- Email: [email protected]
Granulocytes as effective anticancer agent in experimental solid tumor models
Immunobiology 215 (2010) 1015–1020
Contents lists available at ScienceDirect
Immunobiology journal homepage: www.elsevier.de/imbio
Granulocytes as effective anticancer agent in experimental solid tumor models Morana Jaganjac a,n, Marija Poljak-Blazi a, Iva Kirac b, Suzana Borovic a, Rudolf Joerg Schaur c, Neven Zarkovic a a
Department of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, HR-10002 Zagreb, Croatia Department of Surgery, University Hospital Sisters of Charity, Vinogradska 29, HR-10000 Zagreb, Croatia c Institute of Molecular Biosciences University of Graz, Schubertstrasse 1, A-8010 Graz, Austria b
a r t i c l e in fo
abstract
Article history: Received 4 November 2009 Received in revised form 15 January 2010 Accepted 18 January 2010
The aim of the study was to elucidate the effects of murine granulocytes on the growth of solid murine tumors when administrated in the vicinity of W256 carcinoma growing in Sprague Dawley rats, and in the vicinity of Ehrlich ascites tumor (EAT) growing in BALBc mice. The administration of granulocytes significantly improved the survival of W256-bearing rats, and increased the tumor regression incidence from 17% up to 75%. Rats with regressing tumors had 2.5 times increased levels of granulocytes in peripheral blood, which were also cytotoxic in vitro for W256 carcinoma cells. However, blood levels of cytokine-induced neutrophil chemoattractant-2, tumor necrosis factor a and interleukin 6 were similar between rats with regressing tumors and control healthy rats, suggesting that the observed regression of W256 carcinoma was caused by specific anticancer effects of the applied granulocytes. Anticancer effects of granulocytes were also found in BALBc mice bearing solid form of EAT, resulting in a 20% increase of survival in EAT-bearing mice. Therefore, the administration of granulocytes, isolated from healthy animals and applied at the site of solid tumors in rats and in mice, reduced experimental tumor growth, and extended the survival of tumor-bearing animals, while in some rats it even caused a W256 regression. & 2010 Elsevier GmbH. All rights reserved.
Keywords: Ehrlich ascites tumor Granulocytes Sephadex Tumor regression Walker 256 carcinoma
Introduction The role of granulocytes in immune response against cancer cells is not well understood. Several studies have reported cytotoxicity of granulocytes against tumor cells in vitro (Dallegri et al., 1984; Dallegri et al., 1987; Reali et al., 1994; Valerius et al., 1993) and in vivo (Katano and Torisu, 1982). The activation process of granulocytes is accompanied by the intense production of reactive oxygen species (ROS) (Babior, 2000; Sbarra and Karnovsky, 1959), and an extended release of destructive hydrolytic enzymes (Stipancic and Zarkovic, 1997; Niwa et al., 1989). It was demonstrated that the activated granulocytes cause unspecific lysis of tumor cells mediated by ROS (Dallegri et al., 1991; van Kessel et al., 1990; Lichtenstein et al., 1989), hence ROS
Abbreviations: CINC-2, Cytokine-induced neutrophil chemoattractant-2; EAT, Ehrlich ascites tumor; 3H-TdR, 3H-thymidine incorporation assay; IL-6, Interleukin 6; PMN, Polymorpho-nuclear granulocytes; ROS, Reactive oxygen species; TNF-a, Tumor necrosis factor a; W256, Walker 256 carcinoma n Corresponding author at: PhD, Research Associate; Laboratory for Oxidative Stress, Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, HR10002 Zagreb, Croatia. Tel.: + 385 1 457 1212; fax: + 385 1 456 1010. E-mail address: [email protected] (M. Jaganjac). 0171-2985/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2010.01.002
have been identified as effector molecules of the oxygendependent killing of cancer cells by granulocytes (Lichtenstein and Kahle, 1985; Lichtenstein et al., 1989). We have found that oxidative burst of granulocytes is cytotoxic for melanoma B16-F10 (Zivkovic et al., 2007) and for Walker 256 carcinoma (W256) (Zivkovic et al., 2005a). However, in spite of the data showing that granulocytes can participate in destruction of malignant cells, little is known about function of granulocytes in tumor-bearing hosts. In a previous study (Zivkovic et al., 2005a), we have found that functional granulocytes, obtained from the papula of normal, healthy rats injected subcutaneously (s.c.) with Sephadex, act cytotoxically against W256 tumor cells in vitro. Moreover, the granulocytes from the Sephadex papula of the tumor-bearing animals were less reactive in vitro than the granulocytes from the Sephadex papula of the normal healthy animals, indicating that the most potent granulocytes were already engaged in reaction against the W256 in vivo. In an earlier study using murine B16-F10 melanoma (Zivkovic et al., 2007), we have found that s.c. injection of Sephadex distracts the granulocytes from melanoma, allowing faster progression of the tumor, indicating that neutrophils may have an important role in the host defence against malignant cells in
1016
M. Jaganjac et al. / Immunobiology 215 (2010) 1015–1020
the early stage of tumor development. To test further this possibility, in another recently published study, we used the W256 carcinoma model grown on Sprague Dawley rats (Jaganjac et al., 2008). The results showed that innate immunity based on the immune competent granulocytes may be the cause of the well known phenomenon of spontaneous regression of W256 carcinoma in Sprague Dawley rats (Jaganjac et al., 2008). In the same study we have also shown that distraction of granulocytes from the tumor by s.c. Sephadex injection greatly decreased the incidence of W256 regression. Therefore, the aim of the current study was to elucidate the possible anticancer effects of granulocytes administration, isolated from healthy animals, in the vicinity of solid tumors in Sprague Dawley rats bearing W256 and additionally treated with Sephadex and in BALBc mice bearing Ehrlich ascites tumor (EAT).
(PMN group), while other 6 rats were left as a control tumor progressive animals (control group) and injected with sterile 200 mL of saline. Monitoring granulocyte effect in tumor-bearing rats The tumor volume and survival were monitored daily. Tumor growth was evaluated by the use of calliper measuring three diameters of the tumors formed at the site of inoculation. On the 18th day after W256 injection, blood was taken from the tail vein ¨ in EDTA-containing tubes. Leukocytes were counted in a Burker ¨ chamber using Turk’s solution. Blood smears were prepared and ¨ stained with May Grunwald-Giemsa for the differential counting of leukocytes. The remaining blood was used for plasma preparation, while three control healthy rats served as controls for the analyzes of the granulocyte counts and plasma cytokine values.
