Interbacterial competition is prevalent in host-associated microbiota, where it can shape community structure and function, impacting host health in both positive and negative ways. However, the factors that permit bacteria to discriminate among their various neighbors for targeted elimination of competitors remain elusive. We identified a specificity factor in Vibrio species that is used to target specific competitors for elimination. Here, we describe this specificity factor, which is associated with the broadly-distributed type VI secretion system (T6SS), by studying symbiotic Vibrio fischeri, which use the T6SS to compete for colonization sites in their squid host. We demonstrate that a large lipoprotein (TasL) allows V. fischeri cells to restrict T6SS-dependent killing to certain genotypes by selectively integrating competitor cells into aggregates while excluding other cell types. TasL is also required for T6SS-dependent competition within juvenile squid, indicating the adhesion factor is active in the host. Because TasL homologs are found in other host-associated bacterial species, this newly-described specificity factor has the potential to impact microbiome structure within diverse hosts.

Laser speckle contrast imaging (LSCI) has emerged as a promising tool for intraoperative cerebral blood flow (CBF) monitoring because it produces real-time full-field blood flow maps non-invasively and label-free. In this study, we compare LSCI with indocyanine green angiography (ICGA) to assess CBF during aneurysm clipping surgery in humans. LSCI hardware was attached to the surgical microscope prior to the start of each surgery and did not interfere with the sterile draping of the microscope or normal operation of the microscope. LSCI and ICGA were performed simultaneously to visualize CBF in n=4 aneurysm clipping cases, and LSCI was performed throughout each surgery when the microscope was positioned over the patient. To more easily visualize CBF in real-time, LSCI images were overlaid on the built-in microscope white light camera images and displayed to the neurosurgeon in real-time. Blood flow changes before, during, and after an aneurysm clipping were visualized with LSCI and later verified with ICGA. LSCI was performed continuously throughout the aneurysm clipping process, providing the surgeon with immediate actionable information on the success of the clipping. The results demonstrate that LSCI and ICGA provide different, yet complementary information about vessel perfusion.

A fundamental limitation in the development of new therapies to prevent metastatic cancer is a lack of in vitro systems that can accurately recapitulate the steps of cancer cell metastasis. Currently, most assays for examining the steps of metastasis fail to incorporate the biophysical forces experienced by tumor cells due to blood flow, or are low throughput and thereby not amenable to drug screens or high throughput experimentation. We have developed a novel high throughput mesofluidic platform for assaying cell adhesion under flow in a 96-well format. This device functions like a cone and plate viscometer in each well by inducing shear stress on cells cultured in a standard 96-well plate. We validated the fluid flow and alignment of the device and studied the adhesion of cultured leukocytic monocytes (THP-1 cells) and multiple cancer cell lines (MDA-MB-231 and MCF-7 breast cancer cell lines) to purified extracellular matrix molecules (ECM), endothelial cells and immobilized platelets. Assays were carried out under flow (0.5 dynes/cm2 shear stress) and static conditions. Our studies show that adhesion assays performed under flow yield markedly different results from static adhesion assays. Treatment of breast cancer cells with a small library of integrin inhibitors demonstrated that these compounds had minimal effect on cancer cell adhesion to endothelial cells or immobilized platelets under static conditions, whereas under shear conditions many of these compounds significantly reduced adhesion of cancer cells. As well, this experiment elucidated integrins important for breast cancer adhesion to endothelial cells and platelets. A static adhesion assay of breast cancer cells to various types of ECM showed greater adhesion of the less aggressive MCF-7 cell line in comparison to the more aggressive MDA-MB-231 cell line. In contrast, flow incorporating assays showed increased adhesion of the more aggressive MDA-MB-231 breast cancer cell line. Specifically, the shear assay saw a significant increase in adhesion for multiple ECM as well as an increase in the strength of adhesion to laminin. Finally, we performed a high throughput screening experiment using a kinase inhibitor library of 80 compounds and found that the shear based assay yielded notably different results from a similar screen under static conditions for breast cancer cell adhesion to endothelial cells, immune cell adhesion to endothelial cells and breast cancer cell adhesion to platelets. This shear experiment yielded seven "hits", many of which match targets of drugs in clinical trials. In conclusion, our studies show that adhesion assays performed under flow yield markedly different results from static adhesion assays, and are better at identifying both aggressive cancer cells lines and known pathways for circulating cancer and immune cell adhesion. Thus, this high-throughput screening platform may enable the development of novel compounds to inhibit cancer metastasis and facilitate the study of the systems level behavior of cancer-endothelium adhesion. Citation Format: Spencer A, Spruell C, Nandi S, Le V, Crexiell M, Dunn AK, Baker AB. Mesofluidic platform for high throughput screening of inhibitors of metastasis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-18.