Fate Of Individual Cells Research Articles

Optical Control of Cancer Initiation in Zebrafish

Although cancer initiation and evolution have been extensively studied, they are not, as of yet, fully understood. Several models have attempted to answer how cancer arises from individual transformed cells. However, current probes of cancer development are restricted to the collective properties of many thousands of cells. In particular, two outstanding questions, the effectiveness of oncogenic transformation and the role of the local microenvironment on cancer initiation, require the study of the fates of individual cells and their progenies. Recently, we developed a technology that allows for the control of protein activity and gene expression in single cells through light activation. In this work, we utilize this method to activate in a small number of cells in a live zebrafish a typical oncogene, K-RasG12V, and investigate effects of these changes on tumorigenesis under varied genetic backgrounds. We successfully demonstrated the spatiotemporal control of oncogene expression in live zebrafish. Furthermore, we investigated different tumorigenic phenotypes by transiently or permanently activating K-Ras at varied developmental stages. We believe that our studies could shed new light on cancer initiation and growth and provide new tools for target validation and testing of novel anti-cancer drugs.

Abstract IA26: Long-term single cell quantification: New tools for old questions

Abstract Stem cell systems are highly complex and dynamic, and consist of large numbers of different cells expressing many molecules. Despite intensive research, many long-standing questions in stem cell research remain disputed. One major reason is the fact that we usually only analyze populations of cells - rather than individual cells - at very few time points of an experiment. Tracking of individual cells would be an extremely powerful approach to improve our understanding of molecular cell fate control. We are therefore developing imaging systems to follow the fate of individual cells over many generations. We program new software to help recording and displaying the divisional history, position, properties, interaction, etc. of all individual cells over many generations. Our approaches also allow continuous long-term quantification of protein expression or activity in individual living cells. The resulting novel kind of continuous quantitative single cell data is used for the generation of improved models describing the molecular control of stem cell fates. I will discuss how we try to find answers for long standing questions in stem cell research. Citation Format: Timm Schroeder. Long-term single cell quantification: New tools for old questions. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA26.

Cell-marking techniques for cell lineage tracing

A zygote, the only totipotent cell of the developing organism, transforms into a complex, multicellular entity with billions (for humans) of highly specialized cells and tissues. Most adult tissues are maintained by a combination of highly dynamic processes of senescence, apoptosis and rejuvenation with new cells constantly arising from dispersed depots of stem cells. Studying individual cell fates and their intertwined relations thus aids in understanding ontogenetic development as well as pathogenic processes in the body. Direct observations of developing embryos uncovered the fate of single blastomeres of ascidia and nematode. In both cases, research benefited from the simplicity of these objects, because, in ascidia, each blastomere has unique signatures naturally, and, in nematode, transparency of a worm’s 959-cell body allows every cell to be traced through development individually. In most cases, however, studying cellular lineages and identification of stem cells’ subpopulations are a true challenge for investigators. To trace the cell’s fate, novel methods were invented that introduce special tags into cells, the tags that would be inherited during cell divisions. Every descendent of a marked cell bears the same tag and can easily be distinguished from unrelated cellular neighbors. This review focuses on modern methods for cell tracing with dyes and genetic constructs encoding protein reporters that mark cell lineages. Special focus is on genome-integrated tags (genetic labeling), such as viral and cellular barcoding. One chapter of the review describes novel advancements in the field of CRISPR/Cas9-based cellular barcoding.

Lifetime Distributions from Tracking Individual BC3H1 Cells Subjected to Yessotoxin.

This work shows examples of lifetime distributions for individual BC3H1 cells after start of exposure to the marine toxin yessotoxin (YTX) in an experimental dish. The present tracking of many single cells from time-lapse microscopy data demonstrates the complexity in individual cell fate and which can be masked in aggregate properties. This contribution also demonstrates the general practicality of cell tracking. It can serve as a conceptually simple and non-intrusive method for high throughput early analysis of cytotoxic effects to assess early and late time points relevant for further analyzes or to assess for variability and sub-populations of interest. The present examples of lifetime distributions seem partly to reflect different cell death modalities. Differences between cell lifetime distributions derived from populations in different experimental dishes can potentially provide measures of inter-cellular influence. Such outcomes may help to understand tumor-cell resistance to drug therapy and to predict the probability of metastasis.

