Spontaneous Cell Competition in Immortalized Mammalian Cell Lines
Spontaneous Cell Competition in Immortalized Mammalian Cell Lines
Editor: Juan F. Poyatos 0 1
Spanish National Research Council (CSIC) 0 1
SPAIN 0 1
Alfredo I. Penzo-Méndez 0 1
Yi-Ju Chen 0 1
Jinyang Li 0 1
Eric S. Witze 0 1
Ben Z. Stanger 0 1
0 1 Departments of Medicine and Cell and Developmental Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 Department of Cancer Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 3 Abramson Family Cancer Research Institute, Perelman School of Medicine at the University of Pennsylvania , Philadelphia, Pennsylvania , United States of America
1 Funding: This work was supported by the Pew Charitable Trusts and the Abramson Family Cancer Research Institute
Cell competition is a form of cell-cell interaction by which cells compare relative levels of fitness, resulting in the active elimination of less-fit cells, “losers,” by more-fit cells, “winners.” Here, we show that in three routinely-used mammalian cell lines - U2OS, 3T3, and MDCK cells - sub-clones arise stochastically that exhibit context-dependent competitive behavior. Specifically, cell death is elicited when winner and loser sub-clones are cultured together but not alone. Cell competition and elimination in these cell lines is caspase-dependent and requires cell-cell contact but does not require de novo RNA synthesis. Moreover, we show that the phenomenon involves differences in cellular metabolism. Hence, our study demonstrates that cell competition is a common feature of immortalized mammalian cells in vitro and implicates cellular metabolism as a mechanism by which cells sense relative levels of “fitness.”
Competing Interests: The authors have declared
that no competing interests exist.
Tissue growth is influenced by both systemic cues and local cell interactions. In Drosophila,
cell competition is a well-described example of the latter type of interaction, in which the
presence of growth-advantaged “winner” cells triggers apoptosis of otherwise viable, but
growthdisadvantaged “loser” cells [1–3]. One remarkable feature of cell competition is that cellular
responses are triggered by relative rather than absolute growth properties, indicating that cells
are able to sense neighboring cell “fitness” and compare it to their own [1–3]. Several molecular
pathways have been implicated in cell competition in Drosophila, including dMyc and its
ribosomal targets [4–6], components of the Dlg/Lgl/Scrib cell polarity complex [7–9], the BMP
pathway , the Hippo pathway, and the Wnt and JAK/STAT pathways [11, 12]. Differences
in the activity of these signaling pathways across cells result in competition-mediated cell
death, but the mechanisms involved remain poorly understood.
The first evidence for mammalian cell competition came from the study of the Belly spot
and tail (Bst) mutation in the mouse. Bst impairs ribosomal biogenesis and cell growth, but
heterozygous Bst blastocysts still develop into viable animals of normal size. Nonetheless, Bst
blastocysts injected with wild-type embryonic stem (ES) cells grow into embryos derived
mostly from the grafted cells, indicating that Bst cells are outcompeted during embryogenesis
. Two recent studies confirmed that differences in Myc expression drive cell competition in
cultured ES cells as well as in the mouse epiblast [14, 15]. Cell competition has also been
described in adult tissues, as embryonic hepatic progenitors grafted in adult livers expand at
the expense of resident hepatocytes in an age-dependent manner [16, 17]. Moreover,
knockdown of Scribbled results in loser behavior in immortalized epithelial Madin-Darby canine
kidney (MDCK) cells [18, 19].
In this study, we sought to determine whether cell competition could be modeled in vitro.
To this end, we generated sub-clones from commonly cultured cell lines and tested them in
various combinations for competitive interactions. Here we report that such sub-clones display
context-dependent apoptosis in the presence of the parental cell population. Elimination of
loser cells requires direct contact with winners and does not involve transcriptional changes.
Competitive behavior is abrogated by conditions that limit metabolic activity or uncouple ATP
generation from oxidative phosphorylation, indicating that changes in energetic metabolism
underlie cell “fitness.” Taken together, these data indicate that cell competition is active in cell
lines grown under routine culture conditions, reflecting a conserved role in regulating cellular
Cell competition has been shown to occur in the mouse epiblast in response to endogenous
differences in cellular fitness, which are reflected by heterogenous Myc protein levels . Based
on this, we hypothesized that differences in cell fitness might also occur in immortalized cells
grown under standard culture conditions, resulting in the spontaneous emergence of winner
and loser cells in vitro. To test this hypothesis, we transfected U2OS human osteosarcoma cells
with vectors carrying histone 2B-green fluorescent protein (H2B-EGFP), H2B-Venus or
H2BmCherry fusion protein expression cassettes along with a neomycin resistance cassette followed
by G418 (neomycin) selection to generate a large number of stable U2OS sub-lines, or clones.
We then performed pairwise co-cultures of cells from each one of the clones and parental,
“wild-type” (Wt) U2OS cells, using the presence of fluorescent markers in the “clone” cells to
monitor the co-culture cell composition over time. We hoped in this way to identify clones
that would be eliminated from the co-cultures (“losers”) or that would conversely eliminate the
wild type cells and take over the culture (“winners”).
A small subset (9 out of 245) of the screened clones spontaneously exhibited a net decrease
in their numbers upon co-culture with Wt cells (Fig 1A). Staining for cleaved caspase-3 (Cp3)
confirmed that the cells were being eliminated by apoptosis (Fig 1B and 1C). No changes in cell
proliferation were observed in co-cultured cells by phospho-histone H3 immunostaining (Fig
1D). Incubation of co-cultures with the pan-caspase inhibitor Z-VAD-FMK blocked cell
elimination (Fig 1E). Furthermore, incubation with the CDK inhibitor purvalanol A also led to a
dramatic decrease in killing, indicating that cells must be actively proliferating for cell death to
occur (Fig 1F). When grown in mono-culture, growth rates varied widely among these “loser”
clones, but all of them displayed substantially slower growth compared to that of the parental
Wt cells (S1 Fig). Hence, U2OS cells maintained under routine conditions contain sub-clones
that grow more slowly and are selectively killed when co-cultured with more rapidly-dividing
cells, features reminiscent of cell competition in Drosophila. Notably, clones whose growth rate
was similar to that of the parental Wt cells (e.g. clones G1 and 10A in S1 Fig) were not
eliminated upon co-culture and did not show an increase in apoptosis. In addition, no clones
Fig 1. Cell death is triggered by a cell competition-like interaction in clonally-derived mammalian cell
lines. (A) Cell counts showing YFP (“loser” cells) cells first expand, then decline, in the presence of Wt
(“winner”) cells but grow unimpeded when cultured alone. Time is measured from cell seeding (t = 0). (B)
Cleaved caspase-3 (Cp3) immuno-fluorescence (IF) (red) of U2OS cultures showing increased apoptosis in
co-cultured YFP cells (green). Arrows indicate Cp3+ apoptotic YFP cells. Wt cells are counterstained with
Hoescht 33342 (blue). (C) Quantification of apoptosis on immune-stained cultures; x-axis, time in days (d)
Note that the baseline level of apoptosis increases with cell density by day 6 under all culture conditions. (D)
Quantification of cell proliferation in 72-hour U2Os cultures by phospho-histone H3 (PH3)
immunofluorescence. (E) Cp3 IF of 72-hour U2OS cultures treated with the caspase-3 inhibitor Z-VAD-FMK.
