Different Involvement of Vimentin during Invasion by Listeria monocytogenes at the Blood–Brain and the Blood–Cerebrospinal Fluid Barriers In Vitro

The human central nervous system (CNS) is separated from the blood by distinct cellular barriers, including the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CFS) barrier (BCSFB). Whereas at the center of the BBB are the endothelial cells of the brain capillaries, the BCSFB is formed by the epithelium of the choroid plexus. Invasion of cells of either the BBB or the BCSFB is a potential first step during CNS entry by the Gram-positive bacterium Listeria monocytogenes (Lm). Lm possesses several virulence factors mediating host cell entry, such as the internalin protein family—including internalin (InlA), which binds E-cadherin (Ecad) on the surface of target cells, and internalin B (InlB)—interacting with the host cell receptor tyrosine kinase Met. A further family member is internalin (InlF), which targets the intermediate filament protein vimentin. Whereas InlF has been shown to play a role during brain invasion at the BBB, its function during infection at the BCSFB is not known. We use human brain microvascular endothelial cells (HBMEC) and human choroid plexus epithelial papilloma (HIBCPP) cells to investigate the roles of InlF and vimentin during CNS invasion by Lm. Whereas HBMEC present intracellular and surface vimentin (besides Met), HIBCPP cells do not express vimentin (except Met and Ecad). Treatment with the surface vimentin modulator withaferin A (WitA) inhibited invasion of Lm into HBMEC, but not HIBCPP cells. Invasion of Lm into HBMEC and HIBCPP cells is, however, independent of InlF, since a deletion mutant of Lm lacking InlF did not display reduced invasion rates.

Introduction
Listeria monocytogenes (Lm) is a ubiquitous, Gram-positive, facultative anaerobe bacterium primarily recognized as a food contaminant with a high tolerance for various environmental hazards (low temperature, low pH, and high salinity) [1]. It is also an opportunistic pathogen able to enter and survive within various phagocytic and non-phagocytic cells and a causative agent of listeriosis in humans and livestock [1]. Although the overall yearly number of globally reported cases of infection with Lm is relatively small, it is invasion of Lm into HBMEC. Invasion of both HBMEC and HIBCPP cells was independent of InlF. Therefore, invasion of Lm into HIBCPP cells is mainly mediated by InlA and InlB, and possibly further virulence factors, but does not involve an interplay between vimentin and InlF. In contrast, our data point to an important, but InlF-independent, role of vimentin during invasion of HBMEC.

HBMEC and HIBCPP Cells Express Proteins Relevant for Lm Invasion
Lm utilizes proteins of the host cell for its own purposes, many of which are crucial for its successful colonization of and survival within the host [1]. Since the expression of these proteins is not uniform across all the cells of the body, it is necessary to ensure that the cell lines used as models for bacterial interaction, including adherence and invasion experiments, actually express them. Therefore, we analyzed the expression of proteins known to interact with the VFs of Lm investigated in this study (InlA, InlB, and InlF), which are Ecad, Met, and vimentin, respectively, in HBMEC and HIBCPP cells.
It has previously been shown that HIBCPP cells express Met and E-cadherin (Ecad) [10], and there is evidence that HBMEC express Met and vimentin [25,26]. Analysis via semiquantitative RT-PCR and western blotting ( Figure 1) confirmed and demonstrated that while HIBCPP cells express E-cadherin (a marker of epithelial cells), they do not express vimentin (a marker of mesenchymal cells). On the other hand, HBMEC do not express E-cadherin, but do express vimentin (due to being cells of mesenchymal origin). Both cell lines express Met (which is normally expressed in both endothelial and epithelial cells), and both of them express ZO-1 (a tight junction marker protein), although the level of transcription and expression of the two is somewhat different between the two cell lines ( Figure 1). crovascular endothelial cell lines could affect which listerial VFs would be important for the invasion into them [24]. In contrast to the BBB, the role of the BCSFB during InlFmediated CNS invasion has not yet been investigated.
We show that whereas HBMEC possess intracellular and surface vimentin (besides Met), HIBCPP cells do not express vimentin (but Met and Ecad). Accordingly, modulation of surface vimentin via treatment with WitA had no effect on HIBCPP cells but inhibited invasion of Lm into HBMEC. Invasion of both HBMEC and HIBCPP cells was independent of InlF. Therefore, invasion of Lm into HIBCPP cells is mainly mediated by InlA and InlB, and possibly further virulence factors, but does not involve an interplay between vimentin and InlF. In contrast, our data point to an important, but InlF-independent, role of vimentin during invasion of HBMEC.

HBMEC and HIBCPP Cells Express Proteins Relevant for Lm Invasion
Lm utilizes proteins of the host cell for its own purposes, many of which are crucial for its successful colonization of and survival within the host [1]. Since the expression of these proteins is not uniform across all the cells of the body, it is necessary to ensure that the cell lines used as models for bacterial interaction, including adherence and invasion experiments, actually express them. Therefore, we analyzed the expression of proteins known to interact with the VFs of Lm investigated in this study (InlA, InlB, and InlF), which are Ecad, Met, and vimentin, respectively, in HBMEC and HIBCPP cells.
It has previously been shown that HIBCPP cells express Met and E-cadherin (Ecad) [10], and there is evidence that HBMEC express Met and vimentin [25,26]. Analysis via semiquantitative RT-PCR and western blotting ( Figure 1) confirmed and demonstrated that while HIBCPP cells express E-cadherin (a marker of epithelial cells), they do not express vimentin (a marker of mesenchymal cells). On the other hand, HBMEC do not express E-cadherin, but do express vimentin (due to being cells of mesenchymal origin). Both cell lines express Met (which is normally expressed in both endothelial and epithelial cells), and both of them express ZO-1 (a tight junction marker protein), although the level of transcription and expression of the two is somewhat different between the two cell lines ( Figure 1).  Immunostaining and visualization using immunofluorescence microscopy was further used to confirm the expression of Ecad, Met, and vimentin in HIBCPP cells and HBMEC, and to analyze their cellular localization. Whereas HIBCPP cells present the known basolateral localization of Ecad, no staining for Ecad can be detected in HBMEC (Figure 2). In contrast, Met can be visualized in both HIBCPP cells and HBMEC ( Figure 3). Vimentin staining in HBMEC and HIBCPP cells confirms the presence of vimentin in HBMEC and its complete absence in HIBCPP cells ( Figure 4). Analysis of samples which were permeabilized before antibody staining and of samples without permeabilization shows that vimentin is localized both intracellularly and on the cellular surface of HBMEC ( Figure 4). Both HBMEC and HIBCPP cells display an expression of ZO-1 that is indicative of the presence of tight junction strands (Figures 2-4).
of interest in HIBCPP cells and HBMEC via western blotting. Gene names are indicated at the right side of the panels. The presented data were selected as representative of the average outcome of multiple performed experiments.
Immunostaining and visualization using immunofluorescence microscopy was further used to confirm the expression of Ecad, Met, and vimentin in HIBCPP cells and HBMEC, and to analyze their cellular localization. Whereas HIBCPP cells present the known basolateral localization of Ecad, no staining for Ecad can be detected in HBMEC ( Figure 2). In contrast, Met can be visualized in both HIBCPP cells and HBMEC ( Figure  3). Vimentin staining in HBMEC and HIBCPP cells confirms the presence of vimentin in HBMEC and its complete absence in HIBCPP cells ( Figure 4). Analysis of samples which were permeabilized before antibody staining and of samples without permeabilization shows that vimentin is localized both intracellularly and on the cellular surface of HBMEC ( Figure 4). Both HBMEC and HIBCPP cells display an expression of ZO-1 that is indicative of the presence of tight junction strands (Figures 2-4).

Modulation of Surface Vimentin Reduces the Invasion of Lm into HBMEC
Vimentin was implicated as an important target for Lm (strain 10403S) during the invasion of microvascular endothelial cells, including BBB models, in vitro (murine bEnd.3, human hCMEC, and HMEC-1 microvascular endothelial cell lines) and in vivo (murine infection model) and is also a known interaction partner for a number of bacterial pathogens [23,24,27]. To determine its relevance for invasion of Lm into HBMEC and HIB-CPP cells, the cells were infected with wild-type Lm EGD-e after pre-incubation with increasing concentrations of withaferin A (WitA), a known surface vimentin modulator [23,24].

Modulation of Surface Vimentin Reduces the Invasion of Lm into HBMEC
Vimentin was implicated as an important target for Lm (strain 10403S) during the invasion of microvascular endothelial cells, including BBB models, in vitro (murine bEnd.3, human hCMEC, and HMEC-1 microvascular endothelial cell lines) and in vivo (murine infection model) and is also a known interaction partner for a number of bacterial pathogens [23,24,27]. To determine its relevance for invasion of Lm into HBMEC and HIBCPP cells, the cells were infected with wild-type Lm EGD-e after pre-incubation with increasing concentrations of withaferin A (WitA), a known surface vimentin modulator [23,24].
It has previously been shown that Lm can invade HBMEC from the apical side [13], whereas invasion of HIBCPP cells by Lm occurs in a polar fashion from the basolateral side due to the basolateral localization of Ecad and Met [10]. Therefore, apical infection experiments with HBMEC were performed with cells grown in well cultures, whereas basolateral infection of HIBCPP cells was achieved in an inverted cell culture filter insert model, as previously described [10,28,29]. HIBCPP cells grown on cell culture filter inserts develop a strong barrier function, displaying a high transepithelial electrical resistance (TEER) and low permeability for FITC-labelled inulin. Following pre-incubation with WitA (1 µM, 5 µM or 10 µM) for 0.5 h, cells were infected with Lm EGD-e over a time period of 4 h. Subsequently, invasion rates were determined using double immunofluorescence microscopy. Analysis of barrier function of HIBCPP cells showed that average TEER values stayed above 300 Ω × cm 2 , and the average Inulin-FITC flux was below 4% except for HIBCPP cells infected and treated with 10 µM WitA, indicating some impact on barrier function under this condition (Supplementary Figure S1).
The results obtained during the infection experiments show a statistically significant WitA-concentration-dependent decrease in invaded Lm in HBMEC ( Figure 5A). In contrast, treatment of HIBCPP cells with WitA resulted in no statistically significant decrease in invasion with all WitA concentrations applied ( Figure 5B). Analysis via live/dead assay showed that co-incubation with WitA in presence of Lm does not affect the survival of HBMEC or HIBCPP cells (Supplementary Figure S2). It has previously been shown that Lm can invade HBMEC from the apical side [13], whereas invasion of HIBCPP cells by Lm occurs in a polar fashion from the basolateral side due to the basolateral localization of Ecad and Met [10]. Therefore, apical infection experiments with HBMEC were performed with cells grown in well cultures, whereas basolateral infection of HIBCPP cells was achieved in an inverted cell culture filter insert model, as previously described [10,28,29]. HIBCPP cells grown on cell culture filter inserts develop a strong barrier function, displaying a high transepithelial electrical resistance (TEER) and low permeability for FITC-labelled inulin. Following pre-incubation with WitA (1 µ M, 5 µ M or 10 µ M) for 0.5 h, cells were infected with Lm EGD-e over a time period of 4 h. Subsequently, invasion rates were determined using double immunofluorescence microscopy. Analysis of barrier function of HIBCPP cells showed that average TEER values stayed above 300 Ω × cm 2 , and the average Inulin-FITC flux was below 4% except for HIBCPP cells infected and treated with 10 µ M WitA, indicating some impact on barrier function under this condition (Supplementary Figure S1).
The results obtained during the infection experiments show a statistically significant WitA-concentration-dependent decrease in invaded Lm in HBMEC ( Figure 5A). In contrast, treatment of HIBCPP cells with WitA resulted in no statistically significant decrease in invasion with all WitA concentrations applied ( Figure 5B). Analysis via live/dead assay showed that co-incubation with WitA in presence of Lm does not affect the survival of HBMEC or HIBCPP cells (Supplementary Figure S2).

