CXCR4 signaling regulates metastatic onset by controlling neutrophil motility and response to malignant cells

Developing tumors interact with the surrounding microenvironment. Myeloid cells exert both anti- and pro-tumor functions and chemokines are known to drive immune cell migration towards cancer cells. It is documented that CXCR4 signaling supports tumor metastasis formation in tissues where CXCL12, its cognate ligand, is abundant. On the other hand, the role of the neutrophilic CXCR4 signaling in driving cancer invasion and metastasis formation is poorly understood. Here, we use the zebrafish xenotransplantation model to study the role of CXCR4 signaling in driving the interaction between invasive human tumor cells and host neutrophils, supporting early metastasis formation. We found that zebrafish cxcr4 (cxcr4b) is highly expressed in neutrophils and experimental micrometastases fail to form in mutant larvae lacking a functional Cxcr4b. We demonstrated that Cxcr4b controls neutrophil number and motility and showed that Cxcr4b transcriptomic signature relates to motility and adhesion regulation in neutrophils in tumor-naïve larvae. Finally, Cxcr4b deficient neutrophils failed to interact with cancer cells initiating early metastatic events. In conclusion, we propose that CXCR4 signaling supports the interaction between tumor cells and host neutrophils in developing tumor metastases. Therefore, targeting CXCR4 on tumor cells and neutrophils could serve as a double bladed razor to limit cancer progression.

Tumor-microenvironment interactions are crucial in cancer pathogenesis and several signals drive this communication 1 . The composition of cancer microenvironments changes during cancer progression 2 . Fibroblasts, endothelial and immune cells are main components of the tumor stroma, acting in concert with the extracellular matrix (ECM), growth factors, proteases and cytokines 3 . The CXCR4-CXCL12 chemokine signaling axis sustains tumor cell growth and directs the formation of distant metastases. It is established that cancer cells expressing CXCR4 home to secondary organs where CXCL12 is highly secreted, mainly by mesenchymal stromal cells 4 . Moreover, CXCL12 guides the migration of stromal cells that express CXCR4 and locally infiltrate the tumor, providing support by secretion of growth and angiogenic factors, as well as promoting metastasis through activation of epithelial-to-mesenchymal transition (EMT) via mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/Protein kinase B (PI3K/AKT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) pathways 3,5 .
A dual role in either supporting or inhibiting tumor progression has been linked with the immune system 1 . CXCR4-CXCL12 signaling has been associated with the polarization towards an immune-suppressive microenvironment: the possible role of a CXCL12 shield that protects cancer cells from being recognized by cytotoxic lymphocytes and activates regulatory T-cells has recently been described 6 . Polarization of macrophages towards a M2 phenotype has also been associated with tumor survival. Recent studies have pointed at the role of perivascular CXCR4-expressing M2 macrophages in creating tumor vascular networks after chemotherapy, leading to tumor relapse, and confirmed CXCR4 as M2 marker 7 . It has been shown that CXCR4 can also be activated by alternative ligands like MIF (Macrophage Migration Inhibitory Factor) 8 . MIF signalling has been associated to inflammatory diseases. Upon binding to CXCR4 or CXCR2, MIF controls monocyte and T cell chemotaxis and its blockade leads to plaque regression in atherosclerosis 8 . In zebrafish, MIF functions as a neurotrophin during the gression. Inhibitory and supportive functions of the immune system have been associated with tumor growth and metastasis formation. Using the zebrafish embryo model we previously showed that myeloid cells, mainly neutrophils, support the establishment of tumor experimental micrometastasis, when the MAE-FGF2 transformed cell line was inoculated into the blood circulation of 2-day-old embryos 30 . Therefore, we used the same approach to investigate whether zebrafish myeloid cells exert similar tumor supportive functions, when other cell lines were implanted. In particular, we used the osteotropic triple negative breast cancer line MDA-MB-231-B, derived from bone metastases in a mouse xenograft model 32 . The zebrafish embryo model bears the great advantage of studying the contribution of the innate immune system during early metastasis formation separately from the adaptive immunity, which reaches full maturity in 3-4 week old juveniles 33 . To deplete both neutrophils and macrophages, we injected Pu.1/Spi1b morpholino (1 mM) into 1-2 cell stage embryos. Subsequently, the MDA-MB-231-B cell line was inoculated into the blood circulation of 2-day post fertilization (dpf) zebrafish embryos with GFP-expressing neutrophils. The reporter line Tg(mpx:GFP) i114 34 was used to monitor neutrophil depletion, in view of the time-limited efficacy of gene knock-down obtained with morpholino anti-sense oligos. Macrophage depletion was not monitored as it already occurs with lower doses of the same morpholino (0.5 mM) 30 . Tumor phenotype assessment was performed 2-day post implantation (dpi) by quantifying tumor cell invasion in each larva. Depletion of myeloid cells in the Pu.1 morphants resulted in a reduced cancer cell invasion (68%) in the tail fin in proximity of the caudal hematopoietic tissue (CHT) (Fig. 1). As previously found, the CHT, a site of hematopoiesis and analogous to the fetal liver during mammalian development, is a preferential site of early cancer metastasis formation in the zebrafish xenotransplantation model. In conclusion, myeloid cells support triple negative breast cancer early metastasis onset in zebrafish.
