A review on how neutrophils are related with cancer metastasis

Aryesh Acharjee,Pallab Chakraborty

1Department of Zoology,Ramakrishna Mission Vidyamandira,India.

Neutrophils that are mostly related to our body immunity and inflammatory responses are now considered to be an important player in contrast to tumor progression.Along with many controversies,there is much clear evidence that shows neutrophils promote the metastasis process via different modes of actions in a different time frame concerning the advancement of cancer.Not only impacting cell proliferation but also they are found to be helping tumor cells in many events like angiogenesis,immune escape,invasion,and finally it leads to cancer metastasis.Tumor microenvironment,key players of cancer growth and progression are also affected by mostly neutrophils and it's secretory.Tumor associated neutrophils,having certainly different characteristics and expressions,are found high in number around the tumor site and often in pre-metastatic niches.Increased number of premature neutrophils and more neutrophil-extracellular-traps formation is associated with a high degree of metastasis in cancer is observed.As neutrophils has potential role in cancer,more research are warranted in this field to fully understood the overall role of this immune cells and it will helps to open a new way to target neutrophils in therapeutic implications for better cancer treatment.Here we gather currents profile of neutrophils and its role in cancer metastasis and discuss therapeutics strategy.

Key words:Neutrophils,Metastasis,Plasticity of neutrophils,Cancer treatments

Background

To date,we are unable to understand the entire process of metastasis and what are the factors responsible for it[1,2].The chances of getting cured also fall dramatically due to limitations in limited medical treatment options when cells have metastasized and thus making it the real killer in many cancers [3,4].Many of the metastatic directed therapeutic strategies are evolving now [4].To be precise in metastasis,the cells of primary tumor site proliferate and invade the local tissues,enter nearby blood vessels or lymphatic's by intra-vasation,survive and circulate throughout the body and via extravasation enters a new the site,then there they proliferate or remain in dormancy [5].It is around 100 years that,Paget termed metastasis in botanical terms where it was considered the communication between seed and congenial soil [3,6].Many studies of the past have confirmed that inflammations and immune cells do contribute to various cancer metastases [7,8,9,44].The inflammatory milieu creates impacts on the tumor microenvironment and has shown to support cancer cell aggressiveness [10].It is also now demonstrated as one of the hallmarks of cancer [11].Neutrophils are one the critical cell which is responsible for this immune response and is found in numbers in these sites [8,12].They are the earliest cells to react and respond to the specific site of inflammation [1,11].It is found that neutrophils have both pro and anti-tumorigenic role [2,9,11,13].Clinical data show accumulations and increased number of neutrophils in tumors and blood is often related to poor diagnosis and the least chance of survival of the patient [1,2].The heterogeneity of neutrophil cell population,number of neutrophils to the number of lymphocyte ratio,different cytokines and chemokines released by the neutrophils,neutrophil extracellular trap (NET) formation-all are found to be involved in several cancer progressions and metastasis[13,14,15,16,17,18,19].Tumor-associatedneutrophils (TANs) are an important contributor to cancer cell metastasis and are different from normal neutrophils [16,20].Understanding the complex interplay between neutrophils and cancer has opened revealed new scopes on targeting metastasis and to get rid of with the disease [2,11,13].