Different Internalins Are Required for Invasion of Lm into HBMEC and HIBCPP Cells
It was previously reported that listerial invasion into HBMEC requires InlB but not InlA and that Lm uses both InlA and InlB interdependently for efficient invasion into HIB-CPP cells [10,13]. We were now interested in evaluating the requirement of InlF during infection of HBMEC and HIBCPP in comparison to InlA and InlB. For this purpose, HBMEC grown in well cultures and HIBCPP cells cultivated in the inverted cell culture filter insert model were infected with the wild-type bacteria of the EGD-e strain (Lm EGDe wt) as well as with deletion mutants for internalins A, B, and F (Lm EGD-e ΔinlA, Lm

Different Internalins Are Required for Invasion of Lm into HBMEC and HIBCPP Cells
It was previously reported that listerial invasion into HBMEC requires InlB but not InlA and that Lm uses both InlA and InlB interdependently for efficient invasion into HIBCPP cells [10,13]. We were now interested in evaluating the requirement of InlF during infection of HBMEC and HIBCPP in comparison to InlA and InlB. For this purpose, HBMEC grown in well cultures and HIBCPP cells cultivated in the inverted cell culture filter insert model were infected with the wild-type bacteria of the EGD-e strain (Lm EGD-e wt) as well as with deletion mutants for internalins A, B, and F (Lm EGD-e ∆inlA, Lm EGD-e ∆inlB, Lm EGD-e ∆inlAB, and Lm EGD-e ∆inlF). Analysis of barrier function of HIBCPP cells showed that average TEER values remained at or around 200 Ω × cm 2 , and the average Inulin-FITC flux was around or below 4% (Supplementary Figure S3). The more recognizable impact on barrier function by Lm EGD-e wt and Lm EGD-e ∆inlF is probably due to the higher invasion of these strains.
The results confirm that Lm depends strongly on InlB for invasion into the HBMEC ( Figure 6A) and relies interdependently on both InlA and InlB for invasion into the HIBCPP cells ( Figure 6B). Unexpectedly, deletion of InlA also caused reduced invasion into HBMEC, although less pronounced than the deletion of InlB ( Figure 6A). Deletion of InlF did not cause a change in infection rates of either HBMEC or HIBCPP cells ( Figure 6).  Figure S3). The more recognizable impact on barrier function by Lm EGD-e wt and Lm EGD-e ΔinlF is probably due to the higher invasion of these strains. The results confirm that Lm depends strongly on InlB for invasion into the HBMEC ( Figure 6A) and relies interdependently on both InlA and InlB for invasion into the HIB-CPP cells ( Figure 6B). Unexpectedly, deletion of InlA also caused reduced invasion into HBMEC, although less pronounced than the deletion of InlB ( Figure 6A). Deletion of InlF did not cause a change in infection rates of either HBMEC or HIBCPP cells ( Figure 6).

Discussion
The BBB and the BCSFB are potential entry gates for Lm into the human CNS. Invasion of cells of either the BBB or the BCSFB presents a putative first step during brain entry by Lm, which could subsequently continue passing into the CNS. Whereas the microvascular endothelial cells of the brain are the main contributors to the BBB, barrier function at the BCSFB is executed by the choroid plexus epithelium [11,12]. In this study we used HBMEC and HIBCPP cells as established in vitro models of the BBB and the BCSFB, respectively [29][30][31]. An arsenal of VF has been described to be involved during host cell invasion by Lm [17]. Among these, the Inl protein family is of special interest since its members InlA, InlB, and InlF are implicated in the process of CNS entry [2,16]. InlA, InlB, and InlF have been shown to interact with Ecad, Met, and vimentin, respectively, on the surface of host receptors [18,19,23].
When investigating the roles of these Inls during host cell invasion, it is important to analyze whether the employed model systems express their respective interaction partners. Here, we show that HBMEC express Met and vimentin, but not Ecad (Figures 1-3). This confirms previous evidence for the expression of Met and vimentin in HBMEC [25,26]. In contrast, HIBCPP cells expressed Ecad and Met as shown previously [10], but vimentin was not detected (Figures 1-3). There are conflicting results concerning the expression of vimentin in the choroid plexus epithelium [32][33][34]. Primary mouse choroid

Discussion
The BBB and the BCSFB are potential entry gates for Lm into the human CNS. Invasion of cells of either the BBB or the BCSFB presents a putative first step during brain entry by Lm, which could subsequently continue passing into the CNS. Whereas the microvascular endothelial cells of the brain are the main contributors to the BBB, barrier function at the BCSFB is executed by the choroid plexus epithelium [11,12]. In this study we used HBMEC and HIBCPP cells as established in vitro models of the BBB and the BCSFB, respectively [29][30][31]. An arsenal of VF has been described to be involved during host cell invasion by Lm [17]. Among these, the Inl protein family is of special interest since its members InlA, InlB, and InlF are implicated in the process of CNS entry [2,16]. InlA, InlB, and InlF have been shown to interact with Ecad, Met, and vimentin, respectively, on the surface of host receptors [18,19,23].
When investigating the roles of these Inls during host cell invasion, it is important to analyze whether the employed model systems express their respective interaction partners. Here, we show that HBMEC express Met and vimentin, but not Ecad (Figures 1-3). This confirms previous evidence for the expression of Met and vimentin in HBMEC [25,26]. In contrast, HIBCPP cells expressed Ecad and Met as shown previously [10], but vimentin was not detected (Figures 1-3). There are conflicting results concerning the expression of vimentin in the choroid plexus epithelium [32][33][34]. Primary mouse choroid plexus epithelial cells express Ecad, ZO-1 and cytokeratin, but not vimentin, and display a barrier function [35]. In contrast, following immortalization expression of cytokeratin and Ecad is lost. Instead, expression of the mesenchymal marker proteins vimentin and N-cadherin is observed. Additionally, a mouse choroid plexus carcinoma cell line lacked expression of cytokeratin and Ecad and failed to establish junctional complexes, but was positive for vimentin and did not develop a barrier function [35]. We would like to point out that HIBCPP cells resemble the primary mouse choroid plexus epithelial cells since they express Ecad and ZO-1, but not vimentin, and develop a strong barrier function.
The intermediate filament protein vimentin is found primarily in mesenchymal (e.g., endothelial) cells and can be located both intracellularly and extracellularly. It serves multiple roles inside the cell, including but not limited to the provision of flexibility to the cytoskeleton, assistance in the anchoring of cellular organelles, and lipid droplet formation [36,37]. Extracellular vimentin is of special interest during pathogenic host cell invasion since it was found as an interaction partner for several intracellular pathogensdengue virus (via NS4a), E. coli K1 (via IbeA), group B Streptococcus (via BspC), and Lm (via InlF or independently of InlF) [23,24,[38][39][40]. The expression of vimentin by HBMEC, including the detection of surface-located vimentin (Figure 4), raised the question of to which extent vimentin is involved during the invasion of Lm into HBMEC. Treatment of HBMEC with increasing concentrations of withaferin A (WitA), a known surface vimentin modulator, caused a strong decrease in listerial invasion ( Figure 5). Similar findings have been described in human microvascular endothelial cells (HMEC-1) by Bastounis et al. [24], and a dose-dependent decrease in Lm invasion in different cell lines by WitA treatment was also demonstrated by Ghosh et al. [23]. It has also been published that treatment of HBMEC with WitA caused an increasing reduction of vimentin levels and blocked invasion by E. coli K1 via IbeA [38].
Analysis of different Inl deletion mutants confirmed previous findings that the deletion of InlA, InlB or both causes a similar decrease in bacterial invasion into HIBCPP cells, implying an interdependent mode of action for InlA and InlB [10]. Invasion into HBMEC was also strongly inhibited by deletion of InlB (Figure 6), again confirming previous findings [13]. Compared to the wildtype, we found a partial decrease in invasion of HBMEC by a ∆InlA mutant. This result differs from the data of Greiffenberg et al., who found no impact of InlA deletion on invasion into HBMEC [13]. Notably, Parida et al. described a partial inhibition of Lm invasion into human umbilical vein endothelial cells (HUVECs) by deletion of InlA [41]. We speculate that the observed variances are due to differences in experimental setups regarding the exact nature of HBMEC and bacterial strains employed. When analyzing the impact of InlF deletion, we found that InlF was not required for invasion of Lm into both HBMEC and HIBCPP cells ( Figure 6). We hypothesize that the remaining capacity of Lm for invasion into HIBCPP cells in absence of InlA and InlB is mediated by listerial VFs other than InlF, such as further Inls (InlC, InlGHE or others) or LLO.
Whereas the role of InlF during infection of choroid plexus epithelial cells has not been investigated before, InlF was implicated as a key listerial factor in interaction with vimentin during invasion of Lm into the brain microvascular endothelial cell line hCMEC/D3 [23]. Interestingly, these experiments were performed under conditions of inhibition of Rhoassociated protein kinases (ROCKs) [23], which are involved in actin cytoskeleton rearrangements [42], since previous data had shown that inhibition of ROCK activity results in cell-type-specific increased host cell binding and entry by Lm [43]. For example, ROCK inhibition led to increased binding and invasion in murine fibroblast (L2) and hepatocyte (TIB 75) cell lines, which was dependent on InlF. ROCK inhibition also increased binding and entry in human fibroblast (WI38) and only binding in epithelial (HeLa) cell lines, but this effect was independent from InlF [43]. In a recent publication, Ling et al. found that InlF contributes to adhesion and invasion of macrophages but was not involved in adherence or invasion during infection of five non-phagocytic cell lines. However, a function of ROCK was not analyzed [44]. Similarly, we observe InlF independent invasion of Lm into HBMEC and HIBCPP cells, but determination of a possible role of ROCKs would require further investigation.
The data of Ghosh et al. pointed to the roles of InlF and vimentin during listerial colonization of the brain [23]. In contrast, the vimentin-mediated infection of HMEC-1 cells described by Bastounis et al. was independent of InlF [24]. The authors noted that possible significant, but as-of-yet unidentified, differences between various human microvascular endothelial cell lines could affect which listerial VFs are important for invasion. Since a decreased amount of surface vimentin caused decreased adhesion of both Lm and the non-pathogenic relative Listeria inoccua, a potential interaction partner could be expressed by both bacterial species. Furthermore, a role of cell surface vimentin as a host cell receptor mediating extracellular matrix stiffness-dependent adhesion of Lm was suggested [24]. Since invasion of Lm into HBMEC was sensitive to WitA but independent of InlF, we speculate that this process might involve a role of vimentin related to regulation of extracellular matrix stiffness.
In summary, we show that HBMEC and HIBCPP cells differentially express target proteins for listerial virulence factors, with vimentin only present on HBMEC. Deletion of InlF shows that Lm does not require InlF for invasion of HBMEC or HIBCPP cells. Since HIBCPP cells do not express vimentin, InlF-and vimentin-independent invasion of Lm is mainly mediated by InlA and InlB and possibly further virulence factors. Entry into HBMEC requires an InlF-independent role of vimentin, which might involve the previously described regulation of extracellular matrix stiffness. This process could require specific virulence factors of Lm, possibly binding to vimentin, further underlining the differences of listerial invasion at the BBB and BCSFB in vitro.
The isogenic strain EGD-e ∆inlF was constructed by generating the 5 (using primers P1 and P2) and the 3 (using primers P3 and P4) flanking regions of inlF (lmo0409). The primers used to generate the flanking regions are shown in Table 1. The PCR fragments were purified and ligated into the temperature-sensitive suicide vector pAUL-A. The presence of the insert was screened by using the primer pair M13 Forward/M100 and M13 Reverse/M101 ( Table 1). The annealing temperature of the PCR reactions was 55 • C. Subsequently, E. coli InvαF electrocompetent cells were transformed with the plasmid containing the correct insert. Plasmid DNA of pAUL-A bearing the fragments was isolated from the recombinant E. coli cells and used to transform L. monocytogenes to generate the chromosomal ∆inlF deletion mutant as described previously [47]. The deletion of inlF was confirmed by sequencing the PCR products by using the flanking primers P7 and P8. All strains were stored in brain heart infusion (BHI) medium (BD, Franklin Lakes, NJ, USA) containing 30% glycerol. In preparation for infection experiments bacteria were incubated in BHI medium for 6 h at 37 • C under slight agitation to mid-logarithmic phase. Subsequently, bacteria were washed two times in serum-free medium (SFM) and adjusted to an optical density at 600 nm (OD 600 ) values corresponding to a concentration of 1 × 10 8 CFU/mL (EGD-e wt, OD 600nm of 0.2; EGD-e ∆inlA, OD 600nm of 0.4; EGD-e ∆inlB, OD 600nm of 0.1; EGD-e ∆inlAB, OD 600nm of 0.6; EGD-e ∆inlF, OD 600nm of 0.2).
For infection experiments HIBCPP cells were grown in the inverted cell culture insert model as previously described [28,29].

