臺灣博碩士論文加值系統

近年來，無論在全世界或台灣，癌症的發生率及死亡率都居高不下，而其中由肺癌所造成的癌症死亡仍然佔了很高的比例。傳統對於肺癌的治療，主要是以放射線治療、化學治療和手術切除為主，而治療後發生轉移以及預後不佳的問題，為致死的主要原因。而癌症轉移對於臨床上更是一個棘手的問題，臨床對於癌症轉移有偵測及診斷上的困難。骨骼為一個易受癌症轉移的組織，而由癌症所發生的骨轉移，會帶給病患很多痛苦，如骨痛、骨折或骨髓壓迫等，更甚者危及生命。目前在臨床上，分子造影技術已被廣泛的應用在許多疾病的診斷上，包括了癌症、神經性疾病、心血管疾病等。
本研究主要針對肺癌進行兩部分的實驗。第一部分是利用幹細胞具有活體趨癌的特性，以人類間葉幹細胞為媒介追蹤由小鼠肉瘤細胞引起之肺臟轉移，並結合蛋白質疫苗針對幹細胞趨癌所誘發體內免疫來抑制腫瘤之生長。經過實驗發現Kp-hMSCs的確能於活體內趨向肺臟腫瘤，而使用Kp-hMSCs和PE(ΔIII)-E7-KDEL3合併進行治療的老鼠，其肺部所產生的腫瘤結節數比對照組少 (P<0.05)，顯示結合此兩種方式的確可以對肺部腫瘤達到抑制的效果。第二部實驗以非侵入性的方式利用高敏感度及專一性的生物冷光造影，監測轉植luciferase基因的人類肺癌細胞，其於活體內轉移的情形。生物冷光影像顯示H441GL細胞存在於肺部以及後肢的位置，在經過組織切片分析的佐證後，可以證明我們所使用的H441GL肺癌細胞，可以經由循環系統轉移到骨頭中，表示的確具有骨轉移的能力。
結合幹細胞和蛋白質疫苗進行癌症治療，改善了疫苗療法只針對其獨特目標的限制，可提供未來臨床上對於不同癌症治療的應用性。而結合報導基因和報導探針策略的非侵入式的分子基因造影，由於其高專一性和敏感性的表現，可以準確的偵測癌症於活體內的生物活動，更可用來偵測初期癌症及微小的癌症轉移，基於專一性分子基因造影的積極發展，期望將來能廣泛應用於癌症診斷、治療，以及抗癌藥物的開發與測試。

Recently, the incidence and mortality of cancer remains very high in the world, in which large proportion of cancer mortality is caused by lung cancer. Traditional treatments of cancer are radiotherapy, chemotherapy and surgery; however unfortunately, primary cancers often metastasize after treatment resulting in poor-prognosis. Metastases of the cancer have become a troublesome problem for clinic therapy and are hard to detect and to diagnose. Bone is a common tissue of several cancer metastases. When bone metastasis occurs, it always brings agonies to patients including bone pain, pathologic fracture, nerve compression, and also leads to death while being more malignant. Current molecular imaging technologies have been widely used for various diseases diagnosis on clinic, including cancer, neuropathic disease, and cardiovascular disease.
In this study we focused on the lung cancer from two parts. For first aspect, previous studies have shown that the adoptively transferred stem cell have characteristics to target and to migrate to tumor lesion in vivo. Therefore, human mesenchymal stem cells (hMSCs) were used to target murine sarcoma-derived lung metastases and viewed as a “Trojan Horse”. Fusion protein vaccine was also used to recognize the hMSCs and then leaded to the inhibition of tumor growth. In our study, the Kp-hMSCs showed homing to lung tumor in vivo, and mice treated with Kp-hMSCs and PE(ΔIII)-E7-KDEL3 showed less lung nodules than the control group (p<0.05). The results show that combination of hMSCs and fusion protein vaccine can efficiently suppress the growth of lung tumor. In the second aspect, highly sensitive and specific bioluminescence imaging was used to non-invasively monitor the metastases of luciferase-transduced human lung cancer cell in vivo. The bioluminescence imaging results show that H441GL cells exist in lung and metastasize to hindlimb in later time point. After histological analyses, it demonstrates that H441GL lung cancer cells can metastasize to skeleton through the circulation and shows the bone metastatic potential.
Combination of stem cells and protein vaccine for cancer therapy could improve the limitation of vaccine therapy that aim at only a unique organ. It provides an alternative clinical application of different cancer therapy in the future. Because of the reporter gene/reporter probe strategies used in highly specific and sensitive expressions of molecular gene imaging, it could accurately detect biological process of cancer in living object and could be used for diagnosis of cancer in early stage and microscopic tumor metastases. Base on the great development of specific molecular gene imaging, we expect that it can be facilitated widely application of cancer diagnosis, cancer therapy, and the development and test of the anticancer drug.

