Evaluation of Wearable Acoustic Sensors and Machine Learning Algorithms for Automated Measurement of Left Ventricular Ejection Fraction.

Left ventricular ejection fraction (EF) is a predictor of mortality and guides clinical decisions. Although transthoracic echocardiography (TTE) is commonly used for measuring EF, it has limitations, such as subjectivity and requires expert personnel. Advancements in biosensor technology and artificial intelligence are allowing systems capable of determining left ventricular function and providing automated measurement of EF. In this study, we tested new wearable automated real-time biosensors (Cardiac Performance System [CPS]) that compute EF using waveform machine learning on cardiac acoustic signals. The primary aim was to compare the accuracy of CPS EF with TTE EF. Adult patients presenting to cardiology, presurgical, and diagnostic radiology clinical settings in an academic center were enrolled. TTE examination was performed by a sonographer, followed immediately by a 3-minute recording of acoustic signals from CPS biosensors placed on the chest by nonexpert personnel. TTE EF was calculated offline using the Simpson biplane method. A total of 81 patients (aged 19 to 88 years, 27 women, 20% to 80% EF) were included. Deming regression and Bland-Altman analysis were performed to assess the accuracy of CPS EF against TTE EF. Both Deming regression (slope 0.9981; intercept 0.03415%) and Bland-Altman analysis (bias -0.0247%; limits of agreement [-11.65, 11.60]%) demonstrated equivalency between CPS EF and TTE EF. The receiver operating characteristic for measuring sensitivity and specificity of CPS in identifying subjects with abnormal EF showed an area under the curve value of 0.974 for identifying EF <35% and 0.916 for detecting EF <50% CPS EF intraoperator and interoperator assessments demonstrated low variability. In conclusion, this technology measuring cardiac function from noninvasive biosensors and machine learning on acoustic signals provides an accurate EF measurement that is automated, real-time, and acquired rapidly by personnel with minimal training.

Propylene Glycol Caprylate-Based Nanoemulsion Formulation of Plumbagin: Development and Characterization of Anticancer Activity.

Plumbagin, a bioactive naphthoquinone, has demonstrated potent antitumor potential. However, plumbagin is a sparingly water-soluble compound; therefore, clinical translation requires and will be facilitated by the development of a new pharmaceutical formulation. We have generated an oil-in-water nanoemulsion formulation of plumbagin using a low-energy spontaneous emulsification process with propylene glycol caprylate (Capryol 90) as an oil phase and Labrasol/Kolliphor RH40 as surfactant and cosurfactant excipients. Formulation studies using Capryol 90/Labrasol/Kolliphor RH40 components, based on pseudoternary diagram and analysis of particle size distribution and polydispersity determined by dynamic light scattering (DLS), identified an optimized composition of excipients for nanoparticle formulation. The nanoemulsion loaded with plumbagin as an active pharmaceutical ingredient had an average hydrodynamic diameter of 30.9 nm with narrow polydispersity. The nanoemulsion exhibited long-term stability, as well as good retention of particle size in simulated physiological environments. Furthermore, plumbagin-loaded nanoemulsion showed an augmented cytotoxicity against prostate cancer cells PTEN-P2 in comparison to free drug. In conclusion, we generated a formulation of plumbagin with high loading drug capacity, robust stability, and scalable production. Novel Capryol 90-based nanoemulsion formulation of plumbagin demonstrated antiproliferative activity against prostate cancer cells, warranting thus further pharmaceutical development.

Assessment of Left Ventricular Diastolic Function using Phonocardiogram Signals: A Comparison with Echocardiography.

This paper presents a fully-automated end-to-end phonocardiogram(PCG)-based wearable system capable of providing echocardiography-like metrics for left ventricular (LV) diastolic function assessment. Proxy metrics for five echocardiographic parameters were calculated based on physiologically-motivated features extracted from PCG signals using noise-subtraction, heartbeat-segmentation, and quality-assurance algorithms. The clinical value of these proxy metrics was evaluated using the latest American Society of Echocardiography/European Association of Cardiovascular Imaging guidelines for evaluation of LV diastolic function. When tested on a group of n=34 patients, proxy metrics successfully identified LV diastolic dysfunction in a n=29 subset with 87.5% accuracy, and elevated LV filling pressures in a n=17 subset with 75% accuracy.

