MCF-7 Xenograft Model Research Articles

Estrogen-related receptor-α antagonist inhibits both estrogen receptor–positive and estrogen receptor–negative breast tumor growth in mouse xenografts

Estrogen-related receptors (ERR) are orphan members of the nuclear receptor superfamily most closely related to estrogen receptors (ER). Although ERalpha is a successful target for treating breast cancer, there remains an unmet medical need especially for estrogen-independent breast cancer. Although estradiol is not an ERR ligand, ER and ERR share many commonalities and overlapping signaling pathways. An endogenous ERR ligand has not been identified; however, novel synthetic ERRalpha subtype-specific antagonists have started to emerge. In particular, we recently identified a novel compound, N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5H dibenzo[a,d][7]annulen-5-amine (termed compound A) that acts specifically as an ERRalpha antagonist. Here, we show that compound A inhibited cell proliferation in ERalpha-positive (MCF-7 and T47D) and ERalpha-negative (BT-20 and MDA-MD-231) breast cancer cell lines. Furthermore, we report the differential expression of 83 genes involved in ERRalpha signaling in MCF-7 and BT-20 breast cancer cell lines. We show that compound A slowed tumor growth in MCF-7 and BT-20 mouse xenograft models, and displayed antagonistic effects on the uterus. Furthermore, a subset of genes involved in ERRalpha signaling in vitro were evaluated and confirmed in vivo by studying uterine gene expression profiles from xenograft mice. These results suggest for the first time that inhibition of ERRalpha signaling via a subtype-specific antagonist may be an effective therapeutic strategy for ER-positive and ER-negative breast cancers.

2-Methoxyoestradiol-3,17-O,O-bis-sulphamate and 2-deoxy-D-glucose in combination: a potential treatment for breast and prostate cancer

Drug combination therapy is a key strategy to improve treatment efficacy and survival of cancer patients. In this study the effects of combining 2-methoxyoestradiol-3,17-O,O-bis-sulphamate (STX140), a microtubule disruptor, with 2-deoxy-D-glucose (2DG) were assessed in MCF-7 (breast) and LNCaP (prostate) xenograft models in vivo. In mice bearing MCF-7 xenografts, daily p.o. administration of STX140 (5 mg kg−1) resulted in a 46% (P<0.05) reduction of tumour volume. However, the combination of STX140 (5 mg kg−1 p.o.) and 2DG (2 g kg−1 i.p.) reduced tumour volume by 76% (P<0.001). 2-Methoxyoestradiol-3,17-O,O-bis-sulphamate also reduced tumour vessel density. 2-Deoxy-D-glucose alone had no significant effect on tumour volume or vessel density. A similar benefit of the combination treatment was observed in the LNCaP prostate xenograft model. In vitro the degree of inhibition of cell proliferation by STX140 was unaffected by oxygen concentrations. In contrast, the inhibition of proliferation by 2DG was enhanced under hypoxia by 20 and 25% in MCF-7 and LNCaP cells, respectively. The combination of STX140 and 2DG in LNCaP cells under normoxia or hypoxia inhibited proliferation to a greater extent than either compound alone. These results suggest that the antiangiogenic and microtubule disruption activities of STX140 may make tumours more susceptible to inhibition of glycolysis by 2DG. This is the first study to show the benefit of combining a microtubule disruptor with 2DG in the two most common solid tumours.

Enzastaurin renders MCF-7 breast cancer cells sensitive to radiation through reversal of radiation-induced activation of protein kinase C

Enzastaurin (LY317615.HCI), a protein kinase C (PKC)-β inhibitor, has a radiosensitising effect on 4T1 murine breast cancer and human glioma cells; however, the exact mechanism of this action has not been evaluated. The present study investigated the effects of enzastaurin and gamma irradiation on PKC activity in MCF-7 human breast cancer cells in vitro and in vivo. Enzastaurin (5μM) in combination with irradiation (2–8Gy) produced a synergistic decline in MCF-7 clonogenic cell survival. Analysis of MCF-7 cells stained with Annexin V and 7-aminoactinomycin D showed a dose-dependent increase in apoptosis in response to enzastaurin (3, 5 and 7μM) and irradiation (10Gy) compared to irradiation alone. This pro-apoptotic effect was confirmed by increases in caspase-3 and -9 activity. In a MCF-7 xenograft model, irradiation with 25Gy increased PKC-α activity by 2.5-fold compared to untreated controls, whereas PKC-ε and -βII activity was increased by 1.8-fold. Radiation-induced activation of all three anti-apoptotic isoforms of PKC was reversed by pre-treatment with enzastaurin (75mg/kg, twice daily for 3 days). We conclude that enzastaurin has a radiosensitising effect on MCF-7 human xenograft tumours through the reversal of anti-apoptotic activation of PKC isoforms.

