Lian Willetts

Homologous recombination into the eosinophil peroxidase locus generates a strain of mice expressing Cre recombinase exclusively in eosinophils

Eosinophils are generally linked to innate host defense against helminths, as well as the pathologies associated with allergic diseases, such as asthma. Nonetheless, the activities of eosinophils remain poorly understood, which in turn, has prevented detailed definitions of their role(s) in health and disease. Homologous recombination in embryonic stem cells was used to insert a mammalianized Cre recombinase in the ORF encoding Epx. This knock‐in strategy overcame previous inefficiencies associated with eosinophil‐specific transgenic approaches and led to the development of a knock‐in strain of mice (eoCRE), capable of mediating recombination of “floxed” reporter cassettes in >95% of peripheral blood eosinophils. We also showed that this Cre expression was limited exclusively to eosinophil‐lineage committed cells with no evidence of Cre‐mediated toxicity. The efficiency and specificity of Cre expression in eoCRE mice were demonstrated further in a cross with a knock‐in mouse containing a “(flox‐stop‐flox)” DTA cassette at the ROSA26 locus, generating yet another novel, eosinophil‐less strain of mice. The development of eoCRE mice represents a milestone in studies of eosinophil biology, permitting eosinophil‐specific gene targeting and overexpression in the mouse as part of next‐generation studies attempting to define eosinophil effector functions.

Agonist Activation of F-Actin-Mediated Eosinophil Shape Change and Mediator Release Is Dependent on Rac2

Background: Tissue recruitment and activation of eosinophils contribute to allergic symptoms by causing airway hyperresponsiveness and inflammation. Shape changes and mediator release in eosinophils may be regulated by mammalian Rho-related guanosine triphosphatases. Of these, Rac2 is essential for F-actin formation as a central process underlying cell motility, exocytosis, and respiratory burst in neutrophils, while the role of Rac2 in eosinophils is unknown.We set out to determine the role of Rac2 in eosinophil mediator release and F-actin-dependent shape change in response to chemotactic stimuli. Methods: Rac2-deficient eosinophils from CD2-IL-5 transgenic mice crossed with rac2 gene knockout animals were examined for their ability to release superoxide through respiratory burst or eosinophil peroxidase by degranulation. Eosinophil shape change and actin polymerization were also assessed by flow cytometry and confocal microscopy following stimulation with eotaxin-2 or platelet-activating factor. Results: Eosinophils from wild-type mice displayed inducible superoxide release, but at a small fraction (4–5%) of human eosinophils. Rac2-deficient eosinophils showed significantly less superoxide release (p < 0.05, 26% less than wild type). Eosinophils lacking Rac2 had diminished degranulation (p < 0.05, 62% less eosinophil peroxidase) and shape changes in response to eotaxin-2 or platelet-activating factor (with 68 and 49% less F-actin formation, respectively; p < 0.02) compared with wild-type cells. Conclusion: These results demonstrate that Rac2 is an important regulator of eosinophil function by contributing to superoxide production, granule protein release, and eosinophil shape change. Our findings suggest that Rho guanosine triphosphatases are key regulators of cellular inflammation in allergy and asthma.

An essential role for Rab27a GTPase in eosinophil exocytosis

Eosinophil degranulation has been implicated in inflammatory processes associated with allergic asthma. Rab27a, a Rab‐related GTPase, is a regulatory intracellular signaling molecule expressed in human eosinophils. We postulated that Rab27a regulates eosinophil degranulation. We investigated the role of Rab27a in eosinophil degranulation within the context of airway inflammation. Rab27a expression and localization in eosinophils were investigated by using subcellular fractionation combined with Western blot analysis, and the results were confirmed by immunofluorescence analysis of Rab27a and the granule membrane marker CD63. To determine the function of eosinophil Rab27a, we used Ashen mice, a strain of Rab27a‐deficient animals. Ashen eosinophils were tested for degranulation in response to PAF and calcium ionophore by measuring released EPX activity. Airway EPX release was also determined by intratracheal injection of eosinophils into mice lacking EPX. Rab27a immunoreactivity colocalized with eosinophil crystalloid granules, as determined by subcellular fractionation and immunofluorescence analysis. PAF induced eosinophil degranulation in correlation with redistribution of Rab27a+ structures, some of which colocalized with CD63+ crystalloid granules at the cell membrane. Eosinophils from mice had significantly reduced EPX release compared with normal WT eosinophils, both in vitro and in vivo. In mouse models, Ashen mice demonstrated reduced EPX release in BAL fluid. These findings suggest that Rab27a has a key role in eosinophil degranulation. Furthermore, these findings have implications for Rab27a‐dependent eosinophil degranulation in airway inflammation.

