Faculty Bibliography

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared to those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (AB) for growth and survival in the brain parenchyma. Melanoma-secreted AB activates surrounding astrocytes to a pro-metastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacological inhibition of AB decreases brain metastatic burden.

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

Gadolinium (Gd) based contrast agents (GBCAs) are widely used in magnetic resonance imaging (MRI) and are paramount to cancer diagnostics and tumor pharmacokinetic analysis. Accurate quantification of gadolinium concentration is essential to monitoring the biodistribution, clearance, and pharmacodynamics of GBCAs. However, current methods of quantifying gadolinium in blood or plasma (biological media) are both low throughput and clinically unavailable. Here, we have demonstrated the use of a sensitized gadolinium chelate, Gd[DTPA-cs124], as an MRI contrast agent that can be used to measure the concentration of gadolinium via luminescence quantification in biological media following transmetalation with a terbium salt. Gd[DTPA-cs124] was synthesized by conjugating carbostyril-124 (cs124) to diethylenetriaminepentaacetic acid (DTPA) and chelating to gadolinium. We report increases in both stability and relaxivity compared to the clinically approved analog Gd[DTPA] (gadopentetic acid or Magnevist). In vivo MRI experiments were conducted using C57BL6 mice in order to further illustrate the performance of Gd[DTPA-cs124] as an MRI contrast agent in comparison to Magnevist. Our results indicate that similar chemical modification to existing clinically approved GBCA may likewise provide favorable property changes, with the ability to be used in a gadolinium quantification assay. Furthermore, our assay provides a straightforward and high-throughput method of measuring gadolinium in biological media using a standard laboratory plate reader.

Background Gliomas account for roughly 27% of all brain tumors and there is an urgent need to develop new therapeutic modalities. A glioblastoma multiforme (GBM) prognosis signifies a survival time of 14-16 months with only 5% of patients surviving more than 5 years. (1) A significant challenge for traditional GBM drug delivery is the inability to: a) treat tumor cells with cytotoxic drugs due to their poor solubility and lack of blood brain barrier (BBB) permeation; b) specifically target tumor cells while avoiding normal tissue with such cytotoxic agents c) stimulate drug release; and d) monitor GBM status and therapy non-invasively. (2) Theranostic agents are being developed for their ability to diagnose disease and improve therapeutic delivery and can address these requirements because treatments specific to GBM do not currently exist. (3) While considerable efforts have been made in developing protein-based systems as drug-delivery carriers or as diagnostic agents (4), we are investigating a fundamental new insight that is helping us develop a single protein-based system combining drug delivery capabilities with the ability to cross the BBB and remain at cancer site due to the enhanced permeation and retention (EPR) effect. This biomaterial also incorporates functional groups detectable via magnetic resonance (MR) spectroscopy and imaging as well as near-infrared fluorescence (NIR) to enable visualization during chemotherapy. The protein-based theranostic agent we have engineered is called fluorinated thermo-responsive assembled protein (F-TRAP) that bears a non-canonical fluorinated amino acid (trifluoroleucine or TFL), can self-assemble into micellar structures, and encapsulate hydrophobic drugs. Methods and Materials Circular dichroism and dynamic light scattering have been performed to observe F-TRAP's secondary structure and micelle formation respectively (2). Additionally, 19F magnetic resonance imaging (MRI) has been carried out to visualize F-TRAP (5) and near infra-red fluorescence imaging (NIRF) has been utilized to determine its pharmacokinetic properties in a glioblastoma (GBM) mouse model. Results Results indicate that F-TRAP has an ?-helical secondary structure and forms micelles 30 nm in size. F-TRAP shows favorable pharmacokinetic data with a half-life of 123 minutes and high plasma retention. Importantly, animal data also reveals the ability of F-TRAP to cross the BBB and to be imaged inside the brain. Conclusions F-TRAP is capable of encapsulating small hydrophobic molecules, such as dox. It crosses the BBB and undergoes EPR effect allowing it to accumulate therein and be visualized NIRF imaging and is capable of undergoing MR imaging due to an appropriate half-life of about 123 min

Maternal care, including by non-biological parents, is important for offspring survival^1-8. Oxytocin^1,2,9-15, which is released by the hypothalamic paraventricular nucleus (PVN), is a critical maternal hormone. In mice, oxytocin enables neuroplasticity in the auditory cortex for maternal recognition of pup distress¹⁵. However, it is unclear how initial parental experience promotes hypothalamic signalling and cortical plasticity for reliable maternal care. Here we continuously monitored the behaviour of female virgin mice co-housed with an experienced mother and litter. This documentary approach was synchronized with neural recordings from the virgin PVN, including oxytocin neurons. These cells were activated as virgins were enlisted in maternal care by experienced mothers, who shepherded virgins into the nest and demonstrated pup retrieval. Virgins visually observed maternal retrieval, which activated PVN oxytocin neurons and promoted alloparenting. Thus rodents can acquire maternal behaviour by social transmission, providing a mechanism for adapting the brains of adult caregivers to infant needs via endogenous oxytocin.

