Marius Ilié

Julien Mazières

Emmanuel Chamorey

Simon Heeke

PhD Jonathan Benzaquen

Brice Thamphya

Jacques Boutros

Angélica Tiotiu

PhD Julien Fayada

Jacques Cadranel

PhD Marius Ilie

Angelica Tiotiu

MD Michel Poudenx

PHD Denis Moro-Sibilot

PhD Fabrice Barlesi

Christelle Juliette Thariat

PhD Pascale Tomasini

PhD Véronique Hofman

Charles-Hugo Marquette

PhD Paul Hofman

MD Christophe Pison

PhD Sylvie Lantuejoul

PhD Olivier Stephanov

Christophe Mendozat

Manel Zaidi

Sandra Coulouvrat

Edwige Col

MD Pascal Chanez

MD Laurent Greillier

PhD Céline Mascaux

PhD Sandrine Jourdan

Aurélie Roger

Julie Biemar

MD François Chabot

MD Angélica Tiotiu

MD Christelle Clement-Duchene

Jean- Michel Vignaud

Stéphanie Lacomme

Sandra Lomazzi

Carine Laurent

Xavier Bulsei

Laura Bischoff

Raymond Rakotonirina

Mehdi Layouni

MD Romain Kessler

MD Michel Vergnon

MD Isabelle Pelissier

Jean François

MD François Steenhouwer

MD Sylvie Leroy

MARQUETTE Charles Hugo

PhD Andrea Mazzette

MD Bernard Padovani

MD Elodie Long-Mira

PhD Sandra Lassalle

PhD Johanna Pradelli

MD Estelle Martinez

Marine Habault

Mélanie Bonnard

Julie Moutarde

Rachida Yatimi

Hakima Labsi

Loïc Gazoppi

Tumorothèque Lpce

Jennifer Griffonnet

Maureen Fontaine

Ariane Guillemart

MD Catherine Butori

Eric Selva

MD Josiane Otto

MD Christophe Hebert

MD Delphine Botchiellini

Soukaina Boudouf

Margaux Menier

Estelle Occeli

Sophie Bellentani

Carine Pion

Centre Elodie Fournier

Antoine Lacassagne De Nice

PhD Radj Gervais

Karim Hamond

François Centre

Sylvain Baclesse À Caen

MD Laurent Marchand-Adam

Gaelle Plantier

Mélanie Fajolle

Rayez

Jacques Cadranel Md-Phd

MD Vincent Fallet

Marie Wislez Md-Phd

Martine Antoine

Jean-François Cote

MD Nouha Chaabane

MD Anne Marie Ruppert

Eliane Bertrand

Anita Rodenas

Gwenaëlle Pontdeme

Nathalie Mathiot

Tamazouzt Ribert

Hôpital Tenon

MD Julien Mazieres

PhD Nicolas Guibert

PhD Damien Rouviere

MD Emilie Bousquet

MD Laurence Bigay-Game

MD Christophe Hermant

MD Gavin Plat

MD Isabelle Rouquette

MD, Solène EVRARD MD Sandrine Gouin

Estelle Taranchon

Inge Clermont

Christelle Dormoy

Anne Coulomb

Véronique Pradine

Lambert

Prospective Multicenter Validation of the Detection of ALK Rearrangements of Circulating Tumor Cells for Noninvasive Longitudinal Management of Patients With Advanced NSCLC

Keywords: ALK, CTCs, immunocytochemistry, FISH, lung adenocarcinoma

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

INTRODUCTION

Non-small cell lung cancer (NSCLC) harboring ALK receptor tyrosine kinase (ALK)rearrangements is a distinct subset of lung cancers, occurring in approximately 4% of patients at an advanced stage. [START_REF] Shaw | ALK in lung cancer: past, present, and future[END_REF] Several ALK inhibitors (ALKi) have been approved by the U.S. Food and Drug Administration and the European Medicines Agency for the treatment of patients with ALK-positive NSCLC at first or latter lines of treatment. [START_REF] Solomon | Final Overall Survival Analysis From a Study Comparing First-Line Crizotinib Versus Chemotherapy in ALK-Mutation-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Peters | Alectinib versus Crizotinib in Untreated ALK-Positive Non-Small-Cell Lung Cancer[END_REF] The eligibility of NSCLC patients to receive ALKi relies on a companion immunohistochemistry (IHC) performed on tumor tissue [START_REF] Planchard | Metastatic non-small cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF] , e.g. IHC or FISH that need a sufficient number of tumor cells to be conclusive. However, procedures providing small biopsy samples (e.g. fine needle aspiration, transthoracic needle biopsy, endobronchial ultrasound, bronchoscopy, bronchial biopsies) may lack a sufficient number of tumor cells. Furthermore, invasive procedures can be related to a relative risk of morbidity. Moreover, because tumors evolve and adapt to targeted therapies, it is becoming increasingly important to monitor the molecular changes occurring in NSCLC patients in "real-time" especially when selecting subsequent lines of treatment. [START_REF] Horn | Monitoring Therapeutic Response and Resistance: Analysis of Circulating Tumor DNA in Patients With ALK+ Lung Cancer[END_REF] In addition, the degree of benefit from ALKi varies widely between NSCLC patients, despite sharing molecular drivers. Longitudinal biopsies upon disease progression have been useful in identifying the molecular mechanisms driving resistance to ALKi. Yet, repeated tissue biopsies are not always feasible and sampling of a single tumor site may not reflect the spatial heterogeneity of resistance mechanisms. [START_REF] Dagogo-Jack | Tracking the Evolution of Resistance to ALK Tyrosine Kinase Inhibitors through Longitudinal Analysis of Circulating Tumor DNA[END_REF] In daily practice, accurate identification of ALK fusions as well as tumor evolution during therapy could have significant impact on the therapeutic strategy likely to have the greatest impact on patient outcome. [START_REF] Dagogo-Jack | Tracking the Evolution of Resistance to ALK Tyrosine Kinase Inhibitors through Longitudinal Analysis of Circulating Tumor DNA[END_REF] Therefore, developing non-invasive surrogate methods suitable to detect ALK rearrangements at baseline and during tumor evolution can be pivotal to improving the success rate of ALKi. It is now well admitted that liquid biopsy (LB) is a simple, non-invasive and easily repeatable alternative to tumor biopsies to assess the tumor's molecular status and to monitor genetic changes in "real-time". [START_REF] Heitzer | Current and future perspectives of liquid biopsies in genomics-driven oncology[END_REF] Among the LB components, circulating tumor cells (CTCs) contain tumorigenic cell clones with high relevance for metastatic dissemination. [START_REF] Krebs | Molecular analysis of circulating tumour cells-biology and biomarkers[END_REF] CTCs are likely released from distinct metastatic sites and may inform on the genomic heterogenic picture of the metastatic disease. In contrast to tumor biopsies, CTCs could allow longitudinal monitoring at different time-points during treatment, thereby having the potential to guide therapeutic decisions in "realtime". [START_REF] Pantel | Liquid biopsy and minimal residual diseaselatest advances and implications for cure[END_REF] We and other groups have reported the feasibility of ALK-rearrangement detection with CTCs collected from patients with ALK-positive NSCLC at baseline by using a filter-based technique for CTC enrichment and ALK testing by FISH and/or immunocytochemistry (ICC). [START_REF] Ilie | ALK-gene rearrangement: a comparative analysis on circulating tumour cells and tumour tissue from patients with lung adenocarcinoma[END_REF][START_REF] Pailler | Detection of Circulating Tumor Cells Harboring a Unique ALK Rearrangement in ALK-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Tan | Concordance of anaplastic lymphoma kinase (ALK) gene rearrangements between circulating tumor cells and tumor in non-small cell lung cancer[END_REF] To confirm these previous retrospective feasibility studies, we performed the first multi-center prospective clinical validation of the use of CTCs as a surrogate for tumor tissue for ALK testing, thus aiding in prognostication and therapy selection at baseline and during treatment with ALKi of patients with stage IIIB/IV NSCLC.

MATERIALS AND METHODS

Patients and samples

This multi-center prospective observational study (NCT02372448) was conducted across 9 French clinical institutions from January 23, 2015 through November 21, 2019 in accordance with the Declaration of Helsinki and the European Directive and Good Clinical Practices. The study was approved by the Ethics Committee (CPP Sud-Méditerranée V) and authorized by the National Security Agency for Medicines and Health Products (ANSM). Informed-written consent was obtained from all patients. Eligible patients had histologically confirmed stage IIIB/IV NSCLC (Table 1). A few number of patients had initially diagnosed stage I-IIIA NSCLC, enrolled in the study at the time of the metastatic relapse (Table 1). For these patients, a re-biopsy was performed to evaluate the ALK status. These patients may have had prior chemotherapy delivered as part of their primary treatment but must have completed primary therapy at least 3 months before their entry in the study. The pre-specified primary outcome was sensitivity and specificity of FISH between tumor tissue and CTCs at baseline as well as a follow-up « visit 1 » after six weeks (V1) and follow-up « visit 2 » after 12 weeks (V2). Pre-specified secondary outcomes included sensitivity and specificity of ICC at Baseline, V1 and V2 as well as association of ALK positive CTCs and clinical outcome.

