Practical Guidance for Developing Small-Molecule Optical Probes for In Vivo Imaging.

The WMIS Education Committee (2019-2022) reached a consensus that white papers on molecular imaging could be beneficial for practitioners of molecular imaging at their early career stages and other scientists who are interested in molecular imaging. With this consensus, the committee plans to publish a series of white papers on topics related to the daily practice of molecular imaging. In this white paper, we aim to provide practical guidance that could be helpful for optical molecular imaging, particularly for small molecule probe development and validation in vitro and in vivo. The focus of this paper is preclinical animal studies with small-molecule optical probes. Near-infrared fluorescence imaging, bioluminescence imaging, chemiluminescence imaging, image-guided surgery, and Cerenkov luminescence imaging are discussed in this white paper.

Molecular imaging can identify the location to perform a frozen biopsy during intraoperative frozen section consultation.

BACKGROUND: Intraoperative frozen section (FS) consultation is an important tool in surgical oncology that suffers from sampling error because the pathologist does not always know where to perform a biopsy of the surgical specimen. Intraoperative molecular imaging is a technology used in the OR to visualize lesions during surgery. We hypothesized that molecular imaging can address this pathology challenge in FS by visualizing the cancer cells in the specimen in the pathology suite. Here, we report the development and validation of a molecular-imaging capable cryostat called Smart-Cut. METHODS: A molecular imaging capable cryostat prototype was developed and tested using a murine model. Tumors grown in mice were targeted with a NIR contrast agent, indocyanine green (ICG), via tail vein injection. Tumors and adjacent normal tissue samples were frozen sectioned with Smart-Cut. Fluorescent sections and non-fluorescent sections were prepared for H&E and fluorescent microscopy. Fluorescent signal was quantified by tumor-to-background ratio (TBR). NIR fluorescence was tested in one patient enrolled in a clinical trial. RESULTS: The Smart-Cut prototype has a small footprint and fits well in the pathology suite. Fluorescence imaging with Smart-Cut identified cancerous tissue in the specimen in all 12 mice. No false positives or false negatives were seen, as confirmed by H&E. The mean TBR in Smart-Cut positive tissue sections was 6.8 (SD±3.8). In a clinical application in the pathology suite, NIR imaging identified two lesions in a pulmonary resection specimen, where traditional grossing only identified one. CONCLUSION: Molecular imaging can be integrated into the pathology suite via the Smart-Cut device, and can detect cancer in frozen tissue sections using molecular imaging in a murine model.

NIR Fluorescent Imaging and Photodynamic Therapy with a Novel Theranostic Phospholipid Probe for Triple-Negative Breast Cancer Cells.

New exogenous probes are needed for both imaging diagnostics and therapeutics. Here, we introduce a novel nanocomposite near-infrared (NIR) fluorescent imaging probe and test its potency as a photosensitizing agent for photodynamic therapy (PDT) against triple-negative breast cancer cells. The active component in the nanocomposite is a small molecule, pyropheophorbide a-phosphatidylethanolamine-QSY21 (Pyro-PtdEtn-QSY), which is imbedded into lipid nanoparticles for transport in the body. The probe targets abnormal choline metabolism in cancer cells; specifically, the overexpression of phosphatidylcholine-specific phospholipase C (PC-PLC) in breast, prostate, and ovarian cancers. Pyro-PtdEtn-QSY consists of a NIR fluorophore and a quencher, attached to a PtdEtn moiety. It is selectively activated by PC-PLC resulting in enhanced fluorescence in cancer cells compared to normal cells. In our in vitro investigation, four breast cancer cell lines showed higher probe activation levels than noncancerous control cells, immortalized human mammary gland cells, and normal human T cells. Moreover, the ability of this nanocomposite to function as a sensitizer in PDT experiments on MDA-MB-231 cells suggests that the probe is promising as a theranostic agent.

Development of fluorinated naphthofluoresceins for Cerenkov imaging.

