Multiple strategies of oxygen supply in Drosophila malignancies identify tracheogenesis as a novel cancer hallmark

Angiogenesis is the term used to describe all the alterations in blood vessel growth induced by a tumour mass following hypoxic stress. The occurrence of multiple strategies of vessel recruitment favours drug resistance, greatly complicating the treatment of certain tumours. In Drosophila, oxygen is conveyed to the internal organs by the tracheal system, a closed tubular network whose role in cancer growth is so far unexplored. We found that, as observed in human cancers, Drosophila malignant cells suffer from oxygen shortage, release pro-tracheogenic factors, co-opt nearby vessels and get incorporated into the tracheal walls. We also found that the parallelisms observed in cellular behaviours are supported by genetic and molecular conservation. Finally, we identified a molecular circuitry associated with the differentiation of cancer cells into tracheal cells. In summary, our findings identify tracheogenesis as a novel cancer hallmark in Drosophila, further expanding the power of the fly model in cancer research.

C ancer is a complex and extremely heterogeneous disease, and huge efforts have been made to devise a conceptual framework in which to gather the recurring phenotypic traits associated with tumour onset and progression 1 . The most part of these traits has been successfully investigated in cellular and animal models; in particular, thanks to the development of specific genetic tools, the fruitfly Drosophila melanogaster has greatly served the purpose. In the last decade, Drosophila models of epithelial cancers have indeed helped elucidate fundamental aspects of tumour biology, such as the mechanisms linking cell polarity to proliferation control 2 . The regulation of epithelial cell polarity is a crucial issue in cancer biology, as its loss correlates with invasive and metastatic abilities of cancer cells and is strongly associated with a poor prognosis 3 . In Drosophila, lethal giant larvae (lgl), discs large (dlg) and scribble (scrib) are a class of tumour suppressor genes (TSGs) that encode functionally conserved proteins involved in the maintenance of epithelial apical-basal polarity as well as in proliferation control 4 . Their loss-of-function (LOF) phenotypes are especially evident in the imaginal discs, larval epithelial organs that give rise to adult structures following metamorphosis, which show unrestrained growth, complete loss of tissue architecture and ability to fuse with nearby tissues, so displaying local invasiveness 4 . Despite the obvious malignant traits shown by the mutant organs, when mutation in one of these genes is induced clonally, that is in single cells within a wild-type tissue, such mutant cells undergo apoptotic death triggered by non cell-autonomous safeguard mechanisms named ''cell competition'' 5 or ''intrinsic tumour suppression'' 6 . Since clonal induction is however the genetic system that best mimics mammalian cancer onset, several models of cooperative tumourigenesis have been established in Drosophila in which such mutations are combined with oncogenic Ras/Raf signalling. This pathway, whose activating mutations are found in a high percentage of human cancers, provides polarity-deficient cells with survival and proliferation properties 7 necessary to overwhelm the surrounding wild-type tissue and form malignant masses [8][9][10] . In the imaginal cells, Ras V12 ectopic expression alone causes instead hyperplastic growth of the tissue 11 ; malignant growth thus requires a functional cooperation between loss of cell polarity and hyperplasia. We previously demonstrated that lgl human orthologue, namely Lgl1 or Hugl-1, functions as a tumour suppressor gene also in humans, and our and other studies described its altered expression/localisation in several forms of cancer [12][13][14] . Two pathways have been mainly implicated in lgldriven malignant growth: the Hippo pathway and the JNK pathway. The Hippo (Hpo) pathway is a highly conserved signalling network that plays a key role in epithelial growth; it is composed of several upstream regulators which activity converge on the transcriptional co-activator Yorkie (Yki -YAP in humans) 15,16 . When the pathway is active, Yki is sequestered in the cytoplasm and, upon pathway deregulation, it translocates into the nucleus and activates the expression of several target genes involved in cell growth, proliferation and resistance to apoptosis both in Drosophila 17 and humans 15,16,18 . In contexts in which the lgl mutation triggers tumour growth, the inactivation of the Hpo pathway is responsible for the major part of the malignant phenotype 2 . The JNK pathway is a conserved stress-induced MAPK cascade that has multiple roles in cellular processes including proliferation, differentiation, morphogenesis and apoptosis, and has been implicated in several aspects of cancer 19 . In lgl mutant cells grown in a wild-type background, which suffer from cell competition, the apoptotic death triggered by the neighbours has