Plumbagin-Serum Albumin Interaction: Spectral, Electrochemical, Structure-Binding Analysis, Antiproliferative and Cell Signaling Aspects with Implications for Anticancer Therapy.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a small molecule with potent anticancer activity. Like other 1,4-naphthoquinones, it exhibits electrophilic reactivity towards biological nucleophiles. We demonstrate that plumbagin and structurally related 1,4-naphthoquinones with at least one unsubstituted quinoid carbon (C2 or C3) bind to albumin, an ubiquitously present nucleophile, with minimum recovery of free drug. Extraction recovery of plumbagin from albumin in solution showed one-phase exponential decline with a half-live of 9.3 min at 10 µmol/L. In the presence of albumin, plumbagin exhibited instant changes in UV/Vis absorption bands. Electrochemical analysis using cyclic voltammetry showed a decrease in redox peak currents over time until electro-inactivity, thus suggesting the formation of a supramolecular adduct inaccessible for electron transfer. The adduct inhibited cell growth and caused cell-cycle arrest of prostate cancer cells, in part by decreasing levels of the cell-cycle regulator RBBP. The conjugate displayed similar cellular effects to those described for plumbagin, such as decreased levels of androgen receptor and protein kinase C epsilon. The effect of plumbagin-albumin on cancer cells was species-specific, suggesting a receptor-mediated mechanism. Furthermore, it was blocked by cathepsin inhibitor pepstatin A, indicating that lysosomal degradation is involved in the processing of plumbagin-albumin adduct. The spontaneously formed adduct of plumbagin with serum albumin is likely to mediate the biological activities of plumbagin in vivo and to fundamentally influence its pharmacodynamics.

Fully-Automated Diagnosis of Aortic Stenosis Using Phonocardiogram-Based Features.

The irreversible damage and eventual heart failure caused by untreated aortic stenosis (AS) can be prevented by early detection and timely intervention. Prior work in the field of phonocardiogram (PCG) signal analysis has provided proof of concept for using heart-sound data in AS diagnosis. However, such systems either require operation by trained technicians, fail to address a diverse subject set, or involve unwieldy configuration procedures that challenge real-world application. This paper presents an end-to-end, fully-automated system that uses noise-subtraction, heartbeat-segmentation and quality-assurance algorithms to extract physiologically-motivated features from PCG signals to diagnose AS. When tested on n=96 patients showing a diverse set of cardiac and non-cardiac conditions, the system was able to diagnose AS with 92% sensitivity and 95% specificity.

Plumbagin-Loaded Nanoemulsion Drug Delivery Formulation and Evaluation of Antiproliferative Effect on Prostate Cancer Cells.

BACKGROUND: Plumbagin, a medicinal plant-derived 5-hydroxy-2-methyl-1,4-naphthoquinone, is an emerging drug with a variety of pharmacological effects, including potent anticancer activity. We have previously shown that plumbagin improves the efficacy of androgen deprivation therapy (ADT) in prostate cancer and it is now being evaluated in phase I clinical trial. However, the development of formulation of plumbagin as a compound with sparing solubility in water is challenging. METHODS: We have formulated plumbagin-loaded nanoemulsion using pneumatically controlled high pressure homogenization of oleic acid dispersions with polyoxyethylene (20) sorbitan monooleate as surfactant. Nanoemulsion formulations were characterized for particle size distribution by dynamic light scattering (DLS). The kinetics of in vitro drug release was determined by equilibrium dialysis. Anticancer activity toward prostate cancer cells PTEN-P2 was assessed by MTS (Owen's reagent) assay. RESULTS: Particle size distribution of nanoemulsions is tunable and depends on the surfactant concentration. Nanoemulsion formulations of plumbagin with 1-3.5% (w/w) of surfactant showed robust stability of size distribution over time. Plumbagin-loaded nanoemulsion with average hydrodynamic diameter of 135 nm showed exponential release of plumbagin with a half-life of 6.1 h in simulated gastric fluid, 7.0 h in simulated intestinal fluid, and displayed enhanced antiproliferative effect toward prostate cancer cells PTEN-P2 compared to free plumbagin. CONCLUSION: High drug-loading capacity, retention of nanoparticle size, kinetics of release under simulated physiological conditions, and increased antiproliferative activity indicate that oleic-acid based nanoemulsion formulation is a suitable delivery system of plumbagin.

