Induction of complementary immunogenic necroptosis and apoptosis cell death pathways inhibits cancer metastasis and relapse.

Interactions of light-sensitive drugs and materials with Cerenkov radiation-emitting radiopharmaceuticals generate cytotoxic reactive oxygen species (ROS) to inhibit localized and disseminated cancer progression, but the cell death mechanisms underlying this radionuclide stimulated dynamic therapy (RaST) remain elusive. Using ROS-regenerative nanophotosensitizers coated with a tumor-targeting transferrin-titanocene complex (TiO2-TC-Tf) and radiolabeled 2-fluorodeoxyglucose (18FDG), we found that adherent dying cells maintained metabolic activity with increased membrane permeabilization. Mechanistic assessment of these cells revealed that RaST activated the expression of RIPK-1 and RIPK-3, which mediate necroptosis cell death. Subsequent recruitment of the nuclear factors kappa B and the executioner mixed lineage kinase domain-like pseudo kinase (MLKL) triggered plasma membrane permeabilization and pore formation, respectively, followed by the release of cytokines and immunogenic damage-associated molecular patterns (DAMPs). In immune-deficient breast cancer models with adequate stroma and growth factors that recapitulate the human tumor microenvironment, RaST failed to inhibit tumor progression and the ensuing lung metastasis. A similar aggressive tumor model in immunocompetent mice responded to RaST, achieving a remarkable partial response (PR) and complete response (CR) with no evidence of lung metastasis, suggesting active immune system engagement. RaST recruited antitumor CD11b+, CD11c+, and CD8b+ effector immune cells after initiating dual immunogenic apoptosis and necroptosis cell death pathways in responding tumors in vivo. Over time, cancer cells upregulated the expression of negative immune regulating cytokine (TGF-ß) and soluble immune checkpoints (sICP) to challenge RaST effect in the CR mice. Using a signal-amplifying cancer-imaging agent, LS301, we identified latent minimal residual disseminated tumors in the lymph nodes (LNs) of the CR group. Despite increased protumor immunogens in the CR mice, RaST prevented cancer relapse and metastasis through dynamic redistribution of ROS-regenerative TiO2 from bones at the early treatment stage to the spleen and LNs, maintaining active immunity against cancer progression and migration. This study reveals the immune-mechanistic underpinnings of RaST-mediated antitumor immune response and highlights immunogenic reprogramming of tumors in response to RaST. Overcoming apoptosis resistance through complementary necroptosis activation paves the way for strategic drug combinations to improve cancer treatment.

Analysis of Stable Chelate-free Gadolinium Loaded Titanium Dioxide Nanoparticles for MRI-Guided Radionuclide Stimulated Cancer Treatment.

Background: Recent studies demonstrate that titanium dioxide nanoparticles (TiO2 NPs) are an effective source of reactive oxygen species (ROS) for photodynamic therapy and radionuclide stimulated dynamic therapy (RaST). Unfortunately tracking the in vivo distribution of TiO2 NPs noninvasively remains elusive. Objective: Given the use of gadolinium (Gd) chelates as effective contrast agents for magnetic resonance imaging (MRI), this study aims to (1) develop hybrid TiO2-Gd NPs that exhibit high relaxivity for tracking the NPs without loss of ROS generating capacity; and (2) establish a simple colorimetric assay for quantifying Gd loading and stability. Methods: A chelate-free, heat-induced method was used to load Gd onto TiO2 NPs, which was coated with transferrin (Tf). A sensitive colorimetric assay and inductively coupled plasma mass spectrometry (ICP-MS) were used to determine Gd loading and stability of the TiO2-Gd-Tf NPs. Measurement of the relaxivity was performed on a 1.4 T relaxometer and a 4.7 T small animal magnetic resonance scanner to estimate the effects of magnetic field strength. ROS was quantified by activated dichlorodihydrofluorescein diacetate fluorescence. Cell uptake of the NPs and RaST were monitored by fluorescence microscopy. Both 3 T and 4.7 T scanners were used to image the in vivo distribution of intravenously injected NPs in tumor-bearing mice. Results: A simple colorimetric assay accurately determined both the loading and stability of the NPs compared with the expensive and complex ICP-MS method. Coating of the TiO2-Gd NPs with Tf stabilized the nanoconstruct and minimized aggregation. The TiO2-Gd-Tf maintained ROS-generating capability without inducing cell death at a wide range of concentrations but induced significant cell death under RaST conditions in the presence of F-18 radiolabeled 2-fluorodeoxyglucose. The longitudinal (r1 = 10.43 mM-1s-1) and transverse (r2 = 13.43 mM-1s-1) relaxivity of TiO2-Gd-Tf NPs were about twice and thrice, respectively, those of clinically used Gd contrast agent (Gd-DTPA; r1 = 3.77 mM-1s-1 and r2 = 5.51 mM-1s-1) at 1.4 T. While the r1 (8.13 mM-1s-1) reduced to about twice that of Gd-DTPA (4.89 mM-1s-1) at 4.7 T, the corresponding r2 (87.15 mM-1s-1) increased by a factor 22.6 compared to Gd-DTPA (r2 = 3.85). MRI of tumor-bearing mice injected with TiO2-Gd-Tf NPs tracked the NPs distribution and accumulation in tumors. Conclusion: This work demonstrates that Arsenazo III colorimetric assay can substitute ICP-MS for determining the loading and stability of Gd-doped TiO2 NPs. The new nanoconstruct enabled RaST effect in cells, exhibited high relaxivity, and enhanced MRI contrast in tumors in vivo, paving the way for in vivo MRI-guided RaST.

