Fusion-negative rhabdomyosarcoma orthotopic tongue xenografts for study of invasion, intravasation and metastasis in live animals.

PAX3/7 Fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Towards achieving a more complete understanding of advanced FN-RMS, we developed an orthotopic tongue xenograft model for studies of molecular basis of FN-RMS invasion and metastasis. The behavior of FN-RMS cells injected into murine tongue was examined using in vivo bioluminescence imaging, non-invasive intravital microscopy (IVM), and histopathology and compared to the prevailing hindlimb intramuscular and subcutaneous xenografts. FN-RMS cells were retained in the tongue and invaded locally into muscle mysial spaces and vascular lumen. While evidence of hematogenous dissemination to the lungs occurred in tongue and intramuscular xenografts, evidence of local invasion and lymphatic dissemination to lymph nodes only occurred in tongue xenografts. IVM and RNA-seq of tongue xenografts reveal shifts in cellular phenotype and differentiation state in tongue xenografts. IVM also shows homing to blood and lymphatic vessels, lymphatic intravasation, and dynamic membrane protrusions. Based on these findings, the tongue orthotopic xenograft of FN-RMS is a valuable model for tumor progression studies at the tissue, cellular and subcellular levels providing insight into kinetics and molecular bases of tumor invasion and metastasis and, hence, new therapeutic avenues for advanced FN-RMS.

Identification of global inhibitors of cellular glycosylation.

Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.

Aberrant protein expression of Appl1, Sortilin and Syndecan-1 during the biological progression of prostate cancer.

Diagnosis and assessment of patients with prostate cancer is dependent on accurate interpretation and grading of histopathology. However, morphology does not necessarily reflect the complex biological changes occurring in prostate cancer disease progression, and current biomarkers have demonstrated limited clinical utility in patient assessment. This study aimed to develop biomarkers that accurately define prostate cancer biology by distinguishing specific pathological features that enable reliable interpretation of pathology for accurate Gleason grading of patients. Online gene expression databases were interrogated and a pathogenic pathway for prostate cancer was identified. The protein expression of key genes in the pathway, including adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (Appl1), Sortilin and Syndecan-1, was examined by immunohistochemistry (IHC) in a pilot study of 29 patients with prostate cancer, using monoclonal antibodies designed against unique epitopes. Appl1, Sortilin, and Syndecan-1 expression was first assessed in a tissue microarray cohort of 112 patient samples, demonstrating that the monoclonal antibodies clearly illustrate gland morphologies. To determine the impact of a novel IHC-assisted interpretation (the utility of Appl1, Sortilin, and Syndecan-1 labelling as a panel) of Gleason grading, versus standard haematoxylin and eosin (H&E) Gleason grade assignment, a radical prostatectomy sample cohort comprising 114 patients was assessed. In comparison to H&E, the utility of the biomarker panel reduced subjectivity in interpretation of prostate cancer tissue morphology and improved the reliability of pathology assessment, resulting in Gleason grade redistribution for 41% of patient samples. Importantly, for equivocal IHC-assisted labelling and H&E staining results, the cancer morphology interpretation could be more accurately applied upon re-review of the H&E tissue sections. This study addresses a key issue in the field of prostate cancer pathology by presenting a novel combination of three biomarkers and has the potential to transform clinical pathology practice by standardising the interpretation of the tissue morphology.

Intravital Subcellular Microscopy of the Mammary Gland.

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676 or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs ≥ 0.7 µm. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 µm or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

Method for Acute Intravital Imaging of the Large Intestine in Live Mice.