Measurement of Barrier Function
TEER measurements of HIBCPP cell layers grown on cell culture inserts was performed at the beginning and the end of experiments with a STX 01 chopstick voltohmmeter electrode connected to a Millicell ® -ERS voltohmmeter (Millipore, Schwalbach, Germany). Measurement of the permeability of HIBCPP cell layers was performed following infection experiments to evaluate the status of the cellular barriers of cells on individual cell culture insert filters. Before the infection of the cells at the start of the experiment FITC-labelled inulin (average molecular weight of 3000 to 6000; Sigma, Deisenhofen, Germany) was added into the upper compartment of the cell culture filter inserts. At the end of the experiment, the passage from the upper compartment to the lower compartment was monitored with a Tecan Infinite M200 Multiwell reader (Tecan, Switzerland) as described previously [10]. Barriers with a permeability below 4% during the course of the experiment were considered stable.

Semiquantitative RT-PCR
For RNA preparation, cells were initially washed with PBS. Subsequently, RNeasy Micro or RNeasy Mini Kits (depending on the available starting material across experiments) were used in accordance with the manufacturer's (Qiagen, Hilden, Germany) instructions for RNA isolation. Following the purification, the concentration and quality of purified RNA were determined with a NanoDrop ® ND1000 spectrophotometer (Peqlab Biotechnology, Erlangen, Germany). For the generation of cDNA, the AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA, USA) was used with 500 ng of total RNA in accordance with the manufacturer's instructions. The obtained cDNA was used as a template for semi-quantitative PCR with the Taq DNA Polymerase Kit (Qiagen, Hilden, Germany). To enable relative assessment of the initial quantity of the gene material in the sample, PCR product was analyzed via gel electrophoresis and ethidium bromide staining after 26 and 30 PCR cycles, respectively. The following PCR conditions were used: initial denaturation (94 • C, 2 min) and subsequently 30 cycles of denaturation (94 • C, 30 s), annealing (58-62 • C, depending on the primer pair, 30 s), extension (72 • C, 1 min). Subsequently, a final extension (72 • C, 7 min) was performed. The following PCR primers were used: vimentin, AGAGAGAGGAAGCCGAAAAC (forward), TGGATTTCCTCTTCGTGGAGTT (reverse); E-cad, CCTGCCAATCCCGATGA (forward), TGCCCCATTCGTTCAAGTA (reverse); Met ATCTTGGGACATCAGAGGGT (forward), TCGTGATCTTCTTCCCAGTGA (reverse); ZO-1, GCCAAGCAATGGCAGTCTC (forward), CTGGGCCGAAGAAATCCCATC (reverse).

Immunoblot
To obtain protein extracts for Western blotting, cells were washed with PBS twice and lysed in RIPA buffer after the removal of all excess PBS. Proteins of 10 to 20 µg of protein extract were separated on 4-12% Bis-Tris (Invitrogen, Karlsruhe, Germany) and subsequently transferred onto nitrocellulose membranes via electroblotting. Membranes were incubated for 1 h in blocking solution (5% milk powder solution in dH 2 O), and proteins were detected with the following antibodies: chicken anti-vimentin (BioLegend, San Diego, CA, USA), goat anti-Met (Abcam, Cambridge, UK), mouse anti-Ecad (BD, Franklin Lakes, NJ, USA), and rabbit anti-ZO-1 (Invitrogen, Carlsbad, CA, USA). The Immobilon Western Kit (Millipore, Schwalbach, Germany) was used for the visualization of detected proteins.

Infection of HBMEC and HIBCPP Cells with Lm
During the infection experiments, bacteria were co-incubated with the cells in either 1% HIBCPP medium or 1% HBMEC medium, depending on the cell line used. HBMEC at a confluency and HIBCPP cells grown in the inverted cell culture insert model with a TEER between 270 and 800 Ω × cm 2 were infected with the different Lm strains at a multiplicity of infection (MOI) of 100 for 4 h. When infecting HIBCPP cells, bacteria were added into the upper compartment of the filter system to allow infection from the basolateral cell side as previously described [28,29]. In some experiments, WitA (Merck, Darmstadt, Germany) was added to the cells in various concentrations 30 min before addition of the bacteria.

Measurement of Cell Viability
A LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells (Invitrogen, Karlsruhe, Germany) was used in experiments applying WitA and bacterial infection to track the viability of HBMEC and HIBCPP cells throughout the experimental time period. The kit was used according to the manufacturer's instructions. For documentation of the results, fluorescence microscopic pictures were taken.

Statistical Analysis
Statistical analysis of the data presented in this study was performed with SAS, release 9.4 (SAS Institute Inc., Cary, NC, USA). For results presented in Figures 5 and 6, a one-way ANOVA was performed to compare mean values of multiple differently treated groups. In case of a significant result, Dunnett's post hoc test was used in order to compare each treated group with the control group or the wild type, respectively.


s2fieldsofstudy: Biology Chemistry

text:
Easily automated radiosynthesis of [18F]P10A-1910 and its clinical translation to quantify phosphodiesterase 10A in human brain

Our previous work showed that [18F]P10A-1910 was a potential radioligand for use in imaging phosphodiesterase 10A (PDE10A). Specifically, it had high brain penetration and specific binding that was demonstrated in both rodents and non-human primates. Here, we present the first automatic cGMP-level production of [18F]P10A-1910 and translational PET/MRI study in living human brains. Successful one-step radiolabeling of [18F]P10A-1910 on a GE TRACERlab FX2N synthesis module was realized via two different methods. First, formulated [18F]P10A-1910 was derived from heating spirocyclic iodonium ylide in a tetra-n-butyl ammonium methanesulfonate solution. At the end of synthesis, it was obtained in non-decay corrected radiochemical yields (n.d.c. RCYs) of 12.4 ± 1.3%, with molar activities (MAs) of 90.3 ± 12.6 μmol (n = 7) (Method I). The boronic pinacol ester combined with copper and oxygen also delivered the radioligand with 16.8 ± 1.0% n. d.c. RCYs and 77.3 ± 20.7 GBq/μmol (n = 7) MAs after formulation (Method II). The radiochemical purity, radionuclidic purity, solvent residue, sterility, endotoxin content and other parameters were all validated for human use. Consistent with the distribution of PDE10A in the brain, escalating uptake of [18F]P10A-1910 was observed in the order of cerebellum (reference region), substantial nigra, caudate and putamen. The non-displaceable binding potential (BP ND) was estimated by simplified reference-tissue model (SRTM); linear regressions demonstrated that BP ND was well correlated with the most widely used semiquantitative parameter SUV. The strongest correlation was observed with SUV(50–60 min) (R 2 = 0.966, p < 0.01). Collectively, these results indicated that a static scan protocol could be easily performed for PET imaging of PDE10A. Most importantly, that [18F]P10A-1910 is a promising radioligand to clinically quantify PDE10A.

