elastin and Breast-Neoplasms

This mini-review summarizes our recent experimental and clinical studies on neutrophil elastase (NE) and cancer based on our original view point. Neoplasms metastasize as a result of a complex series of events. This process requires various degradative enzymes including proteases. NE has broad substrate specificity under physiological conditions, and excessive NE results in digestion of not only elastin, but also other extracellular matrix proteins. Several cell lines from human breast cancer and human lung cancer produce immunoreactive NE. The amount of immunoreactive NE in tumor tissue is an independent prognostic indicator of patients with breast cancer and lung cancer. Furthermore, a specific NE inhibitor completely suppressed growth of cancer cells transplanted into severe combined immunodeficiency mice. The use of NE inhibitor would seem to be a promising way to prevent the invasion and metastasis of cancer.

Topics: Animals; Breast Neoplasms; Elastin; Enzyme Inhibitors; Humans; Leukocyte Elastase; Lung Neoplasms; Neoplasm Metastasis; Prognosis

This paper underlines the interrelations between tumoral cells and the extra-cellular matrix in breast cancers with possible applications for the diagnosis and the prognosis. In mammary carcinomas, the first step of tumoral invasion is characterized by the loss of basement membrane components, particularly type IV collagen and laminin. Immunohistochemical detection of these disruptions of basement membrane is easy and useful for the diagnosis of "in situ" or microinvasive carcinomas. Laminin seems also to facilitate the adhesion of cancer cells to type IV collagen, and the dosage of its fragment P1 in the blood serum may be a good marker for the follow up of the patients. Stromal reaction involves many intricate macromolecules of the extra-cellular matrix. Types I and III collagens are often present in non colloid carcinomas. Rate, turn over of elastin and its prognostic value are still debated. Elastosis is related to well differentiated carcinomas and the presence of estrogen receptors. The stroma of the colloid form of breast cancer is rich in proteoglycans. Malignant and stromal cells, through the intermediary of cytokines, can synthesize these macromolecules. Hyaluronic acid and chondroitin sulfate are abundant in mammary carcinomas and form a favorable substrate for the growth and the migration of malignant cells. However, proteases decrease and limit their action. The presence of fibronectin, principally in the stroma, is difficult to interpret but fibronectin seems to play a role in tumoral retraction.

Topics: Basement Membrane; Breast Neoplasms; Collagen; Elastin; Extracellular Matrix; Female; Fibronectins; Humans; Laminin; Proteoglycans

Topics: Animals; Basement Membrane; Breast Neoplasms; Carcinoma; Cell Communication; Collagen; Elastin; Extracellular Space; Humans; Microbial Collagenase; Neoplasm Invasiveness; Neoplasm Metastasis; Receptors, Immunologic; Receptors, Laminin

The standard in nipple reconstruction remains the autologous skin flap. Unfortunately, the results are not satisfying, with up to 75% loss of nipple projection over time. Existing studies investigated the use of primates as a source of implants. The authors hypothesized that the porcine nipple can serve as a perfect shape-supporting implant because of functional similarities to the human nipple. A decellularization protocol was developed to obtain an acellular nipple scaffold (ANS) for nipple reconstruction.. Tissue samples were collected from eight disease-free female Yorkshire pigs (60 to 70 kg) and then decellularized. The decellularization efficiency and extracellular matrix characterization was performed histologically and quantitatively (DNA, total collagen, elastin, and glycosaminoglycan content). In vitro and in vivo biocompatibility was determined by human dermal fibroblast culture and subcutaneous implantation of six ANSs in a single Yorkshire pig (60 to 70 kg), respectively. Inflammation and adverse events were monitored daily based on local clinical signs.. The authors showed that all cellular structures and 96% of DNA [321.7 ± 57.6 ng DNA/mg wet tissue versus 11.7 ± 10.9 ng DNA/mg wet tissue, in native and ANS, respectively ( P < 0.001)] can be successfully removed. However, this was associated with a decrease in collagen [89.0 ± 11.4 and 58.8 ± 9.6 μg collagen/mg ( P < 0.001)] and elastin [14.2 ± 1.6 and 7.9 ± 2.4 μg elastin/mg ( P < 0.05)] and an increase in glycosaminoglycan content [5.0 ± 0.7 and 6.0 ± 0.8 ng/mg ( P < 0.05)]. ANS can support continuous cell growth in vitro and during preliminary biocompatibility tests in vivo.. This is a preliminary report of a novel promising ANS for nipple reconstruction, but more research is needed to validate results.. Breast cancer is very common among women. Treatment involves mastectomy, but its consequences affect patient mental well-being, and can lead to depression. Nipple-areola complex reconstruction is critical, and existing methods lead to unsatisfactory outcomes.

Topics: Animals; Breast Neoplasms; Collagen; DNA; Elastin; Female; Glycosaminoglycans; Humans; Mammaplasty; Mastectomy; Nipples; Retrospective Studies; Swine

The involvement of the extracellular matrix (ECM) in tumor progression has motivated the development of biomaterials mimicking the tumor ECM to develop more predictive cancer models. Particularly, polypeptides based on elastin could be an interesting approach to mimic the ECM due to their tunable properties. Here, we demonstrated that elastin-like recombinamer (ELR) hydrogels can be suitable biomaterials to develop breast cancer models. This hydrogel was formed by two ELR polypeptides, one containing sequences biodegradable by matrix metalloproteinase and cyclooctyne and the other carrying arginylglycylaspartic acid and azide groups to allow cell adