Combination therapy of Active Hexose Correlated Compound Plus UFT Significantly Reduces the Metastasis of Rat Mammary Adenocarcinoma

Kazuhiro Matsushita, Yasuhiro Kuramitsu, Youichi Ohiro, Manabu Obara, Masanobu Kobayashi, Yong-Qing Li and Masuo Hosokawa

Laboratory of Pathology, Cancer Institute, Hokkaido University School of Medicine, Kita-15 Nishi-7 Kita-ku, Sapporo 060. Japan. Tel: (+81) 11 706 5070; Fax: (+81) 11 709 6468.

Anti-Cancer Drugs 1998, 9, pp. 343-350

Abstract

Synergistic effects of active hexose correlated compound (AHCC) extracted from mushroom on the treatment with UFT against mammary adenocarcinoma, SST-2 cells, In congenitally T cell-depressed spontaneously hypertensive rats (SHR) were observed. AHCC plus UFT had slighted but significant effects on the growth of primary tumors. Pulmonary metastases were not inhibited by the treatment with AHCC plus UFT, whereas metastases to axillay lymph nodes (LN) were obviously inhibited. Combination of AHCC plus UFT showed similar synergistic anti-metastatic effects in SHR rats with accelerated pulmonary metastases following the surgical removal of the primary tumors, in vitro studies demonstrated that AHCC plus UFT enhanced the NK cell activity In tumor-bearing rats, whereas UFT alone depressed the NK cell activity. AHCC plus UFT also enhanced the NO production and cytotoxicity of peritoneal macrophages. In addition, AHCC re-stored the suppressed mRNA expression of interleukin-la and tumor necrosis factor-c induced by the chemotherapy. Taken together, the combination of AHCC plus UFT brought about good therapeutic effects not only on primary tumor growth but also on reducing metastasis and these effects were mediated by host immunity which was restored or activated by AHCC. AHCC may be a good candidate for a biological response modifier. (© 1998 Lippincott-Raven Publishers.)

Introduction

Tumor cells invade from the primary lesion into the blood and/or lymphatic circulation resulting in the formation of metastases in distant organs as well as in the regional lymph nodes (LN).1'2 This property of the tumor cells is mainly responsible for death in cancer, despite advances in cancer treatment. Therefore, treatment of metastases is one of the major targets in cancer treatment. Previous studies demonstrated that chemotherapy with UFT (tegafur and uracil in a 4:1 molar concentration) had significant therapeutic effects on advanced cancers with distant metastases.^3-5 However, therapeutic doses of anti-cancer drugs have been reported to reduce the anti-tumor immune response ^6,7 which inhibits the micro-metastases.^8-10 Therefore, various biological response modifiers (BRMs) have been used with anti-cancer drugs to restore the anti-tumor immune response reduced by chemotherapy.^11-15 Using an in vivo model system, we have reported that UFT plus lentinan decreased the number of pulmonary metastasis of rat mammary adenocarcinoma (SST-2) cells in congenitally T cell-depressed spontaneously hypertensive rats (SHR).^16,17 Active hexose correlated compound (AHCC) is an extract obtained from several kinds of mushrooms which are cultured with liquid medium. The active component is an oligosaccharide and its molecular weight is about 5000 although it contains various kinds of components. Interestingly, as opposed to conventional active components which consist of a b-1, 3-glucan structure as in PSK and lentinan, the oligomer of glucose in AHCC has a-l, 4-linkage structures and some esterized hydroxy groups. We hypothesized that AHCC may function as a BRM in the same way that PSK and lentinan do in the chemotherapy of cancer.

In this study, we examined the effects of AHCC on the growth and metastasis of SST-2 cells in SHR rats in combination with UFT. We also examined the effects of AHCC plus UFT on the metastasis of SST-2 cells after excision of primary tumors. SST-2 is a highly metascatic cell line, which produces 100% pulmonary metastasis in syngeneic rats after s.c. transplantation. As SHR rats have a dysfunction of T cells but rather enhanced macrophage and NK cell activities compared with other strains of rats, ^18-20 SST-2-bearing SHR rats were useful for examining the efficacy of anti-metastatic activities in various agents which function through the activation of macrophages and NK cells.

Materials and methods

Rats

Congenitally T cell-depressed SHR rats were purchased from Nippon Rat (Urawa, Japan). Throughout the experiments, 10 to 12-week-old female rats were used.

