Hyperactive neutrophils infiltrate vital organs of tumor bearing host and contribute to gradual systemic deterioration with tumor progression Authors:

Various studies have addressed the role of neutrophils in cancer wherein the focus has been drawn on the elevated neutrophil count in blood or at tumor loci. However, cancer has a systemic impact which targets various organs thus challenging the overall physiology of the host. So, it is worthwhile to explore whether and how neutrophils contribute to systemic deterioration in cancer. To discern the systemic role of neutrophils, we monitored their number and function at different stages of tumor growth in Dalton’s lymphoma mice model. Notably, we observed a gradual increase in neutrophil count in blood and their infiltration in vital organs of tumor bearing mice. In parallel, we observed damaged histoarchitecture with significant alterations in biochemical parameters that aggravated with tumor progression. We next examined systemic impact of neutrophil by assessing neutrophil elastase, myeloperoxidase, and matrix metalloproteinases (MMP-8 and MMP-9) wherein we found their upregulated expression and activity in tumor condition. Taken together, our results demonstrate high infiltration and hyperactivation of neutrophils which possibly account for gradual systemic deterioration during cancer progression. Our findings thus implicate neutrophils as a potential therapeutic target that may help to reduce the overall fatality rate of cancer.


Introduction
Cancer initially grows as a local disease but eventually turns into a complex systemic disease which targets various organs in the tumor bearing host (Shinko, Diakos et al. 2017). The systemic effects comprise metastasis, inflammation, thrombosis, cachexia that gradually leads to functional impairment of organs. Indeed, these systemic effects cause the majority of cancerrelated deaths, rather than the primary or metastatic tumors (Cedervall, Dimberg et al. 2015, Rutkowski, Svoronos et al. 2015. Importantly, the majority of these systemic manifestations appear to arise from chronic inflammation caused by the cancer cells and aberrant immune response of the host. Substantial evidence suggests that these states of immune dysregulation and excessive inflammation target different organ systems with potentially lethal or highly morbid conditions (Greten and Grivennikov 2019). Hence, the view of cancer as a systemic disease is now emerging into the research spotlight and the quest is to comprehend the underlying mechanism behind the evolution of a local disease to an intricate systemic ailment.
Chronic inflammation is one of the several hallmarks predisposing to cancer growth and progression (Schmidt and Weber 2006). The inflammatory immune component of a developing tumor may include a diverse leukocyte population such as, macrophages, eosinophils, mast cells, lymphocytes and neutrophils which are capable of producing an array of inflammatory mediators (Wahl andKleinman 1998, Kuper, Adami et al. 2001). Neutrophils, which are the most important effector cells of innate immunity, have been shown to play a crucial role in tumor biology. They have recently become the subject of intense research with focus on the association between inflammation and cancer progression. Being the professional phagocytes, they are the first one to migrate at the site of infection, thus constitute the first line of defense. At the site of infection, once neutrophil function is over, their clearance is essential for resolution of inflammation to maintain tissue homeostasis. But, failure in the resolution machinery and prolonged neutrophil accumulation can damage the host tissue and reflect a state of chronic inflammation (Van Leeuwen, Gijbels et al. 2008, Mollinedo 2019. Of note, cancer patients show remarkable increase in peripheral blood neutrophil count and their infiltration in tumors. Based on this observation, neutrophil-to-lymphocyte ratio (NLR), an indicator of inflammation, has been adopted as a prognostic sign of poor survival in cancer patients. High neutrophil infiltration in patients with renal cell carcinoma, bronchi alveolar carcinoma, melanoma and colon cancer has been correlated with increased mortality (Gregory andHoughton 2011, Eruslanov, Bhojnagarwala et al. 2014). Activation of neutrophils, in response to various stimuli can extend their life span which can further influence tumor cells. In turn, the tumor-derived stimuli can induce phenotypic and functional changes in neutrophils which are known to support tumor growth, metastasis and immunosuppression (Zhang, Zhang et al. 2016).
Neutrophils are equipped with multiple mechanisms to eradicate the invading microbes such as respiratory burst, phagocytosis, neutrophils extracellular traps (NET) formation and degranulation (Yin and Heit 2018). In the process of degranulation, neutrophils release an array of proteins stored in their cytosolic granules into the extracellular spaces (Jasper, McIver et al. 2019). These granules play a major role in every step of neutrophil inflammatory response. They have potent anti-microbial properties and regulate inflammation via processing of chemokines, cytokines and various signaling molecules (Harbort, Soeiro-Pereira et al. 2015). These granules include antimicrobial proteins (lysozyme, lactoferrin), serine proteases (neutrophil elastase, cathepsin G), matrixmetalloproteinases (MMP-8, MMP-9) and myeloperoxidase (Borregaard and Cowland 1997). Effector functions of neutrophils largely depend upon the release of these granular cargoes. The controlled mobilization and release of these cargoes allows the transformation of neutrophils from inactive circulating cells to active effector cells of the innate immune system. However, under certain circumstances, unregulated release of these effector molecules can aggravate tissue damage and could be detrimental to the host (Pham 2008). In cancer setting, their excessive release can modulate tissue microenvironment (Coffelt, Wellenstein et al. 2016) and ultimately lead the way for tumor initiation, growth and metastasis (Rawat, Syeda et al. 2021). Their aberrant activation or persistence at inflammatory site is also associated with various inflammatory disorders ranging from chronic obstructive pulmonary disease (COPD) (Meijer, Rijkers et al. 2013), neutrophilic asthma (Chung 2016), rheumatoid arthritis (Apel, Zychlinsky et al. 2018) to the recent pandemic COVID-19 (Ackermann, Anders et al. 2021).
Substantial reports have focused on the role of neutrophils particularly within the tumor microenvironment or at the inflammation site; however, whether they impact systemic milieu or not is still a question. In the present study, we aimed to address this question for which we used a well-accepted Dalton's lymphoma (DL) mice model. DL is a murine non-Hodgkin's T-cell lymphoma which represents an excellent model to examine various parameters of cancer development, signaling mechanisms, and also for therapeutic drug screening (Prasad, Rosangkima et al. 2010, Kumari, Rawat et al. 2017. Here, we first examined the status of neutrophil count in peripheral blood and their infiltration in vital organs of tumor bearing mice at different stages of tumor growth. Further, we monitored the organ functions by evaluating the histo-architecture and biochemical enzymes level with disease progression. In addition, we monitored the status of neutrophil-derived granule cargoes including neutrophil elastase (NE), myeloperoxidase (MPO) and MMPs  in order to evaluate neutrophil function in the systemic environment. Our results demonstrated the potential involvement of neutrophils in mediating systemic effects during tumor progression.