Side-chain modification of collagen-targeting peptide prevents dye aggregation for improved molecular imaging of arthritic joints.

Near-infrared (NIR) dye-peptide conjugates are widely used for tissue-targeted molecular fluorescence imaging of pathophysiologic conditions. However, the significant contribution of both dye and peptide to the net mass of these bioconjugates implies that small changes in either component could alter their photophysical and biological properties. Here, we synthesized and conjugated a type I collagen targeted peptide, RRANAALKAGELYKCILY, to either a hydrophobic (LS1000) or hydrophilic (LS1006) NIR fluorescent dye. Spectroscopic analysis revealed rapid self-assembly of both LS1000 and LS1006 in aqueous media to form stable dimeric/H aggregates, regardless of the free dye's solubility in water. We discovered that replacing the cysteine residue in LS1000 and LS1006 with acetamidomethyl cysteine to afford LS1001 and LS1107, respectively, disrupted the peptide's self-assembly and activated the previously quenched dye's fluorescence in aqueous conditions. These results highlight the dominant role of the octadecapeptide, but not the dye molecules, in controlling the photophysical properties of these conjugates by likely sequestering or extruding the hydrophobic or hydrophilic dyes, respectively. Application of the compounds for imaging collagen-rich tissue in an animal model of inflammatory arthritis showed enhanced uptake of all four conjugates, which retained high collagen-binding affinity, in inflamed joints. Moreover, LS1001 and LS1107 improved the arthritic joint-to-background contrast, suggesting that reduced aggregation enhanced the clearance of these compounds from non-target tissues. Our results highlight a peptide-driven strategy to alter the aggregation states of molecular probes in aqueous solutions, irrespective of the water-solubilizing properties of the dye molecules. The interplay between the monomeric and aggregated forms of the conjugates using simple thiol-modifiers lends the peptide-driven approach to diverse applications, including the effective imaging of inflammatory arthritis joints.

Inactivation of multidrug-resistant bacteria and bacterial spores and generation of high-potency bacterial vaccines using ultrashort pulsed lasers.

Multidrug-resistant organisms (MDROs) represent a continuing healthcare crisis with no definitive solution to date. An alternative to antibiotics is the development of therapies and vaccines using biocompatible physical methods such as ultrashort pulsed (USP) lasers, which have previously been shown to inactivate pathogens while minimizing collateral damage to human cells, blood proteins, and vaccine antigens. Here we demonstrate that visible USP laser treatment results in bactericidal effect (≥3-log load reduction) against clinically significant MDROs, including methicillin-resistant Staphylococcus aureus and extended spectrum beta-lactamase-producing Escherichia coli. Bacillus cereus endospores, which are highly resistant to conventional chemical and physical treatments, were also shown to be effectively inactivated by USP laser treatment, resulting in sporicidal (≥3-log load reduction) activity. Furthermore, we demonstrate that administration of USP laser-inactivated E. coli whole-cell vaccines at dosages as low as 105 cfu equivalents without adjuvant was able to protect 100% of mice against subsequent lethal challenge. Our findings open the possibility for application of USP lasers in disinfection of hospital environments, therapy of drug-resistant bacterial infections in skin or bloodstream via pheresis modalities, and in the production of potent bacterial vaccines.