In this paper, we report the synthesis and characterization of fluorinated derivatives of naphthofluorescein (NF), a fluorescent pH-sensitive probe that can be used for functional Cerenkov imaging. The compounds were prepared using electrophilic fluorination with dilute fluorine gas under acidic conditions. The fluorination of the NF molecule occurred in the ortho positions to the hydroxyl moiety, producing mono-, di-, and tri-substituted derivatives. The properties of the fluorinated naphthofluoresceins were similar to the parent compound, retaining pH sensitivity and fluorescence capability, but showed a more acidic pKa with increasing fluorination degree and a bathochromic shift in both absorbance and fluorescence. NF and its two major fluorinated derivatives were shown to attenuate Cerenkov radiation in the basic form; the greatest attenuation was observed at wavelengths coinciding with the absorption maxima. NF also showed potential as a Cerenkov Radiation Energy Transfer (CRET) probe. Fluorinated naphthofluoresceins provide a new family of molecular imaging probes for the detection of pH in tissue and organs by using a combination of PET and Cerenkov imaging.

Detection and Differentiation of Breast Cancer Sub-Types using a cPLA2α Activatable Fluorophore.

Cytosolic phospholipase A2α (cPLA2α) has been shown to be elevated in breast cancer and is a potential biomarker in the differentiation of molecular sub-types. Using a cPLA2α activatable fluorophore, DDAO arachidonate, we explore its ability to function as a contrast agent in fluorescence-guided surgery. In cell lines ranging in cPLA2α expression and representing varying breast cancer sub-types, we show DDAO arachidonate activates with a high correlation to cPLA2α expression level. Using a control probe, DDAO palmitate, in addition to cPLA2α inhibition and genetic knockdown, we show that this activation is a result of cPLA2α activity. In mouse models, using an ex vivo tumor painting technique, we show that DDAO arachidonate activates to a high degree in basal-like versus luminal-like breast tumors and healthy mammary tissue. Finally, we show that using an in vivo model, orthotopic basal-like tumors give significantly high probe activation compared to healthy mammary fat pads and surrounding tissue. Together we conclude that cPLA2α activatable fluorophores such as DDAO arachidonate may serve as a useful contrast agent for the visualization of tumor margins in the fluorescence-guided surgery of basal-like breast cancer.

Nonprotecting Group Synthesis of a Phospholipase C Activatable Probe with an Azo-Free Quencher.

The near-infrared fluorescent activatable smart probe Pyro-phosphatidylethanolamine (PtdEtn)-QSY was synthesized and observed to selectively fluoresce in the presence of phosphatidylcholine-specific phospholipase C (PC-PLC). PC-PLC is an important biological target as it is known to be upregulated in a variety of cancers, including triple negative breast cancer. Pyro-PtdEtn-QSY features a QSY21 quenching moiety instead of the Black Hole Quencher-3 (BHQ-3) used previously because the latter contains an azo bond, which could lead to biological instability.

Synthesis of pH indicators for Cerenkov imaging by electrophilic substitution of bromine by fluorine in an aromatic system.

Direct electrophilic fluorination using molecular fluorine gas is used in organic synthesis to create novel fluorine-containing compounds with potential beneficial activity that could not be obtained by nucleophilic substitution. In this paper, we report a novel electrophilic substitution of bromine by fluorine in an aromatic system. The mechanism of this type of fluorination was explored using the reaction between bromothymolsulfonphthalein (Bromothymol Blue) and dilute fluorine gas under acidic conditions. Substitution occurs in the bromine atoms located in the ortho-position relative to the hydroxyl group. A similar electrophilic fluorination of thymolsulfonphthalein (Thymol Blue) leads to a substitution of hydrogen atoms in the same position (ortho to hydroxyl). NMR spectroscopy was used to confirm the fluorination sites. NMR spectra of thymolsulfonphthalein and its derivatives under basic conditions can be explained by considering the absence of resonance between the two phenolic rings. Both dibromothymol blue and fluorobromothymol blue revealed intermolecular attenuate Cerenkov radiation selectively near their maximum absorbance in a pH dependent manner.

Near-Infrared Intraoperative Molecular Imaging Can Locate Metastases to the Lung.

