Marianne F S Vogel
email: marianne.vogel@uqat.ca

Hugo Asselin
email: hugo.asselin@uqat.ca

Sébastien Joannin
email: sebastien.joannin@umontpellier.fr

Yves Bergeron
email: yves.bergeron@uqat.ca

Sabrina Leclercq
email: sabrina.leclercq@usherbrooke.ca

Cécile Latapy
email: cecile.latapy@etu.umontpellier.fr

Adam A Ali

Early afforestation on islands of proglacial Lake Ojibway as evidence of post-glacial migration outposts

Keywords: Afforestation, Island biogeography, Plant macro-remains, Postglacial migration, Proglacial Lake Ojibway, XRF analysis

At the end of the last glacial period in the northern hemisphere, meltwater from receding ice sheets accumulated into large proglacial lakes, potentially limiting postglacial afforestation. We explored whether former islands of proglacial Lake Ojibway (Canada) (hilltops in the current landscape) could have acted as migration outposts and thus accelerated the postglacial migration. We extracted sediments from two small lakes located on "paleo-islands" and used XRF to detect changes in soil erosion and vegetation biomass. We also used plant macro-remains and wood charcoal to determine if (and which) tree species colonized the sites and to detect local fire events. Organic sediment accumulation started around 9657 and 9,947 cal. yr BP at Lakes Perché and Despériers, respectively, before the level of Lake Ojibway started to decrease and liberate parts of the studied landscape ca 9,400 cal. yr BP. Lithogenic elements (Ti, K, Sr, Fe, Zr and Rb) decreased between the beginning of organic sediment accumulation and 8,800-8,700 cal. yr BP, indicating reduced soil erosion, possibly due to soil stabilization by vegetation. Then, the S/Ti ratio, a proxy of organic matter accumulation increased around 8,800 and 8,400 cal. yr BP. The earliest tree macro-remains (needles of Larix laricina, Picea spp., and Pinus spp., and seeds of Betula spp.) were found between 9,850 and 9,300 cal. yr BP. Local fires were detected around 9,820, 9,360 and 8,362 cal. yr BP. Early afforestation occurred on the islands of Lake Ojibway, 200 and 450 years before its level started to decrease , confirming the hypothesis that some islands acted as migratory outposts accelerating postglacial migration.

A Introduction

At the end of the Pleistocene, large portions of the northern hemisphere were covered by ice sheets which were progressively retreating to the north. From the beginning of the Holocene (11,700 years ago) to the mid-Holocene (ca 6,000 years ago), the Laurentide Ice Sheet slowly melted over north-eastern Canada [START_REF] Dalton | An updated radiocarbon-based ice margin chronology for the last deglaciation of the North American Ice Sheet Complex[END_REF]. As landscapes became free of ice, they were available for colonization by vegetation from forests located at the southern margins of ice sheets.

Meltwater from receding ice sheets sometimes accumulated at their front and created so-called proglacial lakes [START_REF] Carrivick | Proglacial lakes: character, behaviour and geological importance[END_REF] that prevented vegetation colonization of deglaciated areas for centuries. Several Holocene proglacial lakes have been identified in Canada, including Lake Ojibway, one of the largest proglacial lakes (115,000 km 3 ), covering what is currently western Quebec and eastern Ontario [START_REF] Godbout | A detailed lake-level reconstruction shows evidence for two abrupt lake drawdowns in the late-stage history of the eastern Lake Agassiz-Ojibway basin[END_REF][START_REF] Veillette | Evolution and paleohydrology of glacial lakes Barlow and Ojibway[END_REF][START_REF] Vincent | L'évolution et l'extension des lacs glaciaires Barlow et Ojibway en territoire québécois[END_REF]. Lake Ojibway was formed because the crustal depression beneath the ice caused the adjacent terrain to slope down towards the north, which prevented meltwater from leaving the ice front (Fig. 1). The ice sheet produced meltwater, which was prevented from flowing north by the ice itself acting as a wall. Water accumulated at the ice sheet front and created proglacial Lake Ojibway around 10,570 cal. yr BP (calibrated years before present). Then, as the ice sheet continued its northward retreat, the southern end of James Bay was eventually freed from ice and Lake Ojibway gradually drained into it. Different phases of lake level have been recorded before the final drainage of Lake Ojibway. The level of proglacial Lake Ojibway reached its maximum during the Angliers phase ca 9,400 cal. yr BP [START_REF] Brouard | A framework for the timing of the final meltwater outbursts from glacial Lake Agassiz-Ojibway[END_REF][START_REF] Godbout | A detailed lake-level reconstruction shows evidence for two abrupt lake drawdowns in the late-stage history of the eastern Lake Agassiz-Ojibway basin[END_REF]. Then, lake level gradually decreased and was ca 65 m lower around 9,000 cal. yr BP when the Kinojévis phase started. As the level of Lake Ojibway decreased, more and more islands emerged. The level of Lake Ojibway had lowered ca 35 m more, at the beginning of the La Sarre phase ca 8,300 cal. yr BP [START_REF] Brouard | A framework for the timing of the final meltwater outbursts from glacial Lake Agassiz-Ojibway[END_REF]. Several dates were proposed for the final drainage of proglacial Lake Ojibway, with most studies placing it at ca 8,200 cal. yr BP [START_REF] Brouard | A framework for the timing of the final meltwater outbursts from glacial Lake Agassiz-Ojibway[END_REF][START_REF] Daubois | The drainage of Lake Ojibway in glaciolacustrine sediments of northern Ontario and Quebec, Canada: The drainage of Lake Ojibway, Canada[END_REF][START_REF] Godbout | A detailed lake-level reconstruction shows evidence for two abrupt lake drawdowns in the late-stage history of the eastern Lake Agassiz-Ojibway basin[END_REF][START_REF] Roy | Late-stage phases of glacial Lake Ojibway in the central Abitibi region, eastern Canada[END_REF].

The observed postglacial migration of several tree species in western Quebec was faster than the estimated migration time based on the mean distance dispersal capacities observed nowadays, a phenomenon called Reid's paradox of rapid plant migration [START_REF] Clark | Why trees migrate so fast: confronting theory with dispersal biology and the paleorecord[END_REF]. Indeed, several vegetation reconstructions based on palynological and macro-remain studies in Western Quebec indicated that afforestation closely followed the drainage of Lake Ojibway [START_REF] Ali | Changes in fire regime explain the Holocene rise and fall of Abies balsamea in the coniferous forests of western Québec, Canada[END_REF][START_REF] Carcaillet | Change of fire frequency in the eastern Canadian boreal forests during the Holocene: does vegetation composition or climate trigger the fire regime[END_REF][START_REF] Richard | Histoire postglaciaire de la végétation au sud du lac Abitibi, Ontario et Québec[END_REF]. Such rapid colonization can either be explained (1) by the presence of glacial refugia [START_REF] Brochmann | Glacial survival or tabula rasa? The history of North Atlantic biota revisited[END_REF][START_REF] Varga | Extra-Mediterranean refugia,post-glacial vegetation history and area dynamics in Eastern Central Europe[END_REF]; or (2) by long distance dispersal events [START_REF] Bullock | A synthesis of empirical plant dispersal kernels[END_REF][START_REF] Cain | Long-distance seed dispersal in plant populations[END_REF]. As there is no evidence that glacial refugia were ever present in western Quebec, the second hypothesis can be retained but remains to be supported by empirical data.

During its >2,300 years presence over western Quebec and north-eastern Ontario, Lake Ojibway should have acted as a barrier slowing down postglacial forest migration, unless its islands were colonized early and acted as outposts facilitating colonization of lowlands following the drainage of Lake Ojibway into James Bay. For the islands of Lake Ojibway to be colonized, long-distance dispersal events from the vegetation front south of the lake were necessary. According to [START_REF] Schurr | Long-distance seed dispersal[END_REF], long-distance seed dispersal via abiotic or biotic action happens when ≤1% of seeds travel >1km. Although such events are rare, they can strongly affect plant biogeography and population dynamics [START_REF] Cain | Long-distance seed dispersal in plant populations[END_REF].

If the "paleo-islands" on Lake Ojibway had remained uncolonized, while Lake Ojibway was present, ie., if no long-distance dispersal events succeeded, colonization following the drainage of the proglacial lake would have followed a south-north trajectory limited by average seed dispersal distance, as most tree species in north-eastern Canada are wind-dispersed [START_REF] Burns | Silvics of North America[END_REF]) (Fig. 1 -first hypothesis). This scenario does not explain how sites located several hundreds of kilometers north of the migration front could have been afforested almost immediately after the drainage of Lake Ojibway [START_REF] Ali | Long-term fire frequency variability in the eastern Canadian boreal forest: the influences of climate vs. local factors[END_REF]. An alternative hypothesis would be that some islands on Lake Ojibway were colonized through long-distance seed dispersal before the final drainage of the proglacial lake into James Bay, which would explain how sites far from the postglacial migration front could have been afforested quickly, then our study would enlighten an early colonization of paleo-islands, ie., while the Lake Ojibway still covered Quebec (Fig. 1 -second hypothesis, Fig. 2).

Figure 1: Schematic representation of proglacial Lake Ojibway (LO) creation and hypotheses concerning the colonization of paleo-islands before the drainage of Lake Ojibway and implications on postglacial migration thereafter.

To test the hypothesis of early afforestation of paleo-islands through long-distance dispersal events (second hypothesis), we sampled sediments from two small lakes located on paleo-islands (current hilltops above the maximum shoreline level of Lake Ojibway) [START_REF] Godbout | Cosmogenic 10 Be dating of raised shorelines constrains the timing of lake levels in the eastern Lake Agassiz-Ojibway basin[END_REF], and we radiocarbon-dated bulk sediment and macro-remains to build age-depth models. We used multiple proxies to infer afforestation: X-ray fluorescence (XRF) geochemical analysis to detect variations in soil erosion as a proxy for plant cover; plant macro-remain analysis to attest local vegetation presence; and charcoal analysis to reconstruct local wildfire activity as an indicator of biomass burning. Only a few studies have been conducted so far on the afforestation of sites located near the maximum shoreline levels of proglacial lakes in eastern Canada [START_REF] Richard | Histoire postglaciaire de la végétation au sud du lac Abitibi, Ontario et Québec[END_REF][START_REF] Richard | La végétation postglaciaire du Témiscamingue, Québec, durant l'épisode glaciolacustre Barlow[END_REF] and they did not allow to clearly demonstrate the potential role of paleo-islands on postglacial forest migration. Our study documents environmental conditions above the maximum limit of Lake Ojibway, therefore allowing us to test the hypothesis of early colonization of paleo-islands on a proglacial lake.

