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The rapid depletion of oxygen during irradiation at ultra-high dose rate calls for tissue oximeters capable of high temporal resolution. This study demonstrates a water-soluble phosphorescent nanoprobe and fiber-coupled instrument, which together are used to measure the kinetics of oxygen depletion at 200 Hz during irradiation of in vitro solutions.

Increasingly difficult-to-treat infections by antibiotic-resistant bacteria have become a major public health challenge. Rapid detection of common resistance mechanisms before empiric antibiotic usage is essential for optimizing therapeutic outcomes and containing further spread of resistance to antibiotics among other bacteria. Herein, we present a bioluminogenic probe, D-Bluco, for rapid detection of β-lactamase activity in viable pathogenic bacteria. D-Bluco is a pro-luciferin caged by a β-lactamase-responsive cephalosporin structure and further conjugated with a dabcyl quencher. The caging and quenching significantly decreased the initial background emission and increased the signal-to-background ratio by more than 1200-fold. D-Bluco was shown to detect a broad range of β-lactamases at the femtomolar level. An ultrasensitive RAPID bioluminescence assay using D-Bluco can detect 10(2) to 10(3) colony forming unit per milliliter (cfu/mL) of β-lactamase-producing Enterobacterales in urine samples within 30 min. The high sensitivity and rapid detection make the assay attractive for the use of point-of-care diagnostics for lactam-resistant pathogens.

Glioblastoma (GBM) has a dismal prognosis. Evidence from preclinical tumor models and human trials indicates the role of GBM-initiating cells (GIC) in GBM drug resistance. Here, we propose a new treatment option with tumor enzyme-activatable, combined therapeutic and diagnostic (theranostic) nanoparticles, which caused specific toxicity against GBM tumor cells and GICs. The theranostic cross-linked iron oxide nanoparticles (CLIO) were conjugated to a highly potent vascular disrupting agent (ICT) and secured with a matrix-metalloproteinase (MMP-14) cleavable peptide. Treatment with CLIO-ICT disrupted tumor vasculature of MMP-14–expressing GBM, induced GIC apoptosis, and significantly impaired tumor growth. In addition, the iron core of CLIO-ICT enabled in vivo drug tracking with MR imaging. Treatment with CLIO-ICT plus temozolomide achieved tumor remission and significantly increased survival of human GBM-bearing mice by more than 2-fold compared with treatment with temozolomide alone. Thus, we present a novel therapeutic strategy with significant impact on survival and great potential for clinical translation. Mol Cancer Ther; 16(9); 1909–21. ©2017 AACR.

In vivo tumorigenic assay for GIC derived from pcGBM39

Supplemental Video M6. 18FC-SNAT uptake in drug-treated tumors

Hypoxia plays a key role in tumor resistance to radiotherapy. It is important to study hypoxia dynamics during radiotherapy to improve treatment planning and prognosis. Here, we describe a luminescent nanoprobe, composed of a fluorescent semiconducting polymer and palladium complex, for quantitative longitudinal imaging of tumor hypoxia dynamics during radiotherapy. The nanoprobe was designed to provide high sensitivity and reversible response for the subtle change in hypoxia over a narrow range (0–30 mmHg O2), which spans the oxygen range where tumors have limited radiosensitivity. Following intravenous administration, the nanoprobe efficiently accumulated in and distributed across the tumor, including the hypoxic region. The ratio between emissions at 700 and 800 nm provided quantitative mapping of hypoxia across the entire tumor. The nanoprobe was used to image tumor hypoxia dynamics over 7 days during fractionated radiotherapy and revealed that high fractional dose (10 Gy) was more effective in improving tumor reoxygenation than low dose (2 Gy), and the effect tended to persist longer in smaller or more radiosensitive tumors. Our results also indicated the importance of the reoxygenation efficiency of the first fraction in the prediction of the radiation treatment outcome. In summary, this work has established a new nanoprobe for highly sensitive, quantitative, and longitudinal imaging of tumor hypoxia dynamics following radiotherapy, and demonstrated its value for assessing the efficacy of radiotherapy and radiation treatment planning.Significance:This study presents a novel nanoagent for the visualization and quantification of tumor hypoxia.

SF1: Zeta-potential of the nanoprobe; SF2: Absorption spectrum; SF3: Specificity test; SF4&5: Cytotoxicity test in HeLa and RWPE-1 cells; SF6: Validating probe response to tumor hypoxia; SF7: Bio-distribution study; SF8: Immunofluorescence study on H460 tumor; SF9&10: Hypoxia measurement at different tumor sizes and types; SF11: Reversible response of the nanoprobe to hypoxia; SF12: Stability of the nanoprobe upon radiation; SF13-15: Imaging tumor hypoxia dynamics during RT at different tumor sizes and types; SF16: Correlation between tumor hypoxia and treatment efficacy; SF17&18: Imaging tumor hypoxia dynamics during RT on H1299 tumor; SF19: Monitoring the response of tumor hypoxia upon drug treatment; Supplementary methods: materials, instrument, and procedures for cell uptake and cytotoxicity test, immunofluorescence staining, and in vivo hypoxia imaging.

Supplemental Materials and Methods. A detailed description of the radiosynthesis of each radiotracer, and detailed PET and SPECT imaging protocols.