Quantitative tumor depth determination using dual wavelength excitation fluorescence.

Quantifying solid tumor margins with fluorescence-guided surgery approaches is a challenge, particularly when using near infrared (NIR) wavelengths due to increased penetration depths. An NIR dual wavelength excitation fluorescence (DWEF) approach was developed that capitalizes on the wavelength-dependent attenuation of light in tissue to determine fluorophore depth. A portable dual wavelength excitation fluorescence imaging system was built and tested in parallel with an NIR tumor-targeting fluorophore in tissue mimicking phantoms, chicken tissue, and in vivo mouse models of breast cancer. The system showed high accuracy in all experiments. The low cost and simplicity of this approach make it ideal for clinical use.

Non-invasive monitoring of arthritis treatment response via targeting of tyrosine-phosphorylated annexin A2 in chondrocytes.

BACKGROUND: The development and optimization of therapies for rheumatoid arthritis (RA) is currently hindered by a lack of methods for early non-invasive monitoring of treatment response. Annexin A2, an inflammation-associated protein whose presence and phosphorylation levels are upregulated in RA, represents a potential molecular target for tracking RA treatment response. METHODS: LS301, a near-infrared dye-peptide conjugate that selectively targets tyrosine 23-phosphorylated annexin A2 (pANXA2), was evaluated for its utility in monitoring disease progression, remission, and early response to drug treatment in mouse models of RA by fluorescence imaging. The intraarticular distribution and localization of LS301 relative to pANXA2 was determined by histological and immunohistochemical methods. RESULTS: In mouse models of spontaneous and serum transfer-induced inflammatory arthritis, intravenously administered LS301 showed selective accumulation in regions of joint pathology including paws, ankles, and knees with positive correlation between fluorescent signal and disease severity by clinical scoring. Whole-body near-infrared imaging with LS301 allowed tracking of spontaneous disease remission and the therapeutic response after dexamethasone treatment. Histological analysis showed preferential accumulation of LS301 within the chondrocytes and articular cartilage in arthritic mice, and colocalization was observed between LS301 and pANXA2 in the joint tissue. CONCLUSIONS: We demonstrate that fluorescence imaging with LS301 can be used to monitor the progression, remission, and early response to drug treatment in mouse models of RA. Given the ease of detecting LS301 with portable optical imaging devices, the agent may become a useful early treatment response reporter for arthritis diagnosis and drug evaluation.

Tumor microenvironment-targeted nanoparticles loaded with bortezomib and ROCK inhibitor improve efficacy in multiple myeloma.

Drug resistance and dose-limiting toxicities are significant barriers for treatment of multiple myeloma (MM). Bone marrow microenvironment (BMME) plays a major role in drug resistance in MM. Drug delivery with targeted nanoparticles have been shown to improve specificity and efficacy and reduce toxicity. We aim to improve treatments for MM by (1) using nanoparticle delivery to enhance efficacy and reduce toxicity; (2) targeting the tumor-associated endothelium for specific delivery of the cargo to the tumor area, and (3) synchronizing the delivery of chemotherapy (bortezomib; BTZ) and BMME-disrupting agents (ROCK inhibitor) to overcome BMME-induced drug resistance. We find that targeting the BMME with P-selectin glycoprotein ligand-1 (PSGL-1)-targeted BTZ and ROCK inhibitor-loaded liposomes is more effective than free drugs, non-targeted liposomes, and single-agent controls and reduces severe BTZ-associated side effects. These results support the use of PSGL-1-targeted multi-drug and even non-targeted liposomal BTZ formulations for the enhancement of patient outcome in MM.

Focal dynamic thermal imaging for label-free high-resolution characterization of materials and tissue heterogeneity.