A Material and methods

B Study area

The study area is located in the Abitibi-Témiscamingue region of western Quebec and is part of the clay belt of Quebec and Ontario created by deposits of proglacial Lake Ojibway (Fig. 2a). The Abijévis hills are located within the Aiguebelle National Park, whereas the Kekeko hills are 50 km southward of the park.

Both hill formations originated from marine volcanism and are composed of mafic rocks, ie., mostly noncarbonate rocks such as gabbro and andesite. Surface deposits are glacial till (MERN -Système d'information géominière, 2022; [START_REF] Nutz | Deglacial to paraglacial history of the Lake Saint-Jean lowlands: A geomorphological perspective[END_REF]. The hills were aerially exposed following ice sheet retreat ca 10,650 cal. yr BP [START_REF] Dyke | An outline of North American deglaciation with emphasis on central and northern Canada[END_REF][START_REF] Veillette | Evolution and paleohydrology of glacial lakes Barlow and Ojibway[END_REF]. Although they were rounded by ice sheet action, the hills remain among the highest elevations in Abitibi-Témiscamingue, with maximum elevations of 570 m and 488 m above sea level (a.s.l.) for Abijévis and Kekeko, respectively. They are surrounded by a generally flat topography (between 180 and 350 m a.s.l.).

Ancient shorelines of proglacial Lake Ojibway are still visible on hillsides in the current landscape and allowed reconstructions of lake level history [START_REF] Godbout | Cosmogenic 10 Be dating of raised shorelines constrains the timing of lake levels in the eastern Lake Agassiz-Ojibway basin[END_REF][START_REF] Godbout | A detailed lake-level reconstruction shows evidence for two abrupt lake drawdowns in the late-stage history of the eastern Lake Agassiz-Ojibway basin[END_REF] and the identification of several paleo-islands that became hills following the final drainage of Lake Ojibway ca 8,200 cal. yr BP [START_REF] Richard | Histoire postglaciaire de la végétation au sud du lac Abitibi, Ontario et Québec[END_REF][START_REF] Richard | Histoire postglaciaire de la végétation de la forêt boréale du Québec et du Labrador[END_REF][START_REF] Veillette | Evolution and paleohydrology of glacial lakes Barlow and Ojibway[END_REF]. Lake Ojibway's highest shorelines (ca 9,400 cal. yr BP) are estimated to be slightly below 400 m a.s.l. and 380 m a.s.l. for the Abijévis and Kekeko hills, respectively [START_REF] Godbout | Cosmogenic 10 Be dating of raised shorelines constrains the timing of lake levels in the eastern Lake Agassiz-Ojibway basin[END_REF]. Areas above these elevations are therefore considered to have always been paleo-islands (Fig. 2b).

B Site selection and lake sediment sampling

We selected two small headwater lakes surrounded by steep shores to maximize sediment collection: Lake Perché (formerly known as Lake Edrien) in the Aiguebelle National Park (Abijévis hills) and Lake Despériers on the Kekeko hills (Table I; Fig. 2). Lake Perché is located at 380 m a.s.l. While this is slightly below the highest shoreline of Lake Ojibway in the area (400 m a.s.l.), Lake Perché was formed before the complete drainage of proglacial Lake Ojibway and could have recorded part of the paleo-island's history. Lake Despériers is located at 407 m a.s.l., ie. almost 30 m above the highest shoreline of Lake Ojibway in the area (Table I) We extracted lake sediment cores from the center of each lake with a 60-mm diameter Livingstone corer. We selected headwater lakes to avoid sediment disturbance by incoming water flows. We collected complete gyttja sequences (organic sediments), from the water-sediment interface to the contact with the underlying mineral glacial sediments. We stored sediments in plastic tubes and kept them in cold conditions (4°C) until further analyses.

B Age-depth models

We built age-depth models with the Bayesian statistics-based 'Bacon' v2.2 package in R [START_REF] Blaauw | Flexible paleoclimate age-depth models using an autoregressive gamma process[END_REF] for the bottom part of the sediment sequences at each lake, as we wanted to focus on the period corresponding to the beginning of organic sediment accumulation on the paleo-islands.

Despite our efforts, we found few macro-remains and we only managed to obtain 14 C dates for two levels at lake Perché and one level at lake Despériers (Table II). Hence, we also dated bulk sediment from three levels from each core to complete the age-depth models (Table II). We calibrated the 14 C dates with the IntCal20 calibration curves [START_REF] Reimer | The IntCal20 Northern hemisphere radiocarbon age calibration curve (0-55 cal kBP)[END_REF].

B Geochemical analyses

We performed XRF analyses on the basal sediments corresponding to the first millennium of the paleoislands' existence. To detect semi-quantitative variations of geochemical composition, we used the TestAllGeo analysis mode on the Niton XL3t 900SHE portable scanner and continuously scanned the sediments every 0.5 cm along the cores. Changes in the concentration of several major and minor elements can be used as proxies of ecological phenomena (Croudace and Rothwell, 2015). We used K, Ti, Rb, Sr, Zr and Fe (lithogenic elements) as indicators of erosion from the watershed to the lakes, which we expected to decrease following afforestation of the paleo-islands. In addition to being an erosion indicator, low levels of K indicate extensive vegetation presence [START_REF] Foerster | Climatic change recorded in the sediments of the Chew Bahir basin, southern Ethiopia, during the last 45,000 years -ScienceDirect[END_REF]. While S and Ca have various environmental interpretations (Croudace and Rothwell, 2015), we considered S to be indicative of organic matter presence and Ca to be indicative of biogenic carbonate (algae production).

As plants fix sulfur (S), we used the S/Ti ratio as an indicator of organic matter presence [START_REF] Moreno | A 14kyr record of the tropical Andes: The Lago Chungará sequence (18°S, northern Chilean Altiplano)[END_REF]. The fact that S, Ca and the S/Ti ratio are indicators of organic matter presence compensates for our inability to perform loss-on-ignition (LOI) analysis due to lack of material. For each lake, we applied principal component analysis (PCA) with the 'factoextra' package in R [START_REF] Kassambara | Practical guide to Principal Component Methods in R: PCA, M(CA)[END_REF] to differentiate portions of the sediment sequences based on their chemical signature. We used the Kaiser criterion to select significant principal components (PCs), ie., those with eigenvalues >1 [START_REF] Kaiser | The application of electronic computers to factor analysis[END_REF]. As variations along the PCs were semi-quantitative because of the XRF scanning method and scanner calibration, we could only compare trends in element dynamics (increase or decrease), and not their absolute values.

B Identification of plant macro-remains

We bathed half of each 0.5-cm slice of basal sediments in a hexametaphosphate (Na6P6O18) solution for at least 12 hours under agitation, for deflocculation purposes. We then filtered each sample through a 250-µm sieve to keep plant macro-remains. We identified plant macro-remains under a dissecting microscope using a published key [START_REF] Lévesque | Guide to the identification of plant macrofossils in Canadian peatlands[END_REF] and recorded the presence and absence of taxa for each level studied.

B Charcoal extraction and analysis

To perform charcoal extraction, we cut the basal sediments continuously into 0.5-cm-thick slices. We extracted a 1-cm 3 subsample from the middle of each slice to avoid contamination risk [START_REF] Mustaphi | A classification for macroscopic charcoal morphologies found in Holocene lacustrine sediments[END_REF]. We bathed the subsample in a solution of potassium hydroxide (KOH), sodium hexametaphosphate (Na6P6O18), and sodium hypochlorite (NaClO) for at least 24 hours under agitation to perform deflocculation and to help differentiate charcoal particles from organic matter [START_REF] Whitlock | Charcoal as a fire proxy[END_REF]. We sieved the solution through a 160-µm mesh to extract macro-charcoal particles and measured their surface area under a dissecting microscope using the WinSEEDLE TM image analysis software (Regents Instruments Inc., https://regent.qc.ca/, August 2021).

To reconstruct local fire history, ie., fire events that occurred within the lakes' watersheds and thus on the paleo-islands, we applied the Charcoal Size Distribution (CSD) method [START_REF] Asselin | Detecting local-scale fire episodes on pollen slides[END_REF]Oris et al., 2014a;[START_REF] Remy | Guidelines for the use and interpretation of palaeofire reconstructions based on various archives and proxies[END_REF] to every fire event detected using the CharAnalysis software v1.1 [START_REF] Higuera | Frequent fires in ancient shrub tundra: implications of paleorecords for Arctic environmental change[END_REF]; http://CharAnalysis.googlepages.com). We calculated geometric mean diameters of charcoal particles from the square root of the surface area [START_REF] Clark | Estimating the mass flux of charcoal from sedimentary records: effects of particle size, morphology, and orientation. The Holocene[END_REF] and converted them to log mm. We sorted charcoal particles of each sample according to their size into six classes ranging from -0.9 log mm to 0.3 log mm (every 0.2 log mm). We applied a linear regression to the distribution of the proportion of charcoal particles in each size class (-0.8, -0.6, etc.) for each sample. As large charcoal particles travel less far, local fires are characterized by a greater proportion of large charcoal particles than regional fires, which is reflected by a gentler slope of the size distribution regression [START_REF] Asselin | Detecting local-scale fire episodes on pollen slides[END_REF]. According to this principle, we classified as local fires (on the paleo-island) the samples with a slope more gentle than a threshold fixed at -1.77 based on a previous study (Oris et al., 2014a). In addition, we considered as local fires only the samples having at least one charcoal particle >-0.3 log mm (Supplemental Figure 1). We considered that other fires (extra-local or regional) did not burn on the paleo-islands.

A Results

B Organic sediment accumulation during Lake Ojibway presence

Basal 14 C dates obtained from organic sediments at or close to the contact with the glacial mineral sediments were 9,657 cal. yr BP and 9,947 cal. yr BP for Lakes Perché and Despériers, respectively (Fig. 3). These basal ages are older than the estimated date of Lake Ojibway's final drainage (8,200 cal. yr BP).