Materials and methods Animals Experiments were performed on male, three months old, Sprague Dawley rats and on male, three months old, BALBc mice. Water and food were given ad libitum. All the experiments were performed in accordance with the ILAR Guide for the Care and Use of Laboratory Animals, Council Directive (86/609/EEC) and the Croatian animal welfare law (NN 19/99). Effects of granulocytes in tumor-bearing rats Tumor regression experiments were carried out on 14 tumorbearing Sprague Dawley rats treated with Sephadex. Rats were intramuscularly injected in the right hind limb with 250 mL of RPMI medium containing 107 W256 tumor cells and additionally s.c. with 5 ml Sephadex (Sephadex G-150, Pharmacia Fine Chemicals, Sweden) on each side in the lower dorsal quadrant. Namely, Sephadex distracts functional granulocytes from the blood into the Sephadex papula thus abolishing spontaneous W256 tumor regression in Sprague Dawley rats (Jaganjac et al., 2008). The Sephadex was prepared as described elsewhere (Zivkovic et al., 2005a). On the 4th day after the tumor injection, the granulocytes were administered at the site of tumor, as a bio-therapeutic agent to 8 rats, while the remaining 6 rats served as saline treated control. Preparation of rat granulocytes As a source for granulocytes healthy control (without tumors) rats were used. On the 3rd day healthy control rats were s.c. injected in the abdomen area with Sephadex and 24 h later Sephadex papule were surgically removed. Rats were anesthetized using intraperitoneal injection of chloralhidrate (150 mg/ml). Operating field was prepared according to animal care standards. Skin incision was made above the site where Sephadex papule was formed following subcutaneous Sephadex injection. Underlying Sephadex was then removed with a spatula and skin was sawn with individual stitches. Sephadex papule containing granulocytes were placed into sterile 0,9% NaCl and filtered over filter paper that allows granulocytes to pass. Injection of granulocytes at the site of W256 tumor progression The viability of granulocyte cell suspensions was checked by the trypan blue exclusion test and was found to be Z93%. Isolated granulocytes were immediately re-suspended in sterile 200 mL of saline and injected into the site of tumor progression of 8 tumor-bearing Sephadex treated Sprague Dawley rats
Determination of CINC-2, TNF-a and IL-6 in rat plasma samples The plasma samples were isolated from the whole blood by centrifugation at 3500 rpm for 15 min at 4 1C as described previously (Poljak-Blazi et al., 2009). Plasma samples were then transferred to a 1.5 ml microcentrifuge tube, frozen at 801C, and subsequently used in Luminex analysis. A commercial rat 3-plex luminex kit (R&D Saystems, Minneapolis, USA) was used for determination of cytokine-induced neutrophil chemoattractant-2 (CINC-2), tumor necrosis factor a (TNF-a) and interleukin 6 (IL-6). The assay was performed and plasma samples were diluted 3-fold according to the manufacturer’s instructions. Briefly, the wells of the 1.2 mm filter membrane 96-well microtiter plates were prewetted with assay wash buffer. 50 ml of diluted microparticle mixture were added to each well followed by adding 50 ml of plasma sample, standard and quality control preparations to the relevant wells and incubated for 3 h on a horizontal orbital shaker at room temperature. The plates were washed three times with assay wash buffer and 50 ml of biotinylated detector antibody added per well. Samples were incubated for 1 h at room temperature on the plate shaker. The plates were washed three times with assay wash buffer and 50 ml per well of streptavidin– phycoerythrin solution were added, and plates incubated for further 30 min at room temperature on a plate shaker, protected from direct light. After completion of staining, the microbeads were washed three times with assay wash buffer and re-suspended in 100 ml/well of assay wash buffer. The luminex assay was performed on a luminex-200TM instrument using AtheNA Analyzer LX200 (Full) 2.3 Software. Hundred events per region were collected and median fluorescence intensity measured. Median fluorescence intensity was converted to concentrations using results from a standard cytokine preparation. Effects of rat granulocytes in vitro Rats were anesthetized using intraperitoneal injection of chloralhydrate (150 mg/ml) and sacrificed by bleeding from the heart (Zivkovic et al., 2005b) under ether anaesthesia on the 18th day after the Sephadex treatment and tumor transplantation. Influence of whole blood granulocytes and granulocytes derived from Sephadex papula on the proliferation of W256 cells was measured in vitro by the 3H-thymidine (3H-TdR) incorporation assay as described before (Zarkovic et al., 1997). Granulocytes were obtained from the Sephadex papula by a method preserving their biological activity as described above and from whole blood using dextran sedimentation followed by hypotonic lysis and centrifugation on a Ficoll-Paque gradient as described elsewhere ¨ (Boyum, 1968). Granulocytes from PMN rats with regressing tumors were analyzed and compared to those obtained from control healthy animals. Briefly, the seeding density of
M. Jaganjac et al. / Immunobiology 215 (2010) 1015–1020
1017
granulocytes cultured in 96-well microcytoplates was 2 105 cells per culture in RPMI 1640 medium with 10% FCS. The seeding density of the tumor cells was 2 104 cells per culture irrespective if cultured alone or if added to the granulocytes. The cell cultures were incubated for 24 h at 37 1C in a humidified air atmosphere with 5% CO2. After 24 h, radioactive 3H-TdR was added to each culture and left for additional 48 h. The incorporation of 3H-TdR, determined with a b-liquid-scintillation counter (Beckman, LS 3800 Series), was used as a parameter of tumor cell proliferation (DNA synthesis). Effects of granulocytes in tumor-bearing mice To study the outcome of granulocyte administration in tumor progressive animals, 33 mice were intramuscularly injected in the right hind limb with 100 mL of Hanks balanced salt solution containing 106 EAT cells. Afterwards, 20 mice were administrated with granulocytes, while the remaining 13 mice served as saline treated control. Preparation and injection of mice granulocytes at the site of EAT tumor progression As a source for granulocytes healthy BALBc mice were used. They were treated with Sephadex and 24 h later the papula content was surgically removed and granulocytes isolated as described above. Isolated granulocytes were immediately re-suspended in sterile 100 mL saline and injected into the site of tumor progression at different time points, while control tumor progressive animals were injected solely with sterile 100 mL saline. Fifteen mice received granulocytes on a one-time basis as follows; PMN1 group, consisting of 5 mice, was administrated on the 1st day while the other 10 mice, group PMN6, were administrated with granulocytes on the 6th day. Furthermore, 5 mice (group PMN 1,4,8) were treated with granulocytes three times (on days 1, 4 and 8). The rest of mice (n= 13) were left as a control tumor progressive animals (control group). Monitoring granulocyte effect in tumor-bearing mice Tumor volume growth and survival were monitored. Tumor growth was evaluated by the use of calliper measuring three times a week the three diameters of the tumors formed at the site of inoculation. Mice mortality was recorded daily. Statistics The incidence of tumor regression was evaluated by the Chisquare test, survival rates of tumor-bearing rats were evaluated by the Kaplan Meier test, while the other parameters were analyzed by the two-tailed Mann-Whitney test. The MedCalc software and the SPSS 11.01 for Mircosoft Windows were used. The values of p o0.05 were considered as significant.
