Human breast cancer cell line xenografts as models of breast cancer — The immunobiologies of recipient mice and the characteristics of several tumorigenic cell lines

Summary The ability to maintain and study human tissues in anin vivo environment has proved to be a valuable tool in breast cancer research for several decades. The most widely studied tissues have been xenografts of established human breast cancer cell lines into athymic nude mice. Human breast tumor xenografts provide the opportunity to study various important interactions between the tumor and host tissues, including endocrinologic, immunologic, and tumor-stroma interactions. The nude mouse is not the only immune-deficient recipient system in which to study xenografts. Additional single and combined mutant strains have been used successfully, including mice homozygous for the severe combined immune deficiency mutation (scid), both the beige (bg) and nude (nu) mutations in combination (bg/nu), and mice bearing the combinedbg/nu/xid mutations. The differing immunobiologies are discussed, with particular reference to the immunobiology of breast cancer, as are the characteristics of several of the more frequently utilized breast cancer xenografts and cell lines. The ability of several endocrine treatments to modulate effectors of cell mediated immunity,e.g., estrogens and antiestrogens, and the effect of site of inoculation on tumor take and metastasis, also are described.     

Animal models of breast cancer: experimental design and their use in nutrition and psychosocial research

This is the second Special Issue addressing the diversity and use of animal models of breast cancer. The previous issue (Breast Cancer Res Treat 39:1-135, 1996), dealt with a variety of topics such as the characteristics of chemically- and virally-induced rodent models, immunobiologies of immunedeficient mice, transgenic mouse models, and models of metastasis. In the first part of this second Special Issue, the articles address animal models for studying life-style factors, including psychosocial, exercise, and nutritional research in breast cancer. In the second section, there is emphasis on the controversial area of dietary fat, with other authors addressing caloric restriction and dietary isoflavonoids, retinoids, and monoterpenes in the third part. In the final section, a series of authors provide suggestions for approaching various issues involving experimental design, including nutritional studies, drug screening models, statistical considerations, quantitation of tumor growth kinetics, and animal husbandry. These articles, and some additional issues raised during the previous Special Issue, are briefly discussed in this overview. They include a further evaluation of the relative merits of 7,12-dimethylbenz(a)anthracene and N-nitroso-N-methylurea as carcinogens, and of the use of the AIN76 and AIN93 semipurified diets in studies of mammary carcinogenesis.     

Allelotype of 28 human breast cancer cell lines and xenografts

Heterozygous loss of relatively large chromosomal regions is a hallmark of the inactivation of tumour suppressor genes. Searching for deletions in cancer genomes therefore provides an attractive option to identify new tumour suppressor genes. Here, we have performed a genome-wide survey for regions exhibiting allelic loss in 24 commercially available breast cancer cell lines and four breast cancer xenografts, using microsatellite analysis. The assembled allelotype revealed an average fractional allelic loss of 0.34. A total of 19 arms had low allelic loss frequencies (<25%) and 17 arms had moderate allelic loss frequencies (25-50%). Five chromosomal arms were deleted in more than half of the breast cancer samples (8p, 10q, 13q, 17p, and 17q). Three of these frequently lost chromosomal arms had not been identified as such by comparative genome hybridisation, illustrating the higher sensitivity of microsatellite analysis for the detection of allelic losses. As we present allelic loss data of individual samples, our allelotype should not only aid the identification of new breast cancer genes but also provides a baseline for myriad studies involving these breast cancer cell lines.     

Inhibition of the MUC1-C oncoprotein is synergistic with cytotoxic agents in the treatment of breast cancer cells

Mucin 1 (MUC1) is a heterodimeric glycoprotein that is aberrantly overexpressed in most human breast cancers. The oncogenic MUC1-C subunit promotes survival and blocks the apoptotic response to genotoxic anticancer agents. In the present studies, human MCF-7 and ZR-75-1 breast cancer cells were treated with the MUC1-C inhibitor, GO-203, a cell-penetrating peptide that blocks MUC1-C homodimerization and thereby its oncogenic function. Treatment with GO-203 was found to promote the apoptotic response of MCF-7 and ZR-75-1 cells to the therapeutic drugs taxol and doxorubicin (DOX). This effect was (1) attenuated by a pan-caspase inhibitor, and (2) mediated, at least in part, by activation of the effector caspase-7 and cleavage of the downstream substrate PARP. Further analysis of the interaction between GO-203 and taxol using isobolograms, which evaluate the nature of the interaction of two drugs, demonstrated that the combination is highly synergistic. These results were supported by combination index (CI) analysis with values of less than 1. GO-203 was also highly synergistic with DOX in studies of both MCF-7 and ZR-75-1 breast cancer cells. These findings indicate that blocking MUC1-C function could be effective in combination with taxol and DOX for the treatment of breast cancer.     

