肿瘤干细胞表型CD44和CD24在乳腺癌骨转移模型骨髓中的表达及意义

目的 探讨人乳腺癌细胞转移到人骨的乳腺癌骨转移小鼠模型中骨髓肿瘤干细胞表型CD44和CD24的表达及其意义.方法 50只SCID小鼠随机分成实验组和对照组,其中实验组鼠背部植入人骨后随机均分3亚组:A组(MDA-MB-231干细胞亚群1×105个/只)、B组(同A组细胞1×106个/只)和C组(MDA-MB-231亲代细胞1×106个/只);对照组设为D组(阳性对照,未植入人骨,同C组细胞直接注射)、E组(阴性对照,植入人骨,生理盐水注射).各组8周后取人骨、鼠骨等行常规HE染色及CK、CD44、CD24、CXCR4、OPN免疫组织化学标记.Real-time PCR检测CD44、CXCR4、OPN的mRNA水平.结果 B组骨转移率最高(77.8%,P＜0.05).B组中人骨转移灶CD44、CXCR4、OPN抗原表达高于C、D组骨中的表达(均有P＜0.05);CD24抗原则在A、B组人骨转移灶中低表达与C、D组骨中的高表达无统计学意义(P＞0.05).B组CD44mRNA表达水平是C组的15.2倍、D组的21.1倍;B组CXCR4mRNA表达水平是C组8.4倍、D组28.4倍;B组OPN-mRNA表达水平是C组4.8倍、D组11.6倍;而B组CD24的mRNA表达显著低于C、D组(均为30%).结论 利用MDA-MB-231肿瘤干细胞亚群(CD44+/CD24-)可制备高转移率的"人源性"乳腺癌骨转移模型,其机制可能与CD44高表达有关.骨转移相关基因CXCR4、OPN转录上调可能参与其过程.     

利用RNA干扰抑制CXCR4基因表达对乳腺癌细胞转移和侵袭能力的影响

目的探讨抑制趋化因子受体CXCR4基因对乳腺癌细胞转移和侵袭能力的影响。方法将人乳腺癌细胞株MDA-MB-31分为未干扰组、CXCR4干扰组、B-actin干扰组和空白载体干扰组。设计并合成了针对CXCR4基因的发夹式siRNA模板,体外合成siRNA表达框架（siRNA expression cassettes,SECs）,经脂质体转染CXCR4干扰组细胞,采用Western blot免疫印迹法和逆转录聚合酶链反应检测各组的抑制效果,用Biocdat Mamgel侵袭能力检测仪检测MDA-MB-231细胞的转移侵袭能力。结果SECs在mRNA水平和蛋白水平均明显抑制CXCR4基因的表达。CXCR4表达被抑制后,癌细胞侵袭能力受到明显抑制,与未干扰组相比,差异有统计学意义（P〈0．05）。结论SECs可以高效特异地抑制人乳腺癌细胞中CXCR4基因的表达。CXCR4基因高表达与乳腺癌细胞的转移密切相关,抑制细胞中CXCR4的表达可以抑制乳腺癌细胞转移和侵袭能力。     

Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions.

A very common metastatic site for human breast cancer is bone. The traditional bone metastasis model requires human MDA-MB-231 breast carcinoma cell inoculation into the left heart ventricle of nude mice. MDA-MB-231 cells usually develop osteolytic lesions 3-4 weeks after intracardiac inoculation in these animals. Here, we report a new approach to study the formation of bone metastasis in animals using breast carcinoma cells expressing the bioluminescent jellyfish protein (green fluorescent protein [GFP]). We first established a subclone of MDA-MB-231 cells by repeated in vivo passages in bone using the heart injection model. On stable transfection of this subclone with an expression vector for GFP and subsequent inoculation of GFP-expressing tumor cells (B02/GFP.2) in the mouse tail vein, B02/GFP.2 cells displayed a unique predilection for dissemination to bone. Externally fluorescence imaging of live animals allowed the detection of fluorescent bone metastases approximately 1 week before the occurrence of radiologically distinctive osteolytic lesions. The number, size, and intensity of fluorescent bone metastases increased progressively with time and was indicative of breast cancer cell progression within bone. Histological examination of fluorescent long bones from B02/GFP.2-bearing mice revealed the occurrence of profound bone destruction. Treatment of B02/GFP.2-bearing mice with the bisphosphonate zoledronic acid markedly inhibited the progression of established osteolytic lesions and the expansion of breast cancer cells within bone. Overall, this new bone metastasis model of breast cancer combining both fluorescence imaging and radiography should provide an invaluable tool to study the effectiveness of pharmaceutical agents that could suppress cancer colonization in bone.     