Neutrophilic Cxcr4 signaling is involved in early tumor metastasis initiation. Therapeutic targeting of CXCR4 on tumor cells could be an effective strategy to limit tumor cell growth and metastasis. However, CXCR4 signaling in the tumor microenvironment also plays a central role in cancer and further investigations are needed to fully understand its contribution.
In our model, teleost evolution has led to a cxcr4 gene duplication. cxcr4a and cxcr4b paralogues are expressed by different cell types, although redundant functions have been reported 35  conserved between zebrafish and human: zebrafish Cxcr4 receptors display more than 60% identity with human CXCR4 and zebrafish Cxcl12 ligands have more than 65% identity with human CXCL12 12 . We performed transcriptome analysis of GFP positive, FACS-sorted neutrophils from 5 dpf Tg(mpx:GFP) i114 larvae and RNA deep sequencing revealed high expression levels of the cxcr4 paralogues in neutrophils. In particular, cxcr4a and cxcr4b transcriptomic levels were higher in the GFP + fractions compared to the GFP − populations. Importantly, neutrophilic cxcr4b levels were at least 100-fold higher than neutrophilic cxcr4a, indicating that cxcr4b is the predominant human CXCR4 orthologue in zebrafish larval neutrophils ( Fig. 2A). Therefore, to study if CXCR4 signaling in the tumor microenvironment supports cancer metastasis initiation, we engrafted the triple negative breast cancer cell line MDA-MB-231-B in the cxcr4b t26035 (odysseus or ody) mutant with deficient cxcr4b 36 . Xenogeneic transplantation into the blood circulation via the duct of Cuvier resulted in a strong proliferating and invasive tumor phenotype, characterized by experimental micrometastasis formation in the CHT region in the wild-type (wt) siblings, whereas a significant reduction was observed in the cxcr4b −/− , ody mutants ( Fig. 2B-E). The establishment of early metastatic events defined by tumor mass formation and extravasation followed by local tissue invasion was monitored by fluorescence at 2 (Fig. 2B,C) and 4 ( Fig. 2D,E) days after engraftment and tumor burden was found to be significantly inhibited in ody larvae (22.5% and 40.5% reduction at 2 and 4 dpi, respectively). In order to test whether Cxcr4 signaling inhibition in the microenvironment could affect the metastatic cascade in other tumor types, we engrafted another triple negative breast cancer cell line MDA-MB-157 (Fig. 3A,B), as well as prostate cancer cells PC3-M-Pro4-Luc2 (Fig. 3C,D) and the Ewing sarcoma cell line WE68 (Fig. 3E,F). Tumor early metastasis establishment in the CHT region of 4 dpi zebrafish larvae was impaired in the ody mutant line compared to the wt siblings, when each cell line was inoculated into the blood circulation (reduction of tumor burden was 52%, 38% and 70% in breast, prostate and Ewing sarcoma tumor cell lines, respectively) ( Fig. 3). Therefore, we suggest that neutrophilic Cxcr4 signaling plays a crucial role in the early steps of metastases formation of triple negative breast cancer as well as other tumor types.