The safety parameters of the study on intraductal cytotoxic agent delivery to the breast before mastectomy

Background： Intraductal administration of cytotoxic agents has been shown to inhibit the development of breast cancer in animal models. The object of this study was to demonstrate the safety of intraductal delivery cytotoxic agents in patients prior to mastectomy. This method is hopeful to be developed as a chemoprevention approach in patients with pre-malignant or non-invasive ductal lesions to prevent breast cancer which will be further developed.
Methods： Two drugs, pegylated liposomal doxorubicin（PLD） and carboplatin were administered at three dose levels（PLD： 10, 20, 50 mg and carboplatin 60, 120, 300 mg）. There were five subjects in each group with 15 subjects treated with each drug once. Venous blood samples were obtained for pharmacokinetic analysis. The breast was removed surgically 2-5 days post administration and the treated ducts were marked to enable identification on pathological evaluation.
Results： Intraductal administration was generally well-tolerated with mild, transient breast discomfort. In the carboplatin arm, three women at the 300 mg dose experienced mild nausea and vomiting. In the PLD arm most women had mild erythema and swelling of the breast over the 72 hours following the drug administration. Patients receiving the 50 mg dose experienced local erythema until the time of surgery. Pharmacokinetic analysis showed that carboplatin rapidly entered systemic circulation with an early peak time（T max -30 min） with a corresponding plasma ultrafiltrate area under the curve（AUC） consistent with the Calvert Formula using estimated glomerular filtration rate（GFR）. Total plasma doxorubicin had delayed peak concentration times（T max 48 hours） with a linear dose response and peak concentrations substantially lower than expected from equivalent intravenous injection dosing. No doxorubicinol metabolite was detected in the plasma.
Conclusions： This study demonstrates that cytotoxic drugs can be safely administered into breast ducts with minimal toxicity.     

Endocrine ablation in breast cancer patients who have failed cytotoxic therapy

Between 1972 and 1979, forty-six women underwent endocrine ablative surgery, having failed combinations of chemotherapy, radiation, and surgery (including oophorectomy). All had clinically measurable disease; nearly half were afflicted with bone pain. Each was judged to be a candidate for the procedure by estrogen receptor studies (52%), response to L-dopa (39%), or response to prior oophorectomy (8%). All were followed to their death or to the present, with a minimum of 12 months for those alive. Thirty-one (67%) were improved, and disease was arrested in five (11%) for a median time of 13.5 months. There was no difference in response rates or intervals between estrogen receptor-positive and L-dopa-positive groups. Response was not correlated with disease-free interval or menopausal status. Best results were achieved in those with metastases confined to an organ system, particularly the skeletal complex. The procedure is withheld in those with brain metastases. Postablative chemotherapy appeared to prolong the control interval, though numbers are small. The low morbidity and mortality (one death) of midline adrenaloophorectomy combined with the high incidence of recapture of disease leads us to recommend this procedure in appropriately selected patients who have previously failed other therapeutic modalities.     

A follow-up study of breast and other cancers in families of an unselected series of breast cancer patients

The cancer experience among relatives of an unselected cohort of 402 breast cancer patients was previously reported. Cases and their first degree relatives were flagged at the National Health Service Central Register for continuous notification of cancer registrations and deaths. More than 10 years of follow-up data have been analysed to update cancer risks overall and to estimate breast cancer risk in relatives prospectively according to family history at the time of breast cancer diagnosis in the index case. Significant excesses of breast cancer (RR 2.24, P<0.0001), prostate cancer (RR 1.71, P=0.039) and bone sarcoma (RR 6.564, P=0.042) overall and soft tissue sarcoma in mothers only (RR 15.44, P=0.001) were found. There was no excess of any other cancer, including ovarian. High breast cancer risk in relatives was associated with young age at diagnosis in the index (index <40 years at diagnosis, RR in relatives 3.76, P=0.004). Prospective risk of breast cancer was higher in relatives of index patients who had an affected first degree relative at the time of their diagnosis (no family history, RR 1.87, P=0.012; with a family history, RR 3.72, P=0.015). These prospective risk estimates are valuable in advising relatives of newly diagnosed breast cancer patients.     