Tumors

We used a transplantable adenocarcinoma SST-2 that had originated from a spontaneous mammary adenocarcinoma in a SHR rat. The cells were maintained as a monolayer culture in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS;Gibco/BRL, Gaithersburg, MD). The mean survival time of SST-2 tumor-bearing rats was about 50 days after tumor inoculation and the main cause of death was extensive metastasis to lung (our unpublished data). YAC-1 and SST-2 cells were used as target cells for NK cells and macrophages, respectively. The cells were also maintained in RPMI 1640 medium supplemented with 10% FBS.

Treatment of rats

SHR rats were inoculated s.c. with cultured SST-2 cells (1 x lO6/rat) on day 0. In this experiment, we did not set up the AHCC alone treated group, because our previous data (data not shown) indicated that AHCC alone had little effect on tumor growth and tumor treatment. Administration per os (p.o.) of UFT (15 mg/ kg) (Taiho Pharmaceutical, Tokyo, Japan) was carried out every day from day 3 to 38. AHCC, an extract of several kinds of mushrooms, was supplied by Amino Up Chemical (Sapporo, Japan). According to our preliminary experiments, a rat drinks about 24 ml of water per day; we dissolved AHCC in water at the appropriate concentration of 100 mg/kg/day in 24 ml and let them drink freely from day 3 to 38. Water was administered p.o. to control rats. On day 38, rats were sacrificed, and the tumor weight and metastasis to axillary LN and lung were examined. The tumor size was measured every 3-4 days and the average diameter was estimated by the formula: (length + width)/2.
To examine the death as a result of metastasis to lung, tumor excision was performed on day 21 and all the rats were kept under observation until they died. UFT and AHCC were administered p.o. every day from day 3 to 38.

NK cell activity of spleen cells

Spleens were aseptically removed from rats on day 21, finely minced with scissors and gently teased in a loose-fitting glass homogenizer in RPM1 1640 medium. These cell suspensions were passed through four layers of gauze and washed twice in RPMI 1640 medium by centrifugation at 500 g for 5 min. The NK cell activity of the spleen cells was determined by a 4 h51 Cr-release assay. Target cells of YAC-1 were pre-labeled for 1 h at 37°C with 100 mCi Na51CrO4, washed three times with RPMI 1640 medium and resuspended in RPMI 1640 medium supplemented with 10% FBS. 51Cr-labeled target cells (1 x 104) were incubated with various concentrations of effector cells in triplicate (tested at E: T ratios of 200:1-50:1) in 96-well-round bottomed microtiter plates (Coming Glass Works, Corning, NY) for 4 h at 37°C in a 5% CO; incubator. The radioactivity of the supernatants was counted in a Aloka g-countcr o (ARC-380). The percentage cytocoxicity was calculated by the following formula: % specific releases=[(experi-mental release-spontaneous release)/(maximum re-lease-spontaneous release)] x 100. Spontaneous release was less than 20%. All assays were performed in triplicate. The SD of triplicate wells was consistently less than 10% of the mean.

Activation of peritoneal macrophages

Rats were i.p. administered with 1 Klinische Einheit (KE) of OK-432 (Chugai Pharmaceutical, Tokyo, Japan) in 1 ml phosphate-buffered saline (PBS) for 48 h before sacrifice. The OK-432-activated peritoneal macrophages were harvested with 20 ml PBS from each group of tumor-bearing rats and prepared for further use. Flow cytometric analysis of cylotoxicity of peritoneal macrophages cytotoxicity of peritoneal macrophages on day 21 was examined against syngeneic rat adenocarcinoma SST-2 target-cells by the method as described by Slezak21 with some modifications. Briefly, 1 x 10⁶ SST-2 cells were stained with 4xl0^-5 M PKH-26 (Zynaxis, Malvern, PA) for 2 min. The PKH-26-stained-target cells mixed with freshly prepared effector cells at E: T ratios of 100:1 in 12-well culture dishes (Coming Glass Works) were cultured for 16h at 37°C, and then the cells were detached and harvested. After washing with cold PBS twice, the cells were suspended in 1 ml cold PBS and stained with 5 m1 propidium iodide (PI, 50 mg/ ml) (Sigma, St Louis, MO) for 30 min on ice. The PKH-26 positive and PI negative cells (PKHA⁺ PI^- cells) were identified as living target cells and the PKH-26 positive and PI positive cells (PKH+ PI- cells) were identified as dead target cells; only PKH-26 positive cells including PKH⁺ PI^- cells and PKH⁺ PI^- cells were gated; the PKH-26 negative cells were identified as effector cells which were gated out. All the cells were analyzed by flow cytometric analysis (FACScan, Lysys system; Becton Dickinson, Mountain View, CA). Cytotoxicity were calculated by the following formula: Cytocoxiciry (%)=PKH⁺ PI^- cells/OPKH+ PI-cells + PKH⁺ PI^- cells) x 100.

