Multiscale Estimation of Cell Kinetics

We introduce a methodology based on the Luria–Delbrück fluctuation model for estimating the cell kinetics of clonally expanding populations. In particular, this approach allows estimation of the net cell proliferation rate, the extinction coefficient and the initial (viable) population size. We present a systematic approach based on spatial partitioning, which captures the local fluctuations of the number and sizes of individual clones. However, partitioning introduces measurement error by inflating the number of clones, which is dependent on time and the degree of cell migration. We perform various in silico experiments to explore the properties of the estimators and we show that there exists a direct relationship between precision and observation time. We also explore the trade-off between the measurement error and the estimation accuracy. By exploring different scales of cellular fluctuations, from the entire population down to those of individual clones, we show that this methodology is useful for inferring important parameters in neoplastic progression

Warburg and Crabtree Effects in Premalignant Barrett's Esophagus Cell Lines with Active Mitochondria

<div><p>Background</p><p>Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in the premalignant condition Barrett's esophagus (BE) due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis.</p> <p>Methodology/Principal Findings</p><p>We compared rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with BE (CP-A, CP-B, CP-C and CP-D) in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from patients with more advanced genetically unstable BE have up to two-fold higher glycolysis compared to a cell line derived from a patient with early genetically stable BE; however, all cell lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D) had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose via the Crabtree effect, and demonstrate a greater range of modulation of oxygen consumption.</p> <p>Conclusions/Significance</p><p>Our findings suggest that cells from premalignant Barrett's esophagus tissue may adapt to an ever-changing selective microenvironment through changes in energy metabolic pathways typically associated with cancer cells.</p> </div

Barrett's esophagus progression to esophageal adenocarcinoma involves an intermediate metabolic stage with increased glycolysis and functional mitochondria.

<p>Early-stage BE cells (e.g. CP-A), rely mainly on mitochondrial oxidative phosphorylation for energy needs prior to the glycolytic increase which occurs in late-stage BE cells (CP-B, CP-C and CP-D), which demonstrate elevated ECAR (all) and the Crabtree effect (CP-C and CP-D). Finally in esophageal adenocarcinoma (OE-33) mitochondrial uncoupling occurs with increased OCR and glycolysis.</p

CP-C and CP-D display a lower ECAR increase after mitochondrial uncoupling than other cell lines.

<p>Following addition of 50 µM 2,4-DNP, total (a) ECAR and (b) OCR were measured on the Seahorse XF24 analyzer for each cell line and changes versus untreated baseline are plotted. Error bars represent standard-deviation of means between experiments (N = 2–4). Each experiment consisted of 3–4 replicate wells per cell line with four serial measures performed on each well. p-values (Tukey-Kramer test) of statistically significant differences from CP-A are shown.</p

CP-D displays a greater OCR and ECAR response to glycolytic inhibition than other cell lines.

<p>Following addition of 50 mM 2-DG, total (a) ECAR and (b) OCR were measured on the Seahorse XF24 analyzer for each cell line and changes versus untreated baseline are plotted. Error bars represent standard-deviation of means between experiments (N = 2–4). Each experiment consisted of 3–4 replicate wells per cell line with four serial measures performed on each well. p-values (Tukey-Kramer test) of statistically significant differences from CP-A are shown.</p

CP-C and CP-D demonstrate a stronger Crabtree effect response than other cell lines.

<p>Following the increase of glucose concentration in media from 0 mM to 5 mM, total (a) ECAR and (b) OCR were measured on the Seahorse XF24 analyzer for each cell line and changes versus untreated baseline are plotted. Error bars represent standard-deviation of means between experiments (N = 2–4). Each experiment consisted of 3–4 replicate wells per cell line with four serial measures performed on each well. p-values (Tukey-Kramer test) indicating significant differences from CP-A are shown.</p