YFP cell counts per microscope field are shown at the bottom. Inhibition of apoptosis by Z-VAD-FM K
prevents YFP elimination from Wt:YFP co-cultures. (F) P-H3 IF of U2OS cells cultured for 72 hours in
presence of the Cyclin D1 inhibitor purvalanol A as indicated. Quantification of apoptosis is shown below.
Purvalanol A treatment inhibits proliferation (top) and rescues YFP elimination (bottom). Images were taken
at 100X magnification. Error bars in this and all subsequent figures reflect mean ± SD. *: p<0.05, **: p<0.01
and ***: p<0.001 by Student’s t-test.
growing faster than Wt cells or displaying “winner” behavior in co-culture were observed (data
To determine whether competition-like behavior is generalizable across different cell types
from distinct species, we used the same strategy to screen randomly generated sub-clones from
non-transformed murine fibroblasts (3T3 cells) and canine renal epithelial cells (MDCK cells).
Consistent with our U2OS results, we found two MDCK clones (out of 169) and one 3T3 clone
(out of 230) displaying increased apoptosis and cell elimination upon co-culture with parental
cells (S2 and S3 Figs). Thus, competition-like behavior seems to be a general property of
mammalian cell lines. We conducted all further experiments with U2OS cells.
Elimination of U2OS losers requires direct contact with winners
Cell competition in Drosophila has been reported to be mediated by soluble factors released
when cells are in a competitive environment [4, 20]. To test whether cell competition in U2OS
cells is also mediated by soluble factors, we performed a series of experiments utilizing
transwell filters and live cell imaging. For the trans-well experiments, we co-cultured YFP cells
(which behave as losers) with Wt cells (which behave as winners) on the top of 0.8 μm
Durapore trans-well inserts and placed YFP cells at the bottom of the well (Fig 2A). As expected,
YFP cells in contact with Wt cells in the insert were eliminated (Fig 2B and 2C). By contrast,
YFP cells in the well displayed no increase in apoptosis despite sharing the same medium with
the Wt:YFP co-cultured cells in the insert (Fig 2B). Thus, cell competition in U2OS cells
requires either direct contact or close proximity.
To distinguish between these possibilities, we examined cell interactions by time-lapse
microscopy over a 72 h period (S4 Fig). In order to identify all cells in this experiment, we used
R1 cells (red), which behave as winners when mixed with YFP cells (green). R1 cells were plated
as a “spot” on tissue culture plastic and then overlaid with YFP cells; this created “Inner” and
“Border” areas in which YFP and R1 cells were in frequent contact as well as an “Outer” region
where YFP cells had no contact with R1 cells (Fig 2C and S4 Fig). We then tracked the fate of
individual YFP to R1 cells, counting over 5,000 cells in 3 movies. As expected, YFP cells inside
the R1 spot (“Inner” region) were progressively eliminated by apoptosis, while YFP cells that
remained far from the R1 spot (>10 cell layers, “Outer” region) experienced significantly less
apoptosis over the 72 h period analyzed (Fig 2D; S4 Fig and S1 File).
To more accurately determine the relationship between the fate of a cell and its location, we
binned YFP cells based on their position relative to R1 cells, designating cells as “B0” to “B10”
to reflect the smallest number of YFP cell layers observed between the designated cell and the
nearest R1 cell. Relative cell position was estimated tracking the cell nuclei when cell contours
could not be clearly seen (due to the high cell density reached by U2OS cultures). We observed
that cell death was highly dependent upon designated cell position, with YFP cells in the
immediate vicinity of R1 cells–“Inner” or “B0”–exhibiting significantly higher death rates than YFP
cells that appeared to be even one cell removed from R1 cells. Indeed, B1 to B10 cells exhibited
death rates similar to that observed in cells in the “Outer” region (Fig 2D, middle panel).
Importantly, YFP cell proliferation was unaffected by position relative to R1 cells (Fig 2D, top
panel). These rates of proliferation and death resulted in a net population decrease for cells in
the “Inner” region and a net increase for all other cell populations (Fig 2D, bottom panel).
These results indicate that YFP cell elimination is driven by direct contact with R1 cells.
Furthermore, a stronger competitive response was observed in “inner” cells, in which losers were
surrounded by winners, than in”B0” cells, in which losers might be in contact with only a single
winner cell for only a brief period of time. This result suggests that loser cell elimination is
Fig 2. U2OS cell competition interactions are short-ranged. (A) Schematic representation of U2OS
transwell cultures, Cells shared culture medium but were separated by a 0.8 μm Durapore membrane. (B)
Cp3-IF analysis of apoptosis in transwell cultures. Wt cells induce apoptosis in YFP cells in the insert but not
in YFP cells separated by the transwell. (C) Spot-seeding of YFP and H2B-mCherry expressing clone R1.
Mixed YFP:R1 spots surrounded by pure YFP cell populations were divided in “inner” (I), “border” (B), and
“outer” (O) zones as represented. (D) Time-lapse microscopy tracking of YFP cell fates during 72-hour spot
cultures. The number of cell layers separating each YFP cell from its nearest R1 neighbor was recorded, and
YFP “B” cells were grouped accordingly: for instance, a YFP cell is labeled “B3” if it comes within 3 cell layers
of the nearest R1 cell, while a”B0” YFP cell comes to lie adjacent to an R1 cell at any time during the
observation period. The data summarizes the fate of cells present at the beginning of each experiment and
their immediate progeny, followed over 72 hours. The percentage of followed cells that underwent cell
division is shown at the top; cell death is shown at the middle, and net population size change at the bottom.
Increased apoptosis is observed only in inner YFP cells and “B0” border cells that come in direct contact with
R1 cells. Data in panel D was derived from 3 independent experiments (Supplemental Movie S1-3),
comprising over 5,000 cells counted. *: p<0.05, **: p<0.01 by Student’s t-test; #:p<0.05, ##:p<0.01 by paired
dose-dependent, such that more extensive or longer contact with a winner cells increases the
likelihood of the loser cell undergoing apoptosis.
U2OS cell competition is context-dependent
Context-dependent responses—whereby cells behave as winners in the presence of less-fit
clones, and as losers in the presence of more-fit clones—is a hallmark of cell competition in
Drosophila. Because our screen for clones with varying fitness had only yielded cells that
behaved as losers compared to Wt parental cells, we sought to determine whether this system
exhibits context dependency.
We reasoned that R1 cells, which grow faster than some clones and slower than others (S1
Fig), would be good candidates for assessing whether U2OS competition is context-dependent.
