s2fieldsofstudy: Biology Medicine

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Bone marrow transplantation induces changes in the gut microbiota that chronically increase the cytokine response pattern of splenocytes

Bone marrow transplantation (BMT) involves conditioning regimens which acutely induce side effects, including systemic inflammation, intestinal damage and shifts in the gut microbial composition, some of which may persist chronically. As the gut microbiota affect systemic immune responses, we aimed to investigate whether, post-BMT, the peripheral immune system is modulated as a direct consequence of alterations in the gut microbiota. We show that 24 weeks post-BMT, splenocytes but not peritoneal macrophages display increased cytokine response patterns upon ex-vivo stimulation with various pathogens as compared to untreated controls. The pattern of BMT-induced cytokine responses was transferred to splenocytes, and not to peritoneal macrophages, of healthy controls via co-housing and transferred to germfree mice via transplantation of cecum content. Thus, BMT induces changes in gut microbiota that in their turn increase cytokine responsiveness of splenocytes. Thus, BMT establishes a dominant microbiota that attenuates normalization of the immune-response.

The intestine is not only the site where food digestion takes place, but it is also home to more than 10 14 commensal microorganisms that are collectively called the gut microbiota. The microbiota continuously interact with and provide benefits to the host, as they are involved in processes such as fermentation of indigestible fibres, metabolism of xenobiotics and regulation of the immune system. Studies in germfree mice that lack gut microbiota have provided insight into the role of gut microbiota in regulating the immune system. For example, germfree mice have a dysfunctional mucosal immune system with less and smaller Peyer's patches and mesenteric lymph nodes 1,2 . These mice also have reduced numbers of intra-epithelial lymphocytes with a compromised immune function 3 . The gut microbiota are thus important for shaping a functional mucosal immune system.
In addition to affecting the mucosal immune system, recent studies have demonstrated that the gut microbiota are important in the development and function of the immune system beyond the intestine. For example, the gut microbiota play a role in the pathogenesis of autoimmune disorders such as rheumatoid arthritis and type-1 diabetes 2,4,5 . Clarke et al. 6 reported that bacterial components translocate from the gut into the circulation under basal conditions and serve as mediators that systemically prime neutrophils in the bone marrow. However, the effects of the gut microbiota on other key lymphoid tissues such as the spleen have not been fully characterized. Germfree mice have reduced numbers of CD4+ cells and smaller germinal centers within the spleen as well www.nature.com/scientificreports/ as lower systemic antibody levels 4,7 . Moreover, oral administration of dietary fibres or short-chain fatty acids (SCFA) to mice increases antibody responses by B cells in the spleen 8 , which indicates that metabolites from the gut microbiota affect spleen function. Bone marrow transplantation (BMT) is applied as therapy for patients with specific cancers of the bone marrow or blood, such as multiple myeloma or leukemia. To minimize residual disease, to create space for the transplant, and to achieve immune-ablation, BMT is preceded by conditioning regimens like total body irradiation (TBI) or chemotherapeutic agents. A drawback of these conditioning regimens is that they induce acute and chronic side effects. TBI not only ablates bone marrow cells, but may also cause acute damage to the gastrointestinal tract. This damage promotes the leakage of bacterial components from the gut into the systemic circulation. Bacterial components act as toll-like receptor ligands, activate immune cells and thus cause systemic inflammation 9 . This phenomenon may certainly explain some of the acute side effects of the BMT treatment.
However, BMT treatment not only resets the host immune system but also affects host microbiota 10 . The long term composition and activity of the intestinal microbiota is the result of intricate interactions between bacteria, host and environment. It is more than likely that BMT treatment thus results in permanently altered microbiota that may contribute to changes in the intra-and extra-intestinal immune system of the host 11 .
In the current study, we investigated in mice the effect of BMT on the response patterns of splenocytes and peritoneal macrophages to various pathogenic stimuli as markers of the extra-intestinal immune system. Splenocytes are a mixed population of immune cells and thus represent responses from both the adaptive and innate immune system, whereas peritoneal macrophages represent the innate immune system. The potential role of microbiota in the response patterns of splenocytes and peritoneal macrophages was investigated by co-housing control mice with BMT-treated mice and by transfer of cecum content to germfree mice. Our data show that the BMT-induced increase in the cytokine response pattern of splenocytes to pathogenic stimuli can be transferred via the gut microbiota.

Results
The cytokine response pattern of stimulated splenocytes was increased after BMT and transferred to healthy controls via co-housing. To study the effect of BMT on the response patterns of splenocytes and peritoneal macrophages to various pathogenic stimuli and the potential role of microbiota herein, C57BL/6 mice underwent syngeneic BMT or sham procedure and were co-housed in three combinations to arrive at the following four groups: (1) healthy control mice co-housed with healthy control mice, (2) BMTtreated mice co-housed with other BMT-treated mice, (3) BMT-treated mice co-housed with healthy control mice, and (4) healthy controls co-housed with BMT-treated mice. Group 1 served as control for the other three groups. All mice, both BMT-treated and control mice, received water containing antibiotics and were exposed to the same dietary regimen. After recovery from the BMT procedure, mice were fed a low-fat diet for 16 weeks to determine the chronic effects of microbiota exchange via co-housing on the responses of splenocytes and peritoneal macrophages. At the end of the study, peritoneal macrophages were isolated and the spleen was harvested and cells were immediately used for ex vivo stimulation assays.
To assess the general health condition of the mice after BMT, we monitored body weight during the study. As reported previously [12][13][14] , the BMT procedure reduced body weight already within a few days (Supplementary Fig. S1). The BMT-induced decrease in body weight was not restored by co-housing the mice with healthy controls and vice versa the body weight of the healthy co-housed controls was not affected by co-housing with BMT-treated mice.
Splenocytes of BMT-treated mice showed higher IL-10, IL-22 and TNF-α cytokine release as compared to healthy controls, when stimulated with various pathogenic stimuli such as LPS, Polyinosinic:polycytidylic acid (Poly(I:C)), Candida conidia or Salmonella typhimurium (outer light blue lines versus inner grey lines, Fig. 1a). Similar IL-10, IL-22 and TNF-α cytokine response patterns were seen in the BMT-treated mice that were co-housed with healthy control mice (outer dark blue lines versus inner grey lines, Fig. 1b), suggesting that co-housing with healthy controls did not rescue the hyper-responsive phenotype of the splenocytes induced by BMT. Strikingly, the healthy controls that were co-housed with the BMT-treated mice also showed a similar IL-10, IL-22 and TNF-α response pattern upon pathogenic stimulation (outer red lines versus inner grey lines Fig. 1c), indicating that the BMT-induced responsiveness of the splenocytes was transferred to healthy controls via co-housing.
The cytokine response pattern of stimulated peritoneal macrophages was not affected after BMT. Peritoneal macrophages of BMT-treated mice did not show increased cytokine release upon ex vivo stimulation with various stimuli, except for a tendency towards increased IL-6 release upon stimulation with S. typhimurium stimulation (outer light blue lines versus inner grey lines, Fig. 2a). Peritoneal macrophages of BMT-treated mice that were co-housed with healthy control mice released more IL-6 after stimulation, which was significant for Pam3Cys, poly(I:C), S. typhimurium and Staphylococcus aureus (outer dark blue lines versus inner grey lines, Fig. 2b). Although the cytokine response pattern of splenocytes upon different pathogenic stimuli was similar between the two groups of BMT-treated mice (Fig. 1a,b), the IL-6 cytokine response pattern of macrophages from co-housed BMT-treated mice was different (Fig. 2b). For TNF-α secretion, no differences were observed between the different groups ( Fig. 2a-c). These data indicate that the cytokine response pattern of peritoneal macrophages upon stimulation was not transferred from BMT-treated mice to control mice via co-housing.
The cytokine response pattern after BMT was transferred to splenocytes of germfree mice after transplantation of cecum content. To  www.nature.com/scientificreports/ www.nature.com/scientificreports/ involved in the altered cytokine response pattern by splenocytes after BMT, germfree mice were inoculated with the cecum content of BMT-treated mice co-housed with or without control mice or with the cecum content of co-housed control mice. Splenocytes derived from germfree mice that were colonized with cecum content of BMT-treated mice as compared to those colonized with cecum content of control mice, secreted more cytokines after stimulation with various pathogenic stimuli, which reached significance for LPS (IL-22 and TNF-α), PHA (IL-10 and TNF-α), poly(I:C) (IL-10, IL-22 and TNF-α) and C. conidia (IL10 and TNF-α) (outer light blue lines versus inner grey lines, Fig. 3a). A partly overlapping cytokine response pattern was observed in germfree mice inoculated with cecum content of BMT-treated mice co-housed with BMT mice and in germfree mice inoculated with cecum content of BMT-treated mice co-housed with control mice (outer light and dark blue lines versus inner grey lines, Fig. 3a,b), with similar (trends in) responses of IL-10, IL-22 and TNF-α secretion to PHA stimulation. Remarkably, splenocytes derived from germfree mice colonized with cecum content of control mice co-housed with BMT-treated mice showed a cytokine response pattern very similar to splenocytes from germfree mice that were colonized with cecum content of BMT-treated mice (outer red lines and light blue lines versus inner grey lines, Fig. 3a,c). The IL-10 response to PHA, poly (I:C) and C. conidia were identical, as was the TNF-α response to LPS and C. conidia. These data are in line with the results derived from the co-housing experiment and indicate that the increased responsiveness of splenocytes after BMT can be largely transferred by cecum content transplantation. Peritoneal macrophages of germfree mice inoculated with cecum content of BMT-treated mice and co-housed BMT-treated mice did not show increased IL-6 and TNF-α secretion upon stimulation (outer light blue and dark Spiderplots show IL-6 and TNF-α release in response to various pathogenic stimuli from peritoneal macrophages derived from (a) BMT-treated mice co-housed with BMT mice (outer light blue lines), (b) BMT-treated mice co-housed with healthy control mice (outer dark blue lines), and (c) control co-housed mice co-housed with BMT-treated mice (outer red lines). Cytokine concentrations in response to the stimuli are normalized to the cytokine concentrations of healthy control mice co-housed with control mice (inner grey lines), averaged per group and plotted on a log scale. See legend to Fig. 1  www.nature.com/scientificreports/ www.nature.com/scientificreports/ blue lines versus inner grey lines, Fig. 4a-c). Peritoneal macrophages of germfree mice inoculated with cecum content of co-housed controls only showed a minor increase in IL-6 secretion upon S. aureus incubation (outer red lines versus inner grey lines, Fig. 4c). These data indicate that gut microbiota specifically stimulate cytokine secretion of splenocytes but not of peritoneal macrophages after BMT.
Splenocytes of germfree mice showed lower cytokine secretion compared to conventional mice. To further investigate the role of gut microbiota in the responsiveness of splenocytes and macrophages, we compared cytokine response pattern of ex-vivo stimulated splenocytes and peritoneal macrophages derived from conventionally versus germfree raised mice. Splenocytes from conventional mice secreted more IL-10, IL-22 and TNF-α as compared to untreated germfree mice upon stimulation with LPS, Polyhydroxyalkanoates (PHA) and poly(I:C), and more IL-10 and IL-22 upon stimulation with S. typhimurium and S. aureus (outer pink lines versus inner purple lines, Fig. 5a-c). These data indicate that the presence of gut microbiota increases the cytokine response pattern of splenocytes upon pathogenic stimulation. In contrast to splenocytes, there was no increase of cytokine secretion by ex-vivo stimulated peritoneal macrophages between conventional mice and untreated germfree mice (outer pink lines versus inner purple lines, Fig. 5d,e). These data confirm that cytokine secretion of stimulated peritoneal macrophages is not affected by the presence of gut microbiota. www.nature.com/scientificreports/