How to maintain a zebrafish brain

Neurodevelopment Even in the zebrafish brain, which seems better able than the human brain to generate new neurons, regenerative capacity may not be unlimited. Barbosa et al. mapped the fates of individual neuronal cells in live zebrafish over time. Seen as glowing dots, neural stem cells sustain the population of neurons, although not quite at full replacement rates. After injury to the brain, more of the stem cells were pulled into neuronal pathways, with fewer remaining to feed future replacement. Science , this issue p. [789][1] [1]: /lookup/doi/10.1126/science.aaa2729

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection. The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of <em>Streptococcus iniae</em> Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection. The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Enhancer modeling uncovers transcriptional signatures of individual cardiac cell states in Drosophila.

Here we used discriminative training methods to uncover the chromatin, transcription factor (TF) binding and sequence features of enhancers underlying gene expression in individual cardiac cells. We used machine learning with TF motifs and ChIP data for a core set of cardiogenic TFs and histone modifications to classify Drosophila cell-type-specific cardiac enhancer activity. We show that the classifier models can be used to predict cardiac cell subtype cis-regulatory activities. Associating the predicted enhancers with an expression atlas of cardiac genes further uncovered clusters of genes with transcription and function limited to individual cardiac cell subtypes. Further, the cell-specific enhancer models revealed chromatin, TF binding and sequence features that distinguish enhancer activities in distinct subsets of heart cells. Collectively, our results show that computational modeling combined with empirical testing provides a powerful platform to uncover the enhancers, TF motifs and gene expression profiles which characterize individual cardiac cell fates.

Mutation, clonal fitness and field change in epithelial carcinogenesis.

Developments in lineage tracing in mouse models have revealed how stem cells maintain normal squamous and glandular epithelia. Here we review recent quantitative studies tracing the fate of individual mutant stem cells which have uncovered how common oncogenic mutations alter cell behaviour, creating clones with a growth advantage that may persist long term. In the intestine this occurs by a mutant clone colonizing an entire crypt, whilst in the squamous oesophagus blocking differentiation creates clones that expand to colonize large areas of epithelium, a phenomenon known as field change. We consider the implications of these findings for early cancer evolution and the cancer stem cell hypothesis, and the prospects of targeted cancer prevention by purging mutant clones from normal-appearing epithelia.

Abstract 432: BIN3 is a novel 8p21 tumor suppressor gene that regulates the attachment checkpoint in epithelial cells

Abstract An important characteristic of multicellular organisms is the control that the tissue architecture exerts on the fate of individual cells. Epithelial cells sense their location through interactions with the extracellular matrix (ECM) and remove themselves by programmed cell death (anoikis) when those interactions are disturbed. Importantly, anoikis is a line of defense that has to be circumvented by cancerous epithelial cells to be able to leave their home environment and establish long distance metastases. Here, by combining a genome-wide RNAi screen and a novel algorithm to study copy number alterations (ISAR-DEL), we identify the Bridging Integrator-3 (BIN3) as a novel 8p21 tumor suppressor gene whose inactivation promotes escape from anoikis in epithelial cancers. Mechanistically, we link the tumor suppression function of BIN3 to its ability to relocate to the cell membrane after cell detachment and to induce a proapoptotic cascade. This death signaling is mediated by CDC42 activation of the P38-α stress pathway and the consequent accumulation of the apoptotic facilitator BimEL. Our results identify BIN3 as a novel epithelial tumor suppressor gene, provide novel insights on the mechanisms of attachment tumor suppressor checkpoint and highlight the importance of anoikis escape in driving cell transformation and metastasis in human cancer. Note: This abstract was not presented at the meeting. Citation Format: Netonia Marshall, Felix Sanchez, David Llobet, Ruth Rodriguez Barrueco, Veronica Castro, Dylan Kotlia, Maira Pires, Patricia Villagrasa, Preeti Putcha, Ramon Parsons, Dana Pe'er, Jose Silva. BIN3 is a novel 8p21 tumor suppressor gene that regulates the attachment checkpoint in epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 432. doi:10.1158/1538-7445.AM2014-432

Abstract 348: Multi-Species Genome-Wide Analyses of the Specification of Individual Cardiac Cell Fates

