Stephanie Lejeune

Armelle Le Mee

Laurent Petyt

Antoine Hutt

Rony Sfeir

Laurent Michaud

Pierre Fayoux

Antoine Deschildre

Frederic Gottrand

Caroline Thumerelle
email: caroline.thumerelle@chru-lille.fr

Bronchopulmonary and vascular anomalies are frequent in children with oesophageal atresia

Keywords: CT, Computed tomography congenital malformations, diagnostic imaging, oesophageal atresia, thoracic malformations, contrast-enhanced CT scan

Aim: Oesophageal atresia is frequently associated with other malformations and our study aim was to use computed tomography (CT) to describe intrathoracic malformations in patients with this condition.

Method:

This was retrospective study of children with oesophageal atresia who were born in 1996-2013 and followed up at the French reference centre for rare oesophageal diseases at the University of Lille. CT scans were available for 48 of the 234 patients during follow-up visits and these were reviewed by a thoracic radiologist.

Results: More than two third of the scans were performed to explore persistent respiratory symptoms. Thirteen percent of patients had a pulmonary malformation: four lobar agenesis, one right pulmonary aplasia, and one congenital cystic adenomatoid malformation. CT permitted diagnosis of an unexpected thoracic malformation among 27%: four lobar agenesis, six arteria lusoria, five persistent left superior vena cava, one partial anomalous pulmonary venous return, and confirmed diagnosis of suspected malformations among 10%: one congenital cystic adenomatoid malformation, one pulmonary hypoplasia, two right-sided aortic arch, and one communicating bronchopulmonary foregut malformation.

Conclusion:

Intrathoracic anomalies were frequently associated with oesophageal atresia, and contrast-enhanced chest CT scans should be performed on patients with persistent respiratory symptoms.

KEY NOTES

• Oesophageal atresia is frequently associated with other malformations and we analysed the contribution of chest computed tomography (CT) scans to explore intrathoracic malformations.

• In 48 children, we diagnosed an unexpected thoracic malformations in 13% and confirmed a suspected malformation in 10%, including one congenital cystic adenomatoid malformation and one pulmonary hypoplasia.

• Contrast-enhanced chest CT scans should be performed in oesophageal atresia patients to explain persistent respiratory symptoms.

INTRODUCTION

Oesophageal atresia is a rare congenital anomaly, with an incidence of one in 2,500 to 3,500 births. It is associated with a tracheoesophageal fistula in about 90% of cases, namely Ladd classification types II, III, IV, and V [START_REF] Ladd | Esophageal Atresia and Tracheo-esophageal Fistula[END_REF][START_REF] Sfeir | Esophageal atresia: data from a national cohort[END_REF]. Various associated anomalies have been reported, with one or more associated malformation in 50-62% of cases [START_REF] Ladd | Esophageal Atresia and Tracheo-esophageal Fistula[END_REF][START_REF] Sfeir | Esophageal atresia: data from a national cohort[END_REF][START_REF] Keckler | VACTERL anomalies in patients with esophageal atresia: an updated delineation of the spectrum and review of the literature[END_REF]. These associated anomalies mostly involve the respiratory tract, cardiovascular system, skeleton, gastrointestinal tract and, or, urogenital tract. In many cases there are more than one anomaly, as seen by patients' vertebral defects, anal atresia, cardiac defects, tracheo-oesophageal fistula, renal anomalies, and limb abnormalities (VACTERL) associations [START_REF] Ladd | Esophageal Atresia and Tracheo-esophageal Fistula[END_REF][START_REF] Keckler | VACTERL anomalies in patients with esophageal atresia: an updated delineation of the spectrum and review of the literature[END_REF]. Specific associations between oesophageal atresia and respiratory tract malformations have been described [START_REF] Usui | Anomalies of the tracheobronchial tree in patients with esophageal atresia[END_REF][START_REF] Stark | Triad of tracheoesophageal fistula-esophageal atresia, pulmonary hypoplasia, and duodenal atresia[END_REF][START_REF] Fayoux | Laryngotracheal anomalies associated with esophageal atresia: importance of early diagnosis[END_REF]. Furthermore, rare bronchopulmonary malformations, including communicating bronchopulmonary foregut malformations, which seem to be associated with oesophageal atresia, occur on a relatively frequent basis [START_REF] Clements | Congenital malformations of the lung and airways[END_REF][START_REF] Sugandhi | Esophageal lung: presentation, management, and review of literature[END_REF].

Chest computed tomography (CT) scans of patients with oesophageal atresia are performed in numerous contexts: preoperative evaluations, including assessment before aortopexy, when there are persistent respiratory symptoms and in cases with postoperative complications [START_REF] Garge | The role of preoperative CT scan in patients with tracheoesophageal fistula: a review[END_REF][START_REF] Mahalik | Role of preoperative 3D CT reconstruction for evaluation of patients with esophageal atresia and tracheoesophageal fistula[END_REF][START_REF] Briganti | Tracheomalacia in oesophageal atresia: morphological considerations by endoscopic and CT study[END_REF]. Contrast-enhanced chest CTs provide precise anatomical descriptions of the airways, lung parenchyma and mediastinal vascularisation and can lead to diagnoses of thoracic abnormalities, including tracheobronchial and pulmonary malformations.

The aim of our study was to analyse the contribution of chest CT to the diagnosis of bronchopulmonary and mediastinal vascular anatomical malformations in children with oesophageal atresia treated at our centre. This paper describes the associated malformations, and compares our findings with the existing literature.

PATIENTS AND METHODS

Our observational, retrospective, descriptive study included all 234 patients with oesophageal atresia born between 1996 and 2013, who were followed at our reference centre for rare oesophageal diseases at the University of Lille, France. We focused on the 48 patients who underwent at least one chest CT during follow up available for interpretation.

The patients' past clinical history was collected and oesophageal atresia was based on the Ladd classification [START_REF] Sfeir | Esophageal atresia: data from a national cohort[END_REF]. The existence of an associated chromosomopathy was noted. As recommended, all children diagnosed with oesophageal atresia underwent the following exams: chest radiography, echocardiography, abdominal ultrasound and spinal radiography [START_REF] Ladd | Esophageal Atresia and Tracheo-esophageal Fistula[END_REF]. We noted cases of associated congenital anomalies and whether they were part of a VACTERL association. The patients' past surgical history was also collected when it was available, namely: oesophageal atresia repair, cardiac surgery, aortopexy, fundoplication and laryngotracheal endoscopy results. Finally, we noted cases of tracheomalacia diagnosed with laryngotracheal endoscopy and any history of tracheoesophageal fistula recurrences.

All chest CT scans were collected and re-analysed by a certified thoracic radiologist with 26 years of experience. If multiple CT scans were available for a child, the exam that provided the best quality was retained and analysed. The exam indications were classified into six categories: persistent respiratory symptoms, lung evaluation in tracheoesophageal fistula recurrence, preoperative assessment before aortopexy, evaluation of tracheal stenosis, complications following oesophageal atresia repair, cardiovascular indications and other indications. With the exception of tracheal bronchus, anatomic variants of the tracheobronchial tree were not retained for analysis. We reported cases of tracheal stenosis with regard to tracheal anomalies,. We also reported suspicion of tracheomalacia on CT scans, assessed by indirect signs in free-breathing children. This involved visualisation of a tracheal compression by the aorto innominate complex [START_REF] Briganti | Tracheomalacia in oesophageal atresia: morphological considerations by endoscopic and CT study[END_REF].

Aortic anomalies were searched for in all cases and were described according to the position of the aortic arch. In patients with contrast-enhanced CTs, we reported cases of arteria lusoria: left arch aberrant right subclavian artery. We also reported other vascular anomalies of the pulmonary veins: anomalous venous return and systemic veins, namely the persistent left superior vena cava [START_REF] Lee | MDCT evaluation of thoracic aortic anomalies in pediatric patients and young adults: comparison of axial, multiplanar, and 3D images[END_REF].

The study was approved by the French Data Protection Commission (CNIL: National Commission on Informatics and Liberties; number DEC16-230). All the data were used anonymously and informed consent was not required.

RESULTS

Sample characteristics

There were 234 patients with oesophageal atresia who were born between 1996 and 2013 and followed at our specialist centre. Of these 49 (21%) had undergone at least one chest CT during their follow-up visits (Fig. 1). One CT scan was unavailable for interpretation and this meant that the analyses were based on 48 patients (56% boys). There was one missing value for five of the six associated malformations because the child was not born in our centre and was followed up in the later years.

Table 1 lists the main sample characteristics and shows that 39/48 (81%) had Ladd classification type III oesophageal atresia. A blood karyotype was performed in 24 patients and two were diagnosed with a chromosomal anomaly: one with 22q11.2 deletion and one with chromosome 15q duplication. We found that 13/48 (27%) patients had a VACTERL association. In addition, 15/47 (32%) patients had congenital heart disease diagnosed with echocardiography. These included three patients with complex congenital heart disease, seven with ventricular septal defects, three with atrial septal defects, one case of associated ventricular septal defect with atrial septal defect, two cases of coarctations of the aorta and one with pulmonary valvular stenosis. Those with complex congenital heart disease included one tetralogy of Fallot, one double outlet right ventricle with a right aorta with a diagnosis of 22q11.2 deletion and one case of associated atrial septal defect and right pulmonary artery hypoplasia. There were 14/48 (29%) patients with a congenital disorder of the urogenital system, including nine high-grade vesicoureteral reflux, two multicystic dysplastic kidneys, one unilateral renal agenesis and two other malformations. Just under a third, 14/47 (30%), of the patients had an associated costovertebral anomaly: six anomalies of the ribs, seven vertebral malformations and one case of associated vertebral and costal malformations. We found that 6/47 (13%) patients had another malformation of the gastrointestinal tract: three imperforate anuses, one duodenal atresia, one intestinal malrotation and one small bowel atresia. Then there were 4/47 (8%) patients with a laryngeal supraglottic anomaly: one posterior laryngotracheal cleft, one left vocal cord paralysis, one laryngeal hypoplasia and one laryngocele. Tracheomalacia was noted in 22/30 (73%) of the patients with an available laryngotracheal endoscopy. We also noted that 20/47 (43%) patients had a history of fundoplication, six (13%) of cardiac surgery, and five (10%) of aortopexy.

