Advancement in Cellular Topographic and Nanoparticle Capture Imaging by High Resolution Microscopy Incorporating a Freeze-Drying and Gaseous Nitrogen-based Approach.

Scanning electron microscopy (SEM) offers an unparalleled view of the membrane topography of mammalian cells by using a conventional osmium (OsO) and ethanol-based tissue preparation. However, conventional SEM methods limit optimal resolution due to ethanol and lipid interactions and interfere with visualization of fluorescent reporter proteins. Therefore, SEM correlative light and electron microscopy (CLEM) has been hindered by the adverse effects of ethanol and OsO on retention of fluorescence signals.

Tumor-derived nanoseeds condition the soil for metastatic organotropism.

Primary tumors secrete a variety of factors to turn distant microenvironments into favorable and fertile 'soil' for subsequent metastases. Among these 'seeding' factors that initiate pre-metastatic niche (PMN) formation, tumor-derived extracellular vesicles (EVs) are of particular interest as tumor EVs can direct organotropism depending on their surface integrin profiles. In addition, EVs also contain versatile, bioactive cargo, which include proteins, metabolites, lipids, RNA, and DNA fragments.

Extracellular vesicles and particles impact the systemic landscape of cancer.

Intercellular cross talk between cancer cells and stromal and immune cells is essential for tumor progression and metastasis. Extracellular vesicles and particles (EVPs) are a heterogeneous class of secreted messengers that carry bioactive molecules and that have been shown to be crucial for this cell-cell communication. Here, we highlight the multifaceted roles of EVPs in cancer.

Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids.

We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers.

Autophagic Degradation of NBR1 Restricts Metastatic Outgrowth during Mammary Tumor Progression.

Although autophagy is being pursued as a therapeutic target in clinical oncology trials, its effects on metastasis, the principal cause of cancer mortality, remain unclear. Here, we utilize mammary cancer models to temporally delete essential autophagy regulators during carcinoma progression. Though genetic ablation of autophagy strongly attenuates primary mammary tumor growth, impaired autophagy promotes spontaneous metastasis and enables the outgrowth of disseminated tumor cells into overt macro-metastases.

Non-reversible tissue fixation retains extracellular vesicles for in situ imaging.

Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues.

Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells.

Exosome-Mediated Metastasis: Communication from a Distance.

Metastasis, a critical phase of tumor progression, remains a primary challenge in treating cancer and a major cause of cancer mortality. Cell-cell communication via extracellular vesicles (exosomes and microvesicles) between primary tumor cells and the microenvironment of distant organs is crucial for pre-metastatic niche (PMN) formation and metastasis. Here, we review work on the contribution of exosome cargo to cancer progression, the role of exosomes in PMN establishment, and the function of exosomes in organotropic metastasis.

Tumor Extracellular Vesicles Impede Interferon Alert Responses.

Tumor-derived extracellular vesicles promote metastasis by inducing functional changes in cells at pre-metastatic sites conducive for tumor cell colonization. In this issue of Cancer Cell, Ortiz and colleagues show that type I interferon regulates extracellular vesicle uptake and that modulating this pathway holds promise for treating metastasis.

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation.

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed 'exomeres' (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling.

Autophagy in adhesion and migration.

Autophagy, a pathway for lysosomal-mediated cellular degradation, has recently been described as a regulator of cell migration. Although the molecular mechanisms underlying autophagy-dependent motility are only beginning to emerge, new work demonstrates that selective autophagy mediated by the autophagy cargo receptor, NBR1, specifically promotes the dynamic turnover of integrin-based focal adhesion sites during motility. Here, we discuss the detailed mechanisms through which NBR1-dependent selective autophagy supports focal adhesion remodeling, and we describe the interconnections between this pathway and other established regulators of focal adhesion turnover, such as microtubules.

Pre-Metastatic Niche Formation Has Taken Its TOLL.

Pre-Metastatic Niches (PMNs) result from communications between primary tumors and the microenvironment of future distant metastasis via tumor-derived factors. In this issue of Cancer Cell, Liu et al. show that TLR3 activation in lung epithelial cells by tumor exosomal RNAs triggers neutrophil recruitment, which contributes to PMN formation and metastasis.

NBR1-dependent selective autophagy is required for efficient cell-matrix adhesion site disassembly.

