A novel orthotopic model of breast cancer metastasis to bone

Breast cancer affects approximately one woman in twelve and kills more women than any other cancer. If detected early, patients have a five year survival rate of 66%, but once metastatic disease has developed, there is no effective treatment. About 70% of patients with metastatic disease have bone involvement, while lungs and liver are the other common targets. Bone metastases cause severe pain, pathological fractures and hypercalcaemia and thus are a significant clinical problem. The development of new therapies for metastatic breast carcinoma depends on a better understanding of the mechanism of homing of the tumour cells to bone, liver and lungs and the factors required for their growth in these organs. Research on mechanisms of breast cancer metastasis, particularly to bone, has relied on in vitro studies or on tumour models in which the inoculation route is designed to promote delivery of tumour cells to a specific organ. Metastases in bone are achieved by inoculation into the right ventricle of the heart. To our knowledge there has been no report of a model of metastatic spread from the mammary gland to distant sites which reliably includes bone. In this paper, we describe our recent development of a novel murine model of metastatic breast carcinoma. The new model is unique in that the pattern of metastatic spread closely resembles that observed in human breast cancer. In particular, these murine breast tumours metastasise to bone from the primary breast site and cause hypercalcaemia, characteristics not normally found in murine tumours, but common in human disease. Furthermore, in a preliminary characterisation of this model, we show that secretion of parathyroid hormone-related protein, a role for which has been implicated in breast cancer spread to bone, correlates with metastasis to bone. This model therefore provides an excellent experimental system in which to investigate the factors that control metastatic spread of breast cancer to specific sites, particularly bone. The special advantage of this system is that it involves the whole metastasis process, beginning from the primary site. Existing models consider mechanisms that pertain to growth of tumour once the site has been reached. An understanding of the regulation of these factors by potential therapeutic agents could lead to improvement in therapies designed to combat metastatic disease. For the first time, this development will allow exploration of the molecular basis of site-specific metastasis of breast cancer to bone in a clinically relevant model.     

Stereocomplex micelle loaded with paclitaxel for enhanced therapy of breast cancer in an orthotopic mouse model

Micelles are promising a nano drug carrier for cancer therapy. However, their application is often limited due to the instability of them in vivo. Herein, we reported the development of stereocomplex micelle (SCM) based on amphiphilic dextran-block-polylactide (Dex-b-PLA) that could improve the stability of micelles, reduce the early release of loaded drugs and target the breast cancer through the enhanced permeability and retention (EPR) effect for enhanced breast cancer therapy. The SCM were fabricated from the equimolar mixture of the enantiomeric Dex-b-PLA copolymers. Paclitaxel (PTX) as a model anti breast cancer drug was loaded in the SCM, noted as SCM/PTX. Transmission electron microscopy (TEM) and dynamic laser scattering (DLS) showed the diameter of SCM/PTX was below100?nm, which was suitable sizes for the EPR effect. The release kinetics of SCM/PTX exhibited that the release of PTX was obviously slow down and showed constant release. In the in vitro antitumor test, the SCM/PTX could effectively suppress the viability of 4T1 cells, which was demonstrated by the MTT assay. Moreover, the SCM/PTX could reduce the distribution of PTX at normal organs and obviously increase the accumulation of PTX at tumor sites. The circulation time of SCM/PTX was also obviously enhanced compared to free PTX. In the in vivo antitumor test, the SCM/PTX effectively inhibited the progression of 4T1 breast cancer in the orthotopic mouse model, as demonstrated by decreased tumor growth and increased apoptosis and necrosis areas within tumor tissues. In addition, the toxic side effects of PTX was also alleviated in the SCM/PTX group. This study introduced a stable micelle system that passive targeted the tumor for enhanced breast cancer therapy.     

Modification of the primary tumor microenvironment by transforming growth factor alpha-epidermal growth factor receptor signaling promotes metastasis in an orthotopic colon cancer model