ALK rearrangement was locally tested on tumor tissue by IHC (anti-ALK antibody, pre-diluted, clone D5F3, Roche Diagnostics, Basel, Switzerland) and FISH (Vysis ALK Break Apart Rearrangement Probe Kit; Abott Molecular Inc., Des Plaines, USA), performed according to pre-specified standard operating procedures in ISO 15189 accredited laboratories (Accreditation n° 8-3034; https://tools.cofrac.fr/annexes/sect8/8-3034.pdf). Thirty ml of peripheral blood was collected in EDTA or Streck tubes at baseline (V0) and at 6 (V1) and 12 (V2) weeks on ALKi initiation and/or at disease progression. [START_REF] Ilie | Use of circulating tumor cells in prospective clinical trials for NSCLC patients -standardization of the pre-analytical conditions[END_REF] CTCs were enumerated on ISET® filters (Isolation by Size of Tumor Cells, Rarecells, Paris, France), as previously reported. [START_REF] Ilie | ALK-gene rearrangement: a comparative analysis on circulating tumour cells and tumour tissue from patients with lung adenocarcinoma[END_REF][START_REF] Hofman | Preoperative circulating tumor cell detection using the isolation by size of epithelial tumor cell method for patients with lung cancer is a new prognostic biomarker[END_REF] Briefly, stained filters were examined by a senior thoracic cytopathologist using light microscopy in two steps: (i) screening at × 20 magnification to perform cells localization and detection, then, (ii) observation at × 100 magnification using an oil immersion for detailed cytomorphological analysis. CTCs were defined as cells presenting all the following criteria: (i) nuclear size equal or larger than one pore (i.e., equal or larger than 8 μm in diameter); (ii) irregularity of the nuclear contour; (iii) presence of a well defined and visible cytoplasm; and, (iv) high nuclear-to-cytoplasmic ratio (>0.8). Consequently, CT scans and/or MRIs were performed at baseline and during followup. The response to ALKi (Supplementary Table 1) was evaluated according to the "Response Evaluation Criteria in Solid Tumors" (RECIST) version 1.1. The disease control rate was defined as a complete or partial response or stable disease based on RECIST.

ALK immunocytochemistry and FISH on ISET-enriched CTCs

All ISET filters were sent to the coordinating team at the CHU Nice for centralized ALK analysis. ICC and FISH were performed on CTCs isolated using the ISET method on unstained spots of the corresponding filters containing CTCs detected on four spots colored with May-Grünwald-Giemsa [15]. Three spots were used for ICC and three spots were used for FISH per filter. For ICC, the spots were incubated with a primary antibody against the ALK protein (pre-diluted, clone D5F3; Ventana) for 32 min at room temperature. FISH analysis was carried out using a break-apart probe for the ALK gene (Vysis ALK Break Apart Rearrangement Probe Kit, Abbott Molecular Inc.) in accordance with the manufacturer's instructions. Cells showing at least one split signal or single 3' signals were considered positive for an ALK rearrangement. ISET filters were examined independently and blinded to clinical, IHC, ICC, FISH data and tissue genotype. We used the human NSCLC cell line H2228 obtained from ATCC (Manassas, VA, USA) as an ALK rearrangement positive control in every run. [START_REF] Ilie | ALK-gene rearrangement: a comparative analysis on circulating tumour cells and tumour tissue from patients with lung adenocarcinoma[END_REF]

Statistical analysis

The performance of ALK assessment on ICC or FISH was compared to the paired tumor biopsy and equivalent method using sensitivity and specificity analysis. The same analysis was carried out first on the population with available CTCs (n=185 patients and n=97 analyzed for ICC and FISH respectively) and second on the entire population including patients with no available CTCs (n=203 patients analyzed). In this latter case, ALK assessment was not available by using ICC or FISH methods, we used the worst scenario by considering all ALK positive cases on the biopsy as false negative with ICC and FISH and all ALK negative on the biopsy as false positive with ICC and FISH. The association to clinical variables was assessed using χ 2 , Student-t test and Fisher's exact test analyses. Progression-free survival (PFS) was defined as the time between the date of the start of ALKi and the date of clinical or radiological progression according to RECIST, or death. Overall survival (OS) was defined as the time between the date of the start of ALKi and the date of death. Patients who were progression-free and alive at the time of analysis were censored. All statistical analyses were performed using R (version 3.5.0, R Foundation for Statistical Computing) or Python (version 3.7.2, Python Software Foundation).

RESULTS

Patient characteristics

Peripheral blood was collected from 193 patients with stage IIIB/IV NSCLC and 10 patients with initially diagnosed stage I-IIIA NSCLC enrolled in the study at the time of the metastatic relapse (Table 1). In total, 81 (40%) patients with ALK-positive NSCLC and 122 (60%) patients with ALK-negative NSCLC were included in the study cohort. Most ALK-positive patients were never or former smokers (97%) with adenocarcinoma tumor histology (99%). At baseline (V0), CTCs were detected in 187 (92%) patients (range, 2-621/4 ml; median, 62 CTCs/4 ml; Fig. 1A). A significantly higher number of CTCs were detected in ALK-negative patients than in ALK-positive patients (median CTCs = 71 vs. 21; P = 0.0098) (Fig. 1A). CTCs were not detected in 16/203 (8%) patients, 10/203 (5%) ALK-positive patients and 6/203 (3%) ALK-negative patients. At first visit (V1) during monitoring of ALK-positive patients, 27/81 (33%) patients had CTCs (range, 6-177/ 4 ml; median, 6 CTCs/ 4 ml; Fig. 1B). At the second follow-up visit (V2), we detected CTCs (range, 11-173/ 4 ml; median, 19 CTCs/ 4 ml) in 17/81 (21%) ALK-positive patients (Fig. 1B).

Performance of CTCs for detection of ALK rearrangements

At baseline, there was substantial concordance between the detection of an ALK rearrangement in the tumor tissue (IHC and FISH) and CTCs (Fig. 2). The sensitivity and the specificity for ICC were 94.36% and 89.39%, across all samples, and 100%, respectively, for patients with detectable CTCs at baseline. Lower concordance was observed for FISH with 35.63% sensitivity and 56.89% specificity, across all samples, and 100%, respectively, for patients with detectable CTCs at baseline. Moreover, the levels of CTCs or ALK-positive CTCs significantly decreased with ALKi treatment (Fig. 1B; Supplemental Fig. S1). In all positive samples by FISH and IHC, 100% of detected CTCs were positive in ICC with an intensity score of 3. All but one FISH positive sample were 100% positive for rearranged ALK with one sample at baseline having only 80% FISH positive cells).

Association between ALK-CTCs and clinical parameters or outcome

An exploratory analysis revealed a significant association between the status of ALK-CTCs, younger age and non-smoking status. ALK-positive CTCs were present more often in younger patients (mean, 60 vs. 64 years, P=0.038) and non-smokers (P<0.0001). All the other clinical parameters were not significantly associated with ALK-rearranged CTC counts at baseline. In addition, in ALK-positive patients, the levels of CTCs significantly correlated to age and stage (Table 2). At the first follow-up visit, 25 (31%) patients had disease progression according to RECIST. For the univariate analysis, the level of CTCs at baseline was assigned into low and high groups according to their respective median in ALK-positive patients. We did not observe a significant association between baseline CTCs levels and OS (P=0.244, HR=0.59, 95% CI, 0.24-1.5) or PFS (P=0.591, HR=0.84, 95% CI, 0.44-1.6; Fig. 3). In addition, we considered the dynamic change in the CTC counts under treatment as a possible parameter to evaluate. Patients were assigned into two groups: 13 patients presented stable or increased numbers of ALK-CTCs (median increase, 36; range, 2-290), whereas 37 patients presented a decrease in ALK-CTCs (median decrease, -7; range, -250 to -280). However, we did not observe a statistically significant association between the dynamic change in the CTC number on ALKi and response to treatment with a univariate analysis (P=0.268; Fig. 4, Supplemental Fig. S2, and Supplemental Fig. S3).

DISCUSSION

In this study, we demonstrated that CTCs could be a reliable surrogate to a tissue biopsy for the detection of ALK rearrangements in advanced-stage or recurrent NSCLC patients. Moreover, the decrease in the levels of ALK-positive CTCs could be highly informative of the predictive efficacy of ALKi in ALK-rearranged NSCLC patients. Our study offers a promising perspective into the use of CTCs for real-time monitoring of this population.

Currently, the eligibility of NSCLC patients for ALKi relies on an assay performed on a tumor sample collected by biopsy. [START_REF] Shaw | Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK[END_REF] This invasive procedure is associated with a non-negligible risk of morbidity. [START_REF] Huang | Accuracy and complications of CTguided pulmonary core biopsy in small nodules: a single-center experience[END_REF] The detection of an ALK rearrangement is currently performed on small biopsies or fine-needle aspirates and can be hindered by the limited quantity of tissue and/or the number of tumor cells. Tumor tissue is difficult to obtain from patients with advanced/metastatic lung cancer for whom surgery is rarely a component of the treatment care. Thus, a subset of NSCLC patients can be deprived of potentially more efficient therapies. Finding alternative and more effective means of diagnosing and monitoring ALK rearrangements are critical issues to identify the patients who may benefit from treatment with ALKi. For instance, detection of ALK fusions in circulating tumor DNA (ctDNA) has been previously tested using next-generation sequencing (NGS-, RT-PCR or RNA-Seq with variable sensitivities (70%-80%) for the different assays. [START_REF] Wang | Noninvasive genotyping and monitoring of anaplastic lymphoma kinase (ALK) rearranged non-small cell lung cancer by capturebased next-generation sequencing[END_REF][START_REF] Park | Feasibility of liquid biopsy using plasma and platelets for detection of anaplastic lymphoma kinase rearrangements in non-small cell lung cancer[END_REF] Thus, in contrast to EGFR mutations, detection of ALK rearrangements with ctDNA is rarely implemented in daily practice.

