Rania Ghossoub

Louise Lindbaek

Anahi Mollà-Herman

Søren Alain Schmitt

Tvorup Christensen
email: stchristensen@bio.ku.dk

Alexandre Benmerah
email: alexandre.benmerah@inserm.fr

Anahi Molla-Herman

Søren Tvorup Christensen

Morphological and Functional Characterization of the Ciliary Pocket by Electron and Fluorescence Microscopy

Keywords: Primary cilia, ciliary pocket, clathrin-dependent endocytosis, early endosomes, cellular signaling

INTRODUCTION

Primary cilia are 9+0 microtubule (MT)-based, membrane-enclosed projections that emanate as solitary organelles on the surface of most quiescent cell types in vertebrates [START_REF] Satir | Overview of structure and function of mammalian cilia[END_REF]. The ciliary axoneme is nucleated from the centrosomal mother centriole (basal body), which docks to the plasma membrane via its distal appendage proteins also known as transition fibers [2]. The membrane surrounding the axoneme is continuous with the plasma membrane, but it has a unique lipid composition and a complement of membrane receptors and ion channels that enable the cilium to function as a sensory organelle that relay signals from the extracellular environment to control developmental processes and tissue homeostasis. The sensory capacity of the cilium is maintained through structural and functional barriers above the basal body that set up a transition zone or molecular filter that gates the selective passage of receptors and ion channels into and out of the ciliary compartment. Cilium defects give rise to a growing number of genetic disorders known as ciliopathies [3,4].

In many cell types, the region between the plasma membrane and the ciliary membrane is infolded to produce a ciliary pocket, which comprises an interphase for the actin cytoskeleton and may function as a major site for exo-and endocytic events for the sorting and targeting of proteins to and from the ciliary base [START_REF] Ghossoub | The ciliary pocket: a onceforgotten membrane domain at the base of cilia[END_REF][START_REF] Benmerah | The ciliary pocket[END_REF]. Indeed, the infolded plasma membrane, also known as the periciliary membrane, comprises a unique site for clathrin-dependent endocytosis, which critically regulates a series of signaling events associated with ligand-induced receptor internalization and subsequent recycling or degradation in the late endosomes/lysosomes compartment [START_REF] Mikkelsen | Neuroleptic-withdrawal cachexia[END_REF]. This raises the possibility that endocytosis at the ciliary pocket regulate the level of many different ciliary signaling systems, such as those operated by G protein-coupled receptors [START_REF] Schou | Ins and outs of GPCR signaling in primary cilia[END_REF], Receptor Tyrosine Kinases [START_REF] Christensen | Primary cilia and coordination of receptor tyrosine kinase (RTK) signalling[END_REF], TRP ion channels [START_REF] Phua | An intelligent nano-antenna: Primary cilium harnesses TRP channels to decode polymodal stimuli[END_REF], and receptors for extracellular matrix proteins [START_REF] Seeger-Nukpezah | The extracellular matrix and ciliary signaling[END_REF]. Indeed, we previously showed that Transforming Growth Factor beta (TGFβ) signaling and activation of Smad transcription factors is associated with clathrin-dependent endocytosis of TGFβ receptors at the ciliary pocket in fibroblasts and in stem cells undergoing cardiomyogenesis [START_REF] Clement | TGF-beta signaling is associated with endocytosis at the pocket region of the primary cilium[END_REF].

Here, we provide different protocols for investigating the morphology and function of the ciliary pocket in RPE1 and NT2 cells, which are cell lines commonly used in laboratories worldwide.

MATERIALS

Cells and cell culture media

1. RPE1, a human retinal pigment epithelial cell line that stably expresses human telomerase reverse transcriptase (hTERT-RPE1; CLONTECH Laboratories, Inc.; ATCC CRL4000 TM ).

Equipment

1. Cells are grown in an incubator under standard growth conditions (37°C and 5% CO 2 ).

2. Cells are seeded on glass coverslips, 12 mm (NeuVitro, GG1212).

3. Coverslips are mounted on plain glass microscope slides (1mm, Pearl, 7101). 4. Immunofluorescence staining is performed in a humidity chamber to avoid evaporation of antibody solutions. Humidity chamber is made by placing damp filter paper in the lid of a petri dish, under a layer of Parafilm ® . Coverslips can be placed on top of the Parafilm ® layer, and the bottom of the petri dish can be used as the lid of the chamber.

5. 8 wells µ-slides (Ibidi, catalog #80821).

6. Ultramicrotome (Reichert ultracut S). 12. Observation/acquisition: JEOL 1011 transmision electron microscope with a GATAN CCD camera erlangshen 1000 with GATAN Digital Micrograph Software. Generated images were recorded in DM4 format then converted in TIFF.

Preparation of coverslips for immunofluorescence

For NT2 cell growth, 12 mm coverslips for immunofluorescence analysis are treated with acid prior to growing cells on them (see note 2).

1. Incubate coverslips in 32% HCl for 60 minutes, in a blue cap bottle. Mix frequently by swirling bottle around. Should be performed in the fume hood.

METHODS

Induction of ciliogenesis in RPE1 cells

1. Set down one coverslip per well in a regular 24 wells plate under the hood. Coverslips are previously autoclaved to avoid any contamination.

2. To optimize cell culture and transfection steps, fix your coverslips to the bottom of the wells by filling each well with 0.5 mL of PBS and then aspirate it with a 200µL tip pipet to create a vacuum between the coverslip and the plate.

3. Seed 250.000 cells per well and grow them in basic cell culture conditions for 24 hours. 4. Wash the cells twice in PBS. To avoid cell detachment, bend the plate and use a 200µL tip pipet to aspirate the medium without touching the cells. 5. Add low serum containing media for an additional 24 to 48 hours period to allow primary cilia formation.

6. Monitor the efficiency of primary cilia formation by staining with acetylated α-tubulin or ARL13b antibodies (Tipically ~80% of ciliated cells; see Immunofluorescence section below).

Induction of ciliogenesis in NT2 cells

Seed cells as described for RPE1 cells and grow under basic cell culture conditions. Primary cilia will form when cells reach confluence of approximately 80% or above, and can be visualized using specific antibodies against structural proteins and/or ciliary receptors, e.g. ARL13b. NT2 cells form rather long primary cilia, which may at times connect to cilia from adjacent cells, as shown in Figure 1a.

Transmission Electron Microscopy (TEM) protocol for flat-embedding

1. Seed RPE1 cells on coverslips and grow them in 24 well plates to induce ciliogenesis as described above.

2. Wash cells twice with PBS(Ca/Mg) to avoid cell detachment. 11. Gently remove the coverslip from the wells, pay attention to not break them.

Fix the cells on coverslips

12. Remove the Epon excess on the coverslip by gently holding the coverslip with the forceps and letting it flow into the well, only leaving a thin layer of Epon on the cells.

13. Place the coverslip on a glass slide, with the cells facing up.

14. Cover the coverslip with the Epon-filled capsule. One capsule is usually enough; putting two or more could lead to difficulties later to separate them. It can be also useful to place the capsule where the cells are more concentrated (this can be simply found by placing the slide/coverslip under a binocular).

15. Polymerize at 60°C for 24 hours.

16. Remove the Gelatin capsule from the coverslip by heating the glass slide at 70°C for 5 minutes. 17. Trim the block using a clean razor blade and cut your samples in 90 nm thin sections with an ultramicrotome. Ultrathin sections are put on specimen grids. Do not try to find region of interest by using 5 µm thin sections; the cell monolayer is too thin. 18. Perform serial cutting in the orientation showed in Figure 2a. The better way is to keep the grids in the cutting order for observation.

19. Stain your sections with uranyl acetate (10 minutes, followed by H 2 O 3x5 minutes) and Reynold's lead citrate (5s, followed by H 2 O 3x5 minutes). 20. Perform observation beginning with the region corresponding to the adherent surface of the cells, i.e. the first section, then the following, to be sure to not miss the level where the region where cilia are. They are usually found in the same region close to the nucleus (on the "side") as indicated by our analysis of cilia positioning in RPE1 cells (PMCID: PMC2972276) and as shown in Figure 2.

Transfection of cells with plasmid DNA in RPE1 cells

1. Seed 100,000 cells per well in a 24 well plates containing a coverslip and grow them as indicated above for 24 hours.

2. Prepare the transfection mix. For 4 wells, mix 1µg of DNA with 100µL of Opti-MEM and 3µL of Fugene 6.

3. Wash the cells twice with PBS(Ca/Mg) as described above.

4. Aspirate the PBS and add 500 µl of low serum containing Opti-MEM (0.5% FBS) to each well.

5. Add 25µl of transfection mix per well. Move gently your plate in perpendicular directions in order to disperse the transfection mix in the wells. Avoid circular movements.

6. Incubate the cells with the transfection mix for 24 hours before fixation and/or life imaging analysis.

Transfection of cells with plasmid DNA in NT2 cells

1. Grow cells to approximately 80% density under basic culture conditions.

2. Prepare the transfection mix as described for RPE1 cells.

3. Wash cells in PBS and add 100 µl of DMEM (10% FBS) without antibiotics to each well.

Proceed as above and transfect cells using Fugene 6 and DNA in a 3:1 dilution. Dilute reagents in DMEM without serum or antibiotics.

An example of transient plasmid transfection is shown in Figure 1, where NT2 cells express Cherry-FYVE (Figure 1a) and 2xGFP-FYVE (Figure 1b) that mark early endosomes (EEs) in close proximity to the pocket region of the primary cilium stained with either ARL13b or acetylated α-tubulin. EEs are enriched in phosphatidylinositol-3 phosphate, which anchors FYVE zinc finger domain-containing proteins, such as SMAD anchor for receptor activation (SARA) that conveys SMAD2/3 to the TGF-β-Rs and promotes TGF-β-RI-mediated SMAD2/3 activation (PMID: 20515759).

Transferrin internalization in RPE1 cells

1. Incubate RPE1 cells grown on coverslips (previously transfected or not, see above) for 20 minutes at 37°C in serum free DMEM (see note 3).

2. Wash the cells twice in TIB previously warmed up at 37°C. 4. Wash rapidly your cells in cold PBS (4°C) to stop membrane trafficking. 5. Fix rapidly the cells with 4% PFA (10 minutes at 4°C, then 20 min at RT).

6. Process for immunofluorescence as described above.

An example of RPE1 cells transfected with pEGFP-F, treated to follow transferrin internalization at the ciliary pocket, fixed and stained for acetylated α-tubulin is shown in Figure 3a.

Transferrin internalization in NT2 cells

1. Wash NT2 cells grown on coverslips (previously transfected or not, see above) twice in serum free DMEM, previously warmed to 37°C.

Incubate cells for 20 min at 37°C in serum free DMEM.

3. Wash the cells twice in TIB previously warmed up at 37°C. 4. Incubate the cells for 10 minutes in TIB pre-heated to 37°C containing 15mg/mL Texas Red-conjugated transferrin (Tf), to allow receptor internalization (see note 4). [START_REF] Ghossoub | The ciliary pocket: a onceforgotten membrane domain at the base of cilia[END_REF]. Wash rapidly your cells in TIB pre-heated to 37°C. Let cells sit in the incubator for 10 minutes to allow surface-bound receptor to be internalized. [START_REF] Benmerah | The ciliary pocket[END_REF]. Wash your cells in cold PBS (4°C) to stop membrane trafficking.