been demonstrated to be JNK-dependent 5 . Nevertheless, in lgl 2 ; Ras V12 malignant clones the JNK pathway promotes growth and invasion, this latter mainly through the induction of matrix metalloproteinases (MMP), whose function is to break down the basement membrane underlining the epithelial sheet 20,21 . Cell polarity loss thus results in JNK activation which, in the presence of an active Ras signalling that restrains its pro-apoptotic effects, induces invasive behaviour and stimulates growth. The same effect was observed in imaginal discs in which lgl was downregulated by RNA interference; upon loss of cell polarity, cancer growth was shown to be JNK-dependent 22 . Among the cancer hallmarks described by Hanahan and Weinberg 1 , angiogenesis plays a fundamental role in tumour expansion. Growing masses suffer from oxygen shortage, and low oxygen tension leads to stabilisation and nuclear accumulation of the transcription factor HIF1a (Hypoxia-Inducible Factor 1a) through the inhibition of the prolyl-hydroxylases that prime it for degradation in normoxia 23 . HIF1a is a strong activator of the VEGF (Vascular Endothelial Growth Factor) promoter; secreted VEGF triggers sprouting angiogenesis (SA) by binding its receptor and activating the downstream signalling in endothelial cells 23 . Oncogenic Ras signalling is also able to upregulate VEGF, as well as other endothelial growth factors such as FGF 7 . In Drosophila, oxygen is conveyed to the internal organs through an interconnected tubular network called ''tracheal system'', whose regulation is significantly analogue to that of mammalian angiogenesis, with Drosophila FGF/FGFR (FGF Receptor), encoded by the branchless (bnl) and breathless (btl) genes respectively, carrying out the functions of VEGF/VEGFR 24,25 . Further to drive the formation of the tracheal tree during embryonic and post-embryonic development, the FGF/FGFR signalling is also active during late larval development, when a structure connected to the basal side of the wing imaginal disc, the ''air sac'', emerges from a tracheal branch named ''transverse connective'' and undergoes a morphogenetic process which relies on both proliferation and migration 26 . Drosophila tracheal network is also known to respond to local hypoxic conditions through a mechanism strikingly similar to that of mammalian SA: hypoxia is sensed by prolyl-hydroxylases, that are hence inactivated, and Similar (Sima), the Drosophila HIF1a, is thereby stabilised and in turn contributes to the induction of FGF expression to attract new terminal branches towards the hypoxic cells 25,27 . At all these stages, tracheal remodelling and elongation requires the expression of MMP1 28 . In addition to SA, alternative forms of vascular modification have been described in mammals, i.e. intussusceptive angiogenesis (IA), vascular co-option (VC) and vascular mimicry (VM) 29 . IA was first described in the 90s and consists in the splitting of preexisting vessels with the resulting formation of microcapillaries; it is a process faster than SA, predominantly found in large tumours 30 . VC is a mechanism observed both in primary lesions and in metastases, by which tumours obtain oxygen supply hijacking the pre-existing vasculature; tumour cells migrate along the vessels of the organ and, as the mass increases, the vessels become completely embedded in the tumour 31 . VM is a process mainly detected in aggressive tumours in which cancer cells help form mosaic vessels characterised by alternated tumour and endothelial cell clusters, or compose a capillary network on their own; cancer stem-like cells can also trans-differentiate into endothelial cells 32 . Anti-angiogenic therapies have been developed based on the evidence that SA occurs upon VEGF secretion by hypoxic tumour cells; specific drugs have thus been designed to block VEGF signalling, but the occurrence of all these mechanisms alternative to SA greatly complicates the treatment of certain tumours 33 . There is indeed evidence that both primary tumours and metastases are able to progress without SA; such tumours have been described in lung, liver and lymph nodes 31 . By inducing oncogenesis in Drosophila epithelia we were able to observe several, distinguishable cellular strategies of oxygen supply whose morphological features overlap with those above described in mammalian cancers. We demonstrate that tumour masses are hypoxic, show Sima/HIF1a nuclear accumulation and FGF ectopic expression. Malignant cells are both able to co-opt pre-existing tracheal branches and to differentiate into tracheal cells and participate in the composition of mosaic vessels, recapitulating the behaviours associated with mammalian VC and VM. In addition, our data suggest that the epithelial-to-tracheal switch requires the regulation of Polycomb and STAT92E by the JNK signalling cascade. In summary, our findings identify tracheogenesis as a novel cancer hallmark in Drosophila and support the use of this model for the investigation of genetic and molecular alterations resulting from hypoxic stress in human cancers.