Chest CT analyses

The median age at the time of the CT was four years and five months (range one day to 16 years and two months). Just under two third, 30/48 (63%), of the exams were performed with contrast administration. The indications for these exams were: exploration of persistent respiratory symptoms for 33/48 (69%), lung evaluation in case of suspicion of tracheoesophageal fistula recurrence for six (13%), preoperative assessment before aortopexy for three (6%) and before congenital tracheal stenosis surgery for three (6%) and complications after oesophageal atresia repair for five (10%). In 7 cases there were multiple reasons. The five cases of complication after oesophageal atresia related to: severe dysphagia for two (4%), mediastinitis after oesophageal atresia repair for three (6%), including one case with severe dysphagia, and preoperative assessment before cardiac surgery for one (2%). One CT scan was performed to investigate an antenatal suspicion of congenital cystic adenomatoid malformation.

Lung anomalies

We found 6/48 (13%) congenital pulmonary malformations including four (8%) lobar agenesis, with two middle lobe agenesis and two middle and upper right lobes agenesis (Fig. 2a). There was also one (2%) right pulmonary aplasia and one (2%) congenital cystic adenomatoid malformation affecting the right middle and lower lobes (Fig. 2b). Both the congenital cystic adenomatoid malformation and the pulmonary hypoplasia were suspected during antenatal follow-up visits. Conversely, the four cases of lobar agenesis were only diagnosed from a CT scan.

Mediastinal vascular anomalies

There were 3/48 (6%) patients who were diagnosed with a right-sided aortic arch (Fig. 2c). Of the 30 patients who underwent a contrast-enhanced CT scan, 14 (29%) had at least one vascular anomaly. There were two (7%) patients with coarctations of the aorta that were previously diagnosed with echocardiography, which were associated with one partial anomalous pulmonary venous return and one persistent left superior vena cava. In addition, six (20%) had arteria lusoria (Fig. 2d) and five (17%) had persistent left superior vena cava, including two (7%) associated with arteria lusoria.

When we combined the echocardiography and CT results, 7/47 (15%) patients had an isolated vascular anomaly, eight (17%) had isolated congenital heart disease and seven (15%) had an association, including the two cases of coarctation of the aorta.

Tracheobronchial tree anomalies

We found a tracheobronchial anatomical anomaly in 9/48 (19%) patients, including two (4%) right tracheal bronchus and one (2%) communicating bronchopulmonary foregut malformation, which was a combination of oesophageal atresia with the right main stem bronchus originating from the oesophagus (Fig. 2e). There were seven (15%) patients with a tracheal stenosis, which was confirmed with laryngotracheal endoscopy. Of these, five (10%) were post-intubation focal circumferential strictures. A further two (4%) were congenital stenoses: one laryngeal and upper tracheal hypoplasia without complete rings associated with a right tracheal bronchus and one extended tracheal stenosis with complete rings associated with a right bronchus stenosis. No tracheoesophageal fistula recurrence was seen on the CT scans of the six patients who underwent examinations for lung evaluation before surgery for tracheoesophageal fistula recurrence.

Of note, 32/44 (73%) patients with a history of tracheoesophageal fistula associated with oesophageal atresia , had a residual tracheal diverticulum on CT scans. In addition 15/22 (68%) patients with a confirmed diagnosis of tracheomalacia, were suspected by using indirect signs on CT scans.

Associations between pulmonary anomalies and other malformations

The associated pulmonary, vascular, and cardiac malformations are shown in Fig. 3.

We found no chromosomal anomalies in the six patients with a congenital pulmonary malformation. All were associated with at least one malformation of another organ system: vascular anomalies in five, congenital heart disease in two, tracheal stenosis in one, vertebral anomalies in two, and urogenital system anomalies in three. Three patients had a VACTERL association (Table 2). The patient with communicating bronchopulmonary foregut malformations had no other malformations.

Associated malformations

We found that 26/48 (54%) patients did not display any anatomical intrathoracic malformations on their chest CTs and only nine (19%) presented with isolated oesophageal atresia without any other anatomical anomaly. Indeed, six had congenital heart disease without a vascular anomaly, five had a congenital disorder of the urogenital system, eight had an associated costovertebral anomaly and 12 had at least one anatomical malformation involving another organ system.

Of the 33 patients who presented with recurrent respiratory symptoms, 14 (42%) had no malformations on their CT scans and echocardiography. Seven of these also underwent laryngotracheal endoscopy and four of these were diagnosed with a tracheomalacia.

Proposed decision tree

Computed tomography revealed diagnoses of an unexpected thoracic malformation in 13/48 (27%) patients. There were four lobar agenesis, six arteria lusoria and five persistent left superior vena cava, which were associated with arteria lusoria in two cases. There were confirmed diagnoses of suspected associated malformations in five cases (10%): one congenital cystic adenomatoid malformation, one pulmonary hypoplasia, two right-sided aortic arches and one communicating bronchopulmonary foregut malformation.

Based on these results, we have put together a decision tree that describes the indications for CT scans in children with oesophageal atresia (Fig. 4). An initial chest CT should be performed in a patient with oesophageal atresia with contrast administration, if possible, to search for associated vascular malformations.

DISCUSSION

Our study confirms that oesophageal atresia was frequently associated with other malformations in our cohort. Chest CTs confirmed the diagnosis of associated thoracic malformations in 37% of patients, including 27% who had not been diagnosed with abnormalities following other investigations. CT scans permitted diagnoses of lobar or unilateral pulmonary agenesis in five (10%) patients and a diagnosis of congenital cystic adenomatoid malformation in one (2%).

Because both the oesophagus and respiratory tract derive from the primitive foregut, the association between digestive and respiratory malformations could be linked to a developmental disorder originating with the division of the primitive foregut into the lung bud and oesophagus during the embryonic stage, at weeks 3-4 of gestation [START_REF] Usui | Anomalies of the tracheobronchial tree in patients with esophageal atresia[END_REF][START_REF] Stark | Triad of tracheoesophageal fistula-esophageal atresia, pulmonary hypoplasia, and duodenal atresia[END_REF][START_REF] Clements | Congenital malformations of the lung and airways[END_REF]. Most previous descriptions of oesophageal atresia cases associated with respiratory anomalies have concerned tracheomalacia [START_REF] Fayoux | Laryngotracheal anomalies associated with esophageal atresia: importance of early diagnosis[END_REF][START_REF] Briganti | Tracheomalacia in oesophageal atresia: morphological considerations by endoscopic and CT study[END_REF][START_REF] Thakkar | Bronchoscopy as a screening tool for symptomatic tracheomalacia in oesophageal atresia[END_REF].

In previous studies, the prevalence of pulmonary agenesis and aplasia was estimated at one per 15,000 births, with about 300 descriptions of unilateral lung agenesis [START_REF] Stark | Triad of tracheoesophageal fistula-esophageal atresia, pulmonary hypoplasia, and duodenal atresia[END_REF][START_REF] Berrocal | Congenital anomalies of the tracheobronchial tree, lung, and mediastinum: embryology, radiology, and pathology[END_REF][START_REF] Muensterer | Pulmonary Agenesis and Associated Pulmonary Hypertension: A Case Report and Review on Variability, Therapy, and Outcome[END_REF][START_REF] Stoll | Associated anomalies in cases with esophageal atresia[END_REF].

These rare congenital malformations differed from each other based on the persistence of a rudimentary bronchus limited to a blind-end pouch, without lung tissue in cases of pulmonary aplasia. Of note, Stark et al combined 28 reported cases of pulmonary hypoplasiaor aplasia associated with oesophageal atresia between 1948 and 2005 [START_REF] Stark | Triad of tracheoesophageal fistula-esophageal atresia, pulmonary hypoplasia, and duodenal atresia[END_REF]. In previous descriptions of children with pulmonary agenesis or aplasia, associated congenital anomalies were found in more than one case in two, including oesophageal atresia, structural congenital heart diseases, diaphragm defects and limb anomalies [START_REF] Clements | Congenital malformations of the lung and airways[END_REF][START_REF] Sugandhi | Esophageal lung: presentation, management, and review of literature[END_REF][START_REF] Muensterer | Pulmonary Agenesis and Associated Pulmonary Hypertension: A Case Report and Review on Variability, Therapy, and Outcome[END_REF][START_REF] Stoll | Associated anomalies in cases with esophageal atresia[END_REF]. Associations between oesophageal atresia and lobar agenesis have previously been described, usually affecting the right upper and middle lobes. There were more frequent in cases of VACTERL associations [START_REF] Clements | Congenital malformations of the lung and airways[END_REF][START_REF] Muensterer | Pulmonary Agenesis and Associated Pulmonary Hypertension: A Case Report and Review on Variability, Therapy, and Outcome[END_REF]. In our study, we found five cases of pulmonary or lobar agenesis in oesophageal atresia patients and four of these were diagnosed based on CT scans. All of these were associated with other anomalies, including two with VACTERL associations. Asymptomatic, lobar agenesis frequently remains undiagnosed throughout childhood in oesophageal atresia patients and it may also be involved in the development of restrictive ventilatory defects in adults [START_REF] Sistonen | Repaired esophageal atresia: Respiratory morbidity and pulmonary function in adults in a population-based long-term follow-up study[END_REF].

This paper also describes one case of congenital cystic adenomatoid malformation, suspected before birth and confirmed with a postnatal chest CT scan. This was also associated with multiple malformations and a VACTERL association. To our knowledge, only three similar cases have been reported before and those were all in patients with type III oesophageal atresia [START_REF] De Felice | Congenital cystic adenomatoid malformation of the lung associated with esophageal atresia and tracheoesophageal fistula[END_REF][START_REF] Lima | The role of emergency thoracoscopy in the management of congenital cystic adenomatoid malformation of the lung associated with oesophageal atresia[END_REF].

In addition, three (6%) of our patients had bronchial anomalies and two (4%) had a right tracheal The latter was an anatomic variant that is frequently described in the general population. The estimated frequency ranges from 0.2% for superior lobar bronchi above the carina to 2% for tracheal bronchus as a whole [START_REF] Clements | Congenital malformations of the lung and airways[END_REF][START_REF] Berrocal | Congenital anomalies of the tracheobronchial tree, lung, and mediastinum: embryology, radiology, and pathology[END_REF]. Usui et al performed laryngotracheal endoscopy on 32 patients with oesophageal atresia and found a tracheal bronchus in 13% and an anomaly of the tracheobronchial tree in 47%, including the absence of right upper lobar bronchus in 6% (4). In our study, one patient had a communicating bronchopulmonary foregut malformation affecting the whole right lung, also known as oesophageal lung. This highly rare malformation has been reported in about 20 cases in the literature, half of which were associated with oesophageal atresia, consistent with Srikanth's classification type IA [START_REF] Sugandhi | Esophageal lung: presentation, management, and review of literature[END_REF][START_REF] He | Communicating bronchopulmonary foregut malformation type IA: radiologic anatomy and clinical dilemmas[END_REF][START_REF] Srikanth | Communicating bronchopulmonary foregut malformations: classification and embryogenesis[END_REF].