Fig. 1. (A) Survival curves of W256 tumor-bearing Sephadex treated rats, according to Kaplan Meier method, (B) W256 tumor volume growth dynamics in control group or PMN rats (7 SE per group) and (C) changes in W256 tumor volume during tumor progression or regression for all animals regardless of granulocyte treatment (7SE per group). On the 4th day rats were treated with PMN obtained from healthy donors (PMN) or with physiological solution (control).
rats (increase from 17% up to 75%). Moreover, as is presented in Fig. 1B, in PMN rats tumor growth was significantly slower (p o0.05 from day 7 further) than in control rats where tumor growth was more aggressive. PMN rats had 2.5 times more granulocytes (p o0.05) present in the blood (Fig. 2) than control healthy rats, however, the levels of CINC-2, TNF-a and IL-6 in plasma were not significantly (p 40.05, Mann-Whitney test) different from the cytokine levels in control healthy rats (Fig. 2).
Results Administration of granulocytes in the vicinity of solid tumors of Sprague Dawley rats Survival rate and tumor volume growth dynamics of tumor-bearing Sephadex treated Sprague Dawley rats are presented in Fig. 1. Administration of granulocytes obtained from subcutaneously formed Sephadex papula of healthy rats to the tumor-bearing Sephadex-treated rats (shown as PMN rats) on the fourth day after tumor transplantation significantly (p o0.01) improved the survival (Fig. 1A), causing tumor regression in 6/8
In vitro anti-tumor effects of granulocytes Anti-tumor activity of the PMN rat papula and whole blood granulocytes against W256 cells was further verified in vitro by the 3H-TdR incorporation assay (Fig. 3). A 50% inhibition (p o0.001) of the W256 growth in vitro was observed in the presence of the granulocytes obtained from papula of PMN rats. Peripheral blood granulocytes obtained from PMN rats also significantly reduced W256 growth for 37% (p o0.005), while granulocytes isolated from blood of healthy rats showed mild
1018
M. Jaganjac et al. / Immunobiology 215 (2010) 1015–1020
Fig. 2. Distribution of neutrophils in the peripheral blood of Sprague Dawley rats and the values of cytokines CINC-2, TNF-a and IL-6 in plasma determined on the day 18 (mean 7SE per group): (a) significance P o0.05 in comparison to healthy control.
(17%), but significant inhibitory effect (po0.05) on W256 tumor cell growth in vitro.
Administration of granulocytes in the vicinity of solid tumors of BALBc mice
Fig. 3. Proliferation of W256 tumor cells in vitro measured by 3H-TdR incorporation assay. Tumor cells were co-cultivated with granulocytes and mean values (7 SE per group) are given: (a) significance P o0.05 compared to values of the W256 cells alone or (b) significance Po 0.03 compared to values of the W256 cells co-cultivated with control rat’s granulocytes.
Granulocyte administration at the site of the growing tumor of EAT-bearing BALBc mice resulted in the increase of survival rate and decrease of the tumor growth (Figs. 4A and B). Depending on the granulocyte administration, the survival was prolonged from 13% for mice treated with granulocytes on the 6th day to almost 25% for mice treated repeatedly with granulocytes on days 1, 4 and 8 (p o0.05). Therefore, the best survival rate was found in mice that were treated with granulocytes three times (PMN 1,4,8 group). Furthermore, treatments with granulocytes reduced the tumor growth significantly (Fig. 4B), in particular in mice treated with granulocytes on days 1, 4 and 8 after tumor transplantation.
M. Jaganjac et al. / Immunobiology 215 (2010) 1015–1020
Fig. 4. (A) Survival curves of EAT tumor-bearing mice, according to Kaplan Meier method and (B) tumor volume growth dynamics (7 SE per group). Mice were administrated with PMN obtained from healthy donors or solely with physiological solution (control). PMN1 and PMN6 mice were administrated with PMN on a one-time basis on day 1 or 6, respectively. PMN 1,4,8 mice were treated with PMN three times (on days 1, 4 and 8).
Discussion Our results have shown that spontaneous W256 tumor regression in Sprague Dawley rats was abolished by Sephadex administration, which distracted functional granulocytes from the blood into the Sephadex papula. The restoration of the functional granulocytes obtained by granulocyte administration at the site of tumor, presented in this paper, showed significantly decreased tumor volume growth and improvement of the survival, causing tumor regression in most of the animals. We assume that autologous granulocytes would have lower effect on tumor growth than those collected from normal healthy donor. Namely, we found before that tumor transplantation causes granulocyte migration to the tumor site resulting in the reduced level of functionally active granulocytes circulating in the blood (Zivkovic et al., 2007). On the other hand, granulocytes that migrate to Sephadex papula have less pronounced inhibiting effect on tumor cell proliferation, when isolated on the 4th day after tumor transplantation, than those obtained from healthy animals (Zivkovic et al., 2005a). Since the aim of this study was to evaluate influence of Sephadex derived granulocytes as possible bioactive anticancer agent, we designed experiments accordingly. According to our previous research W256 regression is not followed by any tumor recurrence for more than two months (unpublished data) supporting consideration of granulocytes as effective anticancer bio(immuno)therapy. Earlier, we have reported that the high incidence of spontaneous regression of W256 in rats can be strongly reduced if the animals received Sephadex, which was further reflected in significant difference in survival of the animals (Jaganjac et al., 2008). This finding suggested that regression of the tumor was based on the innate immunity of granulocytes, which are removed from the blood of tumor nearing animals after injection of Sephadex. The results presented in the current paper confirmed this assumption.
