Myc-induced cell mixing is required for competitive tissue invasion and destruction

Cell-cell intercalation is used in several developmental processes to shape the normal body plan1. There is no clear evidence that intercalation is involved in pathologies. Here, we use the proto-oncogene Myc to study a process analogous to early phase of tumour expansion: Myc-induced cell competition2-7. Cell competition is a conserved mechanism5,6,8,9 driving the elimination of slow proliferating cells (so called losers) by faster proliferating neighbours (so called winners) through apoptosis10 and is important to prevent developmental malformations and maintain tissue fitness11. Using long term live imaging of Myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, we show that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser/winner interface morphology can modulate the strength of competition. We further show that elimination of loser clones requires winner/loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilisation of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference of junctional F-actin levels between loser and winner junctions, induced by differential levels of the phosphatidylinositol PIP3. Our results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues.

phenomenon, we systematically counted the proportion of clones fragmented 48 hours after clone induction (Fig. 2c, see Methods and EDFig.4a,b). We observed a two fold increase of the frequency of split clones in losers (wt in tub-dmyc) versus wt in wt controls (Fig. 2d, EDFig.4c). Overexpressing E-cad or active MyoII was sufficient to prevent loser clone splitting while blocking apoptosis or blocking loser fate by silencing fwe lose did not reduce splitting (Fig. 2d, EDFig.4c). Finally, the proportion of split clones was also increased for winner clones either during Myc-driven competition (UAS-myc, UAS-p35, Fig. 2d, EDFig.4c) or during Minute-dependent competition 2 (wt clones in M -/+ background, Fig. 2d, EDFig.4c).
Altogether, this suggested that winner/loser mixing is increased independently of loser cell death or clone size (EDFig.4d) by a factor upstream of fwe, and could be driven by cell-cell intercalation. Accordingly, junction remodelling events leading to disappearance of a loserloser junction were three times more frequent at loser clone boundaries compared to control clone boundaries in the pupal notum ( Fig. 2e,f , p<10 -4 , Video S9). The rate of junction remodelling was higher in loser-loser junctions and in winner-winner junctions compared to winner-loser junctions (EDFig.5a,b). The preferential stabilisation of winner-loser interfaces should increase the surface of contact between winner and loser cells over time. Accordingly, loser clone compactness in the notum was decreasing over time while it remains constant on average for wt clones in wt background (Fig. 2g,h, p<10 -4 ). Similarly, the compactness of clones in the notum was also decreasing over time for conditions showing high frequency of clone splitting in the wing disc, while clone compactness remained constant for conditions rescuing clone splitting (compare Fig. 2d with EDFig.5 d,e, Videos S1-S11). Altogether, we concluded that both Minute and Myc-dependent competition increase loser/winner mixing through cell-cell intercalation.
We then asked what could modulate the rate of junction remodelling during competition. The rate of junction remodelling can be cell-autonomously increased by Myc (EDFig.5c, Video S12). Interestingly, downregulation of the tumour suppressor PTEN is also sufficient to increase the rate of junction remodelling 18 through the upregulation of the phosphatidylinositol PIP3. We reasoned that differences in PIP3 levels could also modulate junction remodelling during competition. Using a live reporter of PIP3 which can detect modulations of PIP3 in the notum (EDFig.6a,b), we observed a significant increase of PIP3 in the apico-lateral membrane of tub-dmyc/tub-dmyc interfaces compared to wt/wt and wt/tub-dmyc interfaces (Fig. 3 a,b). Moreover, increasing/reducing Myc levels in a full compartment of the wing disc was sufficient to increase/decrease the levels of phospho-Akt (a downstream target of PIP3 19 , Fig. 3c) while fwe loseA overexpression had no effect (EDFig.6c). Similarly, levels of phospho-Akt were relatively higher in wt clones compared to the surrounding M -/+ cells (EDFig.6d). Thus differences in PIP3 levels might be responsible for winner-loser mixing. Accordingly, reducing PIP3 levels by overexpressing a PI3K-DN (PI3