Introduction
Phosphodiesterases (PDEs) are widely present in various cells and catalyze the hydrolysis and inactivation of cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP). Moreover, they synergize with both ATP and GTP to regulate the content of these second messengers (Charbonneau et al., 1990;Beavo, 1995;Francis et al., 2011). Therefore, PDEs play a critical role in various physiological processes involving cyclic nucleotide signaling (Bender and Beavo, 2006). In mammals, different cell types express different PDEs, which can be divided into 11 different subtypes according to their distribution and respective base sequences (Omori and Kotera, 2007). One-PDE10A-is expressed less in peripheral system, however, abundantly in striatum and substantia nigra in the central nervous system (CNS) (Loughney et al., 1999;Coskran et al., 2006;Xie et al., 2006). Medium spiny neurons in the striatum are the primary input point to the basal ganglia and they integrate both dopaminergic and glutaminergic signals with cortical reception. Ultimately, this leads to the performance of associated motor and cognitive activities (Kötter, 1994;Girault, 2012). Therefore, abnormal expression and dysfunction of PDE10A would affect the signal transmission in basal ganglia circuit. This would lead to nigrostriatal neuronal pathway-related mental and functional disorders, such as schizophrenia, Huntington's disease (HD), Parkinson's disease (PD) and Alzheimer's disease (AD) (Geerts et al., 2017;Pagano et al., 2019). Thus, PDE10A is considered an attractive biomarker for the diagnosis and treatment of these diseases (Wilson et al., 2017).
As a non-invasive medical evaluation method, positron emission tomography (PET) can provide valuable, in vivo information for exploring a host of physiological and pathological mechanisms. As a result, PET is useful in assessing the progression of related diseases, as well as help accelerate the development of targeted drugs (Gulyás et al., 1996;Cecchin et al., 2021). Given the clinical significance of PDE10A and increasing interest in PET applications, several radioligands targeted PDE10A have been reported (Laere et al., 2013;Barret et al., 2014;Russell et al., 2014;Marques et al., 2016;Mori et al., 2019;Xiao et al., 2022). In particular, a PET ligand named [ 18 F]MNI659 ([ 18 F]1, Figure 1) has been explored in healthy human volunteers and has shown high brain binding specificity in the brain as well as good pharmacokinetics. It has also been shown to have a non-displaceable binding potential (BP ND ) ranging from 3.0 to 5.0 in healthy people aged 29-47 years (Barret et al., 2014). Clinical PET studies using [ 18 F]1 were further conducted on patients with HD (Russell et al., 2014;Russell et al., 2016). However, the [ 18 F] fluoroethoxyl structure in [ 18 F]1 is peculiarly prone to metabolism by cytochrome-P450 monooxygenase system, resulting in unexpected radioactive aggregation in the brain and skull that interferes with imaging quantification (Russell et al., 2014). To compensate for metabolic deficiency, the side chain [ 18 F]fluoroethoxy was replaced by [ 18 F]fluoromethoxy-d 2 . The modified radioligand [ 18 F]2 ( Figure 1) was more stable in vivo, and its corresponding BP ND was also improved (Mori et al., 2019). Nevertheless, the difficulty and complexity of radiolabeling prevented its further development (Stepanov et al., 2018;Mori et al., 2019).

Chemical syntheses of precursors
As shown in Scheme 1, the radiolabeling precursors SCIDY 5 and BPE 6 were prepared from the common intermediate aryl iodide 4, which was obtained efficiently in gram scale according to our previously reported synthetic procedures (Xiao et al., 2022). Particularly, oxone was used to oxidize 4 into the corresponding iodo (III) intermediate in a solution of TFA/CHCl 3 . After evaporation of the acid solvent, the crude mixture was directly dissolved in ethanol, then coupled with SPIAd under basic conditions to deliver the desired SCIDY 5 as a colorless powder in 69% yield. Additionally, BPE 6, a baby pink solid, was synthesized via Pd-catalyzed cross-coupling with bis(pinacolato)diboron at a comparable yield of 62%. When maintained at −78°C, both precursors remained stable radiolabeling efficiency for at least 6 months.

Radiofluorination optimization in a GE TRACERlab FX2N module
We set out the automatic radiolabeling with SCIDY 5 according to our previously reported method (Xiao et al., 2022), resulting in only trace to low non-decay corrected radiochemical yield (n.d.c. RCY, 1~3.2%). This was not enough to meet our demand for clinical use. This unexpected difference in outcomes between manual operation and automated performance inspired us to re-optimize the radiolabeling conditions directly on a GE TRACERlab FX2N module equipped in our facility. As shown in Figure 2A, after adjusting the type of solvent, we found that 0.5 ml MeCN gave the highest yields (8%). Reaction temperatures were then screened and 110°C was found to be stable at the tube sealing condition in the module ( Figure 2B). Our continuous efforts into researching the best SCIDY strategy revealed that basic conditions may play a critical role in the balance between precursor stability and radiolabeling efficiency (Rotstein et al., 2016;Wang et al., 2017;Liang et al., 2019;Wang et al., 2020;Xiao et al., 2022). Given this, two commonly used quaternary alkylammonium salts tetraethylammonium bicarbonate (TEAB) and tetrabutylammonium methanesulfonate (TBAOMs) (Christopher et al., 2012;Inkster et al., 2016;Shi et al., 2016) were further evaluated in automation with different amounts. After, the latter was elected as the best additive at a loading of 12 mg ( Figure 2C). Collectively, we identified that the combination of 12 mg TBAOMs and 2 mg SCIDY 5 in CH 3 CN at SCHEME 1 Syntheses of radiolabeling precursors SCIDY 5 and BPE 6. Reagents and conditions: (i) oxone, TFA/CHCl 3 , room temperature, 1 h; then SPIAd, 10% Na 2 CO 3 (aq), EtOH, room temperature, 1 h, 69% for 5; (ii) Bis(pinacolato)diboron, Pd(dppf) 2 Cl 2 , KOAc, DMSO, 80°C, 16 h, 62% for 6.
Frontiers in Bioengineering and Biotechnology frontiersin.org 110°C for 10 min provided the desired radioligand [ 18 F]3 with stable and high enough automatic labeling efficiencies. Copper-induced nucleophilic 18 F labeling, which was developed by Gouverneur's (Tredwell et al., 2014;Taylor et al., 2017) and Scott's group (Chu and Qing, 2014), is another strategy for radiofluorination of a non-active aromatic ring. There is easy access to the precursor BPE via Miyaura boration (Ishiyama et al., 1995), which would be complementary to SCIDY synthesis (Rong et al., 2021). Because of this, we also systemically evaluated BPE radiolabeling feasibility in our FX2N module. A big challenge in this process was controlling air bubbling; thus, we tested the effect of air and found that the highest RCYs were obtained when the mixture was ventilated three times during the radiolabeling process ( Figure 2D). Heating the reaction at 120°C was also shown to be optimal ( Figure 2E). By elevating the ratio of N, N-dimethylacetamide (DMA) from 50% to 100%, n.d.c. RCYs were improved to 16.5%, while increasing the solvent volume decreased RCYs dramatically ( Figure 2F). Finally, the molar ratios of BPE 6 and copper salt were optimized. Although low RCYs were observed when decreasing the amount of copper salt to ca. 20%, comparable yields were obtained with the ratio ranging from 1:2.5 to 1:1 ( Figure 2G). Together with the above results and considering efforts to minimize consumption of the precursor, we determined that marriage of 2.5 mg BPE 6, 7 mg copper salt (molar ratio was 1:2.5), 0.5 mg TEAB and 0.3 ml DMA at 120°C for 20 min would allow for the best automatic labeling of [ 18 F]3 with the assistance of three-times air bubbling.
Frontiers in Bioengineering and Biotechnology frontiersin.org C18 Sep-Pak with 1.5 ml ethanol and 15.5 ml saline containing 1.55 mg ascorbic acid. The final solution was filtered by a Millex ® -GV filter and collected in a 25 ml sterile vial. The n.d.c. RCYs obtained by these two different labeling methods were 12.4 ± 1.3% and 16.8 ± 1.0%, respectively, both of which met our requirements for further clinical application.

Quality control and acceptance criteria for human use
According to the USP General Chapter <823> and ChP General Chapter <2321>, we conducted a comprehensive and meticulous validation and quality control for the six consecutive batches of [ 18 F]3, three of them prepared from Method I and all others obtained via Method II. The acceptance criteria and detection results are depicted in Table 1. Immediately after formulation and sterile filtration, a series of real-time assessments were performed. The solution was observed through lead glass to ensure that it was either a colorless or light yellow and transparent liquid without any suspended particles. Using standard pH paper, pH values were determined to be between 5 and 6. The retention time of [ 18 F]3 was determined to be 5.3 ± 0.2 min as measured by an analytical HPLC system equipped with a WondaSil C18-WR column (GL Science, 4.6 × 150 mm, 5 μm), and eluted with CH 3 CN/H 2 O (7:3, v/v) with a flow rate of 1.0 ml/min. The tandem UV (λ = 254 nm) and Gamma detectors showed that radiochemical purities were greater than 95%, and the radioligand structure was confirmed by co-injection using standard reference (Supplementary Figures S5, S6). The above system with 20 μL of injection loop was used for cold masses estimation in the formulated solution. Molar activities were determined within a range of either 77.7-104.9 GBq/ μmol (Method I) or 56.6-98.0 GBq/μmol (Method II) as calculated by a standardized curve derived from the reference compounds.
By radioactivity testing every 30 min in a dose calibrator (7-8 total times), the half-life was found to range from 105-115 min. Only the 0.511 MeV and occasionally Frontiers in Bioengineering and Biotechnology frontiersin.org 1.022 MeV energy peaks were observed by spectrometer. No long-lived isotopes were observed by the analysis on a HPGE detector after 18 F-decay. The formulated product was free of pyrogens (Charles River Endosafe PTS) and sterile. No copper residue was detected using ICP-MS with detection limits of 0.1 mg/L, which ensures that the tracer injection has no effect on the physiological copper level in human body (Trumbo et al., 2001;Leong et al., 2020). Volatile organic solvent analysis was conducted using GC-FID disclosing residual acetone, acetonitrile, ethanol and DMA that were well within the limits required by the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use.
Although both methods could deliver qualified radiotracer, considering the radiolabeling period, synthetic cost and quality control convenience, we chose Method I for routine productions in clinics.

Quantitative assessment of PDE10A in living human brains
After signing an informed consent forms, eight healthy volunteers (HVs) aged 24-37 years (women n = 4, men n = 4) underwent brain 3.0 T magnetic resonance imaging (MRI)  scan for anatomical analysis, followed by a dynamic 0-60 min PET/ CT scan with injection of the radioligand [ 18 F]3. Demographic data and radioligand information for each study are summarized in Table 2, and the outline of this translational performance is illustrated in Figure 4A. According to our efforts on automatic radiofluorination, [ 18 F]3 was produced on the day of the PET/CT scan using the GE TRACERlab FX2N module in a well-shielded hot cell. The reconstructed data were shown with averaged standardized uptake values (SUV 0-60min ) and revealed an obvious, heterogenous radioactivity distribution pattern in the human brain. The highest accumulation was found in the caudate nucleus and putamen, followed by the substantia nigra. The lowest accumulation of tracer uptake was detected in the cerebellum, implying that this region had low PDE10A expression ( Figure 4B).
To assess the binding specificity of [ 18 F]3 to PDE10A in human brain, regional time-activity curves (TACs, Figure 4C) were used for kinetic modeling. For this modeling, the cerebellum was used as the reference region Liu et al., 2018) and nondisplaceable binding potential (BP ND ) was estimated by simplified reference-tissue model (SRTM). As shown in Figure 4D, the highest BP ND were observed in the putamen, followed by the caudate nucleus (putamen vs caudate, 5.2 ± 0.9 vs. 2.7 ± 0.4, p < 0.001). Thus, the distribution of [ 18 F]3 in the human brain closely matched the binding patterns that had been previously observed in both rodent and nonhuman primate (NHP) brain (Xiao et al., 2022). As expected, no skull radioactivity accumulation was detected, demonstrating that [ 18 F]3 was more stable than [ 18 F]1. No defluorination occurred in vivo. Promisingly and despite its small size, the nucleus substantia nigra also showed good tracer accumulation with BP ND of 1.3 ± 0.2. This had not been previously observed in our preclinical animal neuroimaging studies (Xiao et al., 2022). To the best of our knowledge, [ 18 F]3 is the first radioligand capable of detecting PDE10A in substantia nigra in living human brain, which may pave the way for the exploration of the earliest pathogenesis in neurodegenerative diseases such as PD and HD (Chinta and Andersen, 2005;Koch and Raymond, 2019;Balestrino and Schapira, 2020).
In terms of clinical PET neuroimaging applications, either a dynamic or long time period scan would be challenging for patients due to physical or emotional causes and inescapable head movements. To verify a static scan with a reasonable duration, we then examined whether 10-min averaged SUV was practical for quantifying a target imaging signal. Comparisons between SUV 10-20min , SUV 20-30min , SUV 30-40min , SUV 40-50min, and SUV 50-60min with specific binding (Supplementary Figure S8) revealed a much better relationship across the three high-uptake brain regions. Of these, the strongest relationship was observed between SUV 50-60min and BP ND ( Figure 4E, R 2 = 0.966, p < 0.01). These results indicated that the widely used SUV PET signal derived from a 10-min static Frontiers in Bioengineering and Biotechnology frontiersin.org scan after 50 min post-injection can be used as a routine protocol for in vivo measurement of PDE10A density using [ 18 F]3.

