Although neutrophils are traditionally considered in the context of their anti-bacterial functions, it is becoming increasingly clear that tumor-associated neutrophils (TANs) and their myeloid precursors (peripheral neutrophils and granulocytic MDSC) in the spleen, bone marrow, and blood play an important role in cancer biology 
. In contrast to the well-described ability of inflammatory neutrophils to engulf bacteria, activate the immune system, and induce tissue damage in infections 
, it appears that myeloid cells can also function as immunosuppressive cells in the context of tumors 
. This property has been very well described in recent years for the MDSCs found in large quantities in the spleens of tumor-bearing animals 
and for tumor-associated macrophages which develop an “M2” or tumor-supportive phenotype 
. Neutrophils make up a significant portion of the inflammatory cell infiltrate in many models of cancer. Previous studies have shown that TAN in untreated tumors can support tumor growth and metastases 
, and this was further supported by our recent work demonstrating that in untreated tumors, TAN develop a pro-tumorigenic (N2) phenotype, largely driven by the presence of TGF-β 
. The full range of mechanisms responsible for this activity have not yet been elucidated, but neutrophils are known to have pathways that can impact angiogenesis, immune surveillance, as well as secretion of chemokines, cytokines and reactive oxygen species 
Since relatively little is yet known about the phenotype of TAN, the goal of the present study was to use an unbiased, discovery-based approach (gene array analysis) to profile and compare the mRNA content of three highly purified granulocytic populations: “naïve” bone-marrow neutrophils from non-tumor-bearing animals (NN) [serving as the “baseline” control population], the granulocytic fraction of myeloid-derived suppressor cells (G-MDSC) in tumor bearing mice, and tumor-associated neutrophils. Our purpose was to gain additional information on the characteristics of TAN, and specifically how much they resembled the other neutrophil populations. A caveat of this study is that we restricted our initial analysis to primarily transcriptomic information. Although we have conducted some functional studies 
, additional experiments, based on this genomic information, will obviously be needed to fully understand the role and functions of TAN.
Using unbiased analyses, our data show that the three populations of neutrophils are significantly different in their mRNA profiles. However, it appears that the NN and G-MDSC are more closely related to each other than to TAN. To better understand the meaning of these differences, we used pathway analyses (using Genomica software, and published gene lists) focusing on several selected pathways and gene groups important for the function and activation of neutrophils including structural genes, respiratory burst, granule proteins, phagocytosis, apoptosis, and immune functions.
NN were relatively enriched in cytoskeleton organization and biogenesis, as well as in pathways related to actin binding and polymerization. This is probably related to the movement needed by neutrophils prior to arriving to their destination 
. Interestingly these gene pathways are down-regulated in TAN, consistent with their loss of ability to leave the tumor microenvironment after infiltrating the tumor.
The two pathways needed to carry out the “anti-bacterial” functions of neutrophils, granule protein production and the respiratory burst 
, appear to be at their highest levels in the NN. These pathways are progressively lost in the G-MDSC, and even more dramatically in the TAN. TAN show a dramatic down-regulation in mRNA levels of all three groups of granule proteins, which are highly expressed in NN. This finding could be consistent with the work by Shen et. al. showing that TGF-β can inhibit neutrophils degranulation 
. Given that the two main mechanisms of cell killing by neutrophils (respiratory burst and granule proteins) are both down-regulated in TAN, these findings are compatible with our previously published functional data showing that TAN had low cytotoxic capabilities for tumor cells 
. An alternative explanation for these findings could be that the mature neutrophils, either G-MDSC or TAN, have finished producing granule contents, and the relevant mRNA are not needed any longer 
, or that the more mature populations have degranulated. If so, these differences may only be a reflection of the fact that NN are less mature. Even if that explains the up-regulation of these mRNAs in NN, there is a further down-regulation in TAN compared to G-MDSC, suggesting a possible effect at the RNA level as well. Again, it is also possible that these differences reflect that G-MDSC are circulatory neutrophils, whereas TAN are in the tissue. The mRNA for neutrophil elastase, a major effector molecule in the activity of neutrophils in general, and specifically in cancer 
, was surprisingly not different between the 3 populations. It is possible that the modifications in the level of this molecule are post-translational, or that it is needed for the proper activity of neutrophils in either compartment. Further research to evaluate actual production and secretion of the different granule proteins is warranted. The only pathway related to respiratory burst where some members were up-regulated in TAN was the toll-like receptor (TLR) family, probably more related to associated immune system changes (see below).
In contrast, we noted no clear changes in the pathways and genes related to phagocytosis, another major function of neutrophils. This was further confirmed by evaluating the genes suggested by Huang et. al. to be phagocytic receptors or genes involved in phagocytic signaling 
(data not shown).
