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.

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.

Microvascular rarefaction, or the decrease in vascular density, has been described in the cerebrovasculature of aging humans, rats, and, more recently, mice in the presence and absence of age-dependent diseases. Given the wide use of mice in modeling age-dependent human diseases of the cerebrovasculature, visualization, and quantification of the global murine cerebrovasculature is necessary for establishing the baseline changes that occur with aging. To provide in vivo whole-brain imaging of the cerebrovasculature in aging C57BL/6 mice longitudinally, contrast-enhanced magnetic resonance angiography (CE-MRA) was employed using a house-made gadolinium-bearing micellar blood pool agent. Enhancement in the vascular space permitted quantification of the detectable, or apparent, cerebral blood volume (aCBV), which was analyzed over 2 years of aging and compared to histological analysis of the cerebrovascular density. A significant loss in the aCBV was detected by CE-MRA over the aging period. Histological analysis via vessel-probing immunohistochemistry confirmed a significant loss in the cerebrovascular density over the same 2-year aging period, validating the CE-MRA findings. While these techniques use widely different methods of assessment and spatial resolutions, their comparable findings in detected vascular loss corroborate the growing body of literature describing vascular rarefaction aging. These findings suggest that such age-dependent changes can contribute to cerebrovascular and neurodegenerative diseases, which are modeled using wild-type and transgenic laboratory rodents.

The most frequently mutated oncogene in cancer is KRAS, which uses alternative fourth exons to generate two gene products (KRAS4A and KRAS4B) that differ only in their C-terminal membrane-targeting region¹. Because oncogenic mutations occur in exons 2 or 3, two constitutively active KRAS proteins-each capable of transforming cells-are encoded when KRAS is activated by mutation². No functional distinctions among the splice variants have so far been established. Oncogenic KRAS alters the metabolism of tumour cells³ in several ways, including increased glucose uptake and glycolysis even in the presence of abundant oxygen⁴ (the Warburg effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes^3-5, it is not known whether there is direct regulation of metabolic enzymes. Here we report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, and thereby establish that HK1 is an effector of KRAS4A. This interaction is unique to KRAS4A because the palmitoylation-depalmitoylation cycle of this RAS isoform enables colocalization with HK1 on the outer mitochondrial membrane. The expression of KRAS4A in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically.

Thermoresponsive hydrogels are used for an array of biomedical applications. Lower critical solution temperature (LCST)-type hydrogels have been observed in nature and extensively studied in comparison to upper critical solution temperature (UCST)-type hydrogels. Of the limited protein-based UCST-type hydrogels reported, none have been composed of a single coiled-coil domain. Here we describe a biosynthesized homopentameric coiled-coil protein capable of demonstrating a UCST. Microscopy and structural analysis reveal that the hydrogel is stabilized by molecular entanglement of protein nanofibers, creating a porous matrix capable of binding the small hydrophobic molecule, curcumin. Curcumin binding increases the α-helical structure, fiber entanglement, mechanical integrity, and thermostability, resulting in sustained drug release at physiological temperature. This work provides the first example of a thermoresponsive hydrogel comprised of a single coiled-coil protein domain that can be used as a vehicle for sustained release and, by demonstrating UCST-type behavior, shows promise in forging a relationship between coiled-coil protein phase behavior and that of synthetic polymer systems.