About neutrophils

Neutrophils are the most abundant (50-70% of circulating leukocytes) white blood cells found in human blood and one of the most essential components of the innate immune system of the body [2,9].These phagocytic cells are known to have an immense impact on protection from various microbial pathogens,showing anti-microbial activity [9,11].Despite having a short lifespan (half-life 7-10 hours in humans),these cells are capable of performing multiple functions and show phenotypic plasticity [14,16].Neutrophils granules are the storage of various proteins (matrix metalloproteinases (MMP)-8,MMP-9,MMP-25,lysozymes,neutrophil elastase,myeloperoxidase,etc.),antibiotics,reactive oxygen species (ROS) [1].They are reported to be the first cell to effect in the site of inflammation and plays a vital role in chronic inflammatory diseases like cancer [1,2,21].After the realization that neutrophils play an important role in cancer,the modern trend of studying neutrophils and their role in this disease has shown an uplift [11,22,23].Increase in the number of neutrophils found to be associated in cancers like-lung,breast,liver,gastric,esophageal,renal,colorectal,cervical,ovarian,skin and many others thus making it one of the hallmarks of the disease [12,14].Many tumors derived factors like granulocyte-colony stimulating factor (G-CSF) are found to be involved in the increased rate of granulopoiesis,which leads to accumulations of immature neutrophils in blood circulation,primary tumor and distant organs where they might form a premetastatic niche [15].Neutrophils release some cytokines and chemokines that induce tumor growth and invasion [1,11].Neutrophil mediated direct cytotoxicity and antibody-dependent cell-mediated cytotoxicity contributes to their anti-tumor role [23,24].Recent discoveries have confirmed that NETs composed of DNA strands,neutrophil granule proteins and are a non- specific immune response can promote cancer progression and facilitate cancer metastasis [18,25].Neutrophils recruitment into the tumor site is mediated via several different cytokines and chemokines [9].CXCR1 and CXCR2 are the 2 most studied chemokine receptors expressed in TANs and these are capable of binding to certain chemokines which are expressed in high amount like CXCL1,CXCL2,CXCL5,CXCL6,CXCL8 and CXCL17 from a growing tumor [1,9,11].IL-8/CXCL8 is the most potent among them and this is found to be highly expressed in leukemia,melanoma,breast cancer,lung cancer [1].Many other cytokines like tumor necrosis factor α and INFγ are found also to trigger neutrophils incorporation into the tumor site from the blood.Besides these there are several growth factors like GCSF,GM-CSF,chemokines receptors like CCR5,CCR7 and exosomal proteins like S100A8,S100A9 also contribute to this event [9,26,27].Macrophages and T cells have shown to also impact on TANs number and recruitment to the tumor [9,11].

Neutrophils related with metastasis

Metastasis is cascades of incidents that are often observed in the later stages of cancer and investigations have revealed a complex interplay between neutrophils and metastasis.Their relationship with cancer is presented here in Figure 1.They are found abundantly in the microenvironment and act as an important factor associated with cancer cell proliferation,tumor development,and metastasis [1,14,13,21].Neutrophils that show both pro and anti-tumorigenic properties,depend upon the products of neutrophils like several enzymes,ROS,growth factors,cytokines,and chemokines,which determines the overall contribution neutrophils.ROS released by neutrophils in the tumor microenvironment usually play a pro-tumor role by damaging the DNA bases resulting in more mutations in tumor cell which can trigger more invasive and proliferative behavior [21].Myeloperoxidase helps in production of hypochlorous acid from hydrogen peroxide,which is initially released by neutrophils and this hypochlorous acid can activate several extra cellular matrix (ECM)-degrading MMPs like MMP-2,MMP-7,MMP-8,and MMP-9 which ultimately leads to tumor progression angiogenesis and metastasis [11].Several enzymes like NE,CG,and MMP-9,secreted by neutrophils also involved in many pro- tumorigenic responses like epithelial to mesenchymal transition(EMT) induction,ECM remodeling,angiogenesis that ultimately stimulates the process of metastasis [1,12].Matrix metalloproteinase-9 (MMP-9/gelatinase B) is believed to help neutrophils in the process of extravasation via degradation of ECM proteins and also has been shown to inhibit apoptosis of tumor cells [11].MMP-9 enhances angiogenesis via VEGF from the ECM.Other's cell the zymogen proMMP-9 is released along with the inhibitor of metalloprotease 1 (TIMP-1),who slows the activation of MMP-9 and inhibit the proteolytic activity of the once activated enzyme [1,11,14,28].TANs-derived cathepsin G may also induce tumor progression,and metastasis.CG degrades ECM molecules such as fibronectin and attenuates binding between integrins and fibronectin that leads to Ecadherin mediated homotypic cell-cell adhesion and this would allow them to survive and disseminate via the circulation to distant sites,establishing metastases[11,29].NE,a serine protease has also been found to promote the migration,causes degradation of ECM proteins and induce tumor invasion and metastasis [1,11].Neutrophil-released TGF-β has also been shown to induce EMT in tumor cells [21].In hepatocellular carcinoma (HCC),neutrophils release hepatocyte growth factor that effects in more invasive characteristics of tumor cells which makes them more prone to metastasize [11,12,30,31].TANs secrete proinflammatory cytokines into the tumor microenvironment,such as IL17,CXC,CC cytokines which is found to be an inducer of stem cell-like features and also in the recruitment of more neutrophils [21].Neutrophils release many inflammatory factors that promote tumor migration [11].In the murine model system,it is well-observed how neutrophils are important in regulating metastasis in several cancers like - Breast cancer,colorectal cancer,etc.[2,17,32,33].The neutrophils number and neutrophils lymphocyte ratio have shown to be related to cancer progression in HCC and non-small cell lung cancer patients [2,34,35].There are many possible ways that neutrophils help in enhancing the metastasis process.

