Please use this identifier to cite or link to this item:
標題: 以流式細胞儀分析健康犬及腫瘤患犬白血球CD45,CD11b及NSA之表現及荷犬乳腺癌細胞株NOD/SCID小鼠之MDSCs改變
Flow cytometric analysis of CD45, CD11b and NSA expression in leukocytes of healthy and tumor bearing dogs and MDSCs changes in canine mammary carcinoma cell line bearing NOD/SCID mice
作者: 黃秉琦
Hwang, Bin-Chi
關鍵字: dogs;犬;tumor;blood;myeloid derived suppressor cells;腫瘤;血液;骨髓來源抑制細胞
出版社: 獸醫學系暨研究所
引用: Arnaout MA. Structure and function of the leukocyte adhesion molecules CD11/CD18. Blood 1990;75:1037-1050. Arnold H. Zea, Paulo C. Rodriguez, Michael B. Atkins, et al. Arginase producing myeloid suppressor cells in renal cell carcinoma patients: a mechanism of tumor evasion. Cancer Res 2005;65:3044-3048. Avery PR and Avery AC. Molecular methods to distinguish reactive and neoplastic lymphocyte expansions and their importance in transitional neoplastic states. Vet clin pathol 2004;33:196-207. Beckhove P, Schutz F, Diel IJ, Solomayer EF, Bastert G, Foerster J, Feuerer M, Bai L, Sinn HP, Umansky V, Schirrmacher V. Efficient engraftment of human primary breast cancer transplants in nonconditioned NOD/Scid mice. Int J Cancer 2003;105: 444-453. Benjamin SA, Lee AC, Saunders WJ. Classification and behavior of canine mammary epithelial neoplasms based on life-span observations in beagles. Vet patho 1999;36:423-436. Comazzi S, Gelain ME. Flow cytometric patterns in blood from dogs with non-neoplastic and neoplastic hematologic diseases using double labeling for CD18 and CD45. Vet Clin Patho 2006;35:47-54. Comazzi S, Gelain ME, Riondato F, et al. Flow cytometric expression of common antigens CD18/CD45 in blood from dogs with lymphoid malignancies: A semi-quantitative study. Vet Immunol Immunopathol 2006;112:243-252. Chang CY , Chiou PP , Chen WJ, Li YH, Yiu JC, Cheng YH, Chen SD, Lin CT, Lai YS. Assessment of the tumorigenesis and drug susceptibility of three new canine mammary tumor cell lines. Res vet sci 2010;88:285-293. De Santo C, Serafini P, Marigo I, et al. Nitroaspirin corrects immune dysfunction in tumor-bearing hosts and promotes tumor eradication by cancer vaccination. Proc Natl Acad Sci USA 2005;102:4185-4190. Diaz-Montero CM, Salem ML, et al. Increased circulating myeloid-derived suppressor cells correlate with clinical cancer stage, metastatic tumor burden, and doxorubicin-cyclophosphamide chemotherapy. Cancer Immunol Immunother 2009;58:49-59. Dolcetti L, Marigo I, et al.Myeloid-derived suppressor cell role in tumor-related inflammation. Cancer Lett 2008;267:216-225. Donkor MK, Lahue E, Hoke TA, Shafer LR, Coskun U, Solheim JC, Gulen D, Bishay J, Talmadge JE. Mammary tumor heterogenicity in the expansion of myeloid-derived suppressor cells. Int immunopharmacol 2009;9:937-948. Finke JH, Rini B, Ireland J, et al. Sunitinib reverses type-1 immune suppression and decreases T-regulatory cells in renal cell carcinoma patients. Clin Cancer Res 2008;14:6674-6682. Fork MA, Escobar HM, Soller JT, Sterenczak KA, Willenbrock S, Winkler S, Dorsch M, Reimann-Berg N, Hedrich HJ, Bullerdiek J, Nolte I. Establishing an in vivo model of canine prostate carcinoma using the new cell line CT1258. BMC Cancer 2008;8:240-247. Fyfe G, Fisher RI, et al. Results of treatment of 255 patients with metastatic renal cell carcinoma who received high-dose recombinant interleukin-2 therapy. J Clin Oncol 1995;13:688-692. Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nature Reviews Immuno 2009;9:162-174. Henry CJ, Higginbotham. Cancer management in small animal practice. Saunders, 2010. Horiuchi Y, Hanazawa A, Nakajima Y, Nariai Y, Asanuma H, Kuwabara M, Yukawa M, Ito H. T-helper (Th) 1/Th2 imbalance in the peripheral blood of dogs with malignant tumor. Microbiol Immunol 2007;51:1135-1138. Horiuchi Y, Tominaga M, et al. Increase of regulatory T cells in the peripheral blood of dogs with metastatic tumors. Microbiol Immunol 2009;53:468-474. Huang, B. et al. Gr-1+CD115 + immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor- bearing host. Cancer Res 2006;66:1123-1131. Itoh H, Horiuchi Y , Nagasaki T, et al. Evaluation of immunological status in tumor-bearing dogs. Veterinary Immunology and Immunopathology 2009;132: 85-90. Jemal A, Bray F, et al. Global cancer statistics. CA: a cancer journal for clinicians 2011;61:69-90. Kindler HL, van Meerbeeck JP. The role of gemcitabine in the treatment of malignant mesothelioma. Semin oncol 2002;29:70-76. Ko JS, Zea AH, Rini BI, et al. Sunitinib Mediates Reversal of Myeloid-Derived Suppressor Cell Accumulation in Renal Cell Carcinoma Patients. Clin Cancer Res 2009;15:2148-2157. Kobie K, Kawabata M, Hioki K, Tanaka A, Matsuda H, Mori T, Maruo K. The tyrosine kinase inhibitor imatinib [STI571] induces regression of xenografted canine mast cell tumors in SCID mice. Res vet sci 2007;82:239-241. Kosarek CE, Kisseberth WC, Gallant SL, Couto CG. Clinical evaluation of gemcitabine in dogs with spontaneously occurring malignancies. J Vet Intern Med 2005;19:81-86. Manegold C. Gemcitabine (Gemzar) in non-small cell lung cancer. Expert rev anticancer aher 2004;4:345-360. Mantovani A, Sozzani S, et al. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends immunol 2002;23:549-555. Marconato L, Lorenzo RM, Abramo F, Ratto A, Zini E. Adjuvant gemcitabine after surgical removal of aggressive malignant mammary tumours in dogs. Vet comp oncol 2008;6: 90-101. Marigo I, Dolcetti L, et al. Tumor-induced tolerance and immune suppression by myeloid derived suppressor cells. Immunol Rev 2008;222:162-179. Mirza N, Fishman M, Fricke I, et al. All-trans-retinoic acid improves differentiation of myeloid cells and immune response in cancer patients. Cancer Res 2006;66:9299-9307. Mortiz A, Walcheck BK, Deye J, Weiss DJ. Effects of short-term racing activity on platelet and neutrophil activation in dogs. Am J Vet Res 2003;64:855-858. Movahedi K, Guilliams M, et al. Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity. Blood 2008;111:4233-4244. Murdoch C, Muthana M, Coffelt SB, et al. The role of myeloid cells in the promotion of tumour angiogenesis. Nature Rev Cancer 2008;8:618-631. Nagaraj S, Gupta K, et al. Altered recognition of antigen is a novel mechanism of CD8+ T cell tolerance in cancer. Nat Med 2007;13:828-835. Nakamura, Y. et al. Nitric oxide in breast cancer: induction of vascular endothelial growth factor-C and correlation with metastasis and poor prognosis. Clin Cancer Res 2006;12:1201-1207. Nicholson JK, Hubbard M, Jones BM. Use of CD45 Fluorescence and side-scatter characteristics for gating lymphocytes when using the whole blood lysis procedure and flow cytornetry. Cytornetry 1996;26:16-21. Ochoa AC, Zea AH, et al. Arginase, prostaglandin, and myeloid-derived suppressor cells in renal cell carcinoma. Clin Cancer Res 2007;13(2 Suppl):721-726. Okada S, Ueno H, Okusaka T, et al. Phase I trial of gemictabine in patients with advanced pancreatic cancer. Jpn J Clin Oncol 2001;31:7-12. Ostrand-Rosengerg S, Sinha P. Myeloid-derived suppressor cells: linking inflammation and cancer. J immunol 2009;182:4499-4506. Patel SRR, Gandhi V, Jenkins J, et al. Phase II clinical investigation of gemcitabine in advanced soft tissue sarcomas and window evaluation of dose rate on gemcitabine triphosphate accumulation. J clin oncol 2001;19:3483-3489. Owens MA and Loken MR. Safty and specimen acquisition, handling, and processing. In: Flow cytometry principles for clinical laboratory practice. Wiley-Liss