Results
Characterisation of the Minute-ENgrailed (MEN)-lgl KD system. lgl knockdown (lgl KD ) has previously been used to induce tumour growth in the posterior (P) compartment of the wing disc under the control of the engrailed (en) promoter 22 through the UAS-Gal4 binary system 34 . In that case, the UAS-lgl-hairpin transgene was used in conjunction with UAS-dcr2 to enhance the effectiveness of RNAi 35 . The authors found that the neoplastic phenotype induced by lgl KD was associated with deregulation of the Hpo pathway and JNK activation, and blocking the JNK signalling was sufficient to rescue both Hpo pathway deregulation and tumour growth 22 . In our hands, the en.lgl-RNAi-dcr2 system did not induce overgrowth at 25uC. At 29uC morphological alterations of the P comparment were evident, but also the en.dcr2 animals presented fragmentation of the P compartment and migratory behaviours of the P cells (not shown). We thus decided to use an alternative method to induce lgl KD -dependent neoplastic growth, that is a Minute background. Minute (M) are a group of dominant mutations in various ribosomal protein genes conferring a growth defect, so that M 1/2 flies are of normal size but their development is delayed with respect to wild-type flies 36 . We chose a mutation in the M(2)24F locus, which encodes the Rpl27A protein, that we successfully used in a previous study 5 . Pupariation of the w: Rpl27A 1 , en-Gal4, UAS-GFP/In(2LR)Gla, Bc strain, hereafter referred to as MEN (from Minute ENgrailed), occurred at 6,5 days After Egg Laying (AEL) at 25uC, as opposed to the 5 days of a wild-type fly. When MEN flies were crossed to UAS-lglRNAi flies, hereafter referred to as lgl KD , the resulting progeny (MEN-lgl KD ) showed a complete depletion of the Lgl protein in the posterior cells of the wing disc ( Supplementary  Fig. 1a) and a larval life lasting for about 11 days AEL at the end of which it died with no signs of differentiation. Figure 1 describes the main molecular features of the MEN-lgl KD cells: the GFP 1 P compartment appeared highly disorganised due to the loss of apical-basal cell polarity, as suggested by the z section in which the distribution of the apical marker aPKC is altered (Fig. 1a); in addition, P cells showed high levels of phosphorylated JNK (pJNK), indicative of pathway activation (Fig. 1a). Yki, the downstream effector of the Hpo pathway 17 , accumulated in many P cells (Fig. 1b, magnification), and its targets dMyc 37,38 and dIAP1 17 resulted consistently overexpressed (Fig. 1c, d). Ras signalling was also activated in our system, as can be inferred by the ectopic expression in the P compartment of its main effectors phosphorylated AKT (pAKT,   Fig. 1f) 7 . The poor correlation between GFP-expressing cells and the nuclear markers analysed in Fig. 1b and 1f is frequently observed in transformed imaginal discs 22 due to a rapid fluorescence decay in some tumour cells. Another recurrent trait of frank malignancies is the capability of cancer cells to degrade the basement mebrane (BM), and, as can be seen in Fig. 1g, the BM-degrading enzyme MMP1 21 was strongly upregulated in the P cells. Finally, tumourigenesis in this genetic system was found to be JNK-dependent, as the expression of a dominant-negative form of basket, the gene encoding the JNK terminal kinase, rescued tumour growth (Fig. 1h). w/w,UASbsk DN ;Rpl27A 1 ,en-Gal4,UAS-GFP/1;UAS-lgl-RNAi/1 individuals differentiated into adult pharates; a small proportion of the pupae (15%, n 5 335) eclosed into adults with no evident phenotypic alterations. To exclude non-specific effects due to saturation of the RNAi machinery, we repeated the same stainings on MEN wing discs in which the GFP expression was knocked down by a shRNA construct and, as can be seen in Supplementary Fig. 2, all the markers analysed showed a wild-type pattern (see figure legend for details).
Altogether, these data demonstrate that the MEN-lgl KD system induces cancer growth through the same pathways as those found in previous studies utilising lgl KD22 or lgl mutations 5 , making it suitable for the investigation of multiple cancer-associated traits.

MEN-lgl KD tumours show a conserved response to hypoxic stress.
