Apoptosis is a mode of programmed cell death in multicellular organisms and plays a central role in controlling embryonic development, growth and differentiation and monitoring the induction of tumor cell death through anticancer therapy. Since the most effective chemotherapeutics rely on apoptosis, imaging apoptotic processes can be an invaluable tool to monitor therapeutic intervention and discover new drugs modulating apoptosis. The most attractive target for developing specific apoptosis imaging probes is caspases, crucial mediators of apoptosis. Up to now, various optical imaging strategies for apoptosis have been developed as an easy and economical modality. However, current optical applications are limited by poor sensitivity and specificity. A subset of molecular imaging contrast agents known as “activatable” or “smart” molecular probes allow for very high signal-to-background ratios compared to conventional targeted contrast agents and open up the possibility of imaging intracellular targets. In this review, we will discuss the unique design strategies and applications of activatable probes recently developed for fluorescence and bioluminescence imaging of caspase activity.
Activatable probes; apoptosis; bioluminescence; caspases; optical imaging
Conventional imaging methods, such as angiography, computed tomography, magnetic resonance imaging and radionuclide imaging, rely on contrast agents (iodine, gadolinium, radioisotopes) that are “always on”. While these agents have proven clinically useful, they are not sufficiently sensitive because of the inadequate target to background ratio. A unique aspect of optical imaging is that fluorescence probes can be designed to be activatable, i.e. only “turned on” under certain conditions. These probes can be designed to emit signal only after binding a target tissue, greatly increasing sensitivity and specificity in the detection of disease. There are two basic types of activatable fluorescence probes; 1) conventional enzymatically activatable probes, which exist in the quenched state until activated by enzymatic cleavage mostly outside of the cells, and 2) newly designed target-cell specific activatable probes, which are quenched until activated in targeted cells by endolysosomal processing that results when the probe binds specific cell-surface receptors and is subsequently internalized. Herein, we present a review of the rational design and in vivo applications of target-cell specific activatable probes. Designing these probes based on their photo-chemical (e.g. activation strategy), pharmacological (e.g. biodistribution), and biological (e.g. target specificity) properties has recently allowed the rational design and synthesis of target-cell specific activatable fluorescence imaging probes, which can be conjugated to a wide variety of targeting molecules. Several different photo-chemical mechanisms have been utilized, each of which offers a unique capability for probe design. These include: self-quenching, homo- and hetero-fluorescence resonance energy transfer (FRET), H-dimer formation and photon-induced electron transfer (PeT). In addition, the repertoire is further expanded by the option for reversibility or irreversibility of the signal emitted using the aforementioned mechanisms. Given the wide range of photochemical mechanisms and properties, target-cell specific activatable probes possess considerable flexibility and can be adapted to specific diagnostic needs. Herein, we summarize the chemical, pharmacological, and biological basis of target-cell specific activatable imaging probes and discuss methods to successfully design such target-cell specific activatable probes for in vivo cancer imaging.
In vivo molecular imaging with target-specific activatable “smart” probes, which only yield fluorescence at the intended target, enables sensitive and specific cancer detection because of high target to background ratios (TBR). Dimerization and fluorescence quenching has been shown to occur in concentrated aqueous solutions of various fluorophores. Here, we hypothesized that fluorophore dimerization and quenching after conjugation to targeting proteins can occur at low concentration, which is reasonable for in vivo imaging probes, because protein molecules can stabilize the fluorophore dimers based on physico-chemical interactions. This dimerization can be exploited as a mechanism for fluorescence activation. Rhodamine derivatives were conjugated to the cancer targeting molecules, avidin and trastuzumab, which target D-galactose receptor and HER2/neu antigen, respectively. After conjugation, a large proportion of R6G and TAMRA formed H-type dimers, even at low concentrations, but could be fully dequenched upon dissociation of the dimers to monomers. Lipophilicity was a potential factor in promoting H-dimer formation. To demonstrate the fluorescence activation effect during in vivo fluorescence endoscopic molecular imaging, a highly quenched probe, avidin-TAMRA or a minimally quenched probe, avidin-Alexa488 was administered into mice with ovarian metastases to the peritoneum. The tumors were clearly visualized with avidin-TAMRA, with low background fluorescence; in contrast, the background fluorescence was high for avidin-Alexa488. Thus, H-dimer formation as a mechanism of fluorescence quenching could be used to develop fluorescence activatable probes for in vivo molecular imaging. Effective activatable optical probes can be designed by focusing on the H-dimer formation of fluorophores.