Differential gene expression induced by anti-cancer agent plumbagin is mediated by androgen receptor in prostate cancer cells.

Treatment of mice harboring PTEN-P2 tumors in the prostate or on prostate tissue in vivo with 5-hydroxy-2-methyl-1,4-naphthoquinone, also known as plumbagin, results in tumor regression in castrated mice, but not in intact mice. This suggested that dihydrotestosterone (DHT) production in the testes may prevent cell death due to plumbagin treatment, but the underlying mechanism is not understood. We performed RNA-seq analysis on cells treated with combinations of plumbagin and DHT, and analyzed differential gene expression, to gain insight into the interactions between androgen and plumbgin. DHT and plumbagin synergize to alter the expression of many genes that are not differentially regulated by either single agent when used alone. These experiments revealed that, for many genes, increases in mRNAs caused by DHT are sharply down-regulated by plumbagin, and that many transcripts change in response to plumbagin in a DHT-dependent manner. This suggests that androgen receptor mediates some of the effects of plumbagin on gene expression.

Plumbagin improves the efficacy of androgen deprivation therapy in prostate cancer: A pre-clinical study.

BACKGROUND: Plumbagin is a candidate drug for the treatment of prostate cancer. Previous observations indicated that it may improve the efficacy of androgen deprivation therapy (ADT). This study evaluates the effectiveness of treatment with combinations of plumbagin and alternative strategies for ADT in mouse models of prostate cancer to support its clinical use. METHODS: Plumbagin was administered per oral in a new sesame oil formulation. Standard toxicology studies were performed in rats. For tumor growth studies, mouse prostate cancer cell spheroids were placed on top of grafted prostate tissue in a dorsal chamber and allowed to form tumors. Mice were separated in various treatment groups and tumor size was measured over time by intra-vital microscopy. Survival studies were done in mice after injection of prostate cancer cells in the prostate of male animals. Androgen receptor (AR) levels were analyzed by Western blot from prostate cancer cells treated with plumbagin. RESULTS: Plumbagin caused a decrease in AR levels in vitro. In mice, plumbagin at 1 mg/kg in sesame oil displayed low toxicity and caused a 50% tumor regression when combined with castration. The combination of plumbagin with various forms of chemical ADT including treatment with a GnRH receptor agonist, a GnRH receptor antagonist, or CYP17A1 inhibitors, outperformed ADT alone, increasing mouse survival compared to the standard regimen of castration alone. In contrast, the combination of plumbagin with AR antagonists, such as bicalutamide and enzalutamide, showed no improvement over AR antagonists alone. Thus, plumbagin is effective in combination with drugs that prevent the synthesis of testosterone or its conversion to dihydrotestosterone, but not with drugs that bind to AR. CONCLUSION: Plumbagin significantly improves the effect of ADT drugs currently used in the clinic, with few side effects in mice.

Use of inertial sensors to predict pivot-shift grade and diagnose an ACL injury during preoperative testing.

