Les données obtenues sur les cancers pulmonaires ont conduit Ren et collaborateurs à déposer en 2008 un brevet d’invention pour les thérapies et diagnostiques utilisant le ciblage de la protéine ou du transcrit HDGF (WO2008054431 A2). Dans cette même initiative, Ren et collaborateurs ont développé un panel d’anticorps ciblant spécifiquement HDGF, afin de déterminer si un anticorps neutralisant HDGF pouvait avoir un effet sur la tumorigénèse dans le cancer du poumon176. Après xénogreffe de cellules tumorales pulmonaires chez la souris immunodéficiente et développement d’une tumeur, les souris ont été traitées avec les différents anticorps neutralisant HDGF. Les auteurs ont ainsi pu observer que la croissance des tumeurs traitées par l’anticorps anti-HDGF est ralentie, avec des effets plus ou moins important selon l’anticorps utilisé176, le plus prometteur induisant une diminution de la taille tumorale supérieure à deux fois. De plus, l’anticorps a pu être détecté dans le noyau des cellules tumorales, laissant supposer qu’il a pu être internalisé pour bloquer l’action de la protéine nucléaire176. Aucune toxicité de cet anticorps neutralisant n’a été mise en évidence dans cette étude.
Lors de l’étude des tumeurs traitées, les chercheurs ont pu mettre en évidence une diminution de la vascularisation après traitement avec les anticorps anti-HDGF176. En effet, bien que le nombre de vaisseaux ne soit pas réduit, les vaisseaux formés dans les tumeurs sont de diamètre et d’épaisseur inférieurs par comparaison avec le groupe non traité. L’anticorps anti-HDGF pourrait donc favoriser le collapse des vaisseaux sanguins alimentant la tumeur.
Enfin, l’association de l’anticorps anti-HDGF avec des traitements plus classiques utilisés chez les patients atteints de cancers du poumon comme le bevacizumab, qui est un anticorps
neutralisant VEGF, ou la gemcitabine, qui est un agent chimiothérapeutique, une synergie d’action a pu être observée entre l’anticorps neutralisant HDGF et les thérapies anti-cancéreuses plus classiques176. Les tumeurs formées sont de plus petite taille et présentent une vascularisation très fortement réduite. De façon probante, des résultats similaires ont pu être obtenus sur des modèles de xénogreffes dérivées de prélèvements de patients177.
Les premiers tests d’anticorps neutralisant la protéine HDGF dans les cancers du poumon sont donc très encourageants. Ce traitement n’est pas encore en cours d’essai clinique, mais il pourrait apporter de nouvelles perspectives thérapeutiques dans le cas des cancers du poumon, et à terme dans les autres cas de cancers surexprimant HDGF.
R
ESULTATS
Au cours de ma thèse, j’ai cherché à mettre en évidence de nouveaux acteurs de la tumorigénèse neuroblastique en étudiant l’impact du microenvironnement embryonnaire sur ce processus. Les modèles d’étude existants ne permettant pas d’aborder cette question biologique de façon exhaustive, nous avons mis au point un nouveau modèle d’étude du neuroblastome.Ce nouveau modèle consiste à greffer des cellules de neuroblastome humaines rendues préalablement fluorescentes au sein de leur microenvironnement d’origine, la crête neurale sympatho-adrénale, chez l’embryon de poulet. Les cellules de neuroblastome sont ainsi mises au contact direct des cellules de la crête neurale sympatho-adrénale endogène de l’embryon de poulet, juste avant leur délamination du tube neural. Ainsi greffées, elles reconnaissent les signaux développementaux de la crête neurale et migrent ventralement selon le trajet de migration stéréotypé des progéniteurs sympatho-adrénaux afin de rejoindre les ganglions sympathiques et la glande médullo-surrénale en développement. Suite à cette première phase, les cellules de neuroblastome forment des masses et prolifèrent au niveau des dérivés sympathiques deux jours après greffe. Une semaine après greffe, les embryons présentent une masse de taille importante au niveau de la glande médullo-surrénale et/ou des ganglions sympathiques associés. De plus, lorsque les cellules greffées proviennent de neuroblastome métastatique, elles ont la capacité de disséminer à distance de la tumeur primaire ; la réciproque étant vraie pour des cas de neuroblastomes localisés. Ce nouveau modèle d’étude permet donc de reproduire la maladie humaine au sein d’un environnement immature.