Purpose: An early readout of tumor response to therapy through measurement of drug or radiation-induced cell death may provide important prognostic indications and improved patient management. It has been shown that the uptake of 18F-C-SNAT can be used to detect early response to therapy in tumors by positron emission tomography (PET) via a mechanism of caspase-3–triggered nanoaggregation.Experimental Design: Here, we compared the preclinical utility of 18F-C-SNAT for the detection of drug-induced cell death to clinically evaluated radiotracers, 18F-FDG, 99mTc-Annexin V, and 18F-ML-10 in tumor cells in culture, and in tumor-bearing mice in vivo.Results: In drug-treated lymphoma cells, 18F-FDG, 99mTc-Annexin V, and 18F-C-SNAT cell-associated radioactivity correlated well to levels of cell death (R2 > 0.8; P < 0.001), with no correlation measured for 18F-ML-10 (R2 = 0.05; P > 0.05). A similar pattern of response was observed in two human NSCLC cell lines following carboplatin treatment. EL-4 tumor uptake of 99mTc-Annexin V and 18F-C-SNAT were increased 1.4- and 2.1-fold, respectively, in drug-treated versus naïve control animals (P < 0.05), although 99mTc-Annexin V binding did not correlate to ex vivo TUNEL staining of tissue sections. A differential response was not observed with either 18F-FDG or 18F-ML-10.Conclusions: We have demonstrated here that 18F-C-SNAT can sensitively detect drug-induced cell death in murine lymphoma and human NSCLC. Despite favorable image contrast obtained with 18F-C-SNAT, the development of next-generation derivatives, using the same novel and promising uptake mechanism, but displaying improved biodistribution profiles, are warranted for maximum clinical utility. Clin Cancer Res; 21(17); 3896–905. ©2015 AACR.

Prostate cancer is one of the most common malignancies worldwide, yet limited tools exist for prognostic risk stratification of the disease. Identification of new biomarkers representing intrinsic features of malignant transformation and development of prognostic imaging technologies are critical for improving treatment decisions and patient survival. In this study, we analyzed radical prostatectomy specimens from 422 patients with localized disease to define the expression pattern of methionine aminopeptidase II (MetAP2), a cytosolic metalloprotease that has been identified as a druggable target in cancer. MetAP2 was highly expressed in 54% of low-grade and 59% of high-grade cancers. Elevated levels of MetAP2 at diagnosis were associated with shorter time to recurrence. Controlled self-assembly of a synthetic small molecule enabled design of the first MetAP2-activated PET imaging tracer for monitoring MetAP2 activity in vivo. The nanoparticles assembled upon MetAP2 activation were imaged in single prostate cancer cells with post-click fluorescence labeling. The fluorine-18–labeled tracers successfully differentiated MetAP2 activity in both MetAP2-knockdown and inhibitor-treated human prostate cancer xenografts by micro-PET/CT scanning. This highly sensitive imaging technology may provide a new tool for noninvasive early-risk stratification of prostate cancer and monitoring the therapeutic effect of MetAP2 inhibitors as anticancer drugs.Significance:This study defines MetAP2 as an early-risk stratifier for molecular imaging of aggressive prostate cancer and describes a MetAP2-activated self-assembly small-molecule PET tracer for imaging MetAP2 activity in vivo.

We report the discovery of a new prodrug, 6-chloro-9-nitro-5-oxo-5H-benzo(a)phenoxazine (CNOB). This prodrug is efficiently activated by ChrR6, the highly active prodrug activating bacterial enzyme we have previously developed. The CNOB/ChrR6 therapy was effective in killing several cancer cell lines in vitro. It also efficiently treated tumors in mice with up to 40% complete remission. 9-Amino-6-chloro-5H-benzo(a)phenoxazine-5-one (MCHB) was the only product of CNOB reduction by ChrR6. MCHB binds DNA; at nonlethal concentration, it causes cell accumulation in the S phase, and at lethal dose, it induces cell surface Annexin V and caspase-3 and caspase-9 activities. Further, MCHB colocalizes with mitochondria and disrupts their electrochemical potential. Thus, killing by CNOB involves MCHB, which likely induces apoptosis through the mitochondrial pathway. An attractive feature of the CNOB/ChrR6 regimen is that its toxic product, MCHB, is fluorescent. This feature proved helpful in in vitro studies because simple fluorescence measurements provided information on the kinetics of CNOB activation within the cells, MCHB killing mechanism, its generally efficient bystander effect in cells and cell spheroids, and its biodistribution. The emission wavelength of MCHB also permitted its visualization in live animals, allowing noninvasive qualitative imaging of MCHB in mice and the tumor microenvironment. This feature may simplify exploration of barriers to the penetration of MCHB in tumors and their amelioration. [Mol Cancer Ther 2009;8(2):333–41]

Methods, Figures S1-21, NMR & MS spectra

Movie of super-resolution imaging of assembled probes in a single cell.

Supplemental Figures S1-4 and Tables 1-2. Supplemental Figure S1. Mechanism of 18F-C-SNAT activation, cyclization and intracellular trapping; Supplemental Figure S2. Flow cytometric analysis of cell death in EL-4 cell mixtures; Supplemental Figure S3. Radiotracer uptake and retention in carboplatin- and vehicle-treated PC9 and A549 non small cell lung cancer cells; Supplemental Figure S4. SPECT imaging of 99mTc-Annexin V uptake in vivo; Supplemental Table 1. Ex vivo biodistribution data for selected tissues in naive and drug-treated mice 90 min after injection; Supplemental Table 2. Radiotracer tumor-to-tissue ratios in naive and drug-treated EL-4 xenografts 90 min after injection.