7. Fix the cells in 4% PFA (15 minutes at 4°C). Proceed with immunofluorescence as described below.

An example of NT2 cells pulsed for 10 minutes with Tf is depicted in Figure 4, where Tf is largely confined to EEs marked by 2xGFP-FYVE. Localization of Tf to EEs is shown both in an interphase cell (Figure 4a) and in a growth-arrested cells (Figure 4b), where Tf and 2xGFP-FYVE partly co-localizes in multiple endosomes around the pocket region of the primary cilium.

Preparation of cells for immunofluorescence microscopy in RPE1 cells

1. Wash the cells twice with PBS as described above (room temperature, RT).

2. Fix the cells in 4% PFA for 20 minutes at 4°C (by placing the plate on ice) followed by a 10 minutes incubation in quenching solution at RT.

Wash the cells twice with PBS.

4. Incubate the cells with primary antibodies in permeabilization buffer for 30-45 minutes at RT. Incubation can be made directly in the well, the minimal mix volume is then 200µL per well to avoid cells to get dry. To save precious antibodies, you can prepare a 40µL drop containing the antibody/permeabilization mix on Parafilm ® attached to the bench, and then gently reverse your coverslip to put the cells in contact with the liquid. 5. Wash the cells twice with blocking buffer at RT avoiding cell detachment. For cells incubated on a drop, put back the coverslips into the wells properly reversed. In case of doubt about the cells' orientation, cells can be seen on the coverslip by holding it with the forceps and by exposing it to a source of light.

6. Incubate mixed secondary antibodies in blocking buffer for 30 minutes at RT in the dark to preserve the fluorescence of secondary antibodies.

7. Wash once with blocking buffer and twice with PBS. 8. Lay down your cells on microscope slides in mounting medium. 9. Add nail polish by putting small drops around the coverslip and then complete the circle once drops dried. Avoid small holes that will favor oil contact with mounting media and the cells.

10. Store at 4°C, keep in the dark.

Preparation of cells for immunofluorescence microscopy in NT2 cells

1. Wash the cells twice with PBS as described above (room temperature, RT).

2. Fix the cells in 4% PFA for 15 minutes at RT (see note 5).

Wash the cells twice with PBS.

4. Incubate your cells in permeabilization buffer for 12 minutes at RT. 5. Incubate your cells in blocking buffer for 30 minutes RT or at 4°C overnight (ON) 6. Incubate your cells in primary antibodies in blocking buffer for 90 minutes at RT or 4°C ON (see note 6).

7. Wash the cells 3 times, each 5 minutes, with blocking buffer at RT avoiding cell detachment. Transfected NT2 cells are easily detached upon mechanical stress. 8. Incubate for 45 minutes at RT in blocking buffer containing mixed secondary antibodies. 9. Wash once (5 minutes) with blocking buffer and twice (2x5 minutes) with PBS. 10. Lay down your cells on microscope slides in mounting media. Apply light pressure, getting rid of excess mounting medium. 11. Add nail polish around the edge of the coverslip. Allow 20 minutes to dry.

12. Store at 4°C, keep in the dark.

An example of immunofluorescence staining with antibodies directed against CALM and clathrin, which marks clathrin-coated pits (CCPs) and vesicles (CCVs) at the ciliary pocket, is shown in Figure 4c-e. As expected, CCVs display little co-localization with EEs as evidenced by co-staining with either 10 minutes Tf pulsing (Figure 4d) or expression of 2xGFP-FYVE (Figure 4e). This gives a good overview on the spatiotemporal machinery of endoctosis at the primary cilium, which can be elaborated by live-cell imaging, e.g. Figure 6.

Fluorescence microscopy and 3D image reconstruction in RPE1 cells

1. Acquire Z-stacks epifluorescence images every 200 nm along the Z axis using a piezoelectric (PIFOC, E662-LR controller, Physik Instrumente) 100x objective (plan-apo; Axiovert 100M, Zeiss) with a micromax camera (Princeton Instruments).

2. Three methods of deconvolution of Z-stacks can be recommended including MetaMorph (3D deconvolution option), Image J plugin "Deconvolutionlab -EPFL » http://bigwww.epfl.ch/algorithms/deconvolutionlab/ or Huygens https://svi.nl/HomePage.

Obtain 3D reconstruction of deconvoluted images with the Imaris software (Bitplane).

Images can be extracted from Imaris and then used to obtain the final pictures used in the figures.

An example of RPE1 cells transfected with pEGFP-F, treated to follow Transferrin internalization, fixed and stained for acetylated tubulin and CALM is shown in 3D reconstruction images in Figure 3b.

Fluorescence microscopy and 3D image reconstruction in NT2 cells

Capture NT2 cell images on a fully motorized Olympus BX63 upright microscope with a DP72 color, 12.8-megapixel, 4,140 × 3,096-resolution camera and differential interference contrast (DIC). The software used include Olympus CellSens dimension, which is able to perform 3D blending projections on captured z stacks, 3D animation videos, and slice views (see Figures 1, 4 and6).

NOTES

1. NT2 cells may be difficult to transfect, however, we find increased efficiency when transfecting in media without antibiotics. Cell density does not appear to be very important for these particular constructs; however, we see a clear positive effect when using 1:3 DNA concentration to Fugene amount.

2. Preparation of coverslips using acid wash is an optional step, often used to increase cell attachment to surfaces. We find that this helps our growth rate and cell dispersion when culturing NT2 cells for immunofluorescence experiments. Further, we find an increase in transfection efficiency, possibly due to a decrease in cell loss during IFM preparation steps. Concentration may be adjusted, and lower amounts may be used. Further, chasing with non-labelled Tf may be included to optimize the distinct visualization of CCPs, CCVs and EEs at the ciliary pocket. 5. Variations in protocol for preparation of IFM slides between RPE1 and NT2 cells are not a reflection of an optimization in protocol. Rather, it is a result of different lab techniques, and one may very likely be interchanged with another. For instance, fixation for 15 minutes RT followed by 12 minutes in permeabilization buffer and 30 minutes in blocking buffer, as we here describe for NT2 cells, is also successfully used for RPE1 cells.

6. 90 minutes or shorter is often sufficient for visualization using a fluorescence microscope, however, some antibodies bind more weakly than others, and optimization steps often includes longer incubation times. Therefore, incubating overnight may increase signal strength.

2 .

 2 NT2, a human pluripotent embryonal carcinoma stem cell line (NTERA-2 cl.D1; ATCC CRL1973 TM ). 3. Dulbecco's Modified Eagle's Medium DMEM-F12 1:1 GlutaMAX supplement (Thermofischer scientific, 31331-028) supplemented by 10% fetal bovine serum (FBS, Thermofischer scientific, 10270-106) for basic RPE1 cell culture conditions and supplemented with 0.5% FBS for low serum containing media. 4. OPTI-MEM (Thermofischer scientific, 31985-047) was used for transfections in RPE1 cells. 5. DMEM (Thermofischer scientific, 41965-039) was used for Transferrin uptake experiments in RPE1 cells and NT2 cells. 6. DMEM (ATCC, 30-2002) supplemented by 10% FBS (Sigma Aldrich, F9665) and 1% pen/strep for basic cell culture conditions in NT2 cells.

7 . 2 . 2

 722 DMEM supplemented by 10% FBS without pen/strep was used for transfections in NT2 cells (see note 1). Buffers and solutions for fluorescence microscopy 1. 1X Phosphate-buffered saline (PBS) (pH 7.4); 2. 1X Phosphate-buffered saline with Ca 2+ and Mg 2+ (PBS(Ca/Mg)) (pH 7.4); PBS with 3. Transferrin internalization buffer (TIB): DMEM with 1mg/mL bovine serum albumin (BSA).4. Fixation solution: 4% Paraformaldehyde (PFA).

5 .

 5 Permeabilization buffer: Triton X-100 (0.1 or 0.2%) and BSA (1 mg/mL) in PBS. Buffer should be passed through a sterile filter to avoid cellular contamination for long-term storage. Permeabilization buffer should be stored at -20°C in aliquots and thawed and kept at 4°C upon use.[START_REF] Benmerah | The ciliary pocket[END_REF]. Blocking buffer: PBS with BSA (1 or 2 mg/mL). 7. Quenching solution: PBS-NH 4 Cl (50 mM). 8. Mounting media for NT2 cells: Add 4 mL glycerol (5g) in a falcon tube and add 500µL 10 X PBS. Fill up to 5 mL with double distilled water (ddH 2 O). Weigh out and add 0.1 g n-Propyl Gallate and dissolve over night at 4°C while rotating. Store covered in tin foil at 4°C. 9. Mounting media for RPE1 cells: PBS-glycerol mix (50/50) using the SlowFade Light Antifade Kit containing DAPI from Molecular Probes (Thermofischer scientific, S36938).

2. 4 4 .

 44 Ligands, antibodies, staining reagents, plasmids, conjugates and transfection reagents 1. Ligand: Transforming growth factor beta-1 (TGFβ-1) (R&D Systems, 240-B-010).) 2. Primary antibodies (the species, reference number and dilution are indicated): Acetylated-α-tubulin (AcTub), mouse monoclonal clone 6-11B-1 (Sigma, T7451) (1:10,000) ADP-ribosylation factor-like 13b (ARL13b), rabbit polyclonal (Proteintech 17711-1-AP) (1:600) Clathrin, rabbit polyclonal (AbCam Ab21679) (1:500) Clathrin assembly lymphoid myeloid leukemia (CALM), goat polyclonal (Santa Cruz, sc-6463) (1:300) Transforming growth factor beta receptor I (TGFβ-RI, V22) rabbit polyclonal (Santa Cruz sc398) (1:200) 3. Secondary antibodies and conjugates: Alexa 350 -conjugated donkey anti-mouse IgG (Invitrogen, A10035) (1:600) Alexa 350 -conjugated donkey anti-rabbit IgG (Invitrogen, A10039) (1:600) Alexa 488 -conjugated donkey anti-mouse IgG (Invitrogen, A21202) (1:600) Alexa 488 -conjugated donkey anti-rabbit IgG (Invitrogen, A21206) (1:600) Alexa 488 -conjugated donkey anti-goat IgG (Invitrogen, A11055) (1:600) Alexa 568 -conjugated donkey anti-goat IgG (Invitrogen, A11057) (1:600) Alexa 568 -conjugated donkey anti-mouse IgG (Invitrogen, A10037) (1:600) Alexa 555 -conjugated Transferrin from Molecular Probes (Thermofischer scientific, T-35352) Texas Red-conjugated Transferrin from Molecular Probes (Thermofischer scientific, T-2875) Expression plasmids (available from our laboratories upon request): Rab8 fused to GFP (Rab8-GFP) and Clathrin light chain fused to DsRed were kindly provided by Arnaud Echard (Institut Pasteur, Paris, France) and Thomas Kirchhausen (Immune Disease Institute, Boston, USA), and were described previously (ref!!!!!!).