BACKGROUND: Pulmonary metastasectomy is widely accepted for many tumor types because it may prolong survival and potentially cure some patients. However, intraoperative localization of pulmonary metastases can be technically challenging. We propose that intraoperative near-infrared (NIR) molecular imaging can be used as an adjunct during disease localization. METHODS: We inoculated 50 C57BL/6 mice with Lewis lung carcinoma (LLC) flank tumors. After flank tumor growth, mice were injected through the tail vein with indocyanine green (ICG) before operation, and intraoperative imaging was used to detect pulmonary metastases. On the basis of these experiments, we enrolled 8 patients undergoing pulmonary metastasectomy into a pilot and feasibility clinical trial. Each patient received intravenous ICG 1 day before operation, followed by wedge or segmental resection. Samples were imaged on the back table with an NIR camera to confirm disease presence and margins. All murine and human tumors and margins were confirmed by pathologic examination. RESULTS: Mice had an average of 4 ± 2 metastatic tumors on both lungs, with an average size of 5.1 mm (interquartile range [IQR] 2.2 mm to 7.6 mm). Overall, 200 of 211 (95%) metastatic deposits were markedly fluorescent, with a mean tumor-to-background ratio (TBR) of 3.4 (IQR 3.1 to 4.1). The remaining tumors had a TBR below 1.5. In the human study, intraoperative NIR imaging identified six of the eight preoperatively localized lesions. Intraoperative back table NIR imaging identified all metastatic lesions, which were confirmed by pathologic examination. The average tumor size was 1.75 ± 1.4 cm, and the mean ex vivo TBR was 3.3 (IQR 3.1 to 3.7). Pathologic examination demonstrated melanoma (n = 4), osteogenic sarcoma (n = 2), renal cell carcinoma (n = 2), chondrosarcoma (n = 1), leiomyosarcoma (n = 1), and colorectal carcinoma (n = 1). CONCLUSIONS: Systemic ICG identifies subcentimeter tumor metastases to the lung in murine models, and this work provides proof of principle in humans. Future research is focused on improving depth of penetration into the lung parenchyma.

Choline kinase alpha-Putting the ChoK-hold on tumor metabolism.

It is well established that lipid metabolism is drastically altered during tumor development and response to therapy. Choline kinase alpha (ChoKα) is a key mediator of these changes, as it represents the first committed step in the Kennedy pathway of phosphatidylcholine biosynthesis and ChoKα expression is upregulated in many human cancers. ChoKα activity is associated with drug resistant, metastatic, and malignant phenotypes, and represents a robust biomarker and therapeutic target in cancer. Effective ChoKα inhibitors have been developed and have recently entered clinical trials. ChoKα's clinical relevance was, until recently, attributed solely to its production of second messenger intermediates of phospholipid synthesis. The recent discovery of a non-catalytic scaffolding function of ChoKα may link growth receptor signaling to lipid biogenesis and requires a reinterpretation of the design and validation of ChoKα inhibitors. Advances in positron emission tomography, magnetic resonance spectroscopy, and optical imaging methods now allow for a comprehensive understanding of ChoKα expression and activity in vivo. We will review the current understanding of ChoKα metabolism, its role in tumor biology and the development and validation of targeted therapies and companion diagnostics for this important regulatory enzyme. This comes at a critical time as ChoKα-targeting programs receive more clinical interest.

Serotonin as a putative scavenger of hypohalous acid in the brain.

Neurodegenerative disorders represent the culmination of numerous insults including oxidative stress. The long etiology of most of these disorders suggests that lessening the effects of one or more of the insults could significantly delay disease onset. Antioxidants have been tested as possible therapeutics for neurodegenerative disorders, but with little success. Here we report that serotonin acts as a scavenger of hypochlorous acid (HOCl) in the brain. Serotonin was shown to prevent the oxidation of 2-thio-5-nitrobenzoate by HOCl in a biphasic manner. The first phase was a partial scavenging that occurred at concentrations of serotonin that exceeded those of HOCl. (1)H-NMR studies indicated that HOCl chlorinates both the aryl and akyl nitrogen atoms of serotonin. Thus, the oxidation of 2-thio-5-nitrobenzoate that occurred during the first phase of scavenging is likely due to the formation of serotonergic chloramines. A second phase of scavenging occurred at concentrations of HOCl that exceeded those of serotonin. Under these conditions, the chlorinated serotonin polymerized and formed inert aggregates. Serotonin was further shown to prevent the loss of cells and cellular α-ketoglutarate dehydrogenase complex activity caused by HOCl. Extracellular concentrations of serotonin in the brain can be elevated with selective serotonin reuptake inhibitors and suggests that such compounds could be used to increase the cerebral antioxidant capacity. Acute administration of selective serotonin reuptake inhibitors to mice treated with endotoxin partially mitigated sickness behavior and protein chlorination in the brain. These observations suggest that serotonin may act to suppress chlorinative stress in the brain.