Intravital microscopy is an imaging technique aimed at the visualization of the dynamics of biological processes in live animals. In the last decade, the development of nonlinear optical microscopy has enormously increased the use of this technique, thus addressing key biological questions in different fields such as immunology, neurobiology and tumor biology. In addition, new upcoming strategies to minimize motion artifacts due to animal respiration and heartbeat have enabled the visualization in real time of biological processes at cellular and subcellular resolution. Recently, intravital microscopy has been applied to analyze different aspect of mucosal immunity in the gut. However, the majority of these studies have been performed on the small intestine. Although crucial aspects of the biology of this organ have been unveiled, the majority of intestinal pathologies in humans occur in the large intestine.Here, we describe a method to surgically expose and stabilize the large intestine in live mice and to perform short-term (up to 2 h) intravital microscopy.

Transduction of Salivary Gland Acinar Cells with a Novel AAV Vector 44.9.

The loss of salivary gland function caused by radiation therapy of the head and neck or autoimmune disease such as Sjögren's syndrome is a serious condition that affects a patient's quality of life. Due to the combined exocrine and endocrine functions of the salivary gland, gene transfer to the salivary glands holds the potential for developing therapies for disorders of the salivary gland and the expression of therapeutic proteins via the exocrine pathway to the mouth, upper gastrointestinal tract, or endocrine pathway, systemically, into the blood. Recent clinical success with viral vector-mediated gene transfer for the treatment of irradiation-induced damage to the salivary glands has highlighted the need for the development of novel vectors with acinar cell tropism able to result in stable long-term transduction. Previous studies with adeno-associated virus (AAV) focused on the submandibular gland and reported mostly ductal cell transduction. In this study, we have screened AAV vectors for acinar cell tropism in the parotid gland utilizing membrane-tomato floxed membrane-GFP transgenic mice to screen CRE recombinase encoding AAV vectors of different clades to rapidly identify capsid isolates able to transduce salivary gland acinar cells. We determined that AAVRh10 and a novel isolate found as a contaminant of a laboratory stock of simian adenovirus SV15, AAV44.9, are both able to transduce parotid and sublingual acinar cells. Persistence and localization of transduction of these AAVs were tested using vectors encoding firefly luciferase, which was detected 6 months after vector administration. Most luciferase expression was localized to the salivary gland compared to that of distal organs. Transduction resulted in robust secretion of recombinant protein in both blood and saliva. Transduction was species specific, with AAVRh10 having stronger transduction activity in rats compared with AAV44.9 or AAV2 but weaker in human primary salivary gland cells. This work demonstrates efficient transduction of parotid acinar cells by AAV that resulted in secretion of recombinant protein in both serum and saliva.

Collective cancer cell invasion requires RNA accumulation at the invasive front.

Localization of RNAs at protrusive regions of cells is important for single-cell migration on two-dimensional surfaces. Protrusion-enriched RNAs encode factors linked to cancer progression, such as the RAB13 GTPase and the NET1 guanine nucleotide exchange factor, and are regulated by the tumor-suppressor protein APC. However, tumor cells in vivo often do not move as single cells but rather utilize collective modes of invasion and dissemination. Here, we developed an inducible system of three-dimensional (3D) collective invasion to study the behavior and importance of protrusion-enriched RNAs. We find that, strikingly, both the RAB13 and NET1 RNAs are enriched specifically at the invasive front of leader cells in invasive cell strands. This localization requires microtubules and coincides with sites of high laminin concentration. Indeed, laminin association and integrin engagement are required for RNA accumulation at the invasive front. Importantly, perturbing RNA accumulation reduces collective 3D invasion. Examination of in vivo tumors reveals a similar localization of the RAB13 and NET1 RNAs at potential invasive sites, suggesting that this mechanism could provide a targeting opportunity for interfering with collective cancer cell invasion.

A role for keratins in supporting mitochondrial organization and function in skin keratinocytes.