Radiocarbon dates based on macro-remains that are coeval with the gradual lowering of the level of Lake Ojibway were recorded 10 cm above the basal dates obtained from bulk sediments. Despite being a little younger than expected, these radiocarbon dates based on macro-remains are the oldest dates obtained for the region. Moreover, they are from the Kinojévis phase (9,000-8,300 cal. yr BP), before the final drainage of Lake Ojibway. Radiocarbon dates based on bulk sediment and on plant macro-remains are consistent, suggesting that no or little reservoir effect altered the age-depth models and basal age estimations (Grimm et al., 2009).

At Lake Perché, the sediment sequence we analyzed spanned from 9,657 to 6,503 cal. yr BP, and at Lake Despériers from 9,947 to 6,850 cal. yr BP (Fig. 3). The mean sedimentation rates were relatively similar at both lakes at the beginning of the Holocene (44 yr.cm -1 for Lake Perché and 53 yr.cm -1 for Lake Despériers). The mineral sediments found below the gyttja were olive-grey colored silt for both lakes, which was markedly different from the clay characterizing sites located below the shorelines of proglacial Lake Ojibway. Silt is indicative of glacial till associated to the direct action of the ice sheet on the bedrock without a proglacial lake intermediary [START_REF] Boulton | Boulder shapes and grain-size distributions of debris as indicators of transport paths through a glacier and till genesis[END_REF][START_REF] Normandeau | Late-Quaternary morphostratigraphy of Lake St-Joseph (southeastern Canadian Shield): Evolution from a semi-enclosed glacimarine basin to a postglacial lake[END_REF]. II) obtained from basal sediments (brown = organic sediments and olive-grey = mineralogic sediments) at a) Lake Perché and b) Lake Despériers. The purple rectangle illustrates the period when the level of proglacial Lake Ojibway gradually decreased until its final drainage ca 8,200 cal. yr BP.

B Geochemical signature of the organic sediments

Two groups were detected by the PCA analysis in the gyttja of Lake Perché: (1) from the silt-gyttja transition at 9,600 cal. yr BP to 9,000 cal. yr BP and (2) between 9,000 and 6,500 cal. yr BP (Fig. 4a). The first two principal components explained 88.66% of the variability in the dataset (PC1 = 73.67% and PC2 = 14.99%). PC1 was positively associated to lithogenic elements (K, Ti, Fe, Rb, Zr, Sr) coming from the steep shores surrounding the lake, while PC2 was positively correlated to S. One element (Ca) was positively related to both PCs (Fig. 4a).

Figure 4: Principal component analysis (PCA) of sediment samples (dots), associated centroids for each cluster (larger dots and triangles), and XRF results (arrows) for a) Lake Perché and b) Lake Despériers. At both sites the sediment samples pre-and post-dating afforestation are clearly separated along PC1.

Variations of PC1 and PC2 loadings, and of the S/Ti ratio across time at c) Lake Perché and d) Lake Despériers. The purple rectangle illustrates the period when the level of proglacial Lake Ojibway gradually decreased until its final drainage ca 8,200 cal. yr BP.. Note that the data for PC2 at Lake Despériers does not respect the Kaiser criterion of eigenvalue >1 and should be interpreted with caution.

The PCA also differentiated two groups in the gyttja of Lake Despériers: (1) from the silt-gyttja transition at 10,000 cal. yr BP to 8,800 cal. yr BP and (2) between 8,800 and 6,800 cal. yr BP (Fig. 4b). The first two principal components explained 94.56% of the variability (PC1 = 87.15% and PC2 = 7.41%). As was the case for Lake Perché, the lithogenic sediments were positively correlated to PC1 at Lake Despériers, S was positively associated to PC2, and Ca was positively related to both PCs.

For Lake Perché, PC1 values (associated with lithogenic elements) quickly decreased until 8,800 cal. yr BP, and then increased until 8,300 cal. yr BP. After 8,250 cal. yr BP, values slowly decreased and reached their minimum until the end of the record, ie. 6,500 cal. yr BP. PC2 values (associated with S) increased between 9,350 and 9,000 cal. yr BP (Fig. 4a,c) and then remained relatively stable (despite short-term variations) between 9,000 and 7,900 cal. yr BP (Fig. 4c). The S/Ti ratio slowly increased with peaks around 8,800 cal. yr BP, between 8,000 and 7,800 cal. yr BP, between 7,700 and 7,500 cal. yr BP, and ca 6,900 cal. yr BP (Fig. 4c). The absence of a distinguishable pattern at 8,200 cal. yr BP indicates that the environment remained stable during the drainage of proglacial Lake Ojibway.

For Lake Despériers, PC1 values (associated with lithogenic elements) gradually decreased from the beginning of gyttja accumulation around 10,000 cal. yr BP until 8,700 cal. yr BP, then they strongly decreased around 8700 cal. yr BP to reach minimum values after 8,200 cal. yr BP. (Fig. 4b,d). PC2 did not respect the Kaiser criterion (eigenvalue = 0.59) and therefore could not be interpreted (Supplemental Table 1). The S/Ti ratio remained stable during the early Holocene at Lake Despériers, with peaks at ca 9,900, 9,300 and 8,800 cal. yr BP (Fig. 4d). Then, ca 8,400 cal. yr BP, the S/Ti ratio started to increase until 7,900 cal. yr BP. Around 7,700 cal. yr BP, the S/Ti ratio decreased and remained stable until 7,000 cal. yr BP, then it increased until 6,500 cal. yr BP (Fig. 4d).

B Plant macro-remains: early presence of local trees

Despite the paucity of macro-remains in the sediments, we were able to extract and identify some of them. The earliest tree macro-remains that we found were larch (Larix laricina [Du Roi] K. Koch) needles dating from around 9,513 cal. yr BP at Lake Perché (Fig. 5a; Supplemental Figure 2a,b). Spruce (Picea spp.) needle fragments were also found ca 8,385 cal. yr BP (Fig. 5a; Supplemental Figure 2c). At Lake Despériers, the earliest tree macro-remains that we found were pine (Pinus spp.) needles, dating from around 9,845 cal. yr BP (Fig. 5b; Supplemental figure 2d). Needles of spruce (Picea spp.) and larch (Larix laricina), as well as birch (Betula spp.) seeds were also found ca 9,584-9,300 cal. yr BP (Fig. 5b; Supplemental Figure 2e-i). Trees were therefore present on the studied paleo-islands at least 1,400 years before Lake Ojibway drainage.

Figure 5: Fire events detected with CHARanalysis (red triangles), and local fire events detected with the Charcoal Size Distribution (CSD) method (blue circles) and oldest tree macro-remains retrieved from the sediments at (a) Lake Perché and (b) Lake Despériers according to the age-depth models (Fig. 3). The purple rectangle illustrates the period when the level of proglacial Lake Ojibway gradually decreased until its final drainage ca 8,200 cal. yr BP.

B Evidence of local forest fires on a paleo-island

CHARanalysis detected 21 and 25 fire events in the Lake Perché and Lake Despériers sediments, respectively (Fig. 5). At Lake Perché, eight events met the conditions to be considered local fires having burned within the watershed, the earliest local fires were dated at 8,362 (Fig. 5a) when charcoal influx was maximal (Supplemental Figure 3), 7,984 and 7,942 cal. yr BP (Fig. 5a) when charcoal influx was lower (Supplemental Figure 3). The earliest local fire, evidenced by burned needle fragments (possibly Picea spp.) (Supplemental Figure 2c), occurred around 160 years before the drainage of proglacial Lake Ojibway. At Lake Despériers, 11 events met the conditions to be considered local fires having burned within the watershed. The earliest local fires at Lake Despériers were dated at 9,820 and 9,360 cal. yr BP, while Lake Ojibway was still present (Fig. 5b).

A Discussion

Our analyses of two sites located 50 km apart revealed that both paleo-islands were colonized by tree species several centuries before the level of Lake Ojibway lowered enough for paleo-islands to become connected with the mainland to the south ca 9,000 cal. yr BP [START_REF] Godbout | A detailed lake-level reconstruction shows evidence for two abrupt lake drawdowns in the late-stage history of the eastern Lake Agassiz-Ojibway basin[END_REF][START_REF] Roy | Late-stage phases of glacial Lake Ojibway in the central Abitibi region, eastern Canada[END_REF]. We used proxies suited to reveal local vegetation establishment, and we avoided proxies such as pollen, which can be influenced by extra-local vegetation inputs.

B Early afforestation of islands on proglacial Lake Ojibway

Gyttja started to accumulate at the bottom of small lakes on paleo-islands while Lake Ojibway was still at its maximum level , ie., about 250-550 years before it started to gradually decrease. The presence of silt rather than clay beneath the gyttja at both sites is evidence that they were never flooded by proglacial Lake Ojibway. Radiocarbon dates obtained from plant macro-remains testify that shrubs and trees were present on the paleo-islands at least 140-260 years before the final drainage of proglacial Lake Ojibway (Table II). According to the age-depth models, the oldest tree macro-remains we found were (indirectly) dated to ca 9,600 cal. yr BP and 9,850 cal. yr BP at Lakes Perché and Despériers, respectively, 200 and 450 years before the level of Lake Ojibway first started to decrease [START_REF] Brouard | A framework for the timing of the final meltwater outbursts from glacial Lake Agassiz-Ojibway[END_REF][START_REF] Godbout | A detailed lake-level reconstruction shows evidence for two abrupt lake drawdowns in the late-stage history of the eastern Lake Agassiz-Ojibway basin[END_REF]. Hence, while deglaciation time has been estimated at 10,570 cal. yr BP in the study area [START_REF] Daubois | The drainage of Lake Ojibway in glaciolacustrine sediments of northern Ontario and Quebec, Canada: The drainage of Lake Ojibway, Canada[END_REF][START_REF] Godbout | A detailed lake-level reconstruction shows evidence for two abrupt lake drawdowns in the late-stage history of the eastern Lake Agassiz-Ojibway basin[END_REF][START_REF] Roy | Late-stage phases of glacial Lake Ojibway in the central Abitibi region, eastern Canada[END_REF], our results indicate that the afforestation process needed about 1,000 years to unfold after the glacier retreat. This is much longer than what has been documented at recently deglaciated sites in Alaska, where primary succession took less than 200 years to complete [START_REF] Fastie | Causes and ecosystem consequences of multiple pathways of primary succession at Glacier Bay, Alaska[END_REF]. The difference would likely be due to the distance to seed sources having been much greater at our sites (80 km over mostly water) compared to those in Alaska (<30 km over ground).