To check for oxygen shortage in growing cancers we took advantage of Pimonidazole, a chemical widely used for detection of hypoxia in mammalian tissues 39 . As can be seen in Fig. 2a, the FITC-conjugated anti-pimonidazole antibody (see Methods for details) specifically stained the P compartment (RFP 1 cells) of MEN-lgl KD wing discs. In the hypoxic environment, the Drosophila orthologue of the mammalian HIF1a, Similar (Sima), accumulated in the P cells (Fig. 2b, magnification), where it likely contributed to the activation of the bnl/FGF promoter (Fig. 2c, arrow). As the PI3K pathway is also implicated in Sima/HIF1a nuclear localisation 40 , the active pAKT signal (Fig. 1e) may also contribute to its accumulation. As expected, the control MEN-GFP KD individuals did not show any signs of hypoxia ( Supplementary Fig. 3a). Supplementary Fig. 1b Yki accumulation is visible in the P compartment (magnification). (c-g) Ectopic expression of dMyc, dIAP1, pAKT, dpERK and MMP1 respectively is evident in the P compartment. (h) Imaginal wing discs from w, UAS-bsk DN ; Rpl27A 1 , en-Gal4, UAS-GFP/1; UAS-lgl-RNAi/1 individuals. As can be seen, disc structure, growth and invasiveness (MMP1 staining, red) are almost entirely rescued. The A/P border is dotted in green and disc contour is outlined in white. The genotype of the P compartment is indicated in each image. shows the physiological source of Bnl/FGF in MEN wing discs (arrowhead), that was also found unaltered in MEN-lgl KD samples as it does not fall within the engrailed domain ( Fig. 1c, arrowhead). Ras is required for FGF signalling during development 41 and, since its main downstream effectors appeared upregulated in our system (Fig. 1e, f), we knocked it down through RNAi in MEN-lgl KD animals. This however resulted in growth inhibition of the P compartment and production of small wing discs (data not shown), thus it was not possible in this context to analyse the role of Ras signalling in FGF-dependent phenotypes.
These results show that Drosophila tumour cells carry out a hypoxic response that involves the same molecules as those found in mammalian cancers.

MEN-lgl KD tumours show migratory and tracheogenic behaviours.
During cancer progression in the MEN-lgl KD system, GFP 1 cells escaped developmental constraints, trespassed the A/P boundary and colonised the entire disc. Figure 3a represents a wing disc from an individual at the extreme end of the larval life with diffuse MMP1 expression, a mark of high invasive potential. As MMP1 expression is also required for tracheal remodelling 28 , from this figure on it will also be used to identify neo-tracheal structures in alternative to/together with junctional proteins. As can be seen in Fig. 3b, at this stage cancer cells (GFP 1 ) became highly migratory. Figure 3b9 zooms on a cluster of tumour cells engaged in a directional migration towards a tracheal tube, whose cells project several filopodia in the direction of the incoming cancer cells. As a control, the tracheal structure associated with control MEN wing discs is shown in Supplementary Fig. 1c (arrow). As described in the Introduction, mammalian cancer cells have been reported to participate in the formation of new vessels or to form vascular tubes on their own, a phenomenon called vascular mimicry (VM) 32 . We observed an analogous phenomenon: as can be noticed in Fig. 3c, tumour cells (GFP 1 ) could form long branches; the arrowheads indicate GFP-positive nuclei of the cells forming the ectopic structures. Another mechanism correlated to oxygen deprivation in mammalian cancers is vascular co-option (VC), which begins with the migration of cancer cells along a vascular scaffold; as cancer growth proceeds, the vessel results completely embedded in the tumour mass, supplying it with oxygen 31 . As can be seen in Fig. 3d, GFP-positive cells could migrate along the tracheal vessels and fuse them to those of nearby structures; in this case, cancer cells coming from the haltere/leg 3 discs migrated along a tracheal vessel that appeared continuous with that of the wing disc (arrowhead). Finally, we report a phenomenon we named ''transmigration'', that is the emission of GFP 1 branched structures by one disc of the thoracic triad towards the GFP 1 compartment of a nearby disc. Figures 3e and 3e9 show the final step of a haltere-to-wing transmigration, with GFP-positive cells coming from the haltere disc entering the P compartment of the wing disc. The whole sequence is shown in Supplementary Movie 1, where arrowheads indicate GFP-positive cells coming out of the haltere disc (arrow 1), migrating along the tracheal scaffold (arrow 2) and entering the P compartment of the wing disc (arrow 3). The biological significance of this phenomenon has not been addressed, but we speculate that it may represent a rapid way to deliver oxygen to a fast-growing tissue, with cancer cells moving towards an ectopic FGF source such that of the transformed P compartment of the wing disc (Fig. 2c). In principle, each cellular mass growing beyond a certain size should suffer from oxygen shortage, hence we also analysed l(2)gl 4 mutant wing discs, that during larval development reach considerable dimensions 4 . As illustrated in Supplementary Fig. 4a, a large fraction of the mutant