To this end, we co-cultured R1 cells with either G1 cells, which have a growth rate similar to
Wt cells and do not compete with them (a “non-competing” clone), or with YFP cells, which
grow more slowly. Co-culture of R1 and G1 cells led to a marked increase in R1 apoptosis, but
no increase in G1 apoptosis (Fig 3A and 3B). Co-culture of R1 cells with three additional
noncompeting GFP clones yielded similar results (data not shown). By contrast, co-culture of R1
and YFP cells led to a marked increase in YFP apoptosis but no increase in R1 apoptosis (Fig
3A and 3B). Co-culture of the remaining GFP-expressing “loser” clones (S1 Fig) resulted in
either non-competitive growth or the GFP-expressing cells still being eliminated (data not
shown). Based on these results, we conclude that spontaneous cell competition in U2OS cells is
context-dependent such that cells behave as winners or losers depending on the milieu in
Fig 3. U2OS cell “fitness” is context-dependent. (A) Aspect of U2OS G1, R1, and YFP co-cultures in
fluorescence microscopy. Cells were plated at a 1:1 ratio as indicated (R1:G1 or R1:YFP) and allowed to
grow for 1, 3 or 6 days. (B) Cp3-IF apoptosis quantification. R1 cells behave as “winners” in R1:YFP cultures
but as “losers” in the presence of G1 cells, indicating that R1 cells assume “winner” or “loser” status
depending on the properties of their co-culture partners. *: p<0.001 by Student’s t-test.
which they find themselves. Further pairwise assays demonstrated three “competition groups”
in which higher proliferation rates and saturation densities were correlated with higher fitness
(see color coding in S1 Fig). These data suggest that winner or loser status is not an intrinsic
property of a cell, but is rather determined when cells confront their neighbors and relative
levels of cell “fitness” are determined.
U2OS cell competition does not require de novo RNA synthesis
In Drosophila, competitive fitness is affected by the activity of several genes, most notably Myc,
the components of the Hippo pathway, and the members of the Scribd apico-basal polarity
complex [4, 6–9, 21–24]. However, mosaic overexpression or knock-down of MYC, YAP and
SCRIB did not alter cell competitive fitness in Wt U2OS cells (S5 and S6 Figs). Thus,
competitive behavior in US02 cells is likely mediated by molecular mechanisms distinct from those
that underlie such behavior in Drosophila S2 cells.
We reasoned that the determinants that confer relative level of fitness could reflect either a
gain or a loss of information; if this were the case, then “loser status” might be expected to
behave as either a dominant or a recessive trait, respectively. To address this idea, we carried
out a series of cell fusion experiments in which heterokaryons were generated between various
winner and loser U2OS clones. This fusion experiment resulted in diverse behaviors, with
heterokaryons exhibiting winner status, loser status, or an intermediate phenotype depending on
the identity of the parental clones (S7 Fig), suggesting that competitive “fitness” reflects the
integration of multiple genetic or epigenetic factors.
We next compared the transcriptional profiles of Wt, YFP, and R1 cells in an attempt to
identify genes driving cell competition. We expected that transcripts acting as fitness
determinants would be differentially expressed in mono-cultured cells, most likely in a way that reflects
their relative fitness (i.e., Wt>R1>YFP or vice versa). On the other hand, transcripts
implicated in sensing or responding to fitness determinants might not be expected to differ in
abundance in mono-cultures, but instead be differentially expressed only in actively-competing
loser and winner cells (Fig 4A).
U2OS RNA expression levels were characterized by whole-transcriptome RNA microarray
hybridization and compared between mono-cultured or competing Wt, R1 or YFP cells (Fig
4A). Unexpectedly, no transcriptional changes were found to be induced by competitive
coculture in Wt and R1 cells. Six transcripts were found to be expressed at higher levels in YFP
cells when co-cultured with Wt cells. Of these, three were also up-regulated in YFP cells
co-cultured with R1 cells (Fig 4B and S1 Table). However, the nature of these transcripts (encoding
for the olfactory receptor 51B4, the interleukin 13 receptor subunit A2, and the gametocyte
specific factor 1), and the fact that their expression was unchanged in loser R1 cells strongly
suggest that changes observed in YFP cells are not related to competition.
Overall, these results suggested that competitive interactions do not spur unique
transcriptional changes, raising the possibility that competition does not require the synthesis of new
transcripts. To address this possibility, we blocked RNA and protein synthesis in Wt:YFP
cocultures using actinomycin D and cycloheximide, respectively. Despite a complete blockade in
transcription following actinomycin D treatment (Fig 4C), YFP cells continued to be killed in
the presence of Wt cells (Fig 4D). This result demonstrates that U2OS cell competition does
not require de novo transcription. By contrast, cycloheximide treatment abolished the
difference in apoptosis levels of control and co-cultured YFP cells (Fig 4D), indicating that cell
competition requires protein translation. However, cycloheximide treatment also led to a
proliferative arrest (S8 Fig), raising the possibility that cycloheximide blocks cell competition
as a secondary consequence of its inhibition of cell cycle progression.
Fig 4. Cell competition in U2OS cells is mediated by post-transcriptional mechanism. (A) Microarray
RNA analysis experimental design. Cells were grown in mono- or co-cultures for 48h as indicated and sorted
by flow cytometry before RNA extraction. (B) Venn diagram distribution of RNAs displaying >2-fold
expression change across indicated sample groups. Only six transcripts were found to be differentially
expressed in mono-cultured and competing YFP cells, while no transcription changes were observed in
response to competition in Wt and R1 cells. (C) Immuno-staining of metabolite-labeled nascent RNA and
protein chains in Wt U2OS cells treated with actinomycin D or cycloheximide for 24 hours. Ethynil-uridine
(EU) and homopropargyl-glycin (HGP) were added to label nascent RNA and peptide chains 2 h before
staining. Complete inhibition of transcription and translation was obtained with actinomycin D and
cycloheximide, respectively. (D) Cp3-IF of Wt:YFP 72-hour cultures grown in presence or absence of
actinomycin D- and cycloheximide. Cycloheximide completely abolished Wt-induced YFP apoptosis.
Actinomycin D treatment resulted in a partial rescue to a degree consistent with its inhibition of cell division
(S10 Fig). *”p<0.05, **: p<0.01 by Student’s t-test.
Differences in metabolism underlie U2OS cell competition
We next turned our attention to potential fitness determinants. Comparison of transcriptional
profiles across pooled mono- and co-cultured samples identified 656 genes that were
differentially expressed in Wt, R1 and YFP cells. Of these, only 82 were expressed across Wt, R1, and
YFP in a way correlating with cell fitness (S9 Fig) and gene ontology term representation
analysis indicated no significant functional class enrichment except for the presence of 7
metalloprotease-encoding genes in the group (S2 Table). Importantly, further analysis of gene expression
across the full panel of winners and losers (S1 Fig) failed to support the correlation in
expression levels seen with Wt, R1, and YFP (data not shown). Thus, even among those transcripts
which were differentially expressed between winners and losers, this transcriptional analysis
did not lead to the emergence of strong candidates that might constitute determinants, sensors,
or mediators of cell fitness and competition.
Monopolization of trophic factors by metabolically advantaged “winners” was among the
first mechanisms proposed to underlie cell competition. To test whether U2OS cells compete
for trophic factors, we performed competition assays under culture conditions in which we
varied the degree to which such factors might be limiting. Increasing serum concentration to 20%
had no effect on loser cell elimination; nor did reducing or removing serum altogether,
indicating that U2OS cells do not compete for serum-borne factors (S10 Fig). By contrast, hypoxia
was found to completely block cell competition when either YFP or R1 cells were co-cultured
with Wt cells (Fig 5A). As many of the effects hypoxia are promoted via activation of the Hif1a
transcription factor , we sought to determine whether Hif1a was required for the oxygen
dependency of U2OS cell competition. To this end, we performed the cell competition
experiment in the presence of two different shRNAs directed against Hif1a or a non-silencing
shRNA control (NSC). Hif1a knockdown did not protect YFP and R1 cells from cell
competition under normoxia, nor did it reverse the protective effect of hypoxia (Fig 5B). These results
strongly suggest that the ability of hypoxia to block cell competition is Hif1a-independent.