General considerations and study design
All commercial reagents were purchased from commercially available vendors and used as received. Unless otherwise noted, solvents were freshly dried and degassed according to the purification handbook Purification of Laboratory Chemicals before use. Analytical thin-layer chromatography (TLC) was performed on pre-coated glass-backed plates (EMD TLC Silica gel 60 F254) and visualized using either a UV lamp (254 nm) or potassium permanganate. Silica gel for flash chromatography was 300-400 mesh. NMR spectra were recorded on Bruker 400/ 500 MHz on Bruker spectrometers, and resonances chemical shifts (δ) were given in parts per million (ppm) relative residual solvent. Peak multiplicities are abbreviated by the following symbols: s, singlet; d, doublet; t, triplet; hept, heptahedron; m, multiplet; dd, doublet of doublets. HRMS spectra were measured on a Thermo Scientific LTQ Qrbitrap XL using ESI+.
[ 18 F]Fluoride was produced by the GE Qilin MINITrace cyclotron via the 18 O(p, n) 18 F reaction. The automatic radiolabeling was conducted in an GE TRACERlab FX2N module. Radiochemical purity and molar activity of the radioligand were determined by the HPLC (Shimadzu, Japan) with an analytical column (GL Science, WondaSil C18-WR, 4.6 × 150 mm). Radionuclide purity was tested on an energy disperse spectroscopy (Beijing Sunvic Co., Ltd.). The radioactivity of the product at the end of the synthesis was measured using a dose calibrator (Capintec CRC-55tR). The solvent and copper residue in the formulated product were tested by GC-FID (Agilent 6820) and ICP-MS (Agilent 7700x), respectively.
Clinical studies were performed in accordance with Good Clinical Practice standards, as well as in adherence with the ethical standards of the 1964 Helsinki declaration and amendments. The clinical study was approved by the Institutional Review Board (IRB) of the First Affiliated Hospital of Jinan University (Approval Letter No. KY-2022-007) (Guangdong Province Pharmaceutical Association, 2020). The healthy volunteers used in this clinical study signed informed consent forms before undergoing PET imaging with the right to know their legal guardians and welfare, as well as the nature of the study and the potential risks.
C18 2 (position 5): Sep-Pak Plus Light C18 (Part No. WAT023501) activated with 5 ml EtOH, 10 ml sterile water and 10 ml air. 1) 18 F − in Vessel 1 was transferred to REACTOR 1 by vacuum pump, then passed through the QMA installed in position 4 by helium flow, where the 18 F − was trapped. The basic solution in Vial 6 was used for eluting 18 F − from QMA to REACTOR 2, where the radioactive solution was heated to 85°C for 5 min. The mixture in REACTOR 2 was again dried azeotropically by addition of 1 ml anhydrous CH 3 CN, preloaded into Vial 7, at 85°C for 3 min, then at 110°C for 5 min under helium flow and vacuum. 2) After REACTOR 2 was cooled down to room temperature, the precursor solution in Vial 8 was added. The mixture in REACTOR 2 was heated at 110°C for 10 min (Method I), or at 120°C for 20 min (Method II) during which REACTOR 2 was pumped into negative pressure and the air was passed through V25 and V37 three times.
3) The mixture was cooled to 40°C, then transferred to TUBE 2 through valves V16, VZ2, and V33. The liquid in Vial 9 was added, washing the radioactive residues from REACTOR 2 to TUBE 2 through the pipeline. 4) The crude mixture in TUBE 2 was injected into HPLC equipped with a semi-preparative column (OS∧K∧ SOD∧, CAPCELL PAK C18, UG80 5 μm, 250 × 10 mm) and eluted with CH 3 CN/H 2 O (55/45,v/v,Method I;60/40,v/v,Method II) in Eluent 1 at a flow rate of 5 ml/min (Method I) or 3.5 ml/ min (Method II) for radioligand purification. The semipreparative HPLC chromatograms for both Methods are shown in Figure 3. 5) The indicated product portion was collected and diluted in the bottle of Vessel 2, then captured by Light C18 SPE in position 5. The cartridge was washed with 10 ml sterile water preloaded in Vial 12 to remove HPLC mobile phase and 18 F − . 6) The radioligand on Light C18 cartridge was eluted with 1.5 ml EtOH in Vial 13 into collection Vessel 3, followed by 15.

Quality control and analysis
An analytical HPLC (SHIMADZU, SPD-16) was performed on the system using a UV detector (λ = 254 nm), a gamma detector (Eckert & Ziegler, BGO Detector, B-FC-4100) and analysis software (LabSolutions Essentia). The radiochemical purity and molar activity were determined by an analytical column (GL Science, WondaSil C18-WR, 4.6 × 150 mm) with CH 3 CN: H 2 O = 7:3 (v/v) at a flow rate of 1.0 ml/min. Refer to the Supporting Information for the standard curve and calculation of molar activity (Supplementary Figure S7).
Solvent residual testing: Either the FFAP column (30 m, 0.25 mm, 0.5 μm) or INNOWAX column (30 m, 0.53 mm, 0.5 μm) was used with a constant hydrogen flow rate of 1.5 ml/min. Oven temperature was ramped up starting from 50°C and the front inlet was set to 240°C. A split ratio of 10:1 was used. The elution time for ethanol was approximately 1.63 min, acetonitrile was approximately 1.95 min, acetone was approximately 1.39 min, and DMA was approximately 2.23 min.
Determination of copper residue: Sample processing including the following: 0.5 ml was diluted to 50 ml with 2% nitric acid. Instrument conditions: RF power: 1.55 kW; Feedback power < 10 W; Sampling depth: 10 mm; Nebulizer: MicroMist; Nebulizer chamber temperature: 2°C; Argon gas flow: plasma gas 15.0 L/min, auxiliary gas 0.8 L/min, carrier gas 0.8 L/min, compensation gas 0.4 L/min; collision gas: helium, 4.3 ml/min; scanning mode: peak skipping; online internal standard element Ge, concentration 0.5 mg/L. According to these methods, the limit of quantification was determined to be 0.1 mg/L (data in house).

PET-MRI study in living human brain
Each volunteer participating in this study signed an informed consent form. All participants underwent a complete physical exam before the study, including medical history, electrocardiogram (ECG), blood routine and chemical tests. The volunteer first underwent a brain magnetic resonance imaging examination ( flip angle, 20). Preliminary judgement from the anatomical level was conducted by two senior radiologists to make sure only healthy volunteers (HV) with normal brain function and structures were enrolled in the study. HVs were placed in a supine position on a GE Discovery 690 PET/CT Elite scanner [ 18 F]P10A-1910 (ca. 0.12 mCi/kg, mass dose < 10 μg) was injected into the body from the cubital vein within 20 s, followed by dynamic brain PET collection in a 3D list mode for 60 min. Reconstruction of PET raw data was 32 frames (6 × 20 s, 8 × 30 s, 4 × 1 min, 5 × 2 min, 5 × 4 min, 4 × 5 min). A specialist monitored the volunteers' electrocardiograms (ECG) and blood pressure throughout the entire scan. After the scan, all HVs were advised to stay away from people, especially children and pregnant women, and to drink plenty of water to facilitate radioactivity excretion.

Statistical analyses
Continuous variables were presented as mean ± standard deviation (SD). Student's t test and one way analysis of variance (one-way ANOVA) tests were used for group comparisons of continuous variables. Strength and direction of associations were assessed by Spearman's rank-order correlation. Linear regressions were performed as appropriate. A p-value was considered the significance level of the statistical analysis results, with p < 0.05 representing statistical significance. All statistical data were analyzed using GraphPad Prism (version 8.0.1).

Conclusion
We have realized the first automatic radiolabeling of a novel PDE10A PET tracer [ 18 F]3 (designated [ 18 F]P10A-1910). Using the GE TRACERlab FX2N as our operation module, either the spirocyclic iodonium ylide tailing with an adamantyl auxiliary (5, Method I) or the boronic pinacol ester (6) coupled with copper salt and oxygen (Method II) delivered the product with comparable radiolabeling yields and molar activities. Moreover, the quality control of both was fully validated for human use. A subsequent imaging study in the living human brain revealed that the radioactivity accumulated in the caudate, putamen, as well as the substantia nigra. BP ND was strongly correlated with the PET signal indicated by SUV 50−60min . This easy, automatic radiolabeling, good in vivo stability and binding potential, as well as practical protocols for use in imaging scan in clinics would facilitate widespread use of [ 18 F] 3. Critically, its use would contribute to future exploration of PDE10A-related CNS pathologies in large-scale, multicenter trials.

Data availability statement
The original contributions presented in the study are included in the article/SupplementaryMaterial, further inquiries can be directed to the corresponding authors.

Ethics statement
The studies involving human participants were reviewed and approved by IRB of the First Affiliated Hospital of Jinan University (KY-2022-007). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Author contributions
All the authors contributed to this manuscript and have approved its final version. LW and JW proposed the study and conceived the project. HW, JW, and LW wrote the manuscript. HX and LW modified the paper. HW, JW, and SZ synthesized the radioligand. HW, JW, SZ, SD, GL, WR, CD, WZ, CC, and WL carried out experiments. ZD, JW, and LW designed and guided experiments.


The role of MT1-MMP in the progression and metastasis of osteosarcoma

The dysregulation of Membrane type 1 matrix metalloproteinase (MT1-MMP) has been extensively studied in numerous cancer types, and plays key roles in angiogenesis, cancer progression, and metastasis. MT1-MMP is a predictor of poor prognosis in osteosarcoma (OS), yet the molecular mechanisms of disease progression are unclear. This review provides a summary of the literature relating to the gene and protein expression of MT1-MMP (MMP-14) in OS clinical samples, evaluates the expression in cell lines and experimental models, and analyses its potential role in the progression and metastasis of OS. In addition, the therapeutic potential of MT1-MMP as a drug target has been assessed. Due to the biological complexity of MMPs, inhibition has proven to be challenging. However, exploiting the expression and proteolytic capacity of MT1-MMP could open new avenues in the search for novel, safer and selective drugs for use in OS.