Evolution from static to dynamic label-free thermal imaging has improved bulk tissue characterization, but fails to capture subtle thermal properties in heterogeneous systems. Here, we report a label-free, high speed, and high-resolution platform technology, focal dynamic thermal imaging (FDTI), for delineating material patterns and tissue heterogeneity. Stimulation of focal regions of thermally responsive systems with a narrow beam, low power, and low cost 405 nm laser perturbs the thermal equilibrium. Capturing the dynamic response of 3D printed phantoms, ex vivo biological tissue, and in vivo mouse and rat models of cancer with a thermal camera reveals material heterogeneity and delineates diseased from healthy tissue. The intuitive and non-contact FDTI method allows for rapid interrogation of suspicious lesions and longitudinal changes in tissue heterogeneity with high-resolution and large field of view. Portable FDTI holds promise as a clinical tool for capturing subtle differences in heterogeneity between malignant, benign, and inflamed tissue.

Intracranial glioma xenograft model rapidly reestablishes blood-brain barrier integrity for longitudinal imaging of tumor progression using fluorescence molecular tomography and contrast agents.

SIGNIFICANCE: The blood-brain barrier (BBB) is a major obstacle to detecting and treating brain tumors. Overcoming this challenge will facilitate the early and accurate detection of brain lesions and guide surgical resection of tumors. AIM: We generated an orthotopic brain tumor model that simulates the pathophysiology of gliomas at early stages; determine the BBB integrity and breakdown over the time course of tumor progression using generic and cancer-targeted near-infrared (NIR) fluorescent molecular probes. APPROACH: We developed an intracranial tumor xenograft model that rapidly reestablished BBB integrity and monitored tumor progression by bioluminescence imaging. Sham control mice were injected with phosphate-buffered saline only. Fluorescence molecular tomography (FMT) was used to quantify the uptake of tumor-targeted and passive NIR fluorescent imaging agents in orthotopic glioma (U87-GL-GFP PDE7B H217Q cells) tumor model. Cancer-induced and transient (with focused ultrasound, FUS) disruption of BBB integrity was monitored with NIR fluorescent dyes. RESULTS: Stereotactic injection of 50,000 cells into mouse brain allowed rapid reestablishment of BBB integrity within a week, as determined by the inability of both tumor-targeted and generic NIR imaging agents to extravasate into the brain. Tumor-induced BBB disruption was observed 7 weeks after tumor implantation. FUS achieved a similar effect at any time point after reestablishing BBB integrity. While tumor uptake and retention of the passive NIR dye, indocyanine green, was negligible, both actively tumor-targeting agents exhibited selective accumulation in the tumor region. The tumor-targeting molecular probe that clears rapidly from nontumor brain tissue exhibits higher contrast than the analogous vascular-targeting agent and helps delineate tumors from sham control. CONCLUSIONS: We highlight the utility of FMT imaging for longitudinal assessment of brain tumors and the interplay between the stages of BBB disruption and molecular probe retention in tumors, with potential application to other neurological diseases.

Selective imaging of solid tumours via the calcium-dependent high-affinity binding of a cyclic octapeptide to phosphorylated Annexin A2.

The heterogeneity and continuous genetic adaptation of tumours complicate their detection and treatment via the targeting of genetic mutations. However, hallmarks of cancer such as aberrant protein phosphorylation and calcium-mediated cell signalling provide broadly conserved molecular targets. Here, we show that, for a range of solid tumours, a cyclic octapeptide labelled with a near-infrared dye selectively binds to phosphorylated Annexin A2 (pANXA2), with high affinity at high levels of calcium. Because of cancer-cell-induced pANXA2 expression in tumour-associated stromal cells, the octapeptide preferentially binds to the invasive edges of tumours and then traffics within macrophages to the tumour's necrotic core. As proof-of-concept applications, we used the octapeptide to detect tumour xenografts and metastatic lesions, and to perform fluorescence-guided surgical tumour resection, in mice. Our findings suggest that high levels of pANXA2 in association with elevated calcium are present in the microenvironment of most solid cancers. The octapeptide might be broadly useful for selective tumour imaging and for delivering drugs to the edges and to the core of solid tumours.

Calcium carbonate nanoparticles stimulate tumor metabolic reprogramming and modulate tumor metastasis.