2 . 5

 25 pEGFP-F plasmid (Clontech, 6074-1) expressing farnesylated green fluorescent protein (GFP-F). GFP-2xFYVE (kindly provided by Harald Stenmark (Institute for Cancer Research, Oslo, Norway)). Cherry-2xFYVE (kindly provided by Harald Stenmark (Institute for Cancer Research, Oslo, Norway)). 5. Transfection reagents: Fugene 6 (Promega, E2691) 6. Nuclear staining: 4',6-diamidino-2-phenylindole dihydrochlorid (DAPI) (Invitrogen, D1306) 7. Nail polish (electron microscopy science, ref 72180) Electron microscopy 1. Fixation: H2O is double-distilled apyrogenic sterile water rinsing and irrigation (Dominique Dutscher Products : ref 069802A). Phosphate buffer is Sorensen's Sodium-Potassium Phosphate Buffer, prepared as followed: Solution A: 35.61g Na2HPO4.2H2Omake up to 1000 mL with distilled H2O, stir until dissolved. Solution B: 27.6g NaH2PO4.H2Omake up to 1000 mL with distilled H2O, stir until dissolved. Add 40.5 mL of solution A to 9.5 mL of solution B to give 50 mL 0.2M phosphate buffer pH should be 7.4. Glutaraldehyde EM grade 25% (Electron Microscopy Science, EMS ref 16210) should be diluted in PBS Osmium tetroxide 4% (EMS ref 19180) stock should be diluted 1:1 in H2O then diluted 1:1 in phosphate buffer 0.2M to to obtain Osmium 1% in phosphate buffer 0.1M perform post fixation Phosphate buffer 0.1M is used for washing coverslips after post fixation 2. Embedding: Epon is prepared with Mollenhauer's Kit with Epon-812 (EMS ref 13940). Embedding was done using gelatin capsule type 04 (EMS ref 70105).

3 .

 3 Sectionning: Ultrathin sections are performed on a reichert ultracut S (Leica Microsystem) with a Diatome Diamond Knife 2mm 45°. 10. Specimen grids: Gilder 200 mesh Standard Square Mesh Nickel Grids (EMS ref G200Ni). 11. Miscelleanous: 7% Uranyl acetate (EMS ref 22400) in water. Lead citrate (EMS ref 17800).

2 .

 2 Discard HCl (can be saved and re-used) and add water, continue working in the fume hood. Swirl and discard. You can now move out of the hood for a total of 15 washes in ddH 2 O. 3. Subsequently, coverslips are washed 15 times in 96% ethanol. 4. Store in 70% ethanol.

3 .

 3 Incubate the cells for 1 minute at 37°C in TIB containing 6 µg/mL Alexa 555 -conjugated transferrin (Tf) to allow accumulation of receptor-bound Tf into clathrin-coated pits and vesicles (CCP/CCV).

3 .

 3 This step eliminates endogenous receptor-bound transferrin, and therefore increases binding and endocytosis of fluorophore-conjugated transferrin. 4. For NT2 cell experiments, we have used Texas Red-conjugated transferrin. This can be replaced by Alexa 555 -conjugated transferrin used in our RPE1 experiments.

Fig. 1 .

 1 Fig. 1. Fluorescence microscopy analysis on the localization of early endosomes (EEs) around the ciliary pocket region in NT2 cells. (a) The localization of endosomes was observed in cells transiently transfected with plasmids expressing either Cherry-FYVE (a) or 2xGFP-FYVE (b)and primary cilia were stained with either anti-ARL13b or anti-acetylated α-tubulin (AcTub). In some cases, the nucleus was stained with DAPI (b). In some cases, the tip of primary cilia from adjacent cells connected to each other as shown in (a). Endosome localization is shown both with normal epifluorescence (b, upper panel), and with 3D reconstruction (lower panels in a and b).

Fig. 2 .

 2 Fig. 2. Transmission electron microscopy analysis on the ciliary pocket in RPE1 cells, which were grown on coverslips to confluence, then serum-starved, fixed and treated for microscopy. (a) Sections were realized parallel to the adherent surface as depicted in the scheme and explained in the methods. (b) Examples of cilia from cells starved for 24 (left) or 72 hours (right) in which both the basal body (BB) and the axoneme (Ax) are clearly visible. All observed cilia in RPE1 cells present a similar organization, with the basal body not at the same plane as the plasma membrane but inside the cytoplasm and a membrane domain around the proximal region of the axoneme. (c) A representative TEM image is showed to highlight the shape and organization of the ciliary pocket (CiPo) relative to the axoneme (Ax) and the basal body (BB). The extracellular distal part of the axoneme is stressed by arrowheads. Scale bars: 500 nm in (b), 100 nm in (c). Adapted from figures previously published in (PMID: 20427320).

Fig. 3 .

 3 Fig. 3. Fluorescence microscopy analysis on the localization of clathrin-coated pits (CCPs) at the ciliary pocket in RPE1 cells, which were transiently transfected with the farnesylated green fluorescent protein (GFP-F) encoding plasmid for 24 hours and then incubated 2 minutes at 37°C with Alexa 555 -conjugated transferrin (Tf). Cells were immediately fixed and processed for immunofluorescence using antibodies against acetylated--tubulin (AcTub) to stain the axoneme, and CALM to stain clathrin-coated pits (CCPs). Epifluorescence (a) and 3D reconstruction (b) images of a representative cilium are shown. CiPo-associated CCPs present along the axoneme

Fig. 4 .

 4 Fig. 4. Fluorescence microscopy analysis on clathrin-coated pits (CCPs), clathrin-coated vesicles (CCVs) and early endosomes (EEs) at the ciliary pocket region in NT2 cells. NT2 cells were transiently transfected with the plasmid expressing 2xGFP-FYVE followed by pulsing with Texas red-conjugated transferrin (Tf) for 10 minutes. Cells were quickly fixed and prepared for IFM. Following Tf incubation, the endocytic pathway can be visualized. (a) Epifluorescence and 3D reconstruction of Tf localization to 2xGFP-FYVE positive EEs in an interphase cell. (b) 3D reconstruction of accumulation of Tf at EEs around the pocket region of the primary cilium. (c) 3D reconstructions of co-staining with CALM shows that CCVs are localized around the pocket region of the primary cilium. (d) 3D reconstruction shows little or no co-localization between CCVs (CALM) and Tf, which after a 10 minutes pulse primarily localizes to EEs. Similarly, CCPs/CCVs show no or little colocalization with EEs marked 2xGFP-FYVE. Primary cilia were stained with either ARL13b or anti-acetylated α-tubulin (AcTub) antibodies.

Fig. 5 .

 5 Fig. 5. Fluorescence microscopy and 3D reconstruction analysis of the localization of Transforming growth factor beta receptor I (TGFβ-RI) to clathrin-coated vesicles (CCVs) at the pocket region of the primary cilium (stained with anti-acetylated α-tubulin, AcTub) in NT2 cells. Stimulation with TGFβ-1 results in accumulation of the receptor around the base of the cilium as well as along microtubules (MTs) that project from the ciliary base into the cytosol. The right panel shows a merged image exclusively with co-localization between the receptor and Clathrin.

Fig. 6 .

 6 Fig. 6. Live-cell imaging of RPE1 cells were transiently co-transfected with plasmids encoding for Rab8-GFP (green) and clathrin-DsRed (red) fusions and analyzed by fluorescence microscopy. Live cells were imaged at 37°C (one picture for each color every 3 seconds for 4 minutes). The first taken picture is shown (left). An image stream of a clathrin spot (red, arrow) which disappeared during the acquisition is shown (right).The initial position of the followed

Figure 6

 6 Figure 6


Kheira Hireche
email: kheira.hireche@yahoo.fr

Youcef Lounes

Christophe Bacri

Laurence Solovei

Charles Marty-Ané

Ludovic Canaud

Pierre Alric

VATS versus Open Lobectomy following Induction Therapy for Stage III NSCLC: A Propensity Score-Matched Analysis

Keywords: lung cancer, stage III, lobectomy, VATS

published or not. The documents may come

Introduction

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Unfortunately, the advanced-stage (stages III and IV) disease accounts for 79% of newly diagnosed patients, and the five-year survival rate ranges from 4 to 28% [START_REF]SEER Stat Fact Sheets: Lung and Bronchus Cancer[END_REF], depending on the disease stage, the patient's medical conditions, and treatment modality. Chemoradiotherapy has traditionally been the mainstay of treatment for advanced NSCLC, while current guidelines recommend therapeutic intent pulmonary resection for patients with resectable stage IIIA disease and oligometastatic stage IV disease [START_REF] Ettinger | NCCN Guidelines Insights: Non-Small Cell Lung Cancer, Version 1[END_REF]. However, recent studies point to the benefits of surgical resection for more advanced-stage cases (which have typically been considered unresectable [START_REF] Albain | Concurrent cisplatin/etoposide plus chest radiotherapy followed by surgery for stages IIIA (N2) and IIIB non-small-cell lung cancer: Mature results of Southwest Oncology Group phase II study 8805[END_REF][START_REF] Bott | Role for Surgical Resection in the Multidisciplinary Treatment of Stage IIIB Non-Small Cell Lung Cancer[END_REF][START_REF] David | Surgical Management of Advanced Non-Small Cell Lung Cancer Is Decreasing But Is Associated With Improved Survival[END_REF][START_REF] Wang | Surgery of primary non-small cell lung cancer with oligometastasis: Analysis of 172 cases[END_REF]) and have indicated that such intervention can offer significant improvements for both hospital mortality and longterm survival.

The benefits of video-assisted thoracoscopic (VATS) lobectomy in terms of reduced morbidity, faster resumption of daily activities [START_REF] Bendixen | Postoperative pain and quality of life after lobectomy via video-assisted thoracoscopic surgery or anterolateral thoracotomy for early stage lung cancer: A randomised controlled trial[END_REF], and even higher OS and RFS [START_REF] Hernandez-Vaquero | Survival After Thoracoscopic Surgery or Open Lobectomy: Systematic Review and Meta-Analysis[END_REF] are well established in patients with early-stage NSCLC. As a result, VATS is now considered to be the standard treatment for early-stage NSCLC [START_REF] Ettinger | NCCN Guidelines Insights: Non-Small Cell Lung Cancer, Version 1[END_REF]. Despite better compliance with adjuvant chemotherapy following a VATS procedure in patients with advanced disease [START_REF] Petersen | Thoracoscopic lobectomy facilitates the delivery of chemotherapy after resection for lung cancer[END_REF] and the expansion of the use of thoracoscopic procedures for more technically challenging operations, the benefits of VATS for patients with advanced-stage NSCLC have yet to be clearly defined. Indeed, several studies have reported the use of VATS lobectomy for locally advanced NSCLC [START_REF] Gonzalez-Rivas | Double sleeve uniportal video-assisted thoracoscopic lobectomy for non-small cell lung cancer[END_REF][START_REF] Gao | Video-Assisted Vs Thoracotomy Sleeve Lobectomy for Lung Cancer: A Propensity Matched Analysis[END_REF][START_REF] Chen | Propensity-matched comparison of video-assisted thoracoscopic with thoracotomy lobectomy for locally advanced non-small cell lung cancer[END_REF][START_REF] Yang | Long-term survival following open versus thoracoscopic lobectomy after preoperative chemotherapy for nonsmall cell lung cancer[END_REF][START_REF] Hennon | Safety of thoracoscopic lobectomy in locally advanced lung cancer[END_REF] and indicate that VATS is both feasible and safe and is associated with a reduced hospital stay and chest tube duration with equal oncologic efficacy as compared to open lobectomy. However, these studies involved a mixture of disease stages, which may have biased the results in favor of the VATS approach, nor did these studies specifically assess the role of VATS in the management of stage III disease. Consequently, the present study was designed to compare the perioperative and oncologic outcomes of VATS and open lobectomy for patients with advanced stage III NSCLC. We performed propensity score matching to create two homogenous groups for comparison, minimize selection bias, and achieve convincing statistical results.

