Clara K. Chan

miR-509-3p is clinically significant and strongly attenuates cellular migration and multi-cellular spheroids in ovarian cancer

Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E–3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E–3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3ps most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers.

Using optical tweezers to study mechanical properties of collagen

The mechanical response of biological molecules at the microscopic level contributes significantly to their function. Optical tweezers are instruments that enable scientists to study mechanical properties at microscopic levels. They are based on a highly focused laser beam that creates a trap for microscopic objects such as dielectric spheres, viruses, bacteria, living cells and organelles, and then manipulates them by applying forces in the picoNewton range (a range that is biologically relevant). In this work, mechanical properties of single collagen molecules are studied using optical tweezers. We discuss the challenges of stretching single collagen proteins, whose length is much less than the size of the microspheres used as manipulation handles, and show how instrumental design and biochemistry can be used to overcome these challenges.

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~10(2) cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

Development and characterization of a eukaryotic expression system for human type II procollagen

BackgroundTriple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required.ResultsHere, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils.ConclusionsUsing a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.

Tumour-suppressor microRNAs regulate ovarian cancer cell physical properties and invasive behaviour

The activities of pathways that regulate malignant transformation can be influenced by microRNAs (miRs). Recently, we showed that increased expression of five tumour-suppressor miRs, miR-508-3p, miR-508-5p, miR-509-3p, miR-509-5p and miR-130b-3p, correlate with improved clinical outcomes in human ovarian cancer patients, and that miR-509-3p attenuates invasion of ovarian cancer cell lines. Here, we investigate the mechanism underlying this reduced invasive potential by assessing the impact of these five miRs on the physical properties of cells. Human ovarian cancer cells (HEYA8, OVCAR8) that are transfected with miR mimics representing these five miRs exhibit decreased invasion through collagen matrices, increased cell size and reduced deformability as measured by microfiltration and microfluidic assays. To understand the molecular basis of altered invasion and deformability induced by these miRs, we use predicted and validated mRNA targets that encode structural and signalling proteins that regulate cell mechanical properties. Combined with analysis of gene transcripts by real-time PCR and image analysis of F-actin in single cells, our results suggest that these tumour-suppressor miRs may alter cell physical properties by regulating the actin cytoskeleton. Our findings provide biophysical insights into how tumour-suppressor miRs can regulate the invasive behaviour of ovarian cancer cells, and identify potential therapeutic targets that may be implicated in ovarian cancer progression.

A large displacement, high frequency, underwater microelectromechanical systems actuator

Here, we demonstrate an in situ electrostatic actuator that can operate underwater across a wide range of displacements and frequencies, achieving a displacement of approximately 10 μm at 500 Hz and 1 μm at 5 kHz; this performance surpasses that of existing underwater physical actuators. To attain these large displacements at such high speeds, we optimized critical design parameters using a computationally efficient description of the physics of low quality (Q) factor underwater electrostatic actuators. Our theoretical model accurately predicts actuator motion profiles as well as limits of bandwidth and displacement.

Irrigation after Laparoscopic Power Morcellation and the Dispersal of Leiomyoma Cells: a Pilot Study

STUDY OBJECTIVE To evaluate if copious irrigation and suctioning after electromechanical power morcellation will reduce myoma cell dissemination and if there is a difference between sterile water and normal saline. DESIGN Prospective single-center cohort pilot study (Canadian Task Force classification II-2). SETTING Academic tertiary referral center. PATIENTS Sixteen women undergoing laparoscopic myomectomy with 1 surgeon between January 1, 2017 and August 31, 2017. INTERVENTIONS Peritoneal washings were collected 3 specific times during surgery: after dissection of myoma(s) and hysterotomy repair but before morcellation, after morcellation, and after irrigation with 3 L normal saline or sterile water. The primary outcome was the detection of benign spindle cells (BSCs) in peritoneal washings. MEASUREMENTS AND MAIN RESULTS Sixteen patients were enrolled in the study. Eight were randomized to the normal saline group and 8 to the sterile water group. In the normal saline group BSCs were detected in 3 of 8 patients (37.5%) after closure of the hysterotomy but before morcellation, in 3 of 8 (37.5%) after morcellation, and in 0 of 8 (0%) after irrigation and suctioning of the peritoneal cavity with 3 L normal saline. In the sterile water group BSCs were detected in 3 of 8 patients (37.5%) after closure of the hysterotomy but before morcellation, 2 of 8 (25%) after morcellation, and in 0 of 8 (0%) after irrigation and suctioning with 3 L sterile water. Thus, no differences were found between the normal saline and sterile water groups. CONCLUSION In this pilot study myoma cells were disseminated before electromechanical morcellation. Irrigation and suctioning with 3 L normal saline or sterile water after morcellation may reduce myoma cell dissemination.

Additional file 1: Figure S1. of Development and characterization of a eukaryotic expression system for human type II procollagen

Results of mass spectrometric analysis on the recombinant human type II procollagen, demonstrating protein sequence coverage of COL2A1 and expected posttranslational modifications. (PDF 70 kb)