Furthermore, cold temperatures during the early Holocene (Viau et al., 2006) likely implied lower vegetation productivity, as evidenced in our age-depth models by lower sediment accumulation rates compared to the middle Holocene.

Geochemical analyses showed that lithogenic elements -therefore erosion [START_REF] Joannin | First pollen record from the Late Holocene forest environment in the Lesser Caucasus[END_REF][START_REF] Lefebvre | Climate-driven fluxes of organic-bound uranium to an alpine lake over the Holocene[END_REF] -decreased rapidly at both sites from the beginning of gyttja accumulation until 8,800 cal. yr BP (Lake Perché) and 8,700 cal. yr BP (Lake Despériers). As soil cohesion and stabilization are supported by plant root systems [START_REF] Easterbrook | Surface processes and landforms[END_REF], it is reasonable to assume that decreasing erosion is an indirect indicator of vegetation colonization on paleo-islands. Indeed, more individual plants produce more roots which stabilize the ground, meaning that less soil erosion is recorded in the lake sediments. Therefore, PC1 variations suggest the presence of vegetation rapidly after the beginning of gyttja accumulation at both lakes. The plateau PC1 values reached for both lakes around the final drainage of proglacial Lake Ojibway ca 8,200 cal. yr BP likely indicate densification of the vegetation.

Increased PC2 values (mostly linked to S fixation by plants) and a peak of the S/Ti ratio around 8,800 cal.

yr BP indicate a strong organic matter input and suggest the arrival (or densification) of local vegetation, coherent with the decrease of the level of Lake Ojibway. Therefore, the variations in the concentrations of lithogenic elements and elements indicating organic matter input into the lake sediments provide indirect support of early vegetation establishment before the level of Lake Ojibway started to decrease and possible densification on the paleo-islands before Lake Ojibway's final drainage.

Macro-remains analysis confirmed local presence of trees at both sites, while Lake Ojibway was still present and before it gradually drained. According to the age-depth model, the earliest tree species identified at Lake Perché is Larix laricina, possibly indicative of wet conditions around the lake ca 9,500 cal. yr BP [START_REF] Johnston | Larix laricina (Du Roi) K. Koch -Tamarack[END_REF]. It was followed by Picea spp. around 8,400 cal. yr BP, at a time when geochemical analyses indicate that vegetation became denser. At Lake Despériers, Pinus spp. was the earliest species found at the paleo-island around 9,850 cal. yr BP and could be indicative of environmental conditions drier than at Lake Perché [START_REF] Td | Pinus banksiana Lamb. jack pine. Silvics of North America[END_REF]. Then, between ca 9,585 cal. yr BP and 9,300 cal. yr BP, Picea spp., Larix laricina and Betula spp. were also present, possibly indicating that environmental conditions had become more mesic, as at Lake Perché.

The fact that a local fire was dated at 8,362 cal. yr BP at lake Perché (burned unidentified needle) is yet another confirmation of early afforestation of the paleo-island, as fire ignition and propagation are fueldependent (ie., biomass-dependent) [START_REF] Finney | Wildland fire behaviour: dynamics, principles and processes[END_REF]. At Lake Despériers, the earliest local fire around 9,820 cal. yr BP (burned unidentified needle fragment, Fig. S.3.3) confirmed biomass presence and afforestation on the paleo-island [START_REF] Finney | Wildland fire behaviour: dynamics, principles and processes[END_REF] before the drainage of proglacial Lake Ojibway started.

B The paleo-island colonization process: an answer to Reid's Paradox

For primary succession to be successful on a bare island on a proglacial lake, several biotic and abiotic barriers must be overcome [START_REF] Crofts | The importance of biotic filtering on boreal conifer recruitment at alpine treeline[END_REF]. Climatic and meteorological factors are at play, substrate characteristics are also important, but distance to seed sources is the main hurdle.

At the time of full extension of proglacial lake Ojibway, the distance between the studied paleo-islands and the continental forest margin was about 80 km, with other islands located in between (Fig. 2b).

Colonization of the studied paleo-islands could have resulted from seeds coming directly from the colonization front and traveling almost 100 km above water. Such long distance dispersal events have been documented before [START_REF] Davis | Dispersal versus climate: Expansion of Fagus and Tsuga into the Upper Great Lakes region[END_REF]. Seeds could also have come from other paleo-islands that could have acted as 'stepping stones' [START_REF] Harris | Long distance dispersal in the assembly of floras: A review of progress and prospects in North America[END_REF]. In either case, our results support the hypothesis of tree population outposts on paleo-islands, far from the main source population of propagules at the southern margin of the proglacial lake, as previously theorized [START_REF] Davis | Detecting a species limit from pollen in sediments[END_REF].

Even with the 'stepping stones' scenario that would have reduced seed traveling distance to steps of ca 15-20 km, such dispersal would be considered very long, as the vast majority of boreal tree seeds are dispersed less than 100 m from the parent tree [START_REF] Asselin | Spatial distribution of late-successional coniferous species regeneration following disturbance in southwestern Québec boreal forest[END_REF][START_REF] Burns | Silvics of North America[END_REF][START_REF] Greene | Wind dispersal of seeds from a forest into a clearing[END_REF]. Estimating the seed rain at distances longer than a few hundred meters is very difficult technically [START_REF] Nathan | Long-distance dispersal of plants[END_REF]. A modelling study estimated the proportion of seeds traveling 1-10 km (long distance dispersal events) at 0.1% [START_REF] Higgins | Predicting plant migration rates in a changing world: the role of long-distance dispersal[END_REF]. Knowing that yearly seed production of most boreal tree species reaches 1-5 million seeds/ha [START_REF] Burns | Silvics of North America[END_REF], between 12,500 and 62,500 seeds could have reached the studied paleo-islands each year. Multiplied by 1000 years between deglaciation and vegetation establishment on paleo-islands and adding the probability of germination and survival, the possibility of colonization through long distance dispersal is, after all, not so unlikely. Moreover, as the level of Lake Ojibway gradually decreased from ca 9,400 cal. yr BP, more and more land became available for colonization, which would have decreased the travel distance between mainland and islands, thus gradually increasing the probability of successful dispersal events and colonization of paleo-islands.

Catabatic winds blowing from the ice sheet to the south could have slowed down the migration process [START_REF] Richard | La végétation postglaciaire du Témiscamingue, Québec, durant l'épisode glaciolacustre Barlow[END_REF]. Reconstructions of paleo-wind directions indicate a dominant wind blowing towards the east or south-east (Veillette, 1996), but do not indicate a predominance of catabatic winds [START_REF] Nutz | Deglacial to paraglacial history of the Lake Saint-Jean lowlands: A geomorphological perspective[END_REF]. Active (biotic) dispersal (eg, by birds) could have contributed to long-distance dispersal, even if most boreal tree species are generally dispersed by wind [START_REF] Higgins | Are long-distance events in plants usually caused by nonstandard means of dispersal?[END_REF][START_REF] Nathan | Mechanisms of long-distance seed dispersal[END_REF][START_REF] Wilkinson | Plant colonization: are wind dispersed seeds really dispersed by birds at larger spatial and temporal scales?[END_REF]. Indeed, nonstandard dispersal of wind-dispersed seeds by omnivorous birds has been hypothesized and later confirmed by empirical data [START_REF] Nogales | Evidence for overlooked mechanisms of long-distance seed dispersal to and between oceanic islands[END_REF][START_REF] Rumeu | Diversity, distribution and conservation status of island conifers: a global review[END_REF]. We do not have palaeoecological proxies confirming or denying bird presence or passage on the paleo-island, but they could have facilitated tree seed dispersal. In the winter, secondary seed dispersal over snow or ice, or on ice rafts in the spring, could have also favored long-distance dispersal [START_REF] Alsos | The role of sea ice for vascular plant dispersal in the Arctic[END_REF][START_REF] Greene | Secondary dispersal of tree seeds on snow[END_REF]. Paleo-island plant colonization could also have occurred with seeds from the Lake Ojibway free water at Lake Perché, as it was located close to the proglacial lake's shoreline. In the spring, melting snow would have flushed plant material (including seeds) into Lake Ojibway, and windrows would have facilitated their buoyancy and transport by wind up to the paleoisland shores [START_REF] Glaser | Transport and deposition of leaves and seeds on tundra: A late-glacial analog[END_REF].

Simulation studies have estimated the migration rate of Picea spp. at about 50 m.yr -1 [START_REF] Snell | Consideration of dispersal processes and northern refugia can improve our understanding of past plant migration rates in North America[END_REF][START_REF] Sulphur | Holocene fire regimes and treeline migration rates in sub-arctic Canada[END_REF], which would mean that trees would have needed ca 4,000 years to migrate the 200 km from south to north of the area covered by proglacial Lake Ojibway following its final drainage around 8,200 cal. yr BP, if it were not for paleo-islands. However, basal dates of bulk sediments sampled in lakes located at the northern end of the area yielded ages very close to drainage time [START_REF] Ali | Long-term fire frequency variability in the eastern Canadian boreal forest: the influences of climate vs. local factors[END_REF][START_REF] El-Guellab | Holocene variations of wildfire occurrence as a guide for sustainable management of the northeastern Canadian boreal forest[END_REF]Oris et al., 2014b). Our demonstration of early afforestation of two paleo-islands implies that they could have acted as migration outpost following the drainage of proglacial Lake Ojibway, hence accelerating colonization of the lowlands. The total area covered by paleo-islands represented ca 3,600 km 2 , ie., about 10% of the area covered by proglacial Lake Ojibway at its maximal extent (Fig. 2a). This means that several possible outposts were available for early afforestation, from near the colonization front in the south, up to the ice sheet margin in the north (Fig. 2b). The contribution of paleo-islands as migration outposts is the most likely mechanism that could explain Reid's paradox of rapid plant migration in western Quebec [START_REF] Clark | Reid's Paradox of rapid plant migration[END_REF].

Figure 2 :

 2 Figure 2: (a) Extent of proglacial Lake Ojibway in western Quebec around 9,600 cal. yr BP (Brooks, 2020), showing the locations of the two studied paleo-islands (yellow stars) according to the lake level at the time. The location of Aiguebelle National Park (ANP) is also shown. The inset shows the location of ANP relative to Hudson Bay (HB) and James Bay (JB), where proglacial Lake Ojibway finally drained around 8,200 cal. yr BP. (b) Close-up of the study area showing paleo-islands (in light to dark orange), including the Abijévis and Kekeko Hills, where Lakes Perché and Despériers were sampled, respectively.