Kinase Dominant Negative) or increasing PIP3 levels by knocking down PTEN (UAS-pten
RNAi) were both sufficient to induce a high proportion of fragmented clones (Fig. 3d,e) and to reduce clone compactness over time in the notum (EDFig.5d,e, Video S13, S14), while increasing PIP3 in loser clones was sufficient to prevent cell mixing (Fig. 3d,e). Moreover, abolishing winner-loser PIP3 differences through larval starvation ( 20 , EDFig.6e-g) prevented loser clone fragmentation (EDFig.6h,i), the reduction of clone compactness over time in the notum (EDFig.5d,e, Video S15) and could rescue wt clone elimination in tub-dmyc background (EDFig.6 j,k). We therefore concluded that differences in PIP3 levels are necessary and sufficient for loser-winner mixing and required for loser cell elimination.
We then asked which downstream effectors of PIP3 could affect junction stability. A relative growth decrease can generate mechanical stress 21,22 that can be released by cell-cell intercalation. Accordingly, growth reduction through Akt downregulation is sufficient to increase clone splitting (EDFig.7 a,b) and could contribute to loser clone splitting. However, Akt is not sufficient to explain winner-loser mixing as unlike PIP3, increasing Akt had no effect on clone splitting (EDFig.7 a,b). PIP3 could also modulate junction remodelling through its effect on cytoskeleton 23 and the modulation of intercellular adhesion or tension 1 .
This effect was specific of Dia as modulating Arp2/3 complex (a regulator of dendritic actin network 13 ) had no effect on clone splitting (Fig.4d, EDFig.9b). Thus, impaired filamentous actin organisation was necessary and sufficient to drive loser-winner mixing. These actin defects were driven by the differences in PIP3 levels between losers and winners (see EDFig.10). Thus Diaphanous could be an important regulator of competition through its effect on cell mixing. Overexpression of Dia was indeed sufficient to significantly reduce loser clone elimination (EDFig.9d) without affecting Hippo/YAP-TAZ pathway 25 (EDFig.9e).
Filamentous actin has been associated with tension regulation 13 . We therefore asked whether junction tension was modified in winner and loser junctions. The maximum speed of relaxation of junction following laser nanoablation (which is proportional to tension 26 ) was significantly reduced in loser-loser and winner-loser junctions compared to winner-winner junctions (Fig. 4e,f, Video S20). This distribution of tension has been proposed to promote cell mixing 27 . Accordingly, decreasing PIP3 in clones reduced tension both in low-PIP3/low-PIP3 and low-PIP3/normal-PIP3 junctions, while overexpressing Diaphanous in loser clones or starvation were both sufficient to abolish differences in tension (Fig. 4f, Videos S21-S23), in agreement with their effect on winner/loser mixing and the distribution of F-actin. Thus the lower tension at winner/loser and loser/loser junctions is responsible for winner/loser mixing.
Altogether, we concluded that the relative PIP3 decrease in losers increases winner-loser mixing through Akt-dependent differential growth and the modulation of tension through Factin downregulation in winner/loser and loser/loser junctions (Fig. 4g).
Several modes of tissue invasion by cancer cells have been described 28 , most of them relying on the departure of the tumour cells from the epithelial layer 29 . This study suggests that some oncogenes may also drive tissue destruction and invasion by inducing ectopic cell intercalation between cancerous and healthy cells, and subsequent healthy cell elimination. Myc dependent invasion could be enhanced by other mutations further promoting intercalation (such as PTEN). Stiffness is increased in many tumours 30 , suggesting that healthy cells/cancer cells mixing by intercalation might be a general process.  Bottom: ratio of average number of dead cells in the ROI over the total number of dead cells in the wing pouch. The white box is the expected ratio for a random distribution (ROI surface/ total wing pouch surface). p=Mann-Whitney test.