Materials and Methods
Leishman's stain, paraformaldehyde, methanol, glycerol, Coomassie brilliant blue (R-250), hematoxylin and eosin were obtained from SRL India. DAB (3,3'-diaminobenzidine), RNA Later, Revert-Aid first strand cDNA synthesis kit was purchased from Thermo Scientific. Poly-L-lysine, H2O2and citrate buffer were from Sigma-Aldrich. DHE (dihydroethidium) dye was from Invitrogen Molecular Probes, D11347.The elastase kit was purchased from Elabscience (Houston, Texas). Anti-Ly6G and goat anti-rat secondary were purchased from Abcam (Cambridge, USA). Anti-MPO, anti-elastase were purchased from cloud clone corp. Xylene was purchased from Fisher scientific, absolute alcohol was from Merck and RNeasy Micro Kit was from Qiagen. DPX, slides, cover slips and other reagents were of the highest analytical grade and were obtained from the common source.

Experimental animals:
Inbred strains of pathogen free BALB/c mice (22-25g) of either sex were obtained from the Animal House facility of Department of Zoology, University of Delhi.
Animals were housed in propylene cages, where fresh and clean drinking water was supplied ad libitum with a standard pellet diet. Throughout the period of the experiment, animals were kept in a constant environment and diet conditions. Temperature was maintained at 18-26°C with light/dark cycles of 12h interval. The study was performed in accordance with the guidance for the care and use of laboratory animals with approval of the University of Delhi and Committee for the Purpose of Control and Suppression of Experiments on Animals (CPCSEA), India.