Recently, the prospective multi-center NILE study demonstrated that ctDNA can rescue biomarker-positive patients with non-diagnostic tissue results, suggesting the clinical utility of ctDNA in newly diagnosed metastatic NSCLC. [START_REF] Leighl | Clinical Utility of Comprehensive Cell-free DNA Analysis to Identify Genomic Biomarkers in Patients with Newly Diagnosed Metastatic Non-small Cell Lung Cancer[END_REF] However, the LB test used was compared to a standard-of-care tissue genotyping test and not the tissue-based NGS, and the results were only applicable to the Guardant360 test and not to other LB tests. [START_REF] Leighl | Clinical Utility of Comprehensive Cell-free DNA Analysis to Identify Genomic Biomarkers in Patients with Newly Diagnosed Metastatic Non-small Cell Lung Cancer[END_REF] In addition, the FoundationOne®Liquid CDx assay, a pancancer ctDNA-based comprehensive genomic profiling assay, was recently approved by the FDA. [START_REF] Woodhouse | Clinical and analytical validation of FoundationOne Liquid CDx, a novel 324-Gene cfDNA-based comprehensive genomic profiling assay for cancers of solid tumor origin[END_REF] However, the number of cases with an ALK rearrangement used for the analytic validation was quite low. [START_REF] Woodhouse | Clinical and analytical validation of FoundationOne Liquid CDx, a novel 324-Gene cfDNA-based comprehensive genomic profiling assay for cancers of solid tumor origin[END_REF] A "LB" of CTCs present in the blood and the characterization of their ALK status is an appealing alternative that meets the urgent needs of these patients. In addition, no CTC-based molecular test is currently available in routine clinical practice. [START_REF]The future of liquid biopsy[END_REF] In this setting, CTCs which have been shown to be detectable by ISET in 80% to 100 % of late stages lung cancer patients represent a non-invasive and easily accessible source of tumor material for assessing ALK rearrangement in a dynamic manner. [START_REF] Hofman | Preoperative circulating tumor cell detection using the isolation by size of epithelial tumor cell method for patients with lung cancer is a new prognostic biomarker[END_REF][START_REF] Hofman | ALK Status Assessment with Liquid Biopsies of Lung Cancer Patients[END_REF] In our study, we did not isolate CTCs in only 8% of patients, which is in the range of sensitivity described for the detection of CTCs by ISET approach in NSCLC patients. [START_REF] Hofman | Preoperative circulating tumor cell detection using the isolation by size of epithelial tumor cell method for patients with lung cancer is a new prognostic biomarker[END_REF][START_REF] Hofman | ALK Status Assessment with Liquid Biopsies of Lung Cancer Patients[END_REF][START_REF] Farace | A direct comparison of CellSearch and ISET for circulating tumour-cell detection in patients with metastatic carcinomas[END_REF][START_REF] Krebs | Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent andindependent approaches[END_REF] The lack of CTC detection in these patients could be related to either technical or biological hurdles. It may be that patients truly present negative for CTCs, or CTCs may be lost at different times during the filtration and staining process, (i) through the pores of 8 μm during the procedure of filtration, and/or (ii) during the sequential washes used during the staining procedure performed after filtration. [START_REF] Farace | A direct comparison of CellSearch and ISET for circulating tumour-cell detection in patients with metastatic carcinomas[END_REF][START_REF] Krebs | Evaluation and prognostic significance of circulating tumor cells in patients with non-small-cell lung cancer[END_REF] The high sensitivity and specificity of ISET were two essential starting points for the feasibility of the present independent and multi-center project. [START_REF] Ilie | ALK-gene rearrangement: a comparative analysis on circulating tumour cells and tumour tissue from patients with lung adenocarcinoma[END_REF] In our first feasibility mono-centric study, we showed excellent concordance for the ALK status between CTCs and the matched tumor tissue sample in a limited series of 87 patients with advanced NSCLC, with only 5 patients harboring an ALK rearrangement. [START_REF] Ilie | ALK-gene rearrangement: a comparative analysis on circulating tumour cells and tumour tissue from patients with lung adenocarcinoma[END_REF] In this multi-center prospective observational study across 9 centers there was perfect concordance (100%) between the tissue and CTC ALK analysis, in all cases with CTCs isolated on filters (92%). In the small proportion of cases without CTCs, the ALK status could not be determined on the filters, suggesting technical challenges rather than biological heterogeneity, as described previously. [START_REF] Farace | A direct comparison of CellSearch and ISET for circulating tumour-cell detection in patients with metastatic carcinomas[END_REF] Our study presents some limitations. One ISET spot contains on average 5,000 white blood cells, mainly leukocytes, and only a small number of CTCs. [START_REF] Chemi | Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse[END_REF] Consequently, the manual analysis of one ISET spot to identify ALK-rearranged CTCs by FISH is laborious, time consuming and operator-dependent.

In this setting, the ALK-ICC or FISH analysis increases the feasibility, precision and reliability of ALK testing of filtration-enriched CTCs and may contribute to the validation and qualification of CTC assays for ALK-rearranged NSCLC patients.

It is worth noting that other non-invasive biological components that can be isolated from peripheral blood, such as ctDNA, circulating exosomes, platelet RNA or ctRNA, are currently being investigated for the detection of gene rearrangements. [START_REF] Park | Feasibility of liquid biopsy using plasma and platelets for detection of anaplastic lymphoma kinase rearrangements in non-small cell lung cancer[END_REF][START_REF] Rolfo | Liquid Biopsy for Advanced Non-Small Cell Lung Cancer (NSCLC): A Statement Paper from the IASLC[END_REF][START_REF] Hofman | Circulating Tumor Cell Detection in Lung Cancer: But to What End?[END_REF] , although the sensitivity and precision of these strategies needs to be further evaluated and compared with those of CTC assays.

Whereas each of these approaches has the potential to provide novel diagnostic information and their exploration is highly encouraged, ctDNA certainly represents the most mature example of the investigation of the LB in clinical practice for NSCLC patients, in particular for gene mutation detection. [START_REF] Horn | Monitoring Therapeutic Response and Resistance: Analysis of Circulating Tumor DNA in Patients With ALK+ Lung Cancer[END_REF][START_REF] Dagogo-Jack | Tracking the Evolution of Resistance to ALK Tyrosine Kinase Inhibitors through Longitudinal Analysis of Circulating Tumor DNA[END_REF][START_REF] Aggarwal | Clinical Implications of Plasma-Based Genotyping With the Delivery of Personalized Therapy in Metastatic Non-Small Cell Lung Cancer[END_REF][START_REF] Zugazagoitia | Clinical utility of plasma-based digital next-generation sequencing in patients with advance-stage lung adenocarcinomas with insufficient tumor samples for tissue genotyping[END_REF] However, the analysis of ctDNA requires extensive deep sequencing of genomic DNA for reliable detection of the chromosomal break-point. [START_REF] Mccoach | Clinical Utility of Cell-Free DNA for the Detection of ALK Fusions and Genomic Mechanisms of ALK Inhibitor Resistance in Non-Small Cell Lung Cancer[END_REF] Moreover, digital-droplet PCR, BEAMing and NGS have been reported to have a promising sensitivity for the detection of ALK gene fusions, but are more variable than that observed for point mutations and indels. [START_REF] Supplee | Sensitivity of next-generation sequencing assays detecting oncogenic fusions in plasma cell-free DNA[END_REF] Further studies should focus on orthogonal comparisons using CTCs, ctDNA and ctRNA to better characterize the presence of ALK rearrangements and to validate the clinical utility of detecting these genomic variants in plasma, notably in naïve treated patients with no tumor tissue accessibility.

In conclusion, this multi-center study is unique as no CTC-based ALK rearrangement assay has yet been independently validated with clinical samples. The development of a non-invasive predictive test through the genomic analysis of CTCs is a clinically relevant goal for non-invasive stratification of cancer patients, avoiding morbidity related to lung biopsy and surgery. Analysis with a blood sample should allow patient eligibility to targeted therapies to be determined. Small-sized samples with a low percentage of tumor cells and poor biopsy quality from NSCLC patients can often jeopardize optimal treatment management; CTC-based ALK testing can be useful to guide the choice of anti-ALK targeted therapy. Table 1. Main clinicopathological data of the 203 NSCLC patients included in this study.

Legend to Tables

Table 2. Correlation analysis between ALK-positive patients with detectable CTCs and ALK-positive patients without detectable CTCs.

Legend to Figures

Figure 1 .

 1 Figure 1. Boxplots showing the levels of CTCs according to (A) the ALK status at baseline « visit » 0 (V0), and (B) during the monitoring of ALK-positive patients at follow-up « visit 1 » after six weeks (V1) and follow-up « visit 2 » after 12 weeks (V2).

Figure 2 .

 2 Figure 2. Representative images of ALK-positive (A-C) and ALK-negative (D-F) NSCLC cases. A. Circulating tumor cells showing malignant cyto-morphological criteria isolated by the ISET method (original magnification ×200; MGG staining; bar: 20 μm). B. Circulating tumor cells showing an intense and cytoplasmic staining (score 3+) (ALK immunostaining using D5F3 mAb, immunoperoxidase; original magnification ×200; bar: 20 μm). C. Circulating tumor cell nuclei hybridized with the Vysis ALK Break Apart Rearrangement Probe Kit. The two probes (3' red; 5' green) show a distinct separation of the red and green signals (arrows) indicating a rearrangement in the 2p23 ALK-gene locus (original magnification ×1000; bar: 20 μm). D. Circulating cells showing malignant cytomorphological criteria isolated by the ISET method (original magnification ×200; MGG staining; bar: 20 μm). E. Circulating tumor cells showing lack of staining (score 0) (D5F3 mAb, immunoperoxidase; original magnification ×200; bar: 20 μm).

F.

  Circulating tumor cell nuclei hybridized with the Vysis ALK Break Apart Rearrangement Probe Kit. The probes give overlapping signals in nuclei without the rearrangement (arrows; original magnification ×1000; bar: 20 μm).

Figure 3 .

 3 Figure 3. Kaplan-Meier survival curves according to the median of CTCs level (n=62 CTCs/4 ml) at baseline from the 81 NSCLC ALK-positive patients. A) Overall survival, and B) Progression-free survival curves. The P-values were calculated using the Log-rank test.

Figure 4 .