The ideal vaccine against viruses such as influenza and SARS-CoV-2 must provide a robust, durable and broad immune protection against multiple viral variants. However, antibody responses to current vaccines often lack robust cross-reactivity. Here we describe a polymeric Toll-like receptor 7 agonist nanoparticle (TLR7-NP) adjuvant, which enhances lymph node targeting, and leads to persistent activation of immune cells and broad immune responses. When mixed with alum-adsorbed antigens, this TLR7-NP adjuvant elicits cross-reactive antibodies for both dominant and subdominant epitopes and antigen-specific CD8+ T-cell responses in mice. This TLR7-NP-adjuvanted influenza subunit vaccine successfully protects mice against viral challenge of a different strain. This strategy also enhances the antibody response to a SARS-CoV-2 subunit vaccine against multiple viral variants that have emerged. Moreover, this TLR7-NP augments antigen-specific responses in human tonsil organoids. Overall, we describe a nanoparticle adjuvant to improve immune responses to viral antigens, with promising implications for developing broadly protective vaccines.

Raman spectroscopy provides excellent specificity forin vivopreclinical imaging through a readout of fingerprint-like spectra. To achieve sufficient sensitivity forin vivoRaman imaging, metallic gold nanoparticles larger than 10 nm were employed to amplify Raman signals via surface-enhanced Raman scattering (SERS). However, the inability to excrete such large gold nanoparticles has restricted the translation of Raman imaging. Here we present Raman-active metallic gold supraclusters that are biodegradable and excretable as nanoclusters. Although the small size of the gold nanocluster building blocks compromises the electromagnetic field enhancement effect, the supraclusters exhibit bright and prominent Raman scattering comparable to that of large gold nanoparticle-based SERS nanotags due to high loading of NIR-resonant Raman dyes and much suppressed fluorescence background by metallic supraclusters. The bright Raman scattering of the supraclusters was pH-responsive, and we successfully performedin vivoRaman imaging of acidic tumors in mice. Furthermore, in contrast to large gold nanoparticles that remain in the liver and spleen, the supraclusters dissociated into small nanoclusters, and 73% of the administered dose to mice was excreted over 4 months. The highly excretable Raman supraclusters demonstrated here offer great potential for clinical applications ofin vivoRaman imaging by replacing non-excretable large gold nanoparticles.

BackgroundHigh-grade meningioma is an aggressive type of brain cancer that is often recalcitrant to surgery and radiotherapy, leading to poor overall survival. Currently, there are no FDA-approved drugs for meningioma, highlighting the need for new therapeutic options, but development is challenging due to the lack of predictive preclinical models.MethodsTo leverage the known overexpression of procaspase-3 in meningioma, PAC-1, a blood-brain barrier penetrant procaspase-3 activator, was evaluated for its ability to induce apoptosis in meningioma cells. To enhance the effects of PAC-1, combinations with either hydroxyurea or temozolomide were explored in cell culture. Both combinations were further investigated in small groups of canine meningioma patients and assessed by MRI, and the novel apoptosis tracer, [18F]C-SNAT4, was evaluated in patients treated with PAC-1 + HU.ResultsIn meningioma cell lines in culture, PAC-1 + HU are synergistic while PAC-1 + TMZ show additive-to-synergistic effects. In canine meningioma patients, PAC-1 + HU led to stabilization of disease and no change in apoptosis within the tumor, whereas PAC-1 + TMZ reduced tumor burden in all three canine patients treated.ConclusionsOur results suggest PAC-1 + TMZ as a potentially efficacious combination for the treatment of human meningioma, and also demonstrate the utility of including pet dogs with meningioma as a means to assess anticancer strategies for this common brain tumor.

Positron emission tomography (PET) imaging of apoptosis can noninvasively detect cell death in vivo and assist in monitoring tumor response to treatment in patients. While extensive efforts have been devoted to addressing this important need, no apoptosis PET imaging agents have yet been approved for clinical use. This study reports an improved 18F-labeled caspase-sensitive nanoaggregation tracer ([18F]-C-SNAT4) for PET imaging of tumor response to chemo- and immunotherapies in preclinical mouse models. We rationally designed and synthesized a new PET tracer [18F]-C-SNAT4 to detect cell death both in vitro and in vivo. In vitro radiotracer uptake studies were performed on drug-sensitive and -resistant NSCLC cell lines (NCI-H460 and NCI-H1299, respectively) treated with cisplatin at different doses. In vivo therapy response monitoring by [18F]-C-SNAT4 PET imaging was evaluated with two treatment modalities—chemotherapy and immunotherapy in two tumor xenografts in mice. Radiotracer uptake in the tumors was validated ex vivo using γ-counting and cleaved caspase-3 immunofluorescence. This [18F]-C-SNAT4 PET tracer was facilely synthesized and displayed improved serum stability profiles. [18F]-C-SNAT4 cellular update was elevated in NCI-H460 cells in a time- and dose-dependent manner, which correlated well with cell death. A significant increase in [18F]-C-SNAT4 uptake was measured in NCI-H460 tumor xenografts in mice. In contrast, a rapid clearance of [18F]-C-SNAT4 was observed in drug-resistant NCI-H1299 in vitro and in tumor xenografts. Moreover, in BALB/C mice bearing murine colon cancer CT26 tumor xenografts receiving checkpoint inhibitors, [18F]-C-SNAT4 showed its ability for monitoring immunotherapy-induced apoptosis and reporting treatment-responding mice from non-responding. The uptake of [18F]-C-SNAT4 in tumors received chemotherapy and immunotherapy is positively correlated with the tumor apoptotic level and the treatment efficacy. [18F]-C-SNAT4 PET imaging can monitor tumor response to two different treatment modalities and predict the therapeutic efficacy in preclinical mouse models.