Tumor induction and maintenance: Dalton's lymphoma (DL) cells were obtained from
Department of Biotechnology, Banaras Hindu University. The cells were maintained in the peritoneum of BALB/c mice by serial intraperitoneal transplantation as described earlier (Manjula Vinayak, 2015). For the experiment purpose DL cells were collected from the donor mice and were immediately suspended in sterile isotonic saline (PBS). The viability of DL cells was confirmed by the trypan blue assay and total number of cells /ml. was counted. The total number of cells was adjusted to 1×10 6 cells/ml. and then injected in the peritoneal cavity of healthy 3-4 months old BALB/c mice.

Experimental Groups:
BALB/c mice of either sex, each weighing 22-25g and aged 3-4 months were divided into 4 groups, with each group consisting of six mice. Group I served as control and in the rest three groups, DL was induced by injecting 1×10 6 cell/ml of tumor cell suspension i.p. The tumor was allowed to grow and mice were sacrificed through cervical dislocation on different time points i.e. on 7, 14 and 21 day post tumor transplantation.

Hematological and biochemical parameters:
Animals were sacrificed at different time points of tumor growth. The blood samples were collected into collection tubes with and without EDTA (anti-coagulant). EDTA containing tubes were used for analysis of polymorphs, lymphocytes and total leukocyte count (TLC). The blood in non-EDTA tubes was allowed to stand for one hour at room temperature and then centrifuged at 1000g for 10 minutes. Serum was obtained which was used for the determination of various biochemical parameters like total protein, urea, albumin, ALT, AST and creatinine using commercially available kits (Erba diagnostic kits).

Histological Examination:
Mice were sacrificed at different time points and organs such as lungs, liver, kidney, spleen, lymph node and peritoneum were collected and rinsed with PBS.
Subsequently, tissues were fixed in neutral buffer formalin (NBF) and then embedded in paraffin for histopathological examination. The thin sections of 6µm were cut and transferred on clean slides. The slides were further processed and then stained with hematoxylin and eosin and observed under a light microscope using NIS Element software.
2.6 Immunohistochemistry: Samples were fixed in 4%PFA, embedded in paraffin and were cut into 5µm sections. Sections were deparaffinized in xylene twice, for 10 minutes each, rehydrated with graded ethanol, 100%, 95%, 80%, 70% and 50%, for 5 minutes each, and transferred to tap water. The endogenous peroxidase activity was blocked by incubating the sections with 3% H2O2 for 20 min. Slides were heated in sodium citrate buffer (pH 6.0) solution at 95°Cfor 20 minutes for antigen retrieval. Non-specific reactivity was blocked by incubating the slides with 5% normal goat serum for 1h. The slides were washed three times in PBST (0.2% Tween-20) and incubated with anti-Ly6G primary antibody (1:200) at 4°C overnight in a humidified chamber.
Sections were washed three times in PBST and incubated with goat anti-rat secondary antibody (1:200) for 2h at room temperature. Sections were then stained with DAB for 5 minutes and subsequently with hematoxylin and observed under a light microscope using NIS Element software.

2.7
Immunofluorescence for Ly6G, NE and MPO: EDTA anticoagulated peripheral blood samples were smeared on glass slides, air dried and fixed in methanol. Slides were permeabilized with 4%PFA for 20 minutes at 4°C. Non-specific reactivity was blocked by incubating the slides with 5% normal goat serum for 1h. Slides were then incubated with anti-Ly6G primary antibody (1:200) at 4°C overnight. Slides were washed three times with PBST (0.2% Tween-20) and incubated with alexa fluor 488 goat anti-rat secondary antibody for 2h in dark at room temperature. Slides were deparaffinized in xylene twice, for 10 minutes each, rehydrated with graded ethanol, 100%, 95%, 80%, 70% and 50%, for 5 minutes each, and transferred to tap water. Slides were heated in sodium citrate buffer (pH 6.0) solution at 95°C for 20 minutes for antigen retrieval. The endogenous peroxidase activity was blocked by incubating the sections with 3% H2O2 for 20 min. Further, non-specific reactivity was blocked by incubating the slides with 5% normal goat serum for 1h. For NE expressions in blood and various tissues, slides were incubated with anti-elastase (1:200) overnight, and FITC-labeled goat anti-rabbit secondary  (1:200) for 30 minutes in dark at room temperature. Finally the cells were resuspended in 500µl PBS and acquired in BDAccuri C6 cytometer.