Apoptosis is required for normal cellular homeostasis and deregulation of the apoptotic process is implicated in various diseases. Previously, we developed a cell-penetrating near-infrared fluorescence (NIF) probe based on an activatable strategy to detect apoptosis-associated caspase activity in vivo. This probe consisted of a cell-penetrating Tat peptide conjugated to an effector recognition sequence (DEVD) that was flanked by a fluorophore-quencher pair (Alexa Fluor 647 and QSY 21). Once exposed to effector caspases, the recognition sequence was cleaved, resulting in separation of the fluorophore-quencher pair and signal generation. Herein, we present biochemical analysis of a second generation probe, KcapQ, with a modified cell-penetrating peptide sequence (KKKRKV). This modification resulted in a probe that was more sensitive to effector caspase enzymes, displayed an unexpectedly higher quenching efficiency between the fluorophore-quencher pair, and was potentially less toxic to cells. Assays using recombinant caspase enzymes revealed that the probe was specific for effector caspases (caspase 3>7>6). Analysis of apoptosis in HeLa cells treated with doxorubicin showed probe activation specific to apoptotic cells. In a rat model of retinal neuronal excitotoxicity, intravitreal injection of N-methyl-D-aspartate (NMDA) induced apoptosis of retinal ganglion cells (RGCs). Eyecup and retinal flat mount images of NMDA-pretreated animals injected intravitreally with KcapQ using a clinically-applicable protocol showed specific and widely-distributed cell-associated fluorescence signals compared to untreated control animals. Fluorescence microscopy images of vertical retinal sections from NMDA-pretreated animals confirmed that activated probe was predominantly localized to RGCs and co-localized with TUNEL labeling. Thus, KcapQ represents an improved effector caspase-activatable NIF probe for enhanced non-invasive analysis of apoptosis in whole cells and live animals.
Caspase; apoptosis; near-infrared fluorescence; cell-penetrating peptide; retinal ganglion cell; NMDA; molecular imaging
Non-invasive detection of caspase-3/7 activity in vivo has provided invaluable predictive information regarding tumor therapeutic efficacy and anti-tumor drug selection. Although a number of caspase-3/7 targeted fluorescence and positron emission tomography (PET) imaging probes have been developed, there is still a lack of gadolinium (Gd)-based magnetic resonance imaging (MRI) probes that enable high spatial resolution detection of caspase-3/7 activity in vivo. Here we employ a self-assembly approach and develop a caspase-3/7 activatable Gd-based MRI probe for monitoring tumor apoptosis in mice. Upon reduction and caspase-3/7 activation, the caspase-sensitive nano-aggregation MR probe (C-SNAM: 1) undergoes biocompatible intramolecular cyclization and subsequent self-assembly into Gd-nanoparticles (GdNPs). This results in enhanced r1 relaxivity—19.0 (post-activation) vs. 10.2 mM−1 s−1 (pre-activation) at 1 T in solution—and prolonged accumulation in chemotherapy-induced apoptotic cells and tumors that express active caspase-3/7. We demonstrate that C-SNAM reports caspase-3/7 activity by generating a significantly brighter T1-weighted MR signal compared to non-treated tumors following intravenous administration of C-SNAM, providing great potential for high-resolution imaging of tumor apoptosis in vivo.
Objectives: Most chemotherapy agents cause tumor cell death primarily by the induction of apoptosis. The ability to noninvasively image apoptosis in vivo could dramatically benefit pre-clinical and clinical evaluation of chemotherapeutics targeting the apoptotic pathway. This study aims to visualize the dynamics of apoptotic process with temporal bioluminescence imaging (BLI) using an apoptosis specific bioluminescence reporter gene. Methods: Both UM-SCC-22B human head and neck squamous carcinoma cells and 4T1 murine breast cancer cells were genetically modified with a caspase-3 specific cyclic firefly luciferase reporter gene (pcFluc-DEVD). Apoptosis induced by different concentrations of doxorubicin in the transfected cells was evaluated by both annexin V staining and BLI. Longitudinal BLI was performed in xenografted tumor models at different time points after doxorubicin or Doxil treatment, to evaluate apoptosis. After imaging, DNA fragmentation in apoptotic cells was assessed in frozen tumor sections using TUNEL staining. Results: Dose- and time-dependent apoptosis induced by doxorubicin in pcFluc-DEVD transfected UM-SCC-22B and 4T1 cells was visualized and quantified by BLI. Caspase-3 activation was confirmed by both caspase activity assay and GloTM luciferase assay. One dose of doxorubicin treatment induced a dramatic increase in BLI intensity as early as 24 h after treatment in 22B-pcFluc-DEVD xenografted tumors. Sustained signal increase was observed for the first 3 days and the fluorescent signal from ex vivo TUNEL staining was consistent with BLI imaging results. Long-term imaging revealed that BLI signal consistently increased and reached a maximum at around day 12 after the treatment with one dose of Doxil. Conclusions: BLI of apoptosis with pcFluc-DEVD as a reporter gene facilitates the determination of kinetics of the apoptotic process in a real-time manner, which provides a unique tool for drug development and therapy response monitoring.