Prostate cancer is one of the most common malignancies worldwide, yet limited tools exist for prognostic risk stratification of the disease. Identification of new biomarkers representing intrinsic features of malignant transformation and development of prognostic imaging technologies are critical for improving treatment decisions and patient survival. In this study, we analyzed radical prostatectomy specimens from 422 patients with localized disease to define the expression pattern of methionine aminopeptidase II (MetAP2), a cytosolic metalloprotease that has been identified as a druggable target in cancer. MetAP2 was highly expressed in 54% of low-grade and 59% of high-grade cancers. Elevated levels of MetAP2 at diagnosis were associated with shorter time to recurrence. Controlled self-assembly of a synthetic small molecule enabled design of the first MetAP2-activated PET imaging tracer for monitoring MetAP2 activity in vivo. The nanoparticles assembled upon MetAP2 activation were imaged in single prostate cancer cells with post-click fluorescence labeling. The fluorine-18–labeled tracers successfully differentiated MetAP2 activity in both MetAP2-knockdown and inhibitor-treated human prostate cancer xenografts by micro-PET/CT scanning. This highly sensitive imaging technology may provide a new tool for noninvasive early-risk stratification of prostate cancer and monitoring the therapeutic effect of MetAP2 inhibitors as anticancer drugs. Significance: This study defines MetAP2 as an early-risk stratifier for molecular imaging of aggressive prostate cancer and describes a MetAP2-activated self-assembly small-molecule PET tracer for imaging MetAP2 activity in vivo.

Depletion of mitochondrial copper, which shifts metabolism from respiration to glycolysis and reduces energy production, is known to be effective against cancer types that depend on oxidative phosphorylation. However, existing copper chelators are too toxic or ineffective for cancer treatment. Here we develop a safe, mitochondria-targeted, copper-depleting nanoparticle (CDN) and test it against triple-negative breast cancer (TNBC). We show that CDNs decrease oxygen consumption and oxidative phosphorylation, cause a metabolic switch to glycolysis and reduce ATP production in TNBC cells. This energy deficiency, together with compromised mitochondrial membrane potential and elevated oxidative stress, results in apoptosis. CDNs should be less toxic than existing copper chelators because they favorably deprive copper in the mitochondria in cancer cells instead of systemic depletion. Indeed, we demonstrate low toxicity of CDNs in healthy mice. In three mouse models of TNBC, CDN administration inhibits tumor growth and substantially improves survival. The efficacy and safety of CDNs suggest the potential clinical relevance of this approach.

Tuberculosis (TB) disease is a global epidemic caused by the pathogenic Mycobacterium tuberculosis (Mtb). Tools that can track the replication status of viable Mtb cells within macrophages are vital for the elucidation of host-pathogen interactions. Here, we present a cephalosphorinase-dependent green trehalose (CDG-Tre) fluorogenic probe that enables fluorescence labeling of single live Bacille Calmette-Guerin (BCG) cells within macrophages at concentrations as low as 2 μM. CDG-Tre fluoresces upon activation by BlaC, the β-lactamase uniquely expressed by Mtb, and the fluorescent product is subsequently incorporated within the bacterial cell wall via trehalose metabolic pathway. CDG-Tre showed high selectivity for mycobacteria over other clinically prevalent species in the Corynebacterineae suborder. The unique labeling strategy of BCG by CDG-Tre provides a versatile tool for tracking Mtb in both pre- and postphagocytosis and elucidating fundamental physiological and pathological processes related to the mycomembrane.

The condensation reaction between 6-hydroxy-2-cyanobenzothiazole (CBT) and cysteine has been shown for various applications such as site-specific protein labelling and in vivo cancer imaging. This report further expands the substrate scope of this reaction by varying the substituents on aromatic nitriles and amino thiols and testing their reactivity and ability to form nanoparticles for cell imaging. The structure-activity relationship study leads to the identification of the minimum structural requirement for the macrocyclization and assembly process in forming nanoparticles. One of the scaffolds made of 2-pyrimidinecarbonitrile and cysteine joined by a benzyl linker was applied to design fluorescent probes for imaging caspase-3/7 and β-galactosidase activity in live cells. These results demonstrate the generality of this system for imaging hydrolytic enzymes.

The low magnetic saturation of iron oxide nanoparticles, which are developed primarily as contrast agents for magnetic resonance imaging, limits the sensitivity of their detection using magnetic particle imaging (MPI). Here, we show that FeCo nanoparticles that have a core diameter of 10 nm and bear a graphitic carbon shell decorated with poly(ethylene glycol) provide an MPI signal intensity that is sixfold and fifteenfold higher than the signals from the superparamagnetic iron oxide tracers VivoTrax and Feraheme, respectively, at the same molar concentration of iron. We also show that the nanoparticles have photothermal and magnetothermal properties and can therefore be used for tumour ablation in mice, and that they have high optical absorbance in a broad near-infrared region spectral range (wavelength, 700–1,200 nm), making them suitable as tracers for photoacoustic imaging. As sensitive multifunctional and multimodal imaging tracers, carbon-coated FeCo nanoparticles may confer advantages in cancer imaging and hyperthermia therapy. FeCo nanoparticles with a graphitic carbon shell decorated with poly(ethylene glycol) have photothermal and magnetothermal properties and are sensitive tracers for magnetic particle imaging, magnetic resonance imaging and photoacoustic imaging.