Despite data suggesting that TAN may be longer lasting cells than circulating neutrophils 
, we found that most genes related to apoptosis were expressed at similar levels. However, we did find that several anti-apoptotic members of the NF-κB family were up-regulated in TAN. NF-κB may be, therefore, an important regulator of the anti-apoptotic machinery in TAN and it is possible that this pathway is responsible for the notable longevity of TAN compare to other neutrophils. Interestingly, we found an up-regulation of all the BH3 pro-apoptotic genes in G-MDSC neutrophils, suggesting that these cells might be especially sensitive to apoptosis-mediated by death receptor ligands.
It has become increasingly clear that the contribution of neutrophils to host defense and natural immunity extends well beyond their traditional role as professional phagocytes 
. Neutrophils and their myeloid precursors can be induced to express a number of genes whose products lie at the core of inflammatory and immune responses, suggesting a potential role for these cells in orchestrating the sequential recruitment and activation of distinct leukocyte types to the inflamed tissue 
. Neutrophils from humans and mice are recognized as cellular sources of chemokines in inflammatory responses 
. “Immunosculpting”, i.e. the crosstalk between immune and tumor cells changing the phenotype of tumor biology, is widely recognized 
. However, until recently, the role of neutrophils in this cross-talk has been under-estimated. Our study suggests several potential new pathways by which neutrophils can influence both the innate and the adaptive immune system. Our data are also consistent with the suggestion that a potential source for chemokines in tumors are the intratumoral TAN, which constitute a notable percentage of tumor immune cells.
One area of potential importance is in antigen presentation. Accumulating data from the last decade shows that neutrophils can participate in MHC class I and class II restricted antigen presentation, being capable of collecting and cleaving antigens, forming complexes with MHC-II molecules, and expressing co-stimulatory molecules 
. Our data show that the naïve neutrophils lack many of the gene pathways needed to present antigens, however, both TAN and G-MDSC show increased expression of these genes, suggesting an enhanced capability of functioning as APC's. There was also up-regulation of the co-stimulatory molecules CD80 and CD86. These results are consistent with the abundant data on effects of tumor neutrophils and G-MDSC on T-cells 
. Interestingly, there was no clear difference in this capability between G-MDSC and TAN, possibly suggesting that this is a fundamental part of activation of neutrophils, regardless of their specific role. An interesting and very important issue, which will require functional studies to elucidate, is whether neutrophil antigen presentation induces T cell activation or anergy. It has been recently shown that mature neutrophils can function as professional antigen-presenting cells capable of priming a Th-1 and Th-17-acquired immune response 
The most prominent difference that we found between TAN, and either NN or G-MDSC, was the significant up-regulation of cytokines and chemokines. This change suggests an important role of tumor neutrophils in the recruitment of immunocytes and in the balance between activation and suppression of the immune system. The role of chemokines in the pathogenesis of cancer has been increasingly appreciated 
. Among the broad group of chemokines whose mRNAs were up-regulated in TAN were the CCL chemokines 2, 3, 4, 8, 12, and 17 and the CXCL chemokines 1, 2, 9, and 16. The up-regulation of chemokines in TAN suggests that upon entering the tumor, TAN have a pivotal role in recruiting other cells of the immune system to the tumor. This is similar to the role that “classical neutrophils” would have in wound healing. At least some of the recruited cells are known to support tumor growth, such as macrophages (by CCL-2 and CCL-7) and T-regulatory cells (by CCL-17) 
. The increased secretion of CCL-2, CCL-17 and IL-6 was demonstrated at the protein level as well.