Impaired p53 function leads to centrosome amplification, acquired ERα phenotypic heterogeneity and distant metastases in breast cancer MCF-7 xenografts

In this study, we establish an MCF-7 xenograft model that mimics the progression of human breast carcinomas typified by loss of p53 integrity, development of centrosome amplification, acquired estrogen receptor (ERalpha) heterogeneity, overexpression of Mdm2 and metastatic spread from the primary tumor to distant organs. MCF-7 cells with abrogated p53 function (vMCF-7(Dnp53)) maintained nuclear ERalpha expression and normal centrosome characteristics in vitro. However, following mitogen stimulation, they developed centrosome amplification and a higher frequency of aberrant mitotic spindles. Centrosome amplification was dependent on cdk2/cyclin activity since treatment with the small molecule inhibitor SU9516 suppressed centriole reduplication. In contrast to the parental MCF-7 cells, when introduced into nude mice as xenografts, tumors derived from the vMCF-7(DNp53) cell line developed a strikingly altered phenotype characterized by increased tumor growth, higher tumor histopathology grade, centrosome amplification, loss of nuclear ERalpha expression, increased expression of Mdm-2 oncoprotein and resistance to the antiestrogen tamoxifen. Importantly, while MCF-7 xenografts did not develop distant metastases, primary tumors derived from vMCF-7(DNp53) cells gave rise to lung metastases. Taken together, these observations indicate that abrogation of p53 function and consequent deregulation of the G1/S cell cycle transition leads to centrosome amplification responsible for breast cancer progression.

Bioluminescent Monitoring of NIS-Mediated 131 I Ablative Effects in MCF-7 Xenografts

Optical imaging has made it possible to monitor response to anticancer therapies in tumor xenografts. The concept of treating breast cancers with (131)I is predicated on the expression of the Na(+)/I- symporter (NIS) in many tumors and uptake of I- in some. The pattern of (131)I radioablative effects were investigated in an MCF-7 xenograft model dually transfected with firefly luciferase and NIS genes. On Day 16 after tumor cell implantation, 3 mCi of (131)I was injected. Bioluminescent imaging using d-luciferin and a cooled charge-coupled device camera was carried out on Days 1, 2, 3, 7, 10, 16, 22, 29, and 35. Tumor bioluminescence decreased in (131)I-treated tumors after Day 3 and reached a nadir on Day 22. Conversely, bioluminescence steadily increased in controls and was 3.85-fold higher than in treated tumors on Day 22. Bioluminescence in (131)I-treated tumors increased after Day 22, corresponding to tumor regrowth. By Day 35, treated tumors were smaller and accumulated 33% less (99m)TcO(4)(-) than untreated tumors. NIS immunoreactivity was present in <50% of (131)I-treated cells compared to 85-90% of controls. In summary, a pattern of tumor regression occurring over the first three weeks after (131)I administration was observed in NIS-expressing breast cancer xenografts.

Proceedings of the Fourth International Conference on Recent Advances and Future Directions in Endocrine Manipulation of Breast Cancer: Conference Summary Statement

Proceedings of the Fourth International Conference on Recent Advances and Future Directions in Endocrine Manipulation of Breast Cancer: Conference Summary Statement

Interleukin-1α Promotes Tumor Growth and Cachexia in MCF-7 Xenograft Model of Breast Cancer

Progression of breast cancer involves cross-talk between epithelial and stromal cells. This cross-talk is mediated by growth factors and cytokines secreted by both cancer and stromal cells. We previously reported expression of interleukin (IL)-1 alpha in a subset of breast cancers and demonstrated that IL-1 alpha is an autocrine and paracrine inducer of prometastatic genes in in vitro systems. To understand the role of IL-1 alpha in breast cancer progression in vivo, we studied the growth of MCF-7 breast cancer cells overexpressing a secreted form of IL-1 alpha (MCF-7IL-1 alpha) in nude mice. MCF-7IL-1 alpha cells formed rapidly growing estrogen-dependent tumors compared to parental cells. Interestingly, IL-1 alpha expression alone was not sufficient for metastasis in vivo although in vitro studies showed induction of several prometastatic genes and matrix metalloproteinase activity in response to cross-talk between IL-1 alpha-expressing cancer cells and fibroblasts. Animals implanted with MCF-7IL-1 alpha cells were cachetic, which correlated with increased leptin serum levels but not other known cachexia-inducing cytokines such as IL-6, tumor necrosis factor, or interferon gamma. Serum triglycerides, but not blood glucose were lower in animals with MCF-7IL-1 alpha cell-derived tumors compared to animals with control cell-derived tumors. Cachexia was associated with atrophy of epidermal and adnexal structures of skin; a similar phenotype is reported in triglyceride-deficient mice and in ob/ob mice injected with leptin. Mouse leptin-specific transcripts could be detected only in MCF-7IL-1 alpha cell-derived tumors, which suggests that IL-1 alpha increases leptin expression in stromal cells recruited into the tumor microenvironment. Despite increased serum leptin levels, animals with MCF-7IL-1 alpha cell-derived tumors were not anorexic suggesting only peripheral action of tumor-derived leptin, which principally targets lipid metabolism. Taken together, these results suggest that cancer cell-derived cytokines, such as IL-1 alpha, induce cachexia by affecting leptin-dependent metabolic pathways.