Quantification of nitrite

OK-432-activatcd macrophages (1 x 107) were mixed with SST-2 tumor cells at E: T ratios of 100:1, 50:1 and 25:1 in 12-well culture dishes for 16 h at 37°C. Since secreted NO quickly reacts with oxygen yielding nitrite, nitrite levels in culture supernatants were measured by using the Griess reagent. Briefly, 100 ?1 sample aliquots were mixed with an equal volume of Griess reagent (1% sulfanilamine/0.1% naphthylethy-lene diamine dihydrochloride/2% H3PO4; Sigma) and incubated at room temperature for 10 min. The absorbance at 550 nm was measured in a microplate reader (Corona Electric, Katsuta, Japan). Nitrite concentration was determined using sodium nitrite as a standard and RPMI 1640 medium supplemented with 10% FBS as a blank. Reverse transcription-polymerase chain reaction (RT-PCR) total RNAs were extracted from peritoneal macro-phages on day 21 by the guanidine thiocyanate:phe-nol:chloroform method. Three micrograms of each RNA sample underwent cDNA synthesis in 50 ?l of reaction mixture containing 75 mM KC1, 50 mM Tris-HC1, pH 8.3, 3 mM MgCl2, 10 mM dithiothreiotol. 0.5 mM per each dNTP, 2 ?g/ml random primer and 1000 U M-MLV reverse transcriptase (Gibco/BRL) by incubation at 37°C for 1 h. PCR amplification of cDNA (5 m1) was performed in 50 ml of a reaction mixture containing 50 mM KCI, 10 mM Tris-HCl, pH 9.0, 2.5 mM MgCl2, 0.1% Triton X-100, 200 mM each dNTP, 10 mM per each specific primer and 1 U Taq-polymcrase (Promega, Madison, WI). The following primers were used: GAPDH forward primer, 5'-AC-CACCATGGAGAAGGCTGC-3'; reverse primer, 5'-CT-CAGTGTAGCCCAGGATGC-3'; iNOs forward primer, 5'-AGAGTCCTTCATGAAGCACA-3'; reverse primer, 5'. CCAAATACCGCATACCTGAA-3'; tumor necrosis factor (TNF><a forward primer, 5'-CAAGGAGGAGAAGTTCC. CAA-3'; reverse primer, 5'-CGGACTCCGTGATGTC-TAAG-3'; interleukin (IL)-la forward primer, 5'-GTGGTGGTGTCAGCAACTTC-3'; reverse primer, 5'-CCTTCAGCAACACAGGCTTG-3'.

In principle, the primer sequences were chosen from separate DNA exons of the gene. Expected sizes of amplified DNA fragments were 500, 577, 500 and 412 bp for GAPDH. iNOs, TNF-a and IL-la, respectively. The reactions were run for 35 cycles using a thermal cycler as follows: 1 min at 94°C, 1 min at 60°C and 2 min at 72°C. The 9, ul of each PCR sample was mixed with 1 ml of the sample buffer, electrophoresed through 2% agarose gel and stained with ethidium bromide.

Statistical analysis

Arithmetic means were calculated for each experiment group and tested for statistically significant differences by Student's t-test. Survival curves were analyzed by the generalized Wilcoxon test. Survival ratios were analyzed by contingency table analysis.

Results

Effects of AHCC plus LIFT on the s.c. growth and metastases of SST-2. The difference of tumor sizes between three groups was gradually increased, and, finally, on day 38, there was a significant difference of tumor sizes between the control group and AHCC plus UFT treated group (p < 0.01). The average tumor sizes of the control group and AHCC plus UFT treated group were 52.5±4.23 and 43.9±2.95 mm, respectively. Tumor weights of the control group and AHCC plus UFT treated group were 57.6±4.53 and 35.1±8.95g, respectively. Metastases to axillary LN were inhibited by the treatment with AHCC plus UFT, although the inhibitory effects of AHCC plus UFT on pulmonary metastases were not clear. UFT alone had no effect on lung and axillary LN metastasis or, rather, it enhanced the metastasis

Effects on micro-metastasis to lung after excision of primary tumors
As described above, LIFT plus AHCC decreases the number of metastases. Because the metastases to lung days after tumor implantation would be more accelerated after tumor excision in SST-2 in SHR rats model.^{6, 7} We next investigated whether UFT plus AHCC had inhibitory effects on micro-metastasis after excision of primary tumors. There was no difference in tumor weights and sizes at the period of tumor excision on day 21, but mean survival time of the AHCC plus UFT treated group was significantly prolonged compared with that of the control group and UFT treated group. Three out of five rats survived, suggesting that pulmonary metastases were completely inhibited in these rats.