16S sequencing did not show consistent longitudinal effects of BMT on intestinal microbiota composition.
To investigate potential changes in gut microbiota composition induced by BMT that may explain the increased cytokine response pattern of the splenocytes, we determined the intestinal microbial composition by 16S sequencing. We sequenced fecal samples 24 weeks post-BMT, which was just prior to the ex-vivo splenocyte stimulations and collection of the cecum contents. Bacterial taxa were not significantly different between BMT-treated and control mice, either with or without co-housing (Fig. 6a). There was a large interindividual variation between the individual samples from the different groups as revealed by the PCoA plots of unweighted UniFrac distance (Fig. 6b). Individual samples from the different treatment groups clustered together in three sub-clusters, but no clustering was observed based on treatment. The splenocyte responsiveness could thus not be linked to obvious BMT-induced differences in intestinal microbial composition as determined by 16S sequencing.
Co-housing with BMT-treated mice affected cecum metabolite profiles of healthy control mice. To investigate whether BMT induced alterations in gut microbial function that may explain the cytokine response pattern of the splenocytes, we measured 61 metabolites in cecum content samples by 1H-NMR. Metabolite concentrations were quantitatively measured and plotted as z scores in a heat map after unsupervised clustering of all mice (Fig. 6c). The metabolite profile patterns showed considerable heterogeneity between the four www.nature.com/scientificreports/ experimental groups (Fig. 6c). However, the mice from the control group are largely clustered together and are overall different from the other groups. These data indicate that BMT-treated and healthy control mice modulate each other's metabolites via co-housing. However, none of the metabolites were clearly linked to the cytokine responses pattern of splenocytes of BMT-treated mice.

Discussion
Our results demonstrate that BMT induced chronically increased cytokine responses of splenocytes but not peritoneal macrophages upon ex-vivo stimulation with various pathogenic stimuli. For splenocytes, and not for peritoneal macrophages, the BMT-induced cytokine response patterns were at least partly mediated via the gut microbiota, as the increased response pattern was largely transferred to splenocytes of healthy control mice via co-housing and via cecum content transplantation to germfree mice. In line with a role for microbiota, splenocytes, but not peritoneal macrophages from germfree mice showed lower stimulated cytokine response patterns as compared to conventional mice. The BMT-induced increased splenocyte response pattern could not be linked to obvious BMT-induced differences in gut microbiota composition or metabolites in cecum. Nevertheless, our data clearly show that gut microbiota are an important determinant of the cytokine response pattern of splenocytes, but not of peritoneal macrophages. The differential role of microbiota in the cytokine responses pattern of splenocytes but not peritoneal macrophages may be associated with physical access of (components of) the intestinal content to splenocytes. In contrast to the peritoneal cavity, the spleen is an integral part of the blood circulation. In addition to accessibility, the immune cell composition and function of the spleen differs from peritoneal macrophages. The spleen contains multiple populations of leukocytes from both the innate and adaptive immune system, whereas peritoneal macrophages belong to the innate immune system. The increased secretion of IL-10 and IL-22 by splenocytes upon BMT suggests an elevated adaptive immune response, as these cytokines are mainly secreted by T cells. However, it remains to be investigated whether the microbiota affect specific subsets of immune cells from the adaptive or innate immune system in the spleen.
The higher response pattern of splenocytes after BMT did not coincide with detectable changes in gut microbial composition as determined by 16S sequencing. It is possible that the resolution of the 16S sequencing technique to distinguish bacterial taxa was not sufficient to show such alterations. Metagenomic sequencing in this respect might have revealed differences at the species level. However, the intra-individual variation in microbial composition between the mice at baseline before BMT-treatment was extremely large. This more than likely hampered detection of significant changes in microbial composition induced by the BMT.
To identify mechanisms underlying the higher response pattern of splenocytes induced by gut microbiota, we further focused on alterations in the function of the gut microbiota induced by BMT. We considered that multiple species of bacteria can exert the same function and produce the same metabolites. For instance, multiple bacterial species can produce SCFA which can cross the intestinal border and have extra-intestinal immune modulatory effects in several tissues including the spleen 8,15 . By performing 1H-NMR on the cecum content samples that were used to inoculate the germfree mice and subsequent unsupervised clustering on the mice, we observed significant heterogeneity in the experimental groups. However, the mice from the control group largely clustered together and are overall different from the other groups. These data indicate that both the BMT and co-housing procedure affected metabolite levels in the gut. Although we can conclude that BMT and co-housing led to alterations in microbial function in our study, the underlying mechanisms linking the gut microbiota to splenocyte cytokine response pattern remain to be elucidated.
In addition to bacterial metabolites, bacterial components such as LPS and peptidoglycans can modulate the immune system. BMT conditioning results in intestinal damage with the likely consequence of LPS leaking into the system. Our data show that the response pattern of splenocytes of BMT-treated mice was transferrable to splenocytes of healthy controls which have no damaged intestine. This makes LPS leakage an unlikely cause for the hyper-responsiveness of the splenocytes in the healthy controls co-housed with BMT mice. On the other hand, bacterial peptidoglycans have been reported to translocate from the gut into the circulation also under basal conditions 6 . It remains to be investigated whether peptidoglycans can explain the hyper-responsiveness of splenocytes after BMT.
Although we conclude that the microbiota are at least partly responsible for the long term transfer of splenocyte cytokine response patterns via co-housing and cecum transplantation, we cannot exclude that other factors are involved. Recently, Liu et al. 15 identified microRNAs (MIR) in feces that are produced by epithelial cells in the intestine and affect microbial function by regulating bacterial gene transcription. It is thus possible that BMT-induced alterations in epithelial cell MIR secretion play a role in the altered responsiveness of splenocytes after BMT.
Our study may have clinical implications. Here, we focussed on the role of microbiota in syngeneic BMT. However, in humans allogenic BMT is common practice and is associated with graft versus host disease (GVHD). In GVHD, donor immune cells (mainly the T cells) are activated by recipient cells and cause severe inflammation and damage to skin, liver, hematopoietic system and gut. The initiation of GVHD depends not only on activation of the donor T cells, but also on activation of the recipient's antigen presenting cells and the interaction between these two. As the current study shows, also gut changed microbiota influence the recipient's immune response and could affect GVHD activation and outcome. Further research is needed to monitor gut microbiota dysbiosis and the possibility of restoring the gut microbiome to a healthy state in BMT patients to reduce the risk of GVHD development.
In conclusion, gut microbiota increase the cytokine responses pattern of splenocytes after BMT. This phenotype can be transferred to splenocytes of healthy controls by co-housing or to germfree mice via transfer of cecum content, indicating that they are independent of BMT-induced intestinal damage and microbial leakage. Whether www.nature.com/scientificreports/ the BMT-induced higher cytokine response pattern of splenocytes is due to changes in microbial composition or activity, or other transferable factors, remains to be further investigated.