Photoacoustic (PA) imaging uses light excitation to generate the acoustic signal for detection and improves tissue penetration depth and spatial resolution in the clinically relevant depth of living subjects. However, strong background signals from blood and pigments have significantly compromised the sensitivity of PA imaging with exogenous contrast agents. Here we report a nanoparticle-based probe design that uses light to reversibly modulate the PA emission to enable photoacoustic photoswitching imaging (PAPSI) in living mice. Such a nanoprobe is built with upconverting nanocrystals and photoswitchable small molecules and can be switched on by NIR light through upconversion to UV energy. Reversibly photoswitching of the nanoprobe reliably removed strong tissue background, increased the contrast-to-noise ratio, and thus improved imaging sensitivity. We have shown that PAPSI can image 0.05 nM of the nanoprobe in hemoglobin solutions and 10(4) labeled cancer cells after implantation in living mice using a commercial PA imager.

Longitudinal multimodal imaging presents unique opportunities for noninvasive surveillance and prediction of treatment response to cancer immunotherapy. In this work we first designed a novel granzyme B activated self-assembly small molecule, G-SNAT, for the assessment of cytotoxic T lymphocyte mediated cancer cell killing. G-SNAT was found to specifically detect the activity of granzyme B within the cytotoxic granules of activated T cells and engaged cancer cells

Longitudinal multimodal imaging presents unique opportunities for noninvasive surveillance and prediction of treatment response to cancer immunotherapy. In this work we first designed a novel granzyme B activated self-assembly small molecule, G-SNAT, for quantitative assessment of cytotoxic T lymphocyte mediated cancer cell killingin vivo. In lymphoma tumor bearing mice, the retention of cyanine 5 labeled G-SNAT-Cy5 was shown to be highly correlated to CAR T-cell mediated granzyme B release and tumor eradication. In colorectal tumor-bearing transgenic mice, expressing firefly luciferase in hematopoietic cells, and which received combination treatment of anti-PD-1 and anti-CTLA-4, longitudinal bioluminescence and fluorescence imaging revealed the dynamics of immune cell expansion, trafficking, tumor infiltration, and cytotoxic activity which predicted therapeutic outcome before tumor shrinkage was evident. These results support further development of G-SNAT for imaging early immune response to checkpoint blockade and CAR T-cell therapy in patients and highlight the utility of multimodality imaging for improved mechanistic insights into cancer immunotherapy.

Fully effective vaccines for viruses such as Influenza and SARS-CoV-2 must elicit a diverse repertoire of antibodies against multiple drifted virus strains. However, how to achieve a diverse response has no general solution except to combine multiple strains, which risks diluting the response for all strains included. Here, we describe the synthesis of a universal, toll-like receptor 7 agonist (TLR7)-nanoparticle adjuvant, TLR7-NP, constructed from TLR7 agonist-initiated ring-opening polymerization of lactide and self-assembly with poly(ethylene glycol)-b-poly(lactic-co-glycolic acid). When mixed with Alum-adsorbed antigens, this TLR7-NP adjuvant elicited cross-reactive antibodies for both dominant and subdominant epitopes, as well as antigen-specific CD8+ T cell responses. TLR7-NPs adjuvanted influenza subunit vaccine successfully protected mice from heterologous viral challenge. TLR7-NPs also enhanced the antibody response to a SARS-CoV-2 subunit vaccine against multiple variants and revealed the mobilization of a virus-like response. We further demonstrate enhanced antigen-specific responses in human tonsil organoids with this novel adjuvant.

Cancer immunotherapy has revolutionized the treatment of cancer, but only a small subset of patients benefits from this new treatment regime. Imaging tools are useful for early detection of tumor response to immunotherapy and probing the dynamic and complex immune system. Here, we report a bioluminescence probe (GBLI-2) for non-invasive, real-time, longitudinal imaging of granzyme B activity in tumors receiving immune checkpoint inhibitors. GBLI-2 is made of the mouse granzyme B tetrapeptide IEFD substrate conjugated to D-luciferin through a self-immolative group. GBLI-2 was evaluated for imaging the dynamics of the granzyme B activity and predicting therapeutic efficacy in a syngeneic mouse model of CT26 murine colorectal carcinoma. The GBLI-2 signal correlated with the change in the population of PD-1- and granzyme B-expressing CD8

PURPOSE: There is a clinical need for agents that target glioma cells for non-invasive and intraoperative imaging to guide therapeutic intervention and improve the prognosis of glioma. Matrix metalloproteinase (MMP)-14 is overexpressed in glioma with negligible expression in normal brain, presenting MMP-14 as an attractive biomarker for imaging glioma. In this study, we designed a peptide probe containing a near-infrared fluorescence (NIRF) dye/quencher pair, a positron emission tomography (PET) radionuclide, and a moiety with high affinity to MMP-14. This novel substrate-binding peptide allows dual modality imaging of glioma only after cleavage by MMP-14 to activate the quenched NIRF signal, enhancing probe specificity and imaging contrast. METHODS: MMP-14 expression and activity in human glioma tissues and cells were measured in vitro by immunofluorescence and gel zymography. Cleavage of the novel substrate and substrate-binding peptides by glioma cells in vitro and glioma xenograft tumors in vivo was determined by NIRF imaging. Biodistribution of the radiolabeled MMP-14-binding peptide or substrate-binding peptide was determined in mice bearing orthotopic patient-derived xenograft (PDX) glioma tumors by PET imaging. RESULTS: Glioma cells with MMP-14 activity showed activation and retention of NIRF signal from the cleaved peptides. Resected mouse brains with PDX glioma tumors showed tumor-to-background NIRF ratios of 7.6–11.1 at 4 h after i.v. injection of the peptides. PET/CT images showed localization of activity in orthotopic PDX tumors after i.v. injection of (68)Ga-binding peptide or (64)Cu-substrate-binding peptide; uptake of the radiolabeled peptides in tumors was significantly reduced (p < 0.05) by blocking with the non-labeled-binding peptide. PET and NIRF signals correlated linearly in the orthotopic PDX tumors. Immunohistochemistry showed co-localization of MMP-14 expression and NIRF signal in the resected tumors. CONCLUSIONS: The novel MMP-14 substrate-binding peptide enabled PET/NIRF imaging of glioma models in mice, warranting future image-guided resection studies with the probe in preclinical glioma models.