These results were unexpected, as we had predicted that limiting amounts of O2 would
exacerbate, rather than inhibit, cell competition.
Because hypoxia has several cellular effects—including changes in cell metabolism through
effects on oxidative phosphorylation (OXPHOS) and reactive oxygen species (ROS)
production—we hypothesized that differences in metabolic rate might underlie competitive behavior.
Consistent with this notion, reducing the metabolic activity of cells by reducing the
concentration of glutamine (Gln) in the medium eliminated cell competition in U2OS cells (Fig 5C). We
then tested if competitive behavior might involve the production of ROS by carrying out
competition assays in the presence of two ROS scavenging agents: N-acetyl cysteine (NAD) and
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Neither NAD nor Trolox
prevented loser cell elimination in U2OS co-cultures, indicating that ROS are dispensable for
this process (S11 Fig).We next considered the possibility that ATP production (via OXPHOS)
could be involved in cell competition in U2OS cells. To test this idea, U2OS competition assays
were carried out in presence of carbonyl cyanide m-chloro phenyl hydrazine (CCCP), an agent
that permeabilizes the inner mitochondrial membrane to H+ ions. CCCP thus allows OXPHOS
to proceed but uncouples it from ATP synthesis. CCCP treatment resulted in a dose-dependent
reduction in cell death when either YFP or R1 cells were co-cultured with Wt cells (Fig 5D).
We reasoned that if ATP production underlies cell fitness, ATP levels in monocultured U2OS
clones should reflect their fitness. Accordingly, intracellular ATP levels in monocultured YFP
(loser) cells are lower than those observed in Wt (winner) and R1 (intermediate) cells.
Furthermore, ATP concentration increased to near-Wt levels in YFP cells grown under hypoxia,
correlating with the previous observation that it arrests Wt:YFP competition. Interestingly, R1
cells—the intermediate fitness cells—displayed comparative ATP levelswith WT (winner)
under normxia and hypoxia. It suggests that ATP levels are not the sole metabolic product to
determine cell fitness (Fig 5E). Taken together, these results strongly suggest that differences in
energetic metabolism drive cell competition in U2OS cells and that production of ATP by
OXPHOS is one of the parameters to determinate cell fitness.
Over the past 10 years, the phenomenon of cell competition has emerged as a mechanism by
which cell growth and viability are controlled in diverse biological processes, such as tissue size
regulation, aging, and cancer progression. Although most studies of cell competition have
focused on Drosophila, a growing body of evidence indicates that the process is conserved in
mammals [13–19, 26]. Nevertheless, the functional role and underlying molecular mechanisms
of cell competition remain obscure. The results presented here show that mammalian cell lines
spawn sub-clones that are viable and grow well on their own, but which undergo apoptosis in
the presence of more advantaged cells. Relative cell fitness—the likelihood that a cell will
behave as a winner or a loser—is at least partially associated with growth rate and saturation
Fig 5. Differences in energy metabolism drive cell competition in mammalian cells. (A) Quantification of
apoptosis (Cp3 immunofluorescence) in U2OS cultures grown for 72 hours under normoxic (21% O2) or
hypoxic (1.5% O2) conditions. Hypoxia inhibits cell competition-induced elimination of YFP and R1 cells in
Wt:YFP and Wt:R1 co-cultures. (B) Quantification of Cp3 immunofluorescence in U2OS R1 cells expressing
a nonsense (NSC, non-silencing control) shRNA or shRNAs directed against the Hif1a transcription factor.
Hif1a knockdown in loser cells does not affect cell competition. qPCR analysis of Hif1a in shRNA-expressing
YFP cells is shown on the right. (C) Quantification of Cp3 immunofluorescence in U2OS cultures grown for 72
hours in medium containing standard (4 mM) and reduced (0.4 mM) Glutamin (Gln) concentrations.
Withholding Gln arrests cell competition in U2OS cells. (D) Quantification of Cp3 immunofluorescence in
U2OS cultures treated with a mitochondrial uncoupling agent (carbonyl cyanide m-chlorophenyl hydrazine,
CCCP). Uncoupling respiration from oxidative phosphorylation blocks competition-induced elimination of R1
and YFP cells, indicating that competition is driven by differences in the activity of ATP-generating pathways.
(E) Luciferase analysis of intracellular ATP levels in monocultured U2OS cell. YFP, but not R1 cells, display
reduced ATP levels in when compared to Wt cells. Hypoxia increases ATP levels YFP cells, suggesting that
reduced ATP levels may reflect or underlie YFP cell fitness.*: p<0.001, by one-way ANOVA.
density and is context-dependent. This behavior is thus highly similar to the phenomenon of
cell competition as it has been described in the Drosophila imaginal disc [4, 6, 27] and more
recently in the embryonic mouse epiblast [14, 15].
Our results provide several clues about the determinants of cell completion in mammalian
cells lines. At the cellular level, competition between U2OS cells was found to be mediated by
short-range interactions, as only those loser cells located in the immediate vicinity of winners
experienced increased apoptosis. Furthermore, our results suggest that cell competition
involves dose-dependent interactions, since loser cells surrounded by winners had a greater
likelihood of being eliminated than losers having winners on only one flank. These results
differ somewhat from previous studies, in which cell competition was found to be mediated by
diffusible factors when driven by Myc in Drosophila S2 cells  and by BMP deficiency in
cultured mouse ES cells . However, medium conditioned by competing mouse ES cells with
different Myc expression levels does not elicit a response in naïve, loser cells . Similarly, cell
competiton resulting from mosaic inactivation of the cell polarity gene Scribbled in
MadinDarby canine kidney cells also requires direct winner-loser cell contact. In vivo, apoptosis
resulting from cell competition occurs mostly within a few cell diameters of the winner-loser
interface in the Drosophila imaginal disc [4, 6, 10, 28], the mouse epiblast, and the mouse
liver . These results are all consistent with the notion that distinct cues mediate competitive
interactions in different cell types, organisms, and stages of differentiation.
Two mechanisms have been proposed to describe how extracellular determinants could
trigger competitive cell responses in the Drosophila imaginal disc . In the first, cells
compete for a limited supply of one or more factors required for survival. Similarly, competition
could result from different cell sensitivities to toxins that accumulate in the microenvironment
as cells grow . Several observations argue against a mechanism of this type in the U2OS
cells described in this study. Serum deprivation or enrichment did not affect cell competition
while hypoxia and glutamine deprivation arrested it, indicating that none of these factors is
limiting for growth. Alternatively, competition could involve a struggle for a limited amount of
space on the substrate, in which winner cells displace less-adherent loser cells, thereby resulting
in anoikis. Arguing against this possibility, however, time-lapse microscopy showed that losers
undergo apoptosis before detaching from the substrate, a result inconsistent with an
anoikisbased process. Morevover, YFP loser cells can attach and spread even when overlaid over
confluent R1 winners, which is inconsistent with R1 cells physically displacing YFP cells. Finally,
competition in U2OS cells is active at cell densities 5 to 10 fold below those reached by winners
or losers at the end of their growth in monoculture.