AIM: CaCO3 nanoparticles (nano-CaCO3) can neutralize the acidic pHe of solid tumors, but the lack of intrinsic imaging signal precludes noninvasive monitoring of pH-perturbation in tumor microenvironment. We aim to develop a theranostic version of nano-CaCO3 to noninvasively monitor pH modulation and subsequent tumor response. MATERIALS & METHODS: We synthesized ferromagnetic core coated with CaCO3 (magnetite CaCO3). Magnetic resonance imaging (MRI) was used to determine the biodistribution and pH modulation using murine fibrosarcoma and breast cancer models. RESULTS: Magnetite CaCO3-MRI imaging showed that nano-CaCO3 rapidly raised tumor pHe, followed by excessive tumor-associated acid production after its clearance. Continuous nano-CaCO3 infusion could inhibit metastasis. CONCLUSION: Nano-CaCO3 exposure induces tumor metabolic reprogramming that could account for the failure of previous intermittent pH-modulation strategies to achieve sustainable therapeutic effect.

Shape-Dependent Biodistribution of Biocompatible Silk Microcapsules.

Microcapsules are emerging as promising microsize drug carriers due to their remarkable deformability. Shape plays a dominant role in determining their vascular transportation. Herein, we explored the effect of the shape of the microcapsules on the in vivo biodistribution for rational design of microcapsules to achieve optimized targeting efficiency. Silk fibroin, a biocompatible, biodegradable, and abundant material, was utilized as a building block to construct biconcave discoidal and spherical microcapsules with diameter of 1.8 µm and wall thickness of 20 nm. We have compared the cytocompatibility, cellular uptake, and biodistribution of both microcapsules. Both biconcave and spherical microcapsules exhibited excellent cytocompatibility and internalization into cancer cells. During blood circulation in mice, both microcapsules showed retention in liver and kidney and most underwent renal clearance. However, we observed significantly higher accumulation of biconcave silk microcapsules in lung compared with spherical microcapsules, and the accumulation was found to be stable in lung even after 3 days. The higher concentration of biconcave discoidal microcapsules found in lung arises from pulmonary environment, margination dynamics, and enhanced deformation in bloodstream. Red blood cell (RBC)-mimicking silk microcapsules demonstrated here can potentially serve as a promising platform for delivering drugs for lung diseases.

Bio-inspired imager improves sensitivity in near-infrared fluorescence image-guided surgery.

Image-guided surgery can enhance cancer treatment by decreasing, and ideally eliminating, positive tumor margins and iatrogenic damage to healthy tissue. Current state-of-the-art near-infrared fluorescence imaging systems are bulky and costly, lack sensitivity under surgical illumination, and lack co-registration accuracy between multimodal images. As a result, an overwhelming majority of physicians still rely on their unaided eyes and palpation as the primary sensing modalities for distinguishing cancerous from healthy tissue. Here we introduce an innovative design, comprising an artificial multispectral sensor inspired by the Morpho butterfly's compound eye, which can significantly improve image-guided surgery. By monolithically integrating spectral tapetal filters with photodetectors, we have realized a single-chip multispectral imager with 1000 × higher sensitivity and 7 × better spatial co-registration accuracy compared to clinical imaging systems in current use. Preclinical and clinical data demonstrate that this technology seamlessly integrates into the surgical workflow while providing surgeons with real-time information on the location of cancerous tissue and sentinel lymph nodes. Due to its low manufacturing cost, our bio-inspired sensor will provide resource-limited hospitals with much-needed technology to enable more accurate value-based health care.

Metal-Organic Framework Encapsulation Preserves the Bioactivity of Protein Therapeutics.

Protein therapeutics are prone to lose their structure and bioactivity under various environmental stressors. This study reports a facile approach using a nanoporous material, zeolitic imidazolate framework-8 (ZIF-8), as an encapsulant for preserving the prototypic protein therapeutic, insulin, against different harsh conditions that may be encountered during storage, formulation, and transport, including elevated temperatures, mechanical agitation, and organic solvent. Both immunoassay and spectroscopy analyses demonstrate the preserved chemical stability and structural integrity of insulin offered by the ZIF-8 encapsulation. Biological activity of ZIF-8-preserved insulin after storage under accelerated degradation conditions (i.e., 40 °C) is evaluated in vivo using a diabetic mouse model, and shows comparable bioactivity to refrigeration-stored insulin (-20 °C). It is also demonstrated that ZIF-8-preserved insulin has low cytotoxicity in vitro and does not cause side effects in vivo. Furthermore, ZIF-8 residue can be completely removed by a simple purification step before insulin administration. This biopreservation approach is potentially applicable to diverse protein therapeutics, thus extending the benefits of advanced biologics to resource-limited settings and underserved populations/regions.