Afin de déterminer comment les signalisations microenvironementales impactent la biologie des cellules de neuroblastome et leurs capacités tumorigéniques, nous avons réalisé une étude transcriptomique en comparant l’expression des gènes dans les cellules avant greffe et dans les masses sympathiques formées chez l’embryon de poulet. Nous avons constaté que parmi les 11838 gènes exprimés, 3920 gènes voient leur expression varier entre les cellules avant greffe et les masses sympathiques. Grace à une analyse bioinformatique, nous avons pu classer ces différents gènes selon leurs fonctions répertoriées dans la littérature.
Parmi ces gènes différentiellement exprimés, se trouvent de nombreux gènes intervenant dans le développement embryonnaire, et notamment dans le guidage axonal, thématique phare de l’équipe. Ainsi, j’ai participé à l’étude du rôle de la protéine Sémaphorine 3C dans la tumorigénèse neuroblastique. Nous avons pu démontrer que la protéine Sémaphorine 3C est impliquée dans un switch pro-métastatique des cellules de neuroblastome, et que cet effet est médié par les récepteurs Neuropilline1, Neuropilline2 et PlexineA4.
Microenvironment-driven shift of a cohesion/scattering balance within tumors induces a switch towards metastasis in Neuroblastoma
Céline Delloye-Bourgeois1, Lorette Bertin1, Karine Thoinet1, Loraine Jarrosson2, Karine Kindbeiter1, Thomas Buffet1, Servane Tauszig-Delamasure1, Muriel Bozon1, Aurélien Marabelle3, Valérie Combaret4, Christophe Bergeron5, Edmund Derrington1 and Valérie Castellani1*
1, University of Lyon, University of Lyon 1 Claude Bernard Lyon1, NeuroMyoGene Institute, CNRS UMR5310, INSERM U1217, 16 rue Raphael Dubois, F-69000 Lyon, France
2, OncoFactory SAS, L’Atrium, 43 boulevard du 11 Novembre 1918, 69100 Vileurbanne, France.
3, Drug Development Department (DITEP), Gustave Roussy Cancer Campus (GRCC), INSERM U1015, 114 rue Edouard Vaillant, 94805 Villejuif, France.
4, Departments of Oncology and Clinical Research, Centre Léon Berard and Institut d'Hématologie et d'Oncologie Pédiatrique, Lyon, France.
5, Laboratory of Translational Research, Léon Bérard Centre, Lyon, France
* Corresponding author / lead contact: V. Castellani; EMAIL: valerie.castellani@univ-lyon1.fr; FAX 33-0472442685.
SUMMARY
Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. Disseminated forms have high frequency of multiple tumoral foci whose etiology remains unknown, NB embryonic origin limiting investigations in patients and current models. We developed a novel embryonic model, driving human NB tumorigenesis in tissues homolog to patients. We found that aggressive NBs display a metastatic mode, secondary disseminating via peripheral nerves and aorta. Through tumor transcriptional profiling we found that NB dissemination is induced by shutdown of a pro-cohesive autocrine signal, Sema3C, constraining the tumoral mass. Lowering Sema3C levels shifts the balance towards detachment, triggering NB cells to collectively evade the tumor. Together with patient cohort analysis, this identifies the first microenvironment-driven pro-metastatic switch for NB.
KEYWORDS
Animal model, embryonal cancer, neuroblastoma, microenvironment, neural crest, collective migration, neuroblastoma cell cohesion, metastatic switch
SIGNIFICANCE
Neuroblastoma (NB) is a devastating pediatric cancer with 60% of patients characterized by widely disseminated clinical signs at diagnosis. As for many cancers with an embryonic origin, animal models of NB are highly challenging due to early tumorigenic onset. Consequently, the etiology of NB foci and the molecular mechanisms triggering their emergence remain fully uncharacterized, strongly limiting therapeutic development. Taking advantage of the conservation of vertebrate embryonic morphogenetic programs, we conceived a model in which human NBs,
placed back to their primitive environment in an avian embryo, recapitulate the NB disease. We uncover that NB disseminate viaa metastatic sequence, transcriptionally downregulating a Semaphorin/Neuropilin/Plexin pro-cohesive signaling within the primary tumor, leading to cell dissemination via peripheral nerves and dorsal aorta.
