2022 is the year I start from a blank canvas and get my lab up and running…

Institutional Animal and Care and Use Committee (IACUC) protocol approved

Radiation Safety Committee (RSC) protocol approved

Paula Demétrio de Souza França,  Susanne Kossatz, Christian Brand, Daniella Karassawa Zanoni,  Sheryl Roberts, Navjot Guru, Dauren Adilbay,  Audrey Mauguen,  Cristina Valero Mayor, Wolfgang A. Weber, Heiko Schöder,  Ronald A. Ghossein,  Ian Ganly, Snehal G. Patel, Thomas Reiner*

Purpose Visual inspection and biopsy is the current standard of care for oral cancer diagnosis, but is subject to misinterpretation and consequently to misdiagnosis. Topically applied PARPi-FL is a molecularly specific, fluorescent contrast-based approach that may fulfil the unmet need for a simple, in vivo, non-invasive, cost-effective, point-of-care method for the early diagnosis of oral cancer. Here, we present results from a phase I safety and feasibility study on fluorescent, topically applied PARPi-FL.

Translational Relevance Despite their accessible location, oral cavity cancers are often diagnosed late, especially in low-resource areas where their incidence is typically high. The high prevalence of premalignant and benign oral lesions in these populations contributes to a number of issues that make early detection of oral cancer difficult: even in experienced hands, it can be difficult to differentiate cancer from premalignant or benign lesions during routine clinical examination; and biopsy-based histopathology, the current standard of care, is invasive, prone to sampling error, and requires geographic access to appropriate health care professionals, including a highly trained pathologist. While seemingly impenetrable economic and infrastructure barriers have confounded the early diagnosis of oral cancer for most of the world’s population, these could be circumvented by a simple, in vivo, non-invasive, cost-effective, point-of-care method of diagnosis. We are attempting to address this unmet clinical need by using topically applied PARPi-FL — a molecularly specific, fluorescent contrast-based approach — to detect oral cancer.

Sheryl Roberts, Eshita Khera, Crystal Choi, Tejas Navaratna, Jan Grimm, Greg M. Thurber and Thomas Reiner*

Limitations in current imaging tools have long challenged the imaging of small pancreatic islets in animal models. Here, we report the first development and in vivo validation testing of a broad spectrum and high absorbance near infrared optoacoustic contrast agent, E4_x12-Cy7. Our near infrared tracer (E4_x12-Cy7) is based on the amino acid sequence of exendin-4 and targets the glucagon-like peptide-1 receptor (GLP-1R). Cell assays confirmed that E4_x12-Cy7 has a high binding affinity (IC₅₀ = 4.6 ± 0.8 nM). Using the multi-spectral optoacoustic tomography (MSOT), we imaged E4_x12-Cy7 and optoacoustically visualized β-cell insulinoma xenografts in vivo for the first time. In the future, similar optoacoustic tracers that are specific for β-cells and combines optoacoustic and fluorescence imaging modalities could prove to be important tools for monitoring the pancreas for the progression of diabetes.

Robert J Young, MD*, Paula Demétrio De Souza França, MD, Giacomo Pirovano, DPhil, Anna F Piotrowski, MD, Philip J Nicklin, Christopher C Riedl, MD PhD, Jazmin Schwartz, PhD, Tejus A Bale, MD PhD, Patrick L Donabedian, Susanne Kossatz, Eva M Burnazi, Sheryl Roberts, PhD, Serge K Lyashchenko, PharmD, Alexandra M Miller, MD PhD, Nelson S Moss, MD, Megan Fiasconaro, Zhigang Zhang, PhD, Audrey Mauguen, PhD, Thomas Reiner, PhD, Mark P Dunphy, MD*

Background: We report preclinical and first-in-human-brain-cancer data using a targeted poly(ADP-ribose)polymerase1 (PARP1) binding PET tracer, [ ¹⁸F]PARPi, as a diagnostic tool to differentiate between brain cancers and treatment-related changes.

Methods: We applied a glioma model in p53-deficient nestin/tv-a mice, which were injected with [ ¹⁸F]PARPi and then sacrificed 1 hr post-injection for brain examination. We also prospectively enrolled patients with brain cancers to undergo dynamic [ ¹⁸F]PARPi acquisition on a dedicated PET/MR scanner. Lesion diagnosis was established by pathology when available or by RANO or RANO-BM response criteria. Resected tissue also underwent PARPi-FL staining and PARP1 immunohistochemistry.