Figure 3. Growth curves of SST-2 tumors in SHR rats. SST-2 tumors were implanted s.c. on day 0. UFT (15 mg/kg) was administered p.o. every day from day 3 to 38. AHCC (100 mg/kg/day in 24 ml) was also administered from day 3 to 38. Each group consisted of five rats. Each point presents mean±SD of average tumor diameter. Statistically significant (r<0.05).

Restoration of NK cell activity by AHCC

UFT alone showed a slight but not significant effect on the survival of tumor-bearing rats and metastasis of SST-2 cells. These findings suggest that UFT may have negative effects such as inhibition of host immune response other than direct cytotoxic effects on SST-2 cells. Therefore, we investigated the NK cell activity and cytotoxicity and NO production of peritoneal macrophages in these tumor-bearing rats with or without treatment on day 21. NK cell activity was certainly depressed when UFT alone was administered. However, administration of AHCC in combination with UFT restored the NK cell activity to the comparable level of the control group.

Enhancement of NO production and cytotoxicity of peritoneal macrophages by AHCC

NO production during the effector phase of peritoneal macrophages obtained from AHCC plus UFT treated rats on day 21 was drastically enhanced at every E: T ratio. Cytotoxic activity of peritoneal macrophages was also enhanced by AHCC plus UFT, but only slightly compared with the enhancement of NO production.

Restoration of mRNA expression by AHCC

We next investigated mRNA expression in peritoneal macrophages on day 21. mRNA expression of iNOs was drastically increased by AHCC administration.mRNA expression of TNF-a, which was depressed by UFT, was restored by AHCC in combination with UFT to the level observed in the control group. Almost the same results were observed for mRNA expression of IL-la. GAPDH mRNA expression was the same in all experimental groups.

Discussion

Synergistic effects of AHCC on the treatment with UFT against mammary adenocarcinoma, SST-2 cells, in congenitally T cell-depressed SHR rats were observed. As far as the tumor growth on day 38 after tumor implantation was concerned, there was a significant difference in tumor sizes between the AHCC plus UFT treated and control groups, although not significant between the UFT alone treated and control groups. Although lung mecastases were not inhibited by the treatment with AHCC plus UFT, metastases to axillay LN were obviously inhibited. Similar inhibitory effects of the combination of AHCC and UFT were observed in SHR rats with accelerated pulmonary metastases following the surgical removal of the primary tumor and three out of five rats survived. These results suggest that AHCC has obvious anti-metastatic activ-ities and that it can be used for the treatment of patients with advanced cancer.

In vitro experiments showed that administration of AHCC in combination with UFT restored NK activities depressed by UFT and stimulated peritoneal macro-phage cytotoxicities, NO production and cytokine production. It has been reported that both NK cells and macrophages were involved in the inhibition of tumor metastasis, when they were activated by BRMS.22-28 Gallo-Hendrikx et al. have reported that the metastasis of human pancreatic tumor cells implanted in SCID mice was minimal, whereas the metastasis was found in more than 90% of mice carrying the beige mutation.²⁹ This result suggests that NK cells and macrophages may. be more important than T and B cells in the anti-metastatic host immune system, because the beige mutation resulted in the deficient function of macrophages and NK cells. In our models, UFT alone had no effect on the growth of primary tumors but showed slight prolongation of survival of mice after removal of primary tumors. Because UFT reduced NK activities but stimulated peritoneal macrophage function, UFI' may affect the growth of tumor cells indirectly at the metastatic sites by activating macrophages. The combination of UFT and AHCC significantly improved the prognosis of mice after excision of primary tumors. In vitro experiments showed that AHCC activated the NK cell activity in addition to the activation of macrophages. All together, AHCC brought about therapeutic effects in combination with UFT on primary tumor growth and metastasis, followed by the activation of NK cells and macrophages.