BACKGROUND: The pivot-shift (PS) examination is used to demonstrate knee instability and detect anterior cruciate ligament (ACL) injury. Prior studies using inertial sensors identified the ACL-deficient knee with reasonable accuracy, but none addressed the more difficult problem of using these sensors to determine whether a subject has an ACL deficiency and to correctly assign a PS grade to a patient's knee. HYPOTHESIS: Inertial sensor data recorded during a PS examination can accurately predict ACL deficiency and the PS score assigned by the examining physician. STUDY DESIGN: Cohort study (diagnosis); Level of evidence, 2. METHODS: A total of 32 patients with unilateral ACL deficiency and 29 with intact ACLs in both knees had inertial sensor modules strapped to the tibia and femur of each limb for preoperative PS testing under anesthesia. Support vector machine (SVM) methods assessed PS grades on the basis of these data, with the examiner's clinical grading shift used as ground truth. A fusion of regression and SVM classification techniques diagnosed ACL deficiency. RESULTS: The clinically determined PS grades of all 122 knees were as follows: 0 (n = 69), +1 (n = 23), +2 (n = 27), and +3 (n = 3). The SVM classification analysis was 77% accurate in correctly classifying these grades, with 98% of computed PS grades falling within ±1 grade of the clinically determined value. The system fusion algorithm diagnosed ACL deficiency in an individual with an overall accuracy of 97%. This method yielded 6% false negatives and 0% false positives. CONCLUSION: This study used inertial sensor technology with SVM algorithms to accurately determine clinically assigned PS grades in ACL-intact and ACL-deficient knees. By extending the assessment to a separate group of patients without ACL injury, the inertial sensor data demonstrated highly accurate diagnosis of ACL deficiency.

Measurements of tibial rotation during a simulated pivot shift manoeuvre using a gyroscopic sensor.

PURPOSE: The pivot shift has been correlated with patient-reported outcomes and knee function following ACL injury and reconstruction. Tibial rotation has been recognized as an important component to the pivot shift motion path. However, few methodologies exist to quantify tibial rotation in the clinical setting. The purpose of this study was to validate the use of a wireless gyroscopic sensor to measure axial rotation of the tibia during a manually simulated pivot shift manoeuvre in cadaveric specimens. We hypothesized that integrated gyroscopic measurements of tibial rotation velocity (tibial rotation) would be highly correlated with tibial rotations simultaneously recorded with a rotary potentiometer during a simulated pivot shift motion under intact and ACL-deficient conditions. METHODS: Gyroscopic measurements of rotational velocity were integrated and calibrated to a known arc of rotation. The gyroscope was mounted on the distal tibia with its axis aligned to the tibial shaft. Ten simulations of a pivot shift motion pathway were performed on nine cadaveric knees under intact and ACL-deficient conditions. Logistic regression was used to compare gyroscopic and potentiometer measurements of tibial rotation for both test conditions. RESULTS: Gyroscopic measurements of maximum external tibial rotation during the simulated pivot shift motion pathway were strongly correlated with potentiometer measurements of external tibial rotation in both the intact and ACL-deficient states (R (2) = 0.984). CONCLUSION: The gyroscope evaluated in this cadaveric study was capable of accurately recording tibial rotation during a simulated pivot shift motion pathway.

In vivo proteomic imaging analysis of caveolae reveals pumping system to penetrate solid tumors.

Technologies are needed to map and image biological barriers in vivo that limit solid tumor delivery and, ultimately, the effectiveness of imaging and therapeutic agents. Here we integrate proteomic and imaging analyses of caveolae at the blood-tumor interface to discover an active transendothelial portal to infiltrate tumors. A post-translationally modified form of annexin A1 (AnnA1) is selectively concentrated in human and rodent tumor caveolae. To follow trafficking, we generated a specific AnnA1 antibody that targets caveolae in the tumor endothelium. Intravital microscopy of caveolae-immunotargeted fluorophores even at low intravenous doses showed rapid and robust pumping across the endothelium to enter mammary, prostate and lung tumors. Within 1 h, the fluorescence signal concentrated throughout tumors to exceed the peak levels in blood. This transvascular pumping required the expression of caveolin 1 and annexin A1. Tumor uptake with other antibodies were >100-fold less. This proteomic imaging strategy reveals a unique target, antibody and caveolae pumping system for solid tumor penetration.

Effects of different tissue microenvironments on gene expression in breast cancer cells.