apoptosis; cyclic firefly luciferase; bioluminescence imaging; doxorubicin; caspase-3.
Peptide probes for imaging retinal ganglion cell (RGC) apoptosis consist of a cell-penetrating peptide targeting moiety and a fluorophore-quencher pair flanking an effector caspase consensus sequence. Using ex vivo fluorescence imaging, we previously validated the capacity of these probes to identify apoptotic RGCs in cell culture and in an in vivo rat model of N-methyl- D-aspartate (NMDA)-induced neurotoxicity. Herein, using TcapQ488, a new probe designed and synthesized for compatibility with clinically-relevant imaging instruments, and real time imaging of a live rat RGC degeneration model, we fully characterized time- and dose-dependent probe activation, signal-to-noise ratios, and probe safety profiles in vivo. Adult rats received intravitreal injections of four NMDA concentrations followed by varying TcapQ488 doses. Fluorescence fundus imaging was performed sequentially in vivo using a confocal scanning laser ophthalmoscope and individual RGCs displaying activated probe were counted and analyzed. Rats also underwent electroretinography following intravitreal injection of probe. In vivo fluorescence fundus imaging revealed distinct single-cell probe activation as an indicator of RGC apoptosis induced by intravitreal NMDA injection that corresponded to the identical cells observed in retinal flat mounts of the same eye. Peak activation of probe in vivo was detected 12 hours post probe injection. Detectable fluorescent RGCs increased with increasing NMDA concentration; sensitivity of detection generally increased with increasing TcapQ488 dose until saturating at 0.387 nmol. Electroretinography following intravitreal injections of TcapQ488 showed no significant difference compared with control injections. We optimized the signal-to-noise ratio of a caspase-activatable cell penetrating peptide probe for quantitative non-invasive detection of RGC apoptosis in vivo. Full characterization of probe performance in this setting creates an important in vivo imaging standard for functional evaluation of future probe analogues and provides a basis for extending this strategy into glaucoma-specific animal models.
Multimodality molecular imaging should have potential for compensating the disadvantages and enhancing the advantages of each modality. Nuclear imaging is superior to optical imaging in whole body imaging and in quantification due to good tissue penetration of gamma rays. However, target specificity can be compromised by high background signal due to the always signal ON feature of nuclear probes. In contrast, optical imaging can be superior in target specific imaging by employing target-specific signal activation systems, although it is not quantitative because of signal attenuation. In this study, to take advantage of the mutual cooperation of each modality, multimodality imaging was performed by a combination of quantitative radiolabeled probe and an activatable optical probe. The monoclonal antibodies, panitumumab (anti-HER1) and trastuzumab (anti-HER2) were labeled with 111In and ICG, and tested in both HER1 and HER2 tumor bearing mice by the cocktail injection of radiolabeled and optical probes, and by the single injection of a dual-labeled probe. The optical and nuclear images were obtained over 6 days after the conjugates injection. The fluorescence activation properties of ICG labeled antibodies were also investigated by in vitro microscopy. In vitro microscopy demonstrated that there was no fluorescence signal with either panitumumab-ICG or trastuzumab-ICG, when the probes were bound to cell surface antigens but were not yet internalized. After the conjugates were internalized into the cells, both conjugates showed bright fluorescence signal only in the target cells. These results show both conjugates work as activatable probes. In vivo multimodality imaging by injection of a cocktail of radio-optical probes, only the target specific tumor was visualized by optical imaging. Meanwhile, the biodistribution profile of the injected antibody was provided by nuclear imaging. Similar results were obtained with radio and optical dual labeled probe, and it is confirmed that pharmacokinetic properties did not affect the results above.
Here, we could characterize the molecular targets by activatable optical probes, and visualize the delivery of targeting molecules quantitatively by radioactive probes. Multimodality molecular imaging combining activatable optical and radioactive probe has great potential for simultaneous visualization, characterization, and measurement of biological processes.