As a complement to echocardiography, contrast-enhanced CT may provide precise cartography of both the tracheobronchial tree and mediastinal vessels before surgery [START_REF] Mentessidou | Right or left thoracotomy for esophageal atresia and right aortic arch? Systematic review and surgicoanatomic justification[END_REF]. We found a vascular anomaly in nearly half of the 30 patients who underwent a contrast-enhanced CT: arteria lusoria in 6 cases and a right-sided aortic arch in 3 cases. Most of the descriptions of mediastinal vascular anomalies associated with oesophageal atresia that have been reported in the literature also involved the aortic arch, including cases of double aortic arches [START_REF] Canty Tg | Aortic arch anomalies associated with long gap esophageal atresia and tracheoesophageal fistula[END_REF][START_REF] Berthet | Vascular Anomalies Associated with Esophageal Atresia and Tracheoesophageal Fistula[END_REF]. Arteria lusoria has been previously described in 0.5 to 2% of the general population [START_REF] Ergun | Anatomical variations in branching pattern of arcus aorta: 64-slice CTA appearance[END_REF][START_REF] Jakanani | Frequency of variations in aortic arch anatomy depicted on multidetector CT[END_REF] and in around 8% of patients with oesophageal atresia, rising to 58% of patients with an associated long oesophageal defect and tracheoesophageal fistula [START_REF] Canty Tg | Aortic arch anomalies associated with long gap esophageal atresia and tracheoesophageal fistula[END_REF]. In addition, the frequency of a right-sided aortic arch in the general population is estimated to be around 0.1 to 0.9% and as high as 2.5 to 13% in patients with oesophageal atresia [START_REF] Ladd | Esophageal Atresia and Tracheo-esophageal Fistula[END_REF][START_REF] Briganti | Tracheomalacia in oesophageal atresia: morphological considerations by endoscopic and CT study[END_REF][START_REF] Canty Tg | Aortic arch anomalies associated with long gap esophageal atresia and tracheoesophageal fistula[END_REF][START_REF] Berthet | Vascular Anomalies Associated with Esophageal Atresia and Tracheoesophageal Fistula[END_REF].

One of our patients presented with partial anomalous pulmonary venous return associated with congenital heart disease and lobar pulmonary agenesis and five presented with persistent left superior vena cava. The latter has been the most frequently described intrathoracic venous anomaly in the general population (0.3-2%), with a rate of 10% in patients with congenital heart disease [START_REF] Sheikh | Persistent Left Superior Vena Cava with Absent Right Superior Vena Cava: Review of the Literature and Clinical Implications[END_REF] and an even higher frequency in patients with oesophageal atresia. This was illustrated by a study of a select population of 90 children with oesophageal atresia who had at least one other malformation consistent with VACTERL syndrome, including 5.6% who presented with a persistent left superior vena cava [START_REF] De Jong | Non-VACTERL-type anomalies are frequent in patients with esophageal atresia/tracheoesophageal fistula and full or partial VACTERL association[END_REF].

Tracheal anomalies are very frequent in cases of oesophageal atresia with tracheoesophageal fistulas [START_REF] Fayoux | Laryngotracheal anomalies associated with esophageal atresia: importance of early diagnosis[END_REF][START_REF] Briganti | Tracheomalacia in oesophageal atresia: morphological considerations by endoscopic and CT study[END_REF][START_REF] Thakkar | Bronchoscopy as a screening tool for symptomatic tracheomalacia in oesophageal atresia[END_REF] and can be diagnosed using CT scans, although laryngotracheal endoscopy remains the standard exam [START_REF] Fayoux | Laryngotracheal anomalies associated with esophageal atresia: importance of early diagnosis[END_REF][START_REF] Thakkar | Bronchoscopy as a screening tool for symptomatic tracheomalacia in oesophageal atresia[END_REF]. In our study, some cases of confirmed tracheomalacia and tracheal stenosis were discovered on CT scans. However, CT scans may be useful in cases of tracheal stenosis, to assess the length of stenosis as well as the vascular environment [START_REF] Clements | Congenital malformations of the lung and airways[END_REF][START_REF] Berrocal | Congenital anomalies of the tracheobronchial tree, lung, and mediastinum: embryology, radiology, and pathology[END_REF]. The prevalence of tracheomalacia in oesophageal atresia has been reported to range from 65 to 89% in studies, with reported differences between neonates, young children and teenagers [START_REF] Fayoux | Laryngotracheal anomalies associated with esophageal atresia: importance of early diagnosis[END_REF][START_REF] Briganti | Tracheomalacia in oesophageal atresia: morphological considerations by endoscopic and CT study[END_REF][START_REF] Thakkar | Bronchoscopy as a screening tool for symptomatic tracheomalacia in oesophageal atresia[END_REF]. Although CT scans do not provide dynamic analysis, previous studies have shown that tracheomalacia could be suspected in cases with reduced tracheal lumen on end-expiratory images. These were in children able to perform inspiratory and end-expiratory manoeuvres and in young children under general anaesthesia and during passive breath holding [START_REF] Ngerncham | Tracheobronchomalacia in pediatric patients with esophageal atresia: comparison of diagnostic laryngoscopy/bronchoscopy and dynamic airway multidetector computed tomography[END_REF][START_REF] Wallis | ERS Statement on Tracheomalacia and Bronchomalacia in Children[END_REF]. Using this dynamic approach, the anatomical changes can be arbitrarily described as mild (50 to 75% reduction), moderate (75 to 90% reduction) or severe (>90%) [START_REF] Wallis | ERS Statement on Tracheomalacia and Bronchomalacia in Children[END_REF]. During free breathing, as in our study, tracheomalacia could be suspected using indirect signs [START_REF] Briganti | Tracheomalacia in oesophageal atresia: morphological considerations by endoscopic and CT study[END_REF]. With regard to the recurrences of tracheoesophageal fistula, the standard exam for diagnosis remains methylene blue test during endoscopy. In years to come, high resolution structural magnetic resonance imaging could become a new tool for diagnosing tracheoesophageal fistula, without the associated radiation hazards [START_REF] Wallis | ERS Statement on Tracheomalacia and Bronchomalacia in Children[END_REF].

Our study had some limitations. First, our sample comprised children who were severely affected by oesophageal atresia, had a high frequency of associated malformations and who underwent CT scans based on their symptoms and, or, associated conditions. Second, only 62% of the exams were performed with contrast administration and it was likely that this led to underestimating some vascular anomalies. Finally, the exams were performed at different ages, but this did not alter the diagnosis of associated anatomical anomalies.

CONCLUSION

Our study confirms the frequent association of bronchopulmonary and mediastinal vascular anomalies in patients with oesophageal atresia. Chest CT scans enabled us to diagnose unexpected thoracic malformations in 27% of these patients and confirm major associated malformations in 10%. Further studies are needed to assess the prevalence of these associations in unselected populations with oesophageal atresia. In clinical practice, we recommend performing chest CT scans on patients with oesophageal atresia to check for associated multiple malformations, persistent respiratory symptoms or unexplained restrictive syndrome. Due to the high frequency of associated vascular anomalies, we propose that contrast-enhanced chest CT scans should become the initial exam of choice.

Figure 1 :

 1 Figure 1: Sample flow chart

Figure 2 :

 2 Figure 2: Examples of malformations seen on contrast-enhanced chest CT

Figure 4 :

 4 Figure 4: Proposed decision tree on the role of chest CT scans in patients with repaired

Table 1 : Sample characteristics of the 48 patients Characteristics n (%)

 1 Values reported as frequency (percentage) or median (range).There was one missing value for five of the six associated malformations because the child was not born in our centre and was followed up in the later years. CT, computed tomography.

	Demographic and neonatal characteristics
	-Male	27/48 (56)
	-Preterm birth	16/48 (33)
	-Single umbilical artery	6/48 (13)
	-Age (years) at CT scan,	4 years, 5 months
	median (range)	(1 day-16 years, 2 months)
	Ladd classification of oesophageal atresia *
	-Type I	4/48 (8)
	-Type II	2/48 (4)
	-Type III	39/48 (81)
	-Type IV	3/48 (6)
	Associated syndrome
	-VACTERL association	13/48 (27)
	-22q11.2 deletion	1/24 (4)
	-Chromosome 15q duplication	1/24 (4)
	Associated malformations
	-Congenital heart disease	15/47 (32)
	-Malformation of the urogenital tract	14/48 (29)
	-Malformation of the skeleton	14/47 (30)
	-Malformation of the gastrointestinal tract	6/47 (13)
	-Laryngeal supraglottic malformation	4/47 (8)
	-Other malformation	20/47 (43)
	-Malformations of ≥ 2 organ systems	23/47 (49)
	Tracheomalacia	22/30 (73)

*As defined by Ladd et al

[START_REF] Ladd | Esophageal Atresia and Tracheo-esophageal Fistula[END_REF]

Table 2 : Associated malformations in six patients with congenital pulmonary malformations

 2

	Patient	Type of	Pulmonary	Other anomalies
		oesophageal	malformation
		atresia *
	Patient a	III	Right pulmonary aplasia	Ventricular septal defect -
				arteria lusoria
	Patient b	III	Agenesis of the right upper	VACTERL Association
			and middle lobes	Laryngeal diastema
				Coarctation of the aorta -atrial
				septal defect
				Partial right anomalous
				pulmonary venous return
				Vesicoureteral reflux
				Ear malformation
	Patient c	III	Agenesis of the right upper	Arteria lusoria -persistent left
			and middle lobes	superior vena cava
				Intestinal malrotation
	Patient d	III	Agenesis of the middle lobe VACTERL Association
				Tracheal stenosis
				Arteria lusoria -persistent left
				superior vena cava
				Hemivertebrae
				Rib anomaly
				Right radial polydactyly
	Patient e	III	Agenesis of the middle lobe Duodenal atresia
				Multicystic kidney
	Patient f	III	Congenital cystic	VACTERL Association
			adenomatoid malformation	Coarctation of the aorta -
				persistent left superior vena
				cava
				Congenital block vertebrae
				Imperforate anus
				Multicystic kidney
	* As defined by Ladd et al (1)

This study did not receive any specific funding.