INTRODUCTION
Osteosarcoma (OS) is the most common form of primary malignant bone tumour and exhibits its highest prevalence in children and adolescents, with 80% of cases affecting individuals before the age of 20. OS accounts for less than 1% of all new cancer diagnoses with an age-standardised incidence rate of 0.9 per 100,000 in the UK, affecting 3.4 per million people worldwide each year. The survival rate for those who develop OS, especially for those who have developed metastases, has not significantly improved over the past 30 years, with only 55% of patients surviving for more than 10 years. This is primarily attributed to the lack of novel therapeutics [1,2] .
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play key roles in angiogenesis, cancer progression and metastasis. These proteases are classified based on their structural characteristics and are often subdivided into two major groups; secreted MMPs and membrane-type MMPs (MT-MMPs) [3] . In normal tissues, MMPs are involved in a variety of physiological processes, including mediation of inflammation and tissue remodelling. However, the overexpression of MMPs in cancer has been associated with poor prognosis, and they have been considered as potential diagnostic and prognostic biomarkers in several cancer types, including OS, as exemplified by MT1-MMP (see Figure 1). Technically speaking, MT1-MMP is the name for the protein, whereas MMP-14 refers to the gene, but these terms are often used interchangeably in the literature.
The activation of MT-MMPs, achieved through cleavage of a single protein and pro-peptide domain, promotes their transport to the cell surface where they degrade components of the extracellular matrix and basement membranes -either directly or indirectly through the recruitment of other MMPs -thereby facilitating invasion and metastasis [8] . In a variety of carcinomas, the dysregulation of MT1-MMP has been extensively studied, yet the molecular mechanisms underlying OS disease progression are unclear, but MT1-MMP may play a key role [9] . Initially, this review will examine the expression of MT1-MMP in OS clinical tumour samples and cell lines, followed by a discussion as to the potential role of MT1-MMP in OS progression and metastasis, before finally considering opportunities for therapeutic targeting for the benefit of OS patients.

In OS clinical samples
The clinical expression of secreted MMPs, especially MMP-2 and MMP-9, has been the subject of many reviews. Both proteinases have been identified in tumour samples resected from patients with OS [10,11] . The differential expression of MT1-MMP in tumour tissue samples, when compared to normal tissues, has been widely researched; the data available for the expression of MT1-MMP in OS clinical tissues are summarised in this section.
Uchibori et al. [12] examined 47 patient samples, all of which were taken prior to patients undergoing treatment who presented with no distant metastasis. MT1-MMP, MMP-2, -9, and tissue inhibitor of metalloproteinases 2 (TIMP-2) protein expression and their correlation with prognosis were examined immunohistochemically and by gelatin zymography. Strong expression (positive cell ratio > 50%) of MT1-MMP in 21 (45%) samples was noted, 18 (38%) of which were also positive for MMP-2 expression. The overall survival rate (OAS) for the 18 patients exhibiting strong expression of both MT1-MMP and MMP-2 was 57%, whereas, for the other patients, OAS was 87%. Although many patients within this study remained disease-free up until the follow-up (after 5 years), 15 patients developed metastasis, and 11 patients died of the disease. Localisation of MT1-MMP, MMP-2 and TIMP-2 to the cytoplasm and cell membrane was observed, with a significant association between the strong expression of MT1-MMP, but not MMP-2, and a reduction in patient survival when compared to tumour samples that exhibited weak expression. The eventfree survival rate (EFS) was determined for the surviving 36 patients, all of whom were in remission following neoadjuvant and adjuvant chemotherapy and surgical resection. The EFS at 5 years for those with tumours exhibiting strong MT1-MMP expression and for those with tumours exhibiting weak expression was 60% and 81%, respectively. Of the 36 patients, 13 had tumours that exhibited strong expression of both  [4,5] . (B) MMP-14 expression in various types of sarcoma (http://ist.medisapiens.com/) [6] . (Figure adapted from Figure 1 in Ref 7 [7] ).
MT1-MMP and MMP-2, which was also shown to correlate with poorer prognosis. In a follow-up study, it was found that the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), a substrate of MT1-MMP which aids in MMP stimulation, and the co-expression of EMMPRIN and MT1-MMP were predictors of poor prognosis [13] .
In another study, the expression of MMPs and their endogenous inhibitors were investigated in 11 tumour xenografts and 12 OS biopsies (6 from primary tumours and 6 from metastases) [14] . MMP-14 mRNA, along with MMP-2, TIMP-1 and TIMP-2 mRNA, was identified in the majority of samples, with a consistent pattern of expression of MMP-14, MMP-2 and TIMP-2 in both the xenografts and biopsies. The authors stated that a pattern of MMP and TIMP expression in metastases, when compared with primary tumours, could not be discerned.
Utilising high-throughput sequencing, Xiao et al. [15] screened for 339 genes known to be associated with cancer progression in 10 samples from OS patients from China. It was shown that the 10 samples tested demonstrated on average more than 200 gene mutations, including the MMP-14 gene. Although the findings from this study are in line with previously published studies [16] , a more comprehensive evaluation of the OS genome in larger collections of OS samples is required to identify high-yield mutations.
Whole transcriptosome analysis was performed on 33 OS patient samples. Tumour and normal tissues were collected from 18 bone samples following resection and 15 formalin-fixed paraffin-embedded (FFPE) samples that were retrieved from pathology archives. The genes that were most significantly downregulated and upregulated in the tumour tissues were identified [17] . MMP-14 was shown to be the 7th most upregulated gene in osteosarcoma, with a greater than 2-fold increase in gene expression in 87% of tumour samples when compared to their matched controls. Of these, 47% also showed elevated levels of MMP-2 and/or MMP-9. Enrichment analysis showed that there were significant alterations in the genes shown to regulate collagen degradation, extracellular matrix (ECM) organisation and ECM degradation, including collagens; COL11A1, COL2A1 and COL10A1; among others. In the FFPE samples, it was evident that chemotherapy had not brought about any significant changes in MMP-14, MMP-2 and MMP-9 expression.
A study by Engelholm et al. [18] examined OS patient samples to identify novel bone degradation pathways. Of the 10 samples that were tested, by immunohistochemical analysis, for MT1-MMP protein expression, all were positive to varying degrees; scattered expression was observed in 2 samples, moderate expression (< 50%) in 4 samples and high (> 50%) expression in 4 samples. This study also demonstrated the differential expression of MMPs in primary OS tumours and tumours resulting from carcinomas. OS tumour samples showed high expression of MT1-MMP and other endocytic collagen receptors, whereas, in tumours originating from breast carcinoma, there was little to no expression of these proteins.
Also utilising immunostaining techniques, Biswas et al. [19] , demonstrated that in biopsies collected from patients that had undergone standard MAP chemotherapy -that involves administering high dose methotrexate (M), doxorubicin (A -adriamycin) and cisplatin (P) -more than 90% of samples evaluated expressed the MT1-MMP protein. A reduction in overall survival was associated with an increase in the localisation of MT1-MMP to the cytoplasm (P = 0.0002) and to the nucleus (P < 0.0001). In addition to this, a reduction in the EFS was observed in patients with localisation of MT1-MMP to the nucleus, but not to the cytoplasm. Therefore MT1-MMP localisation could prove to be crucial in predicting patient prognosis and in determining the patients' response chemotherapy.

In experimental models of OS
MMP expression in OS has garnered a lot of attention, but studies primarily focus on the expression of the widely-studied MMP-2 [11,20] or MMP-9 [21,22] [24,25] .
Giambernardi et al. [26] reported the differential gene expression of MMPs in cell lines; a strong expression of MMP-14 was observed in HOS, MG-63 and Saos-2. It has been shown that the transformation of the HOS-Te85 parental cell line to its 143B derivative, brought about an increase in MT1-MMP protein expression, which has been associated with a more metastatic phenotype, whereas a reduction in MT1-MMP expression in the transformed HOS-MNNG cell line played a role in reducing its metastatic capacity [27] .
Hypoxia, resulting from oxygen deprivation, is common in many solid tumours with an associated increase in hypoxia-related markers, such as hypoxia-inducible factors-1α or -2α (HIF-1α/-2α), which often correlates with poorer prognoses. Many genes, including those of MMPs, are transcriptionally regulated by these HIFs [28] . Chan et al. [29] determined, by way of Western blot, that the degree of MT1-MMP protein expression in U2OS cells was equal to that of the positive control cell line HT1080, which exhibits a highlevel expression of the majority of MMPs. Further to this, immunohistochemical staining revealed that, in normoxia, MT1-MMP is primarily located in the cell cytoplasm and was shown to interact with HIF-1αthis interaction was not observed following a reduction in O 2 levels. Hypoxia-induced alterations in the subcellular location of MT1-MMP, with a higher expression of MT1-MMP observed within the nucleus. In normoxia, a proximity ligation assay showed that there is minimal interaction between HIF-2α and MT1-MMP, but when cells were cultured under hypoxic conditions, there was a marked increase in the interaction signals within the nucleus. This interaction was shown to be specific to U2OS cells when compared to bone marrow mesenchymal stem cells.
Transfection of 143B cells, with a luciferase-Tomato reporter gene (pFULT) expressing firefly luciferase 2, allowed for the development of murine orthotopic models of OS that are easily traceable in vivo. Also, the cells were labelled with tdTomato making it possible to distinguish between tumour cells and host cells during immunohistochemical analysis. Histological characterisation of tissue samples generated from this orthotopic model revealed that they mimicked human disease, with a strong expression of MT1-MMP in tumour cells excised from the tibia and from metastatic lesions that had developed in the lungs. In addition to this, utilising Crispr/Cas9 gene editing techniques, the researchers generated MT1-MMP knockout and wildtype cells lines (also expressing luciferase and tdTomato), both of which were screened for MT1-MMP protein expression by western blot and genotyped. Comparison of these cell lines revealed that loss of MT1-MMP expression resulted in a reduction in the ability of 143B cells to degrade cellular collagen. Further investigation showed that there was no difference in the extent of bone degradation or the formation of lung metastases in orthotopic models of the newly derived cell lines, however. This suggests that MT1-MMP does not play a direct role in bone degradation or in the lung colonisation processes in 143B orthotopic models of OS, but rather acts via alternative methods [30] .
The differential expression of MT1-MMP in OS cells and clinical samples, when compared to controls, provides compelling evidence for its involvement in OS development. Many of the clinical studies are limited by small sample sizes, a common problem encountered when evaluating protein expression in OS tumours (given the rarity of the disease), and a more comprehensive study with a larger sample size could provide a better understanding of the role of MT1-MMP in OS. Within the tumour microenvironment, the complex network of pathways, many of which could be involved in the regulation of MT1-MMP, determines the rate of progression and metastasis in OS. These pathways, summarised in Figure 2, will be considered in the following section.