 4 Figure 4. Plots of ALK-CTC counts per 3 ml of blood based on ICC detection in patients who were serially sampled (e.g., paired time points for the same patient) during treatment with ALK inhibitors. A) "Spaghetti" plots depicting the ALK-CTC counts in relation to PD, SD, and PR/CR on separate panels. B) Box-plots depicting ALK-CTC changes in relation to CR, PR, SD, PD from baseline to visit 1, and from visit 1 to visit 2.

Table 2 ALK

 2

		+	ALK +
		CTCs < median	CTCs ≥ median	P-value
		n (%)	n (%)
	Overall	63 (77.7%)	18 (22.3%)
	Age (mean, SD)	61.6 (13.3)	52.3 (12.2)	0.009
	Gender			0.4305
	Male	32 (50.8%)	7 (38.9%)
	Female	31 (49.2%)	11 (61.1%)
	Smoking status			0.749
	Current smoker	4 (6.4%)	2 (11.2%)
	Former smoker	25 (39.7%)	7 (38.8%)
	Never smoker	34 (53.9%)	9 (50%)
	Histology			1
	Adenocarcinoma	61 (96.8%)	18 (100%)
	Squamous cell carcinoma	1 (1.6%)	0 (0%)
	NSCLC NOS	1 (1.6%)	0 (0%)
	Stage			0.02
	Stage I / II	2 (3.2%)	4 (22.2%)
	III / IV	61 (96.8%)	14 (77.8%)

Funding

This study was financially supported by a governmental grant from the National Institute of Cancer (INCa PHRC; STALKLUNG01 13-APN-01), the Cancéropôle PACA and the "Conseil Départemental des Alpes-Maritimes".

Conflicts of Interest:


Claire Baranger

Isabelle Pezron

Laurence Lins

Magali Deleu

Anne Le Goff
email: anne.le-goff@utc.fr

Antoine Fayeulle
email: antoine.fayeulle@utc.fr

A compartmentalized microsystem helps understanding the uptake of benzo[a]pyrene by fungi during soil bioremediation processes

Keywords: Bioavailability, Biodegradation, Biosurfactant, Microfluidic device, Mycoremediation, Polycyclic aromatic hydrocarbons

Hydrophobic organic soil contaminants such as polycyclic aromatic hydrocarbons (PAH) are poorly mobile in the aqueous phase and tend to sorb to the soil matrix, resulting in low bioavailability. Some filamentous fungi are efficient in degrading this kind of pollutants. However, the mechanism of mobilization of hydrophobic compounds by non-motile microorganisms such as filamentous fungi needs investigations to improve pollutant bioavailability and bioremediation efficiency. Usual homogeneous media for microbial growth in the lab are poorly suited to model the soil, which is a compartmentalized and heterogeneous habitat.

A microfluidic device was designed to implement a compartmentalization of the fungal inoculum and the source of the pollutant benzo[a]pyrene (BaP) as a deposit of solid crystals in order to gain a further insight into the mechanisms involved in the access to the contaminant and its uptake in soils.

Thus in this device, two chambers are connected by an array of parallel microchannels that are wide enough to allow individual hyphae to grow through them. Macro-cultures of Talaromyces helicus in direct contact with BaP have shown its uptake and intracellular storage in lipid bodies despite the low propensity of BaP to cross aqueous phases as shown by simulation. Observations of T. helicus in the microfluidic device through laser scanning confocal microscopy indicate preferential uptake of BaP at a close range and through contact with the cell wall. However faint staining of some hyphae before contact with the deposit also suggests an extracellular transport phenomenon. Macro-culture filtrates analyses have shown that T. helicus releases extracellular non-lipidic surface-active compounds able to lower the surface tension of culture filtrates to 49.4 mN/m. Thus, these results highlight the significance of active mechanisms to reach hydrophobic contaminants before their uptake by filamentous fungi in compartmentalized micro-environments and the potential to improve them through biostimulation approaches for soil mycoremediation.

Introduction

Soil is a complex environmental compartment resulting from the weathering of parental rock under the combined action of living organisms, surface water and the atmosphere. As a support for human activities, soil is heavily affected by pollution and notably with persistent pollutants, which is a major source of soil quality alteration [START_REF] Panagos | Contaminated sites in Europe: Review of the current situation based on data collected through a european network[END_REF]. Among these pollutants, some families of organic contaminants including polycyclic aromatic hydrocarbons (PAH), polychlorobiphenyls, and various xenobiotics and pesticides, are highly hydrophobic and persistent in soils. Due to their carcinogenicity and high persistence in the environment, PAH are among the most regulated pollutants in environmental policies [START_REF] Jennings | Worldwide regulatory guidance values for surface soil exposure to carcinogenic or mutagenic polycyclic aromatic hydrocarbons[END_REF]. Such poorly water-soluble contaminants can be present in non-aqueous-phase liquids, tar or solid particles. Moreover, they tend to strongly sorb to soil aggregates and organic matter [START_REF] Ortega-Calvo | Is it possible to increase bioavailability but not environmental risk of PAHs in bioremediation?[END_REF]. These properties make them poorly available for degradation by microorganisms [START_REF] Posada-Baquero | Implementing standardized desorption extraction into bioavailability-oriented bioremediation of PAH-polluted soils[END_REF].

Bioremediation corresponds to the use of biological activities in particular of microorganisms to destroy pollutants or decrease the associated risks for humans and the environment. It is regarded as a low-technology and low-cost approach with a higher degree of public acceptance in comparison to other physicochemical remediation techniques more destructive of the living fraction and the structure of soils such as high temperature incineration, chemical decomposition, solvent extraction or UV oxidation [START_REF] Vidali | Bioremediation. an overview[END_REF]. Thus bioremediation is one of the established methods undergoing the highest development for PAH cleanup in soils (Kuppusamy et al., 2017). One of the parameters that can impair bioremediation efficiency is the low competitiveness and adaptability of microbial inocula [START_REF] Rayu | Emerging technologies in bioremediation: constraints and opportunities[END_REF], which could be prevented by the use of telluric strains. Soil is a divided environment consisting in solid, aqueous and gas phases, hosting a great variety of living organisms, including fungi. Among microorganisms, micromycetes are particular in the sense that they do not need water as a support for dispersal, and can form aerial structures. This allows mycelium to occupy vast volumes of soil and come into contact with several phases. Indeed, soil fungi are known for their ability to form mycelial networks in three dimensions that constitute large exchange surfaces within the soil porosities, and to mobilize nutritious substrates through the release of extracellular lytic enzymes before substrates incorporation [START_REF] Moore | 21st Century Guide to Fungi[END_REF]. Notably, micromycetes play important roles in hydrophobic organic pollutants dynamics within the soil matrices [START_REF] Baranger | Fungal influence on hydrophobic organic pollutants dynamics within the soil matrices[END_REF].

Hydrocarbons can be a source of carbon and energy for fungi. In non-ligninolytic fungi, the main metabolic pathway described as involved in PAH biodegradation is the cytochrome P450 pathway.

Cytochrome P450 mono-oxygenases are a family of intracellular enzymes involved in the oxidation of various hydrophobic substrates [START_REF] Črešnar | Cytochrome P450 enzymes in the fungal kingdom[END_REF]. Cytochrome P450 monooxygenases initiate the oxidation of PAH into arene oxides that are further metabolized into phenol and dihydrodiol derivatives [START_REF] Cerniglia | Degradation of polycyclic aromatic hydrocarbons by fungi[END_REF]. This intracellular degradation pathway implies preliminary uptake of hydrocarbons and notably PAH by fungal cells. Lindley and Heydemann (1986) first described the incorporation of dodecanol into whole cells of Cladosporium resinae by early adsorption to the cell surface followed by active uptake. More recent studies have focused on the uptake of various hydrophobic organic pollutants including polycyclic aromatic hydrocarbons (PAH). PAH have been described to behave in a similar way to neutral lipids, diffusing into lipid membranes [START_REF] Castelli | Calorimetric Approach of the Interaction and Absorption of Polycyclic Aromatic Hydrocarbons with Model Membranes[END_REF] and being incorporated in lipid storage sites in fungal cells [START_REF] Verdin | Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles[END_REF][START_REF] Chang | Trapping toxins within lipid droplets is a resistance mechanism in fungi[END_REF]. Notably, the ubiquitous PAH benzo[a]pyrene (BaP) can be localized in lipid bodies in mycelium that has been cultivated in contact with this pollutant [START_REF] Verdin | Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles[END_REF][START_REF] Fayeulle | Energydependent uptake of benzo[a]pyrene and its cytoskeleton-dependent intracellular transport by the telluric fungus Fusarium solani[END_REF]. However, an active transport mechanism for BaP uptake dependent on the fungal cytoskeleton has been highlighted in Fusarium solani, and active phenomena may be involved in BaP mobilization before its uptake by the same fungus [START_REF] Fayeulle | Energydependent uptake of benzo[a]pyrene and its cytoskeleton-dependent intracellular transport by the telluric fungus Fusarium solani[END_REF]. Interestingly, an active secondary transport mechanism was recently proposed for the uptake of monoaromatic compounds by Phanerochaete chrysosporium [START_REF] Leriche-Grandchamp | Inhibition of phenolics uptake by ligninolytic fungal cells and its potential as a tool for the production of lignin-derived aromatic building blocks[END_REF]. Some micro-organisms are able to release biosurfactants, which can partially solubilize hydrophobic substrates and have been studied for the dispersal of hydrocarbons. In fungi, biosurfactants and emulsifiers are involved in nutrition for the mobilization of fats and adhesion to hydrophobic surfaces [START_REF] Käppeli | Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport[END_REF]. Modulations of cell surface hydrophobicity drive the attachment to surfaces and utilization of hydrophobic substrates (plant leaf wax, oils…) and can affect the fungal interaction with hydrophobic pollutants [START_REF] Puchkov | The mycocidal, membrane-active complex of Cryptococcus humicola is a new type of cellobiose lipid with detergent features[END_REF][START_REF] Arutchelvi | Mannosylerythritol lipids: a review[END_REF][START_REF] Garay | Extracellular fungal polyol lipids: A new class of potential high value lipids[END_REF].