A CTGF-RUNX2-RANKL Axis in Breast and Prostate Cancer Cells Promotes Tumor Progression in Bone

Metastasis to bone is a frequent occurrence in patients with breast and prostate cancers and inevitably threatens the patient's quality of life and survival. Identification of cancer-derived mediators of bone metastasis and osteolysis may lead to novel therapeutic strategies. In this study, we established highly bone-metastatic PC3 prostate and MDA-MB-231 (MDA) breast cancer cell sublines by in vivo selection in mice. In bone-metastatic cancer cells, the expression and secretion of connective tissue growth factor (CTGF) were highly upregulated. CTGF knockdown in bone-metastatic cells decreased invasion activity and MMP expression. RUNX2 overexpression in the CTGF knockdown cells restored the invasion activity and MMP expression. In addition, CTGF increased RUNX2 protein stability by inducing its acetylation via p300 acetyl transferase. The integrin αvβ3 receptor mediated these effects of CTGF. Furthermore, CTGF promoted RUNX2 recruitment to the RANKL promoter, resulting in increased RANKL production from the tumor cells and subsequent stimulation of osteoclastogenesis from precursor cells. In addition, animal model with injection of CTGF knocked-down prostate cancer cells into 6-week old BALB/c male mice showed reduced osteolytic lesions. More importantly, the expression levels of CTGF and RANKL showed a strong positive correlation in human primary breast tumor tissues and were higher in bone metastases than in other site metastases. These findings indicate that CTGF plays crucial roles for osteolytic bone metastasis both by enhancing invasiveness of tumor cells and by producing RANKL for osteoclastogenesis. Targeting CTGF may lead to the development of effective preventive and therapeutic strategies for osteolytic metastasis. © 2019 American Society for Bone and Mineral Research.     

Loss of the Vitamin D Receptor in Human Breast Cancer Cells Promotes Epithelial to Mesenchymal Cell Transition and Skeletal Colonization

Expression of the vitamin D receptor (VDR) is thought to be associated with neoplastic progression. However, the role of the VDR in breast cancer metastasis to bone and the molecular mechanisms underlying this process are unknown. Employing a rodent model (female Balb/c nu/nu mice) of systemic metastasis, we here demonstrate that knockdown of the VDR strongly increases the metastatic potential of MDA-MB-231 human breast cancer cells to bone, resulting in significantly greater skeletal tumor burden. Ablation of VDR expression promotes cancer cell mobility (migration) and invasiveness, thereby facilitating skeletal colonization. Mechanistically, these changes in tumor cell behavior are attributable to shifts in the expression of proteins involved in cell adhesion, proliferation, and cytoskeletal organization, patterns characteristic for epithelial-to-mesenchymal cell transition (EMT). In keeping with these experimental findings, analyses of human breast cancer specimens corroborated the association between VDR expression, EMT-typical changes in protein expression patterns, and clinical prognosis. Loss of the VDR in human breast cancer cells marks a critical point in oncogenesis by inducing EMT, promoting the dissemination of cancer cells, and facilitating the formation of tumor colonies in bone. © 2019 American Society for Bone and Mineral Research.     