Proposed model for winner-loser mixing
Two forces contribute to winner-loser mixing: 1. The relative decrease of growth of loser clones. Relative growth decrease leads to clone stretching 21,22 which could be released by cell-cell intercalation. Accordingly, we have shown that Akt downregulation is sufficient to increase clone splitting (EDFig. 7a,b).
2. The relative decrease of F-actin and tension in the loser-loser and winner-loser junctions (Fig. 4g). Overtime winner-loser junctions should get longer due to the relative lower tension, while winner-winner contacts should shrink due to higher tension. Consequently winner cells in contact with loser cells tend to get intermingled with loser cells, which can sometimes lead to cell-cell intercalation and separation of the winner cell from the rest of the winners.
The combination of the two forces increases the surface of contact between winners and losers and accelerates the elimination of loser cells by increasing the probability to induce loser apoptosis.

Fly stocks and clone induction
The following stocks were used in this study: hs-flp22; tub<dmyc<Gal4; UAS-mcd8::GFP 4 , after clone induction, washed in distilled water, and left on a humidified paper without food for 24h (48h for imaging pupae). Pupae were dissected and mounted as indicated in 43 48 or 72h after clone induction.
Activation of progesterone sensitive gal4 (gal4 switch) was performed by using fly food mixed with RU486 at 1µg/ml or 50µg/ml. The full development was occurring in the hormone containing food (egg laying and larval development). Act<cd2<Gal4 clones were induced with a 8min heat shock.

Generation of Flower loseA ::mcherry knock in.
The Flower knock in fly was made by genomic engineering 41

Pupal notum and wing disc live imaging, death probability calculation
Pupae were collected 48 or 72h after clone induction and dissected 18 to 20h after pupae formation (APF). Pupae were prepared as indicated in 43  For every x-y pixel, the z-plane with the maximal Ecad::GFP intensity (calculated by summing pixel intensity on a 50 by 50 px square at every plane) was kept and also used to retrieve RFP signal in the same plane. The measurement of death probability was performed like in the notum. Imaging of fwe loseA ::mcherry KI was also performed in ex-vivo wing disc using the same projection procedure.
Junction remodelling were manually counted at the interface of the clones. Each event leading to the disappearance of a junction between two RFP positive cells was counted.
Remodelling events were only counted if the new topology was maintained until the end of the movie (10h, Fig. 2f and EDFig.5c). The total number of remodelling events was then divided by the total number of junctions analysed. For EDFig.5a, we counted every remodelling event occurring over 10h for single junctions, and calculate the proportion of junctions undergoing a single remodelling event, and the probability to undergo additional remodelling event (after a first remodelling event). All winner-winner junctions tracked were sharing one vertex with a loser cell, while loser-loser junctions tracked were also sharing one vertex with a winner cell. All winner-loser junctions were tracked.

Image quantification
Clone fragmentation, clone size and clone compactness Clone compactness is defined as followed:

C=4.Π.(Area / Perimeter 2 )
Clone compactness was measured for every clone in the notum by drawing their contour using Fiji at t0 and 10 hours later. The fold change was calculated as followed: (C 10h -C 0h )/C 0h .

Laser ablation
Junction laser ablations were performed in the notum using a two-photon infrared laser on a 2-photon microscope (Two-photon Fluoview 1000 Olympus, Center for Microscopy and Image Analysis, University of Zurich) using a 25X water objective (N.A. 1.05). Imaging and ablation were performed with 950nm wavelength, 1.3% power for imaging, 30% power and 300ms exposition for the ablation scanning along a line perpendicular to the junction.
Relaxation of vertices (visualised with E-cad::GFP) was tracked for at least 10sec (1 frame/0.594sec, 1 frame/0.891sec for Fig. 4f "starved"). Vertices position was then tracked using CellTrack software 50 and the evolution of vertices distance over time was fitted with an exponential function ( A 0 +A 1 .exp(A 2 .t) ) on the ten first time point following ablation using Igor software. The V max were the derivative of the exponential fit at t0.