Acid specific dark quencher QC1 pHLIP for multi-spectral optoacoustic diagnoses of breast cancer

Fig1 copy 2

Sheryl Roberts, Arianna Strome, Crystal Choi, Chrysafis Andreou, Susanne Kossatz, Christian Brand, Travis Williams, Michelle Bradbury, Moritz F. Kircher, Yana K. Reshetnyak, Jan Grimm, Jason S. Lewis & Thomas Reiner*

Breast cancer is the most common type of malignant growth in women. Early detection of breast cancer, as well as the identification of possible metastatic spread poses a significant challenge because of the structural and genetic heterogeneity that occurs during the progression of the disease. Currently, mammographies, biopsies and MRI scans are the standard of care techniques used for breast cancer diagnosis, all of which have their individual shortfalls, especially when it comes to discriminating tumors and benign growths. With this in mind, we have developed a non-invasive optoacoustic imaging strategy that targets the acidic environment of breast cancer. A pH low insertion peptide (pHLIP) was conjugated to the dark quencher QC1, yielding a non-fluorescent sonophore with high extinction coefficient in the near infrared that increases signal as a function of increasing amounts of membrane insertion. In an orthotopic murine breast cancer model, pHLIP-targeted optoacoustic imaging allowed us to differentiate between healthy and breast cancer tissues with high signal/noise ratios. In vivo, the sonophore QC1-pHLIP could detect malignancies at higher contrast than its fluorescent analog ICG-pHLIP, which was developed for fluorescence-guided surgical applications. PHLIP-type optoacoustic imaging agents in clinical settings are attractive due to their ability to target breast cancer and a wide variety of other malignant growths for diagnostic purposes. Intuitively, these agents could also be used for visualization during surgery.

Blocking Glucagon Like Peptide-1 Receptors in the Exocrine Pancreas Improves Specificity for Beta Cells in a Mouse Model of Type 1 Diabetes

Untitled copy

Eshita Khera#, Liang Zhang#, Sheryl Roberts, Ian Nessler, Darleen Sandoval, Thomas Reiner and Greg Thurber*

# Equally contributed

The diabetes community has long desired an imaging agent to quantify the number of insulin-secreting beta (β) cells, beyond just functional equivalents (insulin secretion), to help diagnose and monitor early stages of both type 1 and type 2 diabetes mellitus. Loss in the number of β-cells can be masked by a compensatory increase in function of the remaining cells. Since β-cells only form ~1% of the pancreas and decrease as the disease progresses, only a few imaging agents, such as exendin, have demonstrated clinical potential to detect a drop in the already scarce signal. However, clinical translation of imaging with exendin has been hampered by higher than expected pancreatic uptake in subjects with long-term diabetes who lack β-cells. Exendin binds glucagon-like peptide 1 receptor (GLP-1R), previously thought to be expressed only on β-cells, but recent studies report low levels of GLP-1R on exocrine cells, complicating β-cell mass quantification. Here we use a GLP-1R knockout mouse model to demonstrate that exocrine binding of exendin is exclusively via GLP-1R (~1,000/cell), and not any other receptors. We then use lipophilic Cy-7 exendin to selectively pre-block exocrine GLP-1R in healthy and streptozotocin-induced diabetic (STZ) mice. Sufficient receptors remain on β-cells for subsequent labeling with a fluorescent- or 111In-exendin, which improves contrast between healthy and diabetic pancreata, and provides a potential avenue for achieving the long-standing goal of imaging β-cell mass in the clinic.

Nanoemulsion-based delivery of fluorescent PARP inhibitors in mouse models of small cell lung cancer


Junior Gonzales, Susanne Kossatz, Sheryl Roberts, Giacomo Pirovano, Christian Brand, Carlos Perez-Medina, Patrick Donabedian, M. Jason de la Cruz, Willem J. M. Mulder, and Thomas Reiner*

The preclinical potential of many diagnostic and therapeutic small molecules is limited by their rapid washout kinetics and consequently modest pharmacological performances. In several cases, these could be improved by loading the small molecules into nanoparticulates, improving blood half-life, in vivo uptake and overall pharmacodynamics. In this study, we report a nanoemulsion (NE) encapsulated form of PARPi-FL. As a proof of concept, we used PARPi-FL, which is a fluorescently labeled sensor for olaparib, a FDA-approved small molecule inhibitor of the nuclear enzyme poly(ADP-ribose)polymerase 1 (PARP1). Encapsulated PARPi-FL showed increased blood half-life, and delineated subcutaneous xenografts of small cell lung cancer (SCLC), a fast-progressing disease where efficient treatment options remain an unmet clinical need. Our study demonstrates an effective method for expanding the circulation time of a fluorescent PARP inhibitor, highlighting the pharmacokinetic benefits of nanoemulsions as nanocarriers and confirming the value of PARPi-FL as an imaging agent targeting PARP1 in small cell lung cancer.