Circulating interleukin-8 levels explain breast cancer osteolysis in mice and humans

Skeletal metastases of breast cancer and subsequent osteolysis connote a dramatic change in the prognosis for the patient and significantly increase the morbidity associated with disease. The cytokine interleukin 8 (IL-8/CXCL8) is able to directly stimulate osteoclastogenesis and bone resorption in mouse models of breast cancer bone metastasis. In this study, we determined whether circulating levels of IL-8 were associated with increased bone resorption and breast cancer bone metastasis in patients and investigated IL-8 action in vitro and in vivo in mice. Using breast cancer patient plasma (36 patients), we identified significantly elevated IL-8 levels in bone metastasis patients compared with patients lacking bone metastasis (p < 0.05), as well as a correlation between plasma IL-8 and increased bone resorption (p < 0.05), as measured by NTx levels. In a total of 22 ER + and 15 ER − primary invasive ductal carcinomas, all cases examined stained positive for IL-8 expression. In vitro, human MDA-MB-231 and MDA-MET breast cancer cell lines secrete two distinct IL-8 isoforms, both of which were found to stimulate osteoclastogenesis. However, the more osteolytic MDA-MET-derived full length IL-8(1–77) had significantly higher potency than the non-osteolytic MDA-MB-231-derived IL-8(6–77), via the CXCR1 receptor. MDA-MET breast cancer cells were injected into the tibia of nude mice and 7 days later treated daily with a neutralizing IL-8 monoclonal antibody. All tumor-injected mice receiving no antibody developed large osteolytic bone tumors, whereas 83% of the IL-8 antibody-treated mice had no evidence of tumor at the end of 28 days and had significantly increased survival. The pro-osteoclastogenic activity of IL-8 in vivo was confirmed when transgenic mice expressing human IL-8 were examined and found to have a profound osteopenic phenotype, with elevated bone resorption and inherently low bone mass. Collectively, these data suggest that IL-8 plays an important role in breast cancer osteolysis and that anti-IL-8 therapy may be useful in the treatment of the skeletal related events associated with breast cancer.     

miR-223通过IGF-1R及NFIA调控乳腺癌细胞及破骨细胞功能的研究

目的探索miR-223在乳腺癌骨转移微环境中调控乳腺癌细胞、破骨细胞的功能及其机制。 方法采用micro-CT检测野生型及miR-223基因敲除小鼠股骨骨小梁骨体积分数、骨密度等相关数据,并对股骨组织病理切片染色,观察miR-223缺失对破骨活动影响。利用RANKL诱导RAW 264.7细胞分化为破骨细胞的体外模型,研究miR-223在其中的作用及机制。通过MDA-MB-231细胞实验研究miR-223对乳腺癌细胞增殖、凋亡功能的调控作用及机制。 结果miR-223基因敲除小鼠股骨破骨活动明显活跃;miR-223过表达可以通过NFIA基因抑制RNAKL诱导的破骨细胞分化成熟,还可通过抑制IGF-1R及PI3K/Akt信号通路,抑制乳腺癌细胞增殖并促进其凋亡。 结论miR-223是骨转移微环境中抑制乳腺癌骨转移发生发展的保护性因子,可通过抑制破骨细胞分化成熟、破骨活动、乳腺癌细胞增殖及促进乳腺癌细胞凋亡来调节乳腺癌骨转移微环境。     

5-杂氮-2′-脱氧胞苷对实验性乳腺癌肺转移的影响

目的探讨5-杂氮-2′-脱氧胞苷(5-Aza-CdR)对乳腺癌MDA-MB-231细胞实验性肺转移的影响及可能的机制。方法将MDA-MB-231细胞分为对照组与5-Aza-CdR处理组,通过半定量RT-PCR(SqRT-PCR)与甲基化特异性PCR(MSP)方法检测两组细胞的乳腺癌转移抑制基因-1(BRMS1),CXC趋化因子受体-4(CXCR4)基因的mRNA表达及启动子区甲基化的状况。分别将两组细胞通过边缘尾静脉注射至BALB/c nu/nu裸鼠体内(每组5只)。5周后,用荧光定量RT-PCR(FqRT-PCR)检测裸鼠肺组织内的目的基因HPRT及内参GAPDH的mRNA丰度。结果对照组和处理组细胞的BRMS1相对灰度值(IDV)分别为0与0.39±0.001,处理组BRMS1 mRNA表达较对照组明显上调(P0.05),5-Aza-CdR使BRMS1启动子区甲基化的CpG岛B完全去甲基化;CXCR4相对IDV分别为0.58±0.003与0.58±0.01,两组间差异无统计学意义(P0.05),CXCR4启动子区CpG岛1的非甲基化状态亦无明显改变。对照组与处理组细胞的裸鼠肺脏HPRT和GAPDH的Ct值分别为:24.75±1.55,16.19±0.69与27.61±1.67,17.48±0.96,2-ΔΔCt=0.34,处理组裸鼠肺脏HPRTmRNA丰度明显低于对照组,肺组织内转移癌较少。结论 5-Aza-CdR通过去甲基化机制重新激活肿瘤转移抑制基因BRMS1的表达,从而降低了MDA-MB-231细胞实验性肺转移能力。     

Low cell motility induced by hsp27 overexpression decreases osteolytic bone metastases of human breast cancer cells in vivo.