中文摘要...........................................................................................................Ⅰ
Abstract..............................................................................................................Ⅲ
第壹章 緒論
第壹節 分子造影...........................................................................................1
第貳節 基因轉植...........................................................................................5
第參節 癌症疫苗療法...................................................................................7
第肆節 癌症...................................................................................................9
第貳章 材料與方法
第壹節 結合人類間葉幹細胞和蛋白質疫苗治療小鼠肉瘤細胞引起之肺臟轉移.............................................................................................12
一、藥物與試劑...........................................................................................12
二、細胞株與動物.......................................................................................15
三、細胞培養...............................................................................................15
四、電轉良質細胞製備...............................................................................16
五、質體DNA轉型作用.............................................................................17
六、小量質體DNA製備..............................................................................17
七、限制酶切割反應...................................................................................18
八、洋菜膠電泳...........................................................................................18
九、大量質體DNA製備.............................................................................18
十、重組慢病毒載體製備...........................................................................19
十一、偵測慢病毒校價...............................................................................20
十二、人類間葉幹細胞之基因轉導...........................................................20
十三、細胞攝取實驗...................................................................................21
十四、Total RNA萃取.................................................................................21
十五、反轉錄酶-聚合酶連鎖效應............................................................22
十六、動物實驗.........................................................................................23
十七、分子造影實驗.................................................................................25
十八、組織切片及蘇木紫/伊紅染色........................................................25
第貳節 冷光造影活體監測人類肺癌細胞之骨轉移潛能.........................27
一、藥物與試劑.........................................................................................27
二、細胞株與動物.....................................................................................28
三、細胞培養.............................................................................................28
四、細胞冷光測試.....................................................................................28
五、動物實驗.............................................................................................29
六、生物冷光造影.....................................................................................29
七、組織切片及染色.................................................................................29
第參章 實驗結果
第壹節 結合人類間葉幹細胞和蛋白質疫苗治療小鼠肉瘤細胞引起之肺臟轉移............................................................................................32
一、利用重組慢病毒載體對人類間葉幹細胞進行基因轉導.................32
(一) 生產重組慢病毒載體......................................................................32
(二) 慢病毒感染人類間葉幹細胞之基因表現......................................33
二、結合人類間葉幹細胞和蛋白質疫苗治療肺部轉移腫瘤.................33
(一) 非侵入性分子造影監測人類間葉幹細胞趨癌特性......................33
(二) 人類間葉幹細胞結合蛋白質疫苗治療肺部腫瘤模式..................34
第貳節 冷光造影活體監測人類肺癌細胞之骨轉移潛能.........................36
一、非侵入性分子造影連續監測人類肺癌細胞於活體內的轉移.........36
二、人類肺癌細胞轉移之組織生物冷光造影.........................................37
三、組織染色分析人類肺癌之骨轉移現象.............................................38
第肆章 討論
第壹節 結合人類間葉幹細胞和蛋白質疫苗治療小鼠肉瘤細胞引起之肺臟轉移............................................................................................40
第貳節 冷光造影活體監測人類肺癌細胞之骨轉移潛能.........................42
第伍章 結論.....................................................................................................46
第陸章 參考文獻.............................................................................................47
圖與表...............................................................................................................56
圖一、限制酶切割片段確認重組慢病毒所使用之質體DNA的洋菜膠電泳圖...........................................................................................................57
圖二、重組慢病毒所使用之質體DNA共同轉染TE671細胞之轉染效率與病毒校價測試.......................................................................................58
圖三、基因轉植人類間葉幹細胞...................................................................59
圖四、分子造影活體監測人類間葉幹細胞標的腫瘤...................................60
圖五、結合人類間葉幹細胞和蛋白質疫苗治療肺部腫瘤...........................62
圖六、反轉錄酶-聚合酶連鎖反應偵測HSV1-tk和HPV16 E6/E7基因表現...........................................................................................................64
圖七、體外偵測人類肺癌細胞之報導基因之表現.......................................65
圖八、生物冷光造影活體監測人類肺癌細胞轉移.......................................66
圖九、多型式分子造影活體監測人類肺癌細胞轉移...................................67
圖十、人類肺癌細胞轉移組織之生物冷光造影...........................................68
圖十一、組織切片染色分析人類肺癌細胞之轉移I.....................................69
圖十二、組織切片染色分析人類肺癌細胞之轉移II....................................70
圖十三、組織切片染色分析人類肺癌細胞之轉移II....................................72
附錄
附錄一、分子基因造影之兩種型式................................................................73
附錄二、運用於第一型泡疹病毒胸腺嘧啶激酶基因分子造影之報導探針......................................................................................................73
附錄三、製備重祖慢病毒載體之質體DNA.................................................74
附錄四、結合人類間葉幹細胞和蛋白質疫苗治療肺部種瘤之動物實驗示意圖.......................................................................................................75
附錄五、分子造影監測人類肺癌細胞轉移之動物實驗示意圖...................76