[18F]FE-OTS964: a Small Molecule Targeting TOPK for In Vivo PET Imaging in a Glioblastoma Xenograft Model


Giacomo Pirovano, Sheryl Roberts, Christian Brand, Patrick L. Donabedian, Christian Mason, Paula Demétrio de Souza, Geoff S. Higgins & Thomas Reiner*

Purpose: Lymphokine-activated killer T cell-originated protein kinase (TOPK) is a fairly new cancer biomarker with great potential for clinical applications. The labeling of a TOPK inhibitor with F-18 can be exploited for positron emission tomography (PET) imaging allowing more accurate patient identification, stratification, and disease monitoring.

Procedures: [18F]FE-OTS964 was produced starting from OTS964, a preclinical drug which specifically binds to TOPK, and using a two-step procedure with [18F]fluoroethyl p-toluenesulfonate as a prosthetic group. Tumors were generated in NSG mice by subcutaneous injection of U87 glioblastoma cells. Animals were injected with [18F]FE-OTS964 and PET imaging and ex vivo biodistribution analysis was carried out.

Results: [18F]FE-OTS964 was successfully synthesized and validated in vivo as a PET imaging agent. The labeling reaction led to 15.1 ± 7.5 % radiochemical yield, 99 % radiochemical purity, and high specific activity. Chemical identity of the radiotracer was confirmed by co-elution on an analytical HPLC with a cold-labeled standard. In vivo PET imaging and biodistribution analysis showed tumor uptake of 3.06 ± 0.30 %ID/cc, which was reduced in animals co-injected with excess blocking dose of OTS541 to 1.40 ± 0.42 %ID/cc.

Conclusions: [18F]FE-OTS964 is the first TOPK inhibitor for imaging purposes and may prove useful in the continued investigation of the pharmacology of TOPK inhibitors and the biology of TOPK in cancer patients.

Sonophore-enhanced nanoemulsions for optoacoustic imaging of cancer


Sheryl Roberts#, Chrysafis Andreou#, Crystal Choi, Patrick Donabedian, Madhumitha Jayaraman, Edwin C. Pratt, Jun Tang, Carlos Pérez-Medina, M. Jason de la Cruz, Willem J. M. Mulder, Jan Grimm, Moritz Kircher and Thomas Reiner*

# Equally contributed

Optoacoustic imaging offers the promise of high spatial resolution and, at the same time, penetration depths well beyond the conventional optical imaging technologies, advantages that would be favorable for a variety of clinical applications. However, similar to optical fluorescence imaging, exogenous contrast agents, known as sonophores, need to be developed for molecularly targeted optoacoustic imaging. Despite numerous optoacoustic contrast agents that have been reported, there is a need for more rational design of sonophores. Here, using a library screening approach, we systematically identified and evaluated twelve commercially available near-infrared (690–900 nm) and highly absorbing dyes for multi-spectral optoacoustic tomography (MSOT). In order to achieve more accurate spectral deconvolution and precise data quantification, we sought five practical mathematical methods, namely direct classical least squares based on UV-Vis (UV/Vis-DCLS) or optoacoustic (OA-DCLS) spectra, non-negative LS (NN-LS), independent component analysis (ICA) and principal component analysis (PCA). We found that OA-DCLS is the most suitable method, allowing easy implementation and sufficient accuracy for routine analysis. Here, we demonstrate for the first time that our biocompatible nanoemulsions (NEs), in combination with near-infrared and highly absorbing dyes, enable non-invasive in vivo MSOT detection of tumors. Specifically, we found that NE-IRDye QC1 offers excellent optoacoustic performance and detection compared to related near-infrared NEs. We demonstrate that when loaded with low fluorescent or dark quencher dyes, NEs represent a flexible and new class of exogenous sonophores suitable for non-invasive pre-clinical optoacoustic imaging.

Calcium sensor for photoacoustic imaging


Sheryl Roberts, Markus Seeger, Yuanyuan Jiang, Anurag Mishra, Felix Sigmund, Anja Stelzl, Antonella Lauri, Panagiotis Symvoulidis, Hannes Rolbieski, Matthias Preller, X. Luís Deán-Ben, Daniel Razansky, Tanja Orschmann, Sabrina C. Desbordes, Paul Vetschera, Thorsten Bach, Vasilis Ntziachristos, and Gil G. Westmeyer*

We introduce a selective and cell-permeable calcium sensor for photoacoustics (CaSPA), a versatile imaging technique that allows for fast volumetric mapping of photoabsorbing molecules with deep tissue penetration. To optimize for Ca2+-dependent photoacoustic signal changes, we synthesized a selective metallochromic sensor with high extinction coefficient, low quantum yield, and high photobleaching resistance. Micromolar concentrations of Ca2+ lead to a robust blueshift of the absorbance of CaSPA, which translated into an accompanying decrease of the peak photoacoustic signal. The acetoxymethyl esterified sensor variant was readily taken up by cells without toxic effects and thus allowed us for the first time to perform live imaging of Ca2+ fluxes in genetically unmodified cells and heart organoids as well as in zebrafish larval brain via combined fluorescence and photoacoustic imaging.