Preparation of tissue sections for O2 detection:
The exteriorized tissues were rinsed immediately in chilled PBS and snap frozen in liquid nitrogen. 20µm thick cryosections were obtained using cryostat and placed on clean poly L-lysine coated slides. DHE was suspended in DMSO at stock concentration of 10mM and diluted for a final working concentration of 10µm in PBS. DHE was topically applied to each tissue section and slides were incubated in a lightprotected humidified incubator at 37°C for 15 minutes. Slides were then mounted with 5% glycerol and analyzed under fluorescence microscope (Nikon). Minimum six slides per condition were analyzed for ROS quantification.

Detection of NE by ELISA:
Blood samples were collected in microcentrifuge tubes under sterile conditions. Samples were allowed to clot and centrifuged at 1000g for 10 minutes. Serum was separated and stored in aliquots at -80°C. Ascitic fluid was also collected from tumor bearing mice and stored at -80°C. The tissues were removed and rinsed in sterile PBS, blotted on tissue (to remove excess buffer) and weighed. 600µl of RIPA lysis buffer containing protease inhibitor cocktail was added to 5mg tissue and homogenized using electric homogenizer. After homogenization, samples were kept on ice for 30 minutes before transfer into pre-labeled micro centrifuge tubes. Samples were centrifuged at 4°C, 10000g for 10 minutes. Supernatants from each sample were removed, aliquoted and stored at -80°Cfor further analysis. The levels of NE in serum, ascites and tissue specimen were detected using Elisa kit according to the manufacturer's protocol (Elabscience) and the absorbance was measured by a multi detection microplate reader (Biotek). recombinant protein was also included. Gels were then incubated in 2% Triton X-100 solution at room temperature for 1h on shaker to remove the SDS and allow protein renaturation. Gels were subsequently incubated at 37°C for 48h in the developing buffer. Thereafter, gels were stained with 0.25% Coomassie brilliant Blue (R-250) for 30 minutes and destained with 30% methanol, 10% acetic acid solution. Gelatinase activity was detected as a white band against a blue background. Gel images were captured using Amersham imager 600 (GE Healthcare) and band densitometry was assessed using ImageJ software to obtain a semi-quantitative presentation of enzymatic activity.

RNA extraction and PCR analysis:
Total RNA from various tissues was extracted using RNeasy Micro Kit (Qiagen) as per the manufacturer's protocol. The quality of RNA was checked by Nanodrop; 260/280 nm absorbance ratio close to 2.0 was accepted as 'pure' RNA.
1ug RNA was treated with RNase free DNaseI (Thermo scientific) and reverse transcribed using Revert-Aid first strand cDNA synthesis kit (Thermo scientific, K1622). The cDNA was used to perform semi-quantitative PCR for NE, MPO, MMP-8, MMP-9 and GAPDH. Primers were designed using NCBI primer blast.

Neutrophil count increases in peripheral blood and vital organs of tumor bearing mice.
To explore the involvement of neutrophils in mediating systemic effect with tumor progression, we first evaluated their presence in the peripheral blood of DL bearing mice ( fig. 2A). DL cells (1×10 6 ) were intraperitoneally transplanted in healthy mice and the tumor was allowed to grow. increased to 81.6%. As we observed a gradual increase in neutrophil count in peripheral blood we speculated that the presence of neutrophils in different tissues could be a more relevant determinant and marker of persistent inflammation than the circulating neutrophils. Therefore, we next examined the presence of neutrophils in vital organs of tumor bearing mice. The organs selected for the study were liver, lungs, spleen, peritoneum, kidney and lymph nodes. We performed immunohistochemistry wherein 6µm thick paraffin embedded tissue sections were processed and incubated with primary anti-Ly6G. Incubation with appropriate secondary antibody was followed by direct DAB staining and counterstained with hematoxylin.
Interestingly, we found a systemic presence of neutrophils as the number of positive cells for   is applicable to all panels. Every image is the representative of six sections analyzed per condition. C) Assessment of ALT (alanine transaminase), AST (aspartate transaminase), urea, total protein, albumin and creatinine in serum of tumor bearing mice at different time points of tumor growth. The results represent three independent experiments and are expressed as the mean ±SD; statistical significance between the groups was determined by one-way analysis of variance (ANOVA) followed by a Tukey post hoc test where p<0.05(*), p<0.02(**) and p<0.001(***).