Cxcr4b signaling inhibition attenuates neutrophil basal motility and development. CXCR4 signaling has been found to play an important role in regulating neutrophil retention in the CHT in the WHIM syndrome, where neutropenia has been linked to increased susceptibility to infection in patients as well as in the zebrafish model 37,38 . Therefore, as neutrophils express high levels of cxcr4b, we investigated whether the impairment of Cxcr4 signaling affects the motility of neutrophils in the CHT region, altering their ability to prepare the metastatic niche. Neutrophil migration under physiological conditions was recorded for 30 min as previously described 30 . Neutrophils displayed reduced motility when Cxcr4b signaling was impaired in the ody mutant compared to the wt siblings ( Fig. 4A-C). We have previously shown that neutrophils prepare the metastatic niche by creating paths into the collagen, during the transmigration from the CHT to the tail fin. Hence, we hypothesized that path formation is linked to metalloprotease activity. Therefore, we quantified mmp9 expression in ody and wt siblings (whole body) and found decreased mRNA levels upon Cxcr4b inhibition (Fig. 4D). Next, neutrophil number in the CHT and whole body was verified at 6 days post fertilization when metastasis formation was assessed. Neutrophil number was lower in the CHT of ody mutants compared to wt siblings (Fig. 4E,F). Moreover, at the same time point, the total body count of neutrophils was found to be lower (Fig. 4G), suggesting that Cxcr4b controls neutrophil development.
During zebrafish development, primitive and definitive waves of hematopoiesis can be distinguished. In a transition phase, between 24 and 36 hpf, neutrophils originate from the posterior blood island (PBI), which, with the onset of the definitive wave, is replaced by the CHT 39 . Recent studies in zebrafish have revealed that CXCR4 signaling has a direct link with the development of HSPCs, mainly affecting their ability to colonize the CHT, which functions as an intermediate hematopoietic site 40 . In the same study, the use of the CXCR4 antagonist replicates. FACS-sorted neutrophils were obtained from 5 dpf Tg(mpx:GFP) i114 larvae. cxcr4a and cxcr4b gene expression was enriched in neutrophils compared to GFP negative cells in zebrafish larvae (~4-fold and ~10fold, respectively). cxcr4b was highly expressed in neutrophils compared to cxcr4a (~100-fold increased gene expression). (B) Relative metastatic tumor burden of MDA-MB-231-B-DsRed cells was quantified in ody and wt siblings at 2 dpi. Data are mean ± SEM of two independent experiments (wt: n = 64, ody: n = 57). Un-paired t-test ****p < 0.0001. (C) MDA-MB-231-B tumor cells established a secondary tumor mass, with initiation of single cell extravasation, in wt larvae, whereas a phenotype inhibition was found in ody mutants at 2 dpi (22.5% reduction). (D) MDA-MB-231-B tumor burden was measured in wt and cxcr4b null mutants at 4 dpi, at the metastatic site where secondary growth began at 2 dpi. A 40.5% reduction in tumor burden was observed. Data are mean ± SEM of two independent experiments (wt: n = 59, ody: n = 43). Un-paired t-test, with Welch's correction ****p < 0.0001. (E) Highly invasive cancer cells displayed aggressive and metastatic features in wt siblings, whereas few cells remained in the CHT region of 4 dpi ody larvae. Scale bars: 50 µm. Micrographs are acquired using a Leica MZ16FA fluorescent microscope coupled to a DFC420C camera. AMD3100, between 48 and 72 hpf, decreased cmyb/runx + HSPCs numbers. Because neutrophils develop first in the PBI, independently from the HSPCs, and subsequently in the CHT, dependently on the HSPCs with self-renewal potential, we investigated whether the development of neutrophils could be affected in a host with a non-functional Cxcr4b signaling. Neutrophil number was quantified during earlier stages of development (1 dpf), before HSPCs colonize the CHT and initiate the definitive wave of hematopoiesis. An increase in neutrophil number was found in the CHT of ody embryos, compared to wt siblings, whereas no difference was detected on whole embryo level ( Fig. S1A-C). Subsequently, neutrophil number was quantified in the whole zebrafish embryo, as well as in the CHT region, in between the dorsal aorta and caudal vein, starting from the end of the yolk extension, in 2 day old cxcr4b −/− and cxcr4b +/+ Tg(mpx:GFP) i114 embryos. We identified an increase (31%) in neutrophil number in the CHT region of ody mutants compared to wt siblings (Fig. S1D,E) at 2 dpf. At the same time, no difference in total neutrophil number was observed (Fig. S1F). These findings suggest that Cxcr4b controls neutrophil motility and development, in a putative HSPCs-dependent and independent manner.
The transcriptomic signature of Cxcr4b-deficient neutrophils links to defective cell motility.