Adsorption of hydrophobic substrates on the cell surface is thus thought to be a determining step prior to potential uptake by the cells [START_REF] Fayeulle | Etude des mécanismes intervenant dans la biodégradation des Hydrocarbures Aromatiques Polycycliques par les champignons saprotrophes telluriques en vue d'applications en bioremédiation fongique de sols pollués[END_REF][START_REF] Al-Hawash | Removal and biodegradation of different petroleum hydrocarbons using the filamentous fungus Aspergillus sp. RFC-1[END_REF]. It is known that surfactants are released in the aqueous phase and accumulate at solid/water and air/water interfaces.

However the interaction of fungal surfactants with hydrophobic soil contaminants and their potential contribution to pollutant mobilization is poorly understood.

Talaromyces helicus is a filamentous soil ascomycete with a cosmopolitan distribution, found on several continents in temperate to tropical climates [START_REF] Huang | Ohio ascomycete notes II. Talaromyces from soils of southern Ohio[END_REF][START_REF] Romero | Wild soil fungi able to degrade the herbicide isoproturon[END_REF][START_REF] Scervino | Soil fungal isolates produce different organic acid patterns involved in phosphate salts solubilization[END_REF][START_REF] Olagoke | Amylase activities of some thermophilic fungi isolated from municipal solid wastes and palm-kernel stack[END_REF]Wu et al., 2016;[START_REF] Fayeulle | PAH biodegradation by telluric saprotrophic fungi isolated from aged PAH-contaminated soils in mineral medium and historically contaminated soil microcosms[END_REF]. It can grow at moderate temperatures (20°C) and presents relatively fine hyphae of 1 to 3 µm in diameter [START_REF] Baranger | Microfluidic monitoring of the growth of individual hyphae in confined environments[END_REF] able to penetrate microporosities and narrow cracks in the substrate, thus potentially covering large volumes of soil. This species has been isolated from contaminated soils, showing the resistance of some strains to pollutions, and its adaptation to the soil environment [START_REF] Fayeulle | PAH biodegradation by telluric saprotrophic fungi isolated from aged PAH-contaminated soils in mineral medium and historically contaminated soil microcosms[END_REF]. Thus, T. helicus has been identified as holding potential for the bioremediation of multiple contaminations due to the bioaccumulation of heavy metals and the biodegradation of several organic pollutants including isoproturon, biphenyl and benzo[a]pyrene [START_REF] Romero | Biotransformation of biphenyl by the filamentous fungus Talaromyces helicus[END_REF][START_REF] Romero | Wild soil fungi able to degrade the herbicide isoproturon[END_REF][START_REF] Romero | Benzo[a]pyrene degradation by soil filamentous fungi[END_REF][START_REF] Fayeulle | PAH biodegradation by telluric saprotrophic fungi isolated from aged PAH-contaminated soils in mineral medium and historically contaminated soil microcosms[END_REF]. The particular efficiency of T. helicus to improve PAH biodegradation in industrial soils with aged mixed contaminations was also highlighted at the microcosm scale [START_REF] Fayeulle | PAH biodegradation by telluric saprotrophic fungi isolated from aged PAH-contaminated soils in mineral medium and historically contaminated soil microcosms[END_REF].

In this study, we aim to investigate the biodegradation of BaP by T. helicus at the microscopic scale, which corresponds both to the size of fungal hyphae and to that of soil aggregates, and is therefore a natural scale to study soil microbial processes (Wilpiskeski et al., 2019). Microfluidics offer the possibility to design model microenvironments with a good control of their geometry and chemical properties, taking advantage of the specific properties of small scale fluid flows to finely tune mechanical forces and mass transfers [START_REF] Squires | Microfluidics: Fluid physics at the nanoliter scale[END_REF]. Transparent microfluidic devices, usually made of glass or silicon rubber, can be used as soil models [START_REF] Aleklett | Build your own soil: exploring microfluidics to create microbial habitat structures[END_REF] and accommodate the growth of plant roots [START_REF] Grossmann | The RootChip: An Integrated Microfluidic Chip for Plant Science[END_REF], bacterial communities [START_REF] Alnahhas | Spatiotemporal Dynamics of Synthetic Microbial Consortia in Microfluidic Devices[END_REF] or fungi [START_REF] Held | Microfluidics structures for probing the dynamic behaviour of filamentous fungi[END_REF]. Parameters such as hyphal growth velocity can be measured by videomicroscopy and can be used to assess the ability of a fungal strain to colonize a polluted porous matrix [START_REF] Baranger | Microfluidic monitoring of the growth of individual hyphae in confined environments[END_REF].

In this work, we used a microfluidic device in which the model pollutant and the fungus were introduced in two separate compartments. The aim of such an experiment is to elucidate whether a filamentous fungus needs to grow in contact to the pollutant source for its uptake and degradation, or whether prior extracellular mobilization mechanisms are required to enhance the pollutant bioavailability in the aqueous phase. The investigation of this biological process is important for future optimizations of PAH mycoremediation protocols in soils through biostimulation approaches.

This study is the first one to our knowledge to use a microfluidic device in order to study pollutants mobilization mechanisms by microorganisms in soil.

Materials and methods

Fungal strain

A strain of the filamentous fungus Talaromyces helicus from our laboratory collection and previously isolated from an industrial contaminated soil from North of France was used for this study.

Identification of the strain occurred through molecular approach by BCCM™/MUCL (Louvain-la-Neuve, Belgium) based on sequencing ITS region, elongation factor gene, or β-tubulin gene, in complement of the macro-and micro-morphological features of pure cultures. The strain was maintained on MYEA solid medium (malt extract 20 g/L -Condalab, Madrid, Spain; yeast extract 2 g/L -VWR, Fontenaysous-Bois, France; microbiological grade agar 15 g/L -Becton Dickinson, Rungis, France), at 22°C with a 12 h -12 h light-dark cycle, and transplanted onto fresh medium every ten days.

Fresh mycelium was produced in a rich medium (MYPC) containing malt extract 10 g/L, yeast extract 4 g/L, soy peptone 10 g/L (Merck, Darmstadt, Germany) and casamino-acids 2 g/L. Shaking flasks 6 containing 50 mL MYPC were inoculated with spores of T. helicus to reach a final concentration of 10 4 spores/mL.

Off-ship incubation with benzo[a]pyrene and lipid staining

T. helicus cultures with BaP were prepared in mineral medium supplemented with glucose (MMG) at pH 5.5, as described by [START_REF] Fayeulle | PAH biodegradation by telluric saprotrophic fungi isolated from aged PAH-contaminated soils in mineral medium and historically contaminated soil microcosms[END_REF]. MMG is comprised of KCl 0.25 g/L, NaH2PO4.2H2O 1.54 g/L, Na2HPO4 8 mg/L, MgSO4.7H2O 0.25 g/L, NH4NO3 1 g/L, ZnSO4.7H2O 1 mg/L, MnCl2.H2O 0.1 mg/L, FeSO4.7H2O 1 mg/, CuSO4.5H2O 0.5 mg/L, CaCl2.2H2O 0.1 mg/L, MoO3 0.2 mg/L and glucose 20 g/L.

Mineral salts and glucose were purchased from Thermo Fisher Scientific (Illkirch-Graffenstaden, France). Prior to adding the medium, 50 µg of BaP (Sigma Aldrich, Haverhill, United Kingdom) was introduced in each flask by pipetting the correct amount of a BaP stock solution in acetone (0.8 g/L), and allowing the acetone to evaporate under a fume hood. Shaking flasks were inoculated with fresh mycelium of T. helicus that was pre-grown in MYPC, and were then incubated for 24h. After incubation, mycelium pellets were mounted on a microscope slide and observed in bright field and epifluorescence microscopy or in laser scanning confocal microscopy.

Lipids were stained in vivo on fresh mycelium samples taken from liquid cultures, using BODIPY 500/510 lipid dye (Thermo Fischer Scientific -Illkirch-Graffenstaden, France). The stock solution of BODIPY in DMSO was pipetted directly onto mycelium samples mounted on a glass microscope slide, achieving a final dye concentration of 0.1 µM/mL.

Production and detection of surface-active compounds

Shaking flasks containing MMG were inoculated with spores of T. helicus to reach a final concentration of 10 4 spores/mL. 50 mL flasks containing 10 mL medium were used for the growth kinetics experiment, and 250 mL flasks containing 50 mL medium were used for filtrates production before characterization. Cultures were incubated at 22°C with orbital shaking and a 12 h/12 h lightdark cycle. The mycelium was harvested by vacuum-filtration on quantitative filter paper (VWR 434) with a Büchner funnel. Filtrates were collected, filtered again on a 0.2 µm syringe filter to sterilize them and remove any remaining particle, and frozen at -20°C until use. To quantify fungal growth in 10 mL cultures, the paper filters with mycelium cake were rinsed with distilled water and dried in a 105°C oven for 24 h, then weighted. For the growth kinetics experiments, 10 mL cultures were done in triplicate and filtered separately.

Surface tension in filtrates was measured on a tensiometer (Krüss K-100) with the Wilhelmy plate method. Each measurement was taken 10 minutes after immersing the plate into the sample, to allow the meniscus to equilibrate at room temperature. All measurements were performed on triplicate samples, and results are presented as average values and standard deviations for each triplicate.

High-Performance Thin-Layer Chromatography

High-Performance Thin-Layer Chromatography (HPTLC) analysis of culture filtrates was conducted by the CAP DELTA laboratory at Chromacim (Grabels, France) with the CAMAG method. Soy lecithin (1 g/L), bovine serum albumin (1 g/L) and glucose (1 g/L) were used as positive controls for the detection of lipids, proteins and reducing sugars respectively. 10 µL of each control was deposited on the plate, as well as 50 µL of the crude filtrate. Samples were deposited on a TLC plate coated with F254 silica gel with an application spray ATS 4 and migrated vertically in a saturated ADC2 chamber over 20 min (migration distance of 70 mm). The mobile phase was a cosolvent mixture containing CHCl3:CH3OH:H2O:NH3 32 % (32:15:2:1 in volume).