cells showed bnl/FGF promoter activation. In response to this, the tracheal network showed several ectopic branches visible at both the basal ( Supplementary Fig. 4b) and the apical ( Supplementary Fig. 4d) sides of the disc. Tracheal vessels were also included in the disc itself ( Supplementary Fig. 4c). From all these data it appears evident that Drosophila tumour cells undertake a series of mechanisms in response to oxygen starvation that closely resemble those reported in mammals. l(2)gl 4 Ras V12 tumour cells suffer from hypoxia, express Bnl/FGF and form branched neo-structures. The MEN-lgl KD system has proven valuable in the characterisation of the impressive tracheogenic processes that cancer cells carry out following hypoxic stress. However, cancer is by definition a clonal disease, and before proceeding with further analyses we shifted to a cooperative system, widely used to model clonal carcinogenesis, in which the l(2)gl 4 loss-of-function mutation is combined with the oncogenic form of Ras, Ras V129,42,43 . We first confirmed the data collected in the previous system: as can be appreciated in Fig. 4a, the pimonidazole assay gave a strong signal in the clonal areas (marked by the absence of Lgl, arrowheads). As a control, Supplementary Fig. 3 shows the results of the same assay performed on l(2)gl 4 ( Supplementary Fig. 3b) and Ras V12 (Supplementary Fig. 3c) clones; as can be seen, in both cases the pimonidazole assay failed to detect hypoxic cells. Figure 4b shows nuclear accumulation of Sima/HIF1a inside the clones (GFP 1 , see magnification), and many cells within the GFP 1 clone shown in Fig. 4c activated the bnl/FGF promoter. The endogenous FGF source in the wild-type wing disc is shown in Supplementary Fig.  1d (arrowhead). Figure 4d illustrates the evident ectopic structures formed by GFP 1 cells at the basal side of the disc; arrowheads point to branches departing from the central tumour mass. The endogenous tracheal network of a wild-type wing disc is shown for comparison in Supplementary Fig. 1e. In some cases, we observed tube-shaped structures composed of GFP 1 cells whose morphology is shown with greater detail in Supplementary Fig. 5, in which several branches depart from a central mass. One of these branches, as can be seen in the z section, seems to enclose a rudimental lumen ( Supplementary Fig. 5b-b0). In addition, it is worthwhile to mention the phenotype described in Fig. 4e: l(2)gl 4 Ras V12 clones that appeared isolated at the apical side of the disc were frequently found to be interconnected at the basal side by a bridge of tumour cells (GFP 1 ). If such formations have any role in oxygen transport or in clonal expansion is currently unknown. To confirm a causative role for Bnl/FGF expression in tracheogenesis, we downregulated it by RNAi in the l(2)gl 4 Ras V12 clones, but we were not able to obtain viable larvae; as clones are induced randomly in our system, Bnl/FGF deprivation in some body districts possibly impairs larval development. Altogether, these findings point out a clear conservation of the hypoxic response in several genetic systems and identify tracheogenesis as a novel cancer hallmark in Drosophila, considerably similar to mammalian cancer-associated angiogenesis. l(2)gl 4 Ras V12 cells express the tracheal marker Trachealess and exhibit behaviours akin to mammalian vascular co-option and vascular mimicry. In about one-half of the wing discs where l(2)gl 4 Ras V12 clones were induced we observed that some GFP 1 cells began to express the tracheal determinant Trachealess (Trh), known to participate in the activation of btl/FGFR transcription in several populations of cells during development 44,45 . These Trh-positive groups of cells were located randomly across the disc (Fig. 5a, arrowhead) but, more interestingly, they were often found close or connected to pre-existing tracheal branches (Fig. 5b, arrowhead) and, as in mammalian VC, wrapped around tracheal vessels (Fig. 5c, d, arrowheads). Trh-positive cells are restricted in the wild-type wing disc to the tracheal structures at the basal side of the disc (Supplementary Fig. 6a, the air sac and the transverse connective are labelled). Finally, l(2)gl 4 Ras V12 cells have also been found to compose mosaic branches together with resident tracheal cells (Fig. 5e, arrows), similar to mammalian cancer cells undergoing VM. Figure 5e9 shows a higher magnification of Fig. 5e. This evidence deserves deeper considerations. The fact that disc cells change fate upon transformation and are found in a different organ is reminiscent of a cancer stem cell behaviour 32 but, before ascribing the observed phenotype to a trans-differentiation process, we must assure that the GFP-positive clones found inside the tracheal branches originated elsewhere in the disc. It is known from the literature that tracheal precursor cells proliferate until 5-7 h AEL, then formation of the larval tracheal network only occurs by cell migration and changes in cell size and shape 25 . In addition, at about 85 h AEL, some larval tracheoblasts begin to proliferate and form the air sac of the wing disc 41 . As clone induction accounts on mitotic recombination in our system, it can only occur in proliferating cells. We induced clones at about 48 h