HIGHLIGHTS
We set a new animal model fully replicating human NB tumorigenesis and dissemination
Stage 4 NB foci arise from metastatic dissemination via peripheral nerves and aorta RNASeq data reveal a NB gene program regulated by the embryonic microenvironment
Sema3C signaling shutdown in NB primary tumors is a master pro-metastatic trigger
INTRODUCTION
Almost one-half of all childhood cancers are suspected to have an embryonal origin, either through transformation of an embryonal cell or through pathologic acquisition of embryonal features in a mature prenatal cell (Marshall et al., 2014). The etiology of childhood cancers and their eventual metastatic dissemination are thus inseparable from this particular embryonal context (Hanahan and Weinberg, 2000),(Pietras and Östman, 2010).
Crucial aspects of pediatric cancers still remain fully ignored mainly because of the difficulties to recapitulate early tumorigenic events in an embryonic context adapted to experimental manipulations. This is particularly true regarding Neuroblastoma (NB), the most frequent extracranial solid cancer in childhood (Brodeur et al., 2014; Maris, 2010). It is depicted as a highly heterogeneous disease with multiple clinical presentations, ranging from localized asymptomatic primary tumors to widely disseminated disease with poor outcome (Brodeur et al., 2014; Maris, 2010). NB tumors mainly form in the abdomen adjacent to the aorta along the sympathetic chain and in the adrenal gland medullary region. NBs arise from a transitory cell population, the neural crest cells (NCCs) of the sympatho-adrenal lineage that during embryogenesis emerge from the dorsal part of the neural tube and adopt a stereotyped collective ventral migration to reach presumptive sympathetic territories (Le Douarin C, 1999),(Gammill and Roffers-Agarwal, 2010).
While many molecular cues controlling NCC delamination, pathfinding and stop in final embryonal territories have been decrypted, a significant gap is left regarding the nature of NBs interactions with the embryonal environment (Kasemeier-Kulesa et al., 2006),(Gammill et al., 2006),(Rohrer et al., 2009). Indeed, while most aggressive NBs are found already disseminated at diagnosis, the timing and the nature of the events
leading NB cells to disseminate –ie, whether NB is a real metastatic disease rather than a multifocal one - are still a mystery.
Here, we developed a novel in vivo model to explore the etiology of stage 4 NB disseminated foci, consisting in grafting human NB cells back to their original neural crest environment, using the chick embryo as a host. By fully recapitulating NB disease from both cell lines and patient biopsies, our model allowed us to (i) demonstrate that disseminated NBs arise through a metastatic sequence, (ii) to identify metastatic routes and (iii) to characterize a pro-metastatic genetic switch engaging NB cells in the primary tumor towards broad dissemination.
RESULTS
A novel in vivomodel recapitulating disseminated NB
Here we explored the etiology of multiple foci typical of disseminated NBs. We thought to recapitulate NB tumorigenesis in an embryonic animal that would allow access to early events of NB cell dissemination. We generated several stable NB cell lines –i.e., IGR-N91, SH-SY5Y and SHEP- expressing either GFP or RFP through lentiviral transduction. We grafted these fluorescent NB cells into HH14 chick embryos (Hamburger and Hamilton, 1992) (approximately 52 hours post fertilization), at the sympatho-adrenal crest level, between somite 18 and somite 24 (Le Douarin and Teillet, 1971) (Figures 1A and S1A) and analyzed grafted-embryos at HH25 (approximately 50 hours after the graft), in transverse slices and in 3D-imaging. Remarkably, we found that almost all NB cells migrated away from the graft site and exclusively accumulated at sites that are specifically targeted by endogenous sympatho-adrenal neural crest cells (Le Douarin and Teillet, 1971; Le Douarin C, 1999; Teillet and Le Douarin, 1974): mostly sympathetic ganglia and adrenal gland,