1019
Although PMN rats had significantly increased number of granulocytes, present in the blood, indicating strong inflammatory reaction in hosts during W256 regression, the values of cytokines CINC-2, TNF-a and IL-6 in plasma did not differ from the values in control animals. Therefore, the observed regression of the tumors in PMN rats might be due to the direct anticancer activity of the functional granulocytes applied at the side of growing W256 tumors. Furthermore, PMN rat granulocytes showed a strong inhibitory effect of the W256 growth in vitro. In the previous study (Jaganjac et al., 2008) we have shown that granulocytes had the ability to detect the presence of W256 cells and to migrate to the tumor site causing a prominent inflammatory response, which resulted in the destruction of the tumor cells followed by the healing of the affected normal tissue. The results of the current study add further support to the hypothesis that granulocytes that affect tumor cells in vivo have a crucial role also in the inhibition of the tumor cell proliferation in vitro. Granulocytes isolated from the tumor-bearing animals showed a stronger inhibitory effect on tumor cell proliferation in vitro, indicating that granulocytes have the ability to recognize the type of tumor cells to which the body was earlier exposed. In agreement with the hypothesis proposed already 40 years ago (Klein, 1968), we have shown that granulocytes might have the ability to recognize tumor cells antigens, that enable them to migrate to the site of tumor, and consequently lead to tumor regression. In line with this hypothesis, granulocytes have shown anticancer effects by antibody-dependent cell-mediated cytotoxicity (Di Carlo et al., 2001). To further verify the involvement of granulocytes in tumor growth and survival of the animals, the present study included BALBc mice bearing another non immunogenic tumor. The EATbearing BALBc mice, treated with granulocytes in the vicinity of solid tumors at several time points, showed a significant decrease in tumor volume growth and prolonged survival depending on time course and the number of granulocyte administrations. Hence, these results show that multiple administration of granulocytes as bioactive agent at the site of tumor could be effective for the treatment of the solid form of the EAT tumor. In conclusion, granulocytes might play an important role in defence against cancer, manifested by the inflammatory response, causing oxidative stress at the site of tumor transplantation (Zivkovic et al., 2005a; Zivkovic et al., 2007; Jaganjac et al., 2008). The administration of granulocytes can lead to tumor regression or can slower tumor growth and extend the overall survival. The observed findings encourage further studies on the mechanisms and/or roles of granulocytes in tumor biology, as well as studies of the potential use of granulocytes in biological treatment of cancer.
Conflict of interest The authors have declared that no conflict of interest exists.
Acknowledgements The authors thank to Mrs. Nevenka Hirsl for the excellent technical assistance and to Prof. Dr. Nela Pivac for linguistic revision. The study was supported by the Croatian Ministry of Science, Education and Sports and by the COST B35 Action of the European Union.
1020
M. Jaganjac et al. / Immunobiology 215 (2010) 1015–1020
References Babior, B.M., 2000. Phagocytes and oxidative stress. Am. J. Med. 109, 33–44. ¨ Boyum, A., 1968. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1g. Scand. J. Clin. Lab. Invest. Suppl. 97, 77–89. Dallegri, F., Frumento, G., Ballestrero, A., Goretti, R., Patrone, F., 1987. Relationship between antibody-dependent tumour cell lysis and primary granule exocytosis by human neutrophils. Clin. Exp. Immunol. 70, 479–483. Dallegri, F., Ottonello, L., Ballestrero, A., Dapino, P., Ferrando, F., Patrone, F., Sacchetti, C., 1991. Tumor cell lysis by activated human neutrophils: analysis of neutrophil-delivered oxidative attack and role of leukocyte functionassociated antigen 1. Inflammation 15, 15–30. Dallegri, F., Patrone, F., Frumento, G., Sacchetti, C., 1984. Antibody-dependent killing of tumor cells by polymorphonuclear leukocytes. Involvement of oxidative and nonoxidative mechanisms. J. Natl. Cancer Inst. 73, 331–339. Di Carlo, E., Forni, G., Lollini, P., Colombo, M.P., Modesti, A., Musiani, P., 2001. The intriguing role of polymorphonuclear neutrophils in antitumor reactions. Blood 97, 339–345. Jaganjac, M., Poljak-Blazi, M., Zarkovic, K., Schaur, R.J., Zarkovic, N., 2008. The involvement of granulocytes in spontaneous regression of Walker 256 carcinoma. Cancer Lett. 260, 180–186. Katano, M., Torisu, M., 1982. Neutrophil-mediated tumor cell destruction in cancer ascites. Cancer 50, 62–68. Klein, G., 1968. Tumor-specific transplantation antigenes: G.H.A. Clowes memorial lecture. Cancer Res. 28, 625–635. Lichtenstein, A., Kahle, J., 1985. Anti-tumor effect of inflammatory neutrophils: characteristics of in vivo generation and in vitro tumor cell lysis. Int. J. Cancer 35, 121–127. Lichtenstein, A., Seelig, M., Berek, J., Zighelboim, J., 1989. Human neutrophilmediated lysis of ovarian cancer cells. Blood 74, 805–809. Niwa, Y., Kasama, T., Miyachi, Y., Kanoh, T., 1989. Neutrophil chemotaxis, phagocytosis and parameters of reactive oxygen species in human aging: cross-sectional and longitudinal studies. Life Sci. 44, 1655–1664.
Poljak-Blazi, M., Jaganjac, M., Mustapic, M., Pivac, N., Muck-Seler, D., 2009. Acute immunomodulatory effects of iron polyisomaltosate in rats. Immunobiology 214, 121–128. Reali, E., Guiliani, A.L., Spisani, S., Moretti, S., Gavioli, R., Masucci, G., Gambari, R., Traniello, S., 1994. Interferon-gamma enhances monoclonal antibody 17-1Adependent neutrophil cytotoxicity toward colorectal carcinoma cell line SW11–16. Clin. Immunol. Immunopathol. 71, 105–112. Sbarra, A.J., Karnovsky, M.L., 1959. The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. J. Biol. Chem. 234, 1355–1362. Stipancic, I., Zarkovic, N., 1997. The effect of trauma surgery on immune system function. Lijec. Vjesn. 119, 279–290. Valerius, T., Repp, R., de Wit, T.P., Berthold, S., Platzer, E., Kalden, J.R., Gramatzki, M., van de Winkel, J.G., 1993. Involvement of the high-affinity receptor for IgG (Fc gamma RI; CD64) in enhanced tumor cell cytotoxicity of neutrophils during granulocyte colony-stimulating factor therapy. Blood 82, 931–939. van Kessel, K.P., van Strijp, J.A., van Kats-Renaud, H.J., Miltenburg, L.A., Fluit, A.C., Verhoef, J., 1990. Uncoupling of oxidative and non-oxidative mechanisms in human granulocyte-mediated cytotoxicity: use of cytoplasts and cells from chronic granulomatous disease patient. J. Leukocyte Biol. 48, 359–366. Zarkovic, N., Zarkovic, K., Grainca, S., Kissel, D., Jurin, M., 1997. The Viscum album preparation Isorel inhibits the growth of melanoma B16F10 by influencing the tumour host relationship. Anticancer Drugs 8, S17–S22. Zivkovic, M., Poljak-Blazi, M., Egger, G., Borovic Sunjic, S., Schaur, R.J., Zarkovic, N., 2005a. Oxidative burst and anticancer activities of rat neutrophils. Biofactors 24, 305–312. Zivkovic, M., Poljak-Blazi, M., Zarkovic, K., Mihaljevic, D., Schaur, R.J., Zarkovic, N., 2007. Oxidative burst of neutrophils against melanoma B16-F10. Cancer Lett. 246, 100–108. Zivkovic, M., Zarkovic, K., Skrinjar, Lj., Waeg, G., Poljak-Blazi, M., Borovic, S., Schaur, R.J., Zarkovic, N., 2005b. A new method for detection of HNEhistidine conjugates in rat inflammatory cells. Croat. Chem. Acta 78, 91–98.