We introduce a simple, versatile and robust one-step technique that enables real-time imaging of multiple intracellular caspase activities in living cells without the need for complicated synthetic protocols. Conventional fluorogenic probes or recently reported activatable probes have been designed to target various proteases but are limited to extracellular molecules. Only a few have been applied to image intracellular proteases in living cells because most of these probes have limited cell-permeability. Our platform does not need complicated synthetic processes; instead it involves a straightforward peptide synthesis and a simple mixing step with a commercial transfection agent. The transfection agent efficiently delivered the highly quenched fluorogenic probes, comprised of distinctive pairs of dyes and quenchers, to the initiator caspase-8 and the effector caspase-3 in MDA-MB-435 cells, allowing dual-imaging of the activities of both caspases during the apoptotic process induced by TNF-related apoptosis induced ligand (TRAIL). With the combination of multiple fluorogenic probes, this simple platform can be applied to multiplexed imaging of selected intracellular proteases to study apoptotic processes in pathologies or for cell-based high throughput screening systems for drug discovery.
caspase; activatable probe; fluorescence imaging; peptide; transfection agent
Proteases play important roles during tumor angiogenesis, invasion, and metastasis. Various molecular imaging techniques have been employed for protease imaging: optical (both fluorescence and bioluminescence), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). In this review, we will summarize the current status of imaging proteases in cancer with these techniques. Optical imaging of proteases, in particular with fluorescence, is the most intensively validated and many of the imaging probes are already commercially available. It is generally agreed that the use of activatable probes is the most accurate and appropriate means for measuring protease activity. Molecular imaging of proteases with other techniques (i.e. MRI, SPECT, and PET) has not been well-documented in the literature which certainly deserves much future effort. Optical imaging and molecular MRI of protease activity has very limited potential for clinical investigation. PET/SPECT imaging is suitable for clinical investigation; however the optimal probes for PET/SPECT imaging of proteases in cancer have yet to be developed. Successful development of protease imaging probes with optimal in vivo stability, tumor targeting efficacy, and desirable pharmacokinetics for clinical translation will eventually improve cancer patient management. Not limited to cancer, these protease-targeted imaging probes will also have broad applications in other diseases such as arthritis, atherosclerosis, and myocardial infarction.
Protease; cancer; molecular imaging; activatable probe; optical imaging; positron emission tomography
Due to the important roles of matrix metalloproteinases (MMPs) play in tumor invasion and metastasis, various activatable optical probes have been developed to visualize MMP activities in vitro and in vivo. Our recently developed MMP-13 activatable probe, L-MMP-P12, has been successfully applied to image the expression and inhibition of MMPs in a xenografted tumor model (Zhu L et al., Theranostics. 2011;1:18–27). In this study, to further optimize the in vivo behavior of the proteinase activatable probe, we tracked and profiled the metabolites by a high resolution LC/MS system. Two major metabolites that contributed to the fluorescence recovery were identified: One was specifically cleaved between Glycine (G4) and Valine (V5) by MMP, while the other one was generated by non-specific cleavage between Glycine (G7) and Lysine (K8). In order to visualize the MMP activity more accurately and specifically, a new probe D-MMP-P12 was designed by replacing the L-lysine with D-lysine in the MMP substrate sequence. The metabolic profile of the new probe, D-MMP-P12, was further characterized by in vitro enzymatic assay and no non-specific metabolite was found by LC/MS. Our in vivo optical imaging also demonstrated that D-MMP-12 had significantly higher tumor-to-background ratio (TBR, 5.55 ± 0.75) compared with L-MMP-P12 (3.73 ± 0.31) at 2 h post-injection. The improved MMP activatable probe may have the potential for drug screening, tumor diagnosis and therapy response monitoring. Moreover, our research strategy can be further extended to study other protease activatable probes.
Liquid chromatography–mass spectrometry (LC-MS); activatable probe; matrix metalloproteinases (MMPs); metabolite; near-infrared fluorescence imaging
Molecular magnetic resonance (MR) imaging plays an important role in studying molecular and cellular processes associated with heart disease. Targeted probes that recognize important biomarkers of atherosclerosis, apoptosis, necrosis, angiogenesis, thrombosis and inflammation have been developed.
This review discusses properties of chemically different types of contrast agents including iron oxide nanoparticles, gadolinium based nanoparticles or micelles, discrete peptide conjugates and activatable probes. Numerous examples of contrast agents based on these approaches have been used in preclinical MR imaging of cardiovascular diseases. Clinical applications are still under investigation for some selected agents with highly promising initial results.