Mitochondria fulfill essential roles in ATP production, metabolic regulation, calcium signaling, generation of reactive oxygen species (ROS), and additional determinants of cellular health. Recent studies have highlighted a role for mitochondria during cell differentiation, including in skin epidermis. The observation of oxidative stress in keratinocytes from Krt16 null mouse skin, a model for pachyonychia congenita (PC)-associated palmoplantar keratoderma, prompted us to examine the role of Keratin (K) 16 protein and its partner K6 in regulating the structure and function of mitochondria. Electron microscopy revealed major anomalies in mitochondrial ultrastructure in late stage, E18.5, Krt6a/Krt6b null embryonic mouse skin. Follow-up studies utilizing biochemical, metabolic, and live imaging readouts showed that, relative to controls, skin keratinocytes null for Krt6a/Krt6b or Krt16 exhibit elevated ROS, reduced mitochondrial respiration, intracellular distribution differences, and altered movement of mitochondria within the cell. These findings highlight a novel role for K6 and K16 in regulating mitochondrial morphology, dynamics, and function and shed new light on the causes of oxidative stress observed in PC and related keratin-based skin disorders.

Dynamic polyhedral actomyosin lattices remodel micron-scale curved membranes during exocytosis in live mice.

Actomyosin networks, the cell's major force production machineries, remodel cellular membranes during myriad dynamic processes1,2 by assembling into various architectures with distinct force generation properties3,4. While linear and branched actomyosin architectures are well characterized in cell-culture and cell-free systems3, it is not known how actin and myosin networks form and function to remodel membranes in complex three-dimensional mammalian tissues. Here, we use four-dimensional spinning-disc confocal microscopy with image deconvolution to acquire macromolecular-scale detail of dynamic actomyosin networks in exocrine glands of live mice. We address how actin and myosin organize around large membrane-bound secretory vesicles and generate the forces required to complete exocytosis5-7. We find that actin and non-muscle myosin II (NMII) assemble into previously undescribed polyhedral-like lattices around the vesicle membrane. The NMII lattice comprises bipolar minifilaments8-10 as well as non-canonical three-legged configurations. Using photobleaching and pharmacological perturbations in vivo, we show that actomyosin contractility and actin polymerization together push on the underlying vesicle membrane to overcome the energy barrier and complete exocytosis7. Our imaging approach thus unveils a force-generating actomyosin lattice that regulates secretion in the exocrine organs of live animals.

Pak1 Kinase Promotes Activated T Cell Trafficking by Regulating the Expression of L-Selectin and CCR7.

Normal function of the adaptive immune system requires trafficking of T cells between the blood and lymphoid organs. Lymphocyte homing to lymph nodes requires that they cross endothelial barriers present in blood vessels and lymphatics. This multi-step process requires a remodeling of the lymphocyte plasma membrane, which is mediated by the dynamic re-arrangement of the actin cytoskeleton. Pak1 plays a central role in cell morphology, adhesion and migration in various cell types. Here we demonstrate that Pak1 is required for activated CD4+ T cell trafficking to lymph nodes. Pak1 deficiency in T cells causes a defect in the transcription of CCR7 and L-selectin, thereby altering lymphocyte trafficking. Additionally, we report an increase in L-selectin shedding in Pak1-deficient T cells, which correlates with a decrease in the recruitment of calmodulin to the cytoplasmic tail of L-selectin during T cell activation. Overall, our findings demonstrate that by regulating the expression of two major lymph node homing molecules, L-selectin and CCR7, Pak1 mediates activated CD4+ T cell trafficking.

ONC201 kills breast cancer cells in vitro by targeting mitochondria.

We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

Tumor-Associated Macrophages Derived from Circulating Inflammatory Monocytes Degrade Collagen through Cellular Uptake.

Physiologic turnover of interstitial collagen is mediated by a sequential pathway in which collagen is fragmented by pericellular collagenases, endocytosed by collagen receptors, and routed to lysosomes for degradation by cathepsins. Here, we use intravital microscopy to investigate if malignant tumors, which are characterized by high rates of extracellular matrix turnover, utilize a similar collagen degradation pathway. Tumors of epithelial, mesenchymal, or neural crest origin all display vigorous endocytic collagen degradation. The cells engaged in this process are identified as tumor-associated macrophage (TAM)-like cells that degrade collagen in a mannose receptor-dependent manner. Accordingly, mannose-receptor-deficient mice display increased intratumoral collagen. Whole-transcriptome profiling uncovers a distinct extracellular matrix-catabolic signature of these collagen-degrading TAMs. Lineage-ablation studies reveal that collagen-degrading TAMs originate from circulating CCR2+ monocytes. This study identifies a function of TAMs in altering the tumor microenvironment through endocytic collagen turnover and establishes macrophages as centrally engaged in tumor-associated collagen degradation.