Contents lists available at ScienceDirect
Immunobiology journal homepage: www.elsevier.de/imbio
Granulocytes as effective anticancer agent in experimental solid tumor models Morana Jaganjac a,n, Marija Poljak-Blazi a, Iva Kirac b, Suzana Borovic a, Rudolf Joerg Schaur c, Neven Zarkovic a a
Department of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, HR-10002 Zagreb, Croatia Department of Surgery, University Hospital Sisters of Charity, Vinogradska 29, HR-10000 Zagreb, Croatia c Institute of Molecular Biosciences University of Graz, Schubertstrasse 1, A-8010 Graz, Austria b
a r t i c l e in fo
abstract
Article history: Received 4 November 2009 Received in revised form 15 January 2010 Accepted 18 January 2010
The aim of the study was to elucidate the effects of murine granulocytes on the growth of solid murine tumors when administrated in the vicinity of W256 carcinoma growing in Sprague Dawley rats, and in the vicinity of Ehrlich ascites tumor (EAT) growing in BALBc mice. The administration of granulocytes significantly improved the survival of W256-bearing rats, and increased the tumor regression incidence from 17% up to 75%. Rats with regressing tumors had 2.5 times increased levels of granulocytes in peripheral blood, which were also cytotoxic in vitro for W256 carcinoma cells. However, blood levels of cytokine-induced neutrophil chemoattractant-2, tumor necrosis factor a and interleukin 6 were similar between rats with regressing tumors and control healthy rats, suggesting that the observed regression of W256 carcinoma was caused by specific anticancer effects of the applied granulocytes. Anticancer effects of granulocytes were also found in BALBc mice bearing solid form of EAT, resulting in a 20% increase of survival in EAT-bearing mice. Therefore, the administration of granulocytes, isolated from healthy animals and applied at the site of solid tumors in rats and in mice, reduced experimental tumor growth, and extended the survival of tumor-bearing animals, while in some rats it even caused a W256 regression. & 2010 Elsevier GmbH. All rights reserved.
Keywords: Ehrlich ascites tumor Granulocytes Sephadex Tumor regression Walker 256 carcinoma
Introduction The role of granulocytes in immune response against cancer cells is not well understood. Several studies have reported cytotoxicity of granulocytes against tumor cells in vitro (Dallegri et al., 1984; Dallegri et al., 1987; Reali et al., 1994; Valerius et al., 1993) and in vivo (Katano and Torisu, 1982). The activation process of granulocytes is accompanied by the intense production of reactive oxygen species (ROS) (Babior, 2000; Sbarra and Karnovsky, 1959), and an extended release of destructive hydrolytic enzymes (Stipancic and Zarkovic, 1997; Niwa et al., 1989). It was demonstrated that the activated granulocytes cause unspecific lysis of tumor cells mediated by ROS (Dallegri et al., 1991; van Kessel et al., 1990; Lichtenstein et al., 1989), hence ROS
Abbreviations: CINC-2, Cytokine-induced neutrophil chemoattractant-2; EAT, Ehrlich ascites tumor; 3H-TdR, 3H-thymidine incorporation assay; IL-6, Interleukin 6; PMN, Polymorpho-nuclear granulocytes; ROS, Reactive oxygen species; TNF-a, Tumor necrosis factor a; W256, Walker 256 carcinoma n Corresponding author at: PhD, Research Associate; Laboratory for Oxidative Stress, Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, HR10002 Zagreb, Croatia. Tel.: + 385 1 457 1212; fax: + 385 1 456 1010. E-mail address: [email protected] (M. Jaganjac). 0171-2985/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2010.01.002
have been identified as effector molecules of the oxygendependent killing of cancer cells by granulocytes (Lichtenstein and Kahle, 1985; Lichtenstein et al., 1989). We have found that oxidative burst of granulocytes is cytotoxic for melanoma B16-F10 (Zivkovic et al., 2007) and for Walker 256 carcinoma (W256) (Zivkovic et al., 2005a). However, in spite of the data showing that granulocytes can participate in destruction of malignant cells, little is known about function of granulocytes in tumor-bearing hosts. In a previous study (Zivkovic et al., 2005a), we have found that functional granulocytes, obtained from the papula of normal, healthy rats injected subcutaneously (s.c.) with Sephadex, act cytotoxically against W256 tumor cells in vitro. Moreover, the granulocytes from the Sephadex papula of the tumor-bearing animals were less reactive in vitro than the granulocytes from the Sephadex papula of the normal healthy animals, indicating that the most potent granulocytes were already engaged in reaction against the W256 in vivo. In an earlier study using murine B16-F10 melanoma (Zivkovic et al., 2007), we have found that s.c. injection of Sephadex distracts the granulocytes from melanoma, allowing faster progression of the tumor, indicating that neutrophils may have an important role in the host defence against malignant cells in
1016
M. Jaganjac et al. / Immunobiology 215 (2010) 1015–1020
the early stage of tumor development. To test further this possibility, in another recently published study, we used the W256 carcinoma model grown on Sprague Dawley rats (Jaganjac et al., 2008). The results showed that innate immunity based on the immune competent granulocytes may be the cause of the well known phenomenon of spontaneous regression of W256 carcinoma in Sprague Dawley rats (Jaganjac et al., 2008). In the same study we have also shown that distraction of granulocytes from the tumor by s.c. Sephadex injection greatly decreased the incidence of W256 regression. Therefore, the aim of the current study was to elucidate the possible anticancer effects of granulocytes administration, isolated from healthy animals, in the vicinity of solid tumors in Sprague Dawley rats bearing W256 and additionally treated with Sephadex and in BALBc mice bearing Ehrlich ascites tumor (EAT).
(PMN group), while other 6 rats were left as a control tumor progressive animals (control group) and injected with sterile 200 mL of saline. Monitoring granulocyte effect in tumor-bearing rats The tumor volume and survival were monitored daily. Tumor growth was evaluated by the use of calliper measuring three diameters of the tumors formed at the site of inoculation. On the 18th day after W256 injection, blood was taken from the tail vein ¨ in EDTA-containing tubes. Leukocytes were counted in a Burker ¨ chamber using Turk’s solution. Blood smears were prepared and ¨ stained with May Grunwald-Giemsa for the differential counting of leukocytes. The remaining blood was used for plasma preparation, while three control healthy rats served as controls for the analyzes of the granulocyte counts and plasma cytokine values.