Material and Methods

Patient Selection

We retrospectively analyzed 205 consecutive patients with advanced stage III NSCLC who underwent a lobectomy between January 2013 and December 2020 at the Department of Thoracic and Vascular Surgery, Arnaud de Villeneuve Teaching Hospital, Montpellier. The 8th edition of the Union for International Cancer Control's tumor node metastasis (TNM) staging system was used to determine clinical and pathological stages. The preoperative staging was assessed by a computed tomographic (CT) scan of the chest, abdomen, and pelvis, as well as a positron emission tomographic (PET-CT) scan, brain imaging with a CT scan, or magnetic resonance imaging (MRI), and bronchoscopy. Mediastinoscopy or endobronchial ultrasonography (EBUS) was used for mediastinal staging. Patients with stage I-II NSCLC and those with tumors other than NSCLC were excluded. Patients with central tumors requiring complex bronchovascular reconstruction were offered an open approach.

Peripheral tumors less than 7 cm were managed by VATS. Since 2015, in the case of chest wall invasion, a hybrid approach was performed as previously described [START_REF] Hireche | Combined video-assisted thoracoscopy surgery and posterior midline incision for en bloc resection of non-small-cell lung cancer invading the spine[END_REF]. All patients received induction therapy. The preoperative choice of neoadjuvant-type therapy was based on the patient's conditions, their physician's recommendations, and the availability of induction therapy protocols. A chest CT and/or PET-CT scan were used to evaluate the response. In patients with cN2 (single or bulky) and cN-3 disease, invasive restaging using mediastinoscopy or EBUS was performed after induction therapy and prior to resection. Patients with downstaging and without progression underwent radical resection. Four surgeons with at least 4 years of VATS experience were involved in the study. VATS lobectomy was performed through three ports without rib spreading. Open lobectomy was performed via a posterolateral thoracotomy through the fifth interspace with latissimus dorsi muscle section and rib spreading. Patients were divided into two groups according to the surgical approach adopted: a VATS group and an open group. Systemic lymph node dissection of all hilar (N1) and at least three mediastinal (N2) nodal stations were routinely performed. In postoperative care, all patients were managed according to a standardized postoperative protocol. Chest drains were typically removed when there was no air leakage and their volume was less than 400 cc/day. This study was approved by the Institutional Review Board, with individual patient consent being waived.

Data Extraction

Data were collected on patient demographics, smoking history, comorbidities, pulmonary function test, clinical stage, tumor location and size as measured by CT, neoadjuvant therapy modality, histological type, pathological stage, number of lymph nodes and stations removed, surgical details, chest tube duration, length of hospitalization, reoperation, and postoperative complications included any of the following: pneumonia, atrial fibrillation, prolonged air leak (more than 5 days postoperatively), adult respiratory distress syndrome (ARDS), bronchopleural fistula, pleural effusion, and heart failure. Perioperative mortality was defined as death within 30 days of the operation.

Follow-up data were collected from clinical notes and direct contact with patients and physicians. Overall survival (OS) was defined as the time period between surgery and death from any cause or the last follow-up evaluation. Recurrence-free survival (RFS) was defined as the period between surgery and recurrence or death from any cause.

Statistical Analysis

Statistical analysis was performed using SPSS IBM software version 25 (IBM, Armonk, NY, USA). The baseline characteristics and outcomes of the VATS and open groups were compared using Pearson's _2-test or Fisher's exact test when applicable for categorical variables and Student's unpaired t-test or the Wilcoxon rank-sum test when applicable for continuous variables. Patients who underwent conversions from VATS to open lobectomy were assessed using an intent-to-treat analysis and, for the purposes of our analysis, continued to be attributed to the VATS group. OS and RFS were evaluated using the Kaplan-Meier method and the log-rank test. Propensity score matching (PSM) was performed to balance the confounding factors between the two groups to minimize potential selection bias. Propensity scores were developed and defined as the probability of treatment with the VATS approach versus the open approach conditional on measured covariates. Variables included in the propensity score model were: age, sex, hypertension, coronary artery disease, diabetes, COPD, smoking history, FEV1, clinical tumor size, pathological T and N status, tumor location, histology, and induction chemoradiotherapy. Patients were then matched on the propensity score using a 1:1 nearest neighbor matching algorithm with a caliper distance of 0.01 and no replacement. Following propensity matching, the patient demographics and outcomes were assessed using Pearson's _2-test or Fisher's exact test when applicable for categorical variables and Student's unpaired t-test or the Wilcoxon rank-sum test when applicable for continuous variables. OS and RFS were evaluated using the Kaplan-Meier method and the log-rank test. Furthermore, the Cox proportional hazards model was used to identify the independent prognostic factors of RFS for these patients. All tests were two-sided, using an alpha of < 0.05 to be considered statistically significant.

Results

Unmatched Population

Patient Characteristics

The clinical and pathologic backgrounds are described in Tables 1 and2, respectively. A total of 205 patients were enrolled in the study, of whom seventy-seven had undergone VATS lobectomy and one-hundred twenty-eight underwent open lobectomy. Compared with the open group, patients in the VATS group were older (61.33 ± 8.54 vs. 57.6 ± 9.52, p = 0.031) and less likely to have diabetes (9.1 vs. 13.3%, p = 0.041) and cardiac disease (6.5 vs. 16.4%, p = 0.039). Preoperative forced expiratory volume in one second (FAV1%) was significantly lower in the VATS group (88.94 ± 16.98 vs. 90.50 ± 16.64%, p = 0.008), but diffusion capacity of the lung for carbon monoxide (DLCO%) was similar for both groups. The mean clinical tumor size was smaller in the VATS group than in the open group (31.16 ± 17.33 vs. 54.12 ± 35.77 mm, p = 0.002). There were no significant differences between the two groups in terms of histology, induction therapy, and anatomic distribution of resected lobes. However, patients in the open group had a larger pathological tumor size (42.3 ± 30 vs. 31.4 ± 18.9 mm, p = 0.002) and a higher pathological T (p < 0.001) and N (p = 0.013) status. The number of lymph node stations harvested and the total number of removed lymph nodes were similar for the two procedures. However, the VATS approach achieved a greater resection R0 than the open approach (94.8 vs. 82%, p = 0.012) (Table 2).

Perioperative Outcomes

Perioperative outcomes for the two groups are listed in Table 3. Twelve patients (15.6%) were converted from VATS to open surgery. The reasons for conversions were fibrotic tissue and tight adhesions (six patients), hilar anthracofibrotic nodes (two patients), and extensive pulmonary artery involvement requiring bypass reconstruction (four patients). None of the conversions required more extensive resection than expected, and none led to perioperative death. There were no significant differences in surgery time, intra-operative blood loss, 30day mortality, or overall morbidity, including bronchopleural fistula, prolonged air leak, atrial fibrillation, postoperative pneumonia, and respiratory failure. There was no difference in postoperative chest tube drainage duration between the two groups, but the length of hospital stay was significantly shorter (4 vs. 8 days, p < 0.0001) in the VATS group.

Oncologic Outcomes

The median follow-up period was 23.5 months for the VATS group (range 1-69 months) and 26.3 months (range 1-82 months) for the open group. The five-year OS and RFS was 55.7% (95% CI, 42.3-59.5) and 42.8% (95% CI, 31-53%) in the VATS group, and 51.8% (95% CI, 47.2-59.6) and 31% (95% CI, 29-46%) in the open group, respectively. There was no significant difference in the five-year OS (log-rank test, p = 0.563) or five-year RFS (log-rank test, p = 0.193) between the two groups (Figure 1). In addition, no significant differences in overall, local, regional or distant recurrence were found (p = 0.356, see Table 4).

Matched Population

Patient Characteristics and Perioperative Outcomes

The PSM created 64 cases of VATS lobectomy and 64 cases of open lobectomy. The baseline characteristics of the matched patients are listed in Table 1. Both groups were similar in age, sex, smoking history, FEV1, DLCO, comorbidities, clinical tumor size, pathologic stage, histology, anatomic distribution of resected lobe, and induction chemoradiotherapy. After propensity matching, the incidence of overall postoperative complications was similar (p = 0.109). VATS lobectomy was also associated with less blood loss (140 mL vs. 195 mL, p = 0.023) and shorter length of hospital stay (4 vs. 7 days, p < 0.0001). However, there remained no significant difference in surgery time, chest tube drainage duration, and 30-day mortality between the two groups. After matching, more lymph node stations were harvested in the VATS group than the open group (5.9 ± 1.7 vs. 4.8 ± 1.6, p = 0.011), but the total number of removed lymph nodes was almost similar between the two procedures (12.5 ± 5.3 vs. 11.5 ± 6.3, p = 0.219). There was no difference between the two groups in terms of resection margins R0 (93.7 vs.90.6, p = 0.443, Table 2).

Figure 2. Kaplan-Meier curve for long-term survival after propensity score matching (PSM).

(A) Overall survival; (B) Recurrence-free survival. VATS: Video-assisted thoracoscopic surgery.

Discussion

In this comparative study, we evaluated the perioperative and oncologic outcomes of VATS lobectomy for stage III NSCLC and found that the VATS approach was associated with shorter hospital stays compared with open lobectomy, whereas no significant difference was identified in perioperative outcomes, including perioperative complications and 30-day mortality. Additionally, there was no significant difference in five-year OS and RFS between the groups in both unmatched and matched populations. These results support the noninferiority of VATS over open lobectomy.

