A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Tufts University School of Medicine

Small unilamellar vesicles associated with plasmid DNA showed maximum association efficiency for a cationic mixture of egg phosphatidylcholine (EPC):1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE):di-1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP) (16:8:1 molar ratio) [65%], followed by neutral lipids EPC:1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):cholesterol (Chol) (2:2:1 molar ratio) [30%], and a polymerized formulation 1,2-bis(10,12-tricosadiynoyl)sn-glycero-3-phosphocholine (DC8,9PC):DMPE:Chol (2:2:1 molar ratio) [11%]. The hydrophobicity factor (HF) for these formulations followed the trend DC8,9PC:DMPE:CHOL < EPC:DMPE:Chol < EPC:DOPE DOTAP, and DNA association did not alter this trend. Results suggest that the higher the HF value, the more fluid the membrane and the higher the efficiency of DNA association. On the other hand, no differences were observed in cell toxicity with lipids up to 1 mg/ml in VERO cells.

Liposome/DNA systems: correlation between association, hydrophobicity and cell viability.

MC-C fibrosarcoma and B16F0 melanoma tumors were implanted intradermally in the dorsal region of the foot of mice. Tumor progression was compared to standard implantation in the flank. Although foot tumors only reached 13% (MC-C) and 25% (B16F0) of the mean volume of flank tumors, a more malignant phenotype in terms of histology and survival rate was observed in this type of tumors. Moreover, lung metastases were only detected in hosts bearing foot tumors, in contrast to MC-C and B16F0 populations with tumors growing in the flank. In addition, cellular influx and local immune reaction were higher in the dorsal region of the foot. According to our results, the dermis of the flank allows excessive tumor growth due to its low reactivity. Thus, differences in innate and adaptive immune effectors between the evaluated tumor microenvironments would account for the differences in tumor malignancy. Due to its striking differences with the standard flank inoculation, the tumor implantation model herein introduced could be a valuable tool to study the metastatic potential of different cell lines and the microenvironment components affecting tumor growth.

Alternative site of implantation affects tumor malignancy and metastatic potential in mice: its comparison to the flank model.

Murine melanoma B16F0 cells were transfected with SA:DPPC:DOPE (2:1:1 molar ratio) liposomes associated with a plasmid encoding murine IL-12. Stearylamine, a cationic lipid, showed a greater transfection efficiency compared to DOTAP-containing liposomes. The lipid:DNA ratio was 2:1 (w/w). Control groups were mock transfected or transfected with an empty plasmid (pNeo). pNeo or IL-12 transfected cells and controls were inoculated intradermically into the dorsal region of the foot or the lateral flank of C57BL6 mice. Results showed that IL-12 expression had a marked effect on in vivo growth of B16 melanoma tumors developed in both anatomic sites, significantly retarding their growth and prolonging host survival.

Antitumoral effect of IL-12 gene transfected via liposomes into B16F0 cells.

In this work, we analyzed protein interaction, cell toxicity, and biodistribution of liposome formulation for further possible applications as DNA vehicles in gene-therapy protocols. In relation to protein interaction, cationic liposomes showed the lowest protein interaction, but this parameter was incremented with DNA association. On the other hand, noncharged liposomes presented high protein interaction, but DNA association decreased this parameter. Protein interaction of polymeric liposomes did not change with DNA association. Cell toxicity of these three liposome formulations was low, cell death became present at concentrations higher than 0.5 mg/mL, and these concentrations were higher than those usually used in transfection assays. In the case of noncharged and polymeric liposomes, toxicity increased upon interaction with serum proteins. DNA/liposome-mediated tissue distribution was analyzed in Balb-c female mice. Results indicated that noncharged liposomes were able to deliver DNA to liver after intraperitoneal (i.p.) inoculation, while polymeric liposomes were able to deliver DNA to kidney by using the same inoculation route. Cationic liposomes were able to deliver DNA to a wide range of tissues by the i.p. route (e.g., liver, intestine, kidney, and blood). After subcutaneous inoculation, only cationic liposomes were able to deliver DNA to blood, but not the other two formulations within the detection limits of the method.