Radionuclides transform chemotherapeutics into phototherapeutics for precise treatment of disseminated cancer.

Most cancer patients succumb to disseminated disease because conventional systemic therapies lack spatiotemporal control of their toxic effects in vivo, particularly in a complicated milieu such as bone marrow where progenitor stem cells reside. Here, we demonstrate the treatment of disseminated cancer by photoactivatable drugs using radiopharmaceuticals. An orthogonal-targeting strategy and a contact-facilitated nanomicelle technology enabled highly selective delivery and co-localization of titanocene and radiolabelled fluorodeoxyglucose in disseminated multiple myeloma cells. Selective ablation of the cancer cells was achieved without significant off-target toxicity to the resident stem cells. Genomic, proteomic and multimodal imaging analyses revealed that the downregulation of CD49d, one of the dimeric protein targets of the nanomicelles, caused therapy resistance in small clusters of cancer cells. Similar treatment of a highly metastatic breast cancer model using human serum albumin-titanocene formulation significantly inhibited cancer growth. This strategy expands the use of phototherapy for treating previously inaccessible metastatic disease.

Perfusion-based fluorescence imaging method delineates diverse organs and identifies multifocal tumors using generic near-infrared molecular probes.

Rapid detection of multifocal cancer without the use of complex imaging schemes will improve treatment outcomes. In this study, dynamic fluorescence imaging was used to harness differences in the perfusion kinetics of near-infrared (NIR) fluorescent dyes to visualize structural characteristics of different tissues. Using the hydrophobic nontumor-selective NIR dye cypate, and the hydrophilic dye LS288, a high tumor-to-background contrast was achieved, allowing the delineation of diverse tissue types while maintaining short imaging times. By clustering tissue types with similar perfusion properties, the dynamic fluorescence imaging method identified secondary tumor locations when only the primary tumor position was known, with a respective sensitivity and specificity of 0.97 and 0.75 for cypate, and 0.85 and 0.81 for LS288. Histological analysis suggests that the vasculature in the connective tissue that directly surrounds the tumor was a major factor for tumor identification through perfusion imaging. Although the hydrophobic dye showed higher specificity than the hydrophilic probe, use of other dyes with different physical and biological properties could further improve the accuracy of the dynamic imaging platform to identify multifocal tumors for potential use in real-time intraoperative procedures.

Protonation and Trapping of a Small pH-Sensitive Near-Infrared Fluorescent Molecule in the Acidic Tumor Environment Delineate Diverse Tumors in Vivo.

Enhanced glycolysis and poor perfusion in most solid malignant tumors create an acidic extracellular environment, which enhances tumor growth, invasion, and metastasis. Complex molecular systems have been explored for imaging and treating these tumors. Here, we report the development of a small molecule, LS662, that emits near-infrared (NIR) fluorescence upon protonation by the extracellular acidic pH environment of diverse solid tumors. Protonation of LS662 induces selective internalization into tumor cells and retention in the tumor microenvironment. Noninvasive NIR imaging demonstrates selective retention of the pH sensor in diverse tumors, and two-photon microscopy of ex vivo tumors reveals significant retention of LS662 in tumor cells and the acid tumor microenvironment. Passive and active internalization processes combine to enhance NIR fluorescence in tumors over time. The low background fluorescence allows tumors to be detected with high sensitivity, as well as dead or dying cells to be delineated from healthy cells. In addition to demonstrating the feasibility of using small molecule pH sensors to image multiple aggressive solid tumor types via a protonation-induced internalization and retention pathway, the study reveals the potential of using LS662 to monitor treatment response and tumor-targeted drug delivery.

Binocular Goggle Augmented Imaging and Navigation System provides real-time fluorescence image guidance for tumor resection and sentinel lymph node mapping.