Peripheral blood neutrophils attain a hyperactive state with tumor progression.
We were further interested in knowing the change in the activation state of neutrophils and whether it correlates with the histological changes observed during tumor progression. Interestingly, it has been revealed that inflammation is an early and sensitive event which represents the hyperactive state of neutrophils in peripheral blood (Ling, Chapple et al. 2015). These neutrophils release toxic mediators packed in their distinct granule subsets and thus contribute to tissue injury

Neutrophil infiltration contributes to high NE release in tissues.
We first examined NE levels in serum and ascites (obtained from peritoneum cavity) by ELISA. Serum and ascites were  to GAPDH. E) Immunofluorescent staining using anti-elastase was performed in liver, lungs, spleen and peritoneum as described in materials and methods (magnification, × 100, scale bar, 100 μm). The results represent three independent experiments and are expressed as the mean ±SD; statistical significance between the groups was determined by one-way analysis of variance (ANOVA) followed by a Tukey post hoc test or two-tailed Student's ttest for two groups where p<0.05(*), p<0.02(**) and p<0.001(***).

Neutrophil infiltration enhances MPO and ROS production in tumor bearing mice.
Excess generation of MPO-derived oxidants has been linked to tissue destruction and chronic inflammation (Aratani 2018).  GAPDH was used as an internal control and bar graphs showing densitometric analysis of mRNA expression level compared to GAPDH. C) Immunofluorescent staining using anti-MPO was performed in liver, lungs, spleen and peritoneum as described in materials and methods. D) Cryosections of liver, peritoneum, spleen and lungs were prepared and processed for DHE staining as mentioned in materials and methods. Histogram shows DHE fluorescence intensity in vital organs of mice with and without tumor (magnification, × 100, scale bar, 100 μm). The results represent three independent experiments and are expressed as the mean ±SD; statistical significance between the groups was determined by one-way analysis of variance (ANOVA) followed by a Tukey post hoc test or two-tailed Student's t-test for two groups where p<0.05(*), p<0.02(**) and p<0.001(***).

Enzymatic activity of MMPs increases in tumor bearing mice:
MMPs play a critical role in tumor progression by facilitating tumor cell invasion and show broad catalytic activity against components of ECM (Roy, Yang et al. 2009). Neutrophils are known to be the major source of MMPs (specifically, MMP-8 and -9) (Fligiel, Standiford et al. 2006, Rawat, Syeda et al. 2021.  GAPDH was used as an internal control and bar graphs showing densitometric analysis of mRNA expression level compared to GAPDH. C) Activity of MMP-9 in 2 (liver), 3 (lungs), 4 (peritoneum), 5 (spleen), 6 (ascitic fluid) and 7 (serum) was assessed by gelatin zymography. Lane 1 denotes MMP-9 positive control. The tissue samples were homogenized and the supernatant of proteins were resolved on SDS-PAGE gel containing 1 mg/ml gelatin. Gels were developed as mentioned in the material and methods section to show clear bands of gelatinolytic activity. Bar graph shows band intensities measured using ImageJ software. Lanes marked as C represents the control group whereas lanes marked as T denote the tumor group. The results represent three independent experiments and are expressed as the mean ±SD; statistical significance between the groups was determined by one-way analysis of variance (ANOVA) followed by a Tukey post hoc test where p<0.05(*), p<0.02(**) and p<0.001(***).