The transforming growth factor alpha (TGFalpha)/epidermal growth factor receptor (EGFR) signaling pathway appears to play a critical role in colon cancer progression, but the cellular and molecular mechanisms that contribute to metastasis remain unknown. KM12C colon cancer cell clones expressing high (C9) or negligible (C10) levels of TGFalpha were implanted into the cecal walls of nude mice. C9 tumors formed autocrine and paracrine EGFR networks, whereas C10 tumors were unable to signal through EGFR. The tumor microenvironment of C9, but not C10, contained cells enriched in vascular endothelial growth factor (VEGF) A, interleukin-8, and matrix metalloproteinases-2 and -9 and had a high vascular surface area. C9 tumors recruited a macrophage population that co-expressed F4/80 and lymphatic vessel endothelial hyaluronic acid receptor and produced VEGFC. The mean lymphatic density of C9 tumors was threefold higher than that of C10 tumors. C9, but not C10, tumor cells metastasized to regional lymph nodes in all mice and to the liver in 5 of 10 mice. Forced expression of TGFalpha in C10 tumor cells led to the generation of autocrine and paracrine EGFR signaling, macrophage recruitment, enhanced blood and lymphatic vascular surface areas, and increased lymphatic metastasis. Collectively, these data show that activation of TGFalpha-EGFR signaling in colon cancer cells creates a microenvironment that is conducive for metastasis, providing a rationale for efforts to inhibit EGFR signaling in TGFalpha-positive colon cancers.     

Activity of biphenyl matrix metalloproteinase inhibitor BAY 12-9566 in a human breast cancer orthotopic model

Matrix metalloproteinases (MMPs) have been implicated in the invasion, metastasis, and angiogenesis associated with human cancer by mediating the degradation of extracellular matrix components. In this paper, we report data that show that BAY 12-9566, a novel inhibitor of MMPs, inhibits angiogenesis, tumor regrowth, and the growth of lung metastases. BAY 12-9566, at 15–100 μM, inhibited tubule formation by human endothelial cells in an in vitro model, but did not prevent the proliferation of endothelial and human breast cancer cells. In the MDA-MB-435 human mammary carcinoma xenograft model, in which the primary tumor is transplanted into the murine mammary fat pad, BAY 12-9566, administered daily at a dose of 100 mg/kg/day p.o. after resection of the primary tumor, inhibited local tumor regrowth by 58% without causing any toxic effect. In addition, BAY 12-9566 treatment inhibited the number and volume of lung metastases by 57 and 88%, respectively. These effects were highly correlated with the serum concentration of BAY 12-9566 at the end of treatment. The serum of the treated animals, harvested 24 h after the last treatment, and the tumor regrown at the site of tumor transplant in the treated animals, contained less protein with MMP-9 activity (as measured in a gelatin zymography assay) than the corresponding controls. However, no difference in the activity of MMP-2 was observed. Although all clinical trials in cancer involving BAY 12-9566 have been halted, this MMP inhibitor has never been used in clinical trials in breast cancer. These results suggest that the novel MMP inhibitor BAY 12-9566 maybe a useful and safe oral treatment for breast cancer, adjunctive to surgery. This revised version was published online in July 2006 with corrections to the Cover Date.     

Estradiol increases ER-negative breast cancer metastasis in an experimental model

Breast cancer (BC) is the most common cancer affecting women in the United States and metastatic breast cancer is the leading cause of death. The role estradiol plays in ER-positive BC is well-documented, but the way it contributes to ER-negative BC remains unclear. In the present study, we utilized an experimental model of BC metastasis into lung by injecting ER-negative murine 4T1 cells into mice via the lateral tail vein. A 56 % metastasis occurrence rate following the injection of 5 × 10³ cells was observed, thus this cell number was selected to study the potential stimulatory effect of estradiol on ER-negative BC metastasis. Female ovariectomized mice were randomized into estradiol and control groups with 16 mice per group, and estradiol pellets were implanted subcutaneously in the estradiol group. Results demonstrated that estradiol accelerated BC metastasis as indicated by bioluminescent imaging. In addition, estradiol enhanced metastatic tumor colony formation and increased the size of tumor nodules in the lungs, which were due, in part, to the increase in proliferative cells in the metastatic tumors. In vitro, estradiol increased the motility and invasion of 4T1 cells, and the stimulatory effect on cell motility was not blocked by ICI 182, 780, confirming that ER was not involved in the process. Results from the present study suggest that estradiol plays a role in ER-negative BC metastasis in whole animals.     