Quantification of tumor fluorescence during intraoperative optical cancer imaging.

Intraoperative optical cancer imaging is an emerging technology in which surgeons employ fluorophores to visualize tumors, identify tumor-positive margins and lymph nodes containing metastases. This study compares instrumentation to measure tumor fluorescence. Three imaging systems (Spectropen, Glomax, Flocam) measured and quantified fluorescent signal-to-background ratios (SBR) in vitro, murine xenografts, tissue phantoms and clinically. Evaluation criteria included the detection of small changes in fluorescence, sensitivity of signal detection at increasing depths and practicality of use. In vitro, spectroscopy was superior in detecting incremental differences in fluorescence than luminescence and digital imaging (Ln[SBR] = 6.8 ± 0.6, 2.4 ± 0.3, 2.6 ± 0.1, p = 0.0001). In fluorescent tumor cells, digital imaging measured higher SBRs than luminescence (6.1 ± 0.2 vs. 4.3 ± 0.4, p = 0.001). Spectroscopy was more sensitive than luminometry and digital imaging in identifying murine tumor fluorescence (SBR = 41.7 ± 11.5, 5.1 ± 1.8, 4.1 ± 0.9, p = 0.0001), and more sensitive than digital imaging at detecting fluorescence at increasing depths (SBR = 7.0 ± 3.4 vs. 2.4 ± 0.5, p = 0.03). Lastly, digital imaging was the most practical and least time-consuming. All methods detected incremental differences in fluorescence. Spectroscopy was the most sensitive for small changes in fluorescence. Digital imaging was the most practical considering its wide field of view, background noise filtering capability, and sensitivity to increasing depth.

Synthesis of F-18 labeled resazurin by direct electrophilic fluorination.

We present the synthesis and characterization of F18-labeled fluorinated derivatives of resazurin, a probe for cell viability. The compounds were prepared by direct fluorination of resazurin with diluted [F18]-F2 gas under acidic conditions. The fluorination occurs into the ortho-positions to the hydroxyl group producing various mono-, di-, and trifluorinated derivatives. The properties of the fluorinated resazurins are similar to the parent compound with the addition of fluorine leading to decreased pKa values and a bathochromic shift of the absorption maxima. The fluorinated resazurin derivatives can be used as probes for observation of cell viability in various cells, tissues and organs using a combination of positron emission tomography and direct optical imaging of Cerenkov luminescence.

PLA2-responsive and SPIO-loaded phospholipid micelles.

PLA2-responsive and superparamagnetic iron oxide (SPIO) nanoparticle-loaded phospholipid micelles were developed. The release of a phospholipid-conjugated dye from these micelles was triggered due to phospholipid degradation by phospholipase A2. The high relaxivity of the encapsulated SPIO could enable non-invasive magnetic resonance imaging.

Direct fluorination of phenolsulfonphthalein: a method for synthesis of positron-emitting indicators for in vivo pH measurement.

We report a reaction of direct electrophilic fluorination of phenolsulfonphthalein at mild conditions. This reaction affords the synthesis of novel positron-emitting (18)F-labeled pH indicators. These compounds are useful for non-invasive in vivo pH measurement in biological objects.

Synthesis and characterization of fluorinated derivatives of cresolsulfonphthalein.

The reaction between dilute fluorine gas and cresolsufonphthalein in acetic acid was investigated. The mono-, di-, and trifluorinated cresolsufonphthalein derivatives were isolated and characterized. These compounds possessed the properties of pH indicators with biologically relevant pKa values (6.4-7.5) and the absorption maxima of the basic forms at 582-592 nm. This method can be used for synthesis of positron-emitting 18F-labeled pH indicators with potential application for non-invasive in vivo pH measurement in biological objects.

Synthesis of 18F-labeled phenolphthalein and naphtholphthalein.

The fluorination of phenolphthalein and naphtholphthalein was performed with diluted fluorine gas under acidic conditions. For both compounds we observed an electrophilic fluorination into ortho position to the hydroxyl group. Through the use of this reaction we synthesized and characterized mono-and difluorinated derivatives of phenolphthalein and naphtholphthalein. The compounds were also prepared in the 18F labeled form, which are usable as a new type of probe for in vivo pH measurement in biological objects using Cerenkov imaging or combination of light absorption and PET.

