Migratory Metrics of Wound Healing: A Quantification Approach for in vitro Scratch Assays.

Metastatic dissemination generates an aggressive disease facilitated by enhanced migratory and invasive properties. Experimental approaches employ several in vitro and in vivo assays toward quantification of these functionalities. In vitro assessments of cell motility often employ endpoint assays that rely on the global efficacy of wound closure and thwart quantification of migratory phenotypes observed during metastatic dissemination. Recent studies highlight the distinct signatures associated with individual vs. collective cell migration and necessitate the incorporation of these modalities into routine analyses. Advances in live cell imaging that permit real-time visualization of pathophysiological processes can be employed toward elucidating phenotypic plasticity associated with cell migration to overcome caveats inherent to end-point assays. Herein, we corroborate live cell imaging with the in vitro scratch assay toward quantification of migratory modalities in transformed cells. Our protocol describes a step-by-step approach for live cell setup of the scratch assay, and details analyses employed toward definition of three quantitative metrics viz., displacement, velocity and number of nearest neighbors. The current protocol (from scratch induction to data acquisition) is implemented for ~30 h and provides global/single-cell resolution of migratory phenotypes as opposed to the endpoint assays. Routine application of this protocol in cancer biology can aid the design of therapeutic regimes targeting specific migratory modalities and significantly contribute to the dissection of associated molecular networks

Uncoupling Traditional Functionalities of Metastasis: The Parting of Ways with Real-Time Assays.

The experimental evaluation of metastasis overly focuses on the gain of migratory and invasive properties, while disregarding the contributions of cellular plasticity, extra-cellular matrix heterogeneity, niche interactions, and tissue architecture. Traditional cell-based assays often restrict the inclusion of these processes and warrant the implementation of approaches that provide an enhanced spatiotemporal resolution of the metastatic cascade. Time lapse imaging represents such an underutilized approach in cancer biology, especially in the context of disease progression. The inclusion of time lapse microscopy and microfluidic devices in routine assays has recently discerned several nuances of the metastatic cascade. Our review emphasizes that a complete comprehension of metastasis in view of evolving ideologies necessitates (i) the use of appropriate, context-specific assays and understanding their inherent limitations; (ii) cautious derivation of inferences to avoid erroneous/overestimated clinical extrapolations; (iii) corroboration between multiple assay outputs to gauge metastatic potential; and (iv) the development of protocols with improved in situ implications. We further believe that the adoption of improved quantitative approaches in these assays can generate predictive algorithms that may expedite therapeutic strategies targeting metastasis via the development of disease relevant model systems. Such approaches could potentiate the restructuring of the cancer metastasis paradigm through an emphasis on the development of next-generation real-time assays

Functional balance between Tcf21-Slug defines cellular plasticity and migratory modalities in high grade serous ovarian cancer cell lines.

Cellular plasticity and transitional phenotypes add to complexities of cancer metastasis that can be initiated by single cell epithelial to mesenchymal transition (EMT) or cooperative cell migration (CCM). Our study identifies novel regulatory cross-talks between Tcf21 and Slug in mediating phenotypic and migration plasticity in high-grade serous ovarian adenocarcinoma (HGSC). Differential expression and sub-cellular localization associate Tcf21, Slug with epithelial, mesenchymal phenotypes respectively; however gene manipulation approaches identify their association with additional intermediate phenotypic states, implying the existence of a multi-step epithelial-mesenchymal transition program. Live imaging further associated distinct migratory modalities with the Tcf21/Slug status of cell systems and discerned proliferative/passive CCM, active CCM and EMT modes of migration. Tcf21-Slug balance identified across a phenotypic spectrum in HGSC cell lines, associated with micro-environment induced transitions and the emergence of an epithelial phenotype following drug exposure. Phenotypic transitions and associated functionalities following drug exposure were affirmed to ensue from occupancy of Slug promoter E-box sequences by Tcf21. Our study effectively provides a framework for understanding the relevance of ovarian cancer plasticity as a function of two transcription factors

A monoclonal antibody against annexin A2 targets stem and progenitor cell fractions in tumors.

The involvement of cancer stem cells (CSCs) in driving tumor dormancy and drug resistance is well established. Most therapeutic regimens however are ineffective in targeting these regenerative populations. We report the development and evaluation of a monoclonal antibody, mAb150, which targets the metastasis associated antigen, Annexin A2 (AnxA2) through recognition of a N-terminal epitope. Treatment with mAb150 potentiated re-entry of CSCs into the cell cycle that perturbed tumor dormancy and facilitated targeting of CSCs as was validated by in vitro and in vivo assays. Epigenetic potentiation further improved mAb150 efficacy in achieving total tumor regression by targeting regenerative populations to achieve tumor regression, specifically in high-grade serous ovarian adenocarcinoma

Electronic cigarette inhalation alters innate immunity and airway cytokines while increasing the virulence of colonizing bacteria.