Intravenous KITENIN shRNA injection suppresses hepatic metastasis and recurrence of colon cancer in an orthotopic mouse model

KITENIN (KAI1 C-terminal interacting tetraspanin) promotes invasion and metastasis in mouse colon cancer models. In the present study, we evaluated the effects of KITENIN knockdown by intravenous administration of short hairpin RNAs (shRNAs) in an orthotopic mouse colon cancer model, simulating a primary or adjuvant treatment setting. We established orthotopic models for colon cancer using BALB/c mice and firefly luciferase-expressing CT-26 (CT26/Fluc) cells. Tumor progression and response to therapy were monitored by bioluminescence imaging (BLI). In the primary therapy model, treatment with KITENIN shRNA substantially delayed tumor growth (P = 0.028) and reduced the incidence of hepatic metastasis (P = 0.046). In the adjuvant therapy model, KITENIN shRNA significantly reduced the extent of tumor recurrence (P = 0.044). Mice treated with KITENIN shRNA showed a better survival tendency than the control mice (P = 0.074). Our results suggest that shRNA targeting KITENIN has the potential to be an effective tool for the treatment of colon cancer in both adjuvant and metastatic setting.     

ZNF259促进乳腺癌细胞的侵袭和迁移能力

目的锌指蛋白259(ZNF259)可以结合表皮生长因子受体细胞内的酪氨酸激酶功能域,但其在肿瘤细胞中的生物学功能尚不明确。本研究拟探索其对人乳腺癌细胞生物学行为的调控。方法通过ZNF259特异性小RNA干扰实现在人乳腺癌细胞系中下调ZNF259的蛋白表达水平,利用此细胞模型通过基质胶侵袭和划痕实验来揭示ZNF259对肿瘤细胞的侵袭和转移的调控作用。结果在乳腺癌细胞系中ZNF259表达显著高于其在人正常乳腺上皮细胞系的表达。在乳腺癌细胞系MCF-7和MDA-MB-231中特异性干扰ZNF259的表达可显著抑制肿瘤细胞的侵袭和转移能力。结论 ZNF259促进乳腺癌细胞的侵袭和迁移,参与乳腺癌的发生和发展。     

同种异体气管原位移植模型的改良与观察

背景：气管上皮的再生能有效抑制黏膜下组织增生和管腔闭塞的发生,有研究表明通气能够加快气管上皮的再生。 目的：建立并改良同种异体小鼠原位气管移植模型,进一步观察通气对供体气管的影响。 方法：C57BL/6小鼠的气管作为供体,BALB/c小鼠作为受体。实验分2组,实验组取2个供体气管膜部纵行剖开,缝合成一扩大管腔的气管原位植入受体气管;对照组供体气管原位植入受体气管,28 d后获取标本进行检测。 结果与结论：苏木精-伊红染色显示,与对照组相比,实验组气管管腔内上皮层可见分化良好的纤毛上皮,亦可见少量无纤毛的单层或复层扁平上皮,黏膜下轻度纤维组织增生和炎细胞浸润。形态学定量分析结果示,实验组在气管上皮中纤毛上皮的比例高于对照组(P < 0.05),实验组固有层与软骨的比值,固有膜纤维组织的面积、淋巴细胞浸润度均低于对照组(P < 0.05)。免疫组织化学染色显示,两组移植气管上皮均为为受体上皮表型。结果证实,实验成功建立了改良的小鼠原位气管移植模型,移植段气管通气量增加能够加快气管上皮分化,分化良好的气管上皮可以更好的抑制成纤维细胞的增殖。     

Antimetastatic efficacy of adjuvant gemcitabine in a pancreatic cancer orthotopic model

Gemcitabine is a promising new agent that has been recently studied for palliation of advanced (stage IV) unresectable pancreatic cancer. We hypothesized that adjuvant gemcitabine would reduce recurrence and metastases following surgical resection of pancreatic cancer. To test this hypothesis, we evaluated gemcitabine on a green fluorescent protein (GFP) transductant of the human pancreatic cancer cell line BxPC-3 (BxPC-3-GFP) using surgical orthotopic implantation (SOI) in nude mice. GFP enabled high resolution fluorescent visualization of primary and metastatic growth. Five weeks after SOI, the mice were randomized into three groups: Group I received exploratory laparotomy only. Group II underwent surgical resection of the pancreatic tumor without further treatment. Group III underwent tumor resection followed by adjuvant treatment with gemcitabine, 100 mg/kg every three days for a total of four doses, starting two days after resection. The mice were sacrificed at thirteen weeks following implantation and the presence and location of recurrent tumor was recorded. Gemcitabine reduced the recurrence rate to 28.6% compared to 70.6% with resection only (P=0.02) and reduced metastatic events 58% in the adjuvant group compared to resection only. This study, demonstrating that gemcitabine is effective as adjuvant chemotherapy post-pancreatectomy, suggests this new indication of the drug clinically. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transfection of S100A4 produces metastatic variants of an orthotopic model of bladder cancer