A second mechanism involves a process by which cells sense and compare relative levels of
“fitness.” One example of such a fitness-sensing mechanism is the role in imaginal disc cell
competition played by the flower (Fwe) gene. Upon competition, alternative splicing results in
expression of the FweLoseA/B isoform in “loser” cells, which is necessary and sufficient for
competition-driven cell elimination . Furthermore, expression of mFwe1 –a mammalian
homologue of flower–in the fly confers “loser” status in imaginal disc cells, suggesting a
conserved role in competition . In our study, no changes in the expression of the human
mFwe1 homolog (FLOWER isoform 1) were observed in response to cell competition or across
mono-cultured high and low-fitness clones, suggesting that cell competition in U2OS cells is
Our data therefore point to a novel mechanism of cell competition in U2OS cells—one that
is mediated by in a way that is independent of de novo RNA synthesis. Instead, our results
suggest that differences in cell metabolism underlie the differences between winner and loser
status. We found that hypoxia, which promotes a switch from OXPHOS to fermentation, arrests
cell competition. Similarly, depriving cells of the non-essential amino acid Gln—a key fuel for
the citric acid cycle—blocks cell competition. These results are thus in line with the recent
study of de la Cova et al. , who showed that differences in energetic metabolism—mediated
by Myc and p53 and resulting in a shift in the balance of glycolysis and OXPHOS—are key
determinants of cell competition in Drosophila. In addition, we found that uncoupling
OXPHOS from ATP production also arrests cell competition, indicating that differences in
steady-state ATP levels and/or ATP production rates can drive cell competition. Monocultured
YFP display decreased intracellular ATP levels compared to Wt cells, but ATP levels in YFP
increase when cells are grown under hypoxia, suggesting that ATP levels may be a determinant
of fitness in YFP cells. However, we did not observe similar ATP level changes in R1 cells. A
possible explanation is that ATP levels are result of cell fitness rather than a determinant; R1
levels could be normal in monoculture but decrease upon competition. Alternatively, other
products of energy metabolism could be involved in cell fitness determination. Further studies
will be needed to characterize the role of energy metabolism in cell fitness, for instance by
using living imaging techniques to monitor cell metabolism during cell competition.
In summary, we found that competitive behavior is a general property of common
mammalian cell lines, supporting the notion that this phenomenon plays a role in maintaining the
growth properties of mammalian cell populations by weeding out “unfit” cells. One interesting
feature of our study is that all isolated clones behaved as losers when combined with the parental
population; no spontaneous “supercompetitors” were isolated. This suggests that the process of
immortalization and culture selects for populations of cells which exhibit a high fitness level at
steady-state. Hence, our observations may have implications for cancer, in which tumor
evolution may be driven in part by the eradication of less-fit cells through such interactions.
U2OS cells (American Tissue and Cell Collection, HTB-96) were a kind gift of Dr. John
Hogenesh (University of Pennsylvania) NIH-3T3 (CRL-1658), MDCK (CCL-34) and 293T cells
(CRL-3216) were directly obtained from ATCC. All cell lines were cultured in 10% Dulbecco’s
modified Eagle medium supplemented to 10% decomplemented fetal bovine serum at 37°C,
5% CO2, 21% O2 and 100% humidity unless otherwise indicated. For aminoacid depletion
experiments, dialyzed FBS was used and L-glutamine was supplemented as indicated. Cell lines
were maintained and passaged according to ATCC recommended procedures.
Pharmacological agents was as follows: Purvalanol-A (Sigma P4484), Z-VAD-FMK (EMD Millipore
627610), actinomycin D (Sigma A9415), cycloheximide (Sigma C7698), NAC (Sigma A9165),
Trolox (Santa Cruz Biotech sc-200810) and CCCP (Sigma C2759) were added to cell cultures
12 h after cell seeding at the concentrations indicated and maintained until analysis, changing
the medium every 24 hours. Ethynyl-uridine (Life Technologies E10345) and
homopropargylglycine (Life Technologies C10186) were added at 5 and 50 μM respectively, two hours prior to
incorporation analysis using the Click-iT alkyne detection kit (Life Technologies C10330).
Transfection and selection of stable transfectant clones
Transfection was carried out using Fugene6 (Promega E2691). Selection of stable transfectant
clones was obtained with G418 sulfate at 1 mg/ml (U2OS) or 0.5 mg/ml (3T3, MDCK),or
hygromycin B at 150 μg/ml as appropriate. Stable transfectant clones were individually
recovered from the selection plates using a fine pipette, amplified and cultured under standard
Heterokaryons were obtained by treating confluent cells with 50% PEG 1500 in 75 mM HEPES
pH 8.0 for 1 minute followed by G418 + neomycin selection for 3 days. In contrast to stable
transfection, individual clones were not separated after cell fusion, Heterokaryon sub-lines
thus consisted of mixed populations resulting from many independent fusion events.
Heterokaryon sub-lines were passage 3 times before using them in cell competition assays.
Lentiviral transduction of single shRNAs
Cells were seeded at ~70% confluence and grown for 18 h before adding concentrated viral
particles (described below), and further grown for 72 h. Selection of infected shRNA-exressing
cells was then carried out using puromycin (5 μg/ml) for 48 h.
For U2OS clone growth characterization, cells were seeded at a density of 6,600 cells/cm2. For
U2OS, MDCK and 3T3competition assays, cells were seeded at 66 x 105 cells/cm2, which
results in 95% confluence after attachment and spreading. Cell seeding was considered t = 0 for
all experiments and medium was changed every 48 hours. At indicated timepoints, cells were
trypsinized and counted using an Accuri C6 flow cytometer. For the Z-VAD-FMK apoptosis
inhibition experiments, cells were counted manually in 3 independent 10X microscope field.
Cells were seeded at 66,000 cells/cm2 (t = 0h) as mono-cultures or 1:1 co-cultures, which
results in ~95% confluence after cell spreading in U2OS, 3T3 and MDCK cells. Apoptosis was
analyzed after 1, 3 or 6 days in culture or as otherwise indicated by immunostaining with a
cleaved (Asp175) Caspase-3 rabbit polyclonal antibody (Cell Signaling technologies 9661).
Cleaved Caspase3-positive cells were counted on three independent 10X microscope fields for
each sample and averaged. Growth curves were obtained by counting total cell numbers using
an Accuri C6 cytometer. All assays were carried out in triplicate, and statistical significance of
cell count comparisons was determined by Student’s t test.
U2OS R1 cells were spot-seeded at 106 cells/ml on glass-bottom culture dishes (Mat-Tek
Corporation), allowed to attach, and overlaid with YFP cells (106 cells/ml). Cells were maintained
at 37°C, 0.5% CO2 and 100% humidity while filmed at 15 min./frame using a Leica DM6000 B
Whole-transcriptome expression analysis
U2OS cells were grown as mono- or co-cultures for 48 h and separated on a FACSAria cell
sorter (BD biosciences). RNA was isolated using the RNeasy kit (Qiagen) and hybridized to
Affymatrix Human Gene 1.0 ST arrays at the Perelman School of Medicine Molecular Profiling
Core (University of Pennsylvania). Each experimental condition was carried out in triplicate.