Conclusion

AHCC significantly reduced the metastasis of rot mammary adenocarcinoma in combination with UFT. This effect was mediated by natural host immunity, which was restored or activated by AHCC. AHCC could be used as a biological response modifier like PSK and lentinan for the treatment of advanced cancer.

Acknowledgments

We thank Ms. M Yanome for preparing the manuscript.

Figure 1: Protocol for observing the effect on growth of the primary tumor and metastasis. Rats were divided into three groups. Group 1: rats were administered UFT (15 mg/kg) plus AHCC (100mg/kg). Group 2: rats were administered UFT (15 mg/kg). Group 3: rats were administered water control.

Figure 2: Protocol for observing the death by metastasis. Rats were divided into three groups. Rats were administered UFT or UFT plus AHCC described as in Figure 1. Surgical tumor excision was performed on day 21. All the rats were kept under observation until they died.

Figure 3: Survival curves of rats after surgical tumor excision on day 21. SST-2 tumors were implanted s.c. on day 0. UFT (15mg/kg) was administered p.o. every day from day 3 to 38. AHCC (100 mg/kg/day in 24 ml) was also administered from day 3 to 38. Surgical tumor excision was performed on day 21. Each group consists of live rats. *Statically significant (p,0.05) between the UFT plus AHCC treated group and the other two groups by the generalized Wiicoxon test.

Figure 4: NK cell activity of spleen cells in SST-2-bearing rats. SST-2 tumors were implanted s.c. on day 0. UFT (15mg/kg) was administered p.o. every day from day 3 to 21. AHCC (100 mg/kg/day in 24 ml) was also administered from day 3 to 21. on day 21, rats were scarified and spleens were aseptically removed from three rats in each group and mixed for cytotoxicity assay.
 

Figure 5: Expression of various cytokines and iNOs mRNA in peritoneal macrophages. SST-2 tumors were implanted s.c. on day 0. UFT (15mg/kg) was administered p.o. every day from day 3 to 21. AHCC (100 mg/kg/day in 24 ml) was also administered from day 3 to 21. On day 21, rats were i.p. administered 1 KE of OK-432 in 1 ml PBS. On day 23, OK-432 activated peritoneal macrophages were harvested with 20 ml PBS and total RNAs were extracted by the guandine thiocyanate:phenol:chloroform method. After reverse transcription, cDNA was amplified by PCR for 35 cycle using specific primers to detect the indicated cytokines and iNOs. PCR products were visualized under UV light.