AEL, a time at which both embryonic and larval tracheoblasts are quiescent, and thus not prone to recombination. Supplementary Figure 6b shows a scheme describing the time frame for tracheoblast proliferation (green), clone induction (red) and tissue collection (blue) in wildtype individuals reared at 25uC. However, to exclude system pitfalls, we carried out a control experiment in which l(2)gl 4 Ras V12 clones were induced in the tracheal cells by substituting the ubiquitous tub promoter with the trachea-specific btl/FGFR promoter. To test the system, we induced clones at 0-4 hours AEL, and at the end of the larval life we obtained several individuals bearing tracheal clones; as an example, Supplementary Fig. 6c shows the anterior half of a late larva displaying many tracheal clones; the highlighted clone (arrow) is magnified in Supplementary Fig. 6d. Of note, the l(2)gl 4 and Ras V12 mutations were not able to induce overgrowth in the tracheal cells. We then used the same system to induce clones at 48 h AEL and no GFP-positive cells were observed in about 500 late larvae screened under a fluorescent stereoscope. To get into deeper details, we dissected 58 of these larvae and found no GFP-positive cells in the wing discs and connected tracheal network (Supplementary Fig. 6e). This was convincing evidence that the l(2)gl 4 Ras V12 cells growing in the tracheal walls shown in Fig. 5e, e9 are cells of the disc epithelium which, following malignant transformation, acquired a tracheal fate. Altogether, these results demonstrate that both VC and VM are conserved in Drosophila cancers, emphasising this model as a powerful tool in which to investigate the molecular basis of these still poorly charachterised mechanisms.
The tracheal switch of l(2)gl 4 Ras V12 cells is coupled with changes in Polycomb and STAT expression. In cultured imaginal discs, cellular plasticity was found to be tuned, among others, by proteins of the Polycomb group (PcG) 46 , known to maintain cellular fate by controlling the expression pattern of many developmental regulators 47 .
With the aim to test if Trh-positive and Trh-negative l(2)gl 4 Ras V12 cells associated to the tracheal walls displayed any differences in Pc levels, we performed a double Pc-Trh staining. Figure 6a shows a wing disc in which the outlined GFP 1 clone is growing attached to a tracheal tube (arrow), and part of its cells express Trh. A higher magnification of the region squared in Fig. 6a is shown in Fig. 6b, b9. As can be seen in Fig. 6b, all the cells expressing Trh showed high levels of Pc (arrowheads), while GFP 1 cells lacking Trh expression downregulated Pc (compare Fig. 6b9 to Fig. 6b, arrows). Another example of this behaviour is shown in Fig. 6c, c9: GFP 1 cells that did not express Trh (arrowheads) coherently showed low levels of Pc (outlined in Fig. 6c9). On the other hand, GFP 1 cells expressing Trh (Fig. 6c, arrows and dashes) upregulated Pc (Fig. 6c9, arrows). Trh and Pc co-expression was confirmed by an analysis performed on l(2)gl 4 Ras V12 clones located within or around a tracheal vessel, in which GFP 1 cells were scored for the presence of both markers (Supplementary Fig. 7a). It thus seemed that l(2)gl 4 Ras V12 cells associated to a tracheal tube that did not acquire a tracheal fate remained in an unprogrammed state, marked by Pc downregulation. Possibly, Trh-expressing l(2)gl 4 Ras V12 cells acquired a tracheal fate upon exposure to hypoxia or to other local factors. In mammals, it is documented that the pro-angiogenic factors produced by hypoxic cancer cells can be induced by cooperation between HIF1a and STAT3, the JAK-STAT cascade downstream effector 48 . These molecules are indeed frequently co-upregulated in   49 and have been recently associated with VM in gastric cancer 50 . In Drosophila, STAT92E is the only member of the STAT family 51 . The regulatory regions of several genes involved in tracheal development, including trh, have shown to contain specific enhancers for STAT92E 52 . As can be seen in Fig. 6d, a Trh-expressing clone (arrowhead) showed STAT92E staining, while the Trh-negative clone (arrow) did not. We also monitored STAT activity in l(2)gl 4 Ras V12 clones through a STAT-GFP reporter, whose activity in a wild-type disc is showed in Supplementary Fig. 7b. As can be seen in Fig. 6e, GFP and Trh are co-expressed in the same cells (arrow), while in Fig. 6f a wing disc is shown in which the small clone indicated by the arrow does not show STAT activation and does not express Trh. We then screened a number of l(2)gl 4 Ras V12 clones for the presence of both markers and, as can be seen in Supplementary Fig. 7c, both in the proximal and distal regions of the disc, while several STAT92E-positive clones do not show Trh expression, the most part of the Trh-positive clones are also STAT92E-positive. We then downregulated STAT92E by RNAi in l(2)gl 4 Ras V12 clones and observed that only 6% (n 5 34) of the wing discs examined carried a GFP 1 , Trh-expressing clone, compared to the 50% (n 5 23) observed in the control experiment. These data suggest that STAT92E may be involved in Trh ectopic expression in l(2)gl 4 Ras V12 cells, but additional work is required to confirm a direct implication of the JAK/STAT signalling in Trh regulation.