Results: In a preclinical mouse model, we illustrated that [ ¹⁸F]PARPi crossed the blood-brain barrier and specifically bound to PARP1 overexpressed in cancer cell nuclei. In humans, we demonstrated high [ ¹⁸F]PARPi uptake on PET/MR in active brain cancers and low uptake in treatment-related changes independent of blood-brain barrier disruption. Immunohistochemistry results confirmed higher PARP1 expression in cancerous than in non-cancerous tissue. Specificity was also corroborated by blocking fluorescent tracer uptake with excess unlabeled PARP inhibitor in patient cancer biospecimen.

Conclusions: Although larger studies are necessary to confirm and further explore this tracer, we describe the promising performance of [ ¹⁸F]PARPi as a diagnostic tool to evaluate patients with brain cancers and possible treatment-related changes.

Giacomo Pirovano , Sheryl Roberts , Susanne Kossatz and Thomas Reiner*

With the ability to non-invasively image and monitor molecular processes within tumors, molecular imaging represents a fundamental tool for cancer scientists. In the current review, we describe emergent optical technologies for molecular imaging. We aim to provide the reader with an overview of the fundamental principles on which each imaging strategy is based, to introduce established and future applications, and to provide a rationale for selecting optical technologies for molecular imaging depending on disease location, biology, and anatomy. In order to accelerate clinical translation of imaging techniques, we also describe examples of practical applications in patients. Elevating these techniques into standard-of-care tools will transform patient stratification, disease monitoring and response evaluation.

Paula Demétrio de Souza França, Navjot Guru, Sheryl Roberts, Susanne Kossatz, Christian Mason, Marcio Abrahão, Ronald A. Ghossein, Snehal G. Patel & Thomas Reiner*

Complete removal and negative margins are the goal of any surgical resection of primary oral cavity carcinoma. Current approaches to determine tumor boundaries rely heavily on surgeons’ expertise, and final histopathological reports are usually only available days after surgery, precluding contemporaneous re-assessment of positive margins. Intraoperative optical imaging could address this unmet clinical need. Using mouse models of oral cavity carcinoma, we demonstrated that PARPi-FL, a fluorescent PARP inhibitor targeting the enzyme PARP1/2, can delineate oral cancer and accurately identify positive margins, both macroscopically and at cellular resolution. PARPi-FL also allowed identification of compromised margins based on fluorescence hotspots, which were not seen in margin-negative resections and control tongues. PARPi-FL was further able to differentiate tumor from low-grade dysplasia. Intravenous injection of PARPi-FL has significant potential for clinical translation and could aid surgeons in assessing oral cancer margins in vivo.

Sheryl Roberts, Eshita Khera, Crystal Choi, Tejas Navaratna, Jan Grimm, Greg M. Thurber, Thomas Reiner*

Limitations in current imaging tools have long challenged the imaging of small pancreatic islets in animal models. Here, we report the first development and in vivo validation testing of a broad spectrum and high absorbance near infrared optoacoustic contrast agent, E4_x12-Cy7. Our near infrared tracer (E4_x12-Cy7) is based on the amino acid sequence of exendin-4 and targets the glucagon-like peptide-1 receptor (GLP-1R). Cell assays confirmed that E4_x12-Cy7 has a high binding affinity (IC₅₀ = 4.6 ± 0.8 nM). Using the multi-spectral optoacoustic tomography (MSOT), we imaged E4_x12-Cy7 and optoacoustically visualized ß-cell insulinoma xenografts in vivo for the first time. In the future, similar optoacoustic tracers that are specific for ß-cells and combines optoacoustic and fluorescence imaging modalities could prove to be important tools for monitoring the pancreas for the progression of diabetes.

Heiko Schöder, Paula Demétrio De Souza França, Reiko Nakajima, Eva Burnazi, Sheryl Roberts, Christian Brand, Milan Grkovski, Audrey Mauguen, Mark P. Dunphy, Ronald A. Ghossein, Serge K. Lyashchenko, Jason S. Lewis, Joseph A. O’Donoghue, Ian Ganly, Snehal G. Patel, Nancy Y. Lee and Thomas Reiner*

Purpose. We performed a first-in-human clinical trial. The aim of this study was to determine safety and feasibility of PET imaging with ¹⁸F-PARPi in patients with head and neck cancer. Patients and Methods. Eleven patients with newly diagnosed or recurrent oral and oropharyngeal cancer were injected with ¹⁸F-PARPi (331 ± 42 MBq) and dynamic PET/CT imaging was performed between 0 min and 25 min post-injection. Static PET/CT scans were obtained at 30 min, 60 min and 120 min post injection. Blood samples for tracer concentration and metabolite analysis were collected. Blood pressure, ECG, oxygen levels, clinical chemistry and CBC were obtained before and after tracer administration.