In this study ( Fig. 4A-C) we demonstrated that neutrophil motility is altered in physiological conditions when Cxcr4b signaling is impaired. In order to define the contribution of neutrophilic Cxcr4b signaling axis involved Un-paired t-test ****p < 0.0001. (D) Prostate cancer early metastasis formation, characterized by a solid tumor mass formation in the CHT region of zebrafish larvae, occurred in wt siblings and was significantly decreased when Cxcr4b signaling was impaired in the host. (E) Relative metastatic burden of Ewing sarcoma cell line WE-68 td-tomato was affected in ody mutants compared to wt larvae at 4 dpi (70% reduction in tumor burden in the tail fin). Data are mean ± SEM of two independent experiments (wt: n = 69, ody: n = 39). Un-paired t-test, with Welch's correction ****p < 0.0001. (F) Ewing sarcoma cells formed a compact and expanding tumor mass in the CHT region, between the dorsal aorta and the caudal vein. A reduced tumor cell aggregate was present in the ody mutant line at 4 dpi. Scale bars: 50 µm. Micrographs were acquired using a Leica MZ16FA fluorescent microscope coupled to a DFC420C camera. in metastatic niche preparation and subsequent tumor cell invasion, RNA sequencing was performed from FACS-sorted GFP positive neutrophils after dissociation of cxcr4b +/+ and cxcr4b −/− Tg (mpx:GFP) i144 6 dpf larvae. An overall tendency towards upregulation of differentially expressed genes was found in neutrophils from ody mutants (61% upregulated genes vs 39% downregulated genes) (Fig. 5), when a cutoff was considered (p < 0.05 in DESeq and edgeR). More in details, in ody neutrophils 48% of the up-regulated genes showed an over 10-fold increase, whereas 57% of the down-regulated genes showed an over 10-fold decrease. Pathway analysis was performed in DAVID, after selecting 615 differentially expressed genes (p < 0.05 in DESeq and edgeR) and converting them to human orthologues with gPROFILER. Genes involved in focal adhesion and ECM-receptor interaction were found up-regulated in neutrophils, together with genes involved in axon guidance, suggesting impaired motility and anchoring properties (Table 1). In particular, integrins are involved in adhesion strengthening and arrest of leukocytes on the endothelium, during transendothelial migration 41 . Laminin, fibronectin and collagen are components of the extracellular matrix and increased transcription levels suggest a tighter adhesion ad consequently challenged immune cell motility ( Table 1). Members of the Roundabout signaling pathway (slit1b, sema4gb and srgap1), implicated among others in leukocyte chemotaxis and tumor angiogenesis 42 were found to be up-regulated. Down-regulated genes were found to cluster in the metabolism of xenobiotics by cytochrome p450 pathway. Subsequently, pathway analysis was extended to differentially expressed genes identified through statistical analysis performed in RStudio using the package DESeq2 paired. Overall, the analysis performed in DESeq2 paired confirmed the enriched pathways identified with DESeq and edgeR. However, additional genes were identified, either belonging to previously described pathways (focal adhesion/ECM-Receptor interaction) or clustering in a new pathway (MAPK pathway) ( Table 2). Furthermore, NETRIN-1 (zebrafish netrin1b), belonging to the family of laminin-secreted proteins and involved in neuronal chemotaxis 43,44 and leukocyte migration 45 , was found up-regulated in cxcr4 −/− neutrophils (Log 2 FoldChange = 2.6 and p = 0.00009). NETRIN-1 has previously been linked with reduced neutrophil and macrophage infiltration in a kidney injury model 46 . Taken together, our sequencing data support the above described results that suggest motility alteration in neutrophils bearing a cxcr4b mutation.