  . Current forest vegetation around the sampled lakes is dominated by black spruce (Picea mariana [Mill.] BSP), white spruce (Picea glauca [Moench] Voss), jack pine (Pinus banksiana Lamb.) and balsam fir (Abies balsamea [L.] Mill.). Both lakes are surrounded by rolling hills less than 100 m in height.

Figure 3 :

 3 Figure 3: Calibrated age-depth models based on the radiocarbon dates (TableII) obtained from basal sediments (brown = organic sediments and olive-grey = mineralogic sediments) at a) Lake Perché and b) Lake Despériers. The purple rectangle illustrates the period when the level of proglacial Lake Ojibway gradually decreased until its final drainage ca 8,200 cal. yr BP.

Table I :

 I Main characteristics of Lakes Perché and Despériers.

	Name	Latitude	Longitude	Elevation	Area	Organic	Water	Current local
				(m a.s.l.)	(ha)	sediment	column	vegetation
						core	depth at	by order of
						length	sampling	dominance
						(cm)	location
							(cm)
	Lake Perché 48°30'8.10"N	78°41'56.90"W	380	9.70	554	370	Picea mariana,
								Pinus banksiana
	Lake	48°11'4.22"N	79° 9'38.80"W	407	8.89	292	750	Picea mariana,
	Despériers							Picea glauca,
								Abies balsamea

Table II :

 II Radiocarbon dates obtained from samples near the bottom of the sediment cores from Lakes Perché and Despériers. bark fragment and other unidentified vegetal fragments; b bark fragment, Cristatella mucedo statoblast and other unidentified vegetal fragments; c Larix laricina needle fragment and unidentified seed and vegetal fragments.

	Lake	Sample depth (cm) 14 C yr BP Cal. yr BP	Material dated	Lab. Code
	Perché	512.5 -513	6670 ± 20 7537 ± 30	Bulk sediment	ULA-9479
	Perché	537 -538.5	7225 ± 20 8044 ± 25	Bulk sediment	ULA-9477
	Perché	553 -553.5	7510 ± 30 8341 ± 30 Plant macro-remains a BETA-644328
	Perché	555 -555.5	7680 ± 40 8466 ± 30 Plant macro-remains b BETA-644329
	Perché	564 -564.5	8695 ± 20 9626 ± 30	Bulk sediment	ULA-9478
	Despériers	232.5 -233.5	5975 ± 15 6813 ± 30	Bulk sediment	ULA-10171
	Despériers	260 -261	7055 ± 20 7891 ± 30	Bulk sediment	ULA-10172
	Despériers	276 -277.5	7510 ± 30 8341 ± 30 Plant macro-remains c BETA-638098
	Despériers	289 -289.5	8775 ± 20 9774 ± 40	Bulk sediment	ULA-10173

a

A Acknowledgements

We thank Thibaut Petry and his team at Aiguebelle National Park for their help and support. We also thank Mary Robles, Raynald Julien and Danielle Charron for their help with fieldwork and data processing. We are grateful to the Institut de recherche en mines et en environnement (UQAT) for letting us use their XRF scanner, and to Yvan Poirier and Mathieu Villeneuve for their help with the scanner. We thank Nils Ambec and Alayn Larouche for their help to identify plant macro-remains. We also thank Jean J. Veillette for constructive discussions, and Raphaël Chavardès, Dorian Gaboriau and the proofreading committee "Corrige-moi" who provided helpful comments on a first draft. We thank the anonymous reviewers, whose comments improve our manuscript. This is a contribution of the Laboratoire International de Recherche sur les Forêts Froides and ISEM contribution No XXX.

A Conflict of interest

The authors declare no conflict of interest.

A Funding

This work was supported by Mitacs Acceleration in partnership with Aiguebelle National Park [grant number IT25924].

A Data availability statement

Data used for this article are available online: https://data.mendeley.com/datasets/k8b2t978hd/draft?a=bcbf8880-3a2a-4638-aa92-14aac331ac35

A Supplementary Material

Supplemental Table 1: Eigenvalues for principal components 1 and 2 following principal component analysis on the data for Lake Perché and Lake Despériers (Fig. 3). Eigenvalues >1 met the Kaiser criterion for significance and are indicated in bold.

PC1 eigenvalue

PC2 eigenvalue Lake Perché 5.65 1.38 Lake Despériers 6.97 0.59

Supplemental figures

Supplemental Figure 1: Four examples of linear regressions performed on charcoal distributions and their associated slopes. a) Local fire, as both criteria are respected (slope less steep than the -1.77 threshold and presence of charcoal particles >-0.3 log mm), b) Regional fire as the slope is steeper than the threshold, c) Regional fire as no charcoal particle >-0.3 log mm was found, and d) Regional fire as none of the criteria is respected.

Supplemental Supplemental Figure 3: Charcoal influx for Lake Perché in blue and Lake Despériers in orange.


Klaus Pantel

Catherine Alix-Panabières

Crucial roles of circulating tumor cells in the metastatic cascade and tumor immune escape: biology and clinical translation

INTRODUCTION

The Joint Research Centre (JRC) of the European Commission, in collaboration with the International Agency for Research on Cancer (IARC), has released the estimates of the burden of cancer in each of the European Union (EU-27) countries for 2020. A total of 2.7 million new cases of cancer (excluding non-melanoma skin cancers) and 1.3 million cancer-related deaths have been estimated for 2020 (https://www.iarc.who.int/fr/ news-events/new-cancer-burden-estimatesfor-2020-jrc-iarc-collaborations/). Although experimental studies in animal models have greatly enhanced our knowledge on the basic principles of cancer metastasis, the development in patients is sometimes difficult to model. For example, metastasis can occur in patients with estrogen receptor-positive (ER (+) ) breast cancer more than 10 years after initial diagnosis and surgical resection of the primary tumor and this latency or dormancy period is not easy to mimic in mice that have a normal life span of approximately 2 years. Studies performed in patients with breast cancer indicated that even small tumors are able to release circulating tumor cells (CTCs) that extravasate into the bone marrow (and probably other organs) where they are called disseminated cancer cells (DTCs). [START_REF] Braun | Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer[END_REF] Quantitative estimations have shown that these DTCpositive patients harbor at least one million tumor cells in their bodies despite the fact that they are called 'metastasis-free' by imaging procedures used for tumor staging. Interestingly, only half of these DTC-positive patients relapsed with 10 years of follow-up, [START_REF] Braun | A pooled analysis of bone marrow micrometastasis in breast cancer[END_REF] suggesting that the host is able to control the outgrowth of a significant number of DTCs for many years. Factors promoting or suppressing circulation of CTCs and activation of dormant DTCs are subject of intense investigations. In this context, the immune system might play an important role as 'gate keeper'.

Technological advances over the past decade have allowed the detection and molecular characterization of CTCs in the blood of patients with cancer. Blood is easy to obtain and blood draws are much less invasive than needle biopsies of tissues. CTCs have constituted the core of the so-called liquid biopsy concept that has been introduced more than 10 years ago, [START_REF] Pantel | Circulating tumour cells in cancer patients: challenges and perspectives[END_REF] and rapidly adapted to the analysis of tumor cell products such as cellfree nucleic acids or extracellular vesicles as well as immune cells [START_REF] Pantel | Liquid biopsy: from discovery to clinical application[END_REF] (figure 1). Various clinical studies have indicated that CTC detection contributes to a better definition of the Open access prognosis of the patients, enables the characterization of resistance mechanisms to therapy and tumor heterogeneity, the early detection of relapse in the metastatic setting and the detection of minimal residual disease in the earlier stages, which can contribute to a more personalized medicine in oncology. To get a comprehensive view of blood-borne cancer progression in the context of immunotherapies, we need to consider a broader definition of liquid biopsy including tumor-derived circulating biomarkers such as CTCs or cell-free DNA fragments and circulating components of the immune system (eg, immune cells, cytokines, interleukins) for each patient (figure 1).

Personalized diagnostic and treatment strategies in cancer are highly based on the individual characteristics associated with malignant transformation and progression. [START_REF] Hahn | An expanded universe of cancer targets[END_REF] To date, most patients are categorized at initial diagnosis, and treatment remains based on this initial classification while the tumor might undergo fundamental genotypic and functional changes. Recent research has confirmed that natural and therapy-induced selective pressure leads to clonal dynamics in cancers, leading to considerable heterogeneity and treatment resistance. 6 7 In order to monitor these complex dynamic processes in patients with cancer, sequential monitoring of tumor composition in individual patients with cancer is required to adapt therapies to these changes. Today, these challenges may be addressed by the recent development of high-resolution, scalable blood-based detection methods of CTCs. These represent novel technologies to closely monitor the molecular composition of residual tumor burden following initial therapy. The additional benefit Open access for patients consists in the fact that these procedures-as opposed to repetitive tissue biopsies-are minimal or non-invasive and are highly sensitive to monitor situations of low tumor load ('minimal residual disease', MRD) and discover tumor relapses prior to conventional imaging. 8 9 In this review, we summarized and discussed the role of CTC research for better understanding of cancer metastasis with emphasis on tumor immune escape strategies, and how this knowledge can translate into novel strategies to improve the clinical outcome of patients with cancer.