In metastasis, circulating tumor cells penetrate the walls of blood vessels and enter the metastatic target tissue, thereby becoming exposed to novel and relatively unsupportive microenvironments. In the new microenvironments, the tumor cells often remain in a dormant state indefinitely and must adapt before they are able to successfully colonize the tissue. Very little is known about this adaptive process. We studied temporal changes in gene expression when breast cancer cells adapt to survive and grow on brain, bone marrow, and lung tissue maintained in an in vivo culture system, as models of the metastatic colonization of these tissues. We observed the transient activation of genes typically associated with homeostasis and stress during the initial stages of adaptation, followed by the activation of genes that mediate more advanced functions, such as elaboration of cell morphology and cell division, as the cells adapted to thrive in the host tissue microenvironment. We also observed the temporary induction of genes characteristic of the host tissue, which was particularly evident when tumor cells were grown on brain tissue. These early transient gene expression events suggest potential points of therapeutic intervention that are not evident in data from well-established tumors.

Use of a gyroscope sensor to quantify tibial motions during a pivot shift test.

PURPOSE: The purpose of this preliminary study was to evaluate the use of a gyroscope sensor to record rotations of the tibia about its long axis during a clinical pivot shift examination. METHODS: Ten patients with a unilateral ACL injury were tested under anaesthesia prior to surgery. Each ankle was placed in neutral position, wrapped and stabilized with athletic tape, and a small aluminium plate was taped to the bottom of the foot. A data recovery module was attached to the bottom of each plate using a swivel bracket that allowed alignment of the gyro axis with the long axis of the tibia. The module contained a triaxial gyroscope, battery and circuitry for wireless data broadcast to a laptop computer. Ten pivot shift tests were performed on both knees, and the surgeon's clinical grading of the pivot shift was noted for each limb. Mean values (10 trials) of peak tibial rotational velocity and integrated tibial rotation were compared between knees for each patient during the pivot shift reduction event (external tibial rotation during knee flexion). RESULTS: Five patients (50%) had significantly greater tibial rotation in their injured knee, four showed no difference between knees, and one had significantly greater rotation in the normal knee (p < 0.05). Seven patients (70%) showed greater peak rotational velocity in their injured knee, and three had no difference between the knees (p < 0.05). Correlations of rotation and rotational velocity with clinical pivot shift grade were weak (r2 = 0.09 and 0.19, respectively). CONCLUSIONS: Foot gyroscope measurements did not correctly identify the injured limb in all patients. Peak rotational velocity during the reduction event was a better indicator of ACL deficiency than the integrated rotation. If this technology is to be more useful clinically, gyroscope data may have to be combined with accelerometer data, perhaps with sensors mounted on both the tibia and femur. LEVEL OF EVIDENCE: Diagnostic case-control study, Level III.

The combination of plumbagin with androgen withdrawal causes profound regression of prostate tumors in vivo.

BACKGROUND: Hormonal ablation is the standard treatment for disseminated androgen-dependent prostate cancer. Although tumor growth is controlled at first, the tumor invariably recurs in the form of castration-resistant prostate cancer. This study assessed the efficacy of a new therapeutic strategy that combines plumbagin, a naturally occurring naphthoquinone, with androgen ablation. METHODS: Viewing microscopy chambers were placed in the dorsal skinfold of mice. Syngeneic prostate tissue was grafted within the chambers and allowed to vascularize. H2B-GFP/PTEN-P2 prostate cancer cells were co-implanted on top of the grafted prostate tissue. Androgen ablation was achieved using surgical castration. Intact and castrated mice were administered plumbagin or sham treatment. Tumor growth, mitosis and apoptosis were monitored in real-time using fluorescent Intra-Vital Microscopy. The mechanism of action of plumbagin was explored using human and mouse prostate cancer cells. RESULTS: Whereas both plumbagin and castration alone impeded tumor growth, only the combination of plumbagin and castration caused profound tumor regression in vivo, mostly due to increased apoptosis of the tumor cells. The cytotoxicity of plumbagin was not affected by androgens in vitro, suggesting that microenvironmental factors not present in culture play a crucial role in the combination effect. Plumbagin-induced cell death was mediated, at least in part, by activation of ERK and was due to generation of reactive oxygen species, because it was abolished by the anti-oxidant N-acetyl-L-cysteine. CONCLUSION: Androgen deprivation in combination with plumbagin may provide a significant improvement over androgen deprivation alone and deserves further evaluation.