The cell cycle is a progression of four distinct phases (G1, S, G2, M), with various cycle proteins being essential in regulating this process. In breast cancer, alterations in the cell cycle and uncontrolled proliferation led to several studies interrogating the relationship between cyclins and their counterpart cyclin-dependent-kinases (CDKs). We aimed to develop a radiolabeled CDK4/6 inhibitor for breast cancer imaging. Our transfluorinated analog ([¹⁸F]-CDKi) was evaluated and validated as a novel PET imaging agent to quantify CDK4/6 expression in ER-positive HER2-negative breast cancer. Methods: [¹⁸F]-CDKi was synthesized and assayed for its inhibitory activities against CDK4/6 kinases. [¹⁸F]-CDKi was prepared with a 2-step automated synthetic strategy that yielded the final product with remarkable purity and molar activity. In vitro and in vivo biologic activity and specificity was assessed in a MCF-7 cell line and in mice bearing MCF-7 breast tumors. Non radioactive Palbociclib (Inbrance, Pfizer®) was used as blocking agent to investigate the binding specificity and selectivity of [¹⁸F]-CDKi. Results: To generate [¹⁸F]-CDKi, we introduced an F-18 prosthetic group (¹⁸F-fluorobenzoic acid, [¹⁸F-FBA]), and transformed palbociclib into a different, PET active functional molecule. [¹⁸F]-CDKi was obtained with an overall radiochemical uncorrected yield of 15% and radiochemical purity > 98 %. The total synthesis time from the start of synthesis to final injectable formulated tracer is 70 minutes. The retention time reported for [¹⁸F]-CDKi and [¹⁹F]-CDKi is 27.4 min as demonstrated by co-injection with [¹⁹F]-CDKi in a HPLC. The first experiments aimed to analyze pharmacokinetics (PK) and in vitro activity of [¹⁸F]-CDKi (figure 5, Blood HL). In vivo blood half-life [t1/2(weighted) = 7.03 minutes], and octanol/water phase partition coefficient (logDO/W = 1.91 ± 0.24) showed a mainly lipophilic behavior. [¹⁸F]-CDKi is stable in vitro and in vivo (>98% at 4h post injection) and maintained its potent targeting affinity to CDK4/6 (~¹³NM for CDK4 and ~15nM for CDK6). Cellular uptake experiments performed in MCF-7 breast cancer cell line (ER-positive/HER2-negative) demonstrated specific uptake with a maximum intracellular concentration of ~65% as early as 10 minutes post incubation. The tracer uptake was reduced to <5% when cells were co-incubated with a molar excess of Palbociclib. In vivo imaging and ex-vivo biodistribution of ER-positive/HER-2 negative MCF-7 breast cancer models showed a ~4%ID/g tumor specific uptake (reduced to ~0.3%ID/g with a 50-fold excess of cold palbociclib). A comprehensive biodistribution analysis revealed also a significantly lower activation of CDK4/6 in non-targeting organs. Conclusion: [¹⁸F]-CDKi represents the first ¹⁸F positron emission tomography (PET) CDK4/6 imaging agent and a promising imaging agent for ER-positive HER2-negative breast cancer.

Vascular changes occur early in the development of obstructive airways disease. However, the vascular remodeling and dysfunction due to World Trade Center-Particulate Matter (WTC-PM) exposure are not well described and are therefore the focus of this investigation. C57Bl/6 female mice oropharyngeally aspirated 200 µg of WTC-PM₅₃ or phosphate-buffered saline (PBS) (controls). 24-hours (24-hrs) and 1-Month (1-M) after exposure, echocardiography, micro-positron emission tomography(µ-PET), collagen quantification, lung metabolomics, assessment of antioxidant potential and soluble-receptor for advanced glycation end products (sRAGE) in bronchoalveolar lavage(BAL) and plasma were performed. 24-hrs post-exposure, there was a significant reduction in (1) Pulmonary artery(PA) flow-velocity and pulmonary ejection time(PET) (2) Pulmonary acceleration time(PAT) and PAT/PET, while (3) Aortic ejection time(AET) and velocity time integral(VTI) were increased, and (4) Aortic acceleration time (AAT)/AET, cardiac output and stroke volume were decreased compared to controls. 1-M post-exposure, there was also significant reduction of right ventricular diameter as right ventricle free wall thickness was increased and an increase in tricuspid E, A peaks and an elevated E/A. The pulmonary and cardiac standard uptake value and volume 1-M post-exposure was significantly elevated after PM-exposure. Similarly, α-smooth muscle actin(α-SMA) expression, aortic collagen deposition was elevated 1-M after PM exposure. In assessment of the metabolome, prominent subpathways included advanced glycation end products (AGEs), phosphatidylcholines, sphingolipids, saturated/unsaturated fatty acids, eicosanoids, and phospholipids. BAL superoxide dismutase(SOD), plasma total-antioxidant capacity activity, and sRAGE (BAL and plasma) were elevated after 24-hrs. PM exposure and associated vascular disease are a global health burden. Our study shows persistent WTC-Cardiorespiratory and Vascular Dysfunction (WTC-CaRVD), inflammatory changes and attenuation of antioxidant potential after PM exposure. Early detection of vascular disease is crucial to preventing cardiovascular deaths and future work will focus on further identification of bioactive therapeutic targets.