Active JNK signalling regulates Polycomb and STAT92E expression in l(2)gl 4 Ras V12 cells. Previous work suggested that polaritydeficient cells forming tumours in the wing disc are dependent on JNK signalling for growth and invasiveness [20][21][22] . In the present work, tumourigenesis induced by the MEN-lgl KD system was also found to be JNK-dependent (Fig. 1h). We thus checked JNK activation in the clonal system, and found that many l(2)gl 4 Ras V12 clones showed high pJNK staining (Fig. a, b). Since JNK signalling has been shown to specifically suppress the expression of Polycomb group proteins in Drosophila and mammalian cells 53 , we sought to find a correlation between the two and, as can be inferred by Fig. 7a, b, Pc expression appeared to be correlated to JNK activation. In particular, in Fig. 7a a disc is shown in which all l(2)gl 4 Ras V12 clones activated the JNK signalling and repressed Pc expression (arrowheads), and Fig. 7b shows a disc in which the only clone activating JNK signalling downregulated Pc (arrowhead). The remaining clones, possibly exposed to different combinations of local factors, did not show pJNK staining and expressed Pc (arrows). Strikingly, inhibition of the JNK cascade in l(2)gl 4 Ras V12 clones rescued Pc expression (Fig. 7c, outlined clones) and repressed STAT92E (data not shown) and Trh (Fig. 7d) ectopic expression; Trh staining resulted indeed restricted to the normal wing tracheal network (Fig. 7d9, arrowhead). Altogether, these findings suggest that some l(2)gl 4 Ras V12 cells activate JNK signalling that, in turn, represses Pc expression and activates STAT92E. It has been recently shown that Pc mutation triggers neoplastic growth in the eye imaginal tissue through derepression of the JAK-STAT ligand loci 54 ; this suggests that STAT92E ectopic expression may be modulated by Pc protein levels also in our system.

Discussion
Targeted therapy is the most thoughtful tool so far developed to challenge cancer disease, as it is based on a precise knowledge of the key mechanisms supporting cancer growth. Upon oxygen shortage, cancer cells are known to express pro-angiogenic factors such as VEGF and FGF and this, in turn, stimulates nearby vessels to form new branches to feed the expanding mass. Anti-VEGF molecules have thus been developed to counteract this phenomenon 55 . Unfortunately, cancer cells were found to adopt alternative strategies  to access oxygen sources, such as the seizure of pre-existing vessels or the formation of a capillary-like network on their own, thus becoming resistant to anti-VEGF therapies. The molecular basis of these mechanisms, respectively known as vascular co-option (VC) and vascular mimicry (VM), is largely unknown 31,32 . Future therapies aimed at depriving cancer cells of oxygen might thus be most beneficial if based on multi-target approaches, and the identification of the molecular changes at the basis of each different mechanism of blood supply is of great interest. As Drosophila has an open circulatory system, it has long been considered unsuitable to study cancerassociated angiogenesis, and this trait was so far unexplored in the fly. However, oxygen is spread throughout the body by the tracheal system, an interconnected tubular network whose regulation is sig-nificantly analogue to that of mammalian vascular tree 25 . Furthermore, the functional analogies between the Drosophila tracheal system and the mammalian vascular tree may go well beyond oxygen transportation. In a pivotal study about cooperative oncogenesis, the authors clearly showed GFP 1 cancer cells entrapped within a tracheal tube 9 , and a recent study found that midgut homeostasis is regulated in the adult fly by the Dpp/TGFb supplied by the tracheal cells 56 . These data demonstrate that tracheal function, both in physiological and pathological conditions, is not restricted to air exchange. The tracheal system may also be involved in the production and/or transportation of growth factors acting locally or systemically for interorgan communication, support cancer growth and vehiculate cancer cells during metastatic disease. Our analysis of the tracheal changes occurring throughout tumourigenesis in Drosophila larval epithelia showed that they are closely related to the vascular modifications seen in mammalian cancers. We were able to recognise ectopic tracheal sprouting in FGF-expressing cancer tissues ( Supplementary Fig.  4), migration of cancer cells towards nearby tracheal tubes (Fig. 3b,  b9), tracheal co-option (Fig. 5c, d) and tracheal mimicry ( Fig. 3c and Fig. 4d), frequently associated with differentiation of cancer cells into tracheal cells (Fig. 5). Furthermore, these vascular alterations were supported by the same molecular mechanisms known to respond to hypoxic stress in