After migration, thin layer plates were imaged under white light, UV 254 nm, UV 366 nm and scanned at 200 nm. Then a revelation was done with a primulin reagent (0.1 g/L in acetone:water 80:20 by volume) before imaging under white light, UV 366 nm and scanning at 366 nm for the detection of aliphatic carbon chains corresponding to lipids. Finally a revelation with an anisaldehyde reagent (4methoxybenzaldehyde at 0.5% v/v in CH3OH:CH3COOH:H2SO4 42.5:5:2.5 by volume) was carried out as well as imaging under white light and UV 366 nm for the detection of ketone, aldehyde and alcohol functions corresponding mostly to carbohydrates.

Microfabrication

Microfluidic chips for fungal culture were fabricated by soft lithography as described by [START_REF] Baranger | Microfluidic monitoring of the growth of individual hyphae in confined environments[END_REF]. The pattern was imprinted on a silicon wafer using two layers of SU8 photoresist (Microfactory, IPGG, Paris): a first layer comprised of the microchannel pattern with an average thickness of 5.8 ± 0.3 μm, and a second layer forming the culture chambers, with an average thickness of 124 ± 2 μm. Polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning) mixed with 10% reticulating agent was cast against the wafer and allowed to cure for at least 2 h at 70°C. Inlets were added by punching holes through the PDMS with a biopsy puncher (2 mm for the inoculation well, 1 mm for medium injection inlets). The negatively patterned PDMS slab was bound to a clean glass microscope slide or cover slip after surface oxidization in an oxygen plasma chamber (Harrick) for 60 s.

On-chip fungal culture

All chips were filled with sterile MMG prior to seeding with mycelium. A 2 mm diameter mycelium plug was collected from a solid-medium culture of T. helicus at the edge of the growing colony, and transferred to the inoculation inlet of the chip. The inlet was then closed with a PDMS plug to prevent drying. 1 µL of BaP stock solution (0.8 g/L in acetone) was pipetted into one of the injection inlets on the opposite side to the inoculation chamber right after inoculation. Upon contact with the aqueous culture medium, BaP immediately precipitated into crystals localized at the injection inlet. The microchips were then placed in a sealed Petri dish in a water-saturated atmosphere. The mycelium was allowed to grow at 22°C with a 12 h -12 h light-dark cycle in static conditions. Mycelial growth in the device and fluorescent staining of the cells due to BaP uptake were monitored in the chips for up to 8 days.

Microscopy imaging and image processing

Mycelia of T. helicus grown in liquid cultures with BaP and mounted between slide and coverslip were imaged in bright field and epifluorescence using an OLYMPUS BX60 microscope mounted with a color Infinity 3-6UR camera (Lumenera). Time-lapse epifluorescence microscopy observations were performed using a DMI-8 inverted microscope (Leica) equipped with a motorized stage and a DFC 3000G camera (Leica). The fluorescence of BaP was observed with a standard DAPI emission filter set.

Microchips inoculated with T. helicus and spiked with BaP were imaged using a confocal laser scanning microscope (LSM 710 Axio Observer, Carl Zeiss). The fluorescent signal of BaP was detected using the DAPI filter (excitation 405 nm, emission 453 nm) and that of BODIPY using the FITC filter (excitation 488 nm, emission 563 nm).

All image processing and measurements were carried out using ImageJ. For the BODIPY co-staining experiment, mycelium mounted on a glass slide was imaged in confocal microscopy at 40x magnification with z increments of 0.38 µm, and maximum intensity projections along z were made.

For the on-chip fungal culture experiments, stacks of 11 images with a 1 µm increment were projected along z. Average grey intensity was measured over rectangular areas of 10x100 µm (12x121 pixels) in 3 separate microchannels on each image. Results are presented as mean and standard deviation of the 3 measurements for each time point.

In silico simulation

The penetration of BaP through a model lipid bilayer was simulated using the IMPALA method as first described by [START_REF] Ducarme | IMPALA: A simple restraint field to simulate the biological membrane in molecular structure studies[END_REF]. Briefly, this method is based on a Monte Carlo approach using an implicit description of membrane. The latter is described as a continuous medium whose properties vary along the axis perpendicular to the bilayer plane (z axis). The force field was parameterized to mimic a membrane in aqueous environment by considering (1) the hydrophobic effect between the membrane and a solute (Epho) and ( 2) the perturbation effect of the solute on the lipid acyl chain organization (Elip). The two restraints were calculated and summed at each position of BaP into the implicit membrane and the molecule was systematically moved along the z axis by 1 A steps, from one side of the membrane to the other. The method yields a profile of the interaction energy for each position along the Z axis, the most stable positions being those with minimal energy.

Results

Benzo[a]pyrene diffusion in lipid phases

BaP is highly hydrophobic (log Kow = 6.35) and has an extremely low solubility in water (3.8•10 -3 mg/L at 25°C) (IARC, 2012). This compound forms solid crystals when added to aqueous media such as those used for microbial cultures. In soil, it is associated to solid particles and organic matter, but can also be present in hydrocarbon mixtures as non-aqueous phase liquids [START_REF] Ortega-Calvo | Is it possible to increase bioavailability but not environmental risk of PAHs in bioremediation?[END_REF].

The intracellular uptake of BaP in fungal cells implies that the molecule reaches cell surfaces and transport occurs through the plasma membrane. In order to predict the interaction of BaP with biological membranes, its penetration into an implicit symmetrical lipid bilayer was simulated by the IMPALA method (Fig. 2 a). As expected for a hydrophobic molecule, the profile displayed a sharp contrast between the positions corresponding to the water phase surrounding the membrane and those corresponding to the hydrocarbon core of the membrane. The most stable positions for BaP, displaying negative interaction energies, are located between -14 Å and 14 Å, and correspond to the alkyl core of the model lipid bilayer. In contrast, the interaction energy is maximal above -22 Å and 22 Å, and decreases sharply between (-)22 Å and (-)14 Å. This decrease coincides with positions where the BaP molecule is in contact with the membrane, and still partially surrounded by water.

Indeed, for a 36 Å thick membrane, the hydrated phosphate heads of the amphiphilic lipids are located between 14 and 18 Å.

The energy profile obtained predicts that the BaP molecule is most stable at the core of lipid bilayers, This result is consistent with the low solubility in water of BaP, which tends to minimize its contact surface with aqueous phases.

Intracellular localization of benzo[a]pyrene in Talaromyces helicus

Since BaP emits a blue fluorescent signal when excited with UV light, its incorporation into the fungal biomass can be visualized directly through epifluorescence microscopy. Off-chip observations of T. helicus mycelium pellets incubated with BaP for 24 h were thus carried out. In control flasks without BaP, a slight fluorescence of the mycelium was visible under UV (Fig. 2 b). The fluorescent signal observed without addition of any fluorophore corresponds to the autofluorescence of parietal components. Indeed, fungal hyphae are known to emit blue autofluorescence when exposed to UV light (330-385 nm) due to the presence of chitin in the fungal wall [START_REF] Jabaji-Hare | Autofluorescence of vesicles, arbuscules, and intercellular hyphae of a vesicular-arbuscular fungus in leek (Allium porrum) roots[END_REF][START_REF] Dreyer | Autofluorescence detection of arbuscular mycorrhizal fungal structures in palm roots: an underestimated experimental method[END_REF].

After 24 h of incubation with BaP, the mycelium pellets appeared brightly fluorescent at their core, which could be due to the immediate contact with BaP crystals trapped in the hyphal network. Blue fluorescence was detected along hyphae, with cell walls and septa appearing more strongly stained than the cytoplasm. Many intracellular vesicles were visible in bright field, and appeared brightly fluorescent. In some hyphal tips, these vesicles were distributed in the cytoplasm. In mature hyphae, the vesicles were localized at the periphery of the cytoplasm, while vacuoles were not stained (Fig. 2 b). When mycelium previously incubated with BaP was stained with BODIPY and observed in confocal microscopy, blue and green fluorescent signals co-localized in lipid bodies indicating BaP intracellular storage in these organelles (Fig. 2 c).

Detection of fungal surface-active molecules

Culture filtrates of T. helicus were harvested and analyzed through surface tension measurements to detect potential fungal surfactants (Fig. 3 a). Surface tension in culture filtrates decreased over time and reached a minimum after 6 days, stabilizing until the end of the experiment (after 13 days). The surface tension was lowered to 49.4 ± 0 mN/m, compared to 69 ± 3.7 mN/m in fresh, sterile mineral medium. This result clearly demonstrates the presence of surface-active compounds in the extracellular medium. Extracellular proteins or biosurfactants secreted by the fungus may be responsible for this decrease.

In order to gather more information on the chemical nature of these surfactants, culture filtrates harvested after 13 days of incubation were analyzed in high performance thin-layer chromatography (Fig. 3.b). 13-day filtrates were chosen because they contain the lowest glucose contents, which can interfere with the results and make it difficult to concentrate the samples. After migration, six main spots were detected through 254nm UV illumination, which reveals organic compounds in a nonspecific way. The most intense spot was detected at 0.05 Rf and corresponds to residual glucose in the sample. Five fainter spots were detected at 0.08, 0.16, 0.38, 0.60 and 0.77 Rf. After spraying with a primulin solution to detect lipids and imaging at 266 nm, the only visible spot corresponded to the migration coefficient of glucose. Finally, anisaldehyde revelation was used to reveal carbohydrates. Spots at 0.08, 0.16, 0.60 Rf and the glucose spot reacted with anisaldehyde, but not the spots appearing at 0.60 and 0.77 Rf. In addition, a new spot at 0.72 Rf was visible in white light with anisaldehyde revelation. This compound may be present in concentrations too low for the spot to be visible without prior staining.