Materials and methods Animals Experiments were performed on male, three months old, Sprague Dawley rats and on male, three months old, BALBc mice. Water and food were given ad libitum. All the experiments were performed in accordance with the ILAR Guide for the Care and Use of Laboratory Animals, Council Directive (86/609/EEC) and the Croatian animal welfare law (NN 19/99). Effects of granulocytes in tumor-bearing rats Tumor regression experiments were carried out on 14 tumorbearing Sprague Dawley rats treated with Sephadex. Rats were intramuscularly injected in the right hind limb with 250 mL of RPMI medium containing 107 W256 tumor cells and additionally s.c. with 5 ml Sephadex (Sephadex G-150, Pharmacia Fine Chemicals, Sweden) on each side in the lower dorsal quadrant. Namely, Sephadex distracts functional granulocytes from the blood into the Sephadex papula thus abolishing spontaneous W256 tumor regression in Sprague Dawley rats (Jaganjac et al., 2008). The Sephadex was prepared as described elsewhere (Zivkovic et al., 2005a). On the 4th day after the tumor injection, the granulocytes were administered at the site of tumor, as a bio-therapeutic agent to 8 rats, while the remaining 6 rats served as saline treated control. Preparation of rat granulocytes As a source for granulocytes healthy control (without tumors) rats were used. On the 3rd day healthy control rats were s.c. injected in the abdomen area with Sephadex and 24 h later Sephadex papule were surgically removed. Rats were anesthetized using intraperitoneal injection of chloralhidrate (150 mg/ml). Operating field was prepared according to animal care standards. Skin incision was made above the site where Sephadex papule was formed following subcutaneous Sephadex injection. Underlying Sephadex was then removed with a spatula and skin was sawn with individual stitches. Sephadex papule containing granulocytes were placed into sterile 0,9% NaCl and filtered over filter paper that allows granulocytes to pass. Injection of granulocytes at the site of W256 tumor progression The viability of granulocyte cell suspensions was checked by the trypan blue exclusion test and was found to be Z93%. Isolated granulocytes were immediately re-suspended in sterile 200 mL of saline and injected into the site of tumor progression of 8 tumor-bearing Sephadex treated Sprague Dawley rats
Determination of CINC-2, TNF-a and IL-6 in rat plasma samples The plasma samples were isolated from the whole blood by centrifugation at 3500 rpm for 15 min at 4 1C as described previously (Poljak-Blazi et al., 2009). Plasma samples were then transferred to a 1.5 ml microcentrifuge tube, frozen at 801C, and subsequently used in Luminex analysis. A commercial rat 3-plex luminex kit (R&D Saystems, Minneapolis, USA) was used for determination of cytokine-induced neutrophil chemoattractant-2 (CINC-2), tumor necrosis factor a (TNF-a) and interleukin 6 (IL-6). The assay was performed and plasma samples were diluted 3-fold according to the manufacturer’s instructions. Briefly, the wells of the 1.2 mm filter membrane 96-well microtiter plates were prewetted with assay wash buffer. 50 ml of diluted microparticle mixture were added to each well followed by adding 50 ml of plasma sample, standard and quality control preparations to the relevant wells and incubated for 3 h on a horizontal orbital shaker at room temperature. The plates were washed three times with assay wash buffer and 50 ml of biotinylated detector antibody added per well. Samples were incubated for 1 h at room temperature on the plate shaker. The plates were washed three times with assay wash buffer and 50 ml per well of streptavidin– phycoerythrin solution were added, and plates incubated for further 30 min at room temperature on a plate shaker, protected from direct light. After completion of staining, the microbeads were washed three times with assay wash buffer and re-suspended in 100 ml/well of assay wash buffer. The luminex assay was performed on a luminex-200TM instrument using AtheNA Analyzer LX200 (Full) 2.3 Software. Hundred events per region were collected and median fluorescence intensity measured. Median fluorescence intensity was converted to concentrations using results from a standard cytokine preparation. Effects of rat granulocytes in vitro Rats were anesthetized using intraperitoneal injection of chloralhydrate (150 mg/ml) and sacrificed by bleeding from the heart (Zivkovic et al., 2005b) under ether anaesthesia on the 18th day after the Sephadex treatment and tumor transplantation. Influence of whole blood granulocytes and granulocytes derived from Sephadex papula on the proliferation of W256 cells was measured in vitro by the 3H-thymidine (3H-TdR) incorporation assay as described before (Zarkovic et al., 1997). Granulocytes were obtained from the Sephadex papula by a method preserving their biological activity as described above and from whole blood using dextran sedimentation followed by hypotonic lysis and centrifugation on a Ficoll-Paque gradient as described elsewhere ¨ (Boyum, 1968). Granulocytes from PMN rats with regressing tumors were analyzed and compared to those obtained from control healthy animals. Briefly, the seeding density of
M. Jaganjac et al. / Immunobiology 215 (2010) 1015–1020
1017
granulocytes cultured in 96-well microcytoplates was 2 105 cells per culture in RPMI 1640 medium with 10% FCS. The seeding density of the tumor cells was 2 104 cells per culture irrespective if cultured alone or if added to the granulocytes. The cell cultures were incubated for 24 h at 37 1C in a humidified air atmosphere with 5% CO2. After 24 h, radioactive 3H-TdR was added to each culture and left for additional 48 h. The incorporation of 3H-TdR, determined with a b-liquid-scintillation counter (Beckman, LS 3800 Series), was used as a parameter of tumor cell proliferation (DNA synthesis). Effects of granulocytes in tumor-bearing mice To study the outcome of granulocyte administration in tumor progressive animals, 33 mice were intramuscularly injected in the right hind limb with 100 mL of Hanks balanced salt solution containing 106 EAT cells. Afterwards, 20 mice were administrated with granulocytes, while the remaining 13 mice served as saline treated control. Preparation and injection of mice granulocytes at the site of EAT tumor progression As a source for granulocytes healthy BALBc mice were used. They were treated with Sephadex and 24 h later the papula content was surgically removed and granulocytes isolated as described above. Isolated granulocytes were immediately re-suspended in sterile 100 mL saline and injected into the site of tumor progression at different time points, while control tumor progressive animals were injected solely with sterile 100 mL saline. Fifteen mice received granulocytes on a one-time basis as follows; PMN1 group, consisting of 5 mice, was administrated on the 1st day while the other 10 mice, group PMN6, were administrated with granulocytes on the 6th day. Furthermore, 5 mice (group PMN 1,4,8) were treated with granulocytes three times (on days 1, 4 and 8). The rest of mice (n= 13) were left as a control tumor progressive animals (control group). Monitoring granulocyte effect in tumor-bearing mice Tumor volume growth and survival were monitored. Tumor growth was evaluated by the use of calliper measuring three times a week the three diameters of the tumors formed at the site of inoculation. Mice mortality was recorded daily. Statistics The incidence of tumor regression was evaluated by the Chisquare test, survival rates of tumor-bearing rats were evaluated by the Kaplan Meier test, while the other parameters were analyzed by the two-tailed Mann-Whitney test. The MedCalc software and the SPSS 11.01 for Mircosoft Windows were used. The values of p o0.05 were considered as significant.
