cells with the HaloTag® Technology.

Many fluorescent sensors are currently available for in vitro bio-physiological microscopic imaging. The ability to label cells in living animals with these fluorescent sensors would help translate some of these assays into in vivo applications. To achieve this goal, the first step is to establish a method for selectively labeling target cells with exogenous fluorophores. Here we tested whether the HaloTag® protein tagging system provides specific labeling of xenograft tumors in living animals. After systemic delivery of fluorophore-conjugated ligands, we performed whole animal planar fluorescent imaging to determine uptake in tag-expressing HCT116 xenografts. Our results demonstrate that HaloTag ligands containing red or near-infrared fluorophores have enhanced tumor uptake and are suitable for non-invasive in vivo imaging. Our proof-of-concept results establish feasibility for using HaloTag technology for bio-physiological imaging in living animals.

In vivo imaging of inflammatory phagocytes.

Inflammation contributes to the pathophysiology of many diseases. In this report, we present noninvasive bioluminescence imaging methods that distinguish acute and chronic inflammation in mouse models. Systemic delivery of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils, whose myeloperoxidase (MPO) activity is required for luminol bioluminescence. In contrast, bioluminescence from injection of lucigenin (bis-N-methylacridinium nitrate) closely correlates with late phase and chronic inflammation. Lucigenin bioluminescence is independent of MPO and, instead, requires phagocyte NADPH oxidase (Phox) activity in macrophages. We are able to visualize tissue inflammation resulting from wound healing, bacterial infection, foreign substance implantation, and antitumor immune responses. Given the central role of inflammation in a variety of disorders, we believe these noninvasive imaging methods can help dissect the differential roles of neutrophils and macrophages in a variety of pathological conditions.

Incongruity of imaging using fluorescent 2-DG conjugates compared to 18F-FDG in preclinical cancer models.

PURPOSE: We compared the use of near-infrared conjugates of 2-deoxyglucose (NIR 2-DG) to 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG) for the purposes of imaging tumors, as well as response to therapy. PROCEDURES: Uptake of both 18F-FDG and NIR 2-DG within gastrointestinal stromal tumor xenografts were imaged before and after nilotinib treatment. Confocal microscopy was performed to determine NIR 2-DG distribution in tumors. RESULTS: Treatment with nilotinib resulted in a rapid reduction in 18F-FDG uptake and reduced tumor cell viability which was predictive of long-term antitumor efficacy. In contrast, optical imaging with NIR 2-DG probes was unable to differentiate control from niltonib-treated animals, and microscopic analysis revealed no change in probe distribution as a result of treatment. CONCLUSIONS: These results suggest that conjugation of large bulky fluorophores to 2-DG disrupts the facilitated transport and retention of these probes in cells. Therefore, optical imaging of NIR 2-DG probes cannot substitute for 18F-FDG positron emission tomography imaging as a biomarker of tumor cell viability and metabolism.

Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma.

Histone deacetylase (HDAC) enzymatic activity has been linked to the transcription of DNA in cancers including multiple myeloma (MM). Therefore, HDAC inhibitors used alone and in combination are being actively studied as novel therapies in MM. In the present study, we investigated the preclinical activity of ACY-1215, an HDAC6-selective inhibitor, alone and in combination with bortezomib in MM. Low doses of ACY-1215 combined with bortezomib triggered synergistic anti-MM activity, resulting in protracted endoplasmic reticulum stress and apoptosis via activation of caspase-3, caspase-8, and caspase-9 and poly (ADP) ribosome polymerase. In vivo, the anti-MM activity of ACY-1215 in combination with bortezomib was confirmed using 2 different xenograft SCID mouse models: human MM injected subcutaneously (the plasmacytoma model) and luciferase-expressing human MM injected intravenously (the disseminated MM model). Tumor growth was significantly delayed and overall survival was significantly prolonged in animals treated with the combination therapy. Pharmacokinetic data showed peak plasma levels of ACY-1215 at 4 hours after treatment coincident with an increase in acetylated α-tubulin, a marker of HDAC6 inhibition, by immunohistochemistry and Western blot analysis. These studies provide preclinical rationale for acetylated α-tubulin use as a pharmacodynamic biomarker in future clinical trials.

High-level IGF1R expression is required for leukemia-initiating cell activity in T-ALL and is supported by Notch signaling.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of Notch1 and PI3K-Akt pathways. Although mutations that activate PI3K-Akt signaling have previously been identified, the relative contribution of growth factor-dependent activation is unclear. We show here that pharmacologic inhibition or genetic deletion of insulin-like growth factor 1 receptor (IGF1R) blocks the growth and viability of T-ALL cells, whereas moderate diminution of IGF1R signaling compromises leukemia-initiating cell (LIC) activity as defined by transplantability in syngeneic/congenic secondary recipients. Furthermore, IGF1R is a Notch1 target, and Notch1 signaling is required to maintain IGF1R expression at high levels in T-ALL cells. These findings suggest effects of Notch on LIC activity may be mediated in part by enhancing the responsiveness of T-ALL cells to ambient growth factors, and provide strong rationale for use of IGF1R inhibitors to improve initial response to therapy and to achieve long-term cure of patients with T-ALL.

A class of human proteins that deliver functional proteins into mammalian cells in vitro and in vivo.

We discovered a class of naturally occurring human proteins with unusually high net positive charge that can potently deliver proteins in functional form into mammalian cells both in vitro and also in murine retina, pancreas, and white adipose tissues in vivo. These findings represent diverse macromolecule delivery agents for in vivo applications, and also raise the possibility that some of these human proteins may penetrate cells as part of their native biological functions.