The filtrate is a mixture of several compounds with various affinities with the organic phase, which appears to be predominantly made of carbohydrates, and not lipids. Peptides were not investigated in HPTLC, however protein quantification in the filtrates through the Bradford method show the presence of extracellular protein in low amounts with the highest concentration at 3 mg/L after 10 days of incubation.

Benzo[a]pyrene mobilization in a compartmentalized device

T. helicus was grown in microfluidic chips spiked with BaP, and the mycelium was observed at different growth stages. The mycelium was imaged in bright field to monitor growth and locate the edge of the colony. Chips were then observed in laser scanning confocal microscopy to detect the fluorescent staining of hyphae due to BaP incorporation. Faint and diffuse staining of the hyphae was observed, but not more intense than the natural autofluorescence of the mycelium observed in control chips without BaP. After 5 days, hyphae reached the channels but not BaP crystals yet and the mycelium fluorescence appears still low. After 6 days and even more after 7 days, staining intensity clearly increased and was not uniform across the mycelium, i.e. single stained hyphae were distinguishable and appeared more brightly fluorescent than others in contrast to the control (Fig. 4 a). No distinct stained vesicles were visible at 20x magnification. Microchannels enabled the quantification of grey intensity over time in the same area (Fig 4 c). The average fluorescence intensity appears to increase between 3 and 5 days, whereas it remains stable in the control over the whole experiment, which evokes a BaP uptake before direct contact between hyphae and BaP crystals occurred. However the difference of grey intensities between test with BaP and control starts to be statistically significant after 7 days when direct contact between the mycelium and the BaP deposit occurs.

In order to better locate the fluorescence within hyphae, several chips were observed at greater magnification (40X) after 6 and 7 days. Growth velocity was greatly variable from one chip to the other, as previously assessed in the same device with this fungal strain [START_REF] Baranger | Microfluidic monitoring of the growth of individual hyphae in confined environments[END_REF]. As a result, after 7 days of incubation the mycelium had not reached the BaP deposit in all of the chips (Fig. 5 a andb). In some of the chips where direct contact happened, staining of the mycelium was detected in chamber B. Stained apices were observed at the growth front, in close vicinity to the deposit (Fig. 5 c). In these apices, no lipid bodies were distinguishable and the whole cytoplasm appeared fluorescent. This could be due to the presence of numerous, closely packed lipid droplets that could not be resolved with the camera used. Indeed, the resolution of the images was 0.21 µm/pixel at 40x magnification, while some of the lipid droplets detected in stained apices with the Lumenera camera (used for epifluorescence images) could be as small as 0.2 µm. In mature, ramified hyphae located in the chamber, and directly in contact with the crystals, stained vesicles were clearly visible (Fig. 5 d). Some hyphae in the microchannels were stained as well (not shown). The stained structures were similar to those observed in mycelium grown in liquid medium supplemented with BaP, as described in paragraph 3.2.

Interestingly, in chips imaged before the mycelium had reached the deposit, fluorescent staining different from mycelia grown without BaP was observed as well. Indeed, faintly stained apices were visible at the growth front of the mycelium growing through the microchannels (Fig. 5 a). Some hyphae localized in chamber B, closer to the BaP deposit, displayed a stronger staining and visible intracellular lipid bodies (Fig. 5 b). The cell wall and some septa appeared to be stained as well. The stained lipid bodies were smaller and less numerous than they were in hyphae in contact with BaP crystals (Fig. 5 d).

Discussion

Benzo[a]pyrene storage in lipid bodies

Epifluorescence observations with a co-staining with BODIPY confirm that BaP is absorbed into the cells of T. helicus and stored in lipid bodies. Similar observations were made in the saprotrophic fungus Fusarium solani [START_REF] Verdin | Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles[END_REF][START_REF] Fayeulle | Energydependent uptake of benzo[a]pyrene and its cytoskeleton-dependent intracellular transport by the telluric fungus Fusarium solani[END_REF] as well as other fungal strains including the yeast Saccharomyces cerevisiae, the white rot Phanerochaete chrysosporium [START_REF] Verdin | Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles[END_REF], and the oomycete Pythium ultimum [START_REF] Furuno | Mycelia promote active transport and spatial dispersion of polycyclic aromatic hydrocarbons[END_REF]. Intracellular PAH uptake thus appears to occur in several fungal species regardless of PAH degradation efficiency.

Accumulation of lipophilic toxins in lipid bodies may be a defense mechanism against oxidative stress in fungi. Indeed, [START_REF] Chang | Trapping toxins within lipid droplets is a resistance mechanism in fungi[END_REF] found that the formation of large lipid bodies in Candida albicans was associated with mycotoxin resistance, and that toxins were stored in lipid bodies. The authors propose a protection mechanism against oxidative stress caused by the oxidation of aromatic rings, involving quenching of reactive oxygen species by the triacyglycerols making up the bulk of storage lipids. A similar strategy in Talaromyces helicus may serve as a protection against the adverse effects of BaP and other lipophilic organic compounds.

Additionally, lipid bodies may be a site of intracellular degradation. [START_REF] Delsarte | Lipid metabolism and benzo[a]pyrene degradation by Fusarium solani: an unexplored potential[END_REF] proposed a connection between BaP degradation and neutral lipid cycling in fungal cells, hypothesizing that BaP stored in lipid bodies could be oxidized simultaneously as lipid beta-oxidation occurs in neighboring peroxisomes.

Preferential uptake at a close range

Previous hypotheses on the uptake mechanisms of hydrophobic compounds suggest that growing hyphal tips, which are metabolically active and host intense exchanges with the extracellular environment, are preferential sites of uptake [START_REF] Fayeulle | Energydependent uptake of benzo[a]pyrene and its cytoskeleton-dependent intracellular transport by the telluric fungus Fusarium solani[END_REF]. In accordance with this hypothesis, mycelium incubated with BaP frequently displays stained hyphal tips. Transport and storage in older parts of the mycelium is suspected but not established. Indeed, stained lipid bodies are visible in all parts of the mycelium, indicating that BaP is either incorporated in already formed segments, or brought there from the tips through intrahyphal transport. Intrahyphal translocation of PAH through vesicle streaming was observed in P. ultimum [START_REF] Furuno | Mycelia promote active transport and spatial dispersion of polycyclic aromatic hydrocarbons[END_REF]. However, although cytoplasmic streaming was observed during our time-lapse experiments in bright field microscopy, no evidence of lipid body movement was found in T. helicus with specific staining. Previous studies suggest that lipid bodies in filamentous fungi originate from the endoplasmic reticulum, forming small lipid bodies that later fuse into larger ones [START_REF] Kamisaka | Lipid bodies and lipid body formation in an oleaginous fungus, Mortierella ramanniana var. angulispora[END_REF]. Such early precursors of the mature lipid bodies may be more mobile, but smaller than the camera resolution used for the present experiments. Alternatively, BaP could be solubilized in membranes and be transported along with vesicles to various organelles, in a similar way to neutral lipids and other lipophilic compounds [START_REF] Murphy | Lipid droplet-organelle interactions; sharing the fats[END_REF]. Indeed, the IMPALA simulation results predict that the most stable position for BaP in a lipid bilayer is within the hydrophobic core, which indicate a possible penetration into cell membranes and subsequent lateral diffusion. The insertion of PAH into model lipid membranes was also demonstrated experimentally [START_REF] Castelli | Calorimetric Approach of the Interaction and Absorption of Polycyclic Aromatic Hydrocarbons with Model Membranes[END_REF]. However it is very unlikely for BaP in the molecular form to be present in water surrounding cells and to diffuse freely through the extracellular aqueous medium at concentrations high enough to explain its effective biodegradation.

Thus for BaP to be absorbed into membranes, it would need to be already in close vicinity to the cell surface, either as crystals, or solubilized by surface-active molecules. Transport phenomena, other than passive diffusion, must therefore occur earlier than the cell membrane crossing in the incorporation process to bring the pollutant in contact with the cell surface.

Microscopic observations of T. helicus in spiked microfluidic chips indicate a preferential uptake of BaP at a close range and through contact with the cell wall. Indeed, when BaP is supplied as a fixed solid deposit in microfluidic chambers, fluorescent staining of the mycelium is the most intense after the deposit has been reached. This is consistent with observations in mycelium suspensions as well, since, when BaP crystals are dispersed in the liquid medium through continuous agitation, they can come into direct contact with hyphae and get trapped into the tightly knit mycelial network. As a result, the core of mycelial pellets frequently appears brightly stained as opposed to growing hyphae at the periphery. Moreover, early staining of some hyphae was observed before contact with BaP crystals in microfluidic chips. The staining was most often localized in some hyphal apices, sometimes highlighting the cell wall of older hyphae and lipid bodies in the cytoplasm. These results indicate that a long-range mobilization mechanism is at play, bringing BaP molecules to the fungal cells before hyphae could reach the solid crystals. The staining of cell walls observed on some occasions suggests that BaP adsorption to cell walls is a preliminary step to further internalization. Accumulation of BaP at the cell wall was previously shown in F. solani [START_REF] Fayeulle | Energydependent uptake of benzo[a]pyrene and its cytoskeleton-dependent intracellular transport by the telluric fungus Fusarium solani[END_REF] when the formation of actin filaments is inhibited, preventing endocytosis and vesicle trafficking. In a context of non-inhibited metabolic activity, little to no staining of the cell wall is observed, indicating an equilibrium between cell wall adsorption rates and uptake rates.

