Tension directs cancer cell migration over fiber alignment through energy minimization.

Cell migration during many fundamental biological processes including metastasis requires cells to traverse tissue with heterogeneous mechanical cues that direct migration as well as determine force and energy requirements for motility. However, the influence of discrete structural and mechanical cues on migration remains challenging to determine as they are often coupled. Here, we decouple the pro-invasive cues of collagen fiber alignment and tension to study their individual impact on migration. When presented with both cues, cells preferentially travel in the axis of tension against fiber alignment. Computational and experimental data show applying tension perpendicular to alignment increases potential energy stored within collagen fibers, lowering requirements for cell-induced matrix deformation and energy usage during migration compared to motility in the direction of fiber alignment. Energy minimization directs migration trajectory, and tension can facilitate migration against fiber alignment. These findings provide a conceptual understanding of bioenergetics during migration through a fibrous matrix.

Quantitative assessment of cell contractility using polarized light microscopy.

Cell contractility regulates multiple cell behaviors which contribute to both normal and pathological processes. However, measuring cell contractility remains a technical challenge in complex biological samples. The current state of the art technologies employed to measure cell contractility have inherent limitations that greatly limit the experimental conditions under which they can be used. Here, we use quantitative polarization microscopy to extract information about cell contractility. We show that the optical retardance signal measured from the cell body is proportional to cell contractility in 2-dimensional and 3-dimensional platforms, and as such can be used as a straightforward, tractable methodology to assess cell contractility in a variety of systems. This label-free optical method provides a novel and flexible way to assess cellular forces of single cells and monolayers in several cell types, fixed or live, in addition to cells present in situ in mouse tumor tissue samples. This easily implementable and experimentally versatile method will significantly contribute to the cell mechanics field.

Homozygous KSR1 deletion attenuates morbidity but does not prevent tumor development in a mouse model of RAS-driven pancreatic cancer.

Given the frequency with which MAP kinase signaling is dysregulated in cancer, much effort has been focused on inhibiting RAS signaling for therapeutic benefit. KSR1, a pseudokinase that interacts with RAF, is a potential target; it was originally cloned in screens for suppressors of constitutively active RAS, and its deletion prevents RAS-mediated transformation of mouse embryonic fibroblasts. In this work, we used a genetically engineered mouse model of pancreatic cancer to assess whether KSR1 deletion would influence tumor development in the setting of oncogenic RAS. We found that Ksr1-/- mice on this background had a modest but significant improvement in all-cause morbidity compared to Ksr1+/+ and Ksr1+/- cohorts. Ksr1-/- mice, however, still developed tumors, and precursor pancreatic intraepithelial neoplastic (PanIN) lesions were detected within a similar timeframe compared to Ksr1+/+ mice. No significant differences in pERK expression or in proliferation were noted. RNA sequencing also did not reveal any unique genetic signature in Ksr1-/- tumors. Further studies will be needed to determine whether and in what settings KSR inhibition may be clinically useful.

Clinical doses of radiation reduce collagen matrix stiffness.

Cells receive mechanical cues from their extracellular matrix (ECM), which direct migration, differentiation, apoptosis, and in some cases, the transition to a cancerous phenotype. As a result, there has been significant research to develop methods to tune the mechanical properties of the ECM and understand cell-ECM dynamics more deeply. Here, we show that ionizing radiation can reduce the stiffness of an ex vivo tumor and an in vitro collagen matrix. When non-irradiated cancer cells were seeded in the irradiated matrix, adhesion, spreading, and migration were reduced. These data have ramifications for both in vitro and in vivo systems. In vitro, these data suggest that irradiation may be a method that could be used to create matrices with tailored mechanical properties. In vivo, these suggest that therapeutic doses of radiation may alter tissue mechanics directly.

Regulation of ATP utilization during metastatic cell migration by collagen architecture.

Cell migration in a three-dimensional matrix requires that cells either remodel the surrounding matrix fibers and/or squeeze between the fibers to move. Matrix degradation, matrix remodeling, and changes in cell shape each require cells to expend energy. While significant research has been performed to understand the cellular and molecular mechanisms guiding metastatic migration, less is known about cellular energy regulation and utilization during three-dimensional cancer cell migration. Here we introduce the use of the genetically encoded fluorescent biomarkers, PercevalHR and pHRed, to quantitatively assess ATP, ADP, and pH levels in MDA-MB-231 metastatic cancer cells as a function of the local collagen microenvironment. We find that the use of the probe is an effective tool for exploring the thermodynamics of cancer cell migration and invasion. Specifically, we find that the ATP:ADP ratio increases in cells in denser matrices, where migration is impaired, and it decreases in cells in aligned collagen matrices, where migration is facilitated. When migration is pharmacologically inhibited, the ATP:ADP ratio decreases. Together, our data indicate that matrix architecture alters cellular energetics and that intracellular ATP:ADP ratio is related to the ability of cancer cells to effectively migrate.

Manipulation of in vitro collagen matrix architecture for scaffolds of improved physiological relevance.

Type I collagen is a versatile biomaterial that is widely used in medical applications due to its weak antigenicity, robust biocompatibility, and its ability to be modified for a wide array of applications. As such, collagen has become a major component of many tissue engineering scaffolds, drug delivery platforms, and substrates for in vitro cell culture. In these applications, collagen constructs are fabricated to recapitulate a diverse set of conditions. Collagen fibrils can be aligned during or post-fabrication, cross-linked via numerous techniques, polymerized to create various fibril sizes and densities, and copolymerized into a wide array of composite scaffolds. Here, we review approaches that have been used to tune collagen to better recapitulate physiological environments for use in tissue engineering applications and studies of basic cell behavior. We discuss techniques to control fibril alignment, methods for cross-linking collagen constructs to modulate stiffness, and composite collagen constructs to better mimic physiological extracellular matrix.