The mechanisms controlling the formation of osteolytic bone metastases in patients with breast cancer are still poorly understood. To explore the role of motility in the establishment of osteolytic bone metastases, we have used a model of bone metastasis in which MDA-MB-231 breast cancer cells exhibiting low (hsp27-transfectants) and high (control-transfectant) endogenous cell motility were compared. We found that MDA-MB-231 cells exhibiting low cell motility were less capable of establishing osteolytic lesions. The number and the area of the osteolytic lesions in mice inoculated with low motility cells were both significantly smaller. Histomorphometry of bone lesions also demonstrated less tumor area in mice bearing hsp27 transfectants although there was no difference in the osteoclast number per square millimeter of tumor-bone interface. These data suggest that cell motility may be an important mechanism in the metastatic cascade of breast cancer cells to the bone and that controlling cell motility may be a useful target to prevent the establishment of osteolytic bone metastases.     

CXCL12/CXCR4 signaling activates Akt-1 and MMP-9 expression in prostate cancer cells: the role of bone microenvironment-associated CXCL12

BACKGROUND: Hematopoietic cells home to bone by means of chemo-attraction to marrow chemokines, and interaction of chemokines with their receptors leads to the expression/activation of adhesion molecules and proteases. Recent evidence suggests that similar mechanisms may be active in cancer metastasis. Previously, we showed that metalloproteases (MMPs), and in particular MMP-9, play a role in prostate cancer (PC) expansion in bone. METHODS: We used a variety of methods including RT-PCR, immunohistochemistry, ELISA, gelatin zymography, cellular motility and invasion, and subcellular fractionation of PC cells applied to in vivo and in vitro models. RESULTS: Here we showed that (a) CXCL12/CXCR4 axis is expressed in PC bone metastasis; (b) exogenous CXCL12 induced MMP-9 expression by PC cells; (c) bone stromal cells and bone tissue conditioned media induced the migration of PC cells in a CXCR4-dependent manner; (d) pharmacological inhibition of PI3 kinase and MAP kinase pathways abrogated CXCL12-induced MMP-9 expression and invasion of PC cells; (e) exogenous CXCL12 induced Akt1 phosphorylation is indispensable for proMMP-9 secretion, migration, and invasion of PC cells; (f) CXCR4 was localized to lipid rafts in PC cells and initiated Akt phosphorylation. CONCLUSIONS: These data suggest that chemoattractive mechanisms involve migration of cancer cells towards bone tissue, and that cell signaling induced by binding of the chemokine to its receptor leads to the activation of multiple signaling pathways and subsequent secretion of MMP-9 into the local environment. These findings provide a link between chemoattractive mechanisms, growth of tumor cells in bone, and tumor-enhanced bone matrix turnover.     

乳腺癌裸鼠骨转移模型的建立及评估

目的 建立乳腺癌骨转移动物模型并探讨对该转移模型的最佳评估方法.方法 将带GFP标记的人乳腺癌细胞MDA-MB-231注入裸鼠左心室,建立乳腺癌骨转移模型.6周后使用X线机、活体显像系统和病理学检查对裸鼠骨组织进行评估.结果 6周后病理学检查肿瘤细胞已扩散至裸鼠的脑、骨髓腔、肺等组织,X线影像清晰显示骨质破坏的位置及程度,活体显像系统可以观察到肿瘤细胞在裸鼠体内的分布.结论 X线、活体显像和组织学检查3种方法相结合是分析乳腺癌小鼠骨转移模型的最佳方法之一.     