Until recently, mini-invasive surgery was considered a contraindication for advanced stage NSCLC resection, owing to concerns regarding its safety, the technical challenges of hilar dissection after induction therapy [START_REF] Hanna | Contraindications of video-assisted thoracoscopic surgical lobectomy and determinants of conversion to open[END_REF], and uncertainties related to the completeness of oncologic resection. However, with developments in thoracoscopic equipment and growing clinical experience, some thoracic surgeons have expanded the indications of VATS to advanced stages of lung cancer, thus providing evidence of its safety and efficiency [START_REF] Hennon | Safety of thoracoscopic lobectomy in locally advanced lung cancer[END_REF][START_REF] Gonzalez-Rivas | Is uniportal thoracoscopic surgery a feasible approach for advanced stages of non-small cell lung cancer[END_REF][START_REF] Jeon | Outcomes of Pulmonary Resection and Mediastinal Node Dissection by Video-Assisted Thoracoscopic Surgery Following Neoadjuvant Chemoradiation Therapy for Stage IIIA N2 Non-Small Cell Lung Cancer[END_REF][START_REF] Huang | Feasibility of complete video-assisted thoracoscopic surgery following neoadjuvant therapy for locally advanced nonsmall cell lung cancer[END_REF]. Despite the obvious contribution of these studies to the mainstreaming of VATS, they mostly include only stage II or IIIA patients without applying a propensity analysis. To date, few studies have compared short-and long-term outcomes of VATS versus open lobectomy in a well-balanced population of patients with advanced-stage NSCLC. Cao et al. [START_REF] Cao | A meta-analysis of unmatched and matched patients comparing video-assisted thoracoscopic lobectomy and conventional open lobectomy[END_REF] conducted a large multi-institutional study to construct a propensity score analysis for VATS versus open lobectomy and matched a total of 2916 patients, of whom six-hundred sixty-five had stage IIIA NSCLC. While this analysis concluded that VATS has similar long-term survival outcomes compared to open lobectomy, it failed to compare perioperative and recurrence-free survival data for the two groups. Another retrospective study by Yang et al. [START_REF] Yang | Long-term survival following open versus thoracoscopic lobectomy after preoperative chemotherapy for nonsmall cell lung cancer[END_REF] reviewed 272 patients who had undergone neoadjuvant therapy and matched a total of forty-five patients with stage III-IV NSCLC. The authors reported a 10% conversion rate and no significant differences between the two groups in 30-day mortality, overall morbidity, and 3-year OS and RFS. More recently, Chen et al. [START_REF] Chen | Propensity-matched comparison of video-assisted thoracoscopic with thoracotomy lobectomy for locally advanced non-small cell lung cancer[END_REF] reported the results of 120 pairs of wellmatched VATS and open lobectomy in advanced-stage NSCLC patients, of whom approximately one-third only had stage IIIA disease. This study showed that VATS lobectomy was associated with a significantly shorter hospital stay and better compliance for adjuvant chemotherapy but no significant decrease in blood loss, chest tube drainage duration, or postoperative complications. The present study's findings are largely aligned with those of Chen et al. [START_REF] Chen | Propensity-matched comparison of video-assisted thoracoscopic with thoracotomy lobectomy for locally advanced non-small cell lung cancer[END_REF].

The 15.6% conversion rate was relatively high in our series, which may be attributed to the neoadjuvant therapy. In a large, multi-institutional, propensity-matched study of 2887 patients who underwent either VATS or open lobectomy after neoadjuvant therapy, Yang et al. [START_REF] Yang | A Minimally Invasive Approach to Lobectomy After Induction Therapy Does Not Compromise Survival[END_REF] reported a 20% conversion rate following induction chemotherapy and a 25% rate following induction chemoradiation. In line with our findings, there was no significant difference in perioperative mortality between patients who underwent conversions or open lobectomy. These results may eliminate persistent apprehension about the safety of VATS for advancedstage NSCLC. Nevertheless, it must be acknowledged that the avoidance of intra-operative life-threatening injuries during the VATS procedure for advanced disease, especially following induction therapy, requires a careful selection of patients and proactive conversions to thoracotomy where doubt exists to probable injury. The criteria by which thoracotomy and VATS candidates might be distinguished has yet to be clarified because of the wide spectrum of patients generally involved and also because of the within-stage heterogeneity. We believe that the VATS approach should be reserved for tumors < / =7 cm and without massive hilar invasion. Chest wall involvement or N2 disease should not hamper the VATS procedure, even after induction therapy [START_REF] Hireche | Combined video-assisted thoracoscopy surgery and posterior midline incision for en bloc resection of non-small-cell lung cancer invading the spine[END_REF][START_REF] Jeon | Thoracoscopic Vs Open Surgery Following Neoadjuvant Chemoradiation for Clinical N2 Lung Cancer[END_REF].

In our study, while propensity score matching showed significantly decreased bleeding volume in the VATS group, no differences were identified between the two groups in chest tube duration, overall complications, or early mortality (i.e., within 30 days). This study's most interesting finding was the reduced length of hospital stay for the VATS group, both in matched and unmatched analyses, despite the two groups showing a similar overall rate of postoperative complications and chest tube duration. One possible explanation is the reduced trauma leading to less postoperative pain and lighter psychological burden in the VATS group allowing faster recovery than open lobectomy. While previous studies have highlighted the advantages of this [START_REF] Bendixen | Postoperative pain and quality of life after lobectomy via video-assisted thoracoscopic surgery or anterolateral thoracotomy for early stage lung cancer: A randomised controlled trial[END_REF][START_REF] Agostini | Postoperative pulmonary complications and rehabilitation requirements following lobectomy: A propensity score matched study of patients undergoing video-assisted thoracoscopic surgery versus thoracotomy[END_REF], we believe that a VATS approach should be particularly emphasized for frail patients with an advanced stage of the disease who require multimodality therapy.

The reliability and accuracy of the VATS procedure in achieving adequate lymph node dissection have long been a matter of controversy. All surgeons are aware of the complexity and the prognostic impact of achieving a thorough lymph clearance, especially in patients with advanced-stage disease or lymph node metastasis. Several studies have evaluated the role of VATS lobectomy for stage I NSCLC cN0-pN2 and have reported a similar rate of lymph node upstaging and five-year recurrence-free survival for both approaches [START_REF] Li | Comparison of clinical outcomes for patients with clinical N0 and pathologic N2 non-small cell lung cancer after thoracoscopic lobectomy and open lobectomy: A retrospective analysis of 76 patients[END_REF][START_REF] Zhong | Clinical outcomes of thoracoscopic lobectomy for patients with clinical N0 and pathologic N2 non-small cell lung cancer[END_REF][START_REF] Kim | Outcomes of unexpected pathologic N1 and N2 disease after video-assisted thoracic surgery lobectomy for clinical stage I nonsmall cell lung cancer[END_REF][START_REF] Watanabe | Is video-assisted thoracoscopic surgery a feasible approach for clinical N0 and postoperatively pathological N2 non-small cell lung cancer?[END_REF], which points to the oncologic efficacy of VATS lymphadenectomy. The present study found that the number of lymph node stations harvested by VATS was superior to thoracotomy in the matched population (p = 0.011) without impacting the total number of lymph nodes removed (p = 0.219). Consistent with other studies [START_REF] Wang | Comparative study of systematic thoracoscopic lymphadenectomy and conventional thoracotomy in resectable non-small cell lung cancer[END_REF][START_REF] Medbery | Nodal Upstaging Is More Common with Thoracotomy than with VATS During Lobectomy for Early-Stage Lung Cancer: An Analysis from the National Cancer Data Base[END_REF], these results may be attributed to the magnification of the surgical field by camera resulting in a clearer visualization of the anatomical structures and lymph node stations. The growing skills and experience accumulated in our institution, thanks to years of VATS practice, could also be a factor [START_REF] Marty-Ané | Video-assisted thoracoscopic lobectomy: An unavoidable trend? A retrospective single-institution series of 410 cases[END_REF]. The earlier works of D'Amico et al. [START_REF] D'amico | Efficacy of mediastinal lymph node dissection during lobectomy for lung cancer by thoracoscopy and thoracotomy[END_REF] and Lee et al. [START_REF] Lee | Lobectomy for Non-Small Cell Lung Cancer by Video-Assisted Thoracic Surgery: Effects of Cumulative Institutional Experience on Adequacy of Lymphadenectomy[END_REF] show that cumulative experience may positively affect the accuracy of the nodal resection.

Complete resection is a major prognostic factor and may increase the survival of advancedstage lung cancer [START_REF] Friedel | Phase II Trial of a Trimodality Regimen for Stage III Non-Small-Cell Lung Cancer Using Chemotherapy as Induction Treatment With Concurrent Hyperfractionated Chemoradiation With Carboplatin and Paclitaxel Followed by Subsequent Resection: A Single-Center Study[END_REF][START_REF] Belani | Combined chemoradiotherapy regimens of paclitaxel and carboplatin for locally advanced non-small-cell lung cancer: A randomized phase II locally advanced multimodality protocol[END_REF]. In our study, the negative margin rate of the VATS procedure was significantly higher than that of open lobectomy in the entire cohort, but not after matching. This latter fact is probably related to the smaller tumor size in the VATS group rather than to the procedure itself. Furthermore, in both unmatched and propensity score-matched cohorts, we found no significant differences in five-year OS and RFS between the VATS and open groups. More interesting still, the two groups' recurrence pattern was similar even after propensity matching. A limited number of studies have addressed the seldom-debated question of recurrence patterns following VATS lobectomy [START_REF] Shiraishi | Video-assisted thoracoscopic surgery lobectomy for c-T1N0M0 primary lung cancer: Its impact on locoregional control[END_REF][START_REF] Flores | Patterns of recurrence and incidence of second primary tumors after lobectomy by means of video-assisted thoracoscopic surgery (VATS) versus thoracotomy for lung cancer[END_REF][START_REF] Kim | Pleural recurrence and long-term survival after thoracotomy and thoracoscopic lobectomy[END_REF]. These studies involved patients with early-stage disease, and all reported a similar recurrence rate between VATS and open lobectomy and concluded that the VATS procedure does not increase the risk of procedure-related locoregional recurrence. Our results are strikingly consistent with the above studies, despite our cohorts with more advanced-stage disease. As such, our results may help to dispel lingering doubts regarding the ability of VATS to achieve oncologic outcomes equivalent to those of standard thoracotomy.

Limitations

This study had several limitations. First, this was a single-center retrospective cohort study rather than a randomized controlled trial; this could be a source of unobserved confounding selection bias between the two groups. While we used the PSM to balance the observable variables between the two groups, a number of potential unknown factors could not be adjusted, thus reducing the verification effectiveness of our study. Second, it was not easy to eliminate selection bias since only patients who showed a favorable response and good tolerance to initial treatments were, by definition, sufficiently and medically fit to then undergo surgery. Third, there is an obvious surgeon choice bias privileging an open approach to larger, complicated central tumors. We tried to address this by matching tumor size and location, and given the evident inability to carry out a large-scale randomized study, we believe this propensity analysis minimized bias as much as possible. Fourth, the criteria used for assessing the efficacy of neoadjuvant therapy were not fully consistent; for example, invasive mediastinal restaging was only performed for patients with cN2-N3 prior to induction therapy, which might undermine the oncologic outcomes. Next, given this study's small sample size, we were unable to perform further stratified analysis, which would consider neoadjuvant regimens. Lastly, the follow-up period was relatively short, which limits long-term conclusions from being drawn.

Conclusions

The current study has demonstrated that, in experienced VATS centers, VATS lobectomy for the treatment of advanced stage III NSCLC is safe, reliable, and associated with a shorter hospital stay and equivalent oncological outcomes compared with standard thoracotomy. We believe that in selected patients without huge and/or central tumors, VATS may be a sound alternative to thoracotomy, especially for patients weakened by muti-modality treatment. The patient selection process is also fundamental to avoid untimely conversions

Figure 1 .

 1 Figure 1. Kaplan-Meier curve for the long-term survival of the entire cohort. (A) Overall survival; (B) Recurrence-free survival. VATS: Video-assisted thoracoscopic surgery.

Funding:

This research received no external funding.

Institutional Review Board Statement:

This study was approved by the Institutional Review Board (approval number assigned by the IRB: IRB-MTP_2021_01_202000697).

Informed Consent Statement:

Individual patient consent was waivered.

Data Availability Statement:

Data collected for this study will be made available by the corresponding author upon reasonable request following publication.

Conflicts of Interest:

The authors declare no conflict of interest.