ROLE OF MT1-MMP IN OS PROGRESSION AND METASTASIS
The regulation of MT1-MMP expression by the key SRC/ERK1/2 signalling pathway (a major player in OS progression, Figure 2) in response to WNT5A has been investigated by Wang et al. [31] . They found that WNT5A-induced MG-63 cell migration and invasion was in part due to the activation of SRC/ERK1/2 signalling pathways that were shown to upregulate downstream MT1-MMP. Previous studies have reported that WNT5A-induced invasion in osteosarcoma cell lines (U2OS and Saos-2) is mediated by SRC through upregulation of MMP-13 [32,33] . The authors speculated that activation of the SRC/ERK1/2 signalling pathway is responsible for the increased expression of both MT1-MMP and MMP-13 in response to WNT5A.
The activity of a Wnt co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), has been linked with OS metastasis and poorer prognosis. Canonical Wnt signalling is responsible for the downstream regulation of a broad spectrum of cell cycle regulators, oncogenes and matrix-degrading enzymes. The transfection of a soluble form of LRP5 (sLRP5) into SaOS-2 cells significantly reduced the protein expression of MT1-MMP and MMP-2, with the effect on MMP-2 being more pronounced. MT1-MMP is a known Wnt/β-catenin target gene and membrane-bound activator of MMP-2 [34] . The authors did note that due to the differences in genetic backgrounds and mutational profiles of OS cell lines, the results here in Saos-2 cells cannot be generalised. Therefore further investigation of sLRP5 activity in additional cell lines that are stable following sLRP5 transfection is required [35] .
α1-Antitrypsin Portland (α1-PDX), a furin inhibitor, has been shown to affect the migration and invasion of MG-63 and Saos-2 OS cell lines [36] . This study suggested that α1-PDX may play a key role in the inhibition of migration and invasion through the non-canonical Wnt pathway-mediated downregulation of MT1-MMP expression. It would be interesting to see what further exploration into the effect α1-PDX has on the activity Wnt inhibitory factor 1 (WIF-1) protein levels are often downregulated in OS cell lines, yet the role of WIF-1 in disease progression is largely unknown [37] . WIF-1 expression has been shown to decrease tumorigenesis and metastasis in HOS-143B cells [38] . HOS-143B cell lines are highly aggressive and frequently metastasise to the lung in xenograft models [39,40] . HOS-143B cells transfected with WIF-1 exhibited reduced expression of MT1-MMP, and also MMP-2 and MMP-9, but there was no significant effect on proMMP-2. The authors concluded that the anti-metastatic effect of WIF-1 induction could be of clinical importance in OS patients shown to have aberrant Wnt signalling.
RNA interference techniques (siRNA) have also been used to determine the effect of knockdown of βcatenin on the invasion, migration and sensitivity to chemotherapeutics in OS cells [41] . In line with previous work in U2OS cells [42] , the authors were able to demonstrate that the motility and invasive ability of MG-63 cells were affected by the inhibition of β-catenin, which was attributed to the downregulation of MT1-MMP expression.
In chondrosarcoma, breast and prostate cancer cell lines, pigment-epithelium derived factor (PEDF) has been shown to downregulate MT1-MMP protein expression [43,44] . The mechanism by which PEDF reduces the metastatic capacity of OS cells through MMP regulatory pathways was examined in HOS-143B cells [45] . Investigation revealed that increases in the protein expression of PEDF correlated with an increase in MT1-MMP and collagen I expression, yet a decrease in the levels of MMP-2 was observed. The authors speculated that the antagonistic activity of both PEDF and MT1-MMP would be necessary for bone remodeling, which could explain the differential regulation of MT1-MMP by PEDF across cell lines. The downregulation of OS metastasis in HOS-143B cells was attributed to a decrease in MMP-2 expression, which is not in line with previous studies in breast and prostate cancer cell lines [44] , with the authors noting that the decrease in MMP-2 seen could be attributed to an increase in MMP-2 secretion into the medium which went un-monitored in this study.
The aberrant expression of microRNAs (miRNAs) has been implicated in cancer progression [25] . MiRNAs regulate gene expression through the induction of mRNA degradation or by translational regression. The ability of miRNAs to simultaneously target multiple genes increases their likelihood of success as therapeutic agents. Xie et al. [46] , investigated the expression of miR-302b and the mechanism by which it promotes cell invasion and migration in OS cell lines. The overexpression of miR-146b-5p was shown to suppress cell proliferation and increase apoptosis in OS cell lines (HOS-143B and MG-63) [47] . Mimics of miR-302b were shown to suppress the mRNA and protein levels of MT1-MMP and MMP-2 through the targeting of the Runx2 gene, while the treatment of cells with an miR-302b inhibitor produced the opposite effect.
Alternatively, Elenjord et al. [48] utilised ribozyme-transfected cell clones of OHS to determine the effect of collagen 1 expression on MMP activity in the absence/presence of S100 calcium-binding protein A4 (S100A4). Previous work demonstrated that reducing S100A4 in OS cell lines, both in vitro and in vivo, reduced their invasive and metastatic capacity through downregulation of MMPs and TIMPs [49,50] . In addition, a study by Loennechen et al. [51] showed that colchicine induced MT1-MMP expression in an S100A4-independent manner, with higher levels of MT1-MMP observed upon induction of microtubule rearrangements following exposure to colchicine, as observed in both control and transfected cell lines. The authors were able to demonstrate that activation of proMMP-2 by MT1-MMP was mediated by TIMP-1 in OHS cells (control pHβ-1, II-11a and II-11b). Another study evaluated the effect of R1881, a synthetic androgen analogue, on the expression of several MMPs and their endogenous inhibitors in LNCaP (prostatic adenocarcinoma) and OHS cells. TIMP-2 was shown to be inhibited in a time-dependent manner in MT1-MMP deficient LNCaP cells, which also correlated with an increase in the levels of prostate-specific antigen. In these cells, the mRNA levels of other MMPs (MMP-1, -2, -9, MMP-14 and TIMP-1) were not detected. An elevation in the expression of MMP-14 mRNA was seen when LNCaP cells were co-cultured with OHS cells. In this co-culture, the proteolytic activity of the MT1-MMP/TIMP-2/MMP-2 complex was unaffected by R1881 suggesting that this complex may provide an androgen-independent route for the development of bone metastases from prostate carcinoma [52] .
Lysophosphatidic acid (LPA) plays an important role in tumour cell invasion by promoting ECM degradation through the regulation of downstream targets. Investigation of the effect that LPA has on Rho, Rho-associated protein kinase (ROCK), MMPs and TIMPs, revealed that following high-dose treatment with LPA mRNA and protein levels of Rho, ROCK, MT1-MMP and TIMP-2 were reduced. Interestingly low dose LPA treatment resulted in increased mRNA levels of these proteins. Selective inhibition of Rho prevented LPA-mediated reduction in MT1-MMP and TIMP-2. Matsumoto et al. [27] speculated that the Rho-ROCK pathway might be involved in MMP-2 activation through regulating MT1-MMP and TIMP-2 levels.
MMPs also play an important role in inflammatory processes potentiating the activity of inflammatory proteins, which result in the activation of many downstream signalling pathways, including JNK/c-jun [53] . The ability of Dz13, a DNA enzyme, to downregulate c-Jun has been shown to have a direct effect on OS progression in HOS-143B and SJSA-1 cells and resulted in an increased doxorubicin sensitivity in Saos-2 cells [54][55][56] . Tan et al. [57] investigated the relationship between Dz13 and MMP expression in OS. A dose-dependent decrease in MT1-MMP and MMP-2 expression was observed following exposure to Dz13 in SJSA-1 OS cells, and this is in line with previous studies that have also demonstrated that MMP-2 and MMP-9 expression is regulated by c-Jun [58,59] .
Bisphosphonates have been shown to reduce invasion and metastasis and potentiate the effects of conventional chemotherapy in OS [14,60,61] . Heikkilä et al. [62] investigated the effect of clodronate on MT1-MMP mediated activation of MMP-2 and showed that increasing the concentration of this bisphosphonate reduced MT1-MMP and MMP-2 mRNA and protein expression in MG-63 cells. Previously, it has been shown that alendronate reduced the secretion of MMP-2 and is able to induce apoptosis in OS, while risedronate reduces mRNA and protein levels of MMP-2 and MMP-9 in U2OS and Saos-2 cell lines [63,64] . Unfortunately, the effect of alendronate and risedronate on MT1-MMP levels were not assessed in these studies.
In summary, MT1-MMP plays multiple roles in OS, many of which go beyond the roles associated with tumour migration and invasion, which can be directly attributed to its proteolytic capacity and extracellular matrix degradation (see Table 1). There is still much to be learnt, but as we have discussed, MT1-MMP has a significant impact on multiple intracellular signalling pathways, all of which adds to its importance as a key player in OS progression. The potential for targeting MT1-MMP as a therapeutic strategy, and the challenges associated with it will now be considered.