In a nutshell these includes:(1) firstly they are shown to help in cancer cell proliferation thus cancer aggression via many mechanisms [2].(2) Employment of Neutrophils in tumor microenvironment leads to ECM degradation and re-modeling which helps tumor cells to leave the primary site [1,11].(3) Numerous factors that neutrophils released contribute to cancer cell invasion,intravasation by EMT,and migrations[21].Some of the factors are also important in angiogenesis [2,21].(4) Pre-metastatic niche formation and regulation are also observed to be induced by these cells [11].(5) Neutrophils are also seen to help in immune escape of a circulating tumor cell (CTC) by inducing cluster formation in CTC and providing a physical barrier for other immune cells to act upon [11,13].Another role performed by the neutrophils is immunosuppression of other body immunitycells especially T cells [11,24].(6) Moreover,they are also found to play a major role in tumor cell adhesion to the blood vessel in the distant site [2,13].Neutrophils after their death forms NETs that also support the metastasis[14].

Angiogenesis

It is another very key step in cancer metastasis.Stromal cells found to fund pro-angiogenic factors along with the tumor cells [9].Study shows breast cancer cells can stimulate neutrophils to release oncostatin-M,an IL-6-like cytokine that stimulates breast cancer cells to secrete VEGF via JAK-STAT transduction thus inducing angiogenesis and metastasis [11,12,21,36].VEGF-A is a potent angiogenic factor and TANs are found to express the high amount of VEGF-A directly by more VEGF-A mRNA synthesis or indirectly by releasing various other cytokines that increase VEGF-A release in the tumor microenvironment [13].Neutrophils stimulated with tumor-derived G-CSF,upregulate's expression of Bv8/prokineticin-2 using the Stat3 pathway which stimulates angiogenesis and metastasis [13,14].Studies have shown tumors become highly vascularized,and are more infiltrated by neutrophils in IFNβ deficient mice in comparison to the wild-type mice and depletion of neutrophils co-relates with the lesser rate of angiogenesis in IFNβ-deficient mice [13,37].Neutrophils creates a channel while migrating through the ECM toward a tumor inflammation that used by tumor cells to escape into the vasculature,[1].MMP 9 also promotes angiogenesis by cleaving the substrates and adapts ECM.Other neutrophil-derived factors sustains tumor angiogenesis includes CXCL1,CXCL8,IL1b,and IL6 [13].

Figure 1 Showing various impact of neutrophils on promoting cancer metastasis

Escaping immune attack

Despite being one of the major cells related to our body immunity,Neutrophils are found to protect the cancer cells from the attack of the body's immune system.The study report shows that they also help in tumor cells better survival in blood circulation via immunesuppression,which is another major contribution of neutrophils towards the cancer metastasis [2,14].By attracting infiltrating immune cells that possess immunosuppressive activity like neutrophils,tumors evade anti-tumor immune responses mediated by T cells suppression which is an important attribute to survive while metastasis process in on [1].The IL-1β that trigger release of IL-17α from γδ T-cells,secreted by tumor cells,induced the G-CSF,which assistances neutrophils to show more of their immunosuppressive behavior [2,13,14,24].It is been demonstrated in HCCs,Granulocyte-macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor are highly expressed and these factors enhance neutrophil's ability to suppress T cells via increasing their levels of programmed cell death 1 ligand 1 expression [14,16,34].TANs release ARG1 that degrade extracellular arginine,which is required for proper activation of T cells,thus,exerting another immunosuppressive effect in tumors by inhibiting T cells.ARG1-dependent immunosuppression is also induced by TANs [1,11,14,38].Neutrophils are also marked by their ability to decrease the functionality of NK cells that increase the intraluminal survival of tumor cells [2,14,39].