1.Rubin E, Sanders C, Harvey JC, Beattie EJ. Diagnostic imaging and staging of primary lung cancer. Semin Surg Oncol. 1993;9(2):85-91.

2.Chen JJ, LaFrance ND, Allo MD, Cooper DS, Ladenson PW. Single photon emission computed tomography of the thyroid. J Clin Endocrinol Metab. 1988;66(6):1240-1246.

3.Gambhir SS. Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer. 2002;2(9):683-693.

4.Massoud TF, Gambhir SS. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev. 2003;17(5):545-580.

5.Tjuvajev JG, Stockhammer G, Desai R, et al. Imaging the expression of transfected genes in vivo. Cancer Res. 1995;55(24):6126-6132.

6.Gambhir SS, Barrio JR, Herschman HR, Phelps ME. Assays for noninvasive imaging of reporter gene expression. Nucl Med Biol. 1999;26(5):481-490.

7.Strijckmans K. The isochronous cyclotron: principles and recent developments. Comput Med Imaging Graph. 2001;25(2):69-78.

8.Deloar HM, Watabe H, Aoi T, Iida H. Evaluation of penetration and scattering components in conventional pinhole SPECT: phantom studies using Monte Carlo simulation. Phys Med Biol. 2003;48(8):995-1008.

9.Chatziioannou A, Tai YC, Doshi N, Cherry SR. Detector development for microPET II: a 1 microl resolution PET scanner for small animal imaging. Phys Med Biol. 2001;46(11):2899-2910.

10.Cherry SR, Gambhir SS. Use of positron emission tomography in animal research. Ilar J. 2001;42(3):219-232.

11.Blankenberg FG, Backer MV, Levashova Z, Patel V, Backer JM. In vivo tumor angiogenesis imaging with site-specific labeled (99m)Tc-HYNIC-VEGF. Eur J Nucl Med Mol Imaging. 2006.

12.Lauwers E, Beque D, Van Laere K, et al. Non-invasive imaging of neuropathology in a rat model of alpha-synuclein overexpression. Neurobiol Aging. 16 2006.

13.Deng WP, Wu CC, Lee CC, et al. Serial in vivo imaging of the lung metastases model and gene therapy using HSV1-tk and ganciclovir. J Nucl Med. 2006;47(5):877-884.

14.Deng WP, Yang WK, Lai WF, et al. Non-invasive in vivo imaging with radiolabelled FIAU for monitoring cancer gene therapy using herpes simplex virus type 1 thymidine kinase and ganciclovir. Eur J Nucl Med Mol Imaging. 2004;31(1):99-109.