Methods
Bone marrow transplantation and co-housing experiment with mice. 6 week old male C57Bl/6J mice were purchased from Charles River (Maastricht, The Netherlands) and housed under standard conditions with free access to water and food. After a period of 2 weeks acclimatisation, half of the mice underwent syngeneic BMT. Already before the BMT procedure and during the whole experiment, the BMT-treated mice were co-housed with BMT-treated mice or healthy control mice. This resulted in four different experimental mouse groups: (1) healthy control mice co-housed with healthy control mice, (2) BMT-treated mice co-housed with BMT-treated mice, (3) BMT-treated mice co-housed with healthy controls, (4) healthy controls co-housed with BMT-treated mice. For the BMT procedure, mice received 8 Gy X-ray radiation using an Orthovolt and the day thereafter they received an intravenous injection with donor bone marrow cells in the tail vein. The donor mice were male C57Bl/6J mice of similar age. All mice, both BMT-treated and control mice, received water containing antibiotics (Amphotericin B, Ciprofloxacin, Polymyxin B) from 3 days before until 4 weeks after BMT and were exposed to the same dietary regimen. After 8 weeks recovery on chow diet, mice were fed a low-fat diet (10% energy derived from lard fat; D12450B, Research Diet Services, Wijk bij Duurstede, The Netherlands). Body weight was measured weekly during the entire experiment. Fresh fecal samples from individual mice were obtained 1 week before and 24 weeks after BMT by colon massaging. Each fecal sample was separately stored in a cryovial and snap frozen in liquid nitrogen immediately after collection and subsequently stored at − 80 °C until time of genomic DNA isolation. Mice were euthanized after anesthetization by a subcutaneous injection of a mixture of Neurotranq, Midazolam and Fentanyl. The spleen, peritoneal macrophages and cecum content were collected. This study was part of a larger study of which recently the metabolic characterization was described 14 . All experiments were approved by the animal ethics committee of the Leiden University Medical Center, Netherlands (protocol no. 121031), and conducted in accordance with the European directive 2010/63/UE. This study was carried out in compliance with the ARRIVE guidelines.
Experiments with germfree mice. Eight to twelve weeks old male germfree Swiss/NIH mice were obtained from Taconic Farms and kept in sterile flexible plastic isolators (Standard Safety Equipment) with free access to sterile water and food. Conventional Swiss/NIH mice were obtained from the local Animal Facility at Universidade Federal de Minas Gerais, Brasil. The cecum content of the mice from the four different experimental groups (1-healthy control mice co-housed with healthy control mice, 2-BMT-treated mice co-housed with BMT-treated mice, 3-healthy controls co-housed with BMT-treated mice, 4-BMT-treated mice co-housed with healthy controls. All mice in the four groups received water containing antibiotics (Amphotericin B, Ciprofloxacin, Polymyxin B) from 3 days before until 4 weeks after BMT and were exposed to the same dietary regimen) were diluted in PBS (10% w/v) and administered by intragastric gavage (200 µL per mice) to the germfree mice. After 2 weeks of microbiota reconstitution, the colonized mice were euthanized to collect spleen and peritoneal macrophages. Spleen and peritoneal macrophages were also obtained from germfree mice and conventional mice of similar age.
Ex-vivo stimulations of peritoneal macrophages. Peritoneal macrophages were isolated from mice by injecting 10 mL of ice-cold sterile PBS (pH 7.4) into the peritoneal cavity, as previously described 16 . After centrifugation and washing, cells were resuspended in RPMI-1640 culture medium containing 1 mM pyruvate, 2 mM l-glutamine, and 50 mg/L gentamicin. Cells were counted and cultured in 96-well round-bottom microtiter plates (Costar, Corning, The Netherlands) at 1 × 10 5 cells/well, in a final volume of 200 μL. The stimuli were the same as for the splenocytes except that instead of PHA, Pam3Cys was used at 10 µg/mL. Supernatants were collected after 24 h incubation and stored at − 80 °C for measurement of TNF-α and IL-6.
Cytokine measurements. Cytokine concentrations were measured in supernatants of the ex-vivo stimulation experiments. TNF-α concentrations were determined by a specific radioimmunoassay as previously described 17 . IL-6 was measured using a commercially available ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA). Similarly, IL-22 and IL-10 concentrations were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA). All according to the instructions of the manufacturer.
16S rRNA gene sequencing and data analysis. From  www.nature.com/scientificreports/ 70% ethanol. The DNA samples were sent to the Broad Institute of MIT and Harvard (Cambridge, USA) for 16S rRNA gene sequencing. Microbial 16S rRNA gene was amplified targeting the hyper-variable region V4 using forward primer 515F (5′-GTG CCA GCMGCC GCG GTAA-3′) and the reverse primer 806R (5′-GGA CTA CHVGGG TWT CTAAT-3′). The cycling conditions consisted of an initial denaturation step at 94 °C for 3 min, followed by 25 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 60 s, extension at 72 °C for 5 min, and a final extension at 72 °C for 10 min. Sequencing was performed using the Illumina MiSeq platform generating paired-end reads of 175 bp in length in each direction. Overlapping paired-end reads were subsequently aligned. Details of this protocol have been described previously 18 .

Metabolites measurement by 1H-NMR.
Weighed cecum content samples (28.4 ± 8.9 mg) were mixed with 5 volumes of milliQ water and prepared and measured using 1H-NMR spectroscopy as described previously 20 . The identification of metabolites was performed using the databases from Bruker (Bruker Biospin Ltd.) and Chenomx (Chenomx NMR suite 8.2) and assignments were verified by 2D 1H-NMR experiments of selected samples. The quantification of metabolites was performed with the Chenomx software and quantities were corrected for sample weight 21 . Statistics. Data are presented as means ± SEM. Experimental groups were compared using Mann-Whitney U test. All statistical analyses were performed using GraphPad Prism version 6 (GraphPad software, San Diego, CA, USA).


Regulation of Stanniocalcin Secretion by Calcium and PTHrP in Gilthead Seabream (Sparus aurata)

Simple Summary Calcium regulation in body fluids is a fundamental process in vertebrates, which is exerted by a plethora of hormones. Stanniocalcin (STC) is a hypocalcemic hormone ubiquitously expressed in tetrapods; in bony fishes, it is produced mainly by specific glands called the corpuscles of Stannius. The present study described an ELISA method for the analysis of fish STC. Moreover, it also develops a methodology for ex vivo cultures of Stannius corpuscles of gilthead seabream (Sparus aurata). The results show a direct control of the production of STC by one calcium and two PTHrP (parathyroid-related protein, a hypercalcemic hormone) receptors. This study highlights the tight control of circulating calcium in vertebrates and shows the complexity of the processes involved. Abstract Calcium balance is of paramount importance for vertebrates. In fish, the endocrine modulators of calcium homeostasis include the stanniocalcin (STC), and some members of the parathyroid hormone (PTH) family, such as the PTH-related protein (PTHrP), acting as antagonists. STC is ubiquitously expressed in higher vertebrates. In turn, bony fish exhibit specific STC-producing glands named the corpuscles of Stannius (CS). Previous studies pointed to a calcium-sensing receptor (CaSR) involvement in the secretion of STC, but little is known of the involvement of other putative regulators. The CS provides a unique model to deepen the study of STC secretion. We developed an ex vivo assay to culture CS of fish and a competitive ELISA method to measure STC concentrations. As expected, STC released from the CS responds to CaSR stimulation by calcium, calcimimetics, and calcilytic drugs. Moreover, we uncover the presence (by PCR) of two PTHrP receptors in the CS, e.g., PTH1R and PTH3R. Thus, ex vivo incubations revealed a dose-response inhibition of STC secretion in response to PTHrP at basal Ca2+ concentrations. This inhibition is achieved through specific and reversible second messenger pathways (transmembrane adenylyl cyclases and phospholipase C), as the use of specific inhibitors highlights. Together, these results provide evidence for endocrine modulation between two antagonist hormones, STC and PTHrP.

Introduction
Calcium is of paramount importance for many physiological processes in vertebrates, such as bone and scale mineralization, enzyme control, and regulation of plasma membrane permeability and excitability [1], among other functions. The delicate calcium balance is not subject to purely diffusive processes but is under strict endocrine regulation. In this sense, teleost fish primarily regulate calcium availability by the action of two antagonistic hormones, the parathyroid hormone-related protein (PTHrP) and the stanniocalcin (STC) [2][3][4]. The first is a hypercalcemic hormone [5], while the second has anti-hypercalcemic functions in fish [6].

Animal Maintenance
Seabream (Sparus aurata) immature juveniles (84.2 ± 1.9 g body weight, mean ± SEM, n = 134) were obtained from commercial sources in the Algarve (Portugal) and transported to the Campus facility (CCMAR, Faro-Portugal). Before the experiments, fish were maintained for at least ten days in 500 L tanks in a recirculated seawater circuit (36-37‰) with a biological filter. The water temperature was maintained at 23-25 • C. The fish were exposed to a natural photoperiod (September-December), grouped in tanks at a density of 4.5 kg m −3, and fed twice daily 1% body weight (wet fish weight, Sorgal S.A., São João de Ovar, Portugal; Balance 3). Animals fasted 24 h before the experiments, and there was no mortality during these processes. The experiments conducted comply with the guidelines of the European Union Council (86/609/EU). All animal protocols were performed under a "Group-1 ' license from the Direcção-Geral de Veterinária, Ministério da Agricultura, do Desenvolvimento Rural e das Pescas, Portugal.

Sampling
Fish were captured and anesthetized in 2-phenoxyethanol (0.1% v/v; P-1126, Sigma-Aldrich, St Louis, MO, USA). Blood samples were collected from the caudal peduncle with heparinized syringes, and plasma was obtained by centrifugation (3500× g, 5 min, 4 • C) and stored at −20 • C until analysis. Fish were sacrificed by decapitation, and the CS collected (their location in the caudal end of the kidney can be seen in Figure S1) for ex vivo culture (see below).

Development of an ELISA Method
Stanniocalcin protein levels were quantified by an in-house developed indirect antigen competitive ELISA method, similarly to what was previously described [34]. The assay is based on competition between free STC in standards, or samples and STC immobilized on microtiter plates for the STC-antibodies. Purified seabream STC [3] was employed to generate a specific rabbit antiserum [22], hereafter named Anti-STC. A range of assay optimization procedures was carried out, including assessment of assay buffers; the coating concentration of STC (from CS-homogenates, 0.5-10.0 µg prot well −1 ); titration of primary (1:1500-1:8000), and secondary antisera (1:2000-1:6000) to give a maximal optical density (OD490 nm) equal or higher than one and background not higher than 0.1 (OD490 nm); assay incubation times and washing conditions. ELISA validation included verification of parallelism between samples and the standard curve, the recovery rate of standards added to the assay, and the intra-assay and inter-assay variation coefficients.
Using the optimized procedure, ELISA was carried out in duplicate for standard and samples in 96-well plates (Ref. 655061, Greiner bio-one, Kremsmünster, Austria). In brief, wells were coated for 2 h at 37 • C with 2 µg prot 100 µL −1 well −1 of seabream CShomogenates (in 0.6% w/v NaCl) diluted in bicarbonate buffer (50 mM NaHCO 3 / Na 2 CO 3 and 150 mM NaCl to give a final pH of 9.6). All the wells, including blank wells (without STC coating), were then blocked with 250 µL of 2% (w/v) bovine serum albumin (BSA) in PBS. Anti-STC (1:8000 dilution) was added to the plate along with either standard protein (range from 2000 to 52 ng mL −1 ) or samples (100 µL well −1 ) and incubated 10 min at room temperature with constant stirring, followed by incubation for 30 min at 37 • C. Anti-rabbit IgG peroxidase conjugate (100 µL well −1 , 1:5000 dilution, A9169, Sigma-Aldrich) was added and followed by an incubation similar to the primary antibody. Excess reagents were removed between incubation steps by washing 4 × 5 min with PBS/Tween-20 (0.05% v/v). The color was developed with 200 µL OPD (o-phenylenediamine dihydrochloride, P9187, Sigma-Aldrich) for 13 min in the dark at room temperature. The reaction stopped by adding 50 µL well −1 2 M H 2 SO 4, and the absorbance was read at 490 nm in a microplate reader (Benchmark, Bio-Rad, Hercules, CA, USA). All assays determined the standard curve, non-specific binding, and maximal binding.
Stanniocalcin protein levels in plasma, CS-homogenates, and incubation medium were quantified by the developed ELISA method for STC.
A time-course survey was performed for the CS ex vivo incubations in the same conditions described before to test the STC secretion rate for 3, 6, and 24 h. Incubations with 0.05% DMSO (D-8779, Sigma-Aldrich) were also performed, as this is the maximum percentage of DMSO employed to pre-dilute drugs in the experiments.
All the experimental assays were performed together with a control group, using both CS from a single animal to test experimental vs. control condition ceteris paribus (whenever possible). Repeated independent experiments, with at least 4 to 6 CS per treatment, were also performed to ensure proper statistical robustness of the results.
At the end of the incubation period, CS were transferred to 120 µL 0.6% NaCl and homogenized with a pestle (VWR Pellet Mixer, 431-0100, VWR International), followed by a 10 min, 9000× g centrifugation at 4 • C. The supernatant of the CS-homogenates and incubation medium, separately, were immediately frozen at −20 • C until STC analysis.
STC was analyzed in the incubation medium at the end of the ex vivo incubation period to calculate STC secretion (normalized to incubation time and protein quantity in the CS-homogenate). Moreover, total STC was evaluated as the sum of STC quantity in both the incubation medium (and thus secreted from the CS) and that present in the CS at the end of the ex vivo assay. Total STC was employed as a proxy to evaluate STC production in the CS during the incubation time.