Non-invasive intravital imaging of head and neck squamous cell carcinomas in live mice.

Head and neck squamous cell carcinoma is one of the most common cancers with a 50% 5-year survival rate. Understanding the mechanisms that control development, progression, and spreading of the tumor to distal sites is of paramount importance to develop effective therapies. Here, we describe a minimally invasive procedure, which enables performing intravital microscopy of the mouse tongue in models for oral cancer and provides structural and dynamic information of the tumors at cellular and subcellular level.

Unexpected Cartilage Phenotype in CD4-Cre-Conditional SOS-Deficient Mice.

RAS signaling is central to many cellular processes and SOS proteins promote RAS activation. To investigate the role of SOS proteins in T cell biology, we crossed Sos1f/fSos2-/- mice to CD4-Cre transgenic mice. We previously reported an effect of these mutations on T cell signaling and T cell migration. Unexpectedly, we observed nodules on the joints of greater than 90% of these mutant mice at 5 months of age, especially on the carpal joints. As the mice aged further, some also displayed joint stiffness, hind limb paralysis, and lameness. Histological analysis indicated that the abnormal growth in joints originated from dysplastic chondrocytes. Second harmonic generation imaging of the carpal nodules revealed that nodules were encased by rich collagen fibrous networks. Nodules formed in mice also deficient in RAG2, indicating that conventional T cells, which undergo rearrangement of the T cell antigen receptor, are not required for this phenotype. CD4-Cre expression in a subset of cells, either immune lineage cells (e.g., non-conventional T cells) or non-immune lineage cells (e.g., chondrocytes) likely mediates the dramatic phenotype observed in this study. Disruptions of genes in the RAS signaling pathway are especially likely to cause this phenotype. These results also serve as a cautionary tale to those intending to use CD4-Cre transgenic mice to specifically delete genes in conventional T cells.

Kinetics of milk lipid droplet transport, growth, and secretion revealed by intravital imaging: lipid droplet release is intermittently stimulated by oxytocin.

The lipid droplet (LD) fraction of milk has attracted special attention because it supplies preformed lipids for neonatal development, and the assembled LDs are secreted by a unique apocrine mechanism. Because many aspects of this key process remain uncharacterized, we developed a facile method for the intravital imaging of mammary cells in transgenic mice that express fluorescently tagged marker proteins. Using these techniques, we describe the first kinetic analysis of LD growth and secretion at peak lactation in real time. LD transit from basal to apical regions was slow (0-2 µm/min) and frequently intermittent. Droplets grew by the fusion of preexisting droplets, with no restriction on the size of fusogenic partners. Most droplet expansion took several hours and occurred in apical nucleation centers, either close to or in association with the apical surface. Droplets even continued to expand as they were emerging from the cell. Contrary to expectations, LDs attached to the apical plasma membrane but still associated with the cytoplasm were released after oxytocin-mediated contraction of the myoepithelium. Thus milk LD secretion is an intermittently regulated process. This novel procedure will have broad application for investigating trafficking events within the mammary epithelium in real time.

Sinusoidal ephrin receptor EPHB4 controls hematopoietic progenitor cell mobilization from bone marrow.

Hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow. Stress signals from cancer and other conditions promote HSPC mobilization into circulation and subsequent homing to tissue microenvironments. HSPC infiltration into tissue microenvironments can influence disease progression; notably, in cancer, HSPCs encourage tumor growth. Here we have uncovered a mutually exclusive distribution of EPHB4 receptors in bone marrow sinusoids and ephrin B2 ligands in hematopoietic cells. We determined that signaling interactions between EPHB4 and ephrin B2 control HSPC mobilization from the bone marrow. In mice, blockade of the EPHB4/ephrin B2 signaling pathway reduced mobilization of HSPCs and other myeloid cells to the circulation. EPHB4/ephrin B2 blockade also reduced HSPC infiltration into tumors as well as tumor progression in murine models of melanoma and mammary cancer. These results identify EPHB4/ephrin B2 signaling as critical to HSPC mobilization from bone marrow and provide a potential strategy for reducing cancer progression by targeting the bone marrow.

Direct Regulation of Alternative Splicing by SMAD3 through PCBP1 Is Essential to the Tumor-Promoting Role of TGF-ß.

In advanced stages of cancers, TGF-ß promotes tumor progression in conjunction with inputs from receptor tyrosine kinase pathways. However, mechanisms that underpin the signaling cooperation and convert TGF-ß from a potent growth inhibitor to a tumor promoter are not fully understood. We report here that TGF-ß directly regulates alternative splicing of cancer stem cell marker CD44 through a phosphorylated T179 of SMAD3-mediated interaction with RNA-binding protein PCBP1. We show that TGF-ß and EGF respectively induce SMAD3 and PCBP1 to colocalize in SC35-positive nuclear speckles, and the two proteins interact in the variable exon region of CD44 pre-mRNA to inhibit spliceosome assembly in favor of expressing the mesenchymal isoform CD44s over the epithelial isoform CD44E. We further show that the SMAD3-mediated alternative splicing is essential to the tumor-promoting role of TGF-ß and has a global influence on protein products of genes instrumental to epithelial-to-mesenchymal transition and metastasis.

A CCR2 macrophage endocytic pathway mediates extravascular fibrin clearance in vivo.

Extravascular fibrin deposition accompanies many human diseases and causes chronic inflammation and organ damage, unless removed in a timely manner. Here, we used intravital microscopy to investigate how fibrin is removed from extravascular space. Fibrin placed into the dermis of mice underwent cellular endocytosis and lysosomal targeting, revealing a novel intracellular pathway for extravascular fibrin degradation. A C-C chemokine receptor type 2 (CCR2)-positive macrophage subpopulation constituted the majority of fibrin-uptaking cells. Consequently, cellular fibrin uptake was diminished by elimination of CCR2-expressing cells. The CCR2-positive macrophage subtype was different from collagen-internalizing M2-like macrophages. Cellular fibrin uptake was strictly dependent on plasminogen and plasminogen activator. Surprisingly, however, fibrin endocytosis was unimpeded by the absence of the fibrin(ogen) receptors, αMß2 and ICAM-1, the myeloid cell integrin-binding site on fibrin or the endocytic collagen receptor, the mannose receptor. The study identifies a novel fibrin endocytic pathway engaged in extravascular fibrin clearance and shows that interstitial fibrin and collagen are cleared by different subsets of macrophages employing distinct molecular pathways.

Arp2/3-mediated F-actin formation controls regulated exocytosis in vivo.

The actin cytoskeleton plays crucial roles in many cellular processes, including regulated secretion. However, the mechanisms controlling F-actin dynamics in this process are largely unknown. Through 3D time-lapse imaging in a secreting organ, we show that F-actin is actively disassembled along the apical plasma membrane at the site of secretory vesicle fusion and re-assembled directionally on vesicle membranes. Moreover, we show that fusion pore formation and PIP2 redistribution precedes actin and myosin recruitment to secretory vesicle membranes. Finally, we show essential roles for the branched actin nucleators Arp2/3- and WASp in the process of secretory cargo expulsion and integration of vesicular membranes with the apical plasma membrane. Our results highlight previously unknown roles for branched actin in exocytosis and provide a genetically tractable system to image the temporal and spatial dynamics of polarized secretion in vivo.