15.Hung SC, Deng WP, Yang WK, et al. Mesenchymal stem cell targeting of microscopic tumors and tumor stroma development monitored by noninvasive in vivo positron emission tomography imaging. Clin Cancer Res. 2005;11(21):7749-7756.

16.Hadjantonakis AK, Nagy A. The color of mice: in the light of GFP-variant reporters. Histochem Cell Biol. Jan 2001;115(1):49-58.

17.Falk MM, Lauf U. High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells. Microsc Res Tech. 2001;52(3):251-262.

18.Campbell RE, Tour O, Palmer AE, et al. A monomeric red fluorescent protein. Proc Natl Acad Sci U S A. 2002;99(12):7877-7882.

19.So MK, Xu C, Loening AM, Gambhir SS, Rao J. Self-illuminating quantum dot conjugates for in vivo imaging. Nat Biotechnol. 2006;24(3):339-343.

20.Wetterwald A, van der Pluijm G, Que I, et al. Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease. Am J Pathol. 2002;160(3):1143-1153.

21.Vooijs M, Jonkers J, Lyons S, Berns A. Noninvasive imaging of spontaneous retinoblastoma pathway-dependent tumors in mice. Cancer Res. 2002;62(6):1862-1867.

22.Bhaumik S, Gambhir SS. Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc Natl Acad Sci U S A. 2002;99(1):377-382.

23.Ray P, De A, Min JJ, Tsien RY, Gambhir SS. Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res. 2004;64(4):1323-1330.

24.Ponomarev V, Doubrovin M, Serganova I, et al. A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging. Eur J Nucl Med Mol Imaging. 2004;31(5):740-751.

25.Beyer T, Townsend DW, Brun T, et al. A combined PET/CT scanner for clinical oncology. J Nucl Med. 2000;41(8):1369-1379.

26.Imai E. Gene therapy approach in renal disease in the 21st century. Nephrol Dial Transplant. 2001;16 Suppl 5:26-34.

27.Wilson DR. Viral-mediated gene transfer for cancer treatment. Curr Pharm Biotechnol. 2002;3(2):151-164.

28.Iwakuma T, Cui Y, Chang LJ. Self-inactivating lentiviral vectors with U3 and U5 modifications. Virology. 1999;261(1):120-132.

29.Chang LJ, Urlacher V, Iwakuma T, Cui Y, Zucali J. Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system. Gene Ther. 1999;6(5):715-728.

30.Liao CW, Chen CA, Lee CN, et al. Fusion protein vaccine by domains of bacterial exotoxin linked with a tumor antigen generates potent immunologic responses and antitumor effects. Cancer Res. 2005;65(19):9089-9098.

31.Mehlen P, Puisieux A. Metastasis: a question of life or death. Nat Rev Cancer. 2006;6(6):449-458.

32.Sierra A. Metastases and their microenvironments: linking pathogenesis and therapy. Drug Resist Updat. 2005;8(4):247-257.

33.Pantel K, Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer. 2004;4(6):448-456.

34.Mastro AM, Gay CV, Welch DR. The skeleton as a unique environment for breast cancer cells. Clin Exp Metastasis. 2003;20(3):275-284.

35.Cahill DP, Kinzler KW, Vogelstein B, Lengauer C. Genetic instability and darwinian selection in tumours. Trends Cell Biol. 1999;9(12):M57-60.

36.Perucho M. Tumors with microsatellite instability: many mutations, targets and paradoxes. Oncogene. 2003;22(15):2223-2225.

37.Gu B, Espana L, Mendez O, Torregrosa A, Sierra A. Organ-selective chemoresistance in metastasis from human breast cancer cells: inhibition of apoptosis, genetic variability and microenvironment at the metastatic focus. Carcinogenesis. 2004;25(12):2293-2301.

38.Minn AJ, Kang Y, Serganova I, et al. Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. J Clin Invest. 2005;115(1):44-55.