CHALLENGES AND FUTURE PROSPECTS
There is considerable evidence (both clinical and experimental) to support the role of MT1-MMP in metastasis in several cancers (notably breast and prostate cancers), but this is not the case in OS [9,65,66] . This could be attributed to the rarity of OS, which makes accessing tissue samples for gene mapping and experimentation very difficult. Therefore, utilising the metastatic potential of HOS-MNNG and HOS-143B cell lines could provide a better model by which to study the involvement of MT1-MMP in OS metastasis.
In the UK, the ICONIC project, funded by the Bone Cancer Research Trust, aims to create an OS patient registry, providing clinicians and researchers with better access to patient data and samples, thereby helping them to better understand the disease and develop novel treatments [67] . Similar registries also exist in the USA [68,69] .
The MAP chemotherapy regime remains the standard of care for localised OS [1,70] . It has long been understood that significant side effects are associated with the use of chemotherapeutic agents; this is especially true for HD-MTX. HD-MTX has many adverse side effects resulting from its toxicity, and so requires the administration of a rescue dose of leucovorin following its infusion [71] . In addition to this, studies have shown that in OS, the transport of MTX across the cell membrane is impaired, therefore necessitating the larger doses required to achieve a therapeutic effect [72] .
There is a pressing need for novel therapeutics for OS patients, especially for those with metastatic disease or tumour recurrence, both of which correlate with an unfavourable prognosis, and MT1-MMP is clearly a target worthy of attention. One approach has been the development of synthetic MMP inhibitors (MMPIs), but unfortunately, no molecule has successfully made it to the clinic. Many MMPIs failed to progress beyond Phase III clinical trials due to poor specificity, deregulation of TIMPs and the off-target toxicity that has resulted in suboptimal dosing recommendations. Novel molecular biology techniques have been utilised to rapidly detect the expression profiles of MMP genes in each patient, determine the potential efficacy of MMPIs and therefore take an individualised approach to treatment [73] . The recent advances in MMPIs have been extensively reviewed by Gregg Fields [74] , who highlighted that despite previous failures in clinical trials,   which were attributed to a lack of understanding of the biological complexities of MMPs, there had been some recent clinical successes. MMPIs have wideranging applications, from the treatment of HER2-negative adenocarcinomas to diabetic foot ulcers. Doxycycline and minocycline, which are broad-spectrum MMPIs, have also shown promise in the treatment of patients with relapse-remitting multiple sclerosis. Both were shown to reduce brain lesions and serum levels of MMP-9 and were considered to be effective, safe and well-tolerated. A potent, selective MMPI could make a significant clinical impact on OS therapy.
An alternative approach is to exploit the proteolytic activity of MT1-MMP to activate a prodrug (a drug administered in an inactive form, which is subsequently activated at the target site) in the tumour microenvironment. By adopting this approach, there is potential to overcome the off-target toxicity seen in many chemotherapeutic agents like HD-MTX, since the drug would only be released in the tumour microenvironment, thereby sparing normal tissues of exposure to the drug, while simultaneously enhancing its therapeutic index.
Two such examples of MT1-MMP targeted agents are BT1718 and ICT2588 (see Figure 3), which are currently being evaluated in other cancers, but have significant potential for OS therapy. BT1718, which was developed by researchers at Bicycle Therapeutics Ltd, is currently in Phase I/II clinical trials for the treatment of advanced solid tumours (Clinical Trials. gov Identifier: NCT03486730). The drug conjugate is comprised of a bicyclic peptide covalently linked to DM1, a potent anti-tubulin agent. The peptide sequence has been designed to specifically bind to MT1-MMP through a disulphide linker -taking advantage of its expression profile in many solid tumour models (including various sarcomas, non-small cell lung cancer and triple-negative breast cancer) -thereby targeting the delivery of DM1 [75] . Pre-clinical models have shown that in both cell-derived and patient-derived xenograft tumours, DM1 is released in an MT1-MMP dependent manner, which results in partial or complete tumour regression [76] . Although the clinical outcome is not yet known, this validates MT1-MMP as a target. ICT2588, which is currently being prepared for Phase I clinical trials, consists of 3 components; azademethylcolchicine, a vascular disrupting agent; an MT1-MMP activatable peptide sequence, Arg-Ser-Citrulline (Cit)-Gly-Homophenylalanine (Hof)-Tyr-Leu-Tyr; and a fluorescein isothiocyanate (FITC) based end cap. The release of the azademethylcolchicine warhead is achieved through (1) the selective cleavage of the peptide conjugate at the scissile bond between Gly and Hof; and (2) tumour exopeptidase activity removing the remaining amino acids. In tumour xenograft models, ICT2588 was shown to reduce tumour vasculature by up to 90% and, when co-administration with doxorubicin, led to a significant reduction in relative tumour volume [77] . Furthermore, ICT2588 was shown to lack the cardiotoxicity of the parent colchicine, achieved through selective targeting to the tumour [78] . Both of these approaches have significant potential application to OS, given the differential expression of MT1-MMP.
Alternative prodrug approaches to selectively deliver MTX have also been considered. For example, Cy-SS-MTX (see Figure 3), is a glutathione-activated theranostic prodrug consisting of a cystamine functionalised IR780 (Cy), a near-infrared fluorescent heptamethine dye with tumour targeting properties, conjugated to MTX. In a co-culture of LO2 (normal hepatocyte) and MCF-7 (breast adenocarcinoma) cells, Cy-SS-MTX was shown to selectively accumulate within the mitochondria of MCF-7 cells. In addition, in both in vitro and in vivo models, Cy-SS-MTX was shown to reduce off-target toxicity and improve the anti-tumour efficiency of MTX [79] .
In summary, after assessing the available evidence, it is clear that further scientific investigation is required to determine the full role that MT1-MMP plays in OS progression and metastasis. Its expression and proteolytic capacity are evident. However, MT1-MMP has significant therapeutic potential, particularly through the exploitation of its proteolytic activity, which allows for the selective release of toxic agents at the tumour site, helping to reduce off-target toxicity, which could drastically improve OS patient outcomes and quality-of-life.


Is novel research worth doing? Evidence from peer review at 49 journals

Significance There are long-standing concerns that scientific institutions, which often rely on peer review to select the best projects, tend to select conservative ones and thereby discourage novel research. Using peer review data from 49 journals in the life and physical sciences, we examined whether less novel manuscripts were likelier to be accepted for publication. Measuring the novelty of manuscripts as atypical combinations of journals in their reference lists, we found no evidence of conservatism. Across journals, more novel manuscripts were more likely to be accepted, even when peer reviewers were similarly enthusiastic. The findings suggest that peer review is not inherently conservative, and help explain why researchers continue to do novel work.

. Percent of citation hit papers, by novelty group (columns), computed on three different datesets (rows). "Hit" is defined as having the 8-year citation count fall in the top 5% of that year's citation distribution. Low/high tail and median novelty are values below/above the median.
The MAG-Scopus results match USMJ qualitatively. The ordering of groups by percent of citation hits is the same for MAG-Scopus 2010 and similar for MAG-Scopus 1990-2000, aside from the two middle groups, which are only slightly different from each other in the original USMJ. Across all datasets, the exercise supports USMJ's main claim that papers with high tail novelty and high median conventionality display a hit probability about twice as much as papers that are high on either of the two dimensions. Overall, these results suggest that our novelty measure based on the MAG literature filtered by Scopus journals works similarly to WoS data, and applies to more recent literature.
B. Validation using research articles vs. reviews As one validation check of whether USMJ measures novelty, we consider its distribution by article types expected to be more or less novel: primary research (i.e. research articles) and reviews. We expect reviews, being syntheses of primary research from the recent past, to have lower novelty and higher conventionality than primary research. Data on article type was missing for 7717 submissions across the two Life Sciences journals 2 , and these were excluded from this analysis. Among the observations with a valid article type, 21190 were primary research and 120 were reviews. Figure S1 below displays the boxplot of novelty and conventionality percentiles by article type. As predicted, the novelty of primary research was on average 6.9 percentiles higher than that of reviews (Wilcoxon rank-sums test statistic=2.13, p=0.033) and the conventionality was 13.3 percentiles lower (Wilcoxon rank-sums test statistic=-4.27, p<0.001), supporting the interpretation of USMJ measures as novelty and conventionality.

Figure S1. Boxplot of novelty percentiles (A) and conventionality (B) by article type in the Life
Sciences journals.

C. Additional validation from peer review
Additional support for the interpretation of USMJ as a proxy of novelty comes from (Bornmann et al., 2019). Bornmann et al. calculated a version of USMJ that is revised by Lee et al. (Lee et al., 2015). The Lee et al. revision retains the essence of USMJ, but simplifies the normalization of atypical combinations, i.e. does not randomly rewire the year-specific journal-journal co-citation network as USMJ and the present paper do. Bornmann et al. then compared these revised-USMJ measures to qualitative rankings from members of Faculty of 1000, a post-publication peer review system. A key result is that a one SD-increase in revised-USMJ novelty corresponds to a 7.47% increase in the number of reviewers labeling the paper as presenting a "new finding." This result suggests a correspondence between USMJ and qualitative perceptions of novelty post-publication.

Submitted vs. published versions of Cell manuscripts
While it may be desirable to use metrics derived from the submitted versions of manuscripts to study editorial decision-making, our data include the submitted manuscript files (in PDF or Word format) only for submissions to Cell, and only for two years. Lacking submitted versions of most of the submissions, we thus relied on the published versions of articles as a proxy for their submitted versions. However, for the subset with both submitted and published manuscript versions, we can compare how novelty metrics changed.
This subset consists of 3,717 articles submitted to Cell in 2013-2014. We used the Grobid (Introduction -GROBID Documentation, n.d.) package to parse the manuscript files and extracted references from them. Grobid uses the CrossRef API service to resolve the references in text into DOI. We were able to extract references for 2,778 initial submissions.
Whereas submitted-version novelties may be ideal in theory, in practice, accurately extracting references from PDF and Word files is error-prone. There are three main ways in which novelty calculated from the submitted and published versions may differ: 1. True changes in the references 2. Extraction errors from submitted versions: our pipeline may have failed to successfully identify or parse references in the submitted version. The Grobid documentation shows fscores in the range of 0.75 -0.89 (Introduction -GROBID Documentation, n.d.). 3. References included in the calculation of novelty from of the published version consist of all those with a valid MAG ID, but not necessarily a DOI. In contrast, references included in the calculation of novelty from the initial version consist of all those with a valid DOI (because they use CrossRef), but not necessarily MAG ID.
We display scatter plots of novelties and conventionalities calculated from the published vs. submitted versions of papers in Figure S2. Panel A shows raw novelty (z-scores, calculated as in Uzzi et al. (2013)) of submitted vs. published versions, and Panel B shows percentiles of novelty for published vs. submitted versions. Panels C and D repeat the plots for conventionality. Figure S2. Scatter plot of novelties (top row) and conventionalities (bottom row), using raw versions calculated as in Uzzi et al. (2013)) (left column). In each panel, the x-axis is the measure calculated from the paper's submitted version, and the y-axis is from the published version.
We interpret the much smaller correlation for raw novelties and conventionalities as revealing the sensitivity of these metrics to small changes in references, whether due to true changes or parsing and database errors, and which is evidenced by several extreme outliers. In contrast, percentiles are robust to outliers, and show that novelty and conventionality are relatively consistent across the publication process. Consequently, we rely on percentiles in the rest of our analysis.

Full vs. analytic samples
The primary difference between the full and the (smaller) analytic samples for both the Cell Press and IOP datasets is that the analytic samples include only submissions for which we were able to calculate novelty. Novelty calculation for a submission were often not possible because it was rejected and a published version was not located (e.g. never published) or it was published but not indexed in MAG.

A. Cell Press journals
Tables S2 and S3 below show how key covariates vary between the full and analytical samples for Cell and Cell Reports. The last column of the tables shows the standardized difference, i.e. difference in means divided by the pooled standard deviation. The differences are modest, never exceeding a third of the pooled standard deviation. The largest differences are observed in quality, measured by the mean reviewer recommendation (0=reject, 1=accept or revise-and-resubmit), and the number of references, with both being higher in the analytic sample. This pattern is likely explained by lower quality submissions being more likely to be rejected, and subsequently less likely to be matched to MAG, whether due to never being published or published under a significantly different title.

B. IOP journals
We exclude from the IOP analytic sample manuscript types that are likely to undergo an unconventional review process or not present original research, such as Corrigendum (n=580) and Perspective (n=504). The top 5 manuscript types remaining in the IOP analytics smaple are shown in table S4.

Type Count
Paper 38112 Letter 2309 Special Issue Article 2095 Topical Review 1017 Note 382 Table S4. Manuscript types of submissions to IOP journals in 2018 in the full sample. Table S5 presents the difference between the IOP analytic and full samples on key covariates. The analytic sample is much smaller than the full sample for two main reasons. First, we attempt to match 2018 submissions, whether accepted or rejected, to the mid-2019 MAG and the majority of these (63.1%) were not indexed or located by that time. It is possible they were published and indexed in later versions of MAG, or will be published in the future. Second, an additional 21.5% of submissions were located in MAG but had no data on references.
The standardized differences (last column) are modest, never exceeding a quarter of the pooled standard deviation. The analytic sample has slightly more positive reviewer recommendations and citation counts, suggesting that it consists of slightly higher quality papers. This selection bias is likely due to higher quality papers being more likely to be accepted for publication, leading to higher probability and speed of indexing in MAG.   Note: * p<0.05; ** p<0.01; *** p<0.001 Table S11.