乳腺癌组织中趋化因子CXCL12及其受体CXCR4、CXCR7的表达及临床意义

目的:探讨趋化因子CXCL12及其受体CXCR4、CXCR7在乳腺癌组织中的表达及临床意义。方法:应用qRT-PCR方法检测35例新鲜乳腺癌组织及癌旁正常乳腺组织中CXCL12及其受体CXCR4、CXCR7的mRNA表达,采用免疫组化检测120例乳腺癌细胞组织石蜡标本中CXCL12及其受体CXCR4、CXCR7的蛋白表达,并分析三者的表达与患者临床特征的关系。结果:qRT-PCR结果显示,CXCL12、CXCR4、CXCR7的mRNA在乳腺癌组织中表达量均明显高于癌旁正常乳腺组织(均P0.05)。免疫组化结果显示,CXCL12、CXCR4、CXCR7蛋白在乳腺癌组织中阳性表达率分别为70.8%(85/120)、65.8%(79/120)和63.3%(76/120),三者在均在伴有淋巴结转移及TNM分期较高的患者乳腺癌组织中表达明显升高(均P0.05)。结论:趋化因子CXCL12及其受体CXCR4、CXCR7的高表达可能与乳腺癌淋巴转移及恶性进展密切相关。     

Adhesion and migration of extracellular matrix-stimulated breast cancer

BACKGROUND: Extracellular matrix (ECM) components, such as vitronectin and fibronectin, have been shown to enhance the metastatic potential of breast cancer cells. We hypothesized that ECM binding to integrin receptors on breast cancer cells influenced cellular adhesion and migration. MATERIALS AND METHODS: Adhesion assays were performed using breast cancer cell lines MDA-MB-435 and MDA-MB-231 and various concentrations of vitronectin or fibronectin. Migration assays were performed using the same cell lines and invasion chambers with 8 microm pore polycarbonate membranes. Blocking antibodies and specific peptidomimetic inhibitors to integrin receptors were used to identify the integrin subunits reacting with vitronectin and fibronectin. RESULTS: While both breast cancer cell lines adhered to and migrated toward vitronectin and fibronectin, MDA-MB-435 had a higher maximum binding to vitronectin and MDA-MB-231 had a higher maximum binding to fibronectin. Anti-beta1 antibody inhibited the adhesion and migration of MDA-MB-231 to fibronectin and the adhesion of MDA-MB-231 to vitronectin but had no effect on vitronectin-induced adhesion or migration of MDA-MB-435. The alpha(v)beta3/alpha(v)beta5 antagonist, SB 265123, inhibited MDA-MB-231 and MDA-MB-435 adhesion and migration to vitronectin but had no effect on migration to fibronectin in either cell line. CONCLUSIONS: We conclude that the integrin subunits beta1, alpha(v)beta3, and alpha(v)beta5 can be involved in breast cancer cell adhesion and migration to vitronectin and fibronectin. Because more than one integrin inhibitor was required to block adhesion or migration in the cell lines studied, breast cancer therapy based on integrin antagonists would most likely require concomitant use of multiple agents.     

Breast cancer increases osteoclastogenesis by secreting M-CSF and upregulating RANKL in stromal cells

BACKGROUND: Breast cancer metastasis to bone causes resorption of the mineralized matrix by osteoclasts.Macrophage colony stimulating factor (M-CSF)and receptor activator of the NF-kappaB ligand (RANKL) are produced by stromal cells and are essential for osteoclast formation. The human breast cancer cell line, MDA-MB-231, reliably forms bone metastases in a murine model and stimulates osteoclast formation in culture. We hypothesized that MDA-MB-231 stimulates osteoclast formation through secretion of M-CSF and/or RANKL. MATERIALS AND METHODS: We cocultured MDA-MB-231 and a bone marrow derived cell line, UAMS-33, and evaluated the expression of M-CSF and RANKL mRNA. Osteoclast formation was assessed using these cells added to hematopoietic cell cultures. RESULTS: MDA-MB-231 exhibited constitutive expression of M-CSF mRNA. As expected, addition of recombinant M-CSF (30 ng/ml) and RANKL (30 ng/ml) to hematopoietic osteoclast precursors supported osteoclast formation, while the addition of soluble RANKL alone or MDA-231 without added RANKL did not. Notably, coculture of MDA-231 with hematopoietic cells and added soluble RANKL stimulated significant osteoclast formation, indicating that MDA-231 served as an effective source for M-CSF. MDA-231 did not express RANKL. However, when cocultured with the murine bone marrow stromal cell line UAMS-33, RANKL expression was significantly increased in the latter cells. MDA-231 also stimulated osteoclast formation in coculture with UAMS-33 and hematopoietic cells. CONCLUSIONS: We conclude that MDA-MB-231 increases osteoclast formation by secreting adequate amounts of M-CSF protein and enhancing the expression of RANKL by stromal support cells. The ability to stimulate osteoclasts may explain the ability to metastasize to bone.     

Matrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in the injured liver

Effective mobilization of hematopoietic stem cells (HSCs) in injured organs has not been established. Matrix metalloproteinase-9 (MMP-9) is known to release HSCs from bone marrow (BM) into the peripheral blood, but its role in the recruitment of HSCs to injured organs is unclear. In this study we tried to clarify the role of the host MMP-9 in trafficking of HSCs toward the injured liver, especially the relation of MMP-9 with the chemokine receptor 4 (CXCR4)-chemokine ligand 12 (CXCL12) axis, and to examine whether MMP-9 deficiency affects BM cell trafficking to the injured liver in mice. In vitro, we investigated the effect of MMP-9 on migration activity and CXCR4 expression on lineage-negative (Lin(-)) BM cells. In vivo, we induced acute and chronic liver injury in MMP-9 knockout (KO) and control mice by inoculation of carbon tetrachloride, followed by transplantation of Lin(-) BM cells obtained from enhanced green fluorescent protein (EGFP)-transgenic mice, and counted the BM cells mobilized in the injured liver. In a migration assay, active MMP-9, but not proMMP-9, increased the number of migrated Lin(-) BM cells, which was inhibited by tissue inhibitor of metalloproteinase-1 or a MMP inhibitor. This chemoattractant function by MMP-9 was synergistic when cotreated with CXCL12. CXCR4 expression on Lin(-) BM cells was dose- and time-dependently increased by active MMP-9. At the same time, treatment with MMP-9 enhanced CXCL12 expression, and CXCL12 reciprocally increased MMP-9 expression in BM cells. In in vivo studies, many EGFP(+) cells were seen in control recipient mice. In contrast, few EGFP(+) cells were observed in MMP-9 KO mice. BM cells tended to differentiate into desmin(+) cells. In conclusion, MMP-9 contributes to the mobilization of BM cells in the injured liver by upregulating the expression of CXCR4 on Lin(-) BM cells and attracting BM cells along its gradient of CXCL12. Therefore, host MMP-9 plays an important role in BM cell migration in the injured liver.     

CXCR4/CXCL12信号途径在食管鳞癌浸润转移中的作用机制

目的研究趋化因子受体4(CXCR4)/趋化因子12(CXCL12)信号途径在食管鳞癌浸润转移中的作用机制,为探讨CXCR4成为食管癌治疗新的靶点提供理论依据。 方法取对数生长期的食管鳞癌EC9706细胞,添加趋化因子CXCL12,通过侵袭转移实验、黏附实验分别检测食管鳞癌EC9706细胞株的细胞侵袭、移动和黏附能力。采用RT-PCR和Western Blot技术检测表皮生长因子受体(EGFR)mRNA及蛋白的表达水平。 结果添加不同浓度趋化因子CXCL12(终浓度为5、10 μg/ml),食管鳞癌EC9706细胞株的细胞侵袭、移动和黏附能力均较空白对照组高,并且存在剂量依存关系,即CXCL12浓度越高,细胞的侵袭、移动、黏附能力越强,差异均有统计学意义(P<0.01)。添加不同浓度趋化因子CXCL12,食管鳞癌细胞的EGFR mRNA和蛋白表达水平均较对照组高,并且存在剂量依存关系,即CXCL12浓度越高,EGFR mRNA和蛋白表达水平越高,差异均有统计学意义(P<0.01)。 结论CXCR4/CXCL12信号途径与食管鳞癌细胞的侵袭、转移相关,并且存在剂量依存关系,有可能通过调控EGFR的表达参与食管鳞癌的浸润转移。     

Monitoring metastatic behavior of human tumor cells in mice with species-specific polymerase chain reaction: elevated expression of angiogenesis and bone resorption stimulators by breast cancer in bone metastases.