39.Su JL, Yang CY, Shih JY, et al. Knockdown of contactin-1 expression suppresses invasion and metastasis of lung adenocarcinoma. Cancer Res. 2006;66(5):2553-2561.

40.Kvale PA, Simoff M, Prakash UB. Lung cancer. Palliative care. Chest. 2003;123(1 Suppl):284S-311S.

41.Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100(1):57-70.

42.Orr FW, Lee J, Duivenvoorden WC, Singh G. Pathophysiologic interactions in skeletal metastasis. Cancer. 2000;88(12 Suppl):2912-2918.

43.Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics, 1999. CA Cancer J Clin. 1999;49(1):8-31, 31.

44.Doll SR. Smoking and lung cancer. Am J Respir Crit Care Med. 2000;162(1):4-6.

45.Meuwissen R, Berns A. Mouse models for human lung cancer. Genes Dev. 2005;19(6):643-664.

46.Travis WD, Travis LB, Devesa SS. Lung cancer. Cancer. 1995;75(1 Suppl):191-202.

47.Worden FP, Kalemkerian GP. Therapeutic advances in small cell lung cancer. Expert Opin Investig Drugs. 2000;9(3):565-579.

48.Rubens RD. Bone metastases--the clinical problem. Eur J Cancer. 1998;34(2):210-213.

49.Cooper CR, Chay CH, Gendernalik JD, et al. Stromal factors involved in prostate carcinoma metastasis to bone. Cancer. 2003;97(3 Suppl):739-747.

50.Ikeda I, Miura T, Kondo I. Pyridinium cross-links as urinary markers of bone metastases in patients with prostate cancer. Br J Urol. 1996;77(1):102-106.

51.Bagi CM. Targeting of therapeutic agents to bone to treat metastatic cancer. Adv Drug Deliv Rev. 2005;57(7):995-1010.

52.Reddi AH, Roodman D, Freeman C, Mohla S. Mechanisms of tumor metastasis to the bone: challenges and opportunities. J Bone Miner Res. Feb 2003;18(2):190-194.

53.Alliston T, Choy L, Ducy P, Karsenty G, Derynck R. TGF-beta-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation. Embo J. 2001;20(9):2254-2272.

54.Kang Y, Siegel PM, Shu W, et al. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell. 2003;3(6):537-549.

55.Bruder SP, Fink DJ, Caplan AI. Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem. 1994;56(3):283-294.

56.Jin HK, Carter JE, Huntley GW, Schuchman EH. Intracerebral transplantation of mesenchymal stem cells into acid sphingomyelinase-deficient mice delays the onset of neurological abnormalities and extends their life span. J Clin Invest. 2002;109(9):1183-1191.

57.Spees JL, Olson SD, Ylostalo J, et al. Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad Sci U S A. 2003;100(5):2397-2402.

58.Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest. Feb 2002;109(3):337-346.

59.Orlic D, Kajstura J, Chimenti S, Bodine DM, Leri A, Anversa P. Bone marrow stem cells regenerate infarcted myocardium. Pediatr Transplant. 2003;7 Suppl 3:86-88.

60.Studeny M, Marini FC, Dembinski JL, et al. Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst. Nov 3 2004;96(21):1593-1603.

61.Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, Andreeff M. Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res. 2002;62(13):3603-3608.

62.Wei J, Blum S, Unger M, et al. Embryonic endothelial progenitor cells armed with a suicide gene target hypoxic lung metastases after intravenous delivery. Cancer Cell. 2004;5(5):477-488.

63.Stratton MR, Darling J, Pilkington GJ, Lantos PL, Reeves BR, Cooper CS. Characterization of the human cell line TE671. Carcinogenesis. 1989;10(5):899-905.

64.Yang WK, Ch'ang LY, Koh CK, Myer FE, Yang MD. Mouse endogenous retroviral long-terminal-repeat (LTR) elements and environmental carcinogenesis. Prog Nucleic Acid Res Mol Biol. 1989;36:247-266.

65.Hung SC, Yang DM, Chang CF, et al. Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes. Int J Cancer. 20 2004;110(3):313-319.

