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Thyroid cancer has the fastest growing incidence of any cancer in the United States, as measured by the number of new cases per year. Despite advances in tissue culture techniques, a robust model for thyroid cancer spheroid culture is yet to be developed. Using eight established thyroid cancer cell lines, we created an efficient and cost-effective 3D culture system that can enhance our understanding of in vivo treatment response. We found that all eight cell lines readily form spheroids in culture with unique morphology, size, and cytoskeletal organization. In addition, we developed a high-throughput workflow that allows for drug screening of spheroids. Using this approach, we found that spheroids from K1 and TPC1 cells demonstrate significant differences in their sensitivities to dabrafenib treatment that closely model expected patient drug response. In addition, K1 spheroids have increased sensitivity to dabrafenib when compared to monolayer K1 cultures. Utilizing traditional 2D cultures of these cell lines, we evaluated the mechanisms of this drug response, showing dramatic and acute changes in their actin cytoskeleton as well as inhibition of migratory behavior in response to dabrafenib treatment. Our study is the first to describe the development of a robust spheroid system from established cultured thyroid cancer cell lines and adaptation to a high-throughput format. We show that combining 3D culture with traditional 2D methods provides a complementary and powerful approach to uncover drug sensitivity and mechanisms of inhibition in thyroid cancer.
The third-generation tyrosine kinase inhibitor (TKI) ponatinib has been associated with high rates of acute ischemic events. The pathophysiology responsible for these events is unknown. We hypothesized that ponatinib produces an endothelial angiopathy involving excessive endothelial-associated von Willebrand factor (VWF) and secondary platelet adhesion. In wild-type mice and ApoE mice on a Western diet, ultrasound molecular imaging of the thoracic aorta for VWF A1-domain and glycoprotein-Ibα was performed to quantify endothelial-associated VWF and platelet adhesion. After treatment of wild-type mice for 7 days, aortic molecular signal for endothelial-associated VWF and platelet adhesion were five- to sixfold higher in ponatinib vs sham therapy ( < .001), whereas dasatinib had no effect. In ApoE mice, aortic VWF and platelet signals were two- to fourfold higher for ponatinib-treated compared with sham-treated mice ( < .05) and were significantly higher than in treated wild-type mice ( < .05). Platelet and VWF signals in ponatinib-treated mice were significantly reduced by -acetylcysteine and completely eliminated by recombinant ADAMTS13. Ponatinib produced segmental left ventricular wall motion abnormalities in 33% of wild-type and 45% of ApoE mice and corresponding patchy perfusion defects, yet coronary arteries were normal on angiography. Instead, a global microvascular angiopathy was detected by immunohistochemistry and by intravital microscopy observation of platelet aggregates and nets associated with endothelial cells and leukocytes. Our findings reveal a new form of vascular toxicity for the TKI ponatinib that involves VWF-mediated platelet adhesion and a secondary microvascular angiopathy that produces ischemic wall motion abnormalities. These processes can be mitigated by interventions known to reduce VWF multimer size.
© 2019 by The American Society of Hematology.
Advances in single-cell biology have enabled measurements of >40 protein features on millions of immune cells within clinical samples. However, the data analysis steps following cell population identification are susceptible to bias, time-consuming, and challenging to compare across studies. Here, an ensemble of unsupervised tools was developed to evaluate four essential types of immune cell information, incorporate changes over time, and address diverse immune monitoring challenges. The four complementary properties characterized were (i) systemic plasticity, (ii) change in population abundance, (iii) change in signature population features, and (iv) novelty of cellular phenotype. Three systems immune monitoring studies were selected to challenge this ensemble approach. In serial biopsies of melanoma tumors undergoing targeted therapy, the ensemble approach revealed enrichment of double-negative (DN) T cells. Melanoma tumor-resident DN T cells were abnormal and phenotypically distinct from those found in nonmalignant lymphoid tissues, but similar to those found in glioblastoma and renal cell carcinoma. Overall, ensemble systems immune monitoring provided a robust, quantitative view of changes in both the system and cell subsets, allowed for transparent review by human experts, and revealed abnormal immune cells present across multiple human tumor types.
©2018 American Association for Cancer Research.
This Letter describes the chemical optimization of a new series of muscarinic acetylcholine receptor subtype 1 (M) positive allosteric modulators (PAMs) based on novel tricyclic triazolo- and imidazopyridine lactam cores, devoid of M agonism, e.g., no M ago-PAM activity, in high expressing recombinant cell lines. While all the new tricyclic congeners afforded excellent rat pharmacokinetic (PK) properties (CL < 8 mL/min/kg and t > 5 h), regioisomeric triazolopyridine analogues were uniformly not CNS penetrant ( K < 0.05), despite a lack of hydrogen bond donors. However, removal of a single nitrogen atom to afford imidazopyridine derivatives proved to retain the excellent rat PK and provide high CNS penetration ( K > 2), despite inclusion of a basic nitrogen. Moreover, 24c was devoid of M agonism in high expressing recombinant cell lines and did not induce cholinergic seizures in vivo in mice. Interestingly, all of the new M PAMs across the diverse tricyclic heterocyclic cores possessed equivalent CNS MPO scores (>4.5), highlighting the value of both "medicinal chemist's eye" and experimental data, e.g., not sole reliance (or decision bias) on in silico calculated properties, for parameters as complex as CNS penetration.