Borylated poly(fluorene-benzothiadiazoles) (PF8-BT) are π-conjugated polymers (CPs) with deep-red/near-infrared (NIR) absorption and emission profiles suitable for in vivo optical imaging. A fully ...

Hypoxia plays a key role in tumor resistance to radiotherapy. It is important to study hypoxia dynamics during radiotherapy to improve treatment planning and prognosis. Here, we describe a luminescent nanoprobe, composed of a fluorescent semiconducting polymer and palladium complex, for quantitative longitudinal imaging of tumor hypoxia dynamics during radiotherapy. The nanoprobe was designed to provide high sensitivity and reversible response for the subtle change in hypoxia over a narrow range (0–30 mmHg O2), which spans the oxygen range where tumors have limited radiosensitivity. Following intravenous administration, the nanoprobe efficiently accumulated in and distributed across the tumor, including the hypoxic region. The ratio between emissions at 700 and 800 nm provided quantitative mapping of hypoxia across the entire tumor. The nanoprobe was used to image tumor hypoxia dynamics over 7 days during fractionated radiotherapy and revealed that high fractional dose (10 Gy) was more effective in improving tumor reoxygenation than low dose (2 Gy), and the effect tended to persist longer in smaller or more radiosensitive tumors. Our results also indicated the importance of the reoxygenation efficiency of the first fraction in the prediction of the radiation treatment outcome. In summary, this work has established a new nanoprobe for highly sensitive, quantitative, and longitudinal imaging of tumor hypoxia dynamics following radiotherapy, and demonstrated its value for assessing the efficacy of radiotherapy and radiation treatment planning. Significance: This study presents a novel nanoagent for the visualization and quantification of tumor hypoxia.

Despite considerable progress with our understanding of glioblastoma multiforme (GBM) and the precise delivery of radiotherapy, the prognosis for GBM patients is still unfavorable with tumor recurrence due to radioresistance being a major concern. We recently developed a cross-linked iron oxide nanoparticle conjugated to azademethylcolchicine (CLIO-ICT) to target and eradicate a subpopulation of quiescent cells, glioblastoma initiating cells (GICs), which could be a reason for radioresistance and tumor relapse. The purpose of our study was to investigate if CLIO-ICT has an additive therapeutic effect to enhance the response of GBMs to ionizing radiation. Methods: NSG™ mice bearing human GBMs and C57BL/6J mice bearing murine GBMs received CLIO-ICT, radiation, or combination treatment. The mice underwent pre- and post-treatment magnetic resonance imaging (MRI) scans, bioluminescence imaging (BLI), and histological analysis. Tumor nanoparticle enhancement, tumor flux, microvessel density, GIC, and apoptosis markers were compared between different groups using a one-way ANOVA and two-tailed Mann-Whitney test. Additional NSG™ mice underwent survival analyses with Kaplan-Meier curves and a log rank (Mantel-Cox) test. Results: At 2 weeks post-treatment, BLI and MRI scans revealed significant reduction in tumor size for CLIO-ICT plus radiation treated tumors compared to monotherapy or vehicle-treated tumors. Combining CLIO-ICT with radiation therapy significantly decreased microvessel density, decreased GICs, increased caspase-3 expression, and prolonged the survival of GBM-bearing mice. CLIO-ICT delivery to GBM could be monitored with MRI. and was not significantly different before and after radiation. There was no significant caspase-3 expression in normal brain at therapeutic doses of CLIO-ICT administered. Conclusion: Our data shows additive anti-tumor effects of CLIO-ICT nanoparticles in combination with radiotherapy. The combination therapy proposed here could potentially be a clinically translatable strategy for treating GBMs.

Multimodality imaging involves the use of more imaging modes to image the same living subjects and is now generally preferred in clinics for cancer imaging. Here we present multimodality—Magnetic Particle Imaging (MPI), Magnetic Resonance Imaging (MRI), Photoacoustic, Fluorescent—nanoparticles (termed MMPF NPs) for imaging tumor xenografts in living mice. MMPF NPs provide long-term (more than 2 months), dynamic, and accurate quantification, in vivo, of NPs and in real time by MPI. Moreover, MMPF NPs offer ultrasensitive MPI imaging of tumors (the tumor ROI increased by 30.6 times over that of preinjection). Moreover, the nanoparticle possessed a long-term blood circulation time (half-life at 49 h) and high tumor uptake (18% ID/g). MMPF NPs have been demonstrated for imaging breast and brain tumor xenografts in both subcutaneous and orthotopic models in mice via simultaneous MPI, MRI, fluorescence, and photoacoustic imaging with excellent tumor contrast to normal tissues.