The inability to identify microscopic tumors and assess surgical margins in real-time during oncologic surgery leads to incomplete tumor removal, increases the chances of tumor recurrence, and necessitates costly repeat surgery. To overcome these challenges, we have developed a wearable goggle augmented imaging and navigation system (GAINS) that can provide accurate intraoperative visualization of tumors and sentinel lymph nodes in real-time without disrupting normal surgical workflow. GAINS projects both near-infrared fluorescence from tumors and the natural color images of tissue onto a head-mounted display without latency. Aided by tumor-targeted contrast agents, the system detected tumors in subcutaneous and metastatic mouse models with high accuracy (sensitivity = 100%, specificity = 98% ± 5% standard deviation). Human pilot studies in breast cancer and melanoma patients using a near-infrared dye show that the GAINS detected sentinel lymph nodes with 100% sensitivity. Clinical use of the GAINS to guide tumor resection and sentinel lymph node mapping promises to improve surgical outcomes, reduce rates of repeat surgery, and improve the accuracy of cancer staging.

Shape-based Automatic Detection of Pectoral Muscle Boundary in Mammograms.

The detection of the pectoral muscle boundary in the medio-lateral oblique view of mammograms is essential to improving the computer-aided diagnosis of breast cancer. In this study, a shape-based detection method is proposed for accurately extracting the boundary of the pectoral muscle in mammograms. A shape-based enhancement mask is applied to the mammogram and the initial boundary is then defined using morphological operators. The seed point is then detected on the initial boundary and the pectoral boundary is evolved from candidate points produced using a shape-based growth strategy. A cubic polynomial fitting function is implemented to obtain the final pectoral muscle boundary. The proposed method was applied to 322 mammograms from the mini Mammographic Image Analysis Society database. A 97.2 % acceptable rate from expert radiologists and assessment results based on the false positive rate, false negative rate, and Hausdorff distance demonstrate the robustness and effectiveness of the proposed shape-based detection method.

Utilizing the Multiradionuclide Resolving Power of SPECT and Dual Radiolabeled Single Molecules to Assess Treatment Response of Tumors.

PURPOSE: Single photon emission computed tomography (SPECT) radionuclide pairs having distinct decay rates and different energy maxima enable simultaneous detection of dual gamma signals and real-time assessment of dynamic functional and molecular processes in vivo. Here, we report image acquisition and quantification protocols for a single molecule labeled with two different radionuclides for functional SPECT imaging. PROCEDURES: LS370 and LS734 were prepared using modular solid phase peptide synthesis. Each agent has a caspase-3 cleavable reporting motif, flanked by a tyrosine residue and a chelator at the opposite end of molecule. Cell uptake and efflux were assessed in human MDA-MB-231 breast cancer cells. Biodistribution studies were conducted in tumor naive and orthotopic 4T1 metastatic breast cancer tumor mice. NanoSPECT dual-imaging validation and attenuation correction parameters were developed using phantom vials containing varying radionuclide concentrations. Proof-of-principle SPECT imaging was performed in MMTV-PyMT transgenic mice. RESULTS: LS370 and LS734 were singly or dually radiolabeled with (125)I and (111)In or (99m)Tc. Cell assays demonstrated 11-fold higher percent uptake (P < 0.001) of [(125)I]LS734 (3.6 ± 0.5) compared to [(125)I]LS370 (0.3 ± 0.3) at 2 h. Following chemotherapy, cellular retention of [(125)I]LS734 was 3-fold higher (P < 0.05) than untreated cells. Pharmacokinetics at 1 h postinjection demonstrated longer blood retention (%ID/g) for [(125)I]LS734 (3.2 ± 0.9) compared to [(125)I]LS370 (1.6 ± 0.1). In mice bearing bilateral orthotopic 4T1 tumors, the uptake (%ID/g) was 2.4 ± 0.3 for [(125)I]LS734 and 1.2 ± 0.03 for [(125)I]LS370. The iodinated tyrosine peptide residue label was stable under in vitro conditions for up to 24 h; rapid systemic deiodination (high thyroid uptake) was observed in vivo. Phantom studies using standards demonstrated deconvolution of radionuclide signals based on different gamma ray energies. In MMTV-PyMT mice imaged with dual-labeled [(111)In]-[(125)I]LS734, the gamma signals were separable and quantifiable. CONCLUSIONS: Image processing protocols were developed for quantitative signal separation resulting from a caspase-3 responsive dual-radiolabeled SPECT probe. Crosstalk unmixing was obtained for multiradionuclide NanoSPECT imaging. In vitro and in vivo data demonstrated structure-activity relationships for developing functional agents for ratiometric SPECT imaging.