and to a lesser extent spinal cord periphery, and visceral and pre-aortic ganglia (Figures 1B, 1C, S1B, S1C andMovies S1 and S2). We observed dense sympatho-adrenal masses in the majority of grafted embryos (100% of IGR-N91-, 88% of SH-SY5Y- and 89% of SHEP-grafted embryos). To better characterize the behavior of NB cells, we analyzed their position 13h, 24h, 39h and 50h post-grafting relative to migrating endogenous neural crest cells detected with anti-HNK1 antibody (Figures 1D, 1EandS1D). We found NB cells and endogenous NCCs share common migration routes, in time and space, and confirmed that NB cells adopt a unilateral migratory pathway that ends with their clustering in sympatho-adrenal derivatives (Le Douarin C, 1999; Huber, 2006). Indeed, upon migration, the proportion of NB cells embedded in HNK1 domains increased from 30% 13 hours after the graft to 95% 50 hours after the graft, with 60% and 3% of them located adjacent to HNK1 domains 13h and 50 hours after the graft respectively (Figure 1E). Thus, almost all NB cells show a NCC-like migratory pattern. Moreover, we noticed that while migrating, NB cells appeared connected to each other, forming either small migrating masses (IGR-N91 cells) or chains of migrating cells (SH-SY5Y and SHEP cells) (Figure 1F). To characterize NB cell migratory modalities, we set-up live-imaging of thick slices prepared from embryos previously grafted (a few hours before) with IGR-N91 or SHEP cells. We observed typical directional collective migration of NB cells forming close and transient cell-cell contacts with cycles of protrusions formation and retraction (Figure 1G and Movies S3-S6). Thus NB cells recapitulate a collective migratory behavior that is typical of NCCs (Friedl and Gilmour, 2009; Kulesa and Fraser, 1998; Rørth, 2009; Theveneau and Mayor). We also observed in time course analysis that the great majority of NB cells (80%) express the proliferative marker Ki67 at all examined stages (Figures S1E and S1F). Thus remarkably, when replaced in their original environment, NB cells,
while keeping extensive proliferation competency, endorse an NCC program allowing them to properly sense the embryonic microenvironment and to undergo an oriented migration process typical of their physiological counterparts. Once settled in their target site, NB cells engage in a tumorigenesis program leading them to form dense proliferating tumor-like masses.
This suggested that NB dissemination might follow a metastatic scheme. We thus examined whether our avian model could also allow recapitulation of such metastatic process. We analyzed the localization of IGR-N91, SH-SY5Y and SHEP fluorescent cells 7 days after the graft of HH14 chick embryos. We systematically observed the presence of massive tumors in sympatho-adrenal derivatives (Figures 2A, 2B and S2) indicating the maintenance of NB cells tumorigenic potential over this prolonged period. Moreover, in most cases, NB cells were no longer confined to the primary tumor site but rather disseminated to proximal tissues and organs, as well as to more distant sites. We observed loco-regional invasion of proximal DRGs, sympathetic ganglia and/or paraspinal tissue in most cases (respectively in 67%, 80% and 90% of IGR-N91, SH-SY5Y and SHEP-grafted embryos, Figures 2A, 2B, S2A, S2B, S2D and S2E). Distally, we detected clusters of disseminated cells mainly in the small intestine, colon, coeca and/or abdominal aorta affixed to the digestive tract (distant foci respectively in 44%, 40% and 70% of IGR-N91, SH-SY5Y and SHEP-grafted embryos). Thus, remarkably, our model fully recapitulates key feature of NB secondary metastasis, that is its fast and multi-focal metastatic dissemination. The latter observation of an aortic distal invasion led us to more closely examine the early dissemination steps. Interestingly, focusing on NB cells in the vicinity of the primary tumor, we found that NB cells escaped the tumor mass as clusters of cells invading the wall of adjacent aorta and disseminating in arteries (respectively in 31%, 20% and
90% of IGR-N91, SH-SY5Y and SHEP-grafted embryos). We also detected clusters of NB cells migrating along proximal peripheral nerves (respectively in 25%, 40% and 80% of IGR-N91, SH-SY5Y and SHEP-grafted embryos) (Figures 2C-2E, S2C, S2D and S2F). To further visualize tumors and metastatic dissemination, we took advantage of the light-sheet microscopy (Selective Plan Illumination Microscopy), and imaged robustly cleared whole embryos and organs 7 days after NB cells graft (Figure 2FandMovies S7-S10). 3D-images and optical sections confirmed the localization of cell clusters along nerves, cells scattering from the adrenal gland and reaching the aorta, and NB cells distant dissemination reaching intestinal and colon walls. Thus, our model reveals two etiologic metastatic routes taken by NB cells which were yet ignored and explain their propensity to establish metastasis at multiple foci: (1) the aorta, the major embryonic vessel that irrigates most developing structures, and (2) the perineural niche that gives direct access to distant innervated targets (Figure 2E).