Julien Aimonier-Davat
email: julien.aimonier-davat@univ-nantes.fr

Minh Hoang Dang

Hala Skaf-Molli

Pascal Molli
email: pascal.molli@univ-nantes.fr

DeKaloG: Source Selection for Decentralized Knowledge Graphs

Keywords: Linked Data, Semantic Web, SPARQL query service

As a web search engine can find relevant sources for a keyword query, the web of data needs a source selection engine to find relevant endpoints for a SPARQL query. However, source selection requires getting information about the content of endpoints and currently, it remains difficult to automatically explore the content of endpoints as web robots explore the content of web servers. Thanks to the web preemption principle, we propose to automatically build RDF summaries of endpoints through SPARQL queries. We propose an approach to compute the source selection of a query Q by evaluating a rewriting of Q on summaries. As all queries terminate under the web preemption paradigm,DeKaloG can provide a web automated source selection service relying on preemptable SPARQL servers. We empirically demonstrate that various summaries can be extracted with a data transfer proportional to the size of the summary, and highlight the trade-off between the size of the summaries, the accuracy of source selection, and the execution time of source selection.

Introduction

Context and motivation: Following the Linked Data principles [START_REF] Bizer | Linked data -the story so far[END_REF] hundreds of interconnected knowledge graphs are available through public SPARQL Endpoints [START_REF] Vandenbussche | SPARQLES: monitoring public SPARQL endpoints[END_REF]. However, executing a SPARQL query at the web scale is still challenging. The main issue concerns source selection, i.e. given a SPARQL query, find the minimal number of relevant sources on the web of data to be contacted to execute the query.

Existing Federated Query Engines (FQE) [START_REF] Schwarte | Fedx: Optimization techniques for federated query processing on linked data[END_REF] perform source selection just over a local catalog of sources and not over the web. In these conditions, a federated query engine builds a federated database, not a global data space. As a web search engine can find relevant sources for a keyword query, the web of data needs a source selection engine to find relevant endpoints for a SPARQL query. As web search engines help the web to be decentralized, a source selection engine should help the web of data to be decentralized.

Related works and problem: Source selection has been extensively studied [START_REF] Schwarte | Fedx: Optimization techniques for federated query processing on linked data[END_REF][START_REF] Saleem | Hibiscus: Hypergraph-based source selection for sparql endpoint federation[END_REF]. Efficient source selection requires knowing what are inside endpoints. This can be done by asking endpoints [START_REF] Schwarte | Fedx: Optimization techniques for federated query processing on linked data[END_REF] but this cannot scale to many endpoints. Another option relies on summaries but this supposes that summaries can be obtained or computed in a fully automated and reliable way at web scale [START_REF] Hasnain | SPORTAL: profiling the content of public SPARQL endpoints[END_REF]. There is no equivalent to web robots of web search engines able to crawl the whole set of endpoints. This prevents the automation of indexing of endpoints at the web-scale. Recently, web preemption [START_REF] Minier | Sage: Web preemption for public SPARQL query services[END_REF] allowed SPARQL queries to be executed sliced time, and [START_REF] Grall | Processing sparql aggregate queries with web preemption[END_REF] presented how it can be used to compute aggregates. This opens the door to the traversing of endpoints while collecting summaries.

Approach and Contributions: In this paper, we foster on web preemption to build DeKaloG; a source selection service for SPARQL endpoints.DeKaloG is a SPARQL endpoint hosting an RDF dataset containing summaries of endpoints. Thanks to preemptable servers, summaries of endpoints are obtained by executing summary functions as SPARQL queries over endpoints. The same summary functions can also be applied to any query Q to be executable on DeKaloG. The mappings produced by the execution of a query overDeKaloG allow the computation of the source selection of the query Q over endpoints. The contributions of this paper are the following:

-We define a model for building a source selection service at web scale using only the semantic web technologies RDF and SPARQL. -We demonstrate that summaries can be obtained with a data transfer proportional to the size of the summary and not to the size of data sources. -We demonstrate how source selection can be obtained by executing SPARQL queries on summaries. -We validate empiricallyDeKaloG on a subset of LargeRDFBench. This paper is organized as follows. Section 2 summarizes related works. Section 3 details the approach ofDeKaloG for building summaries and source selection. Section 4 presents our experimental results. Finally, conclusions and future work are outlined in Section 5.

Related Works

The source selection for a SPARQL query, finds the minimal number of relevant sources on the web of data to be contacted to execute the query. In this paper, we focus on RDF data hosted in SPARQL endpoints. SPARQL 1.1 Federated queries [START_REF] Steve | SPARQL 1.1 query language[END_REF] allow executing queries over different SPARQL endpoints. The SERVICE clause advises a federated query processor to execute a portion of SPARQL query against a remote SPARQL endpoint. In this case, the source selection is performed by users when authoring SPARQL queries.

Federated query engines [START_REF] Schwarte | Fedx: Optimization techniques for federated query processing on linked data[END_REF][START_REF] Görlitz | Splendid: Sparql endpoint federation exploiting void descriptions[END_REF] suppose the existence of a catalog of SPARQL endpoints and achieves automatic source selection based on the catalog. In its simplest form, this catalog is just a set of endpoints. Automatic source selection can be seen as the rewriting of a SPARQL query into SPARQL 1.1 federated query is given a catalog of endpoints.

Different techniques for source selection are proposed [START_REF] Saleem | A fine-grained evaluation of sparql endpoint federation systems[END_REF]. We can distinguish two categories of source selection: catalog/index-free and catalog/indexassisted. In catalog/index-free source selection, the federated query engine performs source selection without using any stored data summaries, in contrast, in catalog/index-assisted source selection, the federated query engine uses data summaries that have been collected in a pre-processing step.

FedX [START_REF] Schwarte | Fedx: Optimization techniques for federated query processing on linked data[END_REF] is a representative of index-free SPARQL federated query engine. The source selection relies completely on a simple catalog of URLs of SPARQL endpoints and SPARQL ASK query. For each triple pattern of a query, FedX sends ASK queries to all endpoints in the catalog, and those that pass the SPARQL ASK test are selected. The source selection of FedX does not require storing any data summaries. However, it requires a lot of communication with the endpoints before starting the query execution. For instance, for a catalog of 100 endpoints and a query with 10 triple patterns, FedX sends 100*10=1000 ASK queries to compute source selection before starting the execution of the federated query.

Index-assisted federated query engines improve the source selection but require preprocessing. Different levels of detail of statistics of SPARQL endpoints are pre-computed and used by the federated query engine during query processing. Different techniques and formats are proposed for precomputed indexes. Some approaches require sources to compute and maintain statistics [START_REF] Görlitz | Splendid: Sparql endpoint federation exploiting void descriptions[END_REF][START_REF] Montoya | The odyssey approach for optimizing federated SPARQL queries[END_REF], other do not [START_REF] Quilitz | Querying distributed rdf data sources with sparql[END_REF][START_REF] Saleem | Hibiscus: Hypergraph-based source selection for sparql endpoint federation[END_REF].

Both Odyssey [START_REF] Montoya | The odyssey approach for optimizing federated SPARQL queries[END_REF] Splendid [START_REF] Görlitz | Splendid: Sparql endpoint federation exploiting void descriptions[END_REF] requires data sources to compute and share statistics. Odyssey uses sophisticated statistics for source selection. The statistics detail information about the data provided by remote endpoints and links between them. Splendid [START_REF] Görlitz | Splendid: Sparql endpoint federation exploiting void descriptions[END_REF] relies on VOID statistics and ASK queries to perform source selection. The statistics of VOID descriptions are aggregated in a local index. This index maps triple patterns with a bound predicate to relevant data sources. A SPARQL ASK query is used when the subject or object of the triple pattern is a bound or unbound predicate. WoDQA [START_REF] Akar | Querying the web of interlinked datasets using void descriptions[END_REF] is another federated query engine that uses VOID; more precisely, it uses linksets description to retrieve relevant datasources.

Relying on statistics precomputed by data sources is a promising solution for source selection. However, recent studies [START_REF] Hasnain | SPORTAL: profiling the content of public SPARQL endpoints[END_REF] observed that only a third of public SPARQL endpoints give a static description of their content using VOID vocabularies, and even when they are provided, it is unclear the level of details. To mitigate the burden on publishers to describe of their endpoints', Sportal [START_REF] Hasnain | SPORTAL: profiling the content of public SPARQL endpoints[END_REF] proposes to compute VOID descriptions directly from the endpoints. Sportal defines sources self-description queries that allow computing VOID. Unfortunately, as highlighted in [START_REF] Hasnain | SPORTAL: profiling the content of public SPARQL endpoints[END_REF], Sportal naturally inherits the limitations of SPARQL endpoints, many endpoints do not return results or produce incomplete results.

DARQ [START_REF] Quilitz | Querying distributed rdf data sources with sparql[END_REF] and HiBISCuS [START_REF] Saleem | Hibiscus: Hypergraph-based source selection for sparql endpoint federation[END_REF] pre-compute the indexes directly from data in the endpoints. DARQ uses an index known as service description for source selection. The description includes hand-crafted source description similar to the Vocabulary of Linked Datasets VOID [?]. DARQ source selection matches the predicates in the query against the predicates in the service description. HbiBISCuS [START_REF] Saleem | Hibiscus: Hypergraph-based source selection for sparql endpoint federation[END_REF] proposes a join-aware source selection algorithm. HbiBISCuS relies on the authority fragments of resources URIs to estimate whether combing data from multiple sources can lead to any join results. An ASK query is used for source selection for unbound predicates or common predicates.

Relying on statistics precomputed by federated query engines seems affordable. However, existing federated query engines propose different levels of information and different formats for storing the indexes. Moreover, the index is locked up by the federated query engine and cannot be shared with other federated query engines.

Different summary approaches for source selection with different accuracy and granularity have been proposed [START_REF] Umbrich | Comparing data summaries for processing live queries over linked data[END_REF][START_REF] Cebiric | Summarizing Semantic Graphs: A Survey[END_REF]. Some summaries are approximative such as QTree, while others are exact, such as schema-level indexing.

Overall, many techniques have been proposed to build catalogs for source selection, but currently, there is no reliable method to build and maintain such catalogs over the web. In DeKaloG, we foster on web preemtion [START_REF] Minier | Sage: Web preemption for public SPARQL query services[END_REF] that allows SPARQL queries to terminate. Thanks to preemptable servers, it is possible to compute aggregations as proposed in [START_REF] Grall | Processing sparql aggregate queries with web preemption[END_REF]. This allows relying on endpoints to compute summaries and transfer only summaries. Using the same summary functions, a query Q over endpoints can be rewritten as a query Q s , to be executed on DeKaloG. The provenance of the mappings of Q s contains the source selection of Q.

TheDeKaloG approach

A source selection service for the linked data has the same objective than a keyword search service in the web of documents, i.e., ensuring the findability of sources over a decentralized web of data. The general use-case is the following:

1. A user aims to execute the SPARQL query Q1 of the figure 1a over the whole set of SPARQL endpoints with no prior knowledge. 2. She loads a SPARQL 1.1 query engine in her web browser, as proposed by Communica [START_REF] Taelman | Comunica: A modular SPARQL query engine for the web[END_REF] or SaGe [START_REF] Minier | Sage: Web preemption for public SPARQL query services[END_REF] , and launchs the execution. 3. The query engine contacts a source selection service that returns the set of endpoints to contact per triple pattern. The query engine is free to refine the source selection, optimize the query, and finally rewrites the original query into the SPARQL 1.1 federated query Q2 as in Figure 1a. 4. Finally, the web browser starts the execution of Q2 and returns complete results.