Greater than 2-fold RNA expression changes were identified by 2-way (sample, replicate)
ANOVA. Statistical analysis and hierarchical clustering was carried out using Partek Genomic
Suite software (Partek Inc.). Gene ontology enrichment analysis was performed using the Gene
Set Analysis Toolkit V2 (Gene Ontology Consortium). Microarray data are available in the
ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3204.
H2B-YFP (Venus), H2B-EGFP, and H2B-mCherry sequences were obtained from Addgene
(20971, 11680 and 20972, respectively) and cloned into pcDNA3.1-Neor or pcDNA3.1-Hygror
backbones. These pCMV-H2B-XFP plasmids were used in U2OS YFP, G1-3 and R1 cells. For
U2OS, 3T3 and MDCK clone screens, the promoter of pCMV-H2B-EGFP was replaced with
the Ubiquitin-C promoter (pUB-GFP, Addgene 11155). The LSL-Myc and LSL-Yap plasmids
were created in pcDNA 3.1 using a LoxP-(3x pSV40 polyA)-LoxP cassette, the Myc and Yap
sequences were coded in-frame with the picornavirus 2A self-cleaving peptide and the
H2B-EGFP sequences in order to allow bi-cistronic MYC/YAP and GFP expression. pMC-Cre
has been described , and was obtained from Dr. Eric Brown (University of Pennsylvania).
shRNA lentiviral vectors and viral particle preparation
pGIPZ shRNAs against Hif1a (shRNA1, RHS4430-98513964; shRNA2, RHS4430-101518881),
YAP (RHS4430-98525388), Myc (RHS4430-98853488), Scribbled (RHS4430-98901643) and a
non-silencing control (NSC, RHS4348) clones are part of the pGIPZ human whole
transcriptome shRNA library (GE Dharmacon) and were kindly provided by Dr. Patrick Paddison
(Fred Hutchinson Cancer Center). Lentiviral particles were obtained in 293T cells using the
pSPAX2 (Addgene 12260) and pMD2.G (Addgene 12259) packing vector, following the
standard procedures. For each lentiviral preparation, 60 x 106 cells were transfected and cultured
for 72 hours. Lentiviral particles in the medium were then precipitated using the Lenti-X
concentrator reagent (Clontech 631231) and reconstituted in 1 ml of complete medium.
Myc, YAP, and Scribbled expression was analyzed using the Superscript RT kit (Life
Technologies 18080–051) and the SSoadvanced SYBRGreen qPCR mix (BioRad 172–5260) with the
following primers pairs: MYC (GTAGTGGAAAACCAGCAGCCT, AGAAATACGGCTGCACC
GAG); YAP (TGACCCTCGTTTTGCCATGA, GTTGCTGCTGGTTGGAGTTG) Scribbled
(AGGAGATCTACCGCTACAG, GATCTCAGGGATATCGTTCC); HIF1A (GTGAAGAC
ATCGCGGGGA, GTGGCAACTGATGAGCAAGC); GAPDH (GGTGAAGGTCGGAGTCA
Cells were grown in 12-well plates as monocultures for 72 hours under normoxic (21% O2) or
hypoxic (1.5% O2) conditions. Intracellular ATP was extracted by boiling water method as
described previously by Yang et al.  with modification. Briefly, cells in 12-well plates were
washed with cold PBS twice followed by adding 1 ml of boiling water to the wells directly. After
repeated pipetting, cell extracts were cleared by centrifugation at 12000g for 5 min at 4°C. ATP
levels in the supernatants were measured using the ENLITEN ATP Assay System
Bioluminescence Detection Kit (Promega) following the manufacturer’s instructions. Total protein
concentration was determined using the DC Protein Assay (BioRad) following the manufacturer’s
S1 Fig. Correlation between growth and fitness in U2OS cell clones. Growth curves of U2OS
clones identified in the U2OS screen. Clones are grouped according to the outcome of
co-culture with Wt, R1, and YFP cells (see Fig 3). High-fitness clones are shown in blue. They do not
compete with Wt and behave as winners in the presence of R1. Clones shown in black behave
S2 Fig. Spontaneous cell competition in MDCK cells. (A) Growth curves of wild-type
MDCK cells and H2B-GFP transfectant, single-cell derived 10D2 clone in mono- or co-culture.
(B) Apoptosis quantification by Cp3-IF. 10D2 cells undergo increased apoptosis resulting in
10D2 cell number decrease in 10D2:Wt co-cultures.
S4 Fig. Localized cell competition in U2OS-YFP:R1 spot cultures. (A) Schematic
representation of spotted U2OS-R1 cells overlaid with U2OS-YFP cells. (B) Time-course micrographies
of spot cultures. Detail of areas labeled as “1” and “2” is presented in (C) and (D), respectively.
Progressive elimination of U2OS-YFP cells inside the R1 spot can be observed, but the spot
boundary remains in place, indicating that YFP cells outside are still increasing in number.
S5 Fig. Myc and YAP overexpression does not turn U2OS cells into supercompetitors. (A)
Schematic representation of the inducible LSL-Myc-GFP expression cassette. Cre-mediated
recombination results in excision of the SV40 polyadenylation signal (pA), placing the
Myc2A-GFP coding sequence under control of the CMV-actin hybrid promoter (Caggs). The Myc
and GFP sequences are separated by the picornavirus 2A self-cleaving sequence, resulting in
bi-cistronic MYC and GFP expression. The LSL-YAP-GFP expression cassette was similarly
constructed by replacing the Myc coding sequence with that of the constitutive YAPS117A
mutant. (B) Aspect of LSL-Myc-GFP and LSL-YAP-GFP stable transfectant U2OS cells
transiently transfected with a Cre expression vector (pMC-Cre). Cp3 IF quantification of apoptosis
is shown in (C). Apoptosis rates in untransfected cells is similar or lower than that observed in
Cre-transfected, Myc/YAP-GFP expressing cells, indicating that Myc and YAP expression does
not confer supercompetitor status to U2OS cells.
S6 Fig. Inactivation of Myc, YAP, or Scribbled does not result in competition in U2OS
cells. (A) Growth curves of lentivirus-transduced U2OS cells expressing shRNAs directed
against Myc, YAP, or Scribbled; cultured alone or alongside Wt cells. shRNA-expresing cells
are recognized by means of a GFP expression cassette contained in the lentiviral shRNA vector
(not shown). None of these shRNAs induced cell competition. (B) qPCR analysis of gene
expression showing reduced Myc, YAP, and Scribbled RNA levels in shRNA-expressing cells.
: p<0.001 (Student’s t-test).
S7 Fig. U2OS cell fitness is determined by intrinsic, dose-dependent determinants. (A)
U2OS “winner-loser” cell fusion experimental design. Single-cell derived, H2B-mCherry,
hygror stable transfectant clones (RH) were fused to YFP (neor) cells to generate RY cell lines.