Tumor-stroma interactions are of primary importance in determining the pathogenesis of metastasis. Here, we describe the application of sensitive competitive polymerase chain reaction (PCR) techniques for detection and quantitation of human breast cancer cells (MDA-MB-231) in an in vivo mouse model of experimental metastasis. Human-specific oligonucleotide primers in competitive PCR reactions were used to quantify the amount of MDA-MB-231 cells per tissue per organ. Using this species-specific (semi)quantitative PCR approach, gene expression patterns of (human) tumor cells or (mouse) stromal cells in metastatic lesions in the skeleton or soft tissues were investigated and compared. In all metastatic lesions, MDA-MB-231 cells express angiogenic factors (vascular endothelial growth factors [VEGFs]; VEGF-A, -B, and -C) and bone-acting cytokines (parathyroid hormone-related protein [PTHrP] and macrophage colony-stimulating factor [M-CSF]). In these metastases, PECAM-1-positive blood vessels and stromal cells of mouse origin are detected. The latter express angiogenic factors and markers of sprouting vessels (VEGF receptors flt-1/flk - 1/flk-4 and CD31/PECAM-1). Strikingly, steady-state messenger RNA (mRNA) levels of VEGF-A and -B and the major bone resorption stimulators PTHrP and M-CSF by tumor cells were elevated significantly in bone versus soft tissues (p < or = 0.05, p < or = 0.0001, p < or = 0.001, and p < or = 0.05, respectively), indicating tissue-specific expression of these tumor progression factors. In conclusion, MDA-MB-231 breast cancer cells express a variety of factors in vivo that have been implicated in metastatic bone disease and that correlate with poor survival of patients with breast cancer. We hypothesize that the observed up-regulated expression of angiogenic and bone-resorbing factors by the breast cancer cells in the skeleton underlie the clinically observed osteotropism of breast cancer cells and pathogenesis of osteolytic bone metastases. The application of the species-specific competitive PCR-based assay in vivo can provide new information concerning the involvement of gene families in tumor progression and metastatic disease and greatly facilitates the study of tumor-stroma interactions in cancer invasion and metastasis.     

A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro.

Breast cancer has a predilection for spreading to bone. The mechanism of preferential metastasis of breast cancer to bone is unknown. We hypothesize that breast cancer cells that develop bone metastases have the capacity to facilitate their colonization in bone. To examine this hypothesis, we established bone-seeking (MDA-231BO) and brain-seeking (MDA-231BR) clones of the human breast cancer cell line MDA-MB-231 by repeated sequential passages in nude mice and in vitro of metastatic cells obtained from bone and brain metastases, respectively. These clones were examined for distinguishing biological characteristics and compared with the MDA-231 parental cells (MDA-231P) in vivo and in vitro. Both the MDA-231BR and the MDA-231BO showed identical tumorigenicity to MDA-231P at the orthotopic site. MDA-231P that was inoculated into the heart developed metastases in bone, brain, ovary, and adrenal glands. On the other hand, MDA-231BO exclusively metastasized to bone with larger osteolytic lesions than MDA-231P. MDA-231BR exclusively disseminated to brain and failed to develop bone metastases. In culture, MDA-231BO produced greater amounts of parathyroid hormone-related protein (PTH-rP) than MDA-231BR and MDA-231P in the absence or presence of transforming growth factor beta (TGF-beta). Furthermore, the anchorage-independent growth of MDA- 231BO in soft agar was not inhibited by TGF-beta, whereas TGF-beta profoundly inhibited the growth of MDA-231P and MDA-231BR. Insulin-like growth factor I (IGF-I) markedly promoted the anchorage-independent growth of MDA-231BO, whereas marginal or no stimulation was observed in MDA-231BR or MDA-231P, respectively. Our data suggest that these phenotypic changes allow breast cancer cells to promote osteoclastic bone resorption, survive, and proliferate in bone, which consequently leads to the establishment of bone metastases.