Co-implanting orthotopic tissue creates stroma microenvironment enhancing growth and angiogenesis of multiple tumors.

Tumor models are needed to study cancer. Noninvasive imaging of tumors under native conditions in vivo is critical but challenging. Intravital microscopy (IVM) of subcutaneous tumors provides dynamic, continuous, long-term imaging at high resolution. Although popular, subcutaneous tumor models are often criticized for being ectopic and lacking orthotopic tissue microenvironments critical for proper development. Similar IVM of orthotopic and especially spontaneous tumors is seldom possible. Here, we generate and characterize tumor models in mice for breast, lung, prostate and ovarian cancer by co-engrafting tumor spheroids with orthotopic tissue in dorsal skin window chambers for IVM. We use tumor cells and tissue, both genetically engineered to express distinct fluorescent proteins, in order to distinguish neoplastic cells from engrafted tissue. IVM of this new, two-colored model reveals classic tumor morphology with red tumor cell nests surrounded by green stromal elements. The co-implanted tissue forms the supportive stroma and vasculature of these tumors. Tumor growth and angiogenesis are more robust when tumor cells are co-implanted with orthotopic tissue versus other tissues, or in the skin alone. The orthotopic tissue promotes tumor cell mitosis over apoptosis. With time, tumor cells can adapt to new environments and ultimately even grow better in the non-orthotopic tissue over the original orthotopic tissue. These models offer a significant advance by recreating an orthotopic microenvironment in an ectopic location that is still easy to image by IVM. These "ectopic-orthotopic" models provide an exceptional way to study tumor and stroma cells in cancer, and directly show the critical importance of microenvironment in the development of multiple tumors.

Intravital microscopy in the mouse dorsal chamber model for the study of solid tumors.

Intra-Vital Microscopy (IVM) is used to visualize tumors in animals and analyze various aspects of cancer physiology such as tumor vascularization, cell migration and metastasis. The main advantages of IVM include the real -time analysis of dynamic processes with single-cell resolution. The application of IVM, however, is limited by the availability of animal models that carry visually accessible tumors. These models have evolved over time to become more and more relevant to human tumors. The latest step is the development of a pseudo-orthotopic, syngeneic model for tumor growth and metastasis. In this model, tissue from a variety of mouse organs are grafted in a dorsal skinfold chamber and allowed to revascularize, whereupon tumor cell spheroids are implanted. These spheroids develop into tumors that bear a much closer resemblance to human tumors than xenografts. Unlike xenografts, the vasculature is well-ordered and, because the model is syngeneic, there are no cross-species host immune reactions. The use of fluorescence-tagged pseudo-organs and tumor cells allows IVM analysis and provides real-time access to the development of tumors that closely resemble the real disease. This model can be used to test therapeutics and to image tumor development and stroma-tumor interactions.

Regression of prostate tumors upon combination of hormone ablation therapy and celecoxib in vivo.