Poly(ADP ribose) polymerase (PARP) enzymes generate poly(ADP ribose) post-translational modifications on target proteins for an array of functions centering on DNA and cell stress. PARP isoforms 1 and 2 are critically charged with the surveillance of DNA integrity and are the first line guardians of the genome against DNA breaks. Here we present a novel probe ([18F]-SuPAR) for noninvasive imaging of PARP-1/2 activity using positron emission tomography (PET). [18F]-SuPAR is a radiofluorinated nicotinamide adenine dinucleotide (NAD) analog that can be recognized by PARP-1/2 and incorporated into the long branched polymers of poly(ADP ribose) (PAR). The measurement of PARP-1/2 activity was supported by a reduction of radiotracer uptake in vivo following PARP-1/2 inhibition with talazoparib treatment, a potent PARP inhibitor recently approved by FDA for treatment of breast cancer, as well as ex vivo colocalization of radiotracer analog and poly(ADP ribose). With [18F]-SuPAR, we were able to map the dose- and time-dependent activation of PARP-1/2 following radiation therapy in breast and cervical cancer xenograft mouse models. Tumor response to therapy was determined by [18F]-SuPAR PET within 8 h of administration of a single dose of radiation equivalent to one round of stereotactic ablative radiotherapy.

Magnetic particle imaging (MPI) has recently emerged as a promising non-invasive imaging technique because of its signal linearly propotional to the tracer mass, ability to generate positive contrast, low tissue background, unlimited tissue penetration depth, and lack of ionizing radiation. The sensitivity and resolution of MPI are highly dependent on the properties of magnetic nanoparticles (MNPs), and extensive research efforts have been focused on the design and synthesis of tracers. This review examines parameters that dictate the performance of MNPs, including size, shape, composition, surface property, crystallinity, the surrounding environment, and aggregation state to provide guidance for engineering MPI tracers with better performance. Finally, we discuss applications of MPI imaging and its challenges and perspectives in clinical translation.

Nearly 140 years after Robert Koch discovered Mycobacterium tuberculosis, tuberculosis (TB) remains a global threat and a deadly human pathogen. M. tuberculosis is notable for complex host-pathogen interactions that lead to poorly understood disease states ranging from latent infection to active disease. Additionally, multiple pathologies with a distinct local milieu (bacterial burden, antibiotic exposure, and host response) can coexist simultaneously within the same subject and change independently over time. Current tools cannot optimally measure these distinct pathologies or the spatiotemporal changes. Next-generation molecular imaging affords unparalleled opportunities to visualize infection by providing holistic, 3D spatial characterization and noninvasive, temporal monitoring within the same subject. This rapidly evolving technology could powerfully augment TB research by advancing fundamental knowledge and accelerating the development of novel diagnostics, biomarkers, and therapeutics.

Tuberculosis (TB) disease is a global epidemic caused by the pathogenic Mycobacterium tuberculosis (Mtb). Tools that can track the replication status of viable Mtb cells within macrophages are vital for the elucidation of host-pathogen interactions. Here, we present a cephalosphorinase-dependent green trehalose (CDG-Tre) fluorogenic probe that enables fluorescence labeling of single live Bacille Calmette-Guérin (BCG) cells within macrophages at concentrations as low as 2 μM. CDG-Tre fluoresces upon activation by BlaC, the β-lactamase uniquely expressed by Mtb, and the fluorescent product is subsequently incorporated within the bacterial cell wall via trehalose metabolic pathway. CDG-Tre showed high selectivity for mycobacteria over other clinically prevalent species in the Corynebacterineae suborder. The unique labeling strategy of BCG by CDG-Tre provides a versatile tool for tracking Mtb in both pre- and post-phagocytosis and elucidating fundamental physiological and pathological processes related to the mycomembrane.

“Smart” biomaterials that are responsive to physiological or biochemical stimuli have found many biomedical applications for tissue engineering, therapeutics and molecular imaging. In this work we describe in situ polymerization of activatable biorthogonal small molecules in response to reducing environment change in vivo. We designed a carbohydrate linker- and cyanobenzothiazole-cysteine condensation reaction-based small molecule scaffold that can undergo rapid condensation reaction upon physiochemical changes (such as a reducing environment) to form polymers (pseudopolysaccharide). The fluorescent and photoacoustic properties of fluorophore-tagged condensation scaffold before and after the transformation have been examined with a dual-modality optical imaging method. These results confirmed the in situ polymerization of this probe after both local and systemic administration in living mice.

The π-conjugated polymer poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b0]-dithiophene)-alt-4,7-(2,1,3-benzothiadiazole)] (PCPDTBT) with deep-red/near-infrared (NIR) absorption and emission has been investigated as a contrast agent for in vivo optical and photoacoustic imaging. PCPDTBT is encapsulated within poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG2kDa -PLGA4kDa or PEG5kDa -PLGA55kDa ) micelles or enveloped by the phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2kDa -DPPE), to investigate the formulation effect on imaging performance, biodistribution, and biocompatibility. Nanoparticles that meet the quality requirements for parenteral administration are generated with similar physicochemical properties. Optical phantom imaging reveals that both PEG-PLGA systems exhibit a 30% higher signal-to-background ratio (SBR) than PEG2kDa -DPPE. This trend cannot be observed in a murine HeLa xenograft model following intravenous administration since dramatic differences in biodistribution are observed. PEG2kDa -PLGA4kDa systems accumulate more rapidly in the liver compared to other formulations and PEG2kDa -DPPE demonstrates a higher tumor localization. Protein content in the "hard" corona differs between formulations (PEG2kDa -DPPE < PEG2kDa -PLGA4kDa < PEG5kDa -PLGA55kDa ), although this observation alone does not explain biodistribution patterns. PEG2kDa -PLGA4kDa systems show the highest photoacoustic amplitude in a phantom, but also a lower signal in the tumor due to differences in biodistribution. This study demonstrates that formulations for conjugated polymer contrast agents can have significant impact on both imaging performance and biodistribution.