mammalian cancers. Indeed, in Drosophila cancer cells suffering from oxygen deprivation the hypoxia-inducible factor Sima/HIF1a translocated into the nucleus (Fig. 4b) and the bnl/FGF promoter resulted activated ( Fig. 2c and Fig. 4c). This, in turn, may trigger all the tracheogenic phenotypes above described. Cancer cells expressing the tracheal determinant Trachealess (Trh) were frequently found to compose mosaic branches together with resident tracheal cells (Fig. 5e, e9 and Fig. 6c, c9). VM may indeed lead to mosaic vessels that are characterised in mammals by alternating cancer and endothelial cells in the capillary walls 57 . We investigated the molecular basis of this phenomenon starting from the evidence that cellular plasticity underlying the differentiation process involves reprogramming of cell identity, mediated by local signals and by internal changes at the transcriptional and epigenetic levels. Cell identity is established and maintained in both Drosophila and mammals through the organisation of specific chromatin domains by the Polycomb (Pc) and Trithorax (Trx) group proteins 47 , hence we investigated Pc expression in transformed cells. We found that the undifferentiated state of most cancer cells was marked by Pc downregulation; upon transition from an undifferentiated to a tracheal fate, they started to express Pc again (Fig. 6b-c9 and Supplementary Fig. 7a). We also found that Trh ectopic signal correrelated with STAT92E expression/activity (Fig. 6d-f and Supplementary Fig.  7c), the effector of the JAK-STAT signalling cascade in Drosophila 51 .
As described in the Results section, Trh ectopic expression was severely restricted upon STAT92E downregulation by RNAi. The expression of Pc group proteins has been shown to be specifically suppressed by the JNK cascade in Drosophila and mammalian cells 53 . We were able to show that these mechanisms are conserved during cancer growth, finding a correlation between active JNK signal and Pc repression (Fig. 7a, b). Pc expression was consistently rescued following JNK inhibition (Fig. 7c), as were STAT92E (data not shown) and Trh (Fig. 7d) ectopic signals. In support of our findings, STAT92E was found to be directly activated by JNK signalling in a similar tumour model 10 , and Pc mutation was known to trigger neoplastic growth in the eye imaginal tissue through derepression of the JAK-STAT ligand loci 54 . The tendency of malignant clones to grow around tracheal vessels has also been observed in metastatic tumours induced by combined expression of activated Src and Ras 58 . Such masses express Bnl/FGF and show abnormal tracheal vessels, suggesting they are formed de novo by tumour cells 58 . Ectopic tracheal vessels were also found in a Drosophila model of glioma 59 . These observations suggest that the mechanisms we report, summarised in Fig. 8, are not specific to the genetic systems or to the organs utilised in our study, but rather represent a general feature of cancer growth in Drosophila. The use of the fruitfly for future research on this topic will open new possibilities in the dissection of the genetic and molecular basis of angiogenic strategies in human cancers.
Image analysis. Fluorescent images were taken on a Leica TCS SP2 confocal microscope and entire images were processed with Adobe Photoshop software; images are all from a single xy stack. ImageJ free software from NIH, Bethesda, MD, USA was used to rebuild the projections along the z axis starting from 35-50 xy stacks. Unless otherwise specified, each experiment is based on the observation of 55-80 wing discs, and all figures show a phenotype found in over 75% of the samples, that we consider highly representative.
Pimonidazole conjugation assay. To check for hypoxic tissues, larval carcasses were exposed to a 400mM solution of a bio-reactive drug, Pimonidazole, for 45 minutes in Gibco Schneider's medium prior to regular fixation (http://site.hypoxyprobe.com/ knowledge-center-articles/HP-1-Kit-Insert.pdf). This drug forms adducts with thiolcontaining proteins at pO 2 # 10 mm Hg. Carcasses were then washed in PBS and fixed in 3,7% formaldehyde in PBS. A mouse-FITC-Mab against was added to detect the pimonidazole adducts. . Activated JNK downregulates Pc, thus derepressing STAT92E. Upon hypoxic stress, Sima/HIF1a is stabilised, enters nucleus and activates bnl/FGF transcription, while STAT92Edependent Trh expression may, in turn, strengthen btl/FGFR promoter activation. All these events are likely to force undifferentiated cells to acquire a tracheal fate. The empty arrows represent cellular conditions that are permissive to the expression of the indicated molecules, whereas full arrows and bars represent a direct effect on the expression/activity of the indicated molecules. www.nature.com/scientificreports