CTCs capturing the metastatic cascade-tumor biology CTCs can be derived from a primary and/or metastatic tumor lesion and circulate for a short time in the peripheral blood. Besides their value as diagnostic markers, the analyses of CTCs in patients with cancer (and experimental models) can contribute to understand the complexity of metastatic spread which may eventually lead to a favorable outcome of patients with solid malignancies. [START_REF] Cortés-Hernández | Functional analysis of circulating tumour cells: the key to understand the biology of the metastatic cascade[END_REF] In this context, mathematical modeling might help to encompass the complex process of the metastatic cascade. Dujon et al used simulations (adaptation of the Drake equation) based on published breast cancer data and demonstrated that the survival of CTCs in the bloodstream is a key step in the complex process of the metastatic cascade. [START_REF] Dujon | Is there one key step in the metastatic cascade?[END_REF] The detection of CTCs in lymph node-positive patients allows the analysis of tumor cell dissemination at a subclinical level, 12 13 and contributes to a better understanding of the role of lymph nodes in the metastatic cascade. [START_REF] Naxerova | Origins of lymphatic and distant metastases in human colorectal cancer[END_REF] Monitoring of the genomic make up of CTCs by analysis of sequential blood samples from individual patients allows to get insights into the evolution of metastasis, [START_REF] Joosse | Chromosomal aberrations associated with sequential steps of the metastatic cascade in colorectal cancer patients[END_REF] including the role of intratumor heterogeneity. Moreover, the metabolic characterization of the CTCs has shed new light on the survival mechanisms of CTCs in the harsh blood microenvironment. [START_REF] Ubellacker | Lymph protects metastasizing melanoma cells from ferroptosis[END_REF] Patients with tumor lesions in the brain lesions have a dismal prognosis. Despite the blood-brain barrier CTCs can enter the peripheral blood in these patients, which allows minimally invasive access to brain-derived tumor cells. [START_REF] Müller | Hematogenous dissemination of glioblastoma multiforme[END_REF][START_REF] Riebensahm | Clonality of circulating tumor cells in breast cancer brain metastasis patients[END_REF][START_REF] Garzia | A hematogenous route for medulloblastoma leptomeningeal metastases[END_REF] Besides primary brain tumors, brain metastases cause significant harm to patients with cancer and they pose a major challenge to obtain personalized information because needle biopsy of these lesions is only possible in a few specialized clinical centers. Recent advances in the enrichment of CTCs from brain metastases by multiplexing of capture antibodies to different tumorassociated antigens have resulted in better capture rates [START_REF] Scharpenseel | EGFR and HER3 expression in circulating tumor cells and tumor tissue from non-small cell lung cancer patients[END_REF] and pilot studies on phenotypic characterization of CTCs in patients with brain metastases suggest that these cells might reflect the phenotype of the brain metastasis from the same patient. Nevertheless, experimental in vitro studies suggest that gene expression of tumor cells can change during the travel of CTCs in blood (eg, due to changes in oxygen levels). [START_REF] Bartkowiak | In vitro modeling of reoxygenation effects on mRNA and protein levels in hypoxic tumor cells upon entry into the bloodstream[END_REF] The study of CTC biology opens a new avenue for better understanding the process of blood-borne tumor cell dissemination. The harsh conditions in the blood require that CTCs are fit enough for survival and extravasation into a distant organ site. Interestingly, counter to previous findings in experimental mouse models [START_REF] Chambers | Dissemination and growth of cancer cells in metastatic sites[END_REF] this seems to be an essential step in the metastatic cascade, [START_REF] Dujon | Is there one key step in the metastatic cascade?[END_REF] which is consistent with the fact that the CTC detection at initial diagnosis is a robust and significant factor associated with metastatic relapse in solid tumors, such as breast cancer. To survive in the blood after detachment from their tumor tissue of origin, CTCs must avoid undergoing anoikis, a special form of apoptosis. Numerous studies have shown that the epithelial-to-mesenchymal transition (EMT) favors the dissemination of single CTCs, while the role of EpCAM-one of the most prominent markers for enrichment of CTCs that is found also on metastasiscompetent CTCs-in EMT is still not well understood. [START_REF] Cortés-Hernández | Epithelial cell adhesion molecule: an anchor to isolate clinically relevant circulating tumor cells[END_REF] Overall, the most aggressive metastasis-competent CTCs seem to be those that have a high plasticity. Indeed, when we investigated the main characteristics of the unique existing series of colon CTC lines, which have surprisingly acquired only few mesenchymal features but expressed rather epithelial-related genes, suggesting that metastasis-initiator CTCs require a switch from EMT to mesenchymal-to-epithelial transition (MET). [START_REF] Balcik-Ercin | Epithelial-To-Mesenchymal plasticity in circulating tumor cell lines sequentially derived from a patient with colorectal cancer[END_REF] The role and importance of mesenchymal CTCs remain unsolved. Most data on the prognostic relevance of CTCs are based on assays using epithelial markers. 25 26 Cells classified as 'mesenchymal CTCs' are frequently identified by unspecific markers also expressed on normal blood cells. Further characterization of mesenchymal CTCs is required to determine the degree of mesenchymal attributes of CTCs necessary to survive in the circulation or to initiate overt metastasis at distant sites and whether mesenchymal CTCs have a higher propensity to escape killing by from immune.

Although the majority of CTCs detectable in patients with cancer are single isolated cells, there is increasing evidence that the formation of aggregates among CTCs or clusters between CTCs and blood cells is important to metastatic progression 27 28 (figure 1). For example, leukocytes in heterotypic clusters can epigenetically reprogram the attached neighbor CTCs, which results in enhanced CTC survival and induces proliferation of CTCs. Moreover, blood platelets can shield CTCs from destruction of immune cells, induce EMT programs in CTCs and help CTCs to extravasate. These insights derived from CTC analyses could lead to novel therapeutic strategies to prevent metastatic progression.

Functional CTC analyses depend on suitable models. During the past decade a few cell lines derived from CTCs of patients with different tumor entities could be established. However, these were exceptional cases where the CTC counts were very high at the start of the culture. Nevertheless, these models provide insights into CTC biology and in particular might help to identify pathways specific for metastasis-competent CTCs, discover new CTC markers and unravel mechanisms of drug resistance of these CTCs. 10 29-31 Pantel K, Alix-Panabières C. J Immunother Cancer 2022;10:e005615. doi:10.1136/jitc-2022-005615

Open access

Analysis of CTCs might also lead to new insights whether invasive diagnosis procedures like tumor biopsies or surgery might lead to the mobilization of CTCs during these procedures, and whether this potential mechanical tumor cell dissemination has an influence on the development of metastases. In prostate cancer, preliminary data suggest the possibility that tissue biopsy might lead to an increase in CTC concentrations in some patients associated with a higher rate of biochemical recurrence. [START_REF] Joosse | Tumor-Associated release of prostatic cells into the blood after transrectal ultrasound-guided biopsy in patients with histologically confirmed prostate cancer[END_REF] Studies on larger and well-defined cohorts are needed to validate these provocative data and address the question whether tumor cells released into the blood by mechanical forces are able to survive, extravasate and form metastases at distant organs. Further analyses of CTCs in patients with cancer at the DNA, RNA and protein level offers now the opportunity to answer this question which has led to vivid controversial debates for several decades.

Immune escape mechanisms of CTCs

Blood is a hostile environment for CTCs. Although the primary tumor presumably sheds thousands of cells into the bloodstream every day, [START_REF] Chang | Mosaic blood vessels in tumors: frequency of cancer cells in contact with flowing blood[END_REF] only a very small percentage of these cells survive in the bloodstream and become detectable as CTCs in a blood sample. Here, we will discuss reports dealing with the interaction of CTCs and immune cells mediating escape of CTCs from immune surveillance (figure 2) affecting both the innate and adaptive immune system. The recognition and elimination of CTCs via natural killer (NK)-cell-mediated lysis can be prevented by the interaction between NK-cell receptor D on NK cells and MICA/MICB on CTCs. [START_REF] Cluxton | Suppression of natural killer cell NKG2D and CD226 anti-tumour cascades by platelet cloaked cancer cells: implications for the metastatic cascade[END_REF] T cell-mediated lysis can be inhibited by the interference with T-cell receptor recognition of MHC I molecules on CTCs, shielding by blood platelets that interfere at several levels (eg, induction of EMT or acquisition of a 'pseudonormal' phenotype conferring by the transfer of platelet-derived MHC I molecules), 35 36 the expression of inhibitory immune-checkpoint proteins like programmed cell death-ligand 1 (PD-L1); the expression of CD47 (do not eat me signal) on CTCs 37 38 that binds to its ligand signal-regulatory protein α expressed on macrophages and dendritic cells, inhibiting phagocytosis, [START_REF] Jaiswal | Cd47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis[END_REF] and an altered expression of the apoptotic proteins FAS and/ or FASL. [START_REF] Gruber | Relationship between circulating tumor cells and peripheral T-cells in patients with primary breast cancer[END_REF] In the following paragraphs, we will focus on escape mechanisms mediated by immune checkpoint inhibition as the most prominent mechanism for therapeutic interventions which has widely changed the therapeutic landscape in oncology.

