Multi-modal Profiling of the Extracellular Matrix of Human Fallopian Tubes and Serous Tubal Intraepithelial Carcinomas.

Recent evidence supports the fimbriae of the fallopian tube as one origin site for high-grade serous ovarian cancer (HGSOC). The progression of many solid tumors is accompanied by changes in the microenvironment, including alterations of the extracellular matrix (ECM). Therefore, we sought to determine the ECM composition of the benign fallopian tube and changes associated with serous tubal intraepithelial carcinomas (STICs), precursors of HGSOC. The ECM composition of benign human fallopian tube was first defined from a meta-analysis of published proteomic datasets that identified 190 ECM proteins. We then conducted de novo proteomics using ECM enrichment and identified 88 proteins, 7 of which were not identified in prior studies (COL2A1, COL4A5, COL16A1, elastin, LAMA5, annexin A2, and PAI1). To enable future in vitro studies, we investigated the levels and localization of ECM components included in tissue-engineered models (type I, III, and IV collagens, fibronectin, laminin, versican, perlecan, and hyaluronic acid) using multispectral immunohistochemical staining of fimbriae from patients with benign conditions or STICs. Quantification revealed an increase in stromal fibronectin and a decrease in epithelial versican in STICs. Our results provide an in-depth picture of the ECM in the benign fallopian tube and identified ECM changes that accompany STIC formation. (J Histochem Cytochem XX: XXX-XXX, XXXX).

Multispectral Staining and Analysis of Extracellular Matrix.

Multiplexed immunofluorescent (IF) techniques enable the detection of multiple antigens within the same sample and are therefore useful in situations where samples are rare or small in size. Similar to standard IF, multiplexed IF yields information on both the location and relative amount of detected antigens. While this method has been used primarily to detail cell phenotypes, we have recently adapted it to profile the extracellular matrix (ECM), which provides technical challenges due to autofluorescence and spatial overlap. This chapter details the planning, execution, optimization, and troubleshooting to use multiplexed IF to profile the ECM of human fallopian tube tissue.

Engineering the Extracellular Matrix to Model the Evolving Tumor Microenvironment.

Clinical evidence supports a role for the extracellular matrix (ECM) in cancer risk and prognosis across multiple tumor types, and numerous studies have demonstrated that individual ECM components impact key hallmarks of tumor progression (e.g., proliferation, migration, angiogenesis). However, the ECM is a complex network of fibrillar proteins, glycoproteins, and proteoglycans that undergoes dramatic changes in composition and organization during tumor development. In this review, we will highlight how engineering approaches can be used to examine the impact of changes in tissue architecture, ECM composition (i.e., identity and levels of individual ECM components), and cellular- and tissue-level mechanics on tumor progression. In addition, we will discuss recently developed methods to model the ECM that have not yet been applied to the study of cancer.

The Many Microenvironments of Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is the most common and deadly subtype of ovarian cancer as it is commonly diagnosed after substantial metastasis has already occurred. The past two decades have been an active era in HGSOC research, with new information on the origin and genomic signature of the tumor cell. Additionally, studies have begun to characterize changes in the HGSOC microenvironment and examine the impact of these changes on tumor progression and response to therapies. While this knowledge may provide valuable insight into better prognosis and treatments for HGSOCs, its collection, synthesis, and application are complicated by the number of unique microenvironments in the disease-the initiating site (fallopian tube), first metastasis (ovary), distal metastases (peritoneum), and recurrent/platinum-resistant setting. Here, we review the state of our understanding of these diverse sites and highlight remaining questions.

Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes.

The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 µm long filaments that then 'stitch' together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.

Basal epithelial tissue folding is mediated by differential regulation of microtubules.

The folding of epithelial tissues is crucial for development of three-dimensional structure and function. Understanding this process can assist in determining the etiology of developmental disease and engineering of tissues for the future of regenerative medicine. Folding of epithelial tissues towards the apical surface has long been studied, but the molecular mechanisms that mediate epithelial folding towards the basal surface are just emerging. Here, we utilize zebrafish neuroepithelium to identify mechanisms that mediate basal tissue folding to form the highly conserved embryonic midbrain-hindbrain boundary. Live imaging revealed Wnt5b as a mediator of anisotropic epithelial cell shape, both apically and basally. In addition, we uncovered a Wnt5b-mediated mechanism for specific regulation of basal anisotropic cell shape that is microtubule dependent and likely to involve JNK signaling. We propose a model in which a single morphogen can differentially regulate apical versus basal cell shape during tissue morphogenesis.

Calcium signals drive cell shape changes during zebrafish midbrain-hindbrain boundary formation.

One of the first morphogenetic events in the vertebrate brain is the formation of the highly conserved midbrain-hindbrain boundary (MHB). Specific cell shape changes occur at the point of deepest constriction of the MHB, the midbrain-hindbrain boundary constriction (MHBC), and are critical for proper MHB formation. These cell shape changes are controlled by nonmuscle myosin II (NMII) motor proteins, which are tightly regulated via the phosphorylation of their associated myosin regulatory light chains (MRLCs). However, the upstream signaling pathways that initiate the regulation of NMII to mediate cell shape changes during MHB morphogenesis are not known. We show that intracellular calcium signals are critical for the regulation of cell shortening during initial MHB formation. We demonstrate that the MHB region is poised to respond to calcium transients that occur in the MHB at the onset of MHB morphogenesis and that calcium mediates phosphorylation of MRLC specifically in MHB tissue. Our results indicate that calmodulin 1a (calm1a), expressed specifically in the MHB, and myosin light chain kinase together mediate MHBC cell length. Our data suggest that modulation of NMII activity by calcium is critical for proper regulation of cell length to determine embryonic brain shape during development.