Conflicts of interest

The authors have no conflicts of interest to declare.

This study was presented at the Francophone Hepato-gastroenterology and Digestive

Ao: aorta; Oe: oesophagus; T: trachea


Francois Trompier

Jiří Sádlo

Juraj Michálik

Waclaw Stachowicz

Alejandro Mazal

Isabelle Clairand

Joanna Rostkowska

Wojciech Bulski

Andrzej Kulakowski

Janusz Sluszniak

EPR dosimetry for actual and suspected overexposures during radiotherapy treatment

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Dose results

Sentivity variation due to necrosis effect on bone

*•

  EPR dosimetry for actual and suspected overexposures during radiotherapy treatment F. Trompier 1 , J. Sadlo 2 , J. Michalik 2 , W. Stachowicz 2 , A. Mazal 3 , I. Clairand 1 , J. Rostkowska 4 , W. Bulski 4 , A. Kulakowski 5 , J. Sluszniak 5 , S.Gozdz 5 and A. Wojcik 2,6 1 IRSN, France, 2 Institute of Nuclear Chemistry and Technology, Warsaw, Poland, 3 Institut Curie, Paris, France, 4 Institute of Oncology, Warsaw, Poland, 5 Holy Cross Cancer Center, Kielce, Poland, 6 Institute of Biology, Swietokrzyska Academy, Kielce, Poland Contact : francois.trompier@irsn.fr These two cases show that EPR can provide useful data and that some specific studies on bone dosimetry are neccesary to improve analysis of data used to estimate the doses on rib bone samples removed from actual and suspected overexposed patient during radiotherapy treatment Case n°2: investigation for a suspected overexposure The recognized overexposure occurred on February 27th 2001 in the Bialystok Oncology Center, when five breast-cancer patients undergoing radiotherapy received a single, high dose of 8 MeV electrons generated by a Neptun 10p linear accelerator. The ultimate cause of the accident was a defective safety interlock and an obsolete safety system of the Neptun 10p accelerator leading to a large increase of the dose rate. All patients experienced immediate pain and skin reddening, followed by moist desquamation and development of deep necroses by autumn 2001. In 2005, a patient developed necrosis in the right axillary region formerly irradiated (9MV photon) during "standard upper mantel fields" therapy for an Hodgkin's disease in 1999. A late effect of an overexposure during the treatment was considered as an plausible hypothesis for the necrosis appearance. Doses were assessed by the dose addition method on cortical part of rib bone sample. Post irradiation performed using 9 MV electron (IRSN) and 60 Co gamma (INCT) for Byalistock accident samples and 60 Co gamma (IRSN) for the case n°2. Variation of beam and energy response was taken into account when for dose reconstruction based on EPR data are coming from: •Accuracy of sample position •Fast attenuation of electrons •Accuracy on cumulative dose before accident •Effect of necrosis on ESR signal Correction factor e-/g : 1.11 ** from Accidental overexposure of radiotherapy patients in Bialystok, IAEA, Vienna, 2004 Total dose measured on rib bone by EPR (Gy) Problem for dose reconstruction, no available data to correct measured doses from the loss of EPR signal due to bone remodelling and the influence of necrosis on signal loss and dose sensitivity •No conclusions on radio-induced origin of the pathology *in tissue kerma corrected from energy variation **slope for LR in air kerma, 60 Co


Francois Trompier

Alexander Romanyukha

Lucile Rémy-Kornak

Céline Calas

Blaise Leblanc

Isabelle Clairand

Chad Mitchell

Harold Swartz

Protocol for emergency dosimetry based on EPR spectrometry on nails

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

In case of an accident involving a large number of victims, most of dosimetric techniques are limited by the necessary short delay of the answer or the invasive sampling. In this specific case, it is very important in a first time and as quick as

  EPR dosimetry on materials easily sampled on the victims (hairs or nails) may be a pertinent tool. In this context, the objective of this work was to study the EPR properties of nails to propose a first protocol for fast triageThis study showed that it was possible, starting from EPR measurement of nails to carry out a population triage according to the severity of the exposureDose= ( S total -S native-S MIS ).F(t, T°, H).A with S total = S RIS + S native + S MIS F : fading correction factor depending on time elapsed between irradiation and measurement t, temperature T°a nd humidity H and A: of S total , dose estimation with mean value of S native , Estimation of sample humidity (H): improvment of estimation of F Dose estimation Protocol Treatment in distilled water + EPR measurement Sample drying + mass measurements Determination of real value of S native Irradiation at known doses + EPR measurement Determination of real value of AThe protocol is divided in different stages. A first estimation is done very quickly using averaged value of main characteristics (fading constant, radiation sensitivity, amplitude of background signal) previously determined on a large set of samples. At each next stage, the determination of the real values of the different constants for each sample allows to increase the accuracy of the dose estimation.


M I Tenaillon

E Burban

S Huynh

A Wojcik

Anne-Claire Thuillet

A-C Thuillet

D Manicacci

P R Gérard

K Alix

H Belcram

A Cornille

M Brault

R Ste- Vens

J Lagnel

C Dogimont

Y Vigouroux

S Glémin

Crop domestication as a step towards reproductive isolation

Keywords: Speciation Gene flow, Adaptive introgression, Genomic divergence, Mating system, Genetic incompatibilities, Hybrid breakdown, Domestication load, Crop vulnerability

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

BACKGROUND

Speciation, Darwin's mystery of mysteries, is a continuous process that results in genomic divergence accompanied by the gradual increment of reproductive barriers between lineages. Since the beginning of research on the genetics of speciation, sev-This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/ajb2.16173.

Accepted Article

eral questions have emerged such as: What are the genetic bases of incompatibilities? How many loci are necessary to prevent hybridization and how are they distributed along genomes? Can speciation occur despite gene flow and how common is ecological speciation? Early stages of divergence are key to understand the ecology and genetics of speciation, and semi-isolated species where hybrids can still be produced are particularly relevant. Here we argue that the recent divergence between wild and domesticated lineages is an excellent model to capture the very-first steps of reproductive barriers formation, and will bring novel insights into the speciation process.

WHY IS DOMESTICATION A GOOD MODEL TO STUDY SPECIATION?

Domestication is the process of divergent selection between wild forms undergoing natural selection in their habitats, and domesticates evolving under combined natural and human-mediated selection. It has been increasingly recognized that evolution of domesticated species shows many similarities with evolution in the wild: it results primarily from changing environmental conditions and involves unconscious selection under a protracted process [START_REF] Purugganan | Evolutionary Insights into the Nature of Plant Domestication[END_REF] with selection intensities of the same magnitude or even smaller (Yang et al. 2019). Thus, domestication has been considered as a choice example to study adaptation. Here, we argue that it also offers an excellent opportunity to catch the very-first processes at work in ecological speciation, where adaptive divergence between nascent lineages triggers the onset of reproductive isolation (RI).

Allele differentiation resulting from divergent selection can be measured by F ST . F ST between wild and domestic pairs range between 0.05 in sweet cherry and 0.51 in Tomato (Appendix S1, and references herein; see the Supplementary Data with this article), which cover a wide range of divergence within a "grey zone of speciation" in which barriers to gene flow exist but are not complete [START_REF] Roux | Shedding light on the grey zone of Speciation along a continuum of genomic divergence[END_REF]. Interesting-

Accepted Article

ly, within this continuum, self-fertilizing taxa display greater genetic differentiation than outcrossers (Figure 1). As mating systems are predicted to affect the speciation process, domestication also offers the opportunity to address this question [START_REF] Brochmann | Mating system and speciation I: Accumulation of genetic incompatibilities in allopatry[END_REF]. In contrast, life span seems to have no significant effect on divergence (Figure 1), although annuals and perennials experience contrasted domestication dynamics in many respects [START_REF] Gaut | Genomics and the Contrasting Dynamics of Annual and Perennial Domestication[END_REF].

The existence of reproductive barriers between wild and domesticated plants has been repeatedly documented. Despite the occurrence of wild-cultivated gene flow, the establishment of wild alleles into domesticated populations and reciprocallyintrogressionsis rare [START_REF] Ellstrand | Introgression of crop alleles into wild or weedy populations In: Futuyma DJ, ed[END_REF]. Perhaps the best documented examples come from maize, where the introgression from the mexicana teosinte subspecies has contributed to highland adaptation of maize landraces [START_REF] Calfee | Selective sorting of ancestral introgression in maize and teosinte along an elevational cline[END_REF]; and conversely, introgression from locally-adapted maize has contributed to teosinte adaptation in Europe (Le [START_REF] Corre | Adaptive introgression from maize has facilitated the establishment of a teosinte as a noxious weed in Europe[END_REF]. Interestingly, introgressions in the two directions are removed by selection around domestication genes (Le [START_REF] Corre | Adaptive introgression from maize has facilitated the establishment of a teosinte as a noxious weed in Europe[END_REF][START_REF] Calfee | Selective sorting of ancestral introgression in maize and teosinte along an elevational cline[END_REF]. This points to a prominent role of pre-and post-zygotic genetic barriers in the divergence of wild and domesticated lineages, and some genes involved in reproductive barriers have been identified such as the Tcb1 locus in maize that governs pollen rejection by teosinte [START_REF] Corre | Adaptive introgression from maize has facilitated the establishment of a teosinte as a noxious weed in Europe[END_REF].

Whether partial isolation between wild and domesticated forms will ultimately result in full speciation is unknown. But clearly, partial RI does occur and has contributed to the maintenance of the distinct features between wild and domesticated forms, the so-called domestication syndrome. RI therefore stands as a major component of the domestication syndrome, but has been so far largely ignored [START_REF] Dempewolf | Reproductive isolation during domestication[END_REF]. It is even possible that reinforcement played a role in the establishment

Accepted Article

of the domestication syndrome, which involves the evolution of stronger RI due to the costs associated with producing low-fitness hybrids [START_REF] Rushworth | Conflict over fertilization underlies the transient evolution of reinforcement[END_REF].