66.Spiro SG, Porter JC. Lung cancer--where are we today? Current advances in staging and nonsurgical treatment. Am J Respir Crit Care Med. 2002;166(9):1166-1196.

67.Meriggi F, Zaniboni A. Non-small-cell lung cancer in the elderly. Crit Rev Oncol Hematol. 2006;57(2):183-190.

68.Ohira T, Suga Y, Nagatsuka Y, et al. Early-stage lung cancer: diagnosis and treatment. Int J Clin Oncol. 2006;11(1):9-12.

69.Cheng WF, Hung CF, Pai SI, et al. Repeated DNA vaccinations elicited qualitatively different cytotoxic T lymphocytes and improved protective antitumor effects. J Biomed Sci. 2002;9(6 Pt 2):675-687.

70.Rodriguez AM, Pisani D, Dechesne CA, et al. Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. J Exp Med. 2005;201(9):1397-1405.

71.Abraham EJ, Kodama S, Lin JC, Ubeda M, Faustman DL, Habener JF. Human pancreatic islet-derived progenitor cell engraftment in immunocompetent mice. Am J Pathol. 2004;164(3):817-830.

72.Auguste P, Lemiere S, Larrieu-Lahargue F, Bikfalvi A. Molecular mechanisms of tumor vascularization. Crit Rev Oncol Hematol. 2005;54(1):53-61.

73.Paeslow TG, Stites DP, Terr AI, Imboden JB. Medical Immunology. Lange medical book. 2001;tenth edition.

74.Clifford JL, Walch E, Yang X, et al. Suppression of type I interferon signaling proteins is an early event in squamous skin carcinogenesis. Clin Cancer Res. 2002;8(7):2067-2072.

75.Nakamura K, Ito Y, Kawano Y, et al. Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther. 2004;11(14):1155-1164.

76.Abdel-Wahab Z, Dar M, Osanto S, et al. Eradication of melanoma pulmonary metastases by immunotherapy with tumor cells engineered to secrete interleukin-2 or gamma interferon. Cancer Gene Ther. 1997;4(1):33-41.

77.Xiang J, Chen Y, Moyana T. Combinational immunotherapy for established tumors with engineered tumor vaccines and adenovirus-mediated gene transfer. Cancer Gene Ther. 2000;7(7):1023-1033.

78.Kim YJ, Dubey P, Ray P, Gambhir SS, Witte ON. Multimodality imaging of lymphocytic migration using lentiviral-based transduction of a tri-fusion reporter gene. Mol Imaging Biol. 2004;6(5):331-340.

79.Koehne G, Doubrovin M, Doubrovina E, et al. Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes. Nat Biotechnol. 2003;21(4):405-413.

80.Dodd CH, Hsu HC, Chu WJ, et al. Normal T-cell response and in vivo magnetic resonance imaging of T cells loaded with HIV transactivator-peptide-derived superparamagnetic nanoparticles. J Immunol Methods. 2001;256(1-2):89-105.

81.Kozlow W, Guise TA. Breast cancer metastasis to bone: mechanisms of osteolysis and implications for therapy. J Mammary Gland Biol Neoplasia. 2005;10(2):169-180.

82.Neiva K, Sun YX, Taichman RS. The role of osteoblasts in regulating hematopoietic stem cell activity and tumor metastasis. Braz J Med Biol Res. 2005;38(10):1449-1454.

83.Hannon RA, Eastell R. Bone markers and current laboratory assays. Cancer Treat Rev. 2006;32 Suppl 1:7-14.

84.Lipton A. Biochemical bone markers in breast cancer. Cancer Treat Rev. 2006;32 Suppl 1:20-22.

85.Smith MR. Markers of bone metabolism in prostate cancer. Cancer Treat Rev. 2006;32 Suppl 1:23-26.

86.Kang Y, He W, Tulley S, et al. Breast cancer bone metastasis mediated by the Smad tumor suppressor pathway. Proc Natl Acad Sci U S A. 2005;102(39):13909-13914.

87.Chirgwin JM, Guise TA. Does prostate-specific antigen contribute to bone metastases? Clin Cancer Res. 2006;12(5):1395-1397.