Inc. 1995. Pelan-Mattocks LS, Pesch BA, et al. Flow cytometric analysis of intracellular complexity and CD45 expression for use in rapid expression of leukocytes in bovine blood samples. Am J Vet Res 2001;62:1740-1744. Perry AJ, Thamm DH, Avery AC, Dow SW. Increase monocyte chemotactic protein-1 concentration and monocyte count independently associate with a poor prognosis in dogs with lymphoma. Vet comp onco 2010;9:55-64. Plunkett W, Huang P, Xu YZ, et al. Gemcitabine: metabolism, mechanisms of action, and self-potentiation. Semin oncol 1995;22:3-10. Reggeti F, Bienzle D. Flow Cytometry in Veterinary Oncology. Veterinary Pathology 2011;48:223-235. Rodriguez PC, Zea AH, DeSalvo J, et al. L-Arginine consumption by macrophages modulates the expression of CD3~ chain in T lymphocytes. J Immunol 2003;171:1232-1239. Rodriguez PC, Ernstoff MS, Hernandez C, Atkins M, Zabaleta J, Sierra R, Ochoa OC. Arginase I-producing myeloid-derived suppressor cells in renal cell carcinoma are a subpopulation of activated granulocytes. Cancer Res 2009;69:1553-1560. Ryan CW, Vogelzang NJ. Gemcitabine in the treatment of bladder cancer. Expert opin pharmacother 2000;1:547-553. Serafini P, Meckel K, Kelso M, Noonan K, Califano J, Koch W, Dolcetti L, Bronte V, Borrello I. Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function. J exp med 2006;203:2691-2702. Shultz LD, Schweitzer PA, Christianson SW, Cott B, Schweitzer IB, Tennent B, McKenna S, Mobraaten L, Rajan TV, Greiner DL, Leiter EH. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid Mice. J Immunol 1995;154:180-191. Sinha P, Clements VK, et al. Cross-talk between myeloid-derived suppressor cells and macrophages subverts tumor immunity toward a type 2 response. J Immunol 2007;179:977-983. Stettner N, Brenner O, Eilam R, Harmelin A. Pegylated liposomal doxorubicin as a chemotherapeutic agent for treatment of canine transmissible venereal tumor in murine models. J Vet Med Sci 2005;67:1133-1139. Suzuki E, Kapoor V, Jassar AS, et al. Gemcitabine Selectively Eliminates Splenic Gr-1+/CD11b+ myeloid Suppressor Cells in Tumor-Bearing Animals and Enhances Antitumor Immune Activity. Clin Cancer Res 2005;11:6713-6721. Tripathy D. Overview: gemcitabine as single-agent therapy for advanced breast cancer. Clin breast cancer 2002;3:8-11. Ugel S, Delpozzo F, Desantis G, et al. Therapeutic targeting of myeloid-derived suppressor cells. Current Opinion in Pharmacology 2009;9:470-481. Villiers E, Baines S, et al. Identification of acute myeloid leukemia in dogs using flow cytometry with myeloperoxidase, MAC387, and a canine neutrophil -specific antibody. Vet Clin Patho 2006;35:55-71. Visonneau S, Cesano A, Torosian MH, Miller EJ, Santoli D. Growth Characteristics and metastatic properties of human breast cancer xenografts in immunodeficient mice. American Journal of Pathology 1998;152:1299-1311. Weiss DJ, Welle M, Mortiz A, et al. Evaluation of leukocyte cell surface markers in dogs with septic and nonseptic inflammatory disease. Am J Vet Res 2004;65:59-63. Xia Y, Zweier JL. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci USA 1997;94:6954-6958. Yamashita A, Maruo K, Suzuki K, Shirota K, Kobayashi K, Hioki K. Experimental chemotherapy against canine mammary cancer xenograft in SCID mice and its prediction of clinical effect. J vet med sci 2001;63:831-836. Yang L, DeBusk LM, et al. Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis. Cancer cell 2004;6:409-421. Ye XZ, Yu SC. Contribution of myeloid-derived suppressor cells to tumor induced immune suppression, angiogenesis, invasion and metastasis. J Genet Genomics 2010;37:423−430. Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J immunol 2008;181:5791-5802.