Administration of a targeted cytotoxic analog of luteinizing hormone-releasing hormone inhibits growth of estrogen-independent MDA-MB-231 human breast cancers in nude mice

Receptor targeted chemotherapy is less toxic and more effective than conventional chemotherapy. Receptors for luteinizing hormone-releasing hormone (LH-RH) are found in about 50% of human breast cancers. Highly potent cytotoxic radical 2-pyrrolinodoxorubicin (AN-201) was linked to the agonistic analog [D-Lys⁶]LH-RH to form cytotoxic LH-RH analog AN-207. We evaluated whether AN-207 could be targeted to the hormone-independent MDA-MB-231 human breast cancers. Nude mice bearing MDA-MB-231 tumors were injected i.v. with 250 nmol/kg doses of cytotoxic radical AN-201, cytotoxic LH-RH analog AN-207, the unconjugated mixture of AN-201 and carrier [D-Lys⁶]LH-RH, [D-Lys⁶]LH-RH alone and vehicle (control). The growth of MDA-MB-231 tumors in animals given a single dose of AN-207 was inhibited significantly (p=0.01) for 3 weeks after injection, whereas tumors in all the other groups grew steadily. All cytotoxic compounds produced leukopenia, but the strongest lymphocyte suppression was caused by cytotoxic radical AN-201. Three weeks after treatment, the presence of mRNA for LH-RH receptors was demonstrated by RT-PCR in all the groups and radioreceptor assays demonstrated high-affinity binding sites for LH-RH on tumor cell membranes of control animals and those treated with AN-201, the carrier peptide alone or in combination with AN-201. At this time point binding assays did not reveal the expression of membrane proteins in tumors treated with AN-207, but 60 days after administration of AN-207, high affinity LH-RH binding sites were found again in MDA-MB-231 tumors. These results indicate that cytotoxic LH-RH analog AN-207 could be utilized for receptor targeted chemotherapy of breast cancers expressing receptors for LH-RH.     

Phenformin as prophylaxis and therapy in breast cancer xenografts

Background:

Observations that diabetics treated with biguanide drugs have a reduced risk of developing cancer have prompted an enthusiasm for these agents as anti-cancer therapies. We sought to determine the efficacy of the biguanide phenformin in the chemoprophylaxis and in the treatment of oestrogen receptor (ER)-positive MCF7 and receptor triple-negative MDAMB231 xenografts in immunocompromised mice. We also compared the efficacy of phenformin and metformin in the treatment of MDAMB231.

Methods:

Immunocompromised mice were divided into groups: (1) phenformin administered for 2 weeks prior to cell injection; (2) established tumours treated with phenformin; (3) established tumours treated with metformin (only for MDAMB231 tumours); (4) untreated controls. Post-treatment tumours, liver and spleen were harvested for further analysis.

Results:

Phenformin significantly inhibited both the development and growth of MCF7 and MDAMB231 tumours, and for MDAMB231 at greater efficacy than metformin without murine toxicity. The number of mitotic figures was significantly fewer in xenografts treated with phenformin compared with controls. Results suggested that the mechanism of action of phenformin in vivo is consistent with AMPK activation.

Conclusion:

Phenformin has clinical potential as an antineoplastic agent and should be considered for clinical trials both in ER-positive and triple-negative breast cancer.     

小白菊内酯体内杀伤小鼠乳腺癌肿瘤干细胞

目的： 探讨小白菊内酯（parthenolide,PTL）对小鼠乳腺癌肿瘤干细胞（cancer stem cell,CSC）的杀伤作用,为临床应用PTL治疗乳腺癌提供实验依据。 方法： 采用5-氟尿嘧啶（5-fluorouracil, 5-FU）化疗法制备富含CSC的小鼠4T1细胞乳腺癌模型,随机分为对照组、5-FU组、PTL组。4周后脱颈处死小鼠,检测各组小鼠肿瘤的体积和重量,流式细胞术检测小鼠肿瘤组织中CD44+CD24-/low细胞比例,Hoechst33342染色法检测侧群（side population,SP）细胞的比例,免疫组化法检测CD55和乙醛脱氢酶1（aldehyde dehydrogenase1,ALDH1）蛋白的表达,倒置显微镜观察乳腺癌细胞微球体的形成。 结果： 成功制备富含CSC的小鼠乳腺癌细胞移植瘤模型,PTL可下调小鼠肿瘤组织中CD44+CD24-/low细胞的比例\[（42.5±3.7）% vs （68.7±32）%,P<0.05\],有效降低荷瘤小鼠肿瘤组织中SP细胞的比例\[（39.2±1.8）% vs （61.3±2.6）%,P<0.05\],下调小鼠移植瘤组织中CD55和ALDH1蛋白的表达\[（18.9±1.5）% vs （30.1±1.3）%,（8.1±2.3）% vs （18.0±1.4）%;均P<0.05\],抑制小鼠肿瘤细胞在无血清培养条件下形成微球体,并可抑制小鼠移植瘤的体积和重量\[（0.625±0.159）cm3 vs （1.715±0184）cm3,（1.467±0.373）g vs （3.367±0.398）g;均P<0.05\]。 结论： PTL在荷瘤小鼠体内可以明显降低肿瘤组织CSC含量,提示PTL可用来靶向杀伤乳腺癌CSC。     