Cxcr4b signaling affects the neutrophilic response to cancer cells during early metastasis formation. Considering the involvement of Cxcr4b signaling in driving neutrophil motility and development in tumor-naive conditions, next we investigated the ability of neutrophils to respond to cancer cells in ody mutants. Generally, emergency hematopoiesis is initiated upon systemic infections and neutrophils leave the bone marrow in response to damage and danger signals, during inflammation and infection [47][48][49][50] . Emergency hematopoiesis, dependents on Gcsf-Gcsfr signaling, has also been shown to occur in zebrafish larvae, resulting in expansion of HSPCs and mobilization of neutrophils from the CHT in response to lipopolysaccharide (LPS) injection 51 or bacterial infection 52 . Hence, the number of neutrophils in the CHT was quantified 3-6 hours after MDA-MB-231-B tumor cells were inoculated into the blood circulation of embryos at 2 dpf. We found that the acute response of neutrophils to tumor cell engraftment was characterized by a decreased number of neutrophils in the CHT in the wt siblings and ody embryos, compared to uninjected control groups (Fig. S2A,B). These results suggest that, at 2 dpf, the mobilization of neutrophils from the CHT in response to tumor engraftment is independent from Cxcr4b. As tumor early metastatic events in the CHT region were primarily affected in ody mutants at 4 dpi and the CHT colonization by HSPCs is known to occur at 2 dpf, neutrophil response to cancer cells was also assessed at 4 dpi (6 dpf). Like in 2 dpf embryos, we also observed a reduction of neutrophil number in the CHT of tumor-engrafted wt siblings at 6 dpf, compared to the uninjected controls. In contrast, neutrophil numbers were unchanged in tumor-engrafted ody mutants, compared to uninjected ody larvae (Fig. 6A). Therefore, Cxcr4b signaling is required for the mobilization of neutrophils from the CHT as well tumor-invasive phenotype at 6 dpf.
To further support the evidence that neutrophils display a different response towards cancer cells when Cxcr4b signaling is not functional, we quantified neutrophil motility in the metastatic region at 4 dpi (6 dpf). Neutrophils displayed a motility pattern characterized by lower speed and diminished average distance, in presence of MDA-MB-231-B in the wt siblings, compared to the uninjected controls (Fig. 6B,C,F,G). On the other hand, no differences in neutrophil speed and travelled distance were detected in ody larvae implanted with  MDA-MB-231-B compared to engrafted wt siblings (Fig. 6D-G). In conclusion, Cxcr4b signaling impairment affects neutrophil response to cancer cells initiating early metastatic events.

Discussion
Chemokines are key mediators of directional cell migration and the CXCR4-CXCL12 chemokine axis is well known to display major roles in tumor progression, guiding tumor cell homing to CXCL12 expressing organs 53 . Consequently, targeting the CXCR4 receptor expressed by cancer cells is a pharmacological approach that is currently explored in the clinic to limit tumor spreading and metastases 54 . At the same time, it is important to consider the effect of CXCR4 signaling on the tumor microenvironment, especially in view of the antagonizing or supportive functions that myeloid cells are known to have on tumor progression 55 . We previously showed that the zebrafish xenograft model is a powerful tool to study tumor-microenvironment interactions as CXCR4-based interspecies cross talk takes place 12 and genetic and chemical inactivation of CXCR4 receptor on the engrafted human cancer cells block metastatic onset in zebrafish xenograft model. Moreover, the role of neutrophils in preparing the metastatic niche has been previously described by our group 30 . We found that the non-pathological migration correlate with tumor cell invasion in the caudal hematopoietic tissue (CHT), functionally analogous to the fetal liver in mammalian embryo development. Hence, we hypothesized the involvement of CXCR4 signaling in controlling neutrophil motility and immune-tumor cell interactions involved in the initiation of early metastatic events and micrometastasis formation. First, we found that in zebrafish larvae neutrophils express high levels of cxcr4b, the homolog of human CXCR4 and paralog of zebrafish cxcr4a. Then, we used a cxcr4b homozygote  Table 1. Enriched pathways in cxcr4b −/− neutrophils (analysis performed with DESeq and edgeR). Pathway analysis in Cxcr4b-deficient neutrophils. Genes selected with DESeq (p<0.05) and edgeR (p<0.05) analyses in RStudio (from 21621 to 615 genes) were converted to the human orthologues using g:PROFILER and uploaded in DAVID Bioinformatics. Resources 6.7 for pathway analysis. Up-regulation of genes involved in focal adhesion/ECMReceptor interaction and axon guidance was identified, whereas down-regulation of genes in the metabolism of xenobiotic by P450 was found. Additional analysis was performed using DESeq2 paired ( Table 2). The same pathways were identified with DESeq/edgeR (Table 1) and DESeq2 paired ( Table 2) and the genes listed in Table 2 were in addition to genes described in Table 1 mutant zebrafish (also known as odysseus or ody) and showed that engrafted human tumor cells failed to form micrometastases in the CHT region. Therefore, myeloid cell impairment or a non-functional Cxcr4b signaling led to experimental tumor micrometastasis inhibition.  