CaSR and PTHrP Receptors RT-PCR
Total RNA from the CS was extracted with E.Z.N.A. total RNA isolation kit I (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer's instructions. The quantity and quality were assessed (Nanodrop 1000 Thermo Scientific, Barrington, IL, USA). Prior to cDNA synthesis, RNA was treated with DNase using a DNA-free kit (Ambion, Life Technologies, Paisley, UK), following the supplier's instructions. Reverse transcription of RNA into cDNA was carried out using Revert Aid first-strand cDNA synthesis kit (Fermentas, Thermo Scientific, Waltham, MA, USA) following the manufacturer's instructions, with 500 ng of total RNA in a final reaction volume of 20 µL. Table 1 shows primer sequences, amplicon sizes, and NCBI accession numbers of the products.
PCR amplifications were performed in a final volume of 15 µL with 7.5 µL SsoFast Eva-Green Supermix (Bio-Rad, UK),~15 ng cDNA based on RNA input to reverse transcription), and 0.3 µM of each forward and reverse primers. Amplifications were performed in 96-well plates in a T100 thermal cycle (Bio-Rad, UK) with the following protocol: denaturation and enzyme activation step at 95 • C for 2 min, followed by 40 cycles of 95 • C for 5 s, and primer-pair specific annealing temperature for 10 s (Table 1) at 60-55-53 • C for 10 s and 30 s at 72 • C. This protocol runs a product amplification of 131 bp for casr, 142 bp pth1r, and 250 bp for pth3r. 18S ribosomal RNA (18s), as the reference gene, generated a 139 bp product. Amplified products (15 µL) were analyzed on 1.5% agarose gel stained with ethidium bromide. Gel images were captured using an AlphaImager Gel Imaging System from Alpha Innotech (San Leandro, CA, USA).

Statistics
Differences between groups were tested by a Student s t-test. One-way ANOVA was employed when different conditions related to the same experiment were tested at the same time, considering those groups as factors of variance. Normality was analyzed using the Kolmogorov-Smirnov s test. The homogeneity of variances was analyzed by Levene s test. When ANOVA yielded significant differences, Tukey's post-hoc test was used to identify significantly different groups. If the results of the same treatment from different independent experiments did not show statistical differences between them, they were pooled and further analyzed as one single group. Statistical significance was accepted at p < 0.05. All the results are given as mean ± SEM unless otherwise stated.

ELISA
We developed an indirect competitive ELISA using purified seabream STC as the standard and a primary rabbit polyclonal antisera raised against it. The coating concentration of CS-homogenates was 2 µg total protein, around 25 ng STC 100 µL −1 well −1 . The primary antiserum against STC was used at a dilution of 1:8000, which gave a maximal OD490 nm of 1.3 and a non-specific background of 0.09-0.13 at OD490 nm well −1 . The method is reliable and reproducible as the intra-and inter-assay coefficients of variation were 4%, each, calculated from the analysis of seven samples/two times/microplate for the intra-assay coefficient and six samples/four microplates for the inter-assay coefficient. The lower detection limit of the assay was 2.6 ng STC well −1 (52 ng mL −1 ). Dilution curves constructed with plasma samples and CS-homogenates showed parallel displacements compared to the standard dilution curve ( Figure S2).

STC Secretion and CS Content
The STC secretion rate, when normalized to time and amount of protein, does not present variations in ex vivo incubations of CS for 3, 6, or 24 h ( Figure S3) (one-way ANOVA followed by a Tukey s test, p < 0.05). Since proper dilution of some of the chemicals employed in this study required the addition of DMSO, we also performed ex vivo incubations of CS with a final concentration of 0.05% DMSO (v/v) for 3, 6, and 24 h. The results show that the STC secretion rate decreased during incubation time in vitro, with significant differences between the groups at 3 and 24 h (one-way ANOVA followed by a Tukey s test, p < 0.05). When comparing control versus DMSO treated groups (Student s t-test, p < 0.05), no differences were observed in any of the groups tested. In this line, the secreted quantity of STC after 3 h of incubation edges the detection limits of the ELISA method in some samples. We, therefore, choose an incubation time of 6 h for the experiments. Thus, the average STC secretion rate from the CS of control gilthead seabream (84.2 ± 1.9 g weight individuals) is 0.18 ± 0.03 ng STC µg prot −1 h −1 (n = 52) (mean ± SEM). The average content of STC in the CS at the moment of sampling was 11.17 ± 1.31 ng STC µg prot −1 (n = 4), which is in good agreement with the STC content of the CS of the control groups after six hours of incubation (10.33 ± 0.85 ng STC µg prot −1 , n = 52) (Student s t-test, p = 0.77). Plasma STC concentration in seabream was 1693 ± 81 ng STC mL −1 (n = 42 fish). Assuming that the plasma of gilthead seabream is 2.00% of its total body weight (blood occupies 2.9% of the total body weight [43], while the normal hematocrit for seabream is 31% [44]), we have calculated the total amount of STC within the CS (in both together, from a single fish) of each animal compared to the total circulating STC in the plasma, which results in 9.8 ± 1.0% (n = 42). present variations in ex vivo incubations of CS for 3, 6, or 24 h ( Figure S3) (one-way ANOVA followed by a Tukey´s test, p < 0.05). Since proper dilution of some of the chemicals employed in this study required the addition of DMSO, we also performed ex vivo incubations of CS with a final concentration of 0.05% DMSO (v/v) for 3, 6, and 24 h. The results show that the STC secretion rate decreased during incubation time in vitro, with significant differences between the groups at 3 and 24 h (one-way ANOVA followed by a Tukey´s test, p < 0.05). When comparing control versus DMSO treated groups (Student´s t-test, p < 0.05), no differences were observed in any of the groups tested. In this line, the secreted quantity of STC after 3 h of incubation edges the detection limits of the ELISA method in some samples. We, therefore, choose an incubation time of 6 h for the experiments. Thus, the average STC secretion rate from the CS of control gilthead seabream (84.2 ± 1.9 g weight individuals) is 0.18 ± 0.03 ng STC μg prot −1 h −1 (n = 52) (mean ± SEM). The average content of STC in the CS at the moment of sampling was 11.17 ± 1.31 ng STC μg prot −1 (n = 4), which is in good agreement with the STC content of the CS of the control groups after six hours of incubation (10.33 ± 0.85 ng STC μg prot −1 , n = 52) (Student´s ttest, p = 0.77). Plasma STC concentration in seabream was 1693 ± 81 ng STC mL −1 (n = 42 fish). Assuming that the plasma of gilthead seabream is 2.00% of its total body weight (blood occupies 2.9% of the total body weight [43], while the normal hematocrit for seabream is 31% [44]), we have calculated the total amount of STC within the CS (in both together, from a single fish) of each animal compared to the total circulating STC in the plasma, which results in 9.8 ± 1.0% (n = 42).    Figure 2A shows the percentage (%) of STC secreted by seabream s CS incubated for six hours with normal (1.46 mM Ca 2+ ) and high calcium (2.92 mM Ca 2+ ) levels. A significant increase in STC release (Student s t-test, p < 0.05) was observed when incubating in the presence of 2.92 mM Ca 2+ (657 ± 83% compared to the control-normal calcium group). Figure 2B shows the % of total STC quantity (ng STC µg prot −1 ) present in the CS and the incubation medium, together, for the experimental group respecting the control group. No differences were found between both groups (Student s t-test, p < 0.05).

Effect of CaSR Agonist and Antagonist Molecules
increase in STC release (Student´s t-test, p < 0.05) was observed when incubating in the presence of 2.92 mM Ca 2+ (657 ± 83% compared to the control-normal calcium group). Figure 2B shows the % of total STC quantity (ng STC μg prot −1 ) present in the CS and the incubation medium, together, for the experimental group respecting the control group. No differences were found between both groups (Student´s t-test, p < 0.05). The effect of a CaSR antagonist is shown in Figure 3. Incubation with a high concentration of calcium (2.92 mM Ca 2+ ) and the addition of 0.1 μM of the CaSR antagonist NPS-2143 ( Figure 3A) decreased STC release from the CS to 50 ± 10% (mean ± SEM) of the control group (with 2.92 mM Ca 2+ alone), with significant differences (Student´s t-test, p < 0.05). Moreover, the calcilytic NPS-2143 decreased significantly (Student´s t-test, p < 0.05) the total amount of STC compared to the control-high [Ca 2+ ] group (49 ± 4%) ( Figure 3B). The effect of a CaSR antagonist is shown in Figure 3. Incubation with a high concentration of calcium (2.92 mM Ca 2+ ) and the addition of 0.1 µM of the CaSR antagonist NPS-2143 ( Figure 3A) decreased STC release from the CS to 50 ± 10% (mean ± SEM) of the control group (with 2.92 mM Ca 2+ alone), with significant differences (Student s t-test, p < 0.05). Moreover, the calcilytic NPS-2143 decreased significantly (Student s t-test, p < 0.05) the total amount of STC compared to the control-high [Ca 2+ ] group (49 ± 4%) ( Figure 3B). presence of 2.92 mM Ca 2+ (657 ± 83% compared to the control-normal calcium group). Figure 2B shows the % of total STC quantity (ng STC μg prot −1 ) present in the CS and the incubation medium, together, for the experimental group respecting the control group. No differences were found between both groups (Student´s t-test, p < 0.05). The effect of a CaSR antagonist is shown in Figure 3. Incubation with a high concentration of calcium (2.92 mM Ca 2+ ) and the addition of 0.1 μM of the CaSR antagonist NPS-2143 ( Figure 3A) decreased STC release from the CS to 50 ± 10% (mean ± SEM) of the control group (with 2.92 mM Ca 2+ alone), with significant differences (Student´s t-test, p < 0.05). Moreover, the calcilytic NPS-2143 decreased significantly (Student´s t-test, p < 0.05) the total amount of STC compared to the control-high [Ca 2+ ] group (49 ± 4%) ( Figure 3B). The effect of different CaSR agonists (R-568, neomycin, gadolinium, and spermine) was also tested (Figure 4). Significant increases when compared to the control CS STC release (Student s t-test, p < 0.05) are shown in Figure 4A when incubated with calcimimetics R-568 (260 ± 56%), neomycin (381 ± 104%) and gadolinium (166 ± 6%). Spermine, at a concentration of 500 µM, did not affect the STC secretion rate (Student s t-test, p > 0.05). The calcimimetics, neomycin and gadolinium, also increased significantly the total amount of STC (125 ± 7% and 200 ± 37%, respectively) of the incubated CS when compared to the control group ( Figure 4B, Student s t-test, p < 0.05).
The effect of different CaSR agonists (R-568, neomycin, gadolinium, and spermine) was also tested (Figure 4). Significant increases when compared to the control CS STC release (Student´s t-test, p < 0.05) are shown in Figure 4A when incubated with calcimimetics R-568 (260 ± 56%), neomycin (381 ± 104%) and gadolinium (166 ± 6%). Spermine, at a concentration of 500 μM, did not affect the STC secretion rate (Student´s t-test, p > 0.05). The calcimimetics, neomycin and gadolinium, also increased significantly the total amount of STC (125 ± 7% and 200 ± 37%, respectively) of the incubated CS when compared to the control group ( Figure 4B, Student´s t-test, p < 0.05).   Figure 5A shows the inhibitory effect of increasing PTHrP concentrations on the STC secretion at normal (1.46 mM) calcium levels. PTHrP inhibits STC release from the CS in a dose-dependent manner, with significant differences between those groups submitted to 0 ng PTHrP mL −1 and 100 ng PTHrP mL −1 (one-way ANOVA followed by a Tukey s test, p < 0.05). In this sense, the calculated effective dose 50 (ED50) was 17.51 ng PTHrP mL −1 . The % of total STC ( Figure 5B) showed variations in any of the groups tested (one-way ANOVA followed by a Tukey s test, p > 0.05).