Discussion
Our understanding of the role of neutrophils in cancer has substantially evolved. Neutrophils have recently been shown to play a major role in the various phases of cancer initiation to its metastatic progression (Piccard, Muschel et al. 2012). Accumulating evidences suggest that neutrophil infiltration within tumor microenvironment is associated with poor clinical outcomes in many cancer types such as renal cell carcinoma, human hepatocellular carcinoma, bronchoalveolar carcinomas as well as head and neck squamous cell carcinomas. Moreover, neutrophil infiltration also correlates with tumor grade in human gliomas and pancreatic tumors (Fridlender and Albelda 2012). Importantly, all these reports have focused Cancer is an inflammatory disease wherein neutrophilia is considered to be the most frequent alteration detected in the patients (Howard, Kanetsky et al. 2019). It has been significantly correlated with advanced disease and found to be an independent prognostic factor, associated with reduced survival in human metastatic melanoma, pancreatic carcinoma, and renal patients (Mayadas, Tsokos et al. 2009). In acute respiratory distress syndrome (ARDS) and sepsis, intensity of neutrophil infiltration in lungs has been correlated with organ failure (Adams, Hauser et al. 2001). Likewise, we found a correlation between neutrophil infiltration and histopathological aberration in all the examined organs of tumor bearing mice with disease progression ( fig. 3B). Further, altered biochemical enzyme levels in serum confirmed the tissue injury ( fig. 3C). The serum biochemical tests are mostly used in diagnosis of liver, kidney, cardiovascular and many other diseases as well as monitoring the response to inflammation and toxin exposure (Wang, Feng et al. 2006).The marker enzymes of the liver leak into the blood when there is any damage to the liver and their levels increase in the serum (Zimmerman 1999).We observed elevated serum levels of AST and ALT in tumor bearing mice which indicate liver damage ( fig. 3C).Kind, P. R., et al also reported an increased liver transaminases in tumor-bearing animals (Kind, Gordon et al. 1985). Urea is the main excretory product of protein metabolism and its high level in blood may reflect an imbalance between urea formation by protein catabolism and urea excretion by the kidney. We observed an elevation in serum urea levels of tumor bearing mice that reflects altered kidney functioning. The high level of urea in blood can also be attributed to the inflammatory condition which is known to cause the decline in renal functioning even in persons without renal disease (Harirforoosh and Jamali 2008). In addition, we also observed an increase in albumin and total protein level with tumor progression.
Neutrophil-mediated organ dysfunction has been implicated as playing a causative role in the high rates of morbidity and mortality in SIRS (systemic inflammatory response syndrome) patients (Gando, Kameue et al. 2002). In fact, neutrophils have gained major attention in the ongoing pandemic COVID-19 and are reported to play a crucial role in the development of sepsis, cytokine storm, and multi-organ failure (Arcanjo, Logullo et al. 2020, Tomar, Anders et al. 2020.Increased levels of AST, ALT, albumin, creatinine and urea have also been associated with disease severity and worse prognosis in COVID-19 patients (Lippi and Plebani 2020).
Upon infiltration in the inflamed tissue, neutrophils are activated by a multitude of inflammatory mediators, which triggers the release of its key effector molecules which are encapsulated within their distinct granules (Mantovani, Cassatella et al. 2011). Various reports have discussed the involvement of these effectors mediators in contributing to tissue damage (Wilgus, Roy et al. 2013). NE, a key cargo stored in the primary granules of neutrophils, can hydrolyze a variety of substrates, including elastin and other ECM component (Korkmaz, Moreau et al. 2008). In addition to its role in host defense, evidence suggests an important contribution of NE in various chronic inflammatory diseases, including asthma, respiratory syncytial virus (RSV) (Yasui, Baba et al. 2005) and cancer (Gaida, Steffen et al. 2012 5). NE was also found to be elevated in human ARDS samples, and its inhibition further resulted in reduced epithelial injury in animal models (Sun andYang 2004, Fujino, Kubo et al. 2012). Also, Baines et al. correlated high gene expression of neutrophil elastase with over-activation of neutrophils in neutrophilic asthma (Baines, Simpson et al. 2011).