Releasing the brakes on antitumor immune responses by targeting immune checkpoint molecules is the primary focus of recent immunotherapeutic treatment strategies. [START_REF] Sharma | The future of immune checkpoint therapy[END_REF] Within the immunological synapse, a multitude of inhibitory receptors have been identified. [START_REF] Zou | Inhibitory B7-family molecules in the tumour microenvironment[END_REF] Programmed cell death protein-1 (PD-1) and its ligand, PD-L1, have been one of the most prominent examples to antagonize immune escape mechanisms employed by tumor cells. PD-L1 limits immune effector functions by binding to its cognate receptor PD-1 expressed on tumor-specific T cells. [START_REF] Pardoll | The blockade of immune checkpoints in cancer immunotherapy[END_REF] CTC undergoing EMT appears to be associated with inferior clinical outcome, 44 45 and there is increasing evidence for cross talk between EMT-inducing molecules and PD-L1. [START_REF] Alsuliman | Bidirectional crosstalk between PD-L1 expression and epithelial to mesenchymal transition: significance in claudin-low breast cancer cells[END_REF][START_REF] Chen | Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression[END_REF][START_REF] Wang | Pd-L1 induces epithelial-tomesenchymal transition via activating SREBP-1c in renal cell carcinoma[END_REF] Interestingly, platelets are able to initiate and maintain EMT on CTCs by secretion of transforming growth factor-ß as well as support homing and outgrowth of DTCs in bone marrow (figure 2). [START_REF] Leblanc | Metastasis: new functional implications of platelets and megakaryocytes[END_REF] PD-L1 expression by EMT-CTCs has been shown to be associated with poor survival in curatively patients with resected non-small cell lung cancer (NSCLC). [START_REF] Manjunath | Pd-L1 expression with epithelial mesenchymal transition of circulating tumor cells is associated with poor survival in curatively resected non-small cell lung cancer[END_REF] Another recent study showed that cluster and single PD-L1 + EMT-CTCs subpopulations are of clinical significance in patients with metastatic breast cancer (MBC) and highlighted the importance of CTC phenotyping during treatment with eribulin. [START_REF] Polioudaki | Epithelial/Mesenchymal characteristics and PD-L1 co-expression in CTCs of metastatic breast cancer patients treated with eribulin: correlation with clinical outcome[END_REF] These data suggest that immune-checkpoint inhibitors might mitigate the additive effect of PD-L1 and EMT on clinical outcomes of patients with cancer and also how immune cells secreting EMT-inducing cytokines into the tumor microenvironment affect PD-L1 expression of tumor cells released into the circulation is subject of current investigations. 52 53 The idea of disabling immune escape mechanisms on tumor cells is now being explored in clinical trials by using immune blockade inhibitors. [START_REF] Pardoll | The blockade of immune checkpoints in cancer immunotherapy[END_REF] Efforts to restore latent antitumor immunity focuses on antibodybased interventions targeting cytotoxic T-lymphocytesassociated protein 4 or PD-1 on T lymphocytes and its principal ligand, PD-L1, on tumor cells. [START_REF] Sharma | The future of immune checkpoint therapy[END_REF] [START_REF] Shin | The evolution of checkpoint blockade as a cancer therapy: what's here, what's next?[END_REF] Additional immunomodulatory antibodies tested in clinical trials will further expand the spectrum of immune checkpoint blockade. 55 56 In view of the remarkable costs and the toxicity profiles of these therapies, predictive biomarkers able to discriminate responders from non-responders are urgently needed. In this context, CTC/PD-L1 assays should be tested as liquid biopsy for stratification and monitoring of patients with cancer undergoing immune checkpoint blockade. Our group has shown for the first time in 2015 that CTCs can express PD-L1 in patients with MBC: not all patients showed CTC-PD-L1 + , not all CTCs expressed PD-L1 in the same patients. [START_REF] Mazel | Frequent expression of PD-L1 on circulating breast cancer cells[END_REF] Subsequently, we evaluated in a prospective clinical trial the clinicopathological correlations and prognostic value of CTC-PD-L1 + in a new cohort of 72 patients with MBC. Unlike PD-L1 + tumor, CTC-PD-L1 + correlated to survival in MBC (clinical trial registration: NCT02866149). [START_REF] Jacot | Clinical correlations of programmed cell death ligand 1 status in liquid and standard biopsies in breast cancer[END_REF] More recently, we performed a similar study with 54 patients with advanced NSCLC (clinical trial registration: NCT02866149) and showed again that the presence of CTC-PD-L1 + is associated with poor prognosis (progression free survival and overall survival) in this cohort of patients. [START_REF] Sinoquet | Programmed cell death ligand 1-expressing circulating tumor cells: a new prognostic biomarker in non-small cell lung cancer[END_REF] Reappraisal of the role of PD-L1 expression by tumor tissue and by CTCs under anti-PD-1/PD-L1 treatment is necessary to evaluate its predictive value and potential role as a stratifying factor in strategies and trials for patients with MBC. Considering the detection of PD-L1 on CTCs, the current French clinical trial ALCINA2 (clinical trial Open access registration: NCT02866149) focuses on the clinical relevance of liquid biopsy (CTC-PD-L1 + , exosomes-PD-L1 + , phenotype of immune cells) in patients with NSCLC in the context of immunotherapy. Liquid biopsy should allow classification of patients suitable for checkpoint inhibitor therapy before entering into clinical trials. [START_REF] David | PD-L1 expression by circulating breast cancer cells[END_REF] How far CTCs can functionally take advantage of PD-L1 expression and which other mechanisms of immune escape are effective on CTCs/DTCs is currently under investigation.

Promotion of metastases by immune cells

It is well known that metastasis can be also supported by immune cells. [START_REF] Kitamura | Immune cell promotion of metastasis[END_REF] Here, we will focus on reports that have included measurements of CTCs, describing metastasispromoting tumor-immune cell interactions that can lead to the survival and proliferation of CTCs.

Regulatory T cells (T regs ) appear to also play an important role in tumor cell dissemination and formation of metastasis. [START_REF] Jiang | Elevated chronic inflammatory factors and myeloid-derived suppressor cells indicate poor prognosis in advanced melanoma patients[END_REF] More specifically, the expression of interleukin (IL)-1β and a concurrent increase in T reg frequency in the peripheral blood may be strong predictors of clinical outcome, [START_REF] Wilke | Prognostic significance of regulatory T cells in tumor[END_REF] and disease progression and occurrence of CTCs with recruitment of T regs and Open access myeloid-derived suppressor cells has been reported. 62 64 Thus, suppression of the peripheral antitumor immune response might support CTC survival and promote metastatic progression. A possible link between T regs and CTCs was indicated in MBC: CTC-positive patients had a higher frequency of CD4 + T regs in the peripheral blood together with a stem-like CTC phenotype. Furthermore, CTCs in patients with breast cancer exhibit a downregulation in mTOR, PARP, myc genes, and among others upregulation of FOXO3. [START_REF] Hensler | Gene expression profiling of circulating tumor cells and peripheral blood mononuclear cells from breast cancer patients[END_REF] These genes are potentially involved in induction of immune tolerance (eg, downregulation of PARP can enable increased T reg activity). [START_REF] Zhang | PARP-1 controls immunosuppressive function of regulatory T cells by destabilizing FOXP3[END_REF] Recent improvement in CTC culturing and the establishment of CTC-derived cell lines 31 67 might open new avenues for functional studies to unravel the influence of CTCs on the systemic T reg activation. [START_REF] Joosse | Biology, detection, and clinical implications of circulating tumor cells[END_REF] Neutrophils appear to play an important role for promoting CTC dissemination and survival. CTCs directly adhere on top of neutrophils, indicating that CTCs use immune cells to hitchhike during extravasation. [START_REF] Spicer | Neutrophils promote liver metastasis via Mac-1-mediated interactions with circulating tumor cells[END_REF] Multiple mechanisms have been proposed to be involved in this interaction, including the expression of the intercellular adhesion molecule-1 on CTCs and its binding to Mac-1 or β 2 -integrins on neutrophils, 68 69 with cytokines such as IL-8 as presumable mediators in this crosstalk. 70 71 Neutrophils can even actively entrap CTCs through the production of 'neutrophil extracellular trap' (NET), which describes a structure of extruded DNA and proteins on the surface of neutrophils. NETs can enhance CTC adherence and extravasation in target organs of metastatic spread. 70 72 Interestingly, neutrophils form heterotypic clusters with CTCs and promote their survival and ability to grow out to overt metastases. [START_REF] Szczerba | Neutrophils escort circulating tumour cells to enable cell cycle progression[END_REF] This team identified cell-cell junction and cytokine-receptor pairs that define CTC-neutrophil clusters, representing key vulnerabilities of the metastatic process. Cluster formation might also have influences on MHC-mediated antigen presentation [START_REF] Lin | Circulating tumor cells: biology and clinical significance[END_REF] and provide therefore a more general way to promote survival of CTCs.

Clinical translation-implementation of CTCs into clinical decision-making

The clinical relevance of CTCs in patients with cancer has been extensively reviewed (eg, 4 74 CTCs offer the advantage that information on all components of tumor cells including DNA, RNA and proteins can be obtained. Moreover, the analysis of intact tumor cells rather than fragments of circulating tumor DNA (ctDNA (released from apoptotic tumor cells seems to ensure that the information is really derived from viable tumor cells that can contribute to tumor progression. Here, we will provide a brief summary of the most prominent clinical applications and cite some reports that underline our statements. We apologize to all the authors who published excellent work in this area that we did not cite.

Early detection of cancer

Early detection of cancer is one of the key aims of blood biomarkers. Screening of populations at risk to develop cancer may help to identify and resect small malignant lesions before they have spread to regional lymph nodes or distant organs. Currently, this application is dominated by ultrasensitive methods to detect minute amounts of cell-free DNA in blood plasma, [START_REF] Heitzer | Current and future perspectives of liquid biopsies in genomics-driven oncology[END_REF] while the incidence of CTCs detectable at early cancer stages is usually too low using current technologies. However, increases in the sensitivity of CTC detection might be achieved by the analysis of larger blood volumes. [START_REF] Keller | Unravelling tumour heterogeneity by single-cell profiling of circulating tumour cells[END_REF] For early detection of cancer, the future will be to combine different circulating biomarkers: tumor-derived or tumor-induced but also the immune cells. [START_REF]The future of liquid biopsy[END_REF] Monitoring of MRD-surveillance After initial cancer diagnosis, many patients undergo surgical resection of their primary lesion and subsequent adjuvant radiotherapy or systemic therapy (eg, chemotherapy) aimed to eradicate locoregional or systemic spread of metastatic cells. CTC detection at initial diagnosis in a variety of solid tumors can help to determine the subsequent risk of developing metastatic relapse. 77 78 Moreover, monitoring of patients with cancer ('surveillance') over time after initial adjuvant therapy by repeated blood analyses has opened new avenues for early detection of signs that indicate the presence of active MRD that poses a risk for overt metastatic relapse. CTC counts obtained 2 or 5 years after initial adjuvant therapy in breast cancer can identify patients with MRD who have a higher risk to develop metastatic relapse within the next 5 years of further follow-up. 8 9 Since the life time of CTCs in blood is rather short (hours), these CTCs are not derived from the primary lesion resected many years ago but most likely are derived from small micrometastatic lesions. Further molecular analyses of these CTCs open a new avenue for deciphering the mechanisms that control the outgrowth of these lesions that may have been in a dormant or latent state for several years.