PTHrP Downregulates STC Secretion
Biology 2022, 11, 863 9 of 15 a dose-dependent manner, with significant differences between those groups submitted to 0 ng PTHrP mL -1 and 100 ng PTHrP mL -1 (one-way ANOVA followed by a Tukey´s test, p < 0.05). In this sense, the calculated effective dose 50 (ED50) was 17.51 ng PTHrP mL -1 . The % of total STC ( Figure 5B) showed variations in any of the groups tested (one-way ANOVA followed by a Tukey´s test, p > 0.05).  Values are presented as % of the control group for single independent experiments (mean ± SEM, n = 6 per group). Different letters indicate significant differences between groups (one-way ANOVA followed by a Tukey s post-hoc test, p < 0.05). Figure 6 reveals that the tmAC inhibitor SQ-22536 and the PLC inhibitor U-73122, separately, slightly increased STC secretion ( Figure 6A) when it is inhibited with 100 ng PTHrP mL −1 , without being statistically different from the untreated-control or the PTHrPtreated groups (one-way ANOVA, followed by a Tukey s test, p < 0.05). Moreover, using both inhibitors together abolished the inhibitory effect of PTHrP (107 ± 28% respecting the untreated-control group). No differences in STC production ( Figure 6B) were observed between treatments (one-way ANOVA, followed by a Tukey s test, p > 0.05).
PTHrP mL -1 , without being statistically different from the untreated-control or the PTHrPtreated groups (one-way ANOVA, followed by a Tukey´s test, p < 0.05). Moreover, using both inhibitors together abolished the inhibitory effect of PTHrP (107 ± 28% respecting the untreated-control group). No differences in STC production ( Figure 6B) were observed between treatments (one-way ANOVA, followed by a Tukey´s test, p > 0.05). Figure 6. STC ex vivo secretion (A) and production (B) from the corpuscles of Stannius of the gilthead seabream treated with 100 ng mL -1 PTHrP alone or with combinations of the adenylyl cyclase inhibitor SQ-22536 (100 μM, SQ) and the phospholipase C inhibitor U-73122 (10 μM, U). Control group presents 1.46 mM Ca 2+ and no PTHrP or specific inhibitors (basal STC secretion of 0.19 ± 0.03 ng STC μg prot −1 h −1 , mean ± SEM). Different letters indicate significantly different groups (n = 6 per group, one-way ANOVA followed by a Tukey´s post-hoc test, p < 0.05).  Figure 7 shows the combined effects of high (2.92 mM) calcium levels and increasing PTHrP concentrations. STC secretion ( Figure 7A) or production ( Figure 7B) are not significantly affected by any of the PTHrP concentrations tested (one-way ANOVA followed by a Tukey s test, p > 0.05). Figure 7 shows the combined effects of high (2.92 mM) calcium levels and increasing PTHrP concentrations. STC secretion ( Figure 7A) or production ( Figure 7B) are not significantly affected by any of the PTHrP concentrations tested (one-way ANOVA followed by a Tukey´s test, p > 0.05).

Figure 7.
Dose-response effects of PTHrP on STC ex vivo secretion (A) and production (B) from the corpuscles of Stannius of the gilthead seabream at high (2.92 mM Ca 2+ ) calcium concentration. Basal STC secretion for the 0 ng PTHrP mL −1 group is 0.82 ± 0.17 ng STC μg prot −1 h −1 (mean ± SEM). No significant differences between groups were observed (n = 9 per group, one-way ANOVA followed by a Tukey´s post-hoc test, p > 0.05).

Discussion
In the present study, we demonstrate the regulatory action of calcium and PTHrP on the release of stanniocalcin from the corpuscles of Stannius, a specific bony fish gland and endocrine tissue. These effects involve the participation of a calcium-sensing receptor and imply an unexpected role of the PTHrP receptors, which, based on transcriptomic evidence, are supposedly absent from the CS of a teleost fish [45]. In this sense, the antagonistic hormone of the STC, the PTHrP, exerts a direct down-regulation of the STC secretion through the participation of at least two PTHrP receptors, likely receptor1 and receptor3. Figure 7. Dose-response effects of PTHrP on STC ex vivo secretion (A) and production (B) from the corpuscles of Stannius of the gilthead seabream at high (2.92 mM Ca 2+ ) calcium concentration. Basal STC secretion for the 0 ng PTHrP mL −1 group is 0.82 ± 0.17 ng STC µg prot −1 h −1 (mean ± SEM). No significant differences between groups were observed (n = 9 per group, one-way ANOVA followed by a Tukey s post-hoc test, p > 0.05).

Discussion
In the present study, we demonstrate the regulatory action of calcium and PTHrP on the release of stanniocalcin from the corpuscles of Stannius, a specific bony fish gland and endocrine tissue. These effects involve the participation of a calcium-sensing receptor and imply an unexpected role of the PTHrP receptors, which, based on transcriptomic evidence, are supposedly absent from the CS of a teleost fish [45]. In this sense, the antagonistic hormone of the STC, the PTHrP, exerts a direct down-regulation of the STC secretion through the participation of at least two PTHrP receptors, likely receptor1 and receptor3.
A CaSR is present in the CS glands of the gilthead seabream, an observation that reinforces previous studies in trout [18], flounder [17], and zebrafish [19]. The first two studies described a fast 9-fold and 3-fold increase in the plasma STC levels in response to intraperitoneal injections of the calcimimetics NPS-467 or R-568. The resemblance between these in vivo and our ex vivo results is evident as we show a 6.5-fold increase in ex vivo STC secretion in response to high calcium concentrations for six hours. This result is reinforced by the stimulation ranges of STC secretion of a 1.6 to 3.8-fold increase in response to the tested calcimimetics used in our study. Previous studies with rainbow trout and European eel also show increased STC release to the plasma in response to CaCl 2 administration [46]. As those results came from in vivo experiments, side effects or interfering alternative routes in the regulation of STC secretion need consideration. However, our ex vivo approach negates those circumstances and facilitates a direct understanding of the effects at the level of the gland. Thus, the stimulation of STC secretion not only by Ca 2+ but also through the calcimimetics R-568, neomycin, and gadolinium indicates the direct involvement of CaSR in STC secretion regulation.
In addition, calcimimetics, neomycin and gadolinium, also increase the total percentage of available STC, pointing to a possible enhancement of both, increase in secretion and stimulation of hormone synthesis. Furthermore, the allosteric antagonist of the CaSR NPS-2143 down-regulates STC secretion and the STC synthesis in the CS gland. In this sense, using a calcilytic compound in flounder also decreased plasma STC levels to half of the control after treatment with EGTA [17], supporting our results. Thus, the involvement of CaSR in the secretion and synthesis of STC is with this demonstrated in the seabream.
Previous studies show that STC and PTHrP exert functional antagonistic actions at different levels [3,22], but to date, and as far as we are aware, there is no experimental evidence for a putative reciprocal control of their production/secretion. The CS gland, in this sense, offers a unique model to study interactions between both hormones through the analysis of STC synthesis and release. Through next-generation sequencing techniques, there is a reported lack of PTHrP receptor transcripts in the CS of the Japanese eel [45]. However, in the present study, we have demonstrated with a specific RT-PCR approach the presence of two PTHrP receptors (pth1r and pth3r) in the CS of the seabream. Moreover, ex vivo incubations of the CS with PTHrP revealed its inhibitory action on STC release in a dose-dependent fashion. There are no previous reports of this interaction in fish or mammals. This knowledge gap also extends to the mammalian counterparts of the PTHrP and STC, the PTH, and the calcitonin hormone, respectively. Hence, these results highlight the importance of calcium homeostasis regulation, as its intertwined pathways are mutually regulated. Therefore, we calculated the PTHrP ED50 for STC secretion, 17.51 ng mL −1 . Normal levels of PTHrP in the plasma of seabream range from 2.50 ± 0.29 ng mL −1 [47] to 0.86 ± 0.25 ng mL −1 [48]; therefore, a slight but constant inhibition of STC release from the corpuscles of Stannius in seabream takes place at circulating levels of PTHrP.
The PTHrP effects described in this study are likely achieved through specific receptors and second messenger routes. The inhibitory effects of PTHrP in STC secretion are mediated by the activation of a phospholipase C (PLC) and transmembrane adenylyl cyclase (tmAC), as specific inhibitors of both enzymes rescue the inhibition caused by the PTHrP towards the STC secretion at the same time (but not separately). In this sense, gilthead seabream PTH3R (sbPTH3R) seems only to activate the cAMP pathway [32], thus suggesting that the PTH1R in the CS primarily activates the PLC signaling cascade. Our results are in good agreement with previous studies in other species ranging from mammals to fish, as the PTH/PTHrP receptors are closely linked to PKA and PLC signaling pathways [2,28,31] unless their affinities to one or other second messenger routes depend on the specific type of PTHrP receptor. However, both routes are related to the inhibition of the STC secretion in the CS of the gilthead seabream through the action of the PTHrP. Moreover, in zebrafish, a gene duplication produced two distinct PTH molecules with different receptor affinities [49]. If seabream also presents different effectors of the two PTHRs located in the CS, the overall receptor selectivity may present different action patterns depending on substrate binding.
Interestingly, the CS presents two types of granulated cells [50,51], which opens a possibility for different regulatory mechanisms. To understand if both PTHrP receptors are present in the same pool of STC secretory cells or if their inhibitory actions are related to differentiated pools requires further investigation. However, if differentiated groups of STC secretory cells exist, it may indicate that there are other regulatory agents in addition to calcium. In this sense, earlier work on STC postulated that the nervous system might act as a promotor of STC secretion [46]; however, we should not rule out other unknown external factors. The inhibitory action of the sbPTH3R through the production of cAMP matches with the described inhibition of cAMP accumulation due to CaSR stimulation [25,27]. Thus, the PTH3R effects in this species could be related to inhibiting the STC-secretion stimulated by basal Ca 2+ levels through a tmAC. Moreover, our results also show that at high plasma Ca 2+ concentrations, the PTHrP receptors do not inhibit the STC secretion. The reason for that is still not understood, but it could imply the presence of another mechanism to regulate secretion in the CS.