Table 2. Enriched pathways in cxcr4b −/− neutrophils (analysis performed with DESeq2 paired). Pathway analysis in Cxcr4b-deficient neutrophils. Genes selected with DESeq (p < 0.05) and edgeR (p < 0.05) analyses in RStudio (from 21621 to 615 genes) were converted to the human orthologues using g:PROFILER and uploaded in DAVID Bioinformatics Resources 6.7 for pathway analysis. Up-regulation of genes involved in focal adhesion/ECM-Receptor interaction and axon guidance was identified, whereas down-regulation of genes in the metabolism of xenobiotic by P450 was found. Additional analysis was performed using DESeq2 paired ( Table 2). The same pathways were identified with DESeq/edgeR (Table 1) and DESeq2 paired (Table 2) and the genes listed in Table 2 were in addition to genes described in Table 1. Enriched pathways indicate alteration in motility, as shown by the analysis performed with DESeq and edgeR and reveal members of the MAPK signaling to be differentially expressed. Investigating a potential role of the host Cxcr4b signaling in the formation of early metastasis by affecting immune cell motility was the next approach. We found a downregulation in mmp9 mRNA levels in ody and a reduction in neutrophil motility in tumor-naïve cxcr4b deficient zebrafish embryos. These findings link with our previous work on the role of neutrophil physiological migration in tumor invasion in the tail fin 30 . It has been reported that in addition to its function as a protease, mmp9 plays a role as a chemoattractant. Mmp9 chemotactic properties work in synergy with CXCL12 21 . Therefore, inhibition of CXCR4 signaling could lead to impaired neutrophil motility and ability to respond to tumour cells also as a result of altered mmp9-driven chemotaxis. We next investigated whether Cxcr4b signaling affects neutrophil development. In mammals, CXCR4 and CXCR2 chemokine signaling axes regulate hematopoietic stem cell (HSC) retention in and mobilization from the bone marrow, respectively 27,29 . CXCR4 chemical inhibition upon AMD3100 treatment results in mobilized HSCs 56 . Furthermore, patients affected by WHIM syndrome, characterized by neutropenia and enhanced susceptibility to infection, bear a CXCR4-gain-of-function mutation that causes neutrophil retention in hematopoietic sites, in response to cognate ligand CXCL12, highly expressed in the bone marrow 57 . These findings have been confirmed in a zebrafish model of WHIM syndrome, where neutrophils expressing constitutively active Cxcr4b were retained in the CHT and mobilized only upon cxcl12a knock down 37 . We found that the number of neutrophils in the CHT in ody mutants was higher than in the wt siblings at 2 dpf. Because the overall neutrophil number was not affected by the cxcr4b mutation, a higher number of neutrophils in the CHT surprisingly suggested enhanced neutrophil retention. These findings show that receptor stimulation by cognate ligand is needed to activate cell motility, despite of chemotaxis towards Cxcl12. Retention and reduced motility of neutrophils with impaired Cxcr4b signaling at 2 and 3 dpf, respectively, support the hypothesis that neutrophil physiological behavior plays an important role in cancer micrometastasis formation at early stages.
Next we investigated if neutrophils played an important role in preparing the metastatic niche in later stages of tumor micrometastasis formation, when tumor cell invasion has taken place. Therefore, neutrophil number was counted in 6 dpf zebrafish larvae and, in contrast to the observations at 2 dpf, a reduction in neutrophils localized in the CHT was found in ody mutants, with tendency towards a reduced overall number in a whole larva. We hypothesize that the dichotomy in neutrophil numbers is linked to the role of CXCR4 signaling during hematopoiesis. Using the zebrafish embryo model, it has recently been shown that HSPCs colonize the hematopoietic tissue, interacting with mesenchymal cells and inducing modification in the perivascular niche. In the same study, the mesenchymal cells express cxcl12a, whereas cxcr4b expression is mainly found in the CHT region and treatment with the CXCR4 antagonist AMD3100 reduced the number of runx + hematopoietic progenitors 40 .