Little is known about the in vivo impacts of targeted therapy on melanoma cell abundance and protein expression. Here, 21 antibodies were added to an established melanoma mass cytometry panel to measure 32 cellular features, distinguish malignant cells, and characterize dabrafenib and trametinib responses in BRAF melanoma. Tumor cells were biopsied before neoadjuvant therapy and compared to cells surgically resected from the same site after 4 weeks of therapy. Approximately 50,000 cells per tumor were characterized by mass cytometry and computational tools t-SNE/viSNE, FlowSOM, and MEM. The resulting single-cell view of melanoma treatment response revealed initially heterogeneous melanoma tumors were consistently cleared of Nestin-expressing melanoma cells. Melanoma cell subsets that persisted to week 4 were heterogeneous but expressed SOX2 or SOX10 proteins and specifically lacked surface expression of MHC I proteins by MEM analysis. Traditional histology imaging of tissue microarrays from the same tumors confirmed mass cytometry results, including persistence of NES- SOX10+ S100β+ melanoma cells. This quantitative single-cell view of melanoma treatment response revealed protein features of malignant cells that are not eliminated by targeted therapy.
© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Despite the cost to both individual and society, alcohol use disorders (AUDs) remain a major health risk within society, and both relapse and heavy drinking are still poorly controlled with current medications. Here we demonstrate for the first time that a centrally active and selective negative allosteric modulator for the rat M muscarinic acetylcholine receptor (mAChR), ML375, decreases ethanol self-administration and attenuates cue-induced reinstatement of ethanol seeking in ethanol-preferring (iP) rats. Importantly, ML375 did not affect sucrose self-administration or general locomotor activity indicative of a selective effect on ethanol seeking. Based on the expression profile of M mAChRs in the brain and the distinct roles different aspects of the dorsal striatum have on long-term and short-term ethanol use, we studied whether intra-striatal microinjection of ML375 modulated ethanol intake in rats. We show in iP rats with an extensive history of ethanol intake that intra-dorsolateral (DL), but not intra-dorsomedial, striatal injections of ML375 reduced ethanol self-administration to a similar extent as the nicotinic acetylcholine receptor ligand varenicline, which has preclinical and clinical efficacy in reducing the reinforcing effects of ethanol. These data implicate the DL striatum as a locus for the effects of cholinergic-acting drugs on ethanol seeking in rats with a history of long-term ethanol use. Accordingly, we demonstrate in rats that selectively targeting the M mAChR can modulate both voluntary ethanol intake and cue-induced ethanol seeking and thereby provide direct evidence that the M mAChR is a potential novel target for pharmacotherapies aimed at treating AUDs.
Cardiovascular (CV) health has emerged as an important consideration in patients with chronic myeloid leukemia (CML) because of improved prognosis. Indeed, the success of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has increased the focus on survivorship and late toxicity in oncological care. Survivorship issues in this population include CV disease prevention, given its prevalence in the general population. The introduction of BCR-ABL1 TKIs represented a unique concept of indefinite cancer therapy, only recently evolving to include "treatment-free remission." Importantly, later-generation BCR-ABL1 TKIs have been associated with CV complications. Dasatinib has been associated with pleural/pericardial effusions and pulmonary hypertension, whereas nilotinib and ponatinib have been linked to the development of vascular occlusive events. There is currently a dearth of data with respect to the mechanisms of drug toxicities, the subsets of patients at risk, and prevention and treatment strategies to mitigate CV complications in patients with CML. Nevertheless, optimal patient CV risk assessment needs to become a more central tenet of patient care in CML. We propose several practical considerations for the practicing oncologist relative to the CV health of patients with CML, especially those on chronic TKI therapy.
© 2016 by The American Society of Hematology. All rights reserved.
BRAF and MEK inhibitors have improved clinical outcomes in advanced, BRAF -mutated melanomas. Acquired resistance occurs in most patients, with numerous and diverse drivers. We obtained pretreatment and progression biopsies from a patient who progressed on dabrafenib and trametinib. In addition to a preserved BRAF mutation, an internal deletion (rearrangement) of BRAF was observed in the progression sample. This deletion involved exons 2-8, which includes the Ras-binding domain, and is analogous to previously documented BRAF fusions and splice variants known to reactivate RAS-RAF-MEK-ERK signaling. In a large cohort of melanomas, 10 additional internal deletions were identified (0.4% of all melanomas; nine of which had concurrent BRAF mutations), as well as sporadically in other tumor types. Thus, we describe a novel mechanism of resistance to BRAF and MEK inhibition.
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Endothelial dysfunction, characterized by changes in eNOS, is a common finding in chronic inflammatory vascular diseases. These states are associated with increased infectious complications. We hypothesized that alterations in eNOS would enhance the response to LPS-mediated TLR4 inflammation. Human microvascular endothelial cells were treated with sepiapterin or N-nitro-L-arginine methylester (L-NAME) to alter endogenous NO production, and small interfering RNA to knockdown eNOS. Alterations of endogenous NO by sepiapterin, and L-NAME provided no significant changes to LPS inflammation. In contrast, eNOS knockdown greatly enhanced endothelial IL-6 production and permeability in response to LPS. Knockdown of eNOS enhanced LPS-induced p38. Inhibition of p38 with SB203580 prevented IL-6 production, without altering permeability. Knockdown of p38 impaired NF-κB activation. Physical interaction between p38 and eNOS was demonstrated by immunoprecipitation, suggesting a novel, NO-independent mechanism for eNOS regulation of TLR4. In correlation, biopsy samples in patients with systemic lupus erythematous showed reduced eNOS expression with associated elevations in TLR4 and p38, suggesting an in vivo link. Thus, reduced expression of eNOS, as seen in chronic inflammatory disease, was associated with enhanced TLR4 signaling through p38. This may enhance the response to infection in patients with chronic inflammatory conditions.-Stark, R. J., Koch, S. R., Choi, H., Mace, E. H., Dikalov, S. I., Sherwood, E. R., Lamb, F. S. Endothelial nitric oxide synthase modulates Toll-like receptor 4-mediated IL-6 production and permeability via nitric oxide-independent signaling.