THE GENETIC BASES OF REPRODUCTIVE ISOLATION

The establishment of reproductive barriers can occur through various mechanisms. Selection leading to the fixation of advantageous alleles in different environments, resulting in local adaptation, can cause hybrid offspring to have lower fitness in parental environments, which strengthens isolation as populations adapt to differing conditions. This process may contribute to RI between wild and domesticated forms, and some crops may already be considered as independently evolving lineage once human-mediated cessation of gene flow is complete. Loci involved in such adaptation, those governing domestication traits, display a high degree of differentiation between wild and domesticated forms as well as a pattern of positive selection within forms compared with neutral loci (Figure 2). They contribute to limiting effective gene flow at nearby loci, leading to the progressive buildup of the so-called genomic islands of divergence [START_REF] Wolf | Making sense of genomic islands of differentiation in light of speciation[END_REF]. RI may also be promoted by the buildup of intrinsic barriers from the differential fixation of alleles that are incompatible at two or more interacting loci (Bateson-Dobzhansky-Muller Incompatibilities -BDMIs). Such BDMIs can evolve as a byproduct of local adaptation to contrasting environments or through non-adaptative processes [START_REF] Wolf | Making sense of genomic islands of differentiation in light of speciation[END_REF]. If selection favors distinct mutational steps at several loci in each population, deleterious side effect interactions may arise when brought together in hybrids. These interactions may in turn provoke detrimental symptoms and/or Transmission Ratio Distortions (TRDs) at F2 generation for recessive alleles, contributing to intrinsic post-zygotic isolation.

Accepted Article

In domesticated forms, the accumulation of deleterious mutations through domestication bottlenecks and linked selection may have accelerated the evolution of BDMIs between wild and domesticated forms. The loci underlying BDMIs should display genomic fingerprints that can be similar to those left by selection for habitat adaptation in domestic or wild populations (Figure 2). However, in the absence of intra-form selection, we expect increased divergence between forms while the level of polymorphism is not affected by the cessation of gene flow (Figure 2). This illustrates how the use of different statistics helps to clarify the mechanisms at work in RI [START_REF] Cruickshank | Reanalysis suggests that genomic islands of speciation are due to reduced diversity, not reduced gene flow[END_REF]. There is of course a continuum of scenarios between those presented above: the barrier loci that limit gene flow between wild and domesticates can be directly targeted by selection within one of the two forms.

A particular kind of negative epistatic interaction can emerge as a by-product of coevolution between nuclear and cytoplasmic genomes. If different combinations have coevolved in divergent lineages, this may result in organelle dysfunction and hybrid breakdown when inter-lineage crosses occur [START_REF] Burton | Cytonuclear genomic interactions and hybrid breakdown In: Futuyma DJ, ed[END_REF]. Such negative cytonuclear conflicts often result in asymmetrical reproductive barriers, which can be revealed by reciprocal crosses between wild and domesticated lineages as observed in Citrus [START_REF] Wang | Pan-mitogenomics reveals the genetic basis of cytonuclear conflicts in citrus hybridization, domestication, and diversification[END_REF].

Finally, reproductive barriers may result from parental conflicts generating allelic dosage perturbations. If species have evolved contrasting levels of parental conflicts, it can translate into paternal or maternal excess of gene expression in a hybrid context [START_REF] Florez-Rueda | Genomic imprinting in the endosperm is systematically perturbed in abortive hybrid tomato seeds[END_REF]. As evidenced by transcriptomic comparisons of wild and domestic forms (e.g., common bean, Bellucci et al. 2014;tomato, Sauvage et al. 2017) and simulations [START_REF] Burban | Gene network simulations provide testable predictions for the molecular domestication syndrome[END_REF], domestication led to a profound reorchestration of coexpression networks, which can then cause disruptions in allelic dos-

Accepted Article

age between wild and domestic forms resulting in fitness decline in wild x domesticated crosses.

HYPOTHESES TESTING & CHALLENGES

Do the number of generations since domestication correlate with hybrid defects? Does the mutation load depend on the domestication history and the strength of RI? Do "stronger" domestication syndromes and/or higher genome-wide neutral divergence and/or extent of islands of differentiation induce stronger isolation?

Answering these questions will bring unique insights into the very-first steps of reproductive barriers formation, but detecting barrier genes is a daunting task. Divergent selection and BDMIs among loci create patterns of strong allelic differentiation relative to the genomic background (Figure 2) that together with linked loci, form genomic islands of differentiation. Their detection requires overcoming confounding effects such as local variation in recombination rates and effective population size [START_REF] Wolf | Making sense of genomic islands of differentiation in light of speciation[END_REF]. At the species level, parameters such as mating system, intensity of domestication and changes in effective population size (e.g., due to domestication bottlenecks) determine the extent of selection, genetic drift and linkage disequilibrium, and in turn the expected size and depth of islands of differentiation.

Ultimately, interpretations of genomic differentiation patterns need to be guided by modelling in order to properly estimate the fraction of the genome recalcitrant to gene flow and identify the corresponding regions, which can then be combined to experimental results.

CONCLUSION

The alterations of habitats due to human activities are precious laboratories to explore the mechanisms involved in adaptive divergence and the initial phases of speciation [START_REF] Thompson | Speciation and the City[END_REF][START_REF] Touchard | Urban rendezvous along the seashore: Ports as Darwinian field labs for studying marine evolution in the Anthropocene[END_REF]. The establishment of RI be-

Accepted Article

tween wild and domestic forms is a crucial aspect of domestication that has received little attention. In addition to providing basic information about the processes at work in the early stages of speciation, testing for cross-compatibility between cultivated plants and their wild relatives and detecting the underlying barrier loci are essential for overcoming them. Crops wild relatives have faced continuous environmental challenges in their natural environment and often exhibit greater genetic diversity than their domesticated relatives, so they are a valuable reservoir of adaptive alleles that transferred to crops could help mitigate their vulnerability.

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Acknowledgements section

We are grateful to the editor in chief and two anonymous reviewers for helping us to improve the manuscript.

Data Availability Statement

No new data were generated for this manuscript.

Literature

Cited Bellucci E, Bitocchi E, Ferrarini A, et al. 2014. Decreased nucleotide and expression diversity and modified coexpression patterns characterize domestication in

Authors contribution & Funding

This essay is the result of discussions and close collaboration between all participants in the DomIsol project (ANR-19-CE32-0009-02). [START_REF] Roux | Shedding light on the grey zone of Speciation along a continuum of genomic divergence[END_REF]. F ST values (x-axis) were computed for 61 pairs of animal populations/species across sequenced loci (natural divergence/speciation). The posterior probability of ongoing migration (y-axis) for a given pair reflects the capacity of demographic models that allow for ongoing exchange of migrants between diverging

Accepted Article


Michel Malo

Cécile Charrière-Bertrand

Chafika Chettaoui

Élizabeth Fabre-Guillevin

François Maquerlot

Alexandra Lackmy

Benoît Vallée

Franck Delaplace

Georgia Barlovatz-Meimon

Équipe Dynamic

Microenvironnement cellulaire, PAI-1 et migration cancéreuse

Keywords: PAI-1, Protéine matricellulaire, Migration amiboïde, Non-proportionnalité des effets PAI-1, Matricellular protein, Amoeboid migration, Nonlinear effects

Le processus cancéreux est une dynamique complexe, dont l'intégration de tous les paramètres nécessite de mieux comprendre les événements observés aux différentes échelles et d'en modéliser les interactions. La modification d'assemblages moléculaires « matricellulaires » peut déclencher le processus métastatique. In vitro, un microenvironnement cellulaire, très enrichi en PAI-1 matriciel, entraîne une modification du comportement des cellules qui adoptent le mode de migration amiboïde et présentent toutes les caractéristiques morphologiques de l'état post-transition mésenchymateuse-amiboïde et ses conséquences. PAI-1 apparaît donc comme une protéine matricellulaire capable de déclencher le processus à l'échelle moléculaire, cellulaire, et tissulaire, susceptible d'engager un pronostic concernant l'organisme entier.

Abridged English version

Cancer is a complex process caused by a cellular dysfunction leading to a whole organ or even organism vital perturbation. To better understand this process we need to understand each of the levels involved and what allows the change of scales.

A matricellular protein, called PAI-1 (Plasminogen Activator Inhibitor type I) has been identified as able to induce cell adhesion, reorganization of actin cytoskeleton and morphological changes, and to promote cell migration. PAI-1 participates in a regulation network linking the extracellular matrix and the cell. The molecular bridge formed by this protein and others is able to transduce biochemical and biomechanical signals to the cell. The cell response can be evaluated in terms of mRNA rate constants. It can further be assessed by the evaluation of migration mode or speed. We are using the game theory and the game network theory to model this dynamic at the cell scale.

PAI-1, found closely around the most invasive tumours, represents an independent factor of bad prognosis. Cancer cells, having undergone the epithelialmesenchymal transition (EMT), can use PAI-1 for their migration. However, if the environment is very rich in PAI-1, the cells undergo a second transition, the socalled mesenchymal-amoeboid transition (MAT). Their migration becomes then 'amoeboid' and is very different from the mesenchymal migration, at least by the independence towards integrins and protease activity. This amoeboid state is characterized by a complete reorganisation of actin (with actin rings) and by the activation of the Rho GTPases transduction pathways. Furthermore, in the presence of this particular (i.e. PAI-1-enriched) environment, the cells down-regulate their PAI-I mRNAs rate constants.

From the cellular level to the tissue level

The regulation network occurs at the cellular/microenvironmental level and includes a PAI-I concentration threshold. However, the consequences of a unique cell MAT can be its escape from the initial tumour, i.e. metastatic migration. If this occurs, the moving cancer cell will meet a microenvironment in which PAI-1 progressively decreases, and the cell could return to the mesenchymal condition through an inverse transition (form amoeboid to mesenchymal behaviour). As it is a cancer cell, it will proliferate, produce more PAI-I (a characteristic of the most invasive cells) and perhaps again undergo MAT. The PAI-I negative feedback in terms of RNAs, described earlier, could help this regula-tion to occur. We have perhaps identified one of the conditions for a cell to undergo MAT and return. This might be of importance in the understanding of the metastatic escape. To complete our experimental approach, we used the Game theory to model the molecular network involved in this process (see Manceny et al. [37]).