In line with their findings, we propose that the reduced number of neutrophils in the CHT of 6 dpf ody larvae relates to the reduced number of HSPCs and suggest that further investigations should be carried on to confirm this hypothesis. Importantly, a lower number of neutrophils in the CHT in 6 dpf ody mutants might result in a reduced niche modification due to a lower number of paths traced into the collagen by neutrophils themselves. On the other end, the increased number of neutrophils in earlier stages suggests the potential role of Cxcr4b in the primitive wave of hematopoiesis.
In agreement with our findings, Cxcr4b signature in tumor-naïve zebrafish neutrophils confirmed Cxcr4b role in cell motility and adhesion. Upregulation of the integrins, as well as increased interaction with the ECM and alteration of the cytoskeleton reorganization were found in Cxcr4b deficient neutrophils. Members of the Roundabout signaling pathway were also differentially expressed. Roundabout signaling is associated with axon guidance mechanisms and its role in cancer metastasis has been reported 58 . Importantly, Slit1b, found up-regulated in ody, functions as a repellent molecule that interferes with leukocyte chemotaxis 59 and specifically blocks the ability of circulating neutrophils to migrate directionally 60 . Moreover, we propose netrin1b as a candidate gene that links neutrophil ability to provide trophic signals to cancer cells. NETRIN-1 has been reported to reduce neutrophil infiltration in ischemic acute kidney injury by inhibiting COX-2 and PGE2 production 46 . PGE2 has been identified as the trophic signal that sustains neoplastic transformation in a transgenic zebrafish cancer model 15 .
After investigating the role of Cxcr4b in physiological neutrophil development and motility, we unraveled neutrophil behavior in presence of engrafted tumor cells, able to initiate early metastatic events. An acute response to engrafted cancer cells into the blood circulation of 2 dpf zebrafish embryos resulted in no alteration in Cxc4b-deficient neutrophils. To assess neutrophil acute response in tumor-engrafted larvae, the number of mpx + cells was counted in the CHT of zebrafish embryos few hours after tumor cell inoculation. As neutrophil number decreased in the CHT of engrafted wt or ody embryos compared to uninjected larvae, we propose that neutrophils mount an acute response upon tumor inoculation by leaving the CHT in line with previous observations of demand-driven granulopoiesis upon bacterial infection 52 and that this response occurs in a Cxcr4b independent manner. On the other hand, an altered response was found at later stages. In 6 dpf (4 dpi) zebrafish larvae, tumor cells have formed a secondary tumor mass and initiated local tissue invasion. In response, wt siblings diminished the number of neutrophils in the CHT, increasing their mobilization. Mobilized neutrophils were found to migrate and in the surrounding of metastasizing cancer cells and to slow down and to interact with human malignant cells. In contrast, cxcr4b deficient neutrophils remained in the CHT and failed to localize in the surrounding of tailfin tumor micrometastases, suggesting a possibly diminished inflammatory response (Fig. 7).
In conclusion, we demonstrate that CXCR4 signaling plays a major role in neutrophilic innate immune response to early metastatic events and contributes to the establishment of tumor micrometastases. The development of CXCR4-targeted therapies directed to the tumor microenvironment is therefore essential.

Materials and Methods
Zebrafish husbandry. Zebrafish lines were kept in compliance with the local animal welfare regulations and European directives. The study was approved by the local animal welfare committee (DEC) of the University of Leiden (license number: 10612, protocol 14227). Zebrafish adults were maintained according to standard protocols (zfin.org), in a 10/14-hour dark/light cycle. Embryos were maintained at 28 °C in Egg water (60 µg/ml Ocean salt in distilled water), containing 0.003% PTU (1-phenyl-2-thiourea) to block pigmentation.
Pu.1 knock down. Pu.1 (Spi1b, 1 mM) and standard control morpholino injections (0.1 mM) were performed to deplete neutrophils and macrophages as previously described 30 . Morpholino efficiency was assessed by counting number of Mpx + neutrophils in the Tg(mpx:GFP) i114 zebrafish line.

Xenograft experiments.
Tumor cells were inoculated in the blood circulation of 2 day post fertilization (dpf) zebrafish embryos as previously described 33 . Tumor burden. Zebrafish embryos engrafted with fluorescently labelled tumor cells were screened for correct engraftment 5-6 hours after inoculation into the blood circulation at 2 dpf, using a Leica MZ16FA fluorescent microscope coupled to a DFC420C camera. Larvae were positioned on a Petri dish with 1.5% agarose coating and tumor burden was quantified at 4 dpi, acquiring monographs of the metastatic site, in the CHT region. LAS AF Lite software was used to overlay the GFP and dsRed channels and snapshots were analyzed in Image-Pro Analyzer 7.0 (Media Cybernetics). For each larva tumor burden was calculated based on number of objects multiplied by mean area and mean intensity, generated with a macro designed by H. de Bont (Toxicology, LACDR, Leiden University) and previously used to quantify tumor migration and proliferation 63,64 .