The calcium-binding protein S100A4 induces the metastatic phenotype in rodent models of breast cancer, and its expression strongly correlates with reduced survival in human breast and bladder cancer. We have established an orthotopic model of bladder cancer by injecting a cell line derived from a carcinogen-induced rat bladder tumor into the muscular wall of syngeneic rats. MYU-3L cells produce rapidly growing, invasive tumors in the bladder wall but they fail to metastasize. Transfection of MYU-3L cells with a plasmid vector directing overexpression of the S100A4 gene generates variants in which S100A4 expression is elevated by up to sevenfold in comparison with the untransfected cells. Variants overexpressing S100A4 produce primary tumors at similar frequencies and latencies to the parental cell line, a significant number of which metastasize to the para-aortic lymph nodes or lungs. Expression of S100A4 protein in the primary tumors was heterogeneous, but was stronger and more consistent in the metastases, suggesting that transfectants overexpressing S100A4 possess an enhanced ability to form metastatic lesions. We conclude that overexpression of S100A4 can induce the metastatic phenotype in this rodent model of bladder cancer. Taken together with the results from our parallel studies of human bladder cancer, these data suggest a significant role for S100A4 in bladder cancer metastasis and identify a potential new target for systemic therapy in patients with this disease.     

Oleic acid promotes MMP-9 secretion and invasion in breast cancer cells

Epidemiological and animal studies suggest an association between dietary fatty acids and an increase risk of developing breast cancer. Obesity, which is characterized by hyperlipidemia and an elevation of circulating free fatty acids (FFAs), is also associated with enhanced cancer risk. In breast cancer cells, the FFA oleic acid (OA) induces migration, proliferation, prolong survival, invasion, an increase in cellular Ca²⁺ concentration, MEK1/2, ERK1/2, FAK and Src activation. However, the role of OA on MMP-9 secretion and invasion has not been studied in detail. We demonstrate here that stimulation of MDA-MB-231 breast cancer cells with 200 μM OA induces an increase on MMP-9 secretion through a PKC, Src, and EGFR-dependent pathway, as revealed by gelatin zymography assays. Furthermore, microtubule network mediates MMP-9 secretion induced by OA. In contrast, OA does not induce an increase on MMP-9 secretion in MCF10A cells, whereas it does not induce MMP-9 secretion in MCF12A mammary non-tumorigenic epithelial cells. In addition, OA induces invasion through an EGFR, Gi/Go proteins, MMPs, PKC and Src-dependent pathway, but it is not able to promote invasion in non-invasive MCF-7 breast cancer cells. In summary, our findings demonstrate that OA promotes an increase on MMP-9 secretion and invasion through a PKC, Src, and EGFR-dependent pathway in breast cancer cells.     

CARMA3过表达促进乳腺肿瘤细胞生长

目的构建人CARMA3真核表达载体,探讨其在乳腺癌MCF-7细胞中的表达及其与肿瘤发生发展的关系。方法以T7-CARMA3为模版,利用特异PCR引物扩增CARMA3基因全长编码序列,将其定向克隆至TMp3×FLAG-cmv-10载体,融合蛋白表达载体命名为Flag-CARMA3。将Flag-CARMA3转染到MCF-7乳腺癌细胞系中,Western blot和Real-Time PCR法检测基因和蛋白表达。MTT检测过表达Flag-CARMA3的细胞生长能力。结果成功构建Flag-CARMA3真核表达载体,Flag-CARMA3融合基因和蛋白在MCF-7乳腺癌细胞系中表达。MTT实验证实Flag-CARMA3过表达增强MCF-7乳腺癌细胞生长能力。结论 CARMA3促进乳腺肿瘤细胞生长。     

PEAK1 promotes invasion and metastasis and confers drug resistance in breast cancer

Clinical and Experimental Medicine - Pseudopodium-enriched atypical kinase 1 (PEAK1) has been reported to be upregulated in human malignancies and is correlated with a poor prognosis. Enhanced...     