There are remarkable molecular and embryological similarities in cardiogenesis between Drosophila and vertebrates. Cells comprising the Drosophila heart can be subdivided into individual identities based on differences in morphology, function and gene expression patterns. Recent studies have shown that differential modifications of histone proteins, in vivo transcription factor (TF) binding, and the presence of particular TF binding motifs can be used as predictive signatures of the enhancers that govern cell-specific gene expression. Here we used discriminative training methods within an integrative, multi-species framework to uncover the motifs, enhancers and genes underlying cardiac cell fate decisions. As an initial step, we undertook a large-scale validation of Drosophila heart enhancers, which revealed enhancer activities in distinct subpopulations of cardiac cells. To identify related cell-specific regulatory elements, we used the validated enhancers as a training set in a machine learning approach that integrated TF motifs with ChIP data for both TF binding and histone modifications. Empirical validation of candidate enhancers predicted by this method confirmed activity in the appropriate cardiac cells. By clustering the motifs derived from the individual cardiac classifiers, we identified and validated sequence features which discriminate specific cellular identities. Next, we asked if similar predictive signatures underlie mouse and human cardiomyocyte (CM) differentiation from embryonic stem cells (ESCs). We show that the distribution of histone marks found within differentiating human and mouse ESCs indeed predict genes potentially critical for CM differentiation, with the best predictions provided by the overlapping mouse and human candidates. We evaluated this result in a large-scale RNAi-based screen of Drosophila orthologs of the mammalian genes, which uncovered dozens of novel cardiogenic regulators whose function is being tested in differentiating human ESCs. In total, these results document the utility of computational modeling combined with empirical testing to uncover the enhancers, TF motifs and genes which characterize individual cardiac cell fates in both invertebrate and mammalian species.

Linear amplification mediated PCR--localization of genetic elements and characterization of unknown flanking DNA.

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3'- and 5'- sequences adjacent to the integrated lentiviral vector.

Individual Cell Fate as a Factor of Colony Population Dynamics in Oncogenesis

Cells in multicellular organisms may have distinct cell fates, such as proliferation or differentiation into specializied cell types. The individual cell fate is determined by a range of factors, such as genetic predisposition or epigenetic factors. However, for a substantial number of cells in the organism statistic homogeneity and ballance of the system may be achieved; though the fate of an individual cell may vary. For most cell types these random individual events do not [ influence the final outcome due to a large number of cells of this type. Nevertheless, the switch between cell fate plays a far more important role if we consider stemm cells (SC) or colony-forming units (CFU) as well as cancer stem cells (CSC) because individual cell events determine further development of a colony (metastase) or its elimination. The report presents a mathematical model of the initial stage of cell colony development (for hematopoetic tissue). In order to establish the role of an individual event in the process of oncogenesis and its consequences, as well as to understand the mechanism of population dynamics, it is important to study in detail the interaction of probability distribution of cell events, and feedback regulation. This paper is part of a larger research aimed at elaborating a model of a competing clonal hematopoesis in terms of oncogenesis and oncohematological diseases.

Labour-Efficient In Vitro Lymphocyte Population Tracking and Fate Prediction Using Automation and Manual Review

Interest in cell heterogeneity and differentiation has recently led to increased use of time-lapse microscopy. Previous studies have shown that cell fate may be determined well in advance of the event. We used a mixture of automation and manual review of time-lapse live cell imaging to track the positions, contours, divisions, deaths and lineage of 44 B-lymphocyte founders and their 631 progeny in vitro over a period of 108 hours. Using this data to train a Support Vector Machine classifier, we were retrospectively able to predict the fates of individual lymphocytes with more than 90% accuracy, using only time-lapse imaging captured prior to mitosis or death of 90% of all cells. The motivation for this paper is to explore the impact of labour-efficient assistive software tools that allow larger and more ambitious live-cell time-lapse microscopy studies. After training on this data, we show that machine learning methods can be used for realtime prediction of individual cell fates. These techniques could lead to realtime cell culture segregation for purposes such as phenotype screening. We were able to produce a large volume of data with less effort than previously reported, due to the image processing, computer vision, tracking and human-computer interaction tools used. We describe the workflow of the software-assisted experiments and the graphical interfaces that were needed. To validate our results we used our methods to reproduce a variety of published data about lymphocyte populations and behaviour. We also make all our data publicly available, including a large quantity of lymphocyte spatio-temporal dynamics and related lineage information.

Abstract C49: Using single cell fluorescent cell cycle reporters to study mechanisms of action of selective inhibitors of nuclear export (SINE).