UnlabelledElectronic (e)-cigarette use is rapidly rising, with 20 % of Americans ages 25-44 now using these drug delivery devices. E-cigarette users expose their airways, cells of host defense, and colonizing bacteria to e-cigarette vapor (EV). Here, we report that exposure of human epithelial cells at the air-liquid interface to fresh EV (vaped from an e-cigarette device) resulted in dose-dependent cell death. After exposure to EV, cells of host defense-epithelial cells, alveolar macrophages, and neutrophils-had reduced antimicrobial activity against Staphylococcus aureus (SA). Mouse inhalation of EV for 1 h daily for 4 weeks led to alterations in inflammatory markers within the airways and elevation of an acute phase reactant in serum. Upon exposure to e-cigarette vapor extract (EVE), airway colonizer SA had increased biofilm formation, adherence and invasion of epithelial cells, resistance to human antimicrobial peptide LL-37, and up-regulation of virulence genes. EVE-exposed SA were more virulent in a mouse model of pneumonia. These data suggest that e-cigarettes may be toxic to airway cells, suppress host defenses, and promote inflammation over time, while also promoting virulence of colonizing bacteria.Key messageAcute exposure to e-cigarette vapor (EV) is cytotoxic to airway cells in vitro. Acute exposure to EV decreases macrophage and neutrophil antimicrobial function. Inhalation of EV alters immunomodulating cytokines in the airways of mice. Inhalation of EV leads to increased markers of inflammation in BAL and serum. Staphylococcus aureus become more virulent when exposed to EV

A Simple Mathematical Model Based on the Cancer Stem Cell Hypothesis Suggests Kinetic Commonalities in Solid Tumor Growth

Background: The Cancer Stem Cell (CSC) hypothesis has gained credibility within the cancer research community. According to this hypothesis, a small subpopulation of cells within cancerous tissues exhibits stem-cell-like characteristics and is responsible for the maintenance and proliferation of cancer. Methodologies/Principal Findings: We present a simple compartmental pseudo-chemical mathematical model for tumor growth, based on the CSC hypothesis, and derived using a ‘‘chemical reaction’ ’ approach. We defined three cell subpopulations: CSCs, transit progenitor cells, and differentiated cells. Each event related to cell division, differentiation, or death is then modeled as a chemical reaction. The resulting set of ordinary differential equations was numerically integrated to describe the time evolution of each cell subpopulation and the overall tumor growth. The parameter space was explored to identify combinations of parameter values that produce biologically feasible and consistent scenarios. Conclusions/Significance: Certain kinetic relationships apparently must be satisfied to sustain solid tumor growth and to maintain an approximate constant fraction of CSCs in the tumor lower than 0.01 (as experimentally observed): (a) the rate of symmetrical and asymmetrical CSC renewal must be in the same order of magnitude; (b) the intrinsic rate of renewal and differentiation of progenitor cells must be half an order of magnitude higher than the corresponding intrinsic rates for cancer stem cells; (c) the rates of apoptosis of the CSC, transit amplifying progenitor (P) cells, and terminally differentiate

Immunometabolism at the crossroads of obesity and cancer-a Keystone Symposia report.

peer reviewedImmunometabolism considers the relationship between metabolism and immunity. Typically, researchers focus on either the metabolic pathways within immune cells that affect their function or the impact of immune cells on systemic metabolism. A more holistic approach that considers both these viewpoints is needed. On September 5-8, 2022, experts in the field of immunometabolism met for the Keystone symposium "Immunometabolism at the Crossroads of Obesity and Cancer" to present recent research across the field of immunometabolism, with the setting of obesity and cancer as an ideal example of the complex interplay between metabolism, immunity, and cancer. Speakers highlighted new insights on the metabolic links between tumor cells and immune cells, with a focus on leveraging unique metabolic vulnerabilities of different cell types in the tumor microenvironment as therapeutic targets and demonstrated the effects of diet, the microbiome, and obesity on immune system function and cancer pathogenesis and therapy. Finally, speakers presented new technologies to interrogate the immune system and uncover novel metabolic pathways important for immunity

Fat-Resident Regulatory T Cells Drive Age-Associated Insulin Resistance

Age-associated insulin resistance (IR) and obesity-associated IR are two physiologically distinct forms of adult onset diabetes. While macrophage-driven inflammation is a core driver of obesity-associated IR1-6, the underlying mechanisms of the obesity-independent yet highly prevalent age-associated IR7 are largely unexplored. Comparative adipo-immune profiling (AIP) reveals that fat-resident regulatory T cells, termed fTregs, progressively accumulate in adipose tissue as a function of age, but not obesity. Supporting the existence of two distinct mechanisms underlying age-associated versus obesity-associated IR, mice deficient in fTregs are protected against age-associated IR, yet remain susceptible to obesity-associated IR and metabolic disease. In contrast, selective depletion of fTregs via anti-ST2 antibody treatment increases adipose tissue insulin sensitivity. These findings establish that distinct immune cell populations within adipose tissue underlie aging- and obesity-associated IR and implicate fTregs as adipo-immune drivers and potential therapeutic targets in the treatment of age-associated IR