Molecular MR imaging shows great potential for the detection, characterization of a wide range of cardiovascular diseases and for monitoring response to therapy.
Cardiovascular; molecular imaging; MRI; atherosclerosis; iron oxide nanoparticles; targeted peptides; smart probes; micelles; gadolinium
Fibroblast activation protein-alpha (FAPα) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAPα has also been reported to promote tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAPα inhibitors has led to the discovery of many FAPα-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAPα in vivo has largely lagged behind. Herein we report an activatable near infrared (NIR) fluorescent probe (ANPFAP) for in vivo optical imaging of FAPα. The ANPFAP consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAPα cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANPFAP, high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAPα both in vitro and in vivo. In vitro assay on ANPFAP indicated the specificity of the probe to FAPα. The in vivo optical imaging using ANPFAP showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAPα expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAPα expression, demonstrating the in vivo targeting specificity of the ANPFAP. Ex vivo imaging also demonstrated ANPFAP had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANPFAP could serve as a useful NIR optical probe for early detection of FAPα expressing tumors.
FAPα; NIRF; glioma; activatable probe; optical imaging
Caspase-activatable cell-penetrating peptide (CPP) probes, designed for efficient cell uptake and specificity via cleavable intramolecular quenched-fluorophore strategies, show promise for identifying and imaging retinal ganglion cell apoptosis in vivo. However, initial cell uptake and trafficking events cannot be visualized because the probes are designed to be optically quenched in the intact state. To visualize subcellular activation events in real-time during apoptosis, a new series of matched quenched and non-quenched CPP probes were synthesized. In both native and staurosporine-differentiated RGC-5 cells, probe uptake was time- and concentration-dependent through clathrine-, caveolin- and pinocytosis-mediated endocytic mechanisms. During apoptosis, KcapTR488, a novel dual fluorophore CPP probe, revealed by multi-spectral imaging a temporal coupling of endosomal release and effector caspase activation in RGC-5 cells. The novel CPPs described herein provide new tools to study spatial and temporal regulation of endosomal permeability during apoptosis.
Targeted molecular imaging techniques have become indispensable tools in modern diagnostics because they provide accurate and specific diagnosis of disease information. Conventional non-specific contrast agents suffer from low targeting efficiency, thus, the use of molecularly targeted imaging probes are needed depending on different imaging modalities. Although recent technologies have yielded various strategies for designing smart probes, utilization of peptide-based probes has been most successful. Phage display technology and combinatorial peptide chemistry have profoundly impacted the pool of available targeting peptides for the efficient and specific delivery of imaging labels. To date, selected peptides that target a variety of disease-related receptors and biomarkers are in place. These targeting peptides can be coupled with the appropriate imaging moieties or nanoplatforms on demand with the help of sophisticated bioconjugation or radiolabeling techniques. This review article examines the current trends in peptide-based imaging probes developed for in vivo applications. We discuss the advantage and challenges in developing peptide-based probes, and summarize current systems with respect to their unique design strategies and applications.
Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical ‘death-switch' model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [18F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The ‘death-switch' is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Death-switch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of ‘death-switched' xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [18F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [18F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers.
biomarkers; apoptosis; capase-3; death-switch; proteomics; imaging
In vivo molecularly targeted fluorescence imaging of tumors has been proposed as a strategy for improving cancer detection and management. Activatable fluorophores, which increased their fluorescence by 10-fold after binding tumor cells, result in much higher target to background ratios than conventional fluorophores. We developed an in vivo targeted activatable optical imaging probe based on a fluorophore-quencher pair, bound to a targeting moiety. With this system, fluorescence is quenched by the fluorophore-quencher interaction outside cancer cells, but is activated within the target cells by dissociation of the fluorophore-quencher pair. We selected the TAMRA (fluorophore)-QSY7 (quencher) pair and conjugated it to either avidin (targeting the d-galactose receptor) or trastuzumab (a monoclonal antibody against the human epithelial growth factor receptor type2 (HER2/neu)) and evaluated their performance in mouse models of cancer. Two probes, TAMRA-QSY7 conjugated avidin (Av-TM-Q7) and trastuzumab (Traz-TM-Q7) were synthesized. Both demonstrated better than similar self-quenching probes. In vitro fluorescence microscopic studies of SHIN3 and NIH/3T3/HER2+ cells demonstrated that Av-TM-Q7 and Traz-TM-Q7 produced high intracellular fluorescent signal. In vivo imaging with Av-TM-Q7 and Traz-TM-Q7 in mice enabled the detection of small tumors. This molecular imaging probe, based on a fluorophore-quencher pair conjugated to a targeting ligand, successfully detected tumors in vivo due to its high activation ratio and low background signal. Thus, these activatable probes, based on the fluorophore-quencher system, hold promise clinically for “see and treat” strategies of cancer management.