Results. ¹⁸F-PARPi was well-tolerated by all patients without any safety concerns. Of the 11 patients included in the analysis, ¹⁸F-PARPi had focal uptake in all primary lesions (n = 10, SUV_max = 2.8 ± 1.2) and all ¹⁸F-FDG positive lymph nodes (n = 34). ¹⁸F-PARPi uptake was seen in ¹⁸F-FDG negative lymph nodes of three patients (n = 6). Focal uptake of tracer in primary and metastatic lesions was corroborated by CT alone or in combination with ¹⁸F-FDG. The overall equivalent dose with ¹⁸F-PARPi PET was 3.9 mSv – 5.2 mSv, contrast was high (SUV_max(lesion)/SUV_max(trapezius muscle) = 4.5) and less variable than ¹⁸F-FDG when compared to the genioglossus muscle (1.3 versus 6.0, p = 0.001).

Conclusions. Imaging of head and neck cancer with ¹⁸F-PARPi is feasible and safe. ¹⁸F-PARPi detects primary and metastatic lesions, and retention in tumors is longer than in healthy tissues.

Katja Haedicke, Lilach Agemy, Murad Omar, Andrei Berezhnoi, Sheryl Roberts, Camila Longo-Machado, Magdalena Skubal, Karan Nagar, Hsiao-Ting Hsu, Kwanghee Kim, Thomas Reiner, Jonathan Coleman, Vasilis Ntziachristos, Avigdor Scherz & Jan Grimm*

The monitoring of vascular-targeted therapies using magnetic resonance imaging, computed tomography or ultrasound is limited by their insufficient spatial resolution. Here, by taking advantage of the intrinsic optical properties of haemoglobin, we show that raster-scanning optoacoustic mesoscopy (RSOM) provides high-resolution images of the tumour vasculature and of the surrounding tissue, and that the detection of a wide range of ultrasound bandwidths enables the distinction of vessels of differing size, providing detailed insights into the vascular responses to vascular-targeted therapy. Using RSOM to examine the responses to vascular-targeted photodynamic therapy in mice with subcutaneous xenografts, we observed a substantial and immediate occlusion of the tumour vessels followed by haemorrhage within the tissue and the eventual collapse of the entire vasculature. Using dual-wavelength RSOM, which distinguishes oxyhaemoglobin from deoxyhaemoglobin, we observed an increase in oxygenation of the entire tumour volume immediately after the application of the therapy, and a second wave of oxygen reperfusion approximately 24 h thereafter. We also show that RSOM enables the quantification of differences in neoangiogenesis that predict treatment efficacy.

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Patricia M.R. Pereira^#, Sheryl Roberts^#, Flavio Figueira, Joao P. C. Tome, Thomas Reiner and Jason S. Lewis*

^# Equally contributed

Galectins are carbohydrate-binding proteins overexpressed in bladder cancer (BCa) cells. Dendritic galactose moieties have a high affinity for galectin-expressing tumor cells. We radiolabeled a dendritic galactose carbohydrate with fluorine-18 – ¹⁸F-labeled galactodendritic unit 4 – and examined its potential in imaging urothelial malignancies. Methods: The ¹⁸F-labeled 1st generation galactodendritic unit 4 was obtained from its tosylate precursor. We conducted in vivo studies in galectin-expressing UMUC3 orthotopic BCa model to determine the ability of ¹⁸F-labeled galactodendritic unit 4 to image BCa. Results: Intravesical administration of ¹⁸F-labeled galactodendritic unit 4 allowed specific accumulation of the carbohydrate radiotracer in galectin-1 overexpressing UMUC3 orthotopic tumors when imaged with PET. The ¹⁸F-labeled galactodendritic unit 4 was not found to accumulate in non-tumor murine bladders. Conclusion: The ¹⁸F-labeled galactodendritic unit 4 and similar analogs may be clinically relevant and exploitable for PET imaging of galectin-1 overexpressing bladder tumors.

Paula Demétrio de Souza França , Sheryl Roberts, Susanne Kossatz, Navjot Guru, Christian Mason, Daniella Karassawa Zanoni, Marcio Abrahão, Heiko Schöder, Ian Ganly, Snehal G Patel, Thomas Reiner*