Tumor targeting using anti-her2 immunoliposomes

We have generated anti-HER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2-overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2-overexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and non-immunogenicity of sterically stabilized liposomes. In five different HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox+MAb or liposomal dox+MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes ( P values from <0.0001 to 0.04 in eight separate experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs and non-targeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel mechanism of drug delivery for anti-HER2 ILs. Immunotargeting did not increase tumor tissue levels of ILs vs. liposomes, as both achieved very high tumor localization (7.0–8.5% of injected dose/g tissue) in xenograft tumors. However, histologic studies using colloidal-gold labeled ILs demonstrated efficient intracellular delivery in tumor cells, while non-targeted liposomes accumulated within stroma, either extracellularly or within macrophages. In the MCF7 xenograft model lacking HER2-overexpression, no difference in tumor cell uptake was seen, with both ILs and non-targeted liposomes accumulating within stroma. Thus, anti-HER2 ILs, but not non-targeted liposomes, achieve intracellular drug delivery via receptor-mediated endocytosis, and this mechanism is associated with superior antitumor activity. Based on these results, anti-HER2 immunoliposomes have been developed toward clinical trials. Reengineering of construct design for clinical use has been achieved, including: new anti-HER2 scFv F5 generated by screening of a phage antibody library for internalizing anti-HER2 phage antibodies; modifications of the scFv expression construct to support large scale production and clinical use; and development of methods for large-scale conjugation of antibody fragments with liposomes. We developed a scalable two-step protocol for linkage of scFv to preformed and drug-loaded liposomes. Our final, optimized anti-HER2 ILs-dox construct consists of F5 conjugated to derivatized PEG-PE linker and incorporated into commercially available liposomal doxorubicin (Doxil ®). Finally, further studies of the mechanism of action of anti-HER2 ILs-dox suggest that this strategy may provide optimal delivery of anthracycline-based chemotherapy to HER2-overexpressing cancer cells in the clinic, while circumventing the cardiotoxicity associated with trastuzumab+anthracycline. We conclude that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery, and that this strategy may also be applicable to other receptor targets and/or using other delivered agents.

Tamoxifen increases apoptosis but does not influence markers of proliferation in an MCF-7 xenograft model of breast cancer

Twenty-four nude mice bearing MCF-7 breast cancer cells grown as xenografts and treated with tamoxifen (2.5 mg slow-release pellet) were studied for up to 35 days. Tumour size was measured in 2 dimensions at regular time-intervals and tumours were harvested on each of days 2, 4, 7, 14, 28 and 35 after the start of treatment. Control animals (8) received no treatment and the tumours were harvested after 0 or 35 days. Tumour sections were assessed for prevalence of apoptosis and mitosis and examined immunocytochemically for Ki67(MIB-1) and bcl-2 expression. Tumours increased in size during tamoxifen-treatment, but at a significantly slower rate (max. 2.6-fold) than in the untreated control animals; thus tumours not actually regressing may, nevertheless, be responding significantly to tamoxifen.MIB-1 and bcl-2 immunostaining and mitosis failed to show any consistent change over the period of study. Apoptosis, however, increased progressively and significantly to day-28 in tamoxifen-treated tumours, reaching approximately a 5-fold increase over day-0 values, then decreasing again to nearly 3-fold by day-35 (P= 0.0002). The apoptosis: mitosis ratio in treated tumours also increased to approximately 10-fold on day-28 over day-0 values, decreasing to nearly 4-fold by day-35 (P= 0.037). Within the treated group, apoptosis was significantly inversely correlated with both mitosis (R = −0.38, P= 0.03) and expression of bcl-2 (R = −0.48, P= 0.0056) and strongly positively correlated with both time on tamoxifen (R = +0.63, P= 0.0003) and the % inhibition of growth by tamoxifen (R = +0.58,P = 0.0012) in the 28 individual, treated tumours (estimated relative to the mean growth rate in the controls). The apoptosis: mitosis ratio was also inversely correlated with bcl-2 expression (R = −0.56, P= 0.0021) and positively correlated with both time on tamoxifen (R = +0.50, P= 0.0068) and % inhibition of growth (R = +0.56, P= 0.0019).In this hormone-sensitive tumour model for breast cancer, in which tamoxifen caused inhibition rather than regression, it was not possible to detect significant changes in the marker proteins Ki67and bcl-2, or in the prevalence of mitosis in relation to treatment; these factors may therefore not be accurate indices of response to tamoxifen in all situations. By contrast, however, tamoxifen induced a significant, early increase in the prevalence of apoptosis associated with inhibition of tumour growth and an inverse relationship in both mitosis and bcl-2 expression, suggesting that apoptosis may be an accurate and sensitive early marker of even a moderate response to tamoxifen.