References

1. Poste G. FidIer I). The pathogenesi.s of cancer metastasis. Nature 1980; 139-146).
2. Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer
   metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 1991; 64: 327-36.
3. Kurebayashi J. Sonoo H. Anuproliferative and antimeta-static effects of UFT on MKL-4 human breast cancer cells transplanted into nude mice. Gan To Kagaku Ryoho 1995; 22: 665-70.
4. Otani S, Maeta M, Oka A, et al. Long-term survival of 5 years following initial surgery for gastric cancer and simultaneous disseminated peritoneal metastasis: report of a case. Surg Today 1995; 25: 959-61.
5. Pazdur R. Lassere Y, Rhodes V, et al. Phase II trial of uracil and tegafur plus oral leucovorin: an effective oral regimen in the treatment of metastatic colorectal carcinoma. Clin Oncol 1994; 12: 2296-300.
6. Li XB, Hamada J, Takeichi N, Hosokawa M, Kobayashi H. Timing of UFT administration combined with surgical operation in pulmonary metastasis of rat mammary carcinoma. Gan To Kagaku Ryoho 1989; l6: 2381-5.
7. Komatsumoto M, U S, Hamada J, et at. Inhibitory effect of UFT on postoperative lung metastasis of mammary adenocarcinoma in SHR rats. Gan To Kagaku Ryoho 1988;15:1739-45.
8. Mihich E. Immunosuppression in cancer therapeutics. Transplant Proc 1975; 7: 275-8.
9. Hersh E. Immunosuppressive agents. In Sartoli AC, Johns DG, eds. Antineoptastic and immunosuppressive agent. New York: Springer 1974: 1: 577.
10. Bayon LG, Izquierdo MA, Sirovich I, van Rooijen N, Beelen RH, Meijcr S. Role of Kupffer cells in arresting circulating tumor cells and controlling metastatic growth in the liver. Hepatology 1996; 23: 1224-31.
11. Hrushesky WJ, Grubcr SA, Sothem RB, et al. Natural killer cell activity: age, estrous and circadian-stage dependence and inverse correlation with metastatic potential. J Natl Cancer Inst 1988; 80: 1232-7.
12. Gruber SA, Hoffman RA, Sothcm RB, et al. Splenocyte natural killer cell activity and metastatic potential are inversely dependent on estrous Stage. Surgery 1988; 104:
    398-403.
13. Hasegawa J, Hosokawn M, Okada F, Kobayashi H. Inhibition of mitomycin C-induced sister-chromatid exchange in mouse bone marrow cells by the immunopotentiators Krestin and Lentinan. Mutat Res 1989; 226: 9-12.
14. Mizushima Y, Yuhki N, Hosokawa M, Kobayashi H. Diminution of cyclophosphamide-induced suppression of antitumor immunity by an immunomodulator PS-K and combined therapeutic effects of PS-K and cyclopho-sphamide on transplanted tumor in rats. Cancer Res 1982;42: 5176-80.
15. Akiyama J, Kawamura T, Gotohda E, et al. Immuno-chemotherapy of transplanted KMT-17 tumor in WKA rats by combination of cyclophosphamide and immuno-stimulatory protein-bound polysaccharide isolatled from basidiomycetes. Cancer Res 1977; 37: 3042-5.
16. Hosokawa M, Mizukoshi T, Morikawa K, Xu ZY, Kobayashi H. The therapeutic effects of an immunopotentiator, PS-K, on 3-methylcholanthrene-induced auto-chthonous tumors in C57BL/6 mice in combination with the surgical removal of primary sites. Cancer Immunol Immunother 1986; 22: 181-5.
17. Mokyr MB, Hengst JC, Dray S. Role of antitumor immunity in cyclophosphamide-induced rejection of subcutaneous nonpalpable MOPC-315 tumors. Cancer Res 1982; 42: 974-9.
18. Matsuoka T, Takeichi N, Kobayashi H. Age-related changes of natural antirumor resistance in spontaneously hypertensive rats with T cell depression. Cancer Res 1987; 47: 3410-3.
19. Takeichi N, Ba D, Koga Y, Kobayashi H. Immunologic o suppression of carcinogenesis in spontaneously hyperten-sive rats (SHR) with T-cell depression. / Immunol 1983;
    130:501-5.
20. Koga Y, .Hamada J, Takeichi N, Nakane A, Minagawa T, Kobayashi H. Activation of natural resistance against lung metastasis of an adenocarcinoma in T-cell depressed ..spontaneously hypertensive rats by infection with Listeria monocytogenes. Cancer Immunotherapy 1985;103-8
21. Slezak SE, Horan PK. Cell-mediated cytotoxicity. A highly sensitive and informative flow cytometric assay. J Immunot Methods 1989; 117: 205-14.
22. Yunoki S, Tanaka N, Hizuta A, Orita K. Enhancement of antitumor cytotoxicity of hepatic lymphocytes by oral administration of PSK. Int J Immunopharmacol 1994; 16: 123-30.
23. Brunson KW. Ohashi M, Miller CA, Kitson RP. Basse PH. Goldfarb RH. Interleukin-2 (IL-2) activated natural killer (A-NK) cells: binding to microvascular endothelial cells and BRM enhancement of cytolytic activity. In Vivo 1994; 8:71-5.
24. Uchida A, Kariya Y. Autologous tumor killing (ATK) and tumor metastasis in human cancer patients. Hum Cell 1993; 6:15-20.
25. Wiltrout RH, Herberman RB, Zhang SR, et al. Role of organ-associated NK cells in decreased formation of experimental metastasis in lung and liver. / Immunot 1985; 134:4267-75.
26. Hanna N. Regulation of natural killer cell activation:
    implementation for the control of tumor metastasis. Nat Immun Celt Growth Regut 1983; 3: 22-33.
27. Sakurai T, Ohno N, Suzuki I, Yadomae T. Effect of soluble fungal (l-3>-betaglucan obtained from Sclerotinia sclerotiorum on alveolar macrophage activation. Immunopharmacology 1995; 30: 157-66.
28. Talmadge JE, Fidlcr IJ, Oldham RK. The NCI preclinical screen of biological response modifiers. Behring Inst Mitt 1984; 74: 189-94.
29. Gallo-Hendrikx E, Copps J, Percy D, Croy BA, Wildeman AG. Enhancement of pancreatic tumor metastasis in transgenic immunodeficient mice. Oncogene 1994; 9: 2983-90.

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