Stereotactic radiosurgical treatment of trigeminal neuralgia in a community hospital.

Access to state-of-the-art medical therapy can be difficult for people who live in rural areas far from a major medical center. We report here a case in which trigeminal neuralgia was successfully treated with stereotactic radiosurgery at Southeast Missouri Hospital, a community hospital serving a mostly rural population.

From the combat medic to the forward surgical team: the Madigan model for improving trauma readiness of brigade combat teams fighting the Global War on Terror.

BACKGROUND: Medics assigned to combat units have a notable paucity of trauma experience. Our goal was to provide intense trauma refresher training for the conventional combat medic to better prepare them for combat casualty care in the War on Terror. MATERIALS AND METHODS: Our Tactical Combat Casualty Care Course (TC3) consisted of the following five phases: (1) One and one-half-day didactic session; (2) Half-day simulation portion with interactive human surgical simulators for anatomical correlation of procedures and team building; (3) Half-day of case presentations and triage scenarios from Iraq/Afghanistan and associated skills stations; (4) Half-day live tissue lab where procedures were performed on live anesthetized animals in a controlled environment; and (5) One-day field phase where live anesthetized animals and surgical simulators were combined in a real-time, field-training event to simulate realistic combat injuries, evacuation problems, and mass casualty scenarios. Data collection consisted of surveys, pre- and posttests, and after-action comments. RESULTS: A total of 1317 personnel participated in TC3 from October 2003 through May 2005. Over the overlapping study period from December 2004 to April 2005, 327 soldiers participated in the formal five-phase course. Three hundred four (94%) students were combat medics who were preparing for combat operations in Iraq or Afghanistan. Of those completing the training, 97% indicated their confidence and ability to treat combat casualties were markedly improved. Moreover, of those 140 medics who took the course and deployed to Iraq for 1 year, 99% indicated that the principles taught in the TC3 course helped with battlefield management of injured casualties during their deployment. CONCLUSION: The hybrid training model is an effective method for training medical personnel to deal with modern battle injuries. This course increases the knowledge and confidence of combat medics deploying and fighting the Global War on Terrorism.

Establishing learning curves for surgical residents using Cumulative Summation (CUSUM) Analysis.

BACKGROUND: The assessment of technical proficiency is of paramount importance in the training of surgical residents. The fact that technical proficiency is underrepresented in the context of the ACGME outcomes project is evidenced in that proficiency skills comprise less than 5% of all assessments that evaluate residents. In this study, we use Cumulative Summation Analysis (CUSUM) as a visual objective analytic tool to determine performance accuracy and establish learning curves for PGY-1s in surgery. METHODS: From April 2001 to May 2002, 11 surgical residents completed a 1-month anesthesia rotation. Each resident was asked to complete a preoperative airway assessment followed by endotracheal intubation with induction of anesthesia. Airway assessment was performed independently by a resident and a licensed anesthesiologist or certified anesthetist with the modified Mallampati Score. Data were sequentially collected and plotted for summated successes and failures. RESULTS: The average intern required approximately 19 intubation attempts to complete the learning curve experience. There was no learning curve for airway assessment. CONCLUSIONS: The CUSUM analysis is an effective objective tool to define learning curves for technical skills. Vital information is provided for surgical programs that place residents in positions to manage airways, and limitless potential for defining the learning curves for technical skills is provided.

Is PA-PWRmax truly a preload-independent index of myocardial contractility in anesthetized humans?

This study tested the hypothesis that the preload-adjusted maximal power index (PA-PWRmax) is a load-independent index of human myocardial contractility. Based on the ventricular pressure-volume relationship and derived from stroke work, the index is the product of instantaneous ventricular pressure and volume changes, divided by a correction term of end-diastolic volume (EDV2) or end-diastolic area (EDA3/2) to adjust for preload effects. Echocardiographic measures of instantaneous ventricular area change may be used to obtain PA-PWRmax noninvasively. We prospectively evaluated 28 human subjects undergoing cardiac evacuation before cardiopulmonary bypass procedures. Continuous peripheral arterial pressure, pulmonary arterial pressure, and echocardiographic views of the left ventricle in the transgastric short-axis view were recorded. Simultaneously gated instantaneous fractional shortening (FS) and PA-PWRmax indices were calculated, with FS = (EDA - ESA)/EDA and PA-PWRmax = [MAP (EDA - ESA)]/ EDA3/2, where ESA = end-systolic area and MAP = instantaneous mean arterial pressure. FS decreased uniformly with cardiac evacuation and decreasing pulmonary artery diastolic pressure (t = -5.4; 95% confidence interval, -10 to -0.046; p < 0.001), as did PA-PWRmax (t = -5.8; 95% confidence interval, -2.25 to -1.08; p < 0.001). FS and PA-PWRmax showed a strong downward correlation (r = 0.81). Unlike previous studies of autonomically denervated animals, our study did not find PA-PWRmax to be preload independent, perhaps because of the instantaneous homeostatic mechanisms of the human autonomic nervous system linking contractility to loading conditions.