molecular imaging; FRET; photo-quencher; activatable; cancer
of novel imaging probes for cancer diagnostics remains
critical for early detection of disease, yet most imaging agents are
hindered by suboptimal tumor accumulation. To overcome these limitations,
researchers have adapted antibodies for imaging purposes. As cancerous
malignancies express atypical patterns of cell surface proteins in
comparison to noncancerous tissues, novel antibody-based imaging agents
can be constructed to target individual cancer cells or surrounding
vasculature. Using molecular imaging techniques, these agents may
be utilized for detection of malignancies and monitoring of therapeutic
response. Currently, there are several imaging modalities commonly
employed for molecular imaging. These imaging modalities include positron
emission tomography (PET), single-photon emission computed tomography
(SPECT), magnetic resonance (MR) imaging, optical imaging (fluorescence
and bioluminescence), and photoacoustic (PA) imaging. While antibody-based
imaging agents may be employed for a broad range of diseases, this
review focuses on the molecular imaging of pancreatic cancer, as there
are limited resources for imaging and treatment of pancreatic malignancies.
Additionally, pancreatic cancer remains the most lethal cancer with
an overall 5-year survival rate of approximately 7%, despite significant
advances in the imaging and treatment of many other cancers. In this
review, we discuss recent advances in molecular imaging of pancreatic
cancer using antibody-based imaging agents. This task is accomplished
by summarizing the current progress in each type of molecular imaging
modality described above. Also, several considerations for designing
and synthesizing novel antibody-based imaging agents are discussed.
Lastly, the future directions of antibody-based imaging agents are
discussed, emphasizing the potential applications for personalized
molecular imaging; pancreatic cancer; positron
emission tomography (PET); single-photon emission computed
tomography (SPECT); magnetic resonance imaging (MRI); optical imaging; photoacoustic tomography (PAT); antibodies
The exposure of phosphatidylserine (PS) molecules from the inner to the outer leaflet of the plasma membrane has been recognized as a well-defined molecular epitope of cells undergoing apoptosis. Examination and monitoring of PS exposure is an extensively used molecular marker in non-invasive apoptosis imaging under a variety of clinical conditions, including the assessment of therapeutic anti-cancer agents and myocardial infarction. Herein, we report the identification of a PS-recognizing peptide which was identified by the screening of an M13 phage display peptide library onto PS-coated ELISA plates. Repeated biopanning for a total of four rounds revealed a predominant enrichment of the phage clone displaying peptide sequence, CLSYYPSYC (46%). The identified phage clone evidenced enhanced binding to a number of apoptotic cells over non-apoptotic cells, and this binding was inhibited by both annexin V and synthesized peptide displayed on the phage. The binding of the fluorescein-labelled CLSYYPSYC peptide to apoptotic versus normal cells was assessed by both FACS analysis and fluorescence microscopy. Optical imaging after the systemic administration of fluorescein-labelled CLSYYPSYC peptide to tumour-bearing nude mice (H460 cells xenograft model) treated with a single dose of an anticancer drug (camp-tothecin) indicated peptide homing to the tumour. The histological examination of tumour tissues showed intense staining of the tumour vasculature and apoptotic tumour cells. With these results, the CLSYYPSYC peptide is recognized as a novel PS-recognizing moiety which may possibly be developed into a molecular probe for the imaging of apoptosis in vivo. This application would clearly be relevant to assessments of the efficacy of anticancer therapy in tumours.