Neutrophil number and motility. Neutrophil number was quantified by manual counting, using a Leica MZ16FA fluorescent microscope. Neutrophil basal motility was assessed using a Leica TCS SPE confocal microscope with a HC APO 20x DRY objective (0.7 N.A.). 3 dpf larvae were mounted on a 1% low melting point agarose layer, containing tricaine and covering the glass surface of a Will-Co Dish ® (Pelco ® , Ted Pella, Inc). Egg water containing anesthetic was added on top of each larva. Timelapse was performed for 30 minutes, with 1 minute interval between frames. Maximum projections were generated, tail movements were corrected using Stack Reg plugin and neutrophil tracking was performed using the Manual Tracking plugin in ImageJ-Fiji 65 . Neutrophil motility in response to metastatic cancer cells was quantified with a Nikon A1 confocal laser scanning microscope (Tokyo, Japan) using the 488 and 561 laser lines with 20 × (NA 0.75) lens. Images were acquired every minute during timelapse. Videos were analyzed using NIS-Elements AR and tracking performed for the first 30 frames in ImageJ, with Manual Tracking plugin.
cxcr4b transcriptomic signature in neutrophils: from larval dissociation to RNA sequencing analysis. Zebrafish line cxcr4b t26035 Tg(mpx:GFP) i114 was used to isolate neutrophils from 6 dpf larvae. After harvesting, eggs were kept in Petri dishes (n ≤ 100) at 28.5 °C to allow synchronized embryo development.
Triplicates of GFP positive embryos (100-150 per replicate) were selected for dissociation, performed according to 67 . Dissociation with 0.4 mg/ml collagenase/DPBS (Liberase TL, Roche, #05401020001) was alternatively used. Larvae were transferred directly from Egg water to collagenase solution. Dissociation was obtained mechanically with pipetting and 2 incubation steps at 28.5 °C for 10 min. 10% FCS was added and sample preparation was continued as described in 67 . Sorting was performed with a BD FACSAria ™ III Cell Sorter (BD Biosciences, San Jose, CA, USA) with the BD FACSDiva software (version 6.1.3) and gates defined using GFP negative larvae. After sorting, samples were stored in QIAzol at −80 °C. RNA isolation was performed using miRNeasy Mini kit (# 217004 Qiagen). On-column DNase digestion was performed, using RNase-Free DNase Set (# 79254 Qiagen). Agilent Bioanalyzer 2100, RNA 6000 Pico kit (Agilent, Santa Clara) was used to assess RNA quality. cDNA synthesis and amplification were performed with SMARTer ® Ultra ™ Low Input RNA Kit for Sequencing -v3 (Clontech) and cDNA quality validated, using Agilent 2100 BioAnalyzer and the High Sensitivity DNA Chip from Agilent's High Sensitivity DNA Kit (#5067-4626, Agilent). cDNA shearing, library preparation and validation, and Illumina sequencing (HiSeq2000) were performed as described in 67 by ZF-SCREENS (Leiden, The Netherland). Reads (18.684.327 is an average of 12 samples) were mapped to Ensembl transcripts (GRCz10.80) and statistical analysis based on negative binomial distribution performed in R Studio, using DESEq, DESEq2 paired and EdgeR packages, available at Bioconductor.org. Pathway analysis was performed using DAVID Bioinformatics Resources 6.7. Identification of cxcr4a and cxcr4b expression levels in neutrophils by RNA sequencing shown in Fig. 2 was performed as described in 67 .
Statistics. Statistical analysis was performed using GraphPad Prism (versions 5.0 and 6.0). Un-paired t-test was used in datasets of two groups and Welch's correction applied when group variances were significantly different (p < 0.05). One-way ANOVA with Bonferroni post hoc test was used in datasets of three or more groups (continuous variable) and Kruskal-Wallis with Dunn's post hoc test was used to estimate significant difference in the case of counts (discrete variable).

Data Availability
Data generated or analysed during this study are included in this published article (and its Supplementary Information files). The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.