Long-term culture of primary breast cancer in defined medium

Summary Over a period of 6 1/2 years between January 1986 and May 1992, 135 unselected primary breast cancers were cultured and of these 10 developed into cell lines. Six of the lines grew in defined serum-free medium, while the other four required supplementation with 0.5% fetal calf serum. Two of the lines are from the same breast, being derived from a local excision specimen and from a mastectomy specimen 12 months later. In addition, 12 lymph nodes containing metastatic breast cancer were cultured; one of these cultures became permanent in a defined serum-free medium.Oestrogen receptor (ER) status was negative in all but one of the tumours which grew successfully, and even in this case the derived cell line is ER negative. The epithelial nature of the lines has been confirmed by immunocytochemistry and by electron microscopy (EM), while their malignant nature is shown by morphology, unattached growth, chromosome analysis, and, in the case of the line from a lymph node metastasis, the absence of any benign source of epithelial cells.     

人乳腺癌细胞系RUNX3基因启动子甲基化状态研究

目的：探讨人乳腺癌细胞系中抑癌基因RUNX3启动子甲基化状态,并分析其与RUNX3基因表达的相关性。方法：运用甲基化特异性PCR（MSP）检测5种乳腺癌细胞系和一种人类正常乳腺细胞系中RUNX3启动子甲基化状态,运用RT-PCR和Western印迹检测这些细胞系中RUNX3基因mRNA和蛋白的表达。结果：在6种细胞系中,有两种（T47D、MCF7）呈高甲基化状态,并且这两种细胞系的RUNX3 mRNA和蛋白表达阴性。SKBR3中没有检测出RUNX3启动子区的甲基化,但RUNX3 mRNA和蛋白表达阴性。结论：乳腺癌细胞系中RUNX3基因由于启动子区甲基化而失活,但尚有其它失活机制存在,需进一步研究探讨。     

The impact of bilateral breast cancer on the prognosis of breast cancer: a comparative study with unilateral breast cancer

BACKGROUND: The clinical significance of bilateral breast cancer is unclear and its influence on prognosis is controversial. We assessed the impact of synchronous and metachronous bilateral breast cancer on the prognosis compared with unilateral breast cancer. METHODS: Between January 1, 1960 and December 31, 2001, 1,214 women were treated for primary operable breast cancers. Thirteen (1.1%) had synchronous bilateral breast cancer; 33 (2.7%) had a metachronous contralateral breast cancer. We compared age at operation, menopausal status, clinical stage, tumor size and histology, lymph node status, hormone receptor status, and use of adjuvant chemotherapy or hormone therapy, and we analyzed the impact of these factors on recurrence and survival in the 46 patients with bilateral breast cancer and the 1,168 patients with unilateral breast cancer. RESULTS: The 5-and 10-year disease-free survival rates, respectively, were 65% and 65% in metachronous cases, 85.7% and 64.3% in synchronous cases, and 77.9% and 72.1% in unilateral cases. There was no significant difference in overall survival among the three groups. On multivariate analysis, metachronous bilaterality, tumor size, lymph node status and adjuvant hormone therapy were each independent risk factors for recurrence, whereas bilaterality of breast cancer did not influence overall survival. CONCLUSIONS: Our data suggest that metachronous bilateral breast cancer is associated with shorter disease-free survival than synchronous bilateral or unilateral breast cancer, although overall survival does not differ among the 3 groups. Patients with metachronous bilateral breast cancer should be followed particularly closely in order to detect recurrence early and maximize quality of life.     