A quantitative continuous enzyme assay of intramolecularly quenched fluorogenic phospholipase substrates for molecular imaging.

There has been recent growth in the development of activatable near-infrared (NIR) fluorescent probes for molecular imaging, generally designed by placing fluorochromes on a cleavable substrate in close proximity to one another, such that they self-quench, but fluoresce on separation via enzymatic cleavage of the substrate. Although these probes offer excellent contrast, the detection of enzyme activity has largely only been described qualitatively. In order to assess the effectiveness of a probe, it is useful to have a quantitative measure, such as the enzyme-substrate kinetic parameters. We have developed an assay to determine kinetic parameters and applied it to an intramolecularly quenched molecule, Pyro-PtdEtn-BHQ, a NIR fluorescent probe specific to phosphatidylcholine-specific phospholipase C. The development of this assay includes corrections for intermolecular quenching, calibration, optimization of reaction mixtures, and determination of kinetic and inhibition parameters. This assay can easily be extended to analyze and compare the efficiency of other fluorescent activatable phospholipase probes as suitable molecular imaging agents.

In vivo detection of phospholipase C by enzyme-activated near-infrared probes.

In this article, the characterization of the first near-infrared (NIR) phospholipase-activated molecular beacon is reported, and its utility for in vivo cancer imaging is demonstrated. The probe consists of three elements: a phospholipid (PL) backbone to which the NIR fluorophore, pyropheophorbide a (Pyro), and the NIR Black Hole Quencher 3 (BHQ) were conjugated. Because of the close proximity of BHQ to Pyro, the Pyro-PtdEtn-BHQ probe is self-quenched until enzyme hydrolysis releases the fluorophore. The Pyro-PtdEtn-BHQ probe is highly specific to one isoform of phospholipase C, phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine. Incubation of Pyro-PtdEtn-BHQ in vitro with PC-PLC demonstrated a 150-fold increase in fluorescence that could be inhibited by the specific PC-PLC inhibitor tricyclodecan-9-yl xanthogenate (D609) with an IC(50) of 34 ± 8 µM. Since elevations in phosphocholine have been consistently observed by magnetic resonance spectroscopy in a wide array of cancer cells and solid tumors, we assessed the utility of Pyro-PtdEtn-BHQ as a probe for targeted tumor imaging. Injection of Pyro-PtdEtn-BHQ into mice bearing DU145 human prostate tumor xenografts followed by in vivo NIR imaging resulted in a 4-fold increase in tumor radiance over background and a 2 fold increase in the tumor/muscle ratio. Tumor fluorescence enhancement was inhibited with the administration of D609. The ability to image PC-PLC activity in vivo provides a unique and sensitive method of monitoring one of the critical phospholipase signaling pathways activated in cancer, as well as the phospholipase activities that are altered in response to cancer treatment.

MR-visible lipids and the tumor microenvironment.

MR-visible lipids or mobile lipids are defined as lipids that are observable using proton MRS in cells and tissues. These MR-visible lipids are composed of triglycerides and cholesterol esters that accumulate in neutral lipid droplets, where their MR visibility is conferred as a result of the increased molecular motion available in this unique physical environment. This review discusses the factors that lead to the biogenesis of MR-visible lipids in cancer cells and in other cell types, such as immune cells and fibroblasts. We focus on the accumulations of mobile lipids that are inducible in cultured cells by a number of stresses, including culture conditions, and in response to activating stimuli or apoptotic cell death induced by anticancer drugs. This is compared with animal tumor models, where increases in mobile lipids are observed in response to chemo- and radiotherapy, and to human tumors, where mobile lipids are observed predominantly in high-grade brain tumors and in regions of necrosis. Conducive conditions for mobile lipid formation in the tumor microenvironment are discussed, including low pH, oxygen availability and the presence of inflammatory cells. It is concluded that MR-visible lipids appear in cancer cells and human tumors as a stress response. Mobile lipids stored as neutral lipid droplets may play a role in the detoxification of the cell or act as an alternative energy source, especially in cancer cells, which often grow in ischemic/hypoxic environments. The role of MR-visible lipids in cancer diagnosis and the assessment of the treatment response in both animal models of cancer and human brain tumors is also discussed. Although technical limitations exist in the accurate detection of intratumoral mobile lipids, early increases in mobile lipids after therapeutic interventions may be useful as a potential biomarker for the assessment of treatment response in cancer.