Conclusions
In conclusion, using ex vivo approaches and RT-PCR techniques, we have demonstrated consistent STC release from the CS of gilthead seabream controlled by calcium levels and its antagonist hormone, the PTHrP. Three types of membrane receptors are thus mediating this action: CaSR, PTH1R, and PTH3R. The inhibition exerted by both PTHrP receptors could imply alternative pathways, maybe due to differentiated pools of STC secretory cells. Further studies to elucidate whether or not this hypothesis is true are necessary. The present study demonstrates that the regulation of antagonist hormones such as STC and PTHrP in teleost fish is more closely related than expected.


A Cell-Based Systematic Review on the Role of Annexin A1 in Triple-Negative Breast Cancers

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is often associated with a poorer prognosis and does not respond to hormonal therapy. Increasing evidence highlights the exploitability of Annexin A1 (AnxA1), a calcium dependent protein, as a precision medicine for TNBC. To systematically summarize the role of AnxA1 and its associated mechanisms in TNBC, we performed data mining using three main databases: PubMed, Scopus, and Ovid/Medline. The papers retrieved were based on two different sets of key words such as “Annexin A1” or “Lipocortin 1” and “Breast cancer” or “TNBC”. A total of 388 articles were identified, with 210 chosen for comprehensive screening and 13 papers that met inclusion criteria were included. Current evidence from cell culture studies showed that AnxA1 expression is correlated with NF-κB, which promotes migration by activating ERK phosphorylation. AnxaA1 also activates TGF-β signaling which upregulates MMP-9 and miR196a expression to enhance epithelial-mesenchymal transition and migratory capacity of TNBC cells. AnxA1 can steer the macrophage polarization toward the M2 phenotype to create a pro-tumor immune environment. Existing research suggests a potential role of AnxA1 in the metastasis and immune landscape of TNBC tumors. Preclinical and clinical experiments are warranted to investigate the feasibility and effectiveness of targeting AnxA1 in TNBC.

Introduction
Breast cancer is the world's most prevalent cancer among women [1] with 2,261,419 new cases and 684,996 deaths, according to GLOBOCAN 2020 [2]. TNBC accounts for about 15-20% of all breast cancer subtypes [3][4][5]. It is clinically characterized by the lack of main receptors of breast cancer; estrogen receptor (ER) and progesterone receptor (PR), as well as the amplification of human epidermal growth factor 2 (HER2) [3,6,7]. It is associated with rapid growth, distant metastasis, invasiveness, high risk of relapse and poor prognosis [8,9]. Due to the lack of receptors, TNBC does not benefit from hormonal-based therapies, conventional chemotherapy is the only treatment available as the mainstream therapeutic approach for patients with TNBC, despite the considerable side effects and suboptimal outcomes [3,9]. To reduce the burden of TNBC, it is critical to establish optimum therapeutic options for the treatment of early TNBC [9]. Recently, three targeted therapies for TNBC have been approved by US Food and Drug Administration, including the Poly (ADP-ribose) polymerase (PARP) inhibitors olaparib and talazoparib for germline BRCA mutation-associated breast cancer and the checkpoint inhibitor, atezolizumab in combination with nab-paclitaxel for programmed death-ligand 1 positive TNBC [10,11]. However, understanding the complexity and heterogeneity of the disease, more personalized targeted treatment options are still needed to treat TNBC patients. Therefore, we must continue to explore potential predictors responsible for TNBC progression.
Annexin A1 (AnxA1) also known as lipocortin-1 [12] is a 37kDa annexin superfamily, which is calcium and phospholipid dependent [13]. AnxA1 engages in antiinflammatory effects where it modulates innate immune cell activity, such as neutrophils and macrophages [14], cell differentiation regulation, proliferation, and apoptosis [15]. AnxA1 mediated activity is mainly through the activation of formyl peptide receptors (FPRs) which are the only known receptor of AnxA1 [16][17][18]. To facilitate the externalization and localization of AnxA1, the protein must be activated by glucocorticoids [19].
AnxA1 plays varying roles in tumorigenesis [20]. A recent systematic review concluded a strong association of AnxA1 overexpression with hallmarks of cancer, including uncontrolled proliferation, metastasis, lymphatic invasion and drug resistance according to studies on pancreatic, hepatic, gastric, skin and colorectal cancers [19] The aggressive behaviors are typically found in TNBC, highlighting a potential implication of AnxA1 in the oncogenesis of the poor-prognosis subtype. Thus, to build on the existing knowledge, our systematic review zooms into the role of AnxA1 and its related mechanisms in TNBC to explore new therapeutic options for precision oncology in TNBC.

Methods
Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) were used to design the study [21]. Since this systematic review primarily focuses on in-vitro studies, we did not register the systematic review in PROSPERO, which focuses more on human studies and animal studies.

Search for Relevant Studies
A systematic search was conducted based on three online electronic databases namely PubMed, Ovid/Medline, and Scopus for all relevant articles published from January 2000 until January 2020. The following keywords were used to search the databases: "Annexin A1" or "Lipocortin-1" and "Breast cancer" or "Triple-Negative Breast Cancer". Those articles were imported to Mendeley software and duplicates were removed. The language of publication was decided to be English only.

Selection of Articles
Abstracts and titles of the retrieved papers were screened (first screening), and relevant articles were chosen based on eligibility criteria. Then the full texts of selected articles (second screening) and the articles that fit the inclusion and exclusion were included in this review. The eligibility for inclusion was based on "AnxA1 in TNBC" including in-vitro studies. Concurrently, studies that are not relevant to AnxA1, TNBC, limited data on the biological outcome, poorly developed methodologies, biased outcomes and insufficient sample size are all ruled out. Two independent researchers carried out these procedures, and the differences (between the two researchers) were compared, discussed and the agreement was reached accordingly.

Study Selection
A total of 388 articles were identified, after removal of duplications, 210 articles were chosen for comprehensive screening and 13 articles that met inclusion criteria as described in the "Methods" were included. Figure 1 shows the study selection process using the PRISMA flow diagram. procedures, and the differences (between the two researchers) were compared, discussed and the agreement was reached accordingly.

Study Selection
A total of 388 articles were identified, after removal of duplications, 210 articles were chosen for comprehensive screening and 13 articles that met inclusion criteria as described in the "Methods" were included. Figure 1 shows the study selection process using the PRISMA flow diagram.

Data Extraction and Synthesis
Data extracted included information of authors, name of cell lines, culture conditions whether it is comparable to other studies, study findings, and significant statistical value. A qualitative synthesis of data was employed to summarize the information obtained

Data Extraction and Synthesis
Data extracted included information of authors, name of cell lines, culture conditions whether it is comparable to other studies, study findings, and significant statistical value. A qualitative synthesis of data was employed to summarize the information obtained from the selected articles. A meta-analysis (or quantitative analysis) was not conducted due to insufficient homogeneous outcomes from the selected articles.

Quality Assessment
The Quality Assessment (QA) methods used to determine the likelihood of bias associated with cell culture studies were carried out using criteria recommended by WCRF/UoB [22,23]. For each parameter, the score range 0-6 was assigned (a score of '1' was given to 'yes', '0' to parameters that 'are not met' or 'not reported'. Based on the average score, studies were reported as low (0-2), moderate (3)(4), or (5-6) quality in Table 1. This bias assessment was not used to evaluate the inclusion and exclusion criteria. A summary of the bias assessment is useful to make a conclusion as to whether the research included supporting the biological plausibility of the investigated possible mechanism. Studies receive a score of 0 or 1 for each of the following parameters (a score of 0 was given due to lack of fulfillment of the criteria or failure to report): a Every cell line are validated independently; b culture conditions are similar to other studies; c adequate biological and technical repeats are performed; d proper controls included; e different cell lines are used; f all experiment results are included.

AnxA1 Is a Subtype-Specific Markers in TNBC
In breast cancer, AnxA1 expression shows a distinct subtypes specificity [34]. Compared to other subtypes, TNBC consistently expresses higher levels of AnxA1 which correlates with the disease severity as well as poor prognosis [35].
Clinically, patients with TNBC expressing a high level of AnxA1 and have a poorer survival rate as compared to patients with a low level of AnxA1 [37]. AnxA1 is associated with TNBC cell invasion as well as migration [13,30]. AnxA1 expression is more likely to be elevated in breast metastases, especially those seen in regional lymph nodes, the lung, and the liver [18]. Since AnxA1 in TNBC cells is recognized for its highly aggressive characteristics and therefore blocking this signal is considered a therapeutic strategy for TNBC management. However, understanding the heterogeneity of TNBC with different molecular subtype classification by Lehmann et al., including basal-like (BL) 1, BL2, mesenchymal, mesenchymal stem-like, immunomodulatory, and luminal androgen receptor [38,39], AnxA1 expression in cell lines representing immunomodulatory and luminal androgen receptor subtypes has to be studied so that the result reflects the whole TNBC spectrum.