A nonlinear system

The regulation of a given cancer cell behaviour by its PAI-1 microenvironment could then be considered as a cause leading to consequences at the organ or the organism level. And its critical transition point is that one single molecule of PAI-1 could do the job! In avalanches, it is known that at the critical state, the output is not proportional to the input, that is, the system is highly nonlinear and gives rise to 'non-obvious' effects. Could the PAI-1 swing (i.e. effect of one more or one less molecule of PAI-1) control the 'biological avalanche' of cancer? This hypothesis is illustrated in Fig. 1.

Cancer, migration cellulaire : un changement d'échelle ?

Le processus cancéreux est une dynamique complexe : pour mieux comprendre les événements observés à différentes échelles (moléculaire, cellulaire, tissulaire et de l'organisme) il faut intégrer l'ensemble des paramètres qui nous semblent contrôler la dynamique du système dans un modèle mécanistique global.

À l'échelle de l'organisme, il s'agit d'une pathologie. Celle-ci est issue d'un dysfonctionnement moléculaire qui s'exprime à l'échelle de la cellule (dysfonctionnement d'origine génétique). Cette cellule est elle-même sous l'influence de son environnement immédiat. On retrouve en effet des liens étroits entre le processus cancéreux et le milieu extracellulaire, qui peut induire, permettre, favoriser ou au contraire freiner l'échappement métastatique. Ces liens multiples liant la cellule à son environnement se retrouvent à tous les niveaux : à l'échelle cellulaire, ils sont restreints à l'environnement immédiat de la cellule cancéreuse (c'est le microenvironnement cellulaire), à l'échelle du tissu, la nature des liens est la résultante des différents apports, sécrétions ou présence de cellules tumorales et non tumorales ; à l'échelle de l'organe, ces liens dépendent essentiellement des conséquences systémiques de la pathologie.

Ainsi, à chacune des ces échelles, l'environnement influence les processus biologiques ; mais c'est à l'échelle cellulaire que se déclenche un des processusclés : l'échappement métastatique, qui caractérise l'invasion cancéreuse. Cet échappement nécessite une séquence de phases de migration, puis de prolifération cellulaire. Ce sont, sans doute, les modifications des assemblages moléculaires liant cellules et matrice, ou assemblages matricellulaires, qui vont créer les conditions favorables au déclenchement du processus. Ces conditions incluent l'adoption par les cellules tumorales d'un profil migratoire associé aux conditions de départ du processus. Ce profil peut être caractérisé par des variations moléculaires, morphologiques, « comportementales ».

Un assemblage moléculaire matricellulaire implique l'inhibiteur de l'activateur du plasminogène (PAI-1) sous sa forme matricielle [START_REF] Planus | Binding of urokinase to plasminogen activator inhibitor type-1 mediates cell adhesion and spreading[END_REF]. Lorsque cette molécule est abondante dans le microenvironnement, le comportement migratoire de plusieurs types cellulaires s'en trouve modifié [START_REF] Planus | Binding of urokinase to plasminogen activator inhibitor type-1 mediates cell adhesion and spreading[END_REF][START_REF] Bonavaud | Evidence of a non-conventional role for the urokinase tripartite complex (uPAR/uPA/PAI-1) in myogenic cell fusion[END_REF] en particulier celui de cellules cancéreuses invasives [START_REF] Chazaud | Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations[END_REF]. Il apparaît donc essentiel de déterminer le rôle de PAI-1 matriciel dans le processus cancéreux, d'autant plus que des études cliniques ont montré qu'il représente un facteur indépendant de mauvais pronostic [START_REF] Duffy | The urokinase plasminogen activator system: A rich source of tumour markers for the individualised management of patients with cancer[END_REF] et qu'in vivo, PAI-1 est retrouvé en fortes concentrations dans la matrice, en périphérie des tumeurs [START_REF] Umeda | Cellular localization of urokinase-type plasminogen activator, its inhibitors, and their mRNAs in breast cancer tissues[END_REF].

PAI-1 : une molécule-pivot dans le processus de migration cellulaire ?

Migration cellulaire et processus métastatique

Le caractère métastatique des cancers dépend de la capacité des cellules cancéreuses à quitter le tissu d'origine (la tumeur primaire) et ensuite à s'implanter et proliférer ailleurs. La migration cellulaire accompagne l'invasion tumorale et constitue le point de départ du processus métastatique. Les cellules cancéreuses acquièrent de nouvelles propriétés biochimiques et caractéristiques morphologiques leur permettant de s'échapper de la tumeur principale. Une partie de ces transformations est communément appelé EMT (transition épithéliomésenchymateuse) ; elle se traduit par la disparition des phénotypes épithéliaux au profit de marqueurs mésenchymateux [START_REF] Greenburg | Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells[END_REF]. Les cellules n'expriment plus alors le gène de la E-cadhérine, perdent l'expression de la cytokératine 8 et ré-expriment la vimentine [START_REF] Cano | The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression[END_REF].

Les cellules cancéreuses ayant subi l'EMT utilisent spontanément une migration de type mésenchymateux. Ce type de migration est caractérisé par une protéolyse péricellulaire active, l'établissement de points d'ancrages très résistants et un cytosquelette d'actine struc-turé. Les points focaux d'adhérence à la matrice extracellulaire, par l'intermédiaire des intégrines, participent à l'organisation des filaments d'actine et la formation de protrusions membranaires de types lamellipodes [START_REF] Lauffenburger | Cell migration: a physically integrated molecular process[END_REF].

Mais dans certaines conditions environnementales, les cellules cancéreuses (cellules ayant donc déjà « passé » ou « subi » la transition epithélio-mésenchymateuse) peuvent subir une seconde transition, appelée transition mésenchymateuse-amiboïde (MAT). Dans ce cas, le cytosquelette d'actine se réorganise, abandonnant la formation de fibres de stress, au profit d'une architecture plus légère, avec redistribution diffuse et corticale de l'actine. Cette réorganisation, conduit à la formation d'anneaux d'actine (actin rings) et se trouve sous le contrôle de l'activation de la protéine ROCK-1, un effecteur de la petite GTPase RhoA [START_REF] Sahai | Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis[END_REF].L am o rphologie générale s'arrondit : les cellules présentent de nombreux bourgeonnements ou blebs ; on parle d'une morphologie amiboïde.

Le type de migration de ces cellules cancéreuses devient alors très différent d'une migration mésenchymateuse. Il s'agit d'une migration de type amiboïde dans laquelle les cellules se déforment, leur membrane est animée de mouvements dynamiques liés à la formation/déformation des blebs (on parle de blebbing). Les cellules se faufilent au travers des mailles de la matrice extracellulaire en n'ayant recours, ni à une activité protéasique, ni aux intégrines [START_REF] Friedl | Prespecification and plasticity: shifting mechanisms of cell migration[END_REF][START_REF] Wolf | Compensation mechanism in tumor cell migration: mesenchymalamoeboid transition after blocking of pericellular proteolysis[END_REF] ; en revanche, elles utilisent de nombreux points d'ancrage de faible intensité et des séquences d'adhésion/dé-adhésion très rapides [START_REF] Friedl | CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize beta1 integrinindependent strategies for polarization, interaction with collagen fibers and locomotion[END_REF][START_REF] Brakebusch | Beta1 integrin is not essential for hematopoiesis but is necessary for the T cell-dependent IgM antibody response[END_REF].

Nous avançons l'hypothèse que PAI-1 joue un rôle clé dans l'ensemble de ces processus. Pour comprendre ce rôle, il faut se placer à l'échelle cellulaire et considérer PAI-1 en modulateur des fonctions cellulaires.

PAI-1 est un modulateur des fonctions d'adhésion et de dé-adhésion cellulaires

PAI-1 est le principal inhibiteur de l'uPA ou activateur du plasminogène de type urokinase, une sérine protéase qui convertit le plasminogène en plasmine. PAI-1 existe, soit à l'état soluble, soit associé à la matrice.

Sous sa forme soluble, PAI-1 se lie à l'uPA, ellemême fixée à son récepteur membranaire, l'uPAR. Cette interaction aboutit à l'arrêt de la protéolyse péricellulaire dépendante de l'uPA. La fixation de PAI-1 conduit à l'endocytose du complexe uPAR-uPA-PAI-1 par le biais d'un récepteur spécifique de l'internalisation, le α2MLRP. Le complexe uPA-PAI-1 est dégradé tandis que le récepteur uPAR est recyclé à la membrane [START_REF] Nykjaer | Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes[END_REF][START_REF] Nykjaer | Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes[END_REF].

Sous sa forme associée à la matrice, on parle alors de PAI-1 matriciel (ou PAI-1m), PAI-1 est lié principalement à la vitronectine [START_REF] Keijer | Vitronectin governs the interaction between plasminogen activator inhibitor 1 and tissue-type plasminogen activator[END_REF]. Lorsque le complexe uPAR-uPA-PAI-1 se forme avec le PAI-1matriciel, il constitue alors, un pont moléculaire liant la cellule à la matrice extracellulaire. In vitro, le PAI-1 matriciel module les interactions entre la cellule et la matrice [START_REF] Bonavaud | Evidence of a non-conventional role for the urokinase tripartite complex (uPAR/uPA/PAI-1) in myogenic cell fusion[END_REF][START_REF] Chapman | Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration[END_REF][START_REF] Estreicher | The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes[END_REF][START_REF] Seiffert | Interactions between type-1 plasminogen activator inhibitor, extracellular matrix and vitronectin[END_REF] et plus particulièrement lors des processus d'adhésion et de mobilité [START_REF] Chazaud | Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations[END_REF][START_REF] Chazaud | Involvement of the [uPAR:uPA:PAI-1:LRP] complex in human myogenic cell motility[END_REF].

PAI-1, un modulateur de la réorganisation du cytosquelette d'actine ?

Dans certaines conditions, l'assemblage moléculaire (uPAR-uPA-PAI-1) relie la cellule à la matrice extracellulaire et est à l'origine de contraintes mécaniques générées par la matrice ou par la cellule. Ces contraintes ont pu être mesurées en terme de forces par magnétocytométrie [START_REF] Wang | Urokinase receptor mediates mechanical force transfer across the cell surface[END_REF], elles sont directement transmises au cytosquelette et induisent sa réorganisation. Ainsi, la formation du complexe tripartite uPAR-uPA-PAI-1 serait capable de transduire des signaux biomécaniques à la cellule en augmentant la rigidité du cytosquelette [START_REF] Wang | Urokinase receptor mediates mechanical force transfer across the cell surface[END_REF].