Biodistribution of liposome/DNA systems after subcutaneous and intraperitoneal inoculation.

Vaccine development today encounters a main obstacle, which is the need for effective adjuvants suitable for clinical trials. Aluminum salts, discovered 70 years ago and, very recently, MF59, are the only types of adjuvants currently used in vaccines licensed by the U.S. Food and Drug Administration. Liposomes represent an alternative approach to vaccine adjuvants. In this article, we describe the inflammatory response and biological effect of polymerization and the addition of positive charges in liposome formulations. Nonpolymerized cationic (NP(+)) liposomes significantly reduce metabolism in Vero cells after 24 hours. Correspondingly, both NP(+) and polymerized cationic (P(+)) liposomes reduce cell viability following a 48-hour incubation. Similar results were obtained with cells from the peritoneal cavities of mice. Paradoxically, those liposomes that presented clearly cytostatic or cytotoxic effects in vitro stimulated metabolism and had a mitogenic effect in vivo. Finally, the adjuvant effect was tested by immunization in BALB/c mice. The major effect was obtained with NP(+) liposomes. Accordingly, we also demonstrated that NP(+) liposomes injected into the dermis produced an outstanding inflammatory reaction, showing the histopathological characteristics of an inoculation granuloma. Thus, positive charge would play an important role in the immunoadjuvant effect of liposomes by conferring them cytotoxic capacity.

Relationship between the adjuvant and cytotoxic effects of the positive charges and polymerization in liposomes.

The establishment of hormone target breast cells in the 1970's resulted in suitable models for the study of hormone control of cell proliferation and gene expression using two-dimensional (2D) cultures. However, in order to study mammogenesis and breast tumor development in vitro, cells must be able to organize in three-dimensional (3D) structures like in the tissue. We now report the development of a hormone- sensitive three-dimensional (3D) culture model for the study of mammogenesis and neoplastic development. Hormone-sensitive T47D breast cancer cells respond to estradiol in a dose dependent manner by forming complex epithelial structures. Treatment with the synthetic progestagen promegestone, in the presence of estradiol, results in flat epithelial structures that display cytoplasmic projections, a phenomenon reported to precede side-branching. Additionally, as in the mammary gland, treatment with prolactin in the presence of estradiol induces budding structures. These changes in epithelial organization are accompanied by collagen remodeling. Collagen is the major acellular component of the breast stroma and an important player in tumor development and progression. Quantitative analysis of second harmonic generation of collagen fibers revealed that collagen density was more variable surrounding budding and irregularly shaped structures when compared to more regular structures; suggesting that fiber organization in the former is more anisotropic than in the latter. In sum, this new 3D model recapitulates morphogenetic events modulated by mammogenic hormones in the breast, and is suitable for the evaluation of therapeutic agents.

Hormonal regulation of epithelial organization in a three-dimensional breast tissue culture model.