Abstract SINE are novel small molecule drugs in phase I clinical trials for advanced cancers. SINE inhibit nuclear export through covalent binding to Exportin 1 (XPO1/CRM1), inducing forced nuclear retention of major tumor suppressor proteins including p53, p21, and pRb, resulting in selective cancer cell death. In contrast, non-transformed cells show only a transient reduction of S-phase and once drug is removed, they resume proliferation. In order to study SINE drug effects in individual cells we developed a panel of solid tumor-derived cell lines that stably express dynamic fluorescent ubiquitin cell cycle reporters, and followed individual cell responses and cell fate using continuous fluorescent and phase-contrast microscopy. We report that in fibrosarcoma-derived HT-1080 cells treated with ≥100nM SINE KPT-330 (EC50 100nM) for 48 hours, ≈30% of cells showed a penetrance of cell cycle arrest with >80% in G1- or S-phase and ≤20% in G2-phase. These arrested cells were negative for the proliferation antigen Ki67. By 48 hours, ≈25% of dying cells were from interphase, where >80% died from G1- or S-phase [[Unable to Display Character: –]] and not from G2-phase. Direct analysis showed that cell division (mitosis) occurred in >30% of the treated population. Of these mitotic cells, a small number died while in mitosis, while the rest divided. The duration of cell division was >2-fold longer than normal and we observed chromosome missegregations. Lineage tracking of the daughter cells from these abnormal divisions indicated that ≈50% remain in interphase, mostly in G1- or S-phase, while the remainder progress to cell death, also mainly from G1- or S-phase. In contrast, non-transformed retinal pigment epithelial cells showed very little death, and instead remained in prolonged interphase. At 48 hours, cancer cells lacking p53 showed no difference in cell death, however we observed more dividing cells compared to control. These data indicate that p53 does not affect immediate cell death, but it does have a role in SINE mediated cell cycle arrest. We conclude that SINE drugs are highly effective at arresting cell growth and inducing apoptosis in tumor cells, with most cell death occurring in G1- or S-phase. Importantly, cells that overcome cell cycle arrest and divide are so severally compromised that they either die while in mitosis or the daughter cells subsequently arrest or die. Taken together, KPT-SINE may exert anti-cancer effects by perturbing both interphase and cell division. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C49. Citation Format: Joshua Marcus, Yosef Landesman, Sharon Shacham, James Orth. Using single cell fluorescent cell cycle reporters to study mechanisms of action of selective inhibitors of nuclear export (SINE). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C49.

Arrayed lentiviral barcoding for quantification analysis of hematopoietic dynamics

Our understanding of system dynamics of mixed cell populations in whole organisms has benefited from the advent of individual cell marking by nonarrayed DNA barcodes subsequently analyzed by high-throughput DNA sequencing. However, key limitations include statistical biases compromising quantification and the lack of applicability to deconvolute individual cell fate in vivo after pooling single cells differentially exposed to different conditions ex vivo. Here, we have derived an arrayed lentiviral library of DNA barcodes and obtained a proof-of-concept of its resolving capacity by quantifying hematopoietic regeneration after engraftment of mice with genetically modified autologous cells. This method has helped clarify and bridge the seemingly opposed clonal-succession and continuous-recruitment models of hematopoietic stem cell behavior and revealed that myeloid-lymphoid biases are common occurrences in steady-state hematopoiesis. Arrayed lentiviral barcoding should prove a versatile and powerful approach to deconvolute cell dynamics in vivo with applications in hematology, embryology, and cancer biology.

P53 deficiency enhances mitotic arrest and slippage induced by pharmacological inhibition of Aurora kinases

A number of small-molecule inhibitors of Aurora kinases have been developed and are undergoing clinical trials for anti-cancer therapies. Different Aurora kinases, however, behave as very different targets: while inhibition of Aurora A (AURKA) induces a delay in mitotic exit, inhibition of Aurora B (AURKB) triggers mitotic slippage. Furthermore, while it is evident that p53 is regulated by Aurora kinase-dependent phosphorylation, how p53 may in turn regulate Aurora kinases remains mysterious. To address these issues, isogenic p53-containing and -negative cells were exposed to classic inhibitors that target both AURKA and AURKB (Alisertib and ZM447439), as well as to new generation of inhibitors that target AURKA (MK-5108), AURKB (Barasertib) individually. The fate of individual cells was then tracked with time-lapse microscopy. Remarkably, loss of p53, either by gene disruption or small interfering RNA-mediated depletion, sensitized cells to inhibition of both AURKA and AURKB, promoting mitotic arrest and slippage respectively. As the p53-dependent post-mitotic checkpoint is also important for preventing genome reduplication after mitotic slippage, these studies indicate that the loss of p53 in cancer cells represents a major opportunity for anti-cancer drugs targeting the Aurora kinases.