L'assemblage moléculaire (uPAR-uPA-PAI-1) est également capable de transduire des signaux biochimiques et morphologiques à la cellule. En effet, nous avons montré que des cellules placées dans un environnement enrichi en PAI-1 présentent une forte activation de la petite GTPase RhoA ainsi qu'une morphologie arrondie avec de nombreux bourgeonnements membranaires ou blebs, témoins de l'activation de ROCK-1, un effecteur de RhoA. De plus les matrices enrichies en PAI-1 pourraient déclencher des signaux intracellulaires activant la cascade des Rho GTPases [START_REF] Malo | Matrix PAI-1 directs tumour cell mode of migration[END_REF]. Cette famille regroupant entre autres les kinases Cdc42, Rac1 et RhoA contribue au remodelage du cytosquelette d'actine. En effet Rac1 est impliquée dans la formation des lamellipodes tandis que RhoA stimule l'organisation des filaments d'actine soit en anneaux contractiles soit en fibres de stress [START_REF] Sahai | Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis[END_REF].

Les différentes morphologies induites par la réorganisation du cytosquelette d'actine sont utilisées lors de la migration cellulaire [START_REF] Mitchison | Actin-based cell motility and cell locomotion[END_REF].

PAI-1 serait un modulateur de la migration cellulaire

In vitro, selon le type cellulaire utilisé (cellules mésenchymateuses, épithéliales, normales ou cancéreuses, invasives ou non invasives) et selon les condi-tions de culture (en deux ou trois dimensions, avec ou sans sérum, en présence de l'une ou l'autre protéine matricielle) PAI-1 agit soit en favorisant la migration soit en la défavorisant... [START_REF] Chazaud | Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations[END_REF][START_REF] Waltz | Plasmin and plasminogen activator inhibitor type 1 promote cellular motility by regulating the interaction between the urokinase receptor and vitronectin[END_REF][START_REF] Czekay | Unexpected role of plasminogen activator inhibitor 1 in cell adhesion and detachment[END_REF][START_REF] Lazar | Plasminogen activator inhibitor-1 impairs alveolar epithelial repair by binding to vitronectin[END_REF][START_REF] Wang | Plasminogen activator inhibitor-1 (PAI-1) stimulates human corneal epithelial cell adhesion and migration in vitro[END_REF]. Dans nos expériences, où nous utilisons de très fortes concentrations de PAI-1 (molécule recombinante dont l'activité se maintient 500 h), avec des cellules myogéniques [START_REF] Bonavaud | Evidence of a non-conventional role for the urokinase tripartite complex (uPAR/uPA/PAI-1) in myogenic cell fusion[END_REF], des cellules issues de lignées de cancer du sein [START_REF] Chazaud | Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations[END_REF] ou de cancer colorectal [START_REF] Malo | Matrix PAI-1 directs tumour cell mode of migration[END_REF], PAI-1 réunit les prérequis pour être considéré comme une molécule « promigratoire ». Nous avons pu montrer que son action est d'autant plus efficace qu'on s'adresse à des cellules plus invasives [START_REF] Chazaud | Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations[END_REF], tous les types cellulaires étudiés ayant déjà « passé » ou « subi » la transition épithéliomesenchymateuse (EMT).

Nous avons observé in vitro qu'un environnement très enrichi en PAI-1 entraîne une modification du comportement des cellules cancéreuses. On observe une activation des GTPases de la famille Rho dans des cellules qui adoptent le mode de migration amiboïde et présentent toutes les caractéristiques morphologiques de l'état post-MAT.

Jusque là, seules des conditions de culture en trois dimensions ont permis d'observer à la fois la morphologie et la migration amiboïdes [START_REF] Wolf | Compensation mechanism in tumor cell migration: mesenchymalamoeboid transition after blocking of pericellular proteolysis[END_REF]. En proposant à des cellules cancéreuses colorectales, un environnement très enrichi en PAI-1, nous avons obtenu les mêmes résultats.

PAI-1 à haute concentration apparaît donc comme une protéine capable d'entraîner une transition mésenchymateuse-amiboïde et ses conséquences.

PAI-1 à l'interface matrice/cellule

Pour ses capacités, in vitro, d'induire l'adhésion cellulaire, la réorganisation du cytosquelette d'actine, des changements morphologiques importants et sa participation au processus de migration cellulaire, PAI-1, sous sa forme matricielle, pourrait être considéré comme une protéine matricellulaire [START_REF] Maquerlot | Dual role for PAI-1 (Plas-minogen activator inhibitor type 1) as soluble and as matricellular regulator of epithelial alveolar cell wound healing[END_REF]. La famille des protéines dites matricellullaires englobe plusieurs membres, tels que la thrombospondine 1 et 2, l'ostéonectine et l'ostéopontine, par exemple. Ces protéines sont des constituants de la matrice extra cellulaire, mais elles ne participent pas à l'établissement des propriétés structurales de la matrice comme le font les collagènes, les laminines ou la fibronectine. Les protéines matricellulaires permettent de moduler des fonctions cellulaires comme l'adhésion ou la migration [START_REF] Bornstein | Matricellular proteins: extracellular modulators of cell function[END_REF][START_REF] Bornstein | The role of thrombospondins 1 and 2 in the regulation of cell-matrix interactions, collagen fibril formation, and the response to injury[END_REF].

L'assemblage moléculaire, formé entre PAI-1 lié à la matrice extracellulaire, d'une part, et l'uPA lié à son récepteur membranaire, d'autre part, semble être capable de transmettre des signaux biochimiques et biomécaniques à la cellule.

PAI-1, une molécule pour un changement d'échelle ?

De par sa localisation dans le tissu péritumoral et son rôle de marqueur indépendant de mauvais pronostic, PAI-1 joue évidemment un rôle qui dépasse l'échelle cellulaire. . .

À l'échelle moléculaire : une machinerie auto-régulée

Une boucle de régulation négative transforme un environnement très riche en PAI-1 en environnement moins riche en PAI-1. La formation et la déformation du pont moléculaire uPAR-uPA-PAI-1 induit une réponse cellulaire moléculaire et fonctionnelle. La cellule augmente ou diminue sa migration [START_REF] Chazaud | Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations[END_REF] ; elle pourrait également moduler l'expression de certains messagers du système PAs [START_REF] Malo | Matrix PAI-1 directs tumour cell mode of migration[END_REF][START_REF] Fabre-Guillevin | Synergy between PAI-1 and SNAIL in tumoral process[END_REF]. Ainsi le processus migratoire utilisé par les cellules dépendrait à la fois de la biodisponibilité de PAI-1 et de la régulation des éléments du système PAs.

Nous avons observé également, in vitro, que cet environnement très enrichi en PAI-1 entraîne, dans les cellules cancéreuses, une régulation négative des ARNm de PAI-1 [START_REF] Fabre-Guillevin | Synergy between PAI-1 and SNAIL in tumoral process[END_REF]. Si l'on suppose que le taux protéique de PAI-1 suit l'évolution des ARNm, la présence de PAI-1, entretenue in vivo par les cellules elles-mêmes, devrait diminuer rapidement. Les cellules se retrouveraient alors dans l'équivalent d'un microenvironnement pauvre en PAI-1. Ici le réseau de régulation qui régit le comportement cellulaire pourrait être dépendant de la concentration relative de PAI-1 avec deux points critiques : la concentration de PAI-1 qui engage la cellule dans la transition MAT et celle qui permettrait la transition inverse.

À l'échelle cellulaire : des états réversibles

Une faible variation de cette concentration microenvironnementale de PAI-1 pourrait entraîner des conséquences catastrophiques. Les conséquences envisageables d'une seule transition MAT sont disproportion-nées par rapport à la cause. En effet, la transition MAT offre à la cellule qui s'y engage la possibilité de s'échapper de la tumeur initiale, d'entreprendre une migration métastasique et, grâce à la dynamique du microenvironnement, à la faveur d'une transition inverse, de s'arrêter. Ceci implique, pour la cellule cancéreuse, un retour possible au « comportement » prolifératif initial et éventuellement l'établissement d'une tumeur secondaire. La régulation négative du taux d'ARN messagers de PAI-1 pourrait y contribuer.

Si tel est le cas, la cellule cancéreuse qui a repris sa condition de cellule cancéreuse initiale, non migrante, va proliférer, produire du PAI-1 (une caractéristique de la plupart des cellules cancéreuses invasives) et à nouveau se trouver dans les conditions de la transition MAT. Les points critiques de cette situation seraient des seuils de concentration en PAI-1 matriciel. Ils pourraient être franchis, symétriquement, par la variation (c'est-à-dire l'augmentation ou la diminution relative) de la concentration de PAI-1 dans le microenvironnement de la cellule en question.

Cette dynamique des liens cellule/microenvironnement induit un changement qualitatif du comportement cellulaire pouvant influencer la migration ou la prolifération. À chaque changement des conditions environnementales, la cellule peut se retrouver entraînée dans une nouvelle dynamique.

L'émergence d'un comportement migratoire, le retour vers un comportement non migratoire -possiblement prolifératif -ou la transition vers un autre type de migration est un changement qualitatif, à l'échelle cellulaire. Il peut conduire à des conséquences d'une toute autre échelle : la création d'une métastase. Celleci est en effet le résultat d'une séquence de phases de prolifération, d'une phase de migration suivie au moins d'une nouvelle phase de prolifération. Comprendre le processus métastatique implique donc de comprendre comment une même cellule issue d'une même tumeur initiale peut se comporter, soit en cellule proliférative, soit en cellule migrante -que cette migration soit de type adhésion-extension-dé-adhésion, soit de type amiboïde.