Survival of tumor cells

Neutrophils also help CTCs to survive by inducing tumor cell aggregation.In breast carcinoma cells,direct cell-cell interaction of neutrophils with tumor cells has been demonstrated [11,12,14].Circulating human melanoma cells secrete IL8,which induces expression of b2 integrin (Mac-1) on neutrophils,which results in increases in the binding of melanoma cells to neutrophils and endothelial cells,causing an increased rate of metastasis [13,40].It is also recently discovered that neutrophils help in the anchoring of the CTCs to the vascular endothelium,extravasation clustering of tumor cells directly,or via formation of NETs.Metastatic cancer cells secrete G-CSF and stimulate neutrophil to the NET formation,and a study shows,targeting NETs by DNase I-coated nanoparticles prevented lung metastasis in a certain breast cancer model [2,41].It is been observed that neutrophils are accumulated in a distant site rather than the primary tumor site which is termed as “pre-metastatic niche” and promotes cancer cell recruitment therein as well as play a supporting role in secondary cancer growth at that site [2].VEGFR1-positive bone marrow-derived cells are found in premetastatic niches and these bone marrow-derived cells secrete factors that promote tumor cell growth at the secondary tumor site [11].In breast cancer the accumulated neutrophils helps in the lung metastasis while reduction in their number found to decrease the rate of metastasis [2,13,42].Furthermore,neutrophils also have good impact on preventing metastasis as well.Thus studies are required for proper understanding.

Discussion

Therapeutic implications

Targeting neutrophils in the field of cancer metastasis is now seems warranted for more knowledge towards therapeutic improvements.Several chemotherapies are been able to turn down circulating neutrophil levels,causing neutropenia.As mentioned earlier,the heterogeneous neutrophil population consisting of N1 and N2 cells,both has a very different outcome on cancer and only the N2 cell types are reported to have to promote cancers progression,while N1 cell population plays the very much opposite role.So reducing the N2 population and increase in N1 population may have more desired prognosis outcome rather than reducing the number of neutrophils in general,non-specified way.Inhibition of certain signaling pathways induces pro- tumorigenic nature in neutrophils.For example,inhibition of TGFβ signaling results show increase in anti-tumor responses mediated by TANs [16,9,33].Blocking those chemokines that are responsible for the recruitment of neutrophils in the tumor tissues,is a good therapeutic approach.Like if we design some drug that is able to block CXCL8 (one of the major chemo-attractants of neutrophils),will eventually lead to reduced neutrophil number in the tumor site [33,43].As mentioned earlier,neutrophils have both pro and anti-tumor kind's activities and inducing more anti-tumor activities will help us to fight any certain cancer.For example,if we can stimulate neutrophils and enhance their antibody-dependent cellular cytotoxicity activity [16,7].Trigger neutrophils to release more tumor necrosis factor-related apoptosisinducing ligand that ultimately induces apoptosis in tumor cells also counts for anti-tumor behavior of neutrophils [15] Inhibiting NET formation by neutrophils eventually leads to decrease in rate of successful metastasis.Use of such things like DNase I,that causes lesser NET formation seems to be a wise tactics to prevent metastasis [18,25].Several antagonists are in the ongoing trial with patients and have an impact over neutrophils.But with the advancement of oncology with time,this path will also have a clearer overview of us and might lead us to explore new horizons in cancer pharmacology.

Future insight

To our opinion,neutrophils are one of the key cells that have a very important role in the regulation of cancer and found to be acting on different sites simultaneously in various physiological conditions.More studies and research will give us a more clear view of its role and mode of action.The main focus while doing so will be how to turn up the anti-tumor activities of neutrophils and suppress the pro-tumor activities.More studies that deal with the plasticity of neutrophils specifically all the factors that modulate this heterogeneity and conversion to one phenotype to another,is desired to be done soon.Designing specific drugs that will suppress the N2 cell population hence inhibiting the overall cancer metastasis can be beneficial so need more experiments here also.How the microenvironment,convert the N1 population of neutrophils to towards the N2 type need more clarification to design useful drugs for limiting the pro-tumoral activities of neutrophils.Inhibiting key signaling molecule or the modulation of the factors that related with NET formation can be used to decrease the rate of metastasis but need more detailed study before finalized this.In future,we should also focus on how to use neutrophils in immunotherapy in cancer.

Conclusion

It is now clear that the neutrophils can promote angiogenesis,invasiveness of tumor cells,immune escape of tumor cells,and forming pre-metastatic niches and finally promote in metastasis.Thus successfully targeting neutrophils to get a better result in cancer is still a novel path in oncology.With time all the hidden answers are expected to be answered and eventually lead us to many other new innovative ideas with the potential to cure cancer.