Macroautophagy/autophagy has classically been recognized for its vital role in supporting cellular survival during various stresses. However, emerging work has demonstrated that selective autophagy has an impact on diverse cell biological processes by mediating the degradation of various cellular contents during normal cellular homeostasis. We recently established that selective autophagy supports cell migration by promoting the turnover of integrin-based cell-matrix adhesion sites, or focal adhesions (FAs).

NBR1 enables autophagy-dependent focal adhesion turnover.

Autophagy is a catabolic pathway involving the sequestration of cellular contents into a double-membrane vesicle, the autophagosome. Although recent studies have demonstrated that autophagy supports cell migration, the underlying mechanisms remain unknown. Using live-cell imaging, we uncover that autophagy promotes optimal migratory rate and facilitates the dynamic assembly and disassembly of cell-matrix focal adhesions (FAs), which is essential for efficient motility.

Discovery of novel triazolobenzazepinones as γ-secretase modulators with central Aβ42 lowering in rodents and rhesus monkeys.

Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are presented in this communication. Starting from our azepinone leads, optimization studies toward improving central lowering of Aβ42 led to the discovery of novel benzo-fused azepinones. Several benzazepinones were profiled in vivo and found to lower brain Aβ42 levels in Sprague Dawley rats and transgenic APP-YAC mice in a dose-dependent manner after a single oral dose.

Potent benzoazepinone γ-secretase modulators with improved bioavailability.

The triazolyl amide γ-secretase modulators are potent alternatives to the cinnamyl amides that have entered the clinic for the treatment of Alzheimer's disease. Herein we build on the lead benzoazepinones described in our prior communication with imidazomethoxyarene moiety alternatives that offer opportunities to fine tune physical properties as well as address hERG binding and PK. Both half-life and bioavailability were significantly improved, especially in dog, with robust brain Aβ42 lowering maintained in both transgenic mouse and rat.

Cellular and metabolic functions for autophagy in cancer cells.

Autophagy is a lysosomal degradation pathway that acts as a dynamic regulator of tumorigenesis. Specifically, autophagy has been shown to impede early cancer development while facilitating advanced tumor progression. Recent studies have uncovered several tumor-promoting functions for autophagy; these include the maintenance of multiple metabolic pathways critical for aggressive tumor growth and the promotion of tumor cell survival downstream of the unfolded protein response.

Delayed and Prolonged Histone Hyperacetylation with a Selective HDAC1/HDAC2 Inhibitor.

The identification and in vitro and in vivo characterization of a potent SHI-1:2 are described. Kinetic analysis indicated that biaryl inhibitors exhibit slow binding kinetics in isolated HDAC1 and HDAC2 preparations. Delayed histone hyperacetylation and gene expression changes were also observed in cell culture, and histone acetylation was observed in vivo beyond disappearance of drug from plasma.

Autophagy-dependent production of secreted factors facilitates oncogenic RAS-driven invasion.

Unlabelled: The tumor-promoting functions of autophagy are primarily attributed to its ability to promote cancer cell survival. However, emerging evidence suggests that autophagy plays other roles during tumorigenesis. Here, we uncover that autophagy promotes oncogenic RAS-driven invasion.

Triazoles as γ-secretase modulators.

Synthesis, SAR, and evaluation of aryl triazoles as novel gamma secretase modulators (GSMs) are presented in this communication. Starting from the literature and in-house leads, we evaluated a range of five-membered heterocycles as replacements for olefins commonly found in non-acid GSMs. 1,2,3-C-aryl-triazoles were identified as suitable replacements which exhibited good modulation of γ-secretase activity, excellent pharmacokinetics and good central lowering of Aβ42 in Sprague-Dawley rats.

Autophagy facilitates glycolysis during Ras-mediated oncogenic transformation.

The protumorigenic functions for autophagy are largely attributed to its ability to promote cancer cell survival in response to diverse stresses. Here we demonstrate an unexpected connection between autophagy and glucose metabolism that facilitates adhesion-independent transformation driven by a strong oncogenic insult-mutationally active Ras. In cells ectopically expressing oncogenic H-Ras as well as human cancer cell lines harboring endogenous K-Ras mutations, autophagy is induced following extracellular matrix detachment.

Purine derivatives as potent gamma-secretase modulators.

The development of a novel series of purines as gamma-secretase modulators for potential use in the treatment of Alzheimer's disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based gamma-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Alphabeta42 in an APP-YAC transgenic mouse model.