Similarly, our results reflect the hyper-activation of neutrophils which might have contributed to tissue damage ( fig. 4). Like NE, MPO is another key cargo of primary granules in neutrophils which has been found to enhance the inflammatory response in various inflammatory diseases (Loria, Dato et al. 2008). MPO, being an indicator of neutrophils presence in tissues, has been widely used as an inflammatory marker of both acute and chronic conditions (Faith, Sukumaran et al. 2008). Also, MPO changes have been associated with the severity of many diseases and its level reflects the existence of a systemic inflammation, rather than a local inflammatory condition (Schierwagen, Bylund-Fellenius et al. 1990). Similarly, we observed a high expression of MPO in peripheral blood neutrophils ( fig. 4) (Davies 2020). MPO has been associated with enhancing inflammatory response in COPD patients via producing high reactive oxygen intermediates (Zhu, Ge et al. 2014). MPO catalyzes the formation of reactive oxygen intermediates which play a significant role in disease pathogenesis (Yang, Preston et al. 2001). High amounts of oxygen radicals are found in the bronchoalveolar lavage (BAL) fluid of asthmatic patients as compared to healthy controls (Monteseirin 2009). Similarly in RSV bronchiolitis patients, abundant release of ROS damages the host cellular structures and contributes to lung injury (Bataki, Evans et al. 2005). Our results also resonate with these studies showing an increase in ROS levels in liver, lungs, spleen and peritoneum of tumor bearing mice ( fig. 6C) implicating a crucial role of MPO and ROS in mediating tissue damage.
Neutrophils are known to be the major source of MMPs (specifically, MMP-8 and -9). MMP-8 also known as neutrophil collagenase is highly expressed in neutrophils and stored as proenzyme in specific granules. Activated neutrophils quickly release MMP-8 to ensure its availability at the site of infection or inflammation. Various studies have reported the upregulation of MMP-8 in a wide range of inflammatory disorders including cancer (Van Lint and Libert 2006). It has been reported to be upregulated in the tissues of individuals with periodontitis and considered to represent an appropriate therapeutic target for the prevention of periodontal disease progression (Liu, Hynes et al. 2006). High expression of MMP-8 has been observed in tissue samples of pancreatic adenocarcinoma (Jones, Humphreys et al. 2004) and uterine cancer (Ueno, Yamashita et al. 1999) patients as compared with normal tissues. Likewise, we observed an upregulation of MMP-8 expression in liver, lungs, spleen and peritoneum of tumor bearing mice as compared to the control ( fig. 7B). Sirnio and group showed high serum MMP-8 levels in colorectal cancer patients and suggested a physiological link between MMP-8 and systemic inflammation (Sirniö, Tuomisto et al. 2018). Similarly, MMP-9 is one of the most important contributors of tumor progression and a key player in ECM degradation (Ardi, Kupriyanova et al. 2007). In a murine model of pancreatic ductal adenocarcinoma, neutrophils were found to be the major cell group producing MMP-9 and strikingly these neutrophils were predominantly present at the invasive fronts of metastatic tumours. Interestingly in infectious inflammatory conditions neutrophil entry into tissues relies on the production of MMP-9 by neutrophils. Neutrophil-derived MMP-9 is also known to cause airway remodeling in asthma (Ventura, Vega et al. 2014). High levels of MMP-9 in the sputum and BAL fluid of asthma patients has also been correlated with the extent of cellular infiltration and disease severity (Ventura, Vega et al. 2014). Similarly, high expression of MMP-9 at the gene and protein levels has been found in the bronchial walls of asthma patients (Hoshino, Nakamura et al. 1998). BAL fluid from COPD and emphysematous patients also showed upregulation of neutrophil-derived MMP-9, which caused parenchymal destruction and disease extremity (Finlay, Russell et al. 1997, Vlahos, Wark et al. 2012. We showed that MMP-9 expression and activity was dramatically elevated in the liver, lungs, peritoneum and spleen of tumor bearing mice which was further corroborated by its high gelatinase activity in serum and ascitic fluid obtained from tumor bearing mice ( fig. 7C).The circulating leucocytes were considered to be one of the major sources of elevated MMP-9 in the serum of patients suffering from Kawasaki disease (KD), a multi-systemic type of vasculitis (Takeshita, Tokutomi et al. 2001).Taken together, our findings strongly support the hypothesis that neutrophils might be majorly responsible for the systemic manifestations observed in cancer patients with disease progression.

Conclusion
Various studies have well established the role of neutrophils in influencing the tumor microenvironment. However, we hypothesized that neutrophils also play an important role in modulating the systemic environment during cancer development. We observed elevated count and hyperactivation of neutrophils in the peripheral blood and vital organs of tumor bearing mice. In addition, we also found damaged histoarchitecture and altered biochemical enzyme levels suggesting organ dysfunction. We convincingly showed that high neutrophil infiltration was accompanied with the excessive release of its key effector molecules (NE, MPO, MMP-8 and MMP-9). This excessive release of effector molecules represents the hyperactive response of neutrophils, which might be leading to the tissue damage and subsequent organ dysfunction.
Systemic effects cause the majority of cancer-related deaths therefore; taming neutrophils could be a better approach to prevent systemic deterioration in cancer patients thereby leading to increased survival. Our present study demonstrates that significant systemic impact makes the neutrophils as a potential target and further expands the horizon of neutrophil-centered approach for cancer therapeutics.