phosphatidylserine; apoptosis; phage display; annexin V; peptide
Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) is a master anti-apoptotic regulator and resistance factor that suppresses tumor necrosis factor-α (TNF-α), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, as well as apoptosis triggered by chemotherapy agents in malignant cells. c-FLIP is expressed as long (c-FLIPL), short (c-FLIPS), and c-FLIPR splice variants in human cells. c-FLIP binds to FADD and/or caspase-8 or -10 and TRAIL receptor 5 (DR5) in a ligand-dependent and -independent fashion and forms an apoptosis inhibitory complex (AIC). This interaction in turn prevents death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade. c-FLIPL and c-FLIPS are also known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective and pro-survival signaling proteins including Akt, ERK, and NF-kB. Upregulation of c-FLIP has been found in various tumor types, and its silencing has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Hence, c-FLIP is an important target for cancer therapy. For example, small interfering RNAs (siRNAs) that specifically knockdown the expression of c-FLIPL in diverse human cancer cell lines augmented TRAIL-induced DISC recruitment and increased the efficacy of chemotherapeutic agents, thereby enhancing effector caspase stimulation and apoptosis. Moreover, small molecules causing degradation of c-FLIP as well as decreasing mRNA and protein levels of c-FLIPL and c-FLIPS splice variants have been found, and much effort is focused on developing other c-FLIP-targeted cancer therapies. This review focuses on (1) the anti-apoptotic role of c-FLIP splice variants in preventing apoptosis and inducing cytokine and chemotherapy drug resistance, (2) the molecular mechanisms and factors that regulate c-FLIP expression, and (3) modulation of c-FLIP expression and function to eliminate cancer cells or increase the efficacy of anticancer agents. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”.
c-FLIP; apoptosis; death receptors; cancer; chemotherapy
Neuroblastoma (NB) is the second most common solid childhood tumour, an aggressive disease for which new therapeutic strategies are strongly needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumour cells, but not in normal tissues and therefore represents a valuable candidate in apoptosis-inducing therapies. Caspase-8 is silenced in a subset of highly malignant NB cells, which results in full TRAIL resistance. In addition, despite constitutive caspase-8 expression, or its possible restoration by different strategies, NB cells remain weakly sensitive to TRAIL indicating a need to develop strategies to sensitise NB cells to TRAIL. Histone deacetylase inhibitors (HDACIs) are a new class of anti-cancer agent inducing apoptosis or cell cycle arrest in tumour cells with very low toxicity toward normal cells. Although HDACIs were recently shown to increase death induced by TRAIL in weakly TRAIL-sensitive tumour cells, the precise involved sensitisation mechanisms have not been fully identified.
NB cell lines were treated with various doses of HDACIs and TRAIL, then cytotoxicity was analysed by MTS/PMS proliferation assays, apoptosis was measured by the Propidium staining method, caspases activity by colorimetric protease assays, and (in)activation of apoptotic proteins by immunoblotting.
Sub-toxic doses of HDACIs strongly sensitised caspase-8 positive NB cell lines to TRAIL induced apoptosis in a caspases dependent manner. Combined treatments increased the activation of caspases and Bid, and the inactivation of the anti-apoptotic proteins XIAP, Bcl-x, RIP, and survivin, thereby increasing the pro- to anti-apoptotic protein ratio. It also enhanced the activation of the mitochondrial pathway. Interestingly, the kinetics of caspases activation and inactivation of anti-apoptotic proteins is accelerated by combined treatment with TRAIL and HDACIs compared to TRAIL alone. In contrast, cell surface expression of TRAIL-receptors or TRAIL is not affected by sub-toxic doses of HDACIs.
HDACIs were shown to activate the mitochondrial pathway and to sensitise NB cells to TRAIL by enhancing the amplitude of the apoptotic cascade and by restoring an apoptosis-prone ratio of pro- to anti-apoptotic proteins. Combining HDACIs and TRAIL could therefore represent a weakly toxic and promising strategy to target TRAIL-resistant tumours such as neuroblastomas.
Bioluminescence imaging (BLI) has shown its appeal as a sensitive technique for in vivo whole body optical imaging. However, the development of injectable tumor-specific near-infrared fluorescent (NIRF) probes makes fluorescence imaging (FLI) a promising alternative to BLI in situations where BLI cannot be used or is unwanted (e.g., spontaneous transgenic tumor models, or syngeneic mice to study immune effects).
In this study, we addressed the questions whether it is possible to detect tumor progression using FLI with appropriate sensitivity and how FLI correlates with BLI measurements. In addition, we explored the possibility to simultaneously detect multiple tumor characteristics by dual-wavelength FLI (∼700 and ∼800 nm) in combination with spectral unmixing. Using a luciferase-expressing 4T1-luc2 mouse breast cancer model and combinations of activatable and targeting NIRF probes, we showed that the activatable NIRF probes (ProSense680 and MMPSense680) and the targeting NIRF probes (IRDye 800CW 2-DG and IRDye 800CW EGF) were either activated by or bound to 4T1-luc2 cells. In vivo, we implanted 4T1-luc2 cells orthotopically in nude mice and were able to follow tumor progression longitudinally both by BLI and dual-wavelength FLI. We were able to reveal different probe signals within the tumor, which co-localized with immuno-staining. Moreover, we observed a linear correlation between the internal BLI signals and the FLI signals obtained from the NIRF probes. Finally, we could detect pulmonary metastases both by BLI and FLI and confirmed their presence histologically.