Characterization of microenvironment-driven NB gene regulations in primary tumors
We next explored whether and if so which changes of gene expression occur in NB cells in primary tumors that switch on their metastatic behavior. We micro-dissected tumoral masses in sympathetic ganglia of HH25 chick embryos to compare their transcriptome with that of naive NB cells having not experienced tumorigenesis in the embryonic environment, using RNAseq approach (Figure 3A). We found that 3920 of the 11838 gene transcripts expressed above background, varied, with representative functions in cellular growth, proliferation, survival/death, and movement, and implications in developmental processes (Figure 3B and Table S1). Noteworthy, 46 among 266 genes with reported expression in the neural crest lineage varied, with 24
of them having functions related to cell movement (Figure 3C and Table S2). The Semaphorin gene family, known to regulate axon and cell migrations (Jongbloets and Pasterkamp, 2014; Kruger et al., 2005a) including that of the neural crest (Gammill et al., 2006, 2007; Kawasaki et al., 2002; Schwarz et al., 2009a, 2009b) was well-represented, with changes of expression levels observed for three members of the class 3 –SEMA3C, SEMA3D and SEMA3F-. We confirmed in independent qRT-PCR experiments performed on IGR-N91 tumor masses that SEMA3C, 3D and 3F expression is regulated when NB cells form sympathetic tumors, thus validating the RNASeq data (Figure 3D). Moreover, we found that SEMA3C, SEMA3F and to a lesser extent SEMA3D transcripts were expressed in the majority of human NB cell lines including in primary cell lines generated from patient biopsies (Figures 3E and S3A). Sema3C, a key regulator of cardiac neural crest cell migration (Brown et al., 2001; Feiner et al., 2001; Plein et al., 2015) whose expression was found decreased by 2.19 (±0.74, p=0.093) in RNASeq data and by 3.31 (±1.98, p=0.0079) in qRT-PCR experiments, appeared as promising. We confirmed that this decrease in Sema3C expression was not specific to IGR-N91 cells, since we detected a similar down-regulation in independent graft experiments performed with SH-SY5Y cells (Figure S3B). We thus concentrated on Sema3C and explored its potential function as a pro-metastatic trigger.
Semaphorin3C is a cohesive signal for NB cell population
To evaluate the impact of a two-fold decrease of Sema3C levels in NB cells, we took advantage of an siRNA achieving a 47% (±14%, p=0.0079) and a 45% (±8%, p=0.0286) extinction inIGR-N91 and SH-SY5Y cell lines, respectively Figure S4A). A second siRNA (siRNA Sema3C#2) inducing a 55% (±9%, p=0.0022) decrease of
Sema3C levels in IGR-N91 cells was also used to control the specificity. Fluorescent control and Sema3C siRNA-transfected NB cells were grafted in HH14 chick embryos (Figure S4B). We observed that Sema3C reduction resulted in strong alteration of NB migratory pattern with both Sema3C siRNAs (Figures 4A, 4B and S4C-S4F). We detected sympatho-adrenal tumors in only 44% of IGR-N91::siRNA Sema3C-grafted embryos (compared to 100% of IGR-N91::siRNA scr.-grafted embryos; p=0.0108) (Figures 4A and 4B). In 67% of embryos, Sema3C-low cells were found as individual and/or small groups of cells, dispersed throughout embryonic tissues, a phenotype observed in no more than 20% of control embryos. The collective migration of NB cells and correct targeting of embryonic structures are thus impaired upon Sema3C downregulation. Interestingly in tumor-like masses that still could form, we noticed that in most cases (67%), Sema3C-low cells scattered around the tumor core, in contrast to control cells that mostly remained tightly condensed (10% of embryos with scattering from the tumor site, p=0.023). This scattering/dissemination effect was confirmed in similar graft experiments conducted on IGR-N91 cells with a second Sema3C siRNA (siRNA Sema3C#2) (Figures S4C and S4D, p<0.0001) and in SH-SY5Y cells (Figures S4EandS4F, p=0.0018).
Drop of Sema3C in NB preformed primary tumors triggers collective NB cell detachment and metastatic dissemination
Further conclusions were limited by the premature outcome of Sema3C decrease, already impacting NB migration and, as a result, primary tumor formation. We thus achieved stable and temporally controllable Sema3C down-regulation, by generating stable IGR-N91 cells expressing a Sema3C shRNA under the control of a Doxycycline-inducible promoter (Figure 4C). We confirmed the significant down-regulation of
Sema3C expression in presence of Doxycycline (Figure S4G, p=0.0286). Additional controls were performed with IGR-N91::shRNA scr. cells as slight decrease of Sema3C expression was observed in untreated cells compared to shRNA scr. cells,