骨髓來源抑制細胞(myeloid derived suppressor cells, MDSCs)為一群異型性的不成熟骨髓細胞組成,在腫瘤、感染、敗血症等病理狀況下增多並活化,對於身體的先天及後天免疫均具有抑制作用,為腫瘤細胞躲避免疫反應的機制之一。目前獸醫學無MDSCs的相關發表研究,但有報告指出在轉移腫瘤的犬隻的抗腫瘤免疫反應較弱。我們假設腫瘤會改變MDSCs的表現,因此本研究利用流式細胞儀分析比較健康犬、良性腫瘤及惡性腫瘤犬隻白血球表現人及小鼠MDSCs的標記物CD11b、共同白血球標記物CD45及嗜中性球特異性抗體NSA的差異。並建立犬腫瘤異種移植於NOD/SCID小鼠,評估以gemcitabine治療後能否藉由控制MDSCs影響腫瘤形成。結果呈現在惡性腫瘤患犬,中度至高度CD45表現、細胞大、顆粒性高的細胞群比例顯著少於健康犬。腫瘤患犬表現CD11b的比例顯著少於健康犬。在惡性腫瘤患犬,表現中等強度CD11b的細胞百分比、表現中等強度NSA細胞百分比顯著上升。我們推測目標細胞表現中等或低強度的CD45、中等程度的CD11b及NSA。此外我們建立犬混合性乳腺癌細胞株DE-SF異種移植於NOD/SCID小鼠,顯示NOD/SCID小鼠為良好異種移植的平台。在轉殖DE-SF一個月後,小鼠脾臟及骨髓的MDSCs較無腫瘤小鼠高。研究指出gemcitabine可減少荷腫瘤小鼠脾臟MDSCs。體外試驗中此藥物對DE-SF細胞一半致死濃度近200 μM,不具強細胞殺滅毒性。以60 mg gemcitabine/kg BW腹腔注射於荷DE-SF小鼠後,脾臟及骨髓MDSCs均無下降。推測其可能原因為藥物劑量不足以殺滅NOD/SCID小鼠脾臟MDSCs亦或NOD/SCID小鼠MDSCs對gemcitabine不具感受性。總結本研究,在腫瘤患犬確實與健康犬隻呈現不同的白血球標記物表現,未來可循此路徑進一步找出影響腫瘤生長的MDSCs。NOD/SCID小鼠是否為評估減少MDSCs藥物效果平台需再研究。

Myeloid derived suppressor cells (MDSCs) compromise of a group of heterogeneous immature myeloid cells, which are expanded and activated under pathological conditions, such as cancer, infection, sepsis. MDSCs have suppressive activities on both innate and acquired immunity, and are recognized as one of the mechanisms how tumor escape immune response. At present, although there is no published research regarding to MDSCs in veterinary medicine, studies revealed that there is a decrease in antitumor immune response among dogs with metastatic cancer. We hypothesize that tumor will change circulating MDSCs, therefore, by using flow cytometry, this study compares differences in leukocyte expressions of CD11b, a positive marker for MDSCs in mice and human, common leukocyte marker CD45 and neutrophil specific antibody (NSA) expressions between healthy dogs and benign and malignant tumor bearing dogs. Results revealed that there is a significant decrease in the percentage of large and high granularity cells with medium or increased CD45 fluorescence intensity in dog with malignant tumor comparing with healthy controls. The percentage of CD11b positive leukocytes significantly decreased in tumor bearing dogs. In dogs with malignant tumor, leukocytes expressing medium intensity in CD11b and NSA increased significantly. Taken together, we suppose that our target cells express medium or decreased intensity in CD45, medium intensity in CD11b and NSA. We establish DE-SF, a canine complex mammary carcinoma cell line, xenograft in NOD/SCID mice, which proved that NOD/SCID mice is a good host for xenografts. The MDSCs percentage in both spleen and bone marrow increased comparing with naive control. Researches revealed that gemcitabine reduces the number of MDSCs in the spleen of tumor bearing mice. This drug doesn''t have a strong cytotoxic effect on DE-SF in vitro since the half lethal dose is nearly 200 μM. One day after 60 mg/kg gemcitacine IP injection in DE-SF bearing mice, MDSCs percentage in spleen and bone marrow did not reduce. We suppose that this result might be due to an insufficient dose to eliminate MDSCs in NOD/SCID mice or insensitivity of NOD/SCID mice MDSCs to gemcitabine. In conclusion, tumor bearing dogs shows different leukocyte marker expressions comparing with healthy controls, by which we could make a closer approach to find MDSCs. Whether NOD/SCID mice can be used to evaluate the effect of MDSCs eliminating drugs needs further research.
其他識別: U0005-1608201113481100
Appears in Collections:獸醫學系所

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.