AnxA1 Induces Epithelial-Mesenchymal Transition in TNBC
In cancer cells, epithelial-mesenchymal transition (EMT) is characterized by progressive acquisition of mesenchymal and pro-migratory features with heightened resistance to pro-apoptotic signals and chemotherapeutic assaults [36]. EMT is a key feature of epithelial tumors and plays pivotal role in metastasis [18]. Kang and colleagues investigated the role of AnxA1 in the migration and invasion of TNBC cells [13]. The cell line with high AnxA1 expression, was invasive whereby cells with low or no AnxA1 expression exhibited a poor migratory profile [13,15,40]. The involvement of AnxA1 has been correlated with matrix metalloproteinase 9 (MMP-9) gene expression and its regulation of intrinsic MMP-9 gene transcription and proteolytic activity [13]. Breast cancer cells treated with siAnxA1 have suppressed NF-κB activity, which results in downregulation of MMP-9 expression and impaired cell migration [13]. Among all the MMPs, MMP-2 and MMP-9 (gelatinase B) are highly associated with aggressive, metastatic breast cancer and lung cancer [41,42] as they can cause proteolytic degradation of the extracellular matrix [20,41] and basement membrane [41]. Different biochemical simulators, including growth factors, cytokines, and phorbol 12-myristate 13-acetate (PMA), regulate MMP-9 by activating different intracellular signaling pathways [43]. Studies have shown that MMP2 and MMP9 are crucial for tumor cell invasiveness, and MMP9-mediated invasion inhibition suppresses tumor cell metastasis [13]. As a result, there is a clear association between AnxA1 expression and both cell migration as well as invasion.
The effect of AnxA1 knockdown on cancer cell migration has also been investigated in past studies. Interestingly, results showed that AnxA1 knockdown cells had significantly shorter migration distances which were directly mediated through the mammalian target of the rapamycin (mTOR) signaling pathway [20].
Activation of mTOR-S6 signaling is correlated with AnxA1 in TNBC. Activation of this pathway has been linked to the capacity of cancer to generate a new vasculature network, inflammation, and cancer cell survival [44]. Ribosomal protein S6 RPS6 and S6 kinase (S6k), the downstream effector proteins of the mTOR pathway are required for the metastatic behavior and progression of cancer [20,44] via F-actin modification and MMP-9 up-regulation [20]. S6K is highly expressed in lung and ovarian cancers, and the expression of S6K is associated with poor breast, kidney, and hepatocellular carcinoma prognosis [45] and RPS6, in addition, has been suggested as a potential cancer biomarker [44]. Downregulation of RPS6 alone could significantly reduce the proliferation of TNBC [44]. Furthermore, RPS6-knockdown in non-small cell lung cancer (NSCLC) has been shown to regulate the EMT-related proteins where MMP-9, MMP-2, Vimentin and N-cadherin have been downregulated and E-cadherin has been upregulated [44]. A similar trend in the regulation of vimentin and E-cadherin (hallmarks of EMT) [20] has been observed in a study that examined the DC-STAMP domain containing 1-antisense 1 (DCST1-AS1) in promoting EMT via AnxA1 [33].
High AnxA1 was also linked to Cathepsin D (CatD) expression, with CatD expression in AnxA1 knockdown MDA-MB 231 cells being significantly lower than in normal MDA-MB 231 cells [32]. Cathepsins are a protease superfamily made up of 16 members of aspartyl, serine, and cysteine proteases that are highly expressed in various tumors and associated with metastasis [32,46] and currently CatD is being explored as a potential TNBC marker. Pepstatin A (CatD inhibitor), can reduce the cell growth, invasiveness, and migratory potential of MDA-MB 231 cells. Mechanistically, AnxA1's N-terminal peptides activate the formyl peptide receptors (FPRs), namely FPR1 and FPR2 [32], which are the only known receptors of extracellular AnxA1 [18]. By inhibiting CatD catalytic properties, AnxA1 mediated signaling pathways are hindered. Aside from breast cancer, the AnxA1 and FPR complex has been associated with the invasive nature of numerous cancers, including colon, rectal, gastric, prostate, pancreas, melanoma, and esophageal squamous cell carcinoma [47][48][49].
In addition, in MDA-MB 231 cells, FPR1 was reported to be 4-fold higher than human monocytes while FPR2 was 8-fold higher than MCF-7 cells [27]. The activation of the AnxA1 receptor (FPR2) induces cell proliferation of MDA-MB 231 cells which is attenuated by FPR2 antagonists (WRW4 and Boc2) and AnxA1 knockdown [27]. Additionally, mitogens can promote AnxA1 expression, subsequently activating FPR2 and causing further mitogenesis in TNBC via cyclin D1 upregulation [27]. Such mitogen-induced cell proliferation in MDA-MB 231 is inhibited by FPR2 siRNA silencing, clearly showing the pivotal role of AnxA1-FPR2 signaling in cell cycling of TNBC [27].
On the other hand, FPR1 inhibition in MDA-MB 231 showed a positive association in primary tumors specifically on the expression pattern of AnxA1 and its receptor, FPR1 [30]. Moreover, FPR1 inactivation by Cyclosporin H (CsH) and Cyclosporin A (CsA) drugs can decrease cytosolic calcium levels, increase IL-6 secretion, and reduce the extracellular signal-regulated kinase (ERK) phosphorylation, leading to suppressed cell migratory and invasive capability [30]. Therefore, inhibition of FPR1 signaling by CsH and CsA could also be developed as a potential therapeutic strategy for TNBC management. 4T1, a highly metastatic and invasive mouse TNBC cell line, exhibited increased growth and invasive capability co-cultured with AnxA1 +/+ M2 polarized macrophages, which suggests that macrophages can potentially influence the tumor cell proliferation and migration dependent on AnxA1 [28]. Treatment of macrophages with conditioned media of the breast tumor, especially 4T1, can skew the kinetic profiles to the M2 subset (alternatively activated M2) [28]. AnxA1 is also required for tumor-associated macrophage (TAM) polarization to adopt an M2 phenotype which enhances the breast cancer immunosurveillance escape, migration, and metastasis [16,28]. Tumor-associated macrophages (TAM) are found at a very high level in many tumors, such as ovarian, breast, and pancreatic cancer [50]. TAMs especially M2 macrophages facilitate cancer metastasis through multiple mechanisms including angiogenesis promotion, tumor growth activation, tumor cell migration, and invasion [51][52][53]. M2 phenotype is pro-tumor as its functions include cell division, metastasis, and tumor cell survival [53], and AnnexinA1 is required for the macrophage polarization to an M2 phenotype which promotes the tumor growth and invasion [28].
AnxA1 overexpression together with SDF-1α (stromal-derived factor 1α or also known as chemokine CXCL12) treatment exhibits a significant increase in migration, and together with CXCR4 induces concentration-dependent migration [25]. CXCR4 is a downstream target of NF-κB. In response to chemokine CXCL12, AnxA1 significantly modulated the CXCR4-mediated migration of breast cancer cell lines, suggesting its significance in tissuespecific migration of breast cancer cells [25]. CXCL12 is expressed in organs serving at major sites of metastasis of breast cancer, such as the bone and lung, while its CXCR4 receptor is well expressed in cells of metastatic breast cancer [40]. Intracellular signaling is induced by CXCL12 binding to CXCR4 through a variety of pathways that stimulate chemotaxis, cell survival and/or proliferation, intracellular calcium increase, and gene transcription signal [54].
Targeting AnxA1 that regulates EMT and its related proteins mainly matrix metalloproteases, vimentin and E-cadherin may be a promising marker in inhibiting metastasis, invasion and proliferation of TNBC. The role of AnxA1 in inducing TNBC cell migration, invasion, and proliferation has been summarized in Table 2.

AnxA1 Diminishes Sensitivity of TNBC Cells to Apoptotic Signals
As previously stated, Cathepsin D (CatD) is important not just in migration, invasion, and proliferation, but also in preventing apoptosis in TNBC via AnxA1 cleavage [32]. CatD protects MDA-MB 231 cells from apoptosis and autophagy by enhancing the cleavage of AnxA1 into smaller fragments (35.5 KDa). Not only that, FPR1 inhibition by Cyclosporin H (CsH) and Cyclosporin A (CsA) induced cell cycle arrest and early apoptosis after 24-h treatment in MDA-MB 231 cells without affecting the cell line of MCF-7 and MCF-10A [30]. Additionally, it has been shown that AnxA1 secretion induces autocrine signaling in MDA-MB 231 cells via FPR1 [30]. Therefore treatment (Pepstatin A) that keeps AnxA1 (37 KDa) intact, reduces aggressive behavior in MDA-MB 231 cells and promotes apoptosis and autophagy [32].
One of the hallmarks of cancer is the inhibition of apoptosis [8] and it is regulated by the RIPK1 pathway, which can, in turn, modulate NF-κB, NF-κB is a transcription factor that plays an important role in desensitizing cells to apoptosis by decreasing reactive oxygen species (ROS) and antagonizing the p53 protein [55]. Since AnxA1 expression is significantly associated with NF-κB, thus its modulation either directly or indirectly can disrupt RIPK1 signaling, affecting apoptosis and leading to cancer progression [19].
The role of AnxA1 in desensitizing cells to apoptosis of TNBC has been summarized in Table 3.

AnxA1 and Multidrug Resistance (MRD)
The first evidence for the role of AnxA1 in the multidrug resistance of MDA-MB 231 cells has been reported [29]. As compared to MCF7 (AnxA1 − ), MDA-MB 231 (AnxA1 + ) had a 3 to 20-fold rise in resistance to Adriamycin, Melphalan, and Etoposide. In tumor cells, AnxA1 depletion increases their susceptibility to anti-cancer drugs [29]. However, the exact mechanism by which AnxA1 exerts drug resistance to the anti-tumor drug is still unknown [29].
DC-STAMP domain-containing 1-antisense 1 (DCST1-AS1) is known to regulate the drug resistance of BT-549 cells to doxorubicin and paclitaxel by binding to AnxA1 [33]. Typically, DCST1-AS1 knockdown cells have increased susceptibility to doxorubicin and paclitaxel. However, such drug susceptibility due to the DCST1-AS1 knockdown is lost when AnxA1 is overexpressed.
Resistance to multidrug is one of the biggest challenges that is faced to treat TNBC. The findings together suggest a profound implication of AnxA1 in the aggressiveness of TNBC and susceptibility to treatment that has yet to be thoroughly understood. Hence, modulating the expression of AnxA1 can be a potential strategy to overcome multidrug resistance.