La mise en place de cellules de cancer du sein hormone cibles dans les ann&eacute;es 1970 a entra&icirc;n&eacute; des mod&egrave;les appropri&eacute;s pour l&#39;&eacute;tude du contr&ocirc;le de l&#39;hormone de cellule prolif&eacute;ration et l&#39;expression g&eacute;nique en utilisant des cultures &agrave; deux dimensions (2D). Toutefois, afin d&#39;&eacute;tudier le d&eacute;veloppement de tumeurs du sein et de la mammogenesis in vitro, les cellules doivent pouvoir organiser dans des structures tridimensionnelles (3D) comme dans le tissu. Nous rapportons l&#39;&eacute;laboration d&#39;un mod&egrave;le hormono-sensible en trois dimensions (3D) culture for the study of mammogenesis et d&eacute;veloppement n&eacute;oplasique. Cellules de cancer du sein hormono-T47D r&eacute;pondent &agrave; estradiol dans une fa&ccedil;on dose-d&eacute;pendante, en formant des structures &eacute;pith&eacute;liales complexes. Le traitement par le progestatif synth&eacute;tique promegestone, en pr&eacute;sence d&#39;estradiol, se traduit par des structures &eacute;pith&eacute;liales plates qui affichent des projections cytoplasmiques, un ph&eacute;nom&egrave;ne signal&eacute; &agrave; pr&eacute;c&eacute;der la ramification lat&eacute;rale. En outre, comme dans la glande mammaire, traitement avec la prolactine en pr&eacute;sence d&#39;estradiol induit des structures en herbe. Ces changements dans l&#39;organisation &eacute;pith&eacute;liale sont accompagn&eacute;es de remodelage du collag&egrave;ne. Le collag&egrave;ne est le principal composant acellulaire du stroma du sein et un acteur important dans le d&eacute;veloppement de tumeurs et de la progression. Analyse quantitative des fibres de collag&egrave;ne de deuxi&egrave;me g&eacute;n&eacute;ration harmonique a r&eacute;v&eacute;l&eacute; que la densit&eacute; de collag&egrave;ne est plus variable autour de structures de bourgeonnement et de formes irr&eacute;guli&egrave;re par rapport aux structures plus r&eacute;guli&egrave;res ; sugg&eacute;rant qu&#39;il s&#39;agit d&#39;une organisation de fibre dans l&#39;ancienne plus anisotrope que dans ce dernier. En somme, ce nouveau mod&egrave;le 3D r&eacute;capitule les &eacute;v&eacute;nements morphog&eacute;n&eacute;tiques modul&eacute;es par les hormones de mammogenic dans le sein et est adapt&eacute; pour l&#39;&eacute;valuation des agents th&eacute;rapeutiques.

R&eacute;gulation hormonale de l&#39;organisation &eacute;pith&eacute;liale dans un mod&egrave;le de culture de tissus du sein en trois dimensions.

Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma--epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis.

From single cells to tissues: interactions between the matrix and human breast cells in real time.

Iatrogenic gestational exposure to diethylstilbestrol (DES) induced alterations of the genital tract and predisposed individuals to develop clear cell carcinoma of the vagina as well as breast cancer later in life. Gestational exposure of rodents to a related compound, the xenoestrogen bisphenol-A (BPA) increases the propensity to develop mammary cancer during adulthood, long after cessation of exposure. Exposure to BPA during gestation induces morphological alterations in both the stroma and the epithelium of the fetal mammary gland at 18 days of age. We postulate that the primary target of BPA is the fetal stroma, the only mammary tissue expressing estrogen receptors during fetal life. BPA would then alter the reciprocal stroma-epithelial interactions that mediate mammogenesis. In addition to this direct effect on the mammary gland, BPA is postulated to affect the hypothalamus and thus in turn affect the regulation of mammotropic hormones at puberty and beyond.

Estrogens in the wrong place at the wrong time: Fetal BPA exposure and mammary cancer.

Defining fiber orientation at each voxel within a 3D biomedical image stack is potentially useful for a variety of applications, including cancer, wound healing and tissue regeneration. Current methods are typically computationally intensive or inaccurate. Herein, we present a 3D weighted orientation vector summation algorithm, which is a generalization of a previously reported 2D vector summation technique aimed at quantifying collagen fiber orientations simultaneously at each voxel of an image stack. As a result, voxel-wise fiber orientation information with 4° to 5° accuracy can be determined, and the computational time required to analyze a typical stack with the size of 512x512x100 voxels is less than 5 min. Thus, this technique enables the practical extraction of voxel-specific orientation data for characterizing structural anisotropy in 3D specimens. As examples, we use this approach to characterize the fiber organization in an excised mouse mammary gland and a 3D breast tissue model.

Lucia Speroni

Department of Integrative Physiology and Pathobiology

Tufts University School of Medicine

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

Lucia Speroni

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Integrative Physiology and Pathobiology

Tufts University School of Medicine

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

Department of Integrative Physiology and Pathobiology