Functional assessment of von Willebrand factor expression by cancer cells of non-endothelial origin

Von Willebrand factor (VWF) is a highly adhesive procoagulant molecule that mediates platelet adhesion to endothelial and subendothelial surfaces. Normally it is expressed exclusively in endothelial cells (ECs) and megakaryocytes. However, a few studies have reported VWF detection in cancer cells of non-endothelial origin, including osteosarcoma. A role for VWF in cancer metastasis has long been postulated but evidence supporting both pro- and anti-metastatic roles for VWF has been presented. We hypothesized that the role of VWF in cancer metastasis is influenced by its cellular origin and that cancer cell acquisition of VWF expression may contribute to enhanced metastatic potential. We demonstrated de novo expression of VWF in glioma as well as osteosarcoma cells. Endothelial monolayer adhesion, transmigration and extravasation capacities of VWF expressing cancer cells were shown to be enhanced compared to non-VWF expressing cells, and were significantly reduced as a result of VWF knock down. VWF expressing cancer cells were also detected in patient tumor samples of varying histologies. Analyses of the mechanism of transcriptional activation of the VWF in cancer cells demonstrated a pattern of trans-activating factor binding and epigenetic modifications consistent overall with that observed in ECs. These results demonstrate that cancer cells of non-endothelial origin can acquire de novo expression of VWF, which can enhance processes, including endothelial and platelet adhesion and extravasation, that contribute to cancer metastasis.

Eosinophil shape change and secretion.

The analysis of eosinophil shape change and mediator secretion is a useful tool in understanding how eosinophils respond to immunological stimuli and chemotactic factors. Eosinophils undergo dramatic shape changes, along with secretion of the granule-derived enzyme eosinophil peroxidase (EPX) in response to chemotactic stimuli including platelet-activating factor and CCL11 (eotaxin-1). Here, we describe the analysis of eosinophil shape change by confocal microscopy analysis and provide an experimental approach for comparing unstimulated cells with those that have been stimulated to undergo chemotaxis. In addition, we illustrate two different degranulation assays for EPX using OPD and an enzyme-linked immunosorbent assay technique and show how eosinophil degranulation may be assessed from in vitro as well as ex vivo stimulation.

Quantitative in vivo whole genome motility screen reveals novel therapeutic targets to block cancer metastasis

Metastasis is the most lethal aspect of cancer, yet current therapeutic strategies do not target its key rate-limiting steps. We have previously shown that the entry of cancer cells into the blood stream, or intravasation, is highly dependent upon in vivo cancer cell motility, making it an attractive therapeutic target. To systemically identify genes required for tumor cell motility in an in vivo tumor microenvironment, we established a novel quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos. Utilizing this platform to screen a genome-wide shRNA library, we identified a panel of novel genes whose function is required for productive cancer cell motility in vivo, and whose expression is closely associated with metastatic risk in human cancers. The RNAi-mediated inhibition of these gene targets resulted in a nearly total (>99.5%) block of spontaneous cancer metastasis in vivo.Tumour metastasis is dependent on tumour cell motility. Here, the authors investigate genes required for tumour cell motility by establishing a quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos.

Vesicle-associated membrane protein 7-mediated eosinophil degranulation promotes allergic airway inflammation in mice

Eosinophil degranulation is a determining factor in allergy-mediated airway pathology. Receptor-mediated degranulation in eosinophils requires vesicle-associated membrane protein 7 (VAMP-7), a principal component of the SNARE fusion machinery. The specific contribution of eosinophil degranulation to allergen-induced airway responses remains poorly understood. We generated mice with VAMP-7 gene deficiency exclusively in eosinophils (eoCRE/V7) from a cross using eosinophil-specific Cre recombinase-expressing mice crossed with VAMP-7f/f mice. Eosinophils from eoCRE/V7 mice showed deficient degranulation responses in vitro, and responses continued to be decreased following ex vivo intratracheal adoptive transfer of eoCRE/V7 eosinophils into IL-5/hE2/EPX−/− mice. Consistent with diminished degranulation responses, reduced airway hyperresponsiveness was observed in ovalbumin-sensitized and challenged eoCRE/V7 mice following methacholine inhalation. Therefore, VAMP-7 mediates eosinophil degranulation both in vitro and ex vivo, and this event augments airway hyperresponsiveness.Lian Willetts et al. demonstrate that vesicle-associated membrane protein 7 (VAMP 7), a principal component of the membrane fusion machinery, promotes eosinophil degranulation in allergic airway inflammation. This study suggests VAMP7 as a therapeutic target for ameliorating asthma.

Intravital imaging tumor screen used to identify novel metastasis-blocking therapeutic targets

Cancer cell motility is a key driver of metastasis. Although the intravasation of cancer cells into the blood stream is highly dependent on their motility and metastatic dissemination is the primary cause of cancer related deaths, current therapeutic strategies do not target the genes and proteins that are essential for cell motility. A primary reason for this is because the identification of cell motility-related genes that are relevant in vivo requires the visualization of metastatic lesions forming in an appropriate in vivo model. The cancer research community has lacked an in vivo and intravital metastatic cancer model that could be imaged as motility developed, in real-time. To address this, we developed a novel quantitative in vivo screening platform based on intravital imaging in shell-less ex ovo chick embryos. We applied this imaging approach to screen a human genome-wide short hairpin RNA library (shRNA) versus the highly motile head and neck cancer cells (HEp3 cell line) introduced into the chorioallantoic membrane (CAM) of chick embryos and identified multiple novel in vivo cancer cell motility-associated genes. When the expression of several of the identified genes was inhibited in the HEp3 tumors, we observed a nearly total block of spontaneous cancer metastasis.