Monitoring of therapy efficacy in metastatic disease

Changes in CTC counts during therapy of patients with solid tumors such as breast cancer are associated with response to therapy. In breast cancer, CTC counts predicted progression of patients with metastatic disease much earlier and with high accuracy than the conventional blood serum proteins used as 'tumor markers' (eg, carcinoembryonic antigen (CEA)). [START_REF] Bidard | Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data[END_REF] Similar results were obtained in advanced prostate cancer where rapid declines in CTC counts during chemotherapy or antiandrogen therapies provided information on therapy responses independent from the standard serum marker prostate-specific antigen (PSA). [START_REF] Pantel | Circulating tumor cells in prostate cancer: from discovery to clinical utility[END_REF]

Identification of therapeutic targets and resistance mechanisms

Besides quantitative assessment of CTCs, the molecular characterization of these cells opened a new avenue for Open access the qualitative assessment of tumor burden in individual patients. Metastatic lesions are frequently not easy to probe by needle aspirations, which is a hurdle for getting personalized information of potential targets for therapy or resistance mechanisms. In contrast, blood samples can be easily obtained from every patient. Recent advances have made it possible to characterize CTCs at the DNA, RNA and protein level and obtain-in addition to ctDNA analyses which are restricted to genomic aberrations-a more comprehensive view of the alterations relevant to therapy. [START_REF] Pantel | Liquid biopsy: from discovery to clinical application[END_REF] The advantage of CTCs over ctDNA in the context of immunotherapies is that cell surface proteins relevant for the interaction of tumor cells with immune cells such as MHC antigens and immune check point inhibitors such as PD-L1 can be assessed at the single cell level. Moreover, the direct cross talk in heterotypic clusters of CTCs and immune cells can be studied and it may be important for the response to immunotherapies. This cellular information together with the information derived from DNA analysis (eg, tumor mutational burden) will increase our knowledge about the biology of resistance mechanisms to immunotherapies.

Nevertheless, a considerable fraction of patient with metastatic disease has not enough CTCs for subsequent molecular analysis, which points again to the need to develop and validate technologies that increase the CTC capture rates .

Conclusions and perspectives

We have discussed how research on CTCs can capture the steps of the metastatic cascade in patients with cancer in particular with regard to the interface with immune cells, and how this CTC research has translated into clinical applications that might lead to an improved personalized management of patients with cancer at various stages of their disease. Although most of these applications are so far only for research use in clinical studies and are not part of the current recommendations for clinical use in routine diagnostics, recently completed and ongoing interventional studies [START_REF] Pantel | Liquid biopsy: from discovery to clinical application[END_REF] will provide evidence on the clinical utility relevant for implementation into clinical decisionmaking. For the first time in this field, the METABREAST study (clinical trial registration: NCT01710605) proved the clinical utility of CTCs. In this interventional clinical trial, patients with first-line MBC were randomized between the clinician's choice and CTC count-driven choice. In the CTC arm, patients with ≥5 CTC/7.5 mL received chemotherapy, whereas patients with <5 CTC/7.5 mL received endocrine therapy as first-line treatment. In conclusion, the CTC count is a reliable biomarker method for choosing between chemotherapy and endocrine therapy as the first-line treatment in hormone receptor-positive HER2 -MBC.

Besides its potential value for improving patient's care, CTC research offers unique opportunities to unravel the evolution of metastatic progression in patients with cancer. This knowledge will complement the valuable information derived from experimental studies. In particular, the interactions between CTCs and the cells of the immune system deserves intense future investigation. Many research groups have analyzed either CTCs or profiled immune cells in patients with cancer but studies assessing both the tumor and host response are underrepresented. In this context, the lack of functional studies on CTCs might be overcome now by the establishment of conditions that allow in vitro cultures of CTCs and the availability of CTC-derived cell lines. 31 67 Emerging evidence shows that the immune system may have a dual role on CTCs. On the one hand, CTCs released into the bloodstream might be more vulnerable for immune-mediated elimination, and recent studies have gained new insights into the question how CTCs can counteract immune-mediated killing. On the other hand, immune cells like neutrophils can support the survival and growth of CTCs and thereby support metastatic development. Other components of the 'circulome' such as platelets, endothelial cells or cancer-associated fibroblasts may also affect the survival and/or capacity to extravasate into distant tissues. Thus, the assessment of heterotypic clusters between CTCs and host cells is an exciting area of future investigations. Interestingly, recent data suggest that the release and survival of CTCs in the circulation undergoes circadian changes, 81 82 which might have also important implications for the clinical use of CTCs as biomarkers. The future will tell us whether we identified the ultimate selective filter in cancer progression-CTCs releasing during the rest phase [START_REF] Thomas | Nocturnal circulating tumor cells: the ultimate selective filter in cancer progression?[END_REF] -and more importantly, whether this nocturnal CTC dissemination is due to the role of sleep or circadian cycle. [START_REF] Dauvilliers | Dissemination of circulating tumor cells at night: role of sleep or circadian rhythm?[END_REF] For the clinical implementation of CTCs as liquid biopsy marker, the harmonization and technical validation of the plethora of different technologies is essential. The European CANCER-ID network has carried out such studies [START_REF] Neves | Proficiency testing to assess technical performance for CTC-Processing and detection methods in CANCER-ID[END_REF] which are now continued by the successor consortium called European Liquid Biopsy Society (www. elbs.eu) and will result into guidelines for the use of CTCs in cancer research. These activities are connected to other networks worldwide through the International Alliance for Liquid Biopsy Standardization. [START_REF] Connors | International liquid biopsy standardization alliance white paper[END_REF] A challenge for CTC-based therapeutic decision-making is their infrequent detection. High-blood volume analysis could provide a solution, as exemplified in a recent report on monitoring response to (immuno)therapy in lung cancer using leukapheresis. [START_REF] Tamminga | Leukapheresis increases circulating tumour cell yield in non-small cell lung cancer, counts related to tumour response and survival[END_REF] Diagnostic leukapheresis has been applied in selected CTC research studies [START_REF] Fischer | Diagnostic leukapheresis enables reliable detection of circulating tumor cells of nonmetastatic cancer patients[END_REF] to obtain thousands of CTCs from individual patients with cancer but their use in large scale clinical studies is very challenging. In vivo CTC capture devices may become an alternative in the future [START_REF] Kim | A temporary indwelling intravascular aphaeretic system for in vivo enrichment of circulating tumor cells[END_REF] but they are still at an experimental stage of development.

The availability of sufficient amounts of CTCs can also open new avenues for better understanding the response to immunotherapies. For example, besides correlative studies suggesting that PD-L1 expression on CTCs is more prognostically relevant than the expression on tumor tissue, 58 90 it is not clear whether CTCs are more Open access vulnerable to interventions targeting PD-L1 than cells in solid tumors. Platelet association, which may be more CTC specific, is also cited as providing immune protection. However, it is also unclear whether CTCs lacking platelet association are rapidly cleared by the innate immune system in patients with cancer. Future investigations may focus on these important questions. At present, there is a paucity of direct evidence and that we need more extensive analyses.

Taken together, the combined in-depth analysis of CTCs and circulating immune cells in relation to the changes occurring in the respective tumor microenvironment and clinical outcome of patients with cancer will provide new insights in the role of the immune system in metastatic progression.

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  Pantel K, Alix-Panabières C. J Immunother Cancer 2022;10:e005615. doi:10.1136/jitc-2022-005615

Figure 1

 1 Figure1Tumor-derived or tumor-induced circulating biomarkers and circulating immune and endothelial cells as liquid biopsy for precision medicine. In a non-invasive sample, complementary circulating biomarkers can be isolated from the blood, counted and characterized. In the plasma or serum, extracellular vesicles (EVs), proteins, circulating cell-free tumor nucleic acids (circulating tumor DNA (ctDNA), non-coding and messenger RNAs), tumor-educated platelets (TEPs) can be found. In the cellular fraction, circulating tumor cells (CTCs; single, homotypic and heterotypic microemboli), circulating immune cells (CICs) and circulating endothelial cells (CECs) as well as cancer-associated fibroblasts (CAF) can be detected. The Figure was created with BioRender.com. NK, natural killer.

  Pantel K, Alix-Panabières C. J Immunother Cancer 2022;10:e005615. doi:10.1136/jitc-2022-005615

Figure 2

 2 Figure 2 Immune-escape mechanisms of CTCs and collaboration with blood cells to facilitate their journey and optimize their survival in the peripheral blood. The schematic illustrates some of the main mechanisms of immune escape of CTCs, and the interactions between CTCs and immune cells in the peripheral blood. The interplay between NK cells and CTCs is shown, with the interaction between NKG2D on NK and MICA/MICB on CTCs, preventing recognition and elimination of the cells via NK-cell-mediated lysis. Different strategies of CTC escape: (1) the interference with TCR recognition of MHC I molecules; (2) the maintenance of the EMT phenotype resulting from TGFß secretion by the platelets recovering CTCs; (3) the expression of the inhibitory immune-checkpoint protein PD-L1; (4) the presentation of the 'don't eat me' signaling receptor CD47 and (5) an altered expression of the apoptotic proteins FAS and/or FASL. The Figure was created with BioRender.com. CTC, circulating tumor cell; EMT, epithelial-to-mesenchymal transition; FASL, FAS ligand; MHC I, MHC class I; MICA, MHC I polypeptide-related sequence A; MICB, MHC I polypeptide-related sequence B; NK, natural killer; NKG2D, NK-cell receptor D (also known as NKG2-D type II integral membrane protein); PD-1, programmed cell-death protein 1; PD-L1, programmed cell death-ligand 1; SIRPα, signal-regulatory protein α; TCR, T-cell receptor; TGFβ, transforming growth factor β.

  , 2023 at Univ of Montpellier. Protected by http://jitc.bmj.com/ Pantel K, Alix-Panabières C. J Immunother Cancer 2022;10:e005615. doi:10.1136/jitc-2022-005615

  , 2023 at Univ of Montpellier. Protected by http://jitc.bmj.com/ Pantel K, Alix-Panabières C. J Immunother Cancer 2022;10:e005615. doi:10.1136/jitc-2022-005615

J Immunother Cancer: first published as 10.1136/jitc-2022-005615 on 14 December 2022. Downloaded from

copyright. on October 17, 2023 at Univ of Montpellier. Protected by http://jitc.bmj.com/ J Immunother Cancer: first published as 10.1136/jitc-2022-005615 on 14 December 2022. Downloaded from

Funding CA-P and KP received funding from the European IMI research project CANCER-ID (115749-CANCER-ID), European Union Horizon 2020 Research and Innovation program under the Marie Skłodowska-Curie grant agreement No 765492 and ERA-NET EU/TRANSCAN 2 JTC 2016 PROLIPSY. CA-P is also supported by The National Institute of Cancer (INCa, http://www.e-cancer.fr) and SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553. KP also received funding from Deutsche Krebshilfe (Nr. 70112504), Deutsche Forschungsgemeinschaft (DFG) SPP2084 µBone and ERC Advanced Investigator Grant INJURMET (Nr. 834974).

Competing interests KP and CA-P have received honoraria from Menarini and both authors have patent applications related to CTC technologies.