Substratum stiffness tunes membrane voltage in mammary epithelial cells.

Membrane voltage (Vm) plays a critical role in the regulation of several cellular behaviors, including proliferation, apoptosis and phenotypic plasticity. Many of these behaviors are affected by the stiffness of the underlying extracellular matrix, but the connections between Vm and the mechanical properties of the microenvironment are unclear. Here, we investigated the relationship between matrix stiffness and Vm by culturing mammary epithelial cells on synthetic substrata, the stiffnesses of which mimicked those of the normal mammary gland and breast tumors. Although proliferation is associated with depolarization, we surprisingly observed that cells are hyperpolarized when cultured on stiff substrata, a microenvironmental condition that enhances proliferation. Accordingly, we found that Vm becomes depolarized as stiffness decreases, in a manner dependent on intracellular Ca2+. Furthermore, inhibiting Ca2+-gated Cl- currents attenuates the effects of substratum stiffness on Vm. Specifically, we uncovered a role for cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of Vm by substratum stiffness. Taken together, these results suggest a novel role for CFTR and membrane voltage in the response of mammary epithelial cells to their mechanical microenvironment.

Neutrophil-To-Lymphocyte Ratio as a Predictive Tool for Post-Operative Outcomes in Patients Undergoing Open Lower Extremity Revascularization Procedures.

BACKGROUND: Elevated neutrophil-to-lymphocyte ratio (NLR), a marker of systemic inflammation, has been shown to correlate with worse outcomes in patients undergoing vascular surgery. Limited data exists on the association of NLR and outcomes in patients undergoing lower extremity vascular surgery. We sought to investigate whether preoperative NLR correlates with outcomes in patients undergoing open lower extremity revascularization procedures. METHODS: We conducted a retrospective analysis of a prospectively maintained database of patients who underwent open lower extremity revascularization procedures from January 2011 to January 2017 (N = 535). Preoperative NLR was calculated within 6 months of surgery. Primary outcomes were major adverse limb event (MALE) or death. The maximally-ranked statistic method was used to determine the NLR cut-off point. Kaplan-Meier analyses of death and MALE and NLR were used to compare the groups by NLR cut-off point. We conducted a multivariate analysis of the association between NLR and mortality using Cox proportional hazard models, including confounding variables such as age, smoking status, and diabetes. P-values <0.05 were considered statistically significant. RESULTS: Two hundred and fifty four patients undergoing surgery from January 2011 to January 2013 were analyzed. The median NLR was 3.6 interquartile range [IQR 2.5-6.7]. The analysis showed a negative correlation between elevated NLR and mortality (P < 0.001), but not MALE (P = 0.8). Controlling for multiple comorbidities including gender, age, smoking, body mass index (BMI), diabetes, hyperlipidemia, hypertension, and infection, the NLR cut-off point was a significant independent predictor of mortality (P < 0.0001), but not MALE (P = 0.551). Elevated NLR was also correlated with statistically and clinically significant longer hospital stays (6.5 [IQR 3.0-12.8] days vs. 4.0 [IQR 2.0-8.0] days, P = 0.027). CONCLUSIONS: This study suggests that NLR is an independent predictor of mortality and hospital length of stay in patients undergoing open lower extremity revascularizations. Going forward, we plan to expand this study to include more patients and to compare NLR to other risk assessment tools.

Substratum stiffness signals through integrin-linked kinase and ß1-integrin to regulate midbody proteins and abscission during EMT.

Abscission is the final stage of cytokinesis during which the parent cell physically separates to yield two identical daughters. Failure of abscission results in multinucleation (MNC), a sign of genomic instability and a precursor to aneuploidy, enabling characteristics of neoplastic progression. Induction of epithelial-mesenchymal transition (EMT) causes MNC in mammary epithelial cells cultured on stiff microenvironments that have mechanical properties similar to those found in breast tumors, but not on soft microenvironments reminiscent of the normal mammary gland. Here we report that on stiff microenvironments, EMT signaling through Snail up-regulates the midbody-associated proteins septin-6, Mklp1, and anillin, leading to abscission failure and MNC. To uncover the mechanism by which stiff microenvironments promote MNC in cells undergoing EMT, we investigated the role of cell-matrix adhesion through ß1-integrin and integrin-linked kinase (ILK). We found that ILK expression, but not kinase activity, is required for EMT-associated MNC in cells on stiff microenvironments. Conversely, increasing focal adhesions by expressing an autoclustering mutant of ß1-integrin promotes MNC in cells on soft microenvironments. Our data suggest that signaling through focal adhesions causes failure of cytokinesis in cells actively undergoing EMT. These results highlight the importance of tissue mechanics and adhesion in regulating the cellular response to EMT inducers.

Matrix degradation and cell proliferation are coupled to promote invasion and escape from an engineered human breast microtumor.

Metastasis, the leading cause of mortality in cancer patients, depends upon the ability of cancer cells to invade into the extracellular matrix that surrounds the primary tumor and to escape into the vasculature. To investigate the features of the microenvironment that regulate invasion and escape, we generated solid microtumors of MDA-MB-231 human breast carcinoma cells within gels of type I collagen. The microtumors were formed at defined distances adjacent to an empty cavity, which served as an artificial vessel into which the constituent tumor cells could escape. To define the relative contributions of matrix degradation and cell proliferation on invasion and escape, we used pharmacological approaches to block the activity of matrix metalloproteinases (MMPs) or to arrest the cell cycle. We found that blocking MMP activity prevents both invasion and escape of the breast cancer cells. Surprisingly, blocking proliferation increases the rate of invasion but has no effect on that of escape. We found that arresting the cell cycle increases the expression of MMPs, consistent with the increased rate of invasion. To gain additional insight into the role of cell proliferation in the invasion process, we generated microtumors from cells that express the fluorescent ubiquitination-based cell cycle indicator. We found that the cells that initiate invasions are preferentially quiescent, whereas cell proliferation is associated with the extension of invasions. These data suggest that matrix degradation and cell proliferation are coupled during the invasion and escape of human breast cancer cells and highlight the critical role of matrix proteolysis in governing tumor phenotype.