Extracellular mobilization and transport

The access to the BaP deposits in crystal form relies either on hyphal growth up to the source, or on transport in water. Surface-active compounds may increase BaP partitioning into the water phase by accumulating at the surface of solid BaP particles and promote their fragmentation and dispersion in the medium. This hypothesis seems in accordance with the observation that surfactants enable transfers of hydrophobic molecules between hydrophobic phases of an emulsion stabilized by solid particles [START_REF] Drelich | Mixed O/W emulsions stabilized by solid particles : A model system for controlled mass transfer triggered by surfactant addition[END_REF]. However, the possible role of fungal surfactants in BaP mobilization has not been elucidated so far.

The mechanisms involved in hydrophobic pollutant mobilization could be closely linked to the nutrition behaviour of filamentous fungi. Indeed, hydrophobic compounds in contaminated soils such as BaP are frequently associated with non-aqueous phase liquids and organic matter, which can readily be used by fungi as carbon sources. Some microbial surfactants are able to partially solubilize hydrophobic organic compounds [START_REF] Ron | Biosurfactants and oil bioremediation[END_REF][START_REF] Van Hamme | Physiological aspects: Part 1 in a series of papers devoted to surfactants in microbiology and biotechnology[END_REF], including PAH [START_REF] Garcia-Junco | Biosurfactant-and biodegradation-enhanced partitioning of polycyclic aromatic hydrocarbons from nonaqueous-phase liquids[END_REF][START_REF] Sánchez-Vázquez | Polycyclic aromatic hydrocarbon-emulsifier protein produced by Aspergillus brasiliensis (niger) in an airlift bioreactor following an electrochemical pretreatment[END_REF], thus enhancing their bioavailability.

Sophorolipid biosurfactants excretion has also been linked to the degradation of anthracene sorbed to a solid matrix in several yeast species [START_REF] Romero | Sorbed anthracene degradation by sophorolipid producing yeasts[END_REF]. However in our study with Talaromyces helicus, no extracellular lipids were detected, excluding sophorolipids or other similar glycolipids from biosurfactants candidates. Interestingly, non-lipidic surface-active molecules have been described to play major roles in fungal interactions with hydrophobic substrates such as hydrophobins [START_REF] Berger | Hydrophobins : Multifunctional biosurfactants for interface engineering[END_REF] or exopolysaccharides [START_REF] Mahapatra | Fungal exopolysaccharide : Production, composition and applications[END_REF]. This kind of molecules could be related to the surface tension decrease of culture filtrates observed in our study. Dissolved organic matter itself can serve as a mobilizing agent promoting hydrophobic organic compounds (HOC) dispersion in the water phase [START_REF] Smith | Sorption to humic acids enhances polycyclic aromatic hydrocarbon biodegradation[END_REF][START_REF] Smith | Dissolved organic carbon enhances the mass transfer of hydrophobic organic compounds from nonaqueous phase liquids (NAPLs) into the aqueous phase[END_REF]. Biosurfactants could also promote the dispersal and transport of small solid particles or droplets of organic phases containing BaP. Additionally, fungal hyphae whose growth was oriented towards the source of nutrients following a gradient, may thus come into contact with associated pollutants. Facilitated transport of HOC in small amounts in the aqueous phase would thus be a preliminary step to direct contact and incorporation in greater amounts.

Although the main PAH biodegradation pathway in non-ligninolytic fungi is thought to be intracellular, fungi release a variety of extracellular enzymes involved in nutrition, including oxidoreductases such as laccases, lignin peroxidases, manganese peroxidases, and tyrosinases which catalyze the production of oxidizing agents and free radicals leading to the non-specific oxidation of substrates [START_REF] Tuomela | Oxidative fungal enzymes for bioremediation[END_REF]. The fungal metabolism of aromatic compounds produces substituted derivatives that are more reactive and more soluble than the original pollutants like phenol, phthalate, hydroxyl-, carboxy-and dihydrodiol derivates that may in a second step be conjugated with sugar moieties [START_REF] Boll | Polar metabolites of polycyclic aromatic compounds from fungi are potential soil and groundwater contaminants[END_REF][START_REF] Marco-Urrea | Potential of non-ligninolytic fungi in bioremediation of chlorinated and polycyclic aromatic hydrocarbons[END_REF]. Such metabolites display radically different behaviors compared to the parent PAHs, and tend to partition into the water phase at much higher rates, which could promote their absorption by micro-organisms and facilitate their transport through the cell membrane.

Fungal access to benzo[a]pyrene in compartmentalized environments

Taken together our results enable to propose a two-steps mechanism for the access to BaP by a nonligninolytic fungus before its uptake and intracellular biodegradation in a compartmentalized microenvironment like soil. Firstly, a distant uptake was shown, which highlights an extracellular mobilization step possibly related to the surface-active agents detected in filtrates or to extracellular enzyme activities. This first step may increase the presence of BaP or associated hydrophobic compounds in the water phase and create gradients susceptible to drive growth to the pollutant source. Secondly uptake appears to be more efficient after the mycelium touches a BaP deposit , which could be explained by a better access to the pollutant through a direct chemical partitioning between BaP crystals and hydrophobic portions of the hyphae surfaces as suggested by the simulation. These mechanisms of mobilization and uptake could vary according to the physicochemical properties of the considered PAH and in particular its water solubility. Indeed the phenanthrene and BaP uptakes by F. solani showed different kinetics and were suggested to occur through different mechanisms [START_REF] Fayeulle | Energydependent uptake of benzo[a]pyrene and its cytoskeleton-dependent intracellular transport by the telluric fungus Fusarium solani[END_REF]. The better understanding of these active phenomena to reach hydrophobic pollutants is of relevance to enhance mycoremediation efficiency though biostimulation approaches, since low PAH bioavailability remains one of the major limitations for the development of these techniques [START_REF] Akhtar | Mycoremediation : Expunging environmental pollutants[END_REF]. The elucidation of the mechanisms by which filamentous fungi influence PAH bioavailability in soils [START_REF] Posada-Baquero | Implementing standardized desorption extraction into bioavailability-oriented bioremediation of PAH-polluted soils[END_REF] and transport these molecules through the hyphal network [START_REF] Harms | Untapped potential : Exploiting fungi in bioremediation of hazardous chemicals[END_REF] are also important to understand how they can positively interact with other types of organisms within combined bioremediation strategies [START_REF] Baranger | Fungal influence on hydrophobic organic pollutants dynamics within the soil matrices[END_REF]. Notably positive results in bioremediation of PAH impacted soils have been obtained through combination of mycoremediation and phytoremediation [START_REF] Ma | Soil microbial community succession and interactions during combined plant/white-rot fungus remediation of polycyclic aromatic hydrocarbons[END_REF].

Conclusion

A compartmentalized microchip was designed to observe the incorporation of benzo[a]pyrene by the fungus Talaromyces helicus in a controlled geometry. This is the first time to our knowledge that BaP fungal uptake is directly observed in a compartmentalized system and that a combination of extracellular surfactants and direct contact uptake is proposed to explain this phenomenon. Indeed the fungus is able to develop a mycelium in the confined chambers, and hyphae can grow in direct contact with BaP crystals. Observations through epifluorescence and confocal microscopy confirmed that the pollutant is incorporated into the cells of T. helicus, and co-staining experiments with BODIPY lipid dye show that lipid bodies are a preferential storage site in the cell. BaP uptake in T. helicus occurred even when hyphae were not directly touching the crystals, but to a lower extent than when hyphae were in direct contact with the crystals. Additionally, T. helicus produces extracellular surfaceactive compounds that were detected in culture filtrates. Thus, the use of a microfluidic device in our study brings first pieces of evidence for a two-steps mechanism involved in the access to BaP by a non-ligninolytic soil fungus before its uptake and intracellular biodegradation. Wu, H., Wu, L., Wang, J., Zhu, Q., Lin, S., Xu, J., Zheng, C., Chen, J., Qin, X., Fang, C., Zhang, Z., Azeem, S., Lin, W., 2016. Mixed phenolic

Fig. 4 a

 4 Fig. 4 a shows the evolution of the fluorescent staining over time at two observations points in the

6 Fundings

 6 C.B. benefited from a doctoral grant from the French Ministry of Research. The MycoFlu project has been supported by grants from the CNRS (EC2CO programme MICROBIEN) and Sorbonne Universités (programme Emergence, SU-16-R-EMR-28).

Fig. 1 :Fig. 3 :

 13 Fig. 1: Diagram representing the microfluidic device used for fungal cultivation in presence of BaP. a. channel geometry. b. experimental set-up.

Fig 4 :

 4 Fig 4 : a. Mycelium of T. helicus growing in a microfluidic chip spiked with BaP, observed in LSCM at 20x magnification (maximal intensity projection of image stacks with a 1 µm increment). Left column: Images taken in chamber A (inoculum). Right column: Images taking at the opening of microchannels on the side of chamber B (BaP deposit). b. Diagram representing the microfluidic device used for fungal cultivation in presence of BaP. c. Variation of fluorescence intensity over time, as represented by the average grey intensity measured in three microchannels in the same microchip over a 10x100 µm area (error bars represent the standard deviation of each triplicate; arrows indicate the grey intensity of empty channels, the hyphae entrance in channels and the time after which the direct contact between mycelium and BaP occurred in the considered chip).

Fig. 5 :

 5 Fig. 5: Mycelium of T. helicus growing in microfluidic chips spiked with BaP, observed in LSCM at 40x magnification after 6 to 7 days of incubation. (maximum intensity projection of image stacks with a 1 µm increment). a. Detail of hyphae growing in a microchannel before the mycelium reached the BaP deposit -b. Detail of hyphae growing in chamber B. after the mycelium grew through the channels but not yet reached the BaP deposit. c. Stained hyphal tips in chamber B, close to the BaP deposit. d. Hyphae displaying stained intracellular lipid bodies in chamber B, in direct contact with BaP crystals.

  Towards understanding microbiome interactions at biologically relevant scales. Appl. Environ. Microbiol. 85, e00324-19. https://doi.org/10.1128/AEM.00324-19 61

  acids mediated proliferation of pathogens Talaromyces helicus and Kosakonia sacchari in continuously monocultured Radix

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