乳腺癌患者血浆微泡BCRP mRNA水平的改变及其临床分析

目的：探讨血浆微泡乳腺癌耐药蛋白（BCRP）mRNA水平与乳腺癌化疗疗效及乳腺癌组织相关基因的关系。方法选择接受新辅助化疗的乳腺癌患者60例作为乳腺癌组,选择同时期健康体检者60例作为对照组。检测血浆微泡BCRP mRNA转录水平以及乳腺癌组织相关基因中凋亡基因Caspase-3、Caspase-9和增殖相关基因Survivin、Bcl-2 mRNA含量。结果乳腺癌组患者血浆微泡中BCRP mRNA含量高于对照组,差异有统计学意义（t=31.289,P﹤0.05）;CR/PR组患者血浆微泡中BCRP mRNA含量低于SD/PD组,差异具有统计学意义（P﹤0.05）;乳腺癌组织中凋亡基因Caspase-3、Caspase-9 mRNA含量低于癌旁正常组织,增殖相关基因Survivin、Bcl-2 mRNA含量高于癌旁正常组织,差异有统计学意义（P﹤0.05）。结论血浆微泡中BCRP的mRNA含量与caspase-3、caspase-9 mRNA含量呈负相关,与Survivin、Bcl-2 mRNA含量呈正相关。血浆微泡中BCRP mRNA含量可作为乳腺癌辅助化疗疗效的预测指标,为乳腺癌化疗提供指导依据。     

MicroRNA在乳腺癌中作用的研究进展

MicroRNA（miRNA,miRNAs)是广泛存在于生物中,长度约为20-24个核苷酸的非编码微小RNA分子。目前研究发现,miRNA与肿瘤的发生、进展有紧密联系,且逐渐成为癌症研究的热点。近年来,miRNA在乳腺癌发生与进展中起到的重要作用逐渐被揭示,已迅速发展为乳腺癌等癌症的重要生物标记物。本综述将就微小RNA在乳腺癌的发生、进展、远处转移、早期诊断以及化学治疗耐药性方面的作用进行论述。     

MGBA负载脐带血DC诱导特异性CTL对乳腺癌细胞的杀伤作用

目的：观察乳腺珠蛋白（mammaglobin A, MGBA）负载脐带血来源树突状细胞（dendritic cell, DC）诱导产生的细胞毒性T细胞（cytotoxic T lymphocyte, CTL）对乳腺癌细胞的体外杀伤效果。方法：采集健康剖宫产女性志愿者的脐带血,分离脐带血单个核细胞并诱导DC生成,用MGBA致敏DC与自体淋巴细胞共培养诱导CTL。采用流式细胞术测定DC的表型(CD83、CD86、HLADR)变化,ELISA法测定IL-10、IL-12分泌水平,CCK-8法测定CTL对乳腺癌细胞的杀伤活性。结果：成功培养出形态典型、功能成熟的DC,MGBA负载DC诱导的MGBA特异性CTL对乳腺癌细胞MDA-MB-415 产生显著的杀伤效果（P<0.05）;加入HLA-I 抗体可显著减弱杀伤效果,加入HLA-II 抗体细胞毒活性无显著变化,加入HLA-I 抗体或HLA-II 抗体对正常乳腺细胞的杀伤性均无影响。结论：MGBA 能明显加强脐带血DC 诱导的CTL 对乳腺癌细胞的杀伤活性,该杀伤活性具有MHC限制性。     

Interaction of dense breast patterns with other breast cancer risk factors in a case-control study

The question of interactions between breast density and other breast cancer risk factors is of interest, since it bears upon the use of density as a marker for changes in breast cancer risk. We studied breast parenchymal patterns and 13 other potential risk factors for breast cancer in 172 breast cancer cases and 338 age-matched controls in Singapore. Dense breast patterns were defined as having Tabar parenchymal pattern IV or V. We found significant interactions between dense patterns and ethnic group (P=0.046), and between dense patterns and number of deliveries (P=0.04). Among women with nondense breast patterns, the non-Chinese had lower risk than the Chinese with an odds ratio (OR) of 0.47 (95% CI 0.24, 0.88), whereas in those with dense patterns, the non-Chinese had considerably higher risks (OR=5.34, 95% CI 0.54, 52.51). Alternatively expressed, the increased risk with dense patterns was only observed in the non-Chinese (OR=13.99, 95% CI 1.33, 146.99). Among parous women, the protective effect of three or more deliveries was only observed in those with dense breast patterns (OR=0.21, 95% CI 0.06, 0.70). Suggestive but nonsignificant interactions with dense patterns were observed for ever having delivered, age at first delivery, breast feeding and body mass index. The results are consistent with dense breast patterns as a marker for hormonal modification of breast cancer risk.