PAI-1 est capable d'induire la migration amiboïde, et initie une boucle de rétroaction négative conduisant à une diminution de sa biodisponibilité : il pourrait donc représenter un carrefour de transition. D'ailleurs, des souris dont le gène de PAI-1 a été annulé (KO pour PAI-1), réagissent à l'injection de cellules tumorales en développant des tumeurs moins volumineuses avec un nombre de métastases très diminué, en comparaison avec les souris non mutantes [START_REF] Bajou | Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization[END_REF]. [START_REF] Friedl | Prespecification and plasticity: shifting mechanisms of cell migration[END_REF]). Les cellules cancéreuses ayant subi une transition épithélio-mésenchymateuse sont des cellules qui adhèrent fortement à la matrice extracellulaire : elles présentent des points focaux d'adhésion et une structure profonde et stable du cytosquelette d'actine en câbles de tension, responsables de leur aspect fusiforme (photographie et image de droite). Cet état conditionne soit la prolifération cellulaire, soit une migration cellulaire de type mésenchymateuse mettant en jeu les intégrines et les protéases dans une dynamique d'interactions adhérence/protéolyse avec la matrice extracellulaire. Dans l'état de transition mésenchymateuse-amiboïde, les cellules cancéreuses maintiennent une migration de type amiboïde, qui est indépendante des intégrines et qui n'implique pas de protéolyse. Les cellules sont peu adhérentes à la matrice extracellulaire, leur cytosquelette d'actine s'organise en anneaux corticaux et instables responsable du blebbing (photographie et image de gauche). La migration amiboïde est favorisée par une concentration élevée de PAI-1 matriciel dans le microenvironnement, à l'inverse de la migration mésenchymateuse. Nous avançons l'hypothèse que l' « aiguillage » réversible du type de migration et la conversion de l'état mésenchymateux vers l'état amiboïde dépend de la concentration matricielle en PAI-1. La variation d'une seule molécule de PAI-1 pourrait faire basculer la cellule d'un état à un autre. (NB : les photographies sont réalisées en contraste de phase, les images sont des reconstructions en trois dimensions du cytosquelette d'actine permettant d'en distinguer les structures). Fig. 1. The yin and yang of a cancer-cell migration that underwent the epithelio-mesenchymal transition (inspired from P. Friedl, 2004). These cells adhere strongly at the extracellular matrix: they show focal contacts and a dense and profound organization of the actin cytoskeleton (stress fibres) responsible for their spindle-shape aspect (photograph and picture on the right). Two cell behaviour are dependent on that state. Cell proliferation or cell migration (mesenchymal type) that involves integrins and proteases in a dynamic interaction of adhesion and proteolysis. In the state of mesenchymal-amoeboid transition, cancer cells maintain an amoeboid type migration that is independent of integrins and proteolysis. Cells are loosely adherent to the extracellular matrix, their actin cytoskeleton is organized in instable cortical rings responsible for the 'blebbing' phenomenon (photograph and picture on the left). The amoeboid migration is favoured by a high concentration of matrix PAI-1 in the microenvironment, conversely to mesenchymal migration. We hypothesize that the reversible 'switching' of the migration type and the conversion from the mesenchymal state to the amoeboid state could depend on the concentration of matrix PAI-1. The variation of one single molecule of PAI-1 could make the cell swing from one state to another. (NB: Photographs are shown in phase contrast, pictures are three-dimensional reconstructions of the actin cytoskeleton allowing the observation of organized structures.) La Fig. 1 tente de résumer l'hypothèse de la dynamique pour une cellule, des liens cellule/microenvironnement relatif à PAI-1.

À l'échelle du tissu, puis de l'organisme : une situation d'échappement

En utilisant cet environnement particulier (c'est-àdire très enrichi en PAI-1) nous cherchions en fait, à mimer les conditions dans lesquelles les cellules tumorales se trouvent in vivo. En effet on décrit la présence d'un « bouclier » de PAI-1 à la périphérie des tumeurs très invasives [START_REF] Harbeck | Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I: Novel tumor-derived factors with a high prognostic and predictive impact in breast cancer[END_REF]. Cet environnement de PAI-1 est créé, d'une part, par les cellules tumorales elle mêmes [START_REF] Chazaud | Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations[END_REF] ; il est en effet connu que, plus les cellules sont « invasives », plus elles sécrètent de PAI-1. D'autre part, les cellules stromales produisent également du PAI-1 [START_REF] Pedersen | Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas[END_REF]. Nous abordons ainsi une échelle supérieure, celle de l'environnement de la tumeur, ou échelle tissulaire.

On admet généralement que la probabilité d'échappement métastatique concerne moins d'une cellule tumorale sur 10 000, et que c'est une moindre proportion qui sera à l'origine d'une tumeur secondaire. Il s'agit d'un événement rare, et pourtant essentiel. Dans les avalanches, il est connu que la résultante n'est pas proportionnelle à la cause, c'est-à-dire que le système est non linéaire. C'est d'ailleurs cette non-linéarité des effets par rapport aux causes qui rend nécessaire une modélisation adéquate pour mieux comprendre le processus en cause.

Dans notre exemple, une seule molécule de PAI-1 pourrait-elle jouer le rôle d'amorce de cette avalanche biologique -catastrophique -conduisant à la métastase ? Et inversement ?

Conclusion : la dialectique de la migration d'une cellule cancéreuse

Nos observations, réalisées à l'échelle cellulaire et moléculaire, pourraient rendre compte d'un micro-état (ou état local) du système. Mais c'est à une toute autre échelle de complexité que s'évalue le résultat de l'évolution du processus de migration d'une cellule cancéreuse vers un retour à la phase proliférative. Ce nouvel état, que l'on pourrait qualifier d'émergeant, réunirait les pré-requis du développement d'une métastase. L'ensemble des dynamiques cellulaires et leurs résultantes représenteraient l'état global, ou macro-état.

Pour comprendre le processus métastatique, il ne s'agit pas seulement d'identifier une cascade d'interactions moléculaires, aussi essentielle soit-elle, mais il faut la réinscrire dans la dynamique du microenvironnement et des relations cellules-matrice, puis (ré)intégrer les changements et leurs conséquences à différentes échelles. La conscience des limites de « l'exercice biologique expérimental » pour expliquer des mécanismes aussi complexes que celui de l'échappement métastatique nous incite à utiliser la modélisation mathématique, d'une part, pour tester des lois de comportement qu'on ne peut atteindre qu'après d'innombrables expériences et, d'autre part, pour limiter ces expériences à la validation ou à la réfutation d'un modèle [START_REF] Gatenby | Mathematical oncology: cancer summed up[END_REF].

Il apparaît évident que la généralisation des effets possibles de PAI-1 n'est pas raisonnable en biologie cellulaire classique. microenvironnement spécifique des cancers solides. Ce type de modélisation est présenté dans ce numéro, dans l'article intitulé Modéliser les interactions moléculaires par la théorie des réseaux de jeux, par M. Manceny, C. Chettaoui, M. Malo, G. Barlovatz-Meimon et F. Delaplace (ce numéro, [START_REF] Manceny | Modéliser les interactions moléculaires par la théorie des réseaux de jeux[END_REF]).

Références

  À l'échelle d'une cellule, une boucle d'interactions implique, d'une part la cellule et son microenvironnement, avec les changements morphologiques observés et d'autre part, la membrane et le noyau, avec les régulations des messagers et, sans doute, des protéines.

Fig. 1 .

 1 Fig.1. Le yin et le yang de la migration d'une cellule cancéreuse (hypothèse inspirée de[START_REF] Friedl | Prespecification and plasticity: shifting mechanisms of cell migration[END_REF]). Les cellules cancéreuses ayant subi une transition épithélio-mésenchymateuse sont des cellules qui adhèrent fortement à la matrice extracellulaire : elles présentent des points focaux d'adhésion et une structure profonde et stable du cytosquelette d'actine en câbles de tension, responsables de leur aspect fusiforme (photographie et image de droite). Cet état conditionne soit la prolifération cellulaire, soit une migration cellulaire de type mésenchymateuse mettant en jeu les intégrines et les protéases dans une dynamique d'interactions adhérence/protéolyse avec la matrice extracellulaire. Dans l'état de transition mésenchymateuse-amiboïde, les cellules cancéreuses maintiennent une migration de type amiboïde, qui est indépendante des intégrines et qui n'implique pas de protéolyse. Les cellules sont peu adhérentes à la matrice extracellulaire, leur cytosquelette d'actine s'organise en anneaux corticaux et instables responsable du blebbing (photographie et image de gauche). La migration amiboïde est favorisée par une concentration élevée de PAI-1 matriciel dans le microenvironnement, à l'inverse de la migration mésenchymateuse. Nous avançons l'hypothèse que l' « aiguillage » réversible du type de migration et la conversion de l'état mésenchymateux vers l'état amiboïde dépend de la concentration matricielle en PAI-1. La variation d'une seule molécule de PAI-1 pourrait faire basculer la cellule d'un état à un autre. (NB : les photographies sont réalisées en contraste de phase, les images sont des reconstructions en trois dimensions du cytosquelette d'actine permettant d'en distinguer les structures).

Pour notre part, nous avons évalué les effets de nombreuses concentrations de PAI-1 sur différents types cellulaires, sur le plan biochimique comme sur le plan morpho-dynamique. Mais si nous nous bornons à l'approche expérimentale pour mieux comprendre les lois de « comportement » cellulaire propices à l'échappement métastasique, il nous faudrait utiliser des gammes considérables de concentrations de PAI-1, de temps d'exposition, de densités cellulaires, de types cellulaires d'invasivité croissante ; ensuite il nous faudrait produire puis intégrer une quantité gigantesque de données.La modélisation peut nous faire approcher ces lois de comportement cellulaire en évitant cette inflation expérimentale ; dans un esprit de dialogue interdisciplinaire, nous pourrions ensuite en vérifier « à la paillasse » la pertinence, et progressivement aboutir à la simulation.Une première modélisation, par la théorie des jeux et plus précisément celle des réseaux de jeux, développée par Franck Delaplace, nous a permis de décrire une partie de la dynamique de l'assemblage moléculaire uPAR-uPA-PAI-1 et de calculer deux états stables[START_REF] Chettaoui | Games network and application to PAs system[END_REF]. Sur le plan du comportement cellulaire, ces états stables correspondent, l'un à l'état après MAT observé dans un microenvironnement riche en PAI-1, l'autre à l'état après EMT observé dans un microenvironnement pauvre en PAI-1. Ainsi, nous faisons l'hypothèse que la concentration de PAI-1 matriciel oriente le devenir d'une cellule cancéreuse ayant subi l'EMT, soit en la maintenant dans un état « post-EMT » (faible concentration en PAI-1 matriciel), favorable à une migration conventionnelle et possiblement à un retour à l'état prolifératif, soit en lui faisant subir la MAT (forte concentration en PAI-1 matriciel), favorable à la migration amiboïde et à l'échappement métastatique.Sans prétendre utiliser « la meilleure » ou la seule modélisation adéquate, nous poursuivons, dans une approche interdisciplinaire, une modélisation, par la théorie des réseaux de jeux, des interactions cellule-