Fig. 1. (A) Survival curves of W256 tumor-bearing Sephadex treated rats, according to Kaplan Meier method, (B) W256 tumor volume growth dynamics in control group or PMN rats (7 SE per group) and (C) changes in W256 tumor volume during tumor progression or regression for all animals regardless of granulocyte treatment (7SE per group). On the 4th day rats were treated with PMN obtained from healthy donors (PMN) or with physiological solution (control).
rats (increase from 17% up to 75%). Moreover, as is presented in Fig. 1B, in PMN rats tumor growth was significantly slower (p o0.05 from day 7 further) than in control rats where tumor growth was more aggressive. PMN rats had 2.5 times more granulocytes (p o0.05) present in the blood (Fig. 2) than control healthy rats, however, the levels of CINC-2, TNF-a and IL-6 in plasma were not significantly (p 40.05, Mann-Whitney test) different from the cytokine levels in control healthy rats (Fig. 2).
Results Administration of granulocytes in the vicinity of solid tumors of Sprague Dawley rats Survival rate and tumor volume growth dynamics of tumor-bearing Sephadex treated Sprague Dawley rats are presented in Fig. 1. Administration of granulocytes obtained from subcutaneously formed Sephadex papula of healthy rats to the tumor-bearing Sephadex-treated rats (shown as PMN rats) on the fourth day after tumor transplantation significantly (p o0.01) improved the survival (Fig. 1A), causing tumor regression in 6/8
In vitro anti-tumor effects of granulocytes Anti-tumor activity of the PMN rat papula and whole blood granulocytes against W256 cells was further verified in vitro by the 3H-TdR incorporation assay (Fig. 3). A 50% inhibition (p o0.001) of the W256 growth in vitro was observed in the presence of the granulocytes obtained from papula of PMN rats. Peripheral blood granulocytes obtained from PMN rats also significantly reduced W256 growth for 37% (p o0.005), while granulocytes isolated from blood of healthy rats showed mild
1018
M. Jaganjac et al. / Immunobiology 215 (2010) 1015–1020
Fig. 2. Distribution of neutrophils in the peripheral blood of Sprague Dawley rats and the values of cytokines CINC-2, TNF-a and IL-6 in plasma determined on the day 18 (mean 7SE per group): (a) significance P o0.05 in comparison to healthy control.
(17%), but significant inhibitory effect (po0.05) on W256 tumor cell growth in vitro.
Administration of granulocytes in the vicinity of solid tumors of BALBc mice
Fig. 3. Proliferation of W256 tumor cells in vitro measured by 3H-TdR incorporation assay. Tumor cells were co-cultivated with granulocytes and mean values (7 SE per group) are given: (a) significance P o0.05 compared to values of the W256 cells alone or (b) significance Po 0.03 compared to values of the W256 cells co-cultivated with control rat’s granulocytes.
Granulocyte administration at the site of the growing tumor of EAT-bearing BALBc mice resulted in the increase of survival rate and decrease of the tumor growth (Figs. 4A and B). Depending on the granulocyte administration, the survival was prolonged from 13% for mice treated with granulocytes on the 6th day to almost 25% for mice treated repeatedly with granulocytes on days 1, 4 and 8 (p o0.05). Therefore, the best survival rate was found in mice that were treated with granulocytes three times (PMN 1,4,8 group). Furthermore, treatments with granulocytes reduced the tumor growth significantly (Fig. 4B), in particular in mice treated with granulocytes on days 1, 4 and 8 after tumor transplantation.
M. Jaganjac et al. / Immunobiology 215 (2010) 1015–1020
Fig. 4. (A) Survival curves of EAT tumor-bearing mice, according to Kaplan Meier method and (B) tumor volume growth dynamics (7 SE per group). Mice were administrated with PMN obtained from healthy donors or solely with physiological solution (control). PMN1 and PMN6 mice were administrated with PMN on a one-time basis on day 1 or 6, respectively. PMN 1,4,8 mice were treated with PMN three times (on days 1, 4 and 8).
Discussion Our results have shown that spontaneous W256 tumor regression in Sprague Dawley rats was abolished by Sephadex administration, which distracted functional granulocytes from the blood into the Sephadex papula. The restoration of the functional granulocytes obtained by granulocyte administration at the site of tumor, presented in this paper, showed significantly decreased tumor volume growth and improvement of the survival, causing tumor regression in most of the animals. We assume that autologous granulocytes would have lower effect on tumor growth than those collected from normal healthy donor. Namely, we found before that tumor transplantation causes granulocyte migration to the tumor site resulting in the reduced level of functionally active granulocytes circulating in the blood (Zivkovic et al., 2007). On the other hand, granulocytes that migrate to Sephadex papula have less pronounced inhibiting effect on tumor cell proliferation, when isolated on the 4th day after tumor transplantation, than those obtained from healthy animals (Zivkovic et al., 2005a). Since the aim of this study was to evaluate influence of Sephadex derived granulocytes as possible bioactive anticancer agent, we designed experiments accordingly. According to our previous research W256 regression is not followed by any tumor recurrence for more than two months (unpublished data) supporting consideration of granulocytes as effective anticancer bio(immuno)therapy. Earlier, we have reported that the high incidence of spontaneous regression of W256 in rats can be strongly reduced if the animals received Sephadex, which was further reflected in significant difference in survival of the animals (Jaganjac et al., 2008). This finding suggested that regression of the tumor was based on the innate immunity of granulocytes, which are removed from the blood of tumor nearing animals after injection of Sephadex. The results presented in the current paper confirmed this assumption.
1019
Although PMN rats had significantly increased number of granulocytes, present in the blood, indicating strong inflammatory reaction in hosts during W256 regression, the values of cytokines CINC-2, TNF-a and IL-6 in plasma did not differ from the values in control animals. Therefore, the observed regression of the tumors in PMN rats might be due to the direct anticancer activity of the functional granulocytes applied at the side of growing W256 tumors. Furthermore, PMN rat granulocytes showed a strong inhibitory effect of the W256 growth in vitro. In the previous study (Jaganjac et al., 2008) we have shown that granulocytes had the ability to detect the presence of W256 cells and to migrate to the tumor site causing a prominent inflammatory response, which resulted in the destruction of the tumor cells followed by the healing of the affected normal tissue. The results of the current study add further support to the hypothesis that granulocytes that affect tumor cells in vivo have a crucial role also in the inhibition of the tumor cell proliferation in vitro. Granulocytes isolated from the tumor-bearing animals showed a stronger inhibitory effect on tumor cell proliferation in vitro, indicating that granulocytes have the ability to recognize the type of tumor cells to which the body was earlier exposed. In agreement with the hypothesis proposed already 40 years ago (Klein, 1968), we have shown that granulocytes might have the ability to recognize tumor cells antigens, that enable them to migrate to the site of tumor, and consequently lead to tumor regression. In line with this hypothesis, granulocytes have shown anticancer effects by antibody-dependent cell-mediated cytotoxicity (Di Carlo et al., 2001). To further verify the involvement of granulocytes in tumor growth and survival of the animals, the present study included BALBc mice bearing another non immunogenic tumor. The EATbearing BALBc mice, treated with granulocytes in the vicinity of solid tumors at several time points, showed a significant decrease in tumor volume growth and prolonged survival depending on time course and the number of granulocyte administrations. Hence, these results show that multiple administration of granulocytes as bioactive agent at the site of tumor could be effective for the treatment of the solid form of the EAT tumor. In conclusion, granulocytes might play an important role in defence against cancer, manifested by the inflammatory response, causing oxidative stress at the site of tumor transplantation (Zivkovic et al., 2005a; Zivkovic et al., 2007; Jaganjac et al., 2008). The administration of granulocytes can lead to tumor regression or can slower tumor growth and extend the overall survival. The observed findings encourage further studies on the mechanisms and/or roles of granulocytes in tumor biology, as well as studies of the potential use of granulocytes in biological treatment of cancer.
