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MOV file - 313K, Intravital imaging of tumor cells migration

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MOV file - 313K, Rab25 expression reduces protrusion at the plasma membrane

PDF file - 921K, A. Representative core tissues from human oral cancer tissue array stained using an antibody directed against human CLIC3. Quantitative analysis of the CLIC3 expression. ns = not statistically significant ; t-test ; p values > 0.05, n= number of tissue cores, Normal, T1, T2, T3, T4 = TNM classification at T1 and T2,T3 and T4 respectively. B. Representative core tissues from human cervical cancer tissue array stained with Hematoxylin-Eosin or using an antibody against human Rab25. Quantitative staining score of the Rab25 expression at low, medium and high scores are shown.

MOV file - 4354K, Rab25 expression affects F-actin organization at the plasma membrane

Purpose: Head and neck squamous cell carcinoma (HNSCC) is one of the 10 most common cancers with a 50% five-year survival rate, which has remained unchanged for the past three decades. One of the major reasons for the aggressiveness of this cancer is that HNSCCs readily metastasize to cervical lymph nodes that are abundant in the head and neck region. Hence, discovering new molecules controlling the metastatic process as well as understanding their regulation at the molecular level are essential for effective therapeutic strategies.Experimental Design: Rab25 expression level was analyzed in HNSCC tissue microarray. We used a combination of intravital microscopy in live animals and immunofluorescence in an in vitro invasion assay to study the role of Rab25 in tumor cell migration and invasion.Results: In this study, we identified the small GTPase Rab25 as a key regulator of HNSCC metastasis. We observed that Rab25 is downregulated in HNSCC patients. Next, we determined that reexpression of Rab25 in a metastatic cell line is sufficient to block invasion in a three-dimensional collagen matrix and metastasis to cervical lymph nodes in a mouse model for oral cancer. Specifically, Rab25 affects the organization of F-actin at the cell surface, rather than cell proliferation, apoptosis, or tumor angiogenesis.Conclusion: These findings suggest that Rab25 plays an important role in tumor migration and metastasis, and that understanding its function may lead to the development of new strategies to prevent metastasis in oral cancer patients. Clin Cancer Res; 19(6); 1375–88. ©2013 AACR.

PDF file - 79K, A. Tongue tumor weight and volume from HeLa-O3 expressing venus and venus Rab25 tumors were measured at the samples collection date. B. Lymphatic (Lyve1 staining) and blood vessels (CD31 staining) density from HeLa-O3 expressing venus and venus Rab25 tumors are depicted in arbitrary unit. C. Apoptotic cells and proliferating cells from HeLa-O3 expressing venus and venus Rab25 tumors were detected by cleaved caspase3 (green) and Ki67 (red) antibody, respectively. The percentage of positive cells is shown in the lower panel graphs. ns= not statistically significant ; t-test ; p values > 0.05.

MOV file - 9371K, Intravital imaging of tongue cancer using 930 nm as excitation wavelength.

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Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous cell carcinomas (HNSCC) patients remains relatively unchanged at 50% for the past three decades. HNSCCs often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. Among the multiple dysregulated molecular mechanism in HNSCCs, emerging basic, preclinical, and clinical findings support the importance of the mTOR signaling route in HNSCC progression. Indeed, we observed here that the activation of mTOR is a widespread event in clinical specimens of HNSCCs invading locoregional lymph nodes. We developed an orthotopic model of HNSCC consisting of the implantation of HNSCC cells into the tongues of immunocompromised mice. These orthotopic tumors spontaneously metastasize to the cervical lymph nodes, where the presence of HNSCC cells can be revealed by histologic and immunohistochemical evaluation. Both primary and metastatic experimental HNSCC lesions exhibited elevated mTOR activity. The ability to monitor and quantitate lymph node invasion in this model system enabled us to explore whether the blockade of mTOR could impact HNSCC metastasis. We found that inhibition of mTOR with rapamycin and the rapalog RAD001 diminished lymphangiogenesis in the primary tumors and prevented the dissemination of HNSCC cancer cells to the cervical lymph nodes, thereby prolonging animal survival. These findings may provide a rationale for the future clinical evaluation of mTOR inhibitors, including rapamycin and its analogues, as part of a molecular-targeted metastasis preventive strategy for the treatment of patients with HNSCC. Cancer Res; 71(22); 7103–12. ©2011 AACR.

UNSTRUCTURED Background Achieving the physical activity recommendations of at least 150 to 300 minutes a week of moderate-intensity, or 75 to 150 minutes a week of vigorous-intensity aerobic exercise, is important for reducing cardiometabolic risk, but evidence shows that most people struggle to meet these goals, particularly in the mid to long-term. Objectives The Messages Improving Resting hearT Health (MIRTH) study aims to determine whether or not: (1) sending daily motivational messages via a research app is effective in improving motivation and in promoting adherence to physical activity recommendations in men and women with coronary heart diseases randomized to a 12-month intensive lifestyle intervention, and (2) the time of the day when the message is delivered impacts compliance with exercise training. Design A single center, micro-randomized trial (MRT). Participants will be randomized daily to either receive or not receive motivational messages over two 90-day periods at the beginning (months 4-6) and at the end (10-12) of the LIVEPLUS trial (ACTRN12620001151921). Wrist-worn devices (Fitbit Inspire 2) Bluetooth paired with smartphones will be used to passively collect data for proximal (i.e., physical activity duration, steps walked, heart rate within 180 minutes of receiving messages) and distal (i.e., change values for resting heart rate and total steps walked within and across both Phases 1 and 2 of the trial) outcomes. Setting Participants will be recruited from a large academic Cardiology office practice (Central Sydney Cardiology) and Royal Prince Alfred Hospital (RPAH) Departments of Cardiology and Radiology. All clinical investigations will be undertaken at the Charles Perkins Centre-RPAH clinic. Participants Individuals (n = 58) with stable coronary heart disease who have low attenuation plaque based on a CCTA within the past 3 months and have been randomized to an intensive lifestyle intervention program. Discussion This MIRTH micro-randomization trial will represent the single most detailed and integrated analysis of the effects of a comprehensive lifestyle intervention targeting multiple metabolic pathways, delivered via a customized mHealth App on smart devices, on time-based motivational messaging for patients with coronary heart disease. This study will also help inform future studies optimizing for just-in-time adaptive interventions. Trial Registration ACTRN12622000731796; pre-results Universal Trial Number (UTN) U1111-1269-8610

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.

Actomyosin networks, the cell’s major force production machineries, remodel cellular membranes during myriad dynamic processes(1,2) by assembling into various architectures with distinct force generation properties(3,4). While linear and branched actomyosin architectures are well characterized in cell-culture and cell-free systems(3), 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 exocytosis(5–7). 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 minifilaments(8–10) 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 exocytosis(7). Our imaging approach thus unveils a force-generating actomyosin lattice that regulates secretion in the exocrine organs of live animals.