Owing to the diversity of cancer types and to the spatiotemporal heterogeneity of tumour signals, high-resolution imaging of occult malignancy is challenging. 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) allows for near-universal cancer detection, yet in many clinical scenarios it is hampered by false positives. Here, we report a method for the amplification of imaging contrast in tumours via the temporal integration of the imaging signals triggered by tumour acidosis. The method exploits the catastrophic disassembly, at the acidic pH of the tumour milieu, of pH-sensitive positron-emitting neutral copolymer micelles into polycationic polymers, which are then internalized and retained by the cancer cells. PET imaging of the 64Cu-labelled polymers detected small occult tumours (10–20 mm3) in the brain, head, neck and breast of mice at much higher contrast than FDG, 11C-methionine and pH-insensitive 64Cu-labelled nanoparticles. We also show that the pH-sensitive probes reduce false-positive detection rates in a mouse model of non-cancerous lipopolysaccharide-induced inflammation. This macromolecular strategy for integrating tumour acidosis should enable improved cancer detection, surveillance and staging.

Glioblastoma (GBM) has a dismal prognosis. Evidence from preclinical tumor models and human trials indicates the role of GBM-initiating cells (GIC) in GBM drug resistance. Here, we propose a new treatment option with tumor enzyme-activatable, combined therapeutic and diagnostic (theranostic) nanoparticles, which caused specific toxicity against GBM tumor cells and GICs. The theranostic cross-linked iron oxide nanoparticles (CLIO) were conjugated to a highly potent vascular disrupting agent (ICT) and secured with a matrix-metalloproteinase (MMP-14) cleavable peptide. Treatment with CLIO-ICT disrupted tumor vasculature of MMP-14–expressing GBM, induced GIC apoptosis, and significantly impaired tumor growth. In addition, the iron core of CLIO-ICT enabled in vivo drug tracking with MR imaging. Treatment with CLIO-ICT plus temozolomide achieved tumor remission and significantly increased survival of human GBM-bearing mice by more than 2-fold compared with treatment with temozolomide alone. Thus, we present a novel therapeutic strategy with significant impact on survival and great potential for clinical translation. Mol Cancer Ther; 16(9); 1909–21. ©2017 AACR.

Microglial activation is a key pathological feature of Alzheimer's disease (AD). PET imaging of translocator protein 18 kDa (TSPO) is a strategy to detect microglial activation in vivo. Here we assessed flutriciclamide ([18F]GE-180), a new second-generation TSPO-PET radiotracer, for its ability to monitor response to LM11A-31, a novel AD therapeutic in clinical trials. AD mice displaying pathology were treated orally with LM11A-31 for 3 months. Subsequent [18F]GE-180-PET imaging revealed significantly lower signal in cortex and hippocampus of LM11A-31-treated AD mice compared to those treated with vehicle, corresponding with decreased levels of TSPO immunostaining and microglial Iba1 immunostaining. In addition to detecting decreased microglial activation following LM11A-31 treatment, [18F]GE-180 identified activated microglia in AD mice with greater sensitivity than another second-generation TSPO radiotracer, [18F]PBR06. Together, these data demonstrate the promise of [18F]GE-180 as a potentially sensitive tool for tracking neuroinflammation in AD mice and for monitoring therapeutic modulation of microglial activation.

This work reports a novel caging strategy for designing fluorogenic probes to detect the activity of β-lactamases. The caging strategy uses a thiophenyl linker connected to a fluorophore caged by a good leaving group—dinitrophenyl. The uncaging proceeds in two steps through the sulfa-releasing and subsequent intramolecular substitution. The length of the linker has been examined and optimized to maximize the rate of intramolecular reaction and thus the rate of fluorescence activation. Finally based on this strategy, we prepared a green fluorogenic probe CAT-7 and validated its selectivity for detecting metallo-carbapenemases (VIM-27, IMP-1, NDM-1) in carbapenem-resistant Enterobacteriaceae (CRE) lysates.

The pre-targeted imaging of enzyme activity has not been reported, likely owing to the lack of a mechanism to retain the injected substrate in the first step for subsequent labeling. Herein, we report the use of two bioorthogonal reactions-the condensation reaction of aromatic nitriles and aminothiols and the inverse-electron demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO)-to develop a novel strategy for pre-targeted imaging of the activity of proteases. The substrate probe (TCO-C-SNAT4) can be selectively activated by an enzyme target (e.g. caspase-3/7), which triggers macrocyclization and subsequent in situ self-assembly into nanoaggregates retained at the target site. The tetrazine-imaging tag conjugate labels TCO in the nanoaggregates to generate selective signal retention for imaging in vitro, in cells, and in mice. Owing to the decoupling of enzyme activation and imaging tag immobilization, TCO-C-SNAT4 can be repeatedly injected to generate and accumulate more TCO-nanoaggregates for click labeling.