Collective cell migration over long time scales reveals distinct phenotypes.

INTRODUCTION: Migratory phenotypes of metastasizing tumor cells include single and collective cell migration. While migration of tumor cells is generally less cooperative than that of normal epithelial cells, our understanding of precisely how they differ in long time behavior is incomplete. OBJECTIVES: We measure in a model system how cancer progression affects collective migration on long time scales, and determine how perturbation of cell-cell adhesions, specifically reduced E-cadherin expression, affects the collective migration phenotype. METHODS: Time lapse imaging of cellular sheets and particle image velocimetry (PIV) are used to quantitatively study the dynamics of cell motion over ten hours. Long time dynamics are measured via finite time Lyapunov exponents (FTLE) and changes in FTLE with time. RESULTS: We find that non-malignant MCF10A cells are distinguished from malignant MCF10CA1a cells by both their short time (minutes) and long time (hours) dynamics. In addition, short time dynamics distinguish non-malignant E-cadherin knockdown cells from the control, but long time dynamics and increasing spatial correlations remain unchanged. DISCUSSION: Epithelial sheet collective behavior includes long time dynamics that cannot be captured by metrics that assess cooperativity based on short time dynamics, such as instantaneous speed or directionality. The use of metrics incorporating migration data over hours instead of minutes allows us to more precisely describe how E-cadherin, a clinically relevant adhesion molecule, affects collective migration. We predict that the long time scale metrics described here will be more robust and predictive of malignant behavior than analysis of instantaneous velocity fields alone.

Arginase acts as an alternative pathway of L-arginine metabolism in experimental colon anastomosis.

L-Arginine is the substrate for the nitric oxide synthase (NOS) pathway that is essential for gastrointestinal wound healing. L-Arginine is also the substrate for the enzyme arginase which metabolizes L-arginine to ornithine and subsequently to proline and polyamines both known to interact in cell proliferation and collagen synthesis. Two distinct isoforms of arginase exist. The temporal expression of the L-arginine metabolism in experimental colon anastomosis was investigated. Male Lewis rats underwent laparotomy. A left-sided colotomy was performed and the colon reanastomosed using 6-0 prolene. Sham operation was performed in controls. On days 2, 5, 10, 14, and 28 after the surgery the anastomosis was excised. The tissue at the anastomosis (ANAST) as well as above and below the anastomosis (PDC) and from sham colon was harvested and analyzed for distinct arginase isoform I (AI) and arginase isoform II (AII) activity, protein and mRNA expression as well as immunohistochemistry. iNOS protein and mRNA expression were investigated in parallel. A mean of 3 to 4 separate rats were analyzed per time point. Statistical analysis was performed by student's t-test, significance was reached when P < 0.05. AI activity, protein, and mRNA expression were significantly upregulated at the anastomosis compared to sham controls and PDC colons at all time points. The maximum was achieved at days 10 to 14 after wounding, and decreased to baseline levels thereafter. Inflammatory cells stained positive for AI. AII protein was not detectable. However RT-PCR showed low baseline expression. iNOS expression was upregulated early but for a shorter time period after wounding and reverted quickly to undetectable levels. In anastomotic healing, AI upregulation suggests a prolonged metabolism of arginine via arginase to polyamines and proline to provide substrate for collagen synthesis and cell proliferation. The functional implication of this arginase pathway further needs to be elucidated.

Inhibition of tumor necrosis factor-alpha attenuates wound breaking strength in rats.

Exogenous administration of tumor necrosis factor-alpha has been shown to both enhance and attenuate cutaneous healing in a dose-dependent manner. We examined the effects of tumor necrosis factor inhibition in the healing wound by both systemic and local administration of tumor necrosis factor-binding protein. Male Balb/C mice underwent dorsal skin incision with subcutaneous implantation of 20 mg polyvinyl alcohol sponges (4 per animal). In Experiment I, one group (n = 20) received intraperitoneal injections of tumor necrosis factor-binding protein (3 mg/kg) at the time of wounding, while another group (n = 20) received saline. Four animals from each group were euthanized on days 1, 3, 5, 7, and 14 postwounding. In Experiment II, one group (n = 10) received an intraperitoneal injection of tumor necrosis factor-binding protein (3 mg/kg) at the time of wounding and every third day thereafter. Another group (n = 10) received an intraperitoneal injection of saline at the time of wounding and every third day thereafter. In Experiment III, one group received a single intraperitoneal injection of tumor necrosis factor-binding protein (3 mg/kg) at the time of wounding (n = 7), or on postwounding day 4 (n = 7), or day 7 (n = 7). Another group received saline injections at the time of wounding (n = 7), or on postwounding days 4 or 7 (n = 7, respectively). All animals in Experiments II and III were killed at postwounding day 14. Wound breaking strengths were assessed using a tensiometer. Wound fluid collected from the implanted sponges was assayed for tumor necrosis factor-alpha and tumor necrosis factor-binding protein levels using a biological assay and enzyme-linked immunosorbent assay, respectively. Collagen gene expression in sponge granulomata was assessed by Northern analysis. Collagen deposition in sponges was quantified by measuring hydroxyproline content. Wounds were significantly weaker in the animals that received repeated injections of tumor necrosis factor-binding protein with a mean wound breaking strength of 93.1 g vs. 186.6 g in controls (p < 0.05). Wound breaking strength in groups that received a single injection of tumor necrosis factor-binding protein on either day 0, 4, or 7 postwounding were no different than their respective controls. There was no difference in the mean hydroxyproline content of sponges between any of the tumor necrosis factor-binding protein groups and their respective controls. Northern analysis for collagen I and III expression also revealed no differences. These data indicate that continued systemic administration of tumor necrosis factor-binding protein resulted in significantly weaker wounds with no corresponding differences in wound collagen content, and collagen gene expression. This suggests that tumor necrosis factor-alpha inhibition throughout healing leads to a qualitatively impaired wound without a quantitative alteration in collagen deposition.