Taken together, these data suggest that dual-wavelength FLI is a feasible approach to simultaneously detect different features of one tumor and to follow tumor progression with appropriate specificity and sensitivity. This study may open up new perspectives for the detection of tumors and metastases in various experimental models and could also have clinical applications, such as image-guided surgery.
In vivo molecular imaging has a great potential to impact medicine by detecting diseases in early stages (screening), identifying extent of disease, selecting disease- and patient-specific therapeutic treatment (personalized medicine), applying a directed or targeted therapy, and measuring molecular-specific effects of treatment. Current clinical molecular imaging approaches primarily use PET- or SPECT-based techniques. In ongoing preclinical research novel molecular targets of different diseases are identified and, sophisticated and multifunctional contrast agents for imaging these molecular targets are developed along with new technologies and instrumentation for multimodality molecular imaging. Contrast-enhanced molecular ultrasound with molecularly-targeted contrast microbubbles is explored as a clinically translatable molecular imaging strategy for screening, diagnosing, and monitoring diseases at the molecular level. Optical imaging with fluorescent molecular probes and ultrasound imaging with molecularly-targeted microbubbles are attractive strategies since they provide real-time imaging, are relatively inexpensive, produce images with high spatial resolution, and do not involve exposure to ionizing irradiation. Raman spectroscopy/microscopy has emerged as a molecular optical imaging strategy for ultrasensitive detection of multiple biomolecules/biochemicals with both in vivo and ex vivo versatility. Photoacoustic imaging is a hybrid of optical and ultrasound modalities involving optically-excitable molecularly-targeted contrast agents and quantitative detection of resulting oscillatory contrast agent movement with ultrasound. Current preclinical findings and advances in instrumentation such as endoscopes and microcatheters suggest that these molecular imaging modalities have numerous clinical applications and will be translated into clinical use in the near future.
To evaluate the potential of targeted photoacoustic imaging as a non-invasive method for detection of follicular thyroid carcinoma.
We determined the presence and activity of two members of matrix metalloproteinase family (MMP), MMP-2 and MMP-9, suggested as biomarkers for malignant thyroid lesions, in FTC133 thyroid tumors subcutaneously implanted in nude mice. The imaging agent used to visualize tumors was MMP activatable photoacoustic probe, Alexa750-CXeeeeXPLGLAGrrrrrXK-BHQ3. Cleavage of the MMP activatable agent was imaged after intratumoral and intravenous injections in living mice optically, observing the increase in Alexa750 fluorescence, and photoacoustically, using a dual wavelength imaging method.
Active forms of both MMP2 and MMP-9 enzymes were found in FTC133 tumor homogenates, with MMP-9 detected in greater amounts. The molecular imaging agent was determined to be activated by both enzymes in vitro, with MMP-9 being more efficient in this regard. Both optical and photoacoustic imaging showed significantly higher signal in tumors of mice injected with the active agent than in tumors injected with the control, non-activatable, agent.
With the combination of high spatial resolution and signal specificity, targeted photoacoustic imaging holds great promise as a noninvasive method for early diagnosis of follicular thyroid carcinomas.
Photoacoustic Molecular Imaging; Thyroid follicular cancer; Activatable Photoacoustic Probe; Matrix Metalloproteinase; Optical Imaging
In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2) induces apoptosis in Human Papillomavirus (HPV) positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive), as well as MDA-MB-231 (highly invasive) human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs) isolated from tissue biopsies of patients undergoing breast reduction surgery.
AAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis.
AAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated endonuclease activity and AAV2 genomic hair-pin ends have the potential to induce a cellular DNA damage response, which could act in tandem with c-Myc regulated/sensitized apoptosis induction. In contrast, failure of AAV2 to productively infect nHMECs could be clinically advantageous. Identifying the molecular mechanisms of AAV2 targeted cell cycle regulation of death inducing signals could be harnessed for developing novel therapeutics for weakly invasive as well as aggressive breast cancer types.
Adeno-Associated Virus Type 2; AAV2; Breast cancer; Pro-apoptotic therapeutics; Apoptosis; Cell cycle; Rep proteins; c-Myc