Such a scenario raises many issues: how to build this source selection service? How to contact it? Is the source selection optimal? What is the execution time of the source selection? The objective of DeKaloG is to answer the above questions and makes the above use-case possible.

Preliminaries

We consider three disjoint sets I (IRIs), L (literals) and B (blank nodes) and denote the set T of RDF terms

I ∪ L ∪ B.
An RDF triple(s p o) ∈ (I ∪ B) × I × T connects subject s through predicate p to object o. An RDF graph G is a finite set of RDF triples. We denote V al(G) the set of all values (IRI, blank nodes and literals) in G. A mapping µ from V to T is a partial function µ : V → T , the domain of µ, denoted dom(µ) is the subset of V where µ is defined. Mappings µ 1 and µ 2 are compatible on the variable ?x,

written µ 1 (?x) ∼ µ 2 (?x) if µ 1 (?x)= µ 2 (?x) and ?x ∈ dom(µ 1 ) ∩ dom(µ 2 ).
RDF graphs are published on the web following the Linked data principles [START_REF] Bizer | Linked data -the story so far[END_REF]. These RDF graphs could be accessible through public SPARQL endpoints. A SPARQL endpoint is defined as a couple (E i , G i ), where E i is the U RL of the endpoint and G i is the RDF graph accessible by E i . A SPARQL query has the form Head ← Body where Head is an expression indicating how to construct the answer of the body and the body is a complex RDF graph pattern expression. Assume the existence of an infinite set V of variables, disjoint with previous sets. A SPARQL graph pattern expression P is defined recursively as follows [START_REF] Kaminski | Query nesting, assignment, and aggregation in sparql 1.1[END_REF][START_REF] Pérez | Semantics and complexity of SPARQL[END_REF][START_REF] Schmidt | Foundations of SPARQL query optimization[END_REF]. The evaluation of a graph pattern P over a SPARQL endpoint E i denoted by P Gi returns a set of mappings, called result set. Each element of the result of a query is a set of variable bindings.

1. A tuple from (I ∪ L ∪ V ) × (I ∪ V ) × (I ∪ L ∪ V ) is
We define a federation of SPARQL endpoint F (E, G) as a set of couple of

F (E, G) = {(E 1 , G 1 ), . . . , (E n , G n )} where E = {E 1 , . . . , E n } and G = n i=1 G i , respectively.
For the sake of simplicity, we consider the RDF graphs of endpoints do not have blank nodes. We consider federated SPARQL queries as queries defined over a federation of SPARQL endpoints. Given a SPARQL query Q, a data source E i ∈ E is said contribute to the query Q if at least one of the variable bindings in the result set of Q can be found in E i .

DeKaloG definitions and problem description

In this paper, we focus on a federation of SPARQL endpoints F (E, G). Discovering and maintaining E is out of the scope of the paper. For instance, E could be discovered and maintained by crawling the web as proposed by Google Datasearch [START_REF] Brickley | Google dataset search: Building a search engine for datasets in an open web ecosystem[END_REF]. We define source selection as :

Definition 1 (Source Selection). Given a query Q, a source selection of Q over F is the set of sources E tpi ⊆ E per triple pattern of Q that potentially contributes to the result set of Q.
Ideally, a source selection provides a minimal set of endpoints to contact to produce the complete results of the query. We formalize the problem of source selection service as follows:

Definition 2 (Source Selection Service Problem (SSS-P)). A source selection service S is a SPARQL service hosting an RDF graph SC extracted from G. Given a query Q, S computes the source selection of Q over G by evaluating a rewriting of Q over SC.

Building a source selection service raises critical challenges: SC is constructed thanks to SPARQL queries, following the idea proposed in Sportal [START_REF] Hasnain | SPORTAL: profiling the content of public SPARQL endpoints[END_REF]. Computing summaries based on SPARQL queries overcomes the poor adoption of service description conventions and the impracticability of dumps practices for web automation.

SC is a summary of G . We expect the data transfer from E to SC to be proportional to the size of the summary of graphs and not to the original size of graphs, e.g., extracting a summary of 1000 triples from DBpedia should transfer, ideally 1000 triples.

Source selection is minimal, sound and complete. Source selection returns, as possible, the minimal sources per triple pattern. According to the "accuracy" of the summary, source selection could be overestimated, i.e., contain false positives. However, it should always produce sound and complete answers.

Source selection time complexity. The complexity of the source selection should be proportional to the number of sources selected per triple pattern.

BuildingDeKaloG Summaries

We rely on structural graph summarization to define SC. Structural graph summarization is essentially a reduced version of the original RDF graphs where nodes have been merged according to some notion of structural similarity [START_REF] Cebiric | Summarizing Semantic Graphs: A Survey[END_REF]. Consequently, we consider summaries defined as an RDF graph homomorphism. Definition 4 (DeKaloG summary). Let F (E, G) be a federation of SPARQL endpoints, the summary of E using ψ, SC(ψ) is a set of quads such that:

Definition 3 (RDF graph homomorphism). Let G, G ′ be two RDF graphs. A function ψ : V al(G) → V al(G ′ ) is a homomorphism from G to G' iff for every RDF triple (s, p, o) ∈ G there is an RDF triple (ψ(s), ψ(p), ψ(o)) ∈ G ′ .
SC(ψ, E) = {(ψ(s), ψ(p), ψ(o), g)|(s, p, o) ∈ G i and E i ∈ E}
For a federation F (E, G), we can define summaries with different "accuracy" using different ψ functions. Figure 2 describes summaries of three dummy graphs dba,nyt, and wda.

Identity summary: For this summary, ψ id is defined as the identity function. SC(ψ id , E) is composed of all graphs in the federation. This is the most accurate summary, but unrealistic.

1-triple summary For this summary, ψ 1 is defined as:

(ψ 1 (s), ψ 1 (p), ψ 1 (o)) = (: s1, : p1, : o1)
where : s1, : p1 and : o1 are Literals. A 1-triple summary SC(ψ 1 , E) contains a single triple per endpoint. SC(ψ 1 , E) represents the set of all endpoints in the federation, i.e., the catalog of the endpoints. The size of this summary is proportional to the number of endpoints. predicate-aware summary: ψ p is defined as: 1a) and ψ h summary (Figure 2) This function projects all subjects and objects to two constants : s1 and "lit". SC(ψ p , E) is the set of all predicates of E. In the worst case, if all sources have all predicates, the size of this summary is proportional to the number of predicates (#predicates) multiplied by the number of sources (#sources). authorities-aware summary: psi h function is defined as:

(ψ p (s), ψ p (p), ψ p (o)) = (: s1, p, : s1) if s, o ∈ I) (: s1, p, "lit") if s ∈ I, o ∈ L Table 1: Source selection for query Q 1 (Figure
Q
(ψ h (s), ψ h (p), ψ h (o)) = (auth(s), p, auth(o)) if s, o ∈ I (auth(s), p, "lit") if s ∈ I, o ∈ L
Auth(s) is a function that returns the domain of a URI. ψ h builds a summary inspired by the hibiscus summaries [START_REF] Saleem | Hibiscus: Hypergraph-based source selection for sparql endpoint federation[END_REF]. In the worst case, a predicate could have all authorities (#auth) as subjects and as objects If all sources have a such predicate, then the size of the summary is (#predicates * #auth 2 ) * #sources. suffix-authority-aware summary: ψ s function is defined as:

(ψ s (s), ψ s (p), ψ s (o)) = (cc(auth(s), lt(s, 2)), p, cc(auth(o), lt(o, 2))) if s, o ∈ I (cc(auth(s), lt(s, 2)), p, "lit")) if s ∈ I, o ∈ L
The function lt(string, 2) returns the last two characters of the string, and cc() is the string concatenation function. This summary is an extension of ψ h with suffixes of URIs. The summary is more accurate than the psi h summary. However, its size grows quickly. Considering only 26 different letters, the number of nodes in the summary is now equal to #auth * 26 2 . Therefore, the summary size is bounded by (#predicates * (#auth * 26 2 ) 2 ) * #sources. The summary is more accurate but it is much bigger. The different summaries behave as Russian dolls, for instance:

SC(ψ h , SC(ψ s , E)) = SC(ψ h , E)
The extraction of the most accurate summary allows for building less accurate ones.

SC(ψ 1 , E) ← SC(ψ p , E) ← SC(ψ h , E) ← SC(ψ s , E) ← SC(ψ id , E)

Source selection onDeKaloG summaries

Source selection for a query Q as defined in section 3.2 computes a set of endpoints to contact per a triple pattern of Q.

Triple-pattern based source selection (TPSS) As a triple pattern of Q is defined on G, it cannot be executed directly on the summary to find the endpoints to contact. To make a triple pattern executable of a summary, we need to extend ψ function to handle triple patterns, i.e. summarization functions are defined for RDF triples; they cannot be applied to variables of triple patterns.

In the following, we extend the definition of function ψ h to handle triple patterns. As a triple pattern can have one the following forms [START_REF] Umbrich | Comparing data summaries for processing live queries over linked data[END_REF], where ? denotes variables: (?s 1 ?p 1 ?o 1 ), (s 1 ?p 1 ?o 1 ), (?s 1 p 1 ?o 1 ), (?s 1 ?p 1 o 1 ), (s 1 p 1 ?o 1 ), (s 1 ?p 1 o 1 ),(?s 1 p 1 o 1 ), (s 1 p 1 o 1 ). We distinguish the following cases:

ψ h (s), ψ h (p), ψ h (o)) =                s, p, o if s, p, o ∈ V auth(s), p, o if s ∈ I, o ∈ V auth(s), p, auth(o) if s, o ∈ I auth(s), p, "lit" if s ∈ I, o ∈ L s, p, auth(o) if s ∈ V, o ∈ I s, p, "lit" if s ∈ V, o ∈ L
We define the source selection query for a triple pattern as:

Definition 5 (TPSS query). The source selection query for a triple pattern (s, p, o) on a summary SC(ψ, E) is defined by the query:

SS(ψ, (s, p, o)) = π g (ψ(s), ψ(p), ψ(o), g) and g ∈ V
By abusing of notations, we consider π x (q) as the projection operator on variable x of a quad q. SS(ψ, (s, p, o)) SC(ψ,E) returns the source selection of (s, p, o). Table 1 presents the the results of TPSS of query Q1 defined in (Figure 1a). The source selection has different time complexity according to the form of the triple pattern, and the summary.

For (?s ?p ?o), SS(ψ 1 (?s, ?p, ?o)) returns results in a time complexity proportional to the number of selected sources, i. ((s,p,o)|), but will depend of the selectivity of the constants.

The TPSS overestimates the number of sources to contact because it is not aware of the join variables, i.e.variables shared among triple patterns of the query. For instance, for the triple #tp2 (?s?p?o) of query Q 1 (cf. figure 1a), TPSS selects all the sources present in the summary (Table 1) even if a subset of sources really contributes to the results of the query.

