Development and characterization of a high-throughput in vitro cord formation model insensitive to VEGF inhibition

BACKGROUND: Anti-VEGF therapy reduces tumor blood vessels, however, some vessels always remain. These VEGF insensitive vessels may help support continued tumor growth and metastases. Many in vitro assays examining multiple steps of the angiogenic process have been described, but the majority of these assays are sensitive to VEGF inhibition. There has been little focus on the development of high-throughput, in vitro assays to model the vessels that are insensitive to VEGF inhibition. METHODS: Here, we describe a fixed end-point and kinetic, high-throughput stem cell co-culture model of cord formation. RESULTS: In this system, cords develop within 24 hours, at which point they begin to lose sensitivity to VEGF inhibitors, bevacizumab, and ramucirumab. Consistent with the hypothesis that other angiogenic factors maintain VEGF-independent vessels, pharmacologic intervention with a broad spectrum anti-angiogenic antagonist (suramin), a vascular disrupting agent (combretastatin), or a combination of VEGF and Notch pathway inhibitors reduced the established networks. In addition, we used our in vitro approach to develop an in vivo co-implant vasculogenesis model that connects with the endogenous vasculature to form functional blood vessels. Similar to the in vitro system, over time these vessels become insensitive to VEGF inhibition. CONCLUSION: Together, these models may be used to identify novel drugs targeting tumor vessels that are not sensitive to VEGF inhibition

Single Molecule Imaging Reveals Differences in Microtubule Track Selection Between Kinesin Motors

Molecular motors differentially recognize and move cargo along discrete microtubule subpopulations in cells, resulting in preferential transport and targeting of subcellular cargoes

Personal Genome Project UK (PGP-UK): a research and citizen science hybrid project in support of personalized medicine

Background: Molecular analyses such as whole-genome sequencing have become routine and are expected to be transformational for future healthcare and lifestyle decisions. Population-wide implementation of such analyses is, however, not without challenges, and multiple studies are ongoing to identify what these are and explore how they can be addressed. Methods: Defined as a research project, the Personal Genome Project UK (PGP-UK) is part of the global PGP network and focuses on open data sharing and citizen science to advance and accelerate personalized genomics and medicine. Results: Here we report our findings on using an open consent recruitment protocol, active participant involvement, open access release of personal genome, methylome and transcriptome data and associated analyses, including 47 new variants predicted to affect gene function and innovative reports based on the analysis of genetic and epigenetic variants. For this pilot study, we recruited 10 participants willing to actively engage as citizen scientists with the project. In addition, we introduce Genome Donation as a novel mechanism for openly sharing previously restricted data and discuss the first three donations received. Lastly, we present GenoME, a free, open-source educational app suitable for the lay public to allow exploration of personal genomes. Conclusions: Our findings demonstrate that citizen science-based approaches like PGP-UK have an important role to play in the public awareness, acceptance and implementation of genomics and personalized medicine

Caveolin regulates kv1.5 trafficking to cholesterol-rich membrane microdomains Mol Pharmacol 73

ABSTRACT The targeting of ion channels to cholesterol-rich membrane microdomains has emerged as a novel mechanism of ion channel localization. Previously, we reported that Kv1.5, a prominent cardiovascular K ϩ channel ␣-subunit, localizes to caveolar microdomains. However, the mechanisms regulating Kv1.5 targeting and the functional significance of this localization are largely unknown. In this study, we demonstrate a role for caveolin in the trafficking of Kv1.5 to lipid raft microdomains where cholesterol modulates channel function. In cells lacking endogenous caveolin-1 or -3, the association of Kv1.5 with low-density, detergent-resistant membrane fractions requires coexpression with exogenous caveolin, which can form channel-caveolin complexes. Caveolin is not required for cell surface expression, however, and caveolin-trafficking mutants sequester Kv1.5, but not Kv2.1, in intracellular compartments, resulting in a loss of functional cell surface channel. Coexpression with wild type caveolin-1 does not alter Kv1.5 current density; rather, it induces depolarizing shifts in steady-state activation and inactivation. These shifts are analogous to those produced by elevation of membrane cholesterol. Together, these results show that caveolin modulates channel function by regulating trafficking to cholesterol-rich membrane microdomains

Factors affecting timing of surgery following neoadjuvant chemoradiation for esophageal cancer

Background: Neoadjuvant chemoradiation with esophagectomy is standard management for locally advanced esophageal cancer. Studies have shown that surgical timing following chemoradiation is important for minimizing postoperative complications, however in practice timing is often variable and delayed. Although postoperative impact of surgical timing has been studied, less is known about factors associated with delays. Materials and methods: A retrospective review was performed for 96 patients with esophageal cancer who underwent chemoradiation then esophagectomy between 2018 and 2020 at a single institution. Univariable and stepwise multivariable analyses were used to assess association between social (demographics, insurance) and clinical variables (pre-operative weight, comorbidities, prior cardiothoracic surgery, smoking history, disease staging) with time to surgery (≤8 weeks “on-time” vs. >8 weeks “delayed”). Results: Fifty-one patients underwent esophagectomy within 8 weeks of chemoradiation; 45 had a delayed operation. Univariate analysis showed the following characteristics were significantly different between on-time and delayed groups: weight loss within 3 months of surgery (3.9 ± 5.1 kg vs. 1.5 ± 3.6 kg; P = 0.009), prior cardiovascular disease (29% vs. 49%; P = 0.05), prior cardiothoracic surgery (4% vs. 22%; P = 0.01), history of ever smoked (69% vs. 87%; P = 0.04), absent nodal metastasis on pathology (57% vs. 82%; P = 0.008). Multivariate analysis demonstrated that prior cardiothoracic surgery (OR 8.924, 95%CI 1.67–47.60; P = 0.01) and absent nodal metastasis (OR 4.186, 95%CI 1.50–11.72; P = 0.006) were associated with delayed surgery. Conclusions: Delayed esophagectomy following chemoradiotherapy is associated with prior cardiothoracic surgery and absent nodal metastasis. Further investigations should focus on understanding how these factors contribute to delays to guide treatment planning and mitigate sources of outcome disparities

Immune determinants of Barrett’s progression to esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) develops from Barrett’s esophagus (BE), a chronic inflammatory state that can progress through a series of transformative dysplastic states before tumor development. While molecular and genetic changes of EAC tumors have been studied, immune microenvironment changes during Barrett’s progression to EAC remain poorly understood. In this study, we identify potential immunologic changes that can occur during BE-to-EAC progression. RNA sequencing (RNA-Seq) analysis on tissue samples from EAC patients undergoing surgical resection demonstrated that a subset of chemokines and cytokines, most notably IL6 and CXCL8, increased during BE progression to EAC. xCell deconvolution analysis investigating immune cell population changes demonstrated that the largest changes in expression during BE progression occurred in M2 macrophages, pro–B cells, and eosinophils. Multiplex immunohistochemical staining of tissue microarrays showed increased immune cell populations during Barrett’s progression to high-grade dysplasia. In contrast, EAC tumor sections were relatively immune poor, with a rise in PD-L1 expression and loss of CD8+ T cells. These data demonstrate that the EAC microenvironment is characterized by poor cytotoxic effector cell infiltration and increased immune inhibitory signaling. These findings suggest an immunosuppressive microenvironment, highlighting the need for further studies to explore immune modulatory therapy in EAC