Abstract 3799: Technical validation of a micro-flow cytometry platform for prostate cancer biomarker discovery

The analysis of extracellular vesicles (EVs) in biofluids is an expanding area of biomarker discovery. To be effective, a diagnostic test must differentiate EVs originating from cancer cells versus non-cancerous origins, and also define biomarkers enriched in cancer EVs. Our team has developed a micro-flow cytometry platform using the Apogee A50 to analyze prostate cancer (PCa) EVs in different biofluids. While flow cytometry is powerful tool for quantitative and qualitative evaluation of cellular biomarkers, direct translation of most applications to EVs is challenging. For example, EVs in biofluids typically range in size from 50-1000nm, and are present as low refractive index, polydisperse populations. We have optimized 30 assay parameters for the identification of prostate specific EVs so that disease specific biomarkers may be evaluated. To illustrate the sensitivity of the system, conditioned media from palmitoylated-GFP-LNCaP cells was used as a positive signal sample. When spiked into increasing concentrations of unstained plasma EVs, the GFP spike was detected linearly, even when present as low as 0.03% of the total EV population. Furthermore, the rate of positive signal/uL sample was quantitative after 15-30 seconds of sample analysis. The platform also permits high throughput analysis using a 96 well plate autosampler. To show sample reproducibility, we analyzed both low and high positive-signal samples over an 8hr sampling window. Low positive samples (120events/uL; 0.1% of total EVs) had a 4% CV over 96 samples whereas the high positive signal samples (2300events/uL, 1.0% of total EVs) had a 2.2% CV. Similar CVs have been obtained with CD9 and CD63. Analysis of sample aliquots over multiple days shows less than 6% CV. Conventional flow cytometers acquire tens of thousands of events while the Apogee system is able to collect millions of events with several parameters/event, in a high throughput manner. This generates a significant amount of data for which we have designed a unique machine learning approach to detect specific EV populations within the whole sample. In this manner, we utilize all data from the sample and not simply a subjectively defined area of interest. Using fresh samples from the clinic we have standardized a 2hr arm-freezer protocol for the collection and preservation of serum and plasma. Comparison studies in LNCaps and PC3 cells of 5 antibodies against PSMA, indicated the J591 anti-PSMA monoclonal antibody as a selective marker for prostate cancer EVs. Positive signal for this antibody was linearly titrated in both a LNCaP EV system and plasma EVs from metastatic prostate cancer patients. Specificity was shown by saturating a plasma system with unconjugated J591 and then reacting with PE-conjugated J591. Using the A50 micro-flow cytometry platform, the J591 antibody to identify prostate specific EVs, and a unique machine learning algorithm, we identified a multiple biomarker, diagnostic signature for PCa. Citation Format: Desmond Pink, Robert Paproski, Deborah Sosnowski, Lian Willetts, Eric Hyndman, John D. Lewis. Technical validation of a micro-flow cytometry platform for prostate cancer biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3799. doi:10.1158/1538-7445.AM2017-3799

Abstract 1463: Intravital discovery of miRNA drivers of human cancer cell directional invasion

Metastatic cancer cells use directional ECM cues such as blood vessels or collagen fibers when invading through live tissue. Oncogenic miRNAs have been implicated as key regulators of cancer progression, yet the systemic discovery of miRNAs that drive directional cancer cell invasion has not been achieved. Here we describe the first in vivo quantitative whole human miRNAome screen for miRNA drivers of directional cancer cell invasion that is based on high-resolution intravital imaging. We identified several novel miRNAs that promote cancer cell invasion during the key rate-limiting step of cancer metastasis: the initiation of overt metastatic lesions. In vivo 4D cancer cell tracking revealed that these prometastatic miRNAs are required for successful invasion into collagen-rich tissue and for attachment to the outer surface of the vascular wall. Deregulation of these miRNAs led to formation of loose contacts with the vasculature and chaotic, nondirectional cancer cell invasion patterns in living tissue. Intravital SHG microscopy showed that inhibition of the expression of these miRNAs blocked the ability of cancer cells to rearrange the disorganized collagen network into collagen fiber bundles and stably protrude along these bundles. Further imaging analysis revealed that this miRNA expression specifically blocks vesicular transport of key cell invasion machinery proteins such as MT1-MMP to the cancer cell invadopodia. Human cancer gene expression database analysis showed that our top miRNA candidates are specifically deregulated in invasive pancreatic cancer. Indeed, intravital imaging analysis showed that blocking of metastatic miRNA function inhibited MT1-MMP secretion and ECM degradation by human pancreatic cancer cells. In summary, we identified a novel panel of human miRNAs that are functionally involved in the regulation of directional invasion and metastasis. This work establishes these miRNAs as promising therapeutic targets to block the metastatic spread of lethal cancers. Citation Format: Konstantin V. Stoletov, Lian Willetts, Juan Jovel, Emma Woolner, John D. Lewis. Intravital discovery of miRNA drivers of human cancer cell directional invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 494.