Sex allocation pattern of the diatomCyclotella meneghiniana

Sex allocation is one of the most successful applications of evolutionary game theory. This theory has usually been applied to multicellular organisms; however, conditional sex allocation in unicellular organisms remains an unexplored field of research. Observations at the cellular level are indispensable for an understanding of the phenotypic sex allocation strategy among individuals within clonal unicellular organisms. The diatom Cyclotella meneghiniana, in which the sexes are generated from vegetative cells, is suitable for investigating effects of phenotypic plasticity factors on sex allocation while excluding genetic differences. We designed a microfluidic system that allowed us to trace the fate of individual cells. Sex allocation by individual mother cells was affected by cell lineage, cell size and cell density. Sibling cell pairs tended to differentiate into the same fates (split sex ratio). We found a significant negative correlation between the cell area of the mother cell and sex ratio of the two sibling cells. The male-biased sex ratio declined with higher local cell population density, supporting the fertility insurance hypothesis. Our results characterize multiple non-genetic factors that affect the phenotypic single cell-level sex allocation. Sex allocation in diatoms may provide a model system for testing evolutionary game theory in unicellular organisms.

Abstract 4626: Quantifying erlotinib response variability in EGFR-addicted cells.

Abstract Patients with activating epidermal growth factor receptor (EGFR) mutations display drastic initial responses to erlotinib, but tumor reduction and progression-free survival vary widely. An open question is whether response variation derives, in part, from tumor heterogeneity, i.e., differential composition of cells responding to erlotinib with distinct cell fates (death, division, or quiescence). Conventional assays obscure response heterogeneity by taking average measurements of cell populations. To address this question we developed high-throughput live-cell imaging assays that quantify both subpopulation growth dynamics and cell-to-cell fate variability in response to drug. The Dynamic Colony Growth Assay (DCGA) tracks simultaneously the erlotinib response of hundreds of single-cell-derived colonies from within a cell population. In the PC9 lung cancer cell line (widely used to model oncogene addiction, exon19del EGFR) the DCGA reveals that individual colonies span a wide range of erlotinib response, from continued cell division to massive apoptosis. Plotting net growth rates of 207 colonies shows that PC9 parental is comprised of a normally-distributed aggregate of individual single colonies responding to erlotinib with steady-state growth rates from positive to negative. Notably, the positive-growth colonies lie in the tail of this response distribution, suggesting they do not originate from rare genetic variants or cancer stem cells but, rather, are part of a continuous response distribution that is an attribute of parental PC9. To determine whether isolated PC9 colonies maintain unique rate responses, we expanded random-sampled single cells (without erlotinib selection) into 7 discrete sublines (DS), which remain highly sensitive to erlotinib (IC50 ∼50nM). In each DS, erlotinib induced an initial non-linear growth period (72h) followed by a distinct steady-state growth rate that predicts the long-term (10d) response. DS steady-state growth rates were resolved into individual cell fate composition by Fractional Proliferation Assays (FPA). As expected from random selection, the growth rates of treated DS clustered around the median of the DCGA rate distribution, missing the most extreme, less frequent erlotinib responses. Isolating the greatest variance in erlotinib response should facilitate identification of molecular mechanisms underlying drug response variation. Therefore, we isolated 96 new DS and selected the two with highest (DS-B03) and lowest (DS-C03) erlotinib steady state growth rate. We are currently measuring the activity of hundreds of proteins in these two DS by Microwestern Arrays to discover protein signatures and/or mechanistic models that quantitatively link FPA-resolved cell fates to underlying signaling network events. Thus tumor response variability and recurrence may be explained by a continuous distribution of erlotinib response at the level of single cells within a tumor. Citation Format: Peter L. Frick, Darren R. Tyson, Shawn P. Garbett, Carlos F. Lopez, Zach W. Jones, Vito Quaranta. Quantifying erlotinib response variability in EGFR-addicted cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4626. doi:10.1158/1538-7445.AM2013-4626

Cycling progenitors maintain epithelia while diverse cell types contribute to repair

It has recently been shown that stem and progenitor cells undergo population self-renewal to maintain epithelial homeostasis. The fate of individual cells is stochastic but the production of proliferating and differentiating cells is balanced across the population. This new paradigm, originating in mouse epidermis and since extended to mouse oesophagus and mouse and Drosophila intestine, is in contrast to the long held model of epithelial maintenance by exclusively asymmetric division of stem cells. Recent lineage tracing studies have now shown that wound responses vary between tissues, and that a stem cell reserve is not essential as cycling progenitors and even differentiating cells contribute to regeneration.