BGP-Based source selection query (BGPSS)

For the sake of simplicity, we focus on queries with conjunctive graph patterns (BGP queries). However, any SPARQL query with union graph patterns, optional graph patterns, etc, can be rewritten following the same approach. A BGP query is a set of triple patterns.

Definition 6 (BGPSS Query).

Let Q a BGP query a set of triple patterns (s i , p i , o i ), the source selection query is defined as:

SS(ψ, Q) = π gi 1 (si,pi,oi)∈Q (ψ(s i ), ψ(p i ), ψ(o i ), g i ), g i ∈ V
The evaluation of SS(ψ, Q) over SC(ψ, E) returns for all triple patterns tp i of Q their respective selected sources in g i . As the homomorphic query of the original query is executed on the summary, the source selection is optimal for that summary.

Following this definition, the query Q1 with ψ h can be rewritten as:

SELECT DISTINCT ?g1, ?g2 WHERE { graph ?g1 {?s <sameas> <http://dba.org>} graph ?g2 {?s ?p ?o} }

Then the execution of this query on the ψ h summary of the figure 2, return the result of described in Table 1. As we can see, the BGP-aware source selection is more accurate than the previous TP-based source selection. Therefore, executing SS(ψ id , Q) on SC(ψ id , E) returns the exact source selection for Q. executing SS(ψ 1 , Q) on SC(ψ 1 , E) returns all sources for all triple patterns of Q.

The BGPSS requires executing all triple patterns of the query on the same summary because mappings of join variables are shared among the triple patterns. This is not the case of TPSS where different summaries can be used. In the worst case, each triple pattern scans the whole summary, so complexity is the number of triple patterns multiplied by the size of the summary. For example, executing a source selection on ψ h is now in the worst case in (#predicates * #auth 2 ) * #sources multiplied by the number of triple patterns in the query. However, in an average case, for a bounded authority and a bounded predicate in a triple pattern, the worst time complexity is #auth * #source which is much more tractable. Concretely, there is a trade-off between the accuracy of the source selection and the time for source selection. For example, for the BGP query Q1 of figure 1a, executing the source selection query for Q1 on the ψ s summary returns a better source selection than executing the source selection for Q1 on the ψ h . However, the source selection query for Q 1 on the ψ h , generally, returns the source selection much faster than with ψ s .

Implementing summaries with web preemption

We can implement ψ functions as a SPARQL 1.1 query. For example, computing authorities-aware summary ψ h can be done by executing the following SPARQL query over the SPARQL endpoints of the federation.

CONSTRUCT { ? p s ? p ? po } where { ? s ? p ? o FILTER i s i r i ( ? s ) BIND ( URI (REPLACE(STR( ? s ) , " ^( h t t p s ? : / / ? . * ? ) / . * " , " $1 " ) ) AS ? p s ) BIND ( i f ( i s i r i ( ? o ) , URI (REPLACE(STR( ? o ) , " ^( h t t p s ? : / / ? . * ? ) / . * " , " $1 " ) ) , " l i t " ) a s ? po ) } However, executing this query over an endpoint is challenging:

-A public SPARQL endpoint will interrupt the query after a time quota as reported in [START_REF] Hasnain | SPORTAL: profiling the content of public SPARQL endpoints[END_REF]. -A TPF server [START_REF] Verborgh | Triple pattern fragments: A low-cost knowledge graph interface for the web[END_REF] or SaGe [START_REF] Minier | Sage: Web preemption for public SPARQL query services[END_REF] server return complete results for queries, but FILTERS, BIND and CONSTRUCT operators are executed on client side. Consequently, the query execution first transfers all mappings for the (?s, ?p, ?o) triple pattern from the server to the client, then the summary is computed on client-side. This clearly require to transfer all the RDF graphs of the federation to compute a summary.

An affordable solution is to follow the approach of SaGe-agg [START_REF] Grall | Processing sparql aggregate queries with web preemption[END_REF] to implement this query without interruption and low data transfer. In SaGe-agg, the SaGeserver can compute partial aggregates per quantum, thanks to the decomposability property of aggregate functions.

We extended the SaGe server to handle BIND operation to express summary functions in SPARQL. We also extended the SaGe server to handle CON-STRUCT per quantum, i.e., a graph is constructed during one quantum and transferred to the client at the end of the quantum. As BIND statements summarize subjects and objects, most of the element of the graph is likely to be duplicated. Consequently, the compression of the graph is mostly done on the server side, and data transfer is dramatically reduced, as demonstrated in the experimental study.

The transfer is optimal if all the summary queries can be processed in one quantum. If not, some triples can be transferred from the server to the client several times. This is an overhead intrinsic to the web preemption approach.

This overhead mainly depends on the summary function and the order of scanned triples. To illustrate, suppose we are computing the ψ h summary. It is important to scan triples following a PSO index or POS index. As triples are ordered by the predicate, followed by the subject or object, it is very likely that all duplicates are eliminated during the same quantum. The the following table illustrates this process for the POS index: The result of the CONSTRUCT is only one triple:birthyear lit http://dbp. The ψ s summary is less likely to remove all duplicates in a quantum. SaGe should provide low overhead for authorities-aware summaries.

We want to empirically answer the following questions: (i) What is the data transfer and execution time of computing different kinds of summaries on online SPARQL servers? (ii) How good is the source selection for TP-based and BGPbased source selection? (iii) What is the execution time of the source selection for a source selection service?

We extended the SaGe server to support the execution of summaries functions. The SaGe server now supports CONSTRUCT, REDUCED keyword, BIND operations and custom functions for efficient computation. All extensions and experimental results are available at https://github.com/momo54/semcat.

Dataset and Queries: We consider a workload (SP ) of 14 SPARQL queries extracted from the LargeRDF Benchmark [START_REF] Saleem | Largerdfbench: a billion triples benchmark for sparql endpoint federation[END_REF]. These queries run on the 9 datasets presented in figure 3a (orig).

Summaries and source selection

We compare the performances of the TPSS and BGPSS services on ψ p , ψ h and ψ s summaries, named respectively void, hib and suf . In the experimentations, the TPSS engine uses the same summary for all triples of the query.

Server configuration: We run experimentations on personal computer 4 GHz Intel Core i5 four CPU, 8 Go 2133 MHz LPDDR3.

Evaluation Metrics: (i) Summary Data transfer : is the number of triple transferred from a SPARQL server toDeKaloG to compute a summary. (ii) Summary Execution time: is the time required by DeKaloG to compute the summary per a SPARQL server. (iii) Summary size: is the number of triples in the summary per graph. (iv) SSQ: is the sum of sources selected per query. For example, if a query has two triple patterns tp1 and tp2 and the source selection for tp1 is s1 and the source selection for tp2 is s1, s2, then SSQ is 3. (v) SSt: is the source selection time, i.e., time to perform the source selection of a query.

Building ψ summaries

For this experiment, we set up a SaGe server configured with a quantum of 60s. We ingested the nine datasets and executed the different summary functions as SPARQL queries on the server. We measured the data transfer and the execution time as shown in Figure 3.

Figure 3a presents the number of triples in the summary (unique), the number of triples retrieved to compute this summary (transferred), and the original size of the graph (orig). For most endpoints, the data transfer is optimal; in one quantum, the SaGe server can scan all the graphs and return the summary. For large graphs, several quanta are necessary and duplicates appear for DBpedia and Geonames. However, the overheads remain marginal.

Figure 3b presents the time required to compute the summary. We observe that the time remains slightly the same, whatever the summary. This is normal because computing the summary requires scanning the complete graph and the scan speed remains the same whatever the summary function. Figures 4a, 4b present, respectively, the selected sources by BGPSS engine and TPSS engines. As expected, for both source selection engines, a more accurate summary improves the accuracy of the source selection, i.e. produces less SSQ. For instance, the suf summary (ψ s ), returns the best results. The void summary (ψ p ) makes no difference between engines. For hib and suf , the BGPSS engine improves the source selection compared to TPSS engine. For instance, for S2 using the hib summary SSQ is 7 with TPSS and pruned to 3 with BGPSS, for S4 is pruned from 20 to 5. In total, the SSQ for all queries is improved with BGPSS engine except for S14 and S11. The SSQ of S8 remains unchanged.

The suf summary with BGPSS, as hib with BGPSS, improves most of the queries SSQ. Compared to hib with BGPSS only the SSQ of 4 queries S1, S11, S13 and S6 is improved. Overall, the suf summary with the BGPSS dominates the source selection accuracy.

Execution time Source selection

Figues 4a and 4b present, respectively, the execution time of BGPSS engine and TPSS engine. We run the experiment with RDFLib, Virtuoso (without quota), and SaGe. For space limitations, only the execution time obtained with Virtuoso is presented. All the results are available at *anonymized*.

As in previous experimentations, TPSS uses the same summary for all triple patterns of the query, i.e., do not choose the summary according to the characteristic of the triple pattern.

For both source selection engines, the suf summary is more expensive than the others. This is normal as the size of the suf summary impacts significantly

Conclusions

In this paper, we highlighted the need for a source selection service to make endpoints findable. Such a service requires web automation for its creation and maintenance. Thanks to web preemption, we demonstrated how to query endpoints and collect efficient summaries based on SPARQL queries. We define different summary functions. We presented how a query Q on endpoints can be rewritten as a query Q ′ on summaries that returns the source selection of Q on endpoints. If all endpoints support web preemption, then any federated query terminates and delivers complete results.

We empirically demonstrated the different trade-offs between the accuracy of the source selection, the execution time of the source selection, and the size of the summary. An interesting conjecture could be that one dimension has to be sacrificed to preserve the others.

This approach raises several perspectives. First, it is an open garden, i.e., it exists other summary functions and certainly many different ways to combine summaries. We can imagine a BGP-based source selection combining different summaries. Bindings obtained on one summary can be transformed to be injected into other summaries. Second, in this paper, we focused on source selection; the next step is to extend the summary functions to collect statistics for join ordering.

Fig. 1 :

 1 Fig. 1: SPARQL queries Q 1 and Q 2

Fig. 2 :

 2 Fig. 2: Three RDF Graphs hosted by dba,nyt and wda and their 4 summaries

  birthyear 1967 http://dbp/Bob birthyear 1967 http://dbp/Alice ψ h --→ birthyear lit http://dbp birthyear lit http://dbp

  (a) Summary data transfer and summary size (b) Summary execution time

Fig. 3 :

 3 Fig. 3: Results for a federation of nine SPARQL endpoints using void, hib and suf summaries. orig is the size of the original RDF graph

Fig. 4 :

 4 Fig. 4: Source selection and execution times

  It possible to choose among different summaries according to the form of triple patterns. Summaries that handle constants such ψ h or ψ s cannot answer in O(|SS

e. O(|SS(?s, ?p, ?o)|). For (?s p ?o), SS(ψ p (?s, p, ?o)) returns results in O(|SS((?s, p, ?o)|). For (s p ?o), SS(ψ h (s, p, ?o)) returns the results in the worst case in O(#auth * #sources). For (s p ?o), SS(ψ s (s, p, ?o)) maybe return a more accurate selection but, in the worst case, in O((#auth * 26 2 ) * #sources).

Acknowledgments This work is supported by the ANR DeKaloG (Decentralized Knowledge Graphs) project, ANR-19-CE23-0014 -AAPG2019-program CE23 and cominLabs project MikroloG (The Microdata Knowledge Graph).