Immunomodulatory Role of AnxA1 in TNBC
4T1 breast cancer cells exhibit an AnxA1-dependent M2 macrophage polarization that promotes tumor growth, invasion, and migration [28]. 4T1 (AnxA1 + ) or Ac2-26 (AnxA1 peptide) enhance the ERK and NF-κB activation via formyl peptide receptor 2 (FPR2) and ERK signaling, downstream of AnxA1 which is required for macrophages to polarize into an M2 phenotype [28]. Additionally, high expression of chemokine C-C motif chemokine ligand 5 (CCL5) is also associated with AnxA1 to promote macrophage polarization. Chemokine CCL5 expression is elevated in tissues and plasma of patients with advanced-stage breast carcinoma [28]. Macrophages treated with recombinant CCL5 displayed an increased expression of AnxA1, revealing a new linkage between AnxA1 and CCL5 chemokine which could modulate the intratumoral immune landscape.
Furthermore, a high level of AnxA1 in TNBC induces Treg cell-mediated immunomodulation, where inhibiting AnxA1 can weaken Treg cells resulting in the reduction of tumor size in the in-vivo model [37]. RNA-seq reveals that inhibiting AnxA1 reduces granzyme A expression in Treg cells which can dampen natural killer cells and cytotoxic T lymphocytes. Moreover, the expression of key Treg surface markers such as IL2RA (CD25), CCR8, and PDCD1 has diminished [37]. AnxA1 may exert a profound regulator role in onco-immunity in the tumor microenvironment of TNBC, however, these warrant further investigation involving knockdown studies using a co-culture model.

Mechanism of Action Related to AnxA1
Silencing AnxA1 in MDA-MB 231 cells decreased NF-κB DNA binding, which results in a reduction of IkB kinase (IKK), IκBα, and p65 phosphorylation [25]. Additionally, NEMO and receptor-interacting protein (RIP) are associated with AnxA1 in MDA-MB 231 cells. Disruption of these interactions is disrupted, leading to the inactivation of NF-κB [25]. The NEMO-ANXA1-RIP complex facilitates a survival response through the upregulation of NF-κB. Not only that, AnxA1 knockdown in MDA MB-231 cells could reduce the upregulation of uPA, a target gene of NF-κB which has significant involvement in metastasis [25].
AnxA1 is also involved in the regulation of microRNAs which are a group of noncoding RNAs that have been demonstrated to control many of the genes involved in cellular processes such as proliferation, differentiation, and apoptosis [24]. AnxA1 inhibited the transcription of miR196a which in turn induced a negative feedback loop to enhance breast cancer proliferation, migration, and metastasis [31]. MiR196a is an oncogenic miRNA that is used as a diagnostic biomarker for many cancers including laryngeal, colorectal, and glioma cancers. Additionally, high expression of AnxA1 leads to a decrease in miR26b * and miR562 which enhances NF-κB activity. Conversely, MCF-7 cells, which have lower AnxA1 levels, have a high level of miR26b * and reduced NF-κB activity. Therefore, existing evidence supports a regulatory role of AnxA1 on NF-κB activity via miRNA modulation.
In TNBC cell lines, a higher endogenous level of AnxA1 is associated with higher EGFR, c-Met, and AKT [20]. Additionally, AnxA1 knockdown cells have reduced mTOR and protein S6 phosphorylation [20] but a 1.8-fold times increase in the phosphorylation of a stress-induced protein, AMPKα [20,56]. How AnxA1 interacts with AMPK to control the mTOR signaling pathway remains unclear. One possible mechanism is through the activation of pATM, which is involved in stress response and can activate AMPK [20]. The mammalian target of rapamycin (mTOR) is a major regulator of cell growth in normal circumstances, however, mTOR signaling in tumor cells is alternatively activated to stimulate metastasis and invasion [57]. This AnxA1-AMPK-mTOR-cell migration could explain why AnxA1 levels are linked to a poor prognosis in TNBC.
On the other hand, AnxA1 knockdown inhibited the Smad2 phosphorylation, Smad 4 nuclear translocation, and Smad 3 and 4 driven transcriptional activity caused by transforming growth factor β (TGF-β) [26]. By regulating TGF-β and actin reorganization, AnxA1 promotes epithelial-mesenchymal transition (EMT) and migration of breast cancer cells [26]. Phosphorylation of downstream mediators of TGF-β (Smad2 and Smad3) forms a complex with Smad4 that translocates into the nucleus to modulate gene transcription [58]. Another study showed that the DC-STAMP domain-containing 1-antisense 1 (DCST1-AS1) which is highly expressed in TNBC (MDA-MB 231 and BT-549 cells) directly binds to AnxA1 and promotes EMT through TGF-β/Smad signaling [33]. TGF-β signaling plays a very important role in tumor initiation and progression. TGF-β will increase the tumor growth and invasiveness of breast cancer cells by activating the expression of MMP-2 and MMP-9 [33].
Apart from the different mechanisms explained above, AnxA1 primarily promotes the activation of NF-κB signaling which is a mediator of AnxA1-induced breast cancer cell migration [18]. Nuclear translocation of NF-κB triggers cell proliferation genes (cyclins/CDKs), thus promoting cell growth. Additionally, there is a positive association between NF-κB activation and increased expression of anti-apoptotic members of the Bcl-2 family, resulting in resistance to apoptosis [59]. In HER2-positive tumors and TNBC, the constitutive activation of NF-κB is responsible for cell proliferation, angiogenesis, and inhibition of apoptosis [8]. MDA-MB 231 cells have impaired cell proliferation when NF-κB signals are blocked by mutant IκB [8]. Additionally, it also induced an epithelial-mesenchymal transformation and promote migration capability, involving TGF-β-dependent signaling. Apart from that, NF-κB-induced stimulation of MMP-9 production is inhibited by AnxA1 knockdown in TNBC cells preventing the subsequent cancer cell migration [13].
The mechanism of action related to AnxA1 has been summarized in Table 4 and a schematic diagram has been developed to provide a broad picture of the mechanism of action via different pathways influenced by AnxA1 (Figure 2). Although AnxA1 has been well implicated in a number of TNBC-related mechanisms, however additional research is required to fully comprehend how this protein interacts with common signaling pathways of TNBC. Rubinstein et al. described AnxA1 as an unknown regulator of Wnt/β-catenin signaling and a vital protein in colorectal cancer progression [60]. However, the expression of β-catenin in glioma cells was not affected by AnxA1 expression [61]. Given that AnxA1 is closely associated with the targeted genes of this pathway, MMP-9 and MMP-2 [13] and cyclin-D1 [27], it is possible that AnxA1 also plays a role in the regulation of Wnt/β-catenin signaling in TNBC. Targeting this signal is equally crucial as it plays a significant role in the progression and metastatic nature of TNBC by increasing the MMP-7 expression [62,63].    Based on the studies presented, the aberrant pathways (mTOR, TGF-β and NF-κB signaling) that are affected by the direct or indirect effect of AnxA1 contributes to the progression of TNBC. With the extended involvement of AnxA1 in various signaling, we believe that it has the potential to be developed as a novel multi-targeted protein to inhibit dysregulated pathways in TNBC. However, extensive study is needed to fully understand AnxA1′s role in other TNBC-related key pathways such as Notch, mitogen-activated pro- Based on the studies presented, the aberrant pathways (mTOR, TGF-β and NF-κB signaling) that are affected by the direct or indirect effect of AnxA1 contributes to the progression of TNBC. With the extended involvement of AnxA1 in various signaling, we believe that it has the potential to be developed as a novel multi-targeted protein to inhibit dysregulated pathways in TNBC. However, extensive study is needed to fully understand AnxA1 s role in other TNBC-related key pathways such as Notch, mitogen-activated protein kinase (MAPK) and Hedgehog signaling [8].

Conclusions
Current evidence suggests a unique role of AnxA1 in the progression of TNBC. It should be noted that most findings are derived from TNBC cell lines, whereas in vivo animal experiments and clinical data are lacking. Furthermore, TNBC is a heterogenous breast cancer that can be subclassified into six subtypes, but most of the mechanistic studies on AnxA1 covered only the mesenchymal, BL1 and BL2 TNBC cell lines. Conceivably, the three subtypes represent about 70-80% of all TNBC cases [8], allowing us to infer a decent association between AnxA1 and TNBC. Future mechanistic studies should focus on the role of AnxA1 in the under-represented TNBC molecular subtypes. Disease models with better clinical relevance such as mammosphere culture and mouse tumor xenografts should be used to interrogate the targetability of AnxA1 in highly heterogenous TNBC.
AnxA1 is well-implicated in the modulation of tumor inflammation via NF-κB activation. It can also remodel the macrophage polarization to favor alternatively activated macrophages or tumor-associated macrophages which contributes to the aggressiveness of TNBC. However, the underpinning mechanism remains largely elusive. Further investigation should employ the TNBC-macrophage co-culture models to explore the AnxA1mediated tumor-immune crosstalk, which may reveal the exciting role of AnxA1 in the tumor microenvironment.
Additionally, AnxA1 exhibits a significant involvement in the epithelial-to-mesenchymal transition process via the modulation of TGF-β signaling and extracellular matrix remodeling enzymes. Such activity can confer profound chemoresistance, which is a typical feature of TNBC. Interestingly, AnxA1 ablation restores the sensitivity of colorectal and pancreatic cancers to chemotherapeutic agents through the inhibition of the drug efflux pump, p-glycoprotein [64,65]. Hence, it may be worthwhile to examine the intrinsic functionality of AnxA1 on drug efflux pumps in TNBC. The results can potentially underscore its practicality as an adjunctive targeted therapy to potentiate chemotherapeutic agents in TNBC treatment. Taken together, AnxA1 is implicated in the aggressive behaviors of TNBC, including proliferation, metastasis, invasion, pro-survival, and chemoresistance. The underlying mechanisms are linked to the activation of NF-κB and TGF-β signaling which are key pathways for tumor inflammation and epithelial-to-mesenchymal transition. AnxA1 also promotes the alternative activation of macrophages, suggesting a role in the formation of an immunosuppressive TNBC tumor microenvironment. Although existing evidence supports the pro-tumorigenic role of AnxA1 in TNBC, further validation using preclinical animal models and different subtypes of TNBCs is warranted.