血管内皮细胞生长因子在肥胖乳腺癌患者乳腺癌组织中的表达及临床意义

 摘要：　目的 探讨肥胖乳腺癌患者乳腺癌组织中血管内皮细胞生长因子（vascular endothelial growth factor,VEGF）的表达及临床意义。方法 采集45例肥胖乳腺癌患者,37例正常体重乳腺癌患者,用RT-PCR和免疫组织化学检测肥胖组和正常体重组乳腺癌组织VEGF的mRNA和蛋白表达。结果 肥胖组乳腺癌组织VEGF的mRNA和蛋白表达均高于正常体重组 (P<0.05),并且VEGF mRNA和蛋白表达之间呈正相关(r=0.785,P<0.05);VEGF在肥胖乳腺癌组织中的表达与组织学分级、5年复发转移有关,与患者年龄无关。结论 VEGF在肥胖乳腺癌患者中具有高表达趋势,提示VEGF可能在肥胖相关性乳腺癌的发生、演变及预后中起重要作用。     

Hyaluronidase expression induces prostate tumor metastasis in an orthotopic mouse model

Molecular mechanisms of prostate cancer progression are frequently studied in mice by orthotopic injection of aggressive cell lines, which yield primary tumors that spontaneously metastasize to lymph nodes. In this report, we characterized the human prostate carcinoma cell line 22Rv1 in an orthotopic system and evaluated the functional relevance of the hyaluronidase Hyal1, a correlate of invasive human prostate cancer, to progression in this model. To provide real-time insights into these processes, we first validated use of an epidermal growth factor-conjugated fluorophore to illuminate orthotopic prostate tumors and their metastases in whole animal imaging. Animals receiving intraprostatic injections were tracked throughout a 6-week period. Tumor sizes were correlated 92% with total fluorescence intensities of 22 prostate tumors. In contrast to the highly tumorigenic and metastatic PC3M-LN4 cells, the 22Rv1 line was orthotopically tumorigenic but not metastatic, despite larger tumor sizes. Lymph node metastasis was successfully imaged in animals with PC3M-LN4 tumors on endpoint dissection. Stable transfection of 22Rv1 cells with Hyal1 did not alter growth kinetics of primary orthotopic tumors, but all animals implanted with Hyal1 transfectants exhibited tumor-positive para-aortic lymph nodes. Hyal1 is implicated as an inducer of prostate cancer metastatic progression.     

Switching of vascular phenotypes within a murine breast cancer model induced by angiopoietin‐2

Sustained growth of solid tumours can rely on both the formation of new and the co‐option of existing blood vessels. Current models suggest that binding of angiopoietin‐2 (Ang‐2) to its endothelial Tie2 receptor prevents receptor phosphorylation, destabilizes blood vessels, and promotes vascular permeability. In contrast, binding of angiopoietin‐1 (Ang‐1) induces Tie2 receptor activation and supports the formation of mature blood vessels covered by pericytes. Despite the intense research to decipher the role of angiopoietins during physiological neovascularization and tumour angiogenesis, a mechanistic understanding of angiopoietin function on vascular integrity and remodelling is still incomplete. We therefore assessed the vascular morphology of two mouse mammary carcinoma xenotransplants (M6378 and M6363) which differ in their natural angiopoietin expression. M6378 displayed Ang‐1 in tumour cells but no Ang‐2 in tumour endothelial cells in vivo. In contrast, M6363 tumours expressed Ang‐2 in the tumour vasculature, whereas no Ang‐1 expression was present in tumour cells. We stably transfected M6378 mouse mammary carcinoma cells with human Ang‐1 or Ang‐2 and investigated the consequences on the host vasculature, including ultrastructural morphology. Interestingly, M6378/Ang‐2 and M6363 tumours displayed a similar vascular morphology, with intratumoural haemorrhage and non‐functional and abnormal blood vessels. Pericyte loss was prominent in these tumours and was accompanied by increased endothelial cell apoptosis. Thus, overexpression of Ang‐2 converted the vascular phenotype of M6378 tumours into a phenotype similar to M6363 tumours. Our results support the hypothesis that Ang‐1/Tie2 signalling is essential for vessel stabilization and endothelial cell/pericyte interaction, and suggest that Ang‐2 is able to induce a switch of vascular phenotypes within tumours. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Prognostic value of vascular density and cell proliferation in breast cancer patients

We measured microvessel density and cell proliferation index in 84 breast cancers using survival analysis to find out their prognostic value. Immunohistochemistry was applied using antibody to factor VIII-related antigen as a marker for microvessels and MIB-1 antibody to stain proliferating cells. We were not able to show any difference in survival with a mean follow-up of 10.3 years between patients with high and low microvessel count or cell proliferation index. Vascular density did not correlate with the cell proliferation rate (p = 0.4). However, patient age correlated negatively with the cell proliferation index of the tumor (p = 0.0009). There was no significant difference both in microvessel count and in cell proliferation between patients with lymph node metastasis and node negative patients. This result questions the usefulness of vascular density and cell proliferation rate as prognostic markers in breast cancer.