Conflict of interest The authors have declared that no conflict of interest exists.
Acknowledgements The authors thank to Mrs. Nevenka Hirsl for the excellent technical assistance and to Prof. Dr. Nela Pivac for linguistic revision. The study was supported by the Croatian Ministry of Science, Education and Sports and by the COST B35 Action of the European Union.
1020
M. Jaganjac et al. / Immunobiology 215 (2010) 1015–1020
References Babior, B.M., 2000. Phagocytes and oxidative stress. Am. J. Med. 109, 33–44. ¨ Boyum, A., 1968. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1g. Scand. J. Clin. Lab. Invest. Suppl. 97, 77–89. Dallegri, F., Frumento, G., Ballestrero, A., Goretti, R., Patrone, F., 1987. Relationship between antibody-dependent tumour cell lysis and primary granule exocytosis by human neutrophils. Clin. Exp. Immunol. 70, 479–483. Dallegri, F., Ottonello, L., Ballestrero, A., Dapino, P., Ferrando, F., Patrone, F., Sacchetti, C., 1991. Tumor cell lysis by activated human neutrophils: analysis of neutrophil-delivered oxidative attack and role of leukocyte functionassociated antigen 1. Inflammation 15, 15–30. Dallegri, F., Patrone, F., Frumento, G., Sacchetti, C., 1984. Antibody-dependent killing of tumor cells by polymorphonuclear leukocytes. Involvement of oxidative and nonoxidative mechanisms. J. Natl. Cancer Inst. 73, 331–339. Di Carlo, E., Forni, G., Lollini, P., Colombo, M.P., Modesti, A., Musiani, P., 2001. The intriguing role of polymorphonuclear neutrophils in antitumor reactions. Blood 97, 339–345. Jaganjac, M., Poljak-Blazi, M., Zarkovic, K., Schaur, R.J., Zarkovic, N., 2008. The involvement of granulocytes in spontaneous regression of Walker 256 carcinoma. Cancer Lett. 260, 180–186. Katano, M., Torisu, M., 1982. Neutrophil-mediated tumor cell destruction in cancer ascites. Cancer 50, 62–68. Klein, G., 1968. Tumor-specific transplantation antigenes: G.H.A. Clowes memorial lecture. Cancer Res. 28, 625–635. Lichtenstein, A., Kahle, J., 1985. Anti-tumor effect of inflammatory neutrophils: characteristics of in vivo generation and in vitro tumor cell lysis. Int. J. Cancer 35, 121–127. Lichtenstein, A., Seelig, M., Berek, J., Zighelboim, J., 1989. Human neutrophilmediated lysis of ovarian cancer cells. Blood 74, 805–809. Niwa, Y., Kasama, T., Miyachi, Y., Kanoh, T., 1989. Neutrophil chemotaxis, phagocytosis and parameters of reactive oxygen species in human aging: cross-sectional and longitudinal studies. Life Sci. 44, 1655–1664.
Poljak-Blazi, M., Jaganjac, M., Mustapic, M., Pivac, N., Muck-Seler, D., 2009. Acute immunomodulatory effects of iron polyisomaltosate in rats. Immunobiology 214, 121–128. Reali, E., Guiliani, A.L., Spisani, S., Moretti, S., Gavioli, R., Masucci, G., Gambari, R., Traniello, S., 1994. Interferon-gamma enhances monoclonal antibody 17-1Adependent neutrophil cytotoxicity toward colorectal carcinoma cell line SW11–16. Clin. Immunol. Immunopathol. 71, 105–112. Sbarra, A.J., Karnovsky, M.L., 1959. The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. J. Biol. Chem. 234, 1355–1362. Stipancic, I., Zarkovic, N., 1997. The effect of trauma surgery on immune system function. Lijec. Vjesn. 119, 279–290. Valerius, T., Repp, R., de Wit, T.P., Berthold, S., Platzer, E., Kalden, J.R., Gramatzki, M., van de Winkel, J.G., 1993. Involvement of the high-affinity receptor for IgG (Fc gamma RI; CD64) in enhanced tumor cell cytotoxicity of neutrophils during granulocyte colony-stimulating factor therapy. Blood 82, 931–939. van Kessel, K.P., van Strijp, J.A., van Kats-Renaud, H.J., Miltenburg, L.A., Fluit, A.C., Verhoef, J., 1990. Uncoupling of oxidative and non-oxidative mechanisms in human granulocyte-mediated cytotoxicity: use of cytoplasts and cells from chronic granulomatous disease patient. J. Leukocyte Biol. 48, 359–366. Zarkovic, N., Zarkovic, K., Grainca, S., Kissel, D., Jurin, M., 1997. The Viscum album preparation Isorel inhibits the growth of melanoma B16F10 by influencing the tumour host relationship. Anticancer Drugs 8, S17–S22. Zivkovic, M., Poljak-Blazi, M., Egger, G., Borovic Sunjic, S., Schaur, R.J., Zarkovic, N., 2005a. Oxidative burst and anticancer activities of rat neutrophils. Biofactors 24, 305–312. Zivkovic, M., Poljak-Blazi, M., Zarkovic, K., Mihaljevic, D., Schaur, R.J., Zarkovic, N., 2007. Oxidative burst of neutrophils against melanoma B16-F10. Cancer Lett. 246, 100–108. Zivkovic, M., Zarkovic, K., Skrinjar, Lj., Waeg, G., Poljak-Blazi, M., Borovic, S., Schaur, R.J., Zarkovic, N., 2005b. A new method for detection of HNEhistidine conjugates in rat inflammatory cells. Croat. Chem. Acta 78, 91–98.