BACKGROUND: Hormonal ablation is the standard of treatment for advanced androgen-dependent prostate cancer. Although tumor regression is usually achieved at first, the cancer inevitably evolves toward androgen-independence, in part because of the development of mechanisms of resistance and in part because at the tissue level androgen withdrawal is not fully attained. Current research efforts are focused on new therapeutic strategies that will increase the effectiveness of androgen withdrawal and delay recurrence. We used a syngeneic pseudo-orthotropic mouse model of prostate cancer to test the efficacy of combining androgen withdrawal with FDA-approved COX-2 inhibitor celecoxib. METHODS: GFP-tagged TRAMP-C2 cells were co-implanted with prostate tissue in the dorsal chamber model and tumors were allowed to establish and vascularize. Tumor growth and angiogenesis were monitored in real-time using fluorescent intravital microscopy (IVM). Androgen withdrawal in mice was achieved using surgical castration or chemical hormonal ablation, alone or in combination with celecoxib (15 mg/kg, twice daily). RESULTS: Celecoxib alone decreased the growth of prostate tumors mostly by inducing mitotic failure, which resulted in increased apoptosis. Surprisingly, celecoxib did not possess significant angiostatic activity. Surgical or chemical castration prevented the growth of prostate tumors and this, on the other hand, was associated with disruption of the tumor vasculature. Finally, androgen withdrawal combined with celecoxib caused tumor regression through decreased angiogenesis and increased mitosis arrest and apoptosis. CONCLUSION: Celecoxib, a relatively safe COX-2-selective anti-inflammatory drug, significantly increases the efficacy of androgen withdrawal in vivo and warrants further investigation as a complement therapy for advanced prostate cancer.

Early Metabolic Flare in Squamous Cell Carcinoma after Chemotherapy is a Marker of Treatment Sensitivity In Vitro.

PURPOSE: Early metabolic response with a decrease in glucose demand after cytotoxic treatment has been reported to precede tumor volume shrinkage. However, preclinical studies report of a very early rise in metabolism, a flare, following treatment. To elucidate these observations, we performed an experimental study on early metabolic response with sequential analysis of metabolic changes. METHODS: Three squamous cell carcinoma cell lines and one nontumorigenic cell line were exposed to cisplatin. The uptake of the fluorescent glucose analogue 2-NBDG was examined at days 1-6 using fluorescence microscopy. The relation between 2-NBDG-uptake and cell survival was evaluated. RESULTS: The tumor cells exhibited a high uptake of 2-NBDG, whereas the uptake in the nonmalignant cells was low. The more cisplatin sensitive cell lines exhibited a more pronounced metabolic flare than the less sensitive cell line. CONCLUSION: A metabolic flare was a very early sign of treatment response and potentially it could be used as an early marker of treatment sensitivity.

Designed auto-assembly of nanostreptabodies for rapid tissue-specific targeting in vivo.

Molecular medicine can benefit greatly from antibodies that deliver therapeutic and imaging agents to select organs and diseased tissues. Yet the development of complex and defined composite nanostructures remains a challenge that requires both designed stoichiometric assembly and superior in vivo testing ability. Here, we generate nanostructures called nanostreptabodies by controlled sequential assembly of biotin-engineered antibody fragments on a streptavidin scaffold with a defined capacity for additional biotinylated payloads such as other antibodies to create bispecific antibodies as well as organic and non-organic moieties. When injected intravenously, these novel and stable nanostructures exhibit exquisite targeting with tissue-specific imaging and delivery, including rapid transendothelial transport that enhances tissue penetration. This "tinkertoy construction" strategy provides a very flexible and efficient way to link targeting vectors with reporter and/or effector agents, thereby providing virtually endless combinations potentially useful for multipurpose molecular and functional imaging in vivo as well as therapies.

Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung.

How effectively and quickly endothelial caveolae can transcytose in vivo is unknown, yet critical for understanding their function and potential clinical utility. Here we use quantitative proteomics to identify aminopeptidase P (APP) concentrated in caveolae of lung endothelium. Electron microscopy confirms this and shows that APP antibody targets nanoparticles to caveolae. Dynamic intravital fluorescence microscopy reveals that targeted caveolae operate effectively as pumps, moving antibody within seconds from blood across endothelium into lung tissue, even against a concentration gradient. This active transcytosis requires normal caveolin-1 expression. Whole body gamma-scintigraphic imaging shows rapid, specific delivery into lung well beyond that achieved by standard vascular targeting. This caveolar trafficking in vivo may underscore a key physiological mechanism for selective transvascular exchange and may provide an enhanced delivery system for imaging agents, drugs, gene-therapy vectors and nanomedicines. 'In vivo proteomic imaging' as described here integrates organellar proteomics with multiple imaging techniques to identify an accessible target space that includes the transvascular pumping space of the caveola.