Special attention should be given to our data on neutrophil chemoattractants and their chemokine receptors. Ueha et. al have recently summarized the dynamics of myeloid cells, including neutrophils, from the bone marrow to the circulation and into tumors 
. CXCR-2 and CXCR-4 were shown to cooperatively regulate the release of neutrophils from bone marrow 
. Whereas the expression of CXCR-2 was not evaluated in this array, CXCR-4 was highly expressed in all 3 neutrophil populations. Its expression, however, was mildly down-regulated in the splenic neutrophils and in TAN. Ueha et. al. further suggest that tumor-infiltration by neutrophils is at-least partly mediated by autocrine CXCL-2 production. Indeed, in our data the expression of CXCL-2 was markedly up-regulated in G-MDSC compared to NN (71-fold), and even more in TAN (188-fold compared to NN). Interestingly, we found similar results in two other known neutrophils chemoattractants – CXCL-1 (increased 14-fold in G-MDSC and 140-fold in TAN compared to NN), and CCL-3 (increased 60-fold in G-MDSC and 76-fold in TAN compared to NN). Unfortunately GCP-2/CXCL-6 was not part of the Illumina array we used, and therefore we could not assess changes in this previously-described 
neutrophil chemoattractant. It seems therefore, that the neutrophils begin a positive feedback loop by secreting neutrophil chemoattractants that recruit more neutrophils into the tumor, as previously described in infections 
It is worth considering the relationship between TAN and the granulocytic fraction of myeloid-derived suppressor cells (G-MDSC) in light of our results. MDSC, a heterogeneous population of immune suppressive cells that are produced at high levels in cancer, are defined in mice on the basis of expression of the surface markers CD11b and GR1 and by their ability to inhibit T lymphocyte activation. The CD11b+
MDSC population is comprised of at least two subsets - granulocytic (Ly6G+
) and monocytic cells (Ly6C+
), possibly with different immunosuppressive properties 
. There is substantial agreement on the immunosuppressive activity of the monocytic MDSC subset. However, there is still contradictory evidence on the role of the granulocytic fraction. Whereas some have shown that granulocytic MDSC have immunosuppression properties similar to the monocytic fraction 
, others have recently demonstrated that they are less immunosuppressive 
. It has been previously shown that adoptively transferred MDSC can enter tumors and differentiate to mature macrophages (TAM) or neutrophils (TAN) 
, however little is known in animals about whether MDSC leave the spleen and circulate. It is thus not clear whether the majority of TAN are actually G-MDSC that have been attracted to the tumor or whether they are bone marrow/blood-derived neutrophils that were then converted to N2 TAN by the tumor microenvironment, specifically by the high local concentrations of TGF-β 
. In our previously published work, we saw no effects of TGF-β blockade on the percentage or phenotype of blood neutrophils or splenic MDSC 
. In the current study, we clearly show that TAN are not “tissue-based G-MDSC”, but are a distinct population of neutrophils, differing markedly in their transcriptomic profile from both NN and G-MDSC. Taken together, these data support the idea that tumor TGF-β (and perhaps other factors) changes only the local “education” of neutrophils. However, the studies described here cannot definitively determine if the cells were recruited from the bone marrow/blood pool of neutrophils or the splenic G-MDSC population.
A possible limitation of our study is that it was performed only in one type of tumor, i.e. the mesothelioma cell line AB12. We did confirm some of the results in a different cell line – the non-small cell lung cancer LKR-M. However further analysis is needed in order to establish the generalization of our data to other tumor systems. It is also possible to argue that since cells were collected at different time points during tumor progression, some of the differences in RNA profiles observed may be due to the time-point at which the cells were isolated (earlier or later during tumor growth) and not their location in the spleen or tumor. Since little is known about the kinetics of the development of TAN, and it is well established that G-MDSC arise only at later time in tumor development 
, we decided to do our comparisons using well-established G-MDSC (associated with larger tumor size) and TAN from established, but not necrotic tumors (less than 500 mm3
). However, we also confirmed some of our data in key transcripts, using RT-PCR of neutrophils isolated from tumors and spleens at the same time, both at an early and a late time point (Day 14 and Day 21), and showed that similar differences to those seen in the arrays were noted. The important question of the kinetics of the changes in tumor and spleen neutrophils will be addressed in future research.
Our data confirm that the native tumor microenvironment influences the mRNA program of neutrophils. We have previously shown that these cells have pro-tumor characteristics 
. We have also shown that altering the tumor microenvironment by blocking the effects of TGFβ can further alter the phenotype of TANs to a more “anti-tumor phenotype (N1 TAN). We are currently comparing gene arrays of N1 and N2 TAN and are seeing clear changes in chemokines and cytokines profile between these two populations of neutrophils (manuscript in preparation), suggesting that the specific profile of chemokines secreted is a major characteristic of TAN and a determinant in their polarization. One example is that the T regulatory cell chemoattractant, CCL-17, is up-regulated in N2 versus N1 TAN.
In their recent review on TAN as targets for cancer therapy, Gregory and Houghton argued that changes in TAN are not a switch to a unique transcriptional program, but a heightened state of activation 
. Borregaard et. al. suggested that circulating neutrophils have two major bursts of transcriptional and protein synthetic activities – one in the bone marrow, producing the granules, and the second upon migration into tissues, resulting mainly in the secretion of cytokines and chemokines 
. The data we presented here suggests that neutrophils may have a different program if they reside in the spleen, becoming G-MDSC or upon entering the tumor, becoming TAN. If so, these 3 neutrophil populations do have different transcription programs that separate them. It remains to be seen if the differences noted are merely different activation states that can be reversible.
Significant research has recently been done elucidating the important role of myeloid cells in the cancerous process. Our work adds another important layer to the understanding of neutrophils in cancer by further characterizing the different populations of neutrophils induced by tumors and by pointing out major differences between TAN and other neutrophil populations. Further research on the functional role of different pathways and genes up-regulated in TAN and differences between the different subsets of TAN is currently underway.