RY cell fitness levels were then tested by co-culturing RY cells with either Wt or YFP cells. (B)
Fluorescence micrographs and flow cytometry profiles of U2OS RY1 cells 4 days after fusion.
The result is typical of observed cell fusion outcomes. Separate images of RY1 cell mCherry
and YFP fluorescence are shown on the far right. Flow cytometry profiles showing YFP and
mCherry co-expression in >95% of RY1 cells are displayed at the bottom. (C) Cp3 IF apoptosis
analysis in 72 hour Wt, RH, RY and YFP cultures. Results shown are representative of 3
independent fusion experiments for each RH:YFP pairing. All three RH cell lines behave as
“winners” in the presence of YFP cells. RH1:YFP fusion results in a fully “winner” line indicating
that YFP “loser” status is rescued and therefore results from loss of function. However, RH4:
YFP results in partial rescue and RH7:YFP fusion results in no rescue at all, suggesting that
multiple factors determine U2OS cell “fitness”.
S8 Fig. Expression levels across U2OS-Wt, R1, and YFP correlate with “fitness” level in a
small subset of genes. (A) Venn diagram distribution of transcripts displaying a 2-fold or
greater expression level difference in Wt vs. R1 vs.YFP cells by whole transcriptome microarray
analysis (see Fig 4 for detail of experimental design). (B) Hierarchical clustering of expression
profiles of the 422 transcripts displaying a greater than 2-fold expression level change in the
Wt vs YFP comparison. Three clusters matching the profile Wt>R1>YFP and 2 clusters
matching YFP>R1>Wt were identified (a total of 82 transcripts), suggesting that these
transcripts could play a role as “fitness” determinants.
S9 Fig. Transcription inhibition reduces proliferation in U2OS cells. (A) PH3 IF (red) of 48
h U2OS cell cultures grown in presences or absence of actinomycind D or cycloheximide as
indicated. Actinomycin D treatment partially inhibits proliferation, suggesting that partial
rescue of YFP cell elimination in treated Wt:YFP cultures is due to decreased overall culture
growth (See Fig 5). Cycloheximide treatment results in complete proliferation blockade within
48 hours, along with cell competition arrest (Fig 5).
S10 Fig. U2OS cell competition does not involve serum-borne factors. Cp3 IF analysis of
apoptosis in 72 hour U2OS cultures grown in medium supplemented with fetal bovine serum
at the concentrations indicated. Serum concentration does not affect apoptosis rates in R1 and
YFP cells cultured alone or in alongside with Wt cells.
S11 Fig. U2OS cell competition does not involve production of radical oxygen species
(ROS). Cp3 IF analysis of apoptosis in 72 h U2OS cultures treated with raical ROS scavengers
N-acetyl cysteine (NAC) (A) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox) (B), as indicated. ROS scavengers do not reduce loser cell apoptosis rates in Wt:R1 or
Wt:YFP co-cultures. A slight increase is observed in competing R1 cells at the highest Trolox
concentration. :p<0.05 (Student’s t-test).
S1 File. U2OS cell competition is mediated by short-range cell interactions. Time-lapse
microscospy video of R1:YFP spot co-cultures over 72 hours (1 frame = 15 min). Note that
YFP (green nuclei) are progressively eliminated from the R1 (red nuclei) spot; however, the
YFP cells outside the spot are not eliminated. Also note that in most cases death of YFP cells in
the R1 spot is not preceded by cell detachment.
S1 Table. RNA expression changes mono-cultured vs. competing U2OS YFP cells.
Wholetranscriptome microarray hybridization analysis of RNA expression levels in U2OS YFP cells
grown for 48 hours as monocultures (YFPmono) or as 1:1 co-cultures with WT (YFPco-Wt) or
R1 (YFPco-R1) cells. Expression levels changes were compared as indicated by 2-way ANOVA
(culture condition, replicate). Transcripts displaying a >2-fold change in expression are listed.
S2 Table. Metalloprotease-encoding genes differentially expressed in Wt and YFP cells.
Gene Ontology (GO) term enrichment analysis of the 82-gene potential competition
determinant list. C = 179; O = 7; E = 0.80; R = 8.79; rawP = 1.41e-05; adjP = 0.0007. C, total genes in
the GO category, O, occurrence in subject group, rawP, unadjusted P-value, adjP, false
discovery adjusted P value, FC, fold change.
Conceived and designed the experiments: APM YJC JL ESW BZS. Performed the experiments:
APM YJC JL. Analyzed the data: APM YJC JL BZS. Contributed reagents/materials/analysis
tools: ESW. Wrote the paper: APM BZS.
1. Amoyel M , Bach EA . Cell competition: how to eliminate your neighbours . Development . 2014 ; 141 ( 5 ): 988 - 1000 . PMID: 24550108. doi: 10.1242/dev.079129
2. Levayer R , Moreno E. Mechanisms of cell competition: themes and variations . J Cell Biol . 2013 ; 200 ( 6 ): 689 - 98 . PMID: 23509066. doi: 10.1083/jcb.201301051
3. de Beco S , Ziosi M , Johnston LA . New frontiers in cell competition . Dev Dyn . 2012 ; 241 ( 5 ): 831 - 41 . doi: 10.1002/dvdy.23783 PMID: 22438309.
4. de la Cova C , Abril M , Bellosta P , Gallant P , Johnston LA . Drosophila myc regulates organ size by inducing cell competition . Cell . 2004 ; 117 ( 1 ): 107 - 16 . PMID: 15066286 .
5. Johnston LA . Socializing with MYC: Cell Competition in Development and as a Model for Premalignant Cancer. Cold Spring Harbor perspectives in medicine . 2014 ; 4 ( 4 ). PMID: 24692189.
6. Moreno E , Basler K. dMyc transforms cells into super-competitors . Cell . 2004 ; 117 ( 1 ): 117 - 29 . PMID: 15066287 .
7. Brumby AM , Richardson HE. scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila . EMBO J . 2003 ; 22 ( 21 ): 5769 - 79 . doi: 10.1093/emboj/cdg548 PMID: 14592975 ; PubMed Central PMCID : PMCPMC275405 .
8. Igaki T , Pagliarini RA , Xu T. Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila . Curr Biol . 2006 ; 16 ( 11 ): 1139 - 46 . doi: 10.1016/j.cub. 2006 .04.042 PMID: 16753569.
9. Pagliarini RA , Xu T. A genetic screen in Drosophila for metastatic behavior . Science . 2003 ; 302 ( 5648 ): 1227 - 31 . doi: 10.1126/science.1088474 PMID: 14551319.
10. Moreno E , Basler K , Morata G . Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development . Nature . 2002 ; 416 ( 6882 ): 755 - 9 . doi: 10.1038/416755a PMID: 11961558.
11. Rodrigues AB , Zoranovic T , Ayala-Camargo A , Grewal S , Reyes-Robles T , Krasny M , et al. Activated STAT regulates growth and induces competitive interactions independently of Myc, Yorkie, Wingless and ribosome biogenesis . Development . 2012 ; 139 ( 21 ): 4051 - 61 . doi: 10.1242/dev.076760 PMID: 22992954.
12. Vincent JP , Kolahgar G , Gagliardi M , Piddini E. Steep differences in wingless signaling trigger Mycindependent competitive cell interactions . Dev Cell . 2011 ; 21 ( 2 ): 366 - 74 . doi: 10.1016/j.devcel. 2011 . 06.021 PMID: 21839923.
13. Oliver ER , Saunders TL , Tarlé SA , Glaser T. Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse Minute . Development. 2004 ; 131 ( 16 ): 3907 - 20 . doi: 10.1242/dev.01268 PMID: 15289434.
14. Clavería C , Giovinazzo G , Sierra R , Torres M. Myc-driven endogenous cell competition in the early mammalian embryo . Nature . 2013 ; 500 ( 7460 ): 39 - 44 . doi: 10.1038/nature12389 PMID: 23842495.
15. Sancho M , Di-Gregorio A , George N , Pozzi S , Sánchez JM , Pernaute B , et al. Competitive interactions eliminate unfit embryonic stem cells at the onset of differentiation . Dev Cell . 2013 ; 26 ( 1 ): 19 - 30 . doi: 10. 1016/j.devcel. 2013 .06.012 PMID: 23867226.
16. Dabeva MD , Petkov PM , Sandhu J , Oren R , Laconi E , Hurston E , et al. Proliferation and differentiation of fetal liver epithelial progenitor cells after transplantation into adult rat liver . Am J Pathol . 2000 ; 156 ( 6 ): 2017 - 31 . doi: 10. 1016/s0002-9440(10)65074-2 PMID: 10854224.
17. Oertel M , Menthena A , Dabeva MD , Shafritz DA . Cell competition leads to a high level of normal liver reconstitution by transplanted fetal liver stem/progenitor cells . Gastroenterology . 2006 ; 130 ( 2 ): 507 - 20 ; quiz 90. doi: 10.1053/j.gastro. 2005 .10.049 PMID: 16472603.
18. Norman M , Wisniewska KA , Lawrenson K , Garcia-Miranda P , Tada M , Kajita M , et al. Loss of Scribble causes cell competition in mammalian cells . J Cell Sci . 2012 ; 125 (Pt 1): 59 - 66 . doi: 10.1242/jcs.085803 PMID: 22250205.
19. Tamori Y , Bialucha CU , Tian AG , Kajita M , Huang YC , Norman M , et al. Involvement of Lgl and Mahjong/VprBP in cell competition . PLoS Biol . 2010 ; 8 ( 7 ) :e1000422 . doi: 10.1371/journal. pbio.1000422 PMID: 20644714.
20. Senoo-Matsuda N , Johnston LA . Soluble factors mediate competitive and cooperative interactions between cells expressing different levels of Drosophila Myc . Proc Natl Acad Sci U S A . 2007 ; 104 ( 47 ): 18543 - 8 . doi: 10.1073/pnas.0709021104 PMID: 18000039.
21. Chen CL , Schroeder MC , Kango-Singh M , Tao C , Halder G . Tumor suppression by cell competition through regulation of the Hippo pathway . Proc Natl Acad Sci U S A . 2012 ; 109 ( 2 ): 484 - 9 . doi: 10.1073/ pnas.1113882109 PMID: 22190496; PubMed Central PMCID: PMCPMC3258595.
22. Tyler DM , Li W , Zhuo N , Pellock B , Baker NE . Genes affecting cell competition in Drosophila . Genetics. 2007 ; 175 ( 2 ): 643 - 57 . doi: 10.1534/genetics.106.061929 PMID: 17110495; PubMed Central PMCID: PMCPMC1800612.
23. Neto-Silva RM , de Beco S , Johnston LA. Evidence for a growth-stabilizing regulatory feedback mechanism between Myc and Yorkie, the Drosophila homolog of Yap . Dev Cell . 2010 ; 19 ( 4 ): 507 - 20 . doi: 10. 1016/j.devcel. 2010 .09.009 PMID: 20951343; PubMed Central PMCID: PMCPMC2965774.
24. Ziosi M , Baena-López LA , Grifoni D , Froldi F , Pession A , Garoia F , et al. dMyc functions downstream of Yorkie to promote the supercompetitive behavior of hippo pathway mutant cells . PLoS Genet . 2010 ; 6 ( 9 ) :e1001140 . doi: 10.1371/journal.pgen.1001140 PMID: 20885789; PubMed Central PMCID: PMCPMC2944792.
25. Bacon AL , Harris AL . Hypoxia-inducible factors and hypoxic cell death in tumour physiology . Ann Med . 2004 ; 36 ( 7 ): 530 - 9 . Epub 2004 /10/30. doi: 10.1080/07853890410018231 PMID: 15513303.
26. Bondar T , Medzhitov R. p53-mediated hematopoietic stem and progenitor cell competition . Cell Stem Cell . 2010 ; 6 ( 4 ): 309 - 22 . doi: 10.1016/j.stem. 2010 .03.002 PMID: 20362536.
27. Morata G , Ripoll P. Minutes: mutants of drosophila autonomously affecting cell division rate . Dev Biol . 1975 ; 42 ( 2 ): 211 - 21 . PMID: 1116643 .
28. Lolo FN , Casas-Tintó S , Moreno E. Cell competition time line: winners kill losers, which are extruded and engulfed by hemocytes . Cell Rep . 2012 ; 2 ( 3 ): 526 - 39 . doi: 10.1016/j.celrep. 2012 .08.012 PMID: 22981235.
29. Díaz B , Moreno E. The competitive nature of cells . Exp Cell Res . 2005 ; 306 ( 2 ): 317 - 22 . doi: 10.1016/j. yexcr. 2005 .03.017 PMID: 15925586.
30. Rhiner C , López-Gay JM , Soldini D , Casas-Tinto S , Martín FA , Lombardía L , et al. Flower forms an extracellular code that reveals the fitness of a cell to its neighbors in Drosophila . Dev Cell . 2010 ; 18 ( 6 ): 985 - 98 . doi: 10.1016/j.devcel. 2010 .05.010 PMID: 20627080.
31. Petrova E , López-Gay JM , Rhiner C , Moreno E. Flower-deficient mice have reduced susceptibility to skin papilloma formation . Dis Model Mech . 2012 ; 5 ( 4 ): 553 - 61 . doi: 10.1242/dmm.008623 PMID: 22362363; PubMed Central PMCID: PMCPMC3380718.
32. de la Cova C , Senoo-Matsuda N , Ziosi M , Wu DC , Bellosta P , Quinzii CM , et al. Supercompetitor Status of Drosophila Myc Cells Requires p53 as a Fitness Sensor to Reprogram Metabolism and Promote Viability . Cell Metab . 2014 ; 19 ( 3 ): 470 - 83 . doi: 10.1016/j.cmet. 2014 .01.012 PMID: 24561262; PubMed Central PMCID: PMCPMC3970267.
33. Gu H , Zou YR , Rajewsky K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting . Cell . 1993 ; 73 ( 6 ): 1155 - 64 . PMID: 8513499 .
34. Yang NC , Ho WM , Chen YH , Hu ML . A convenient one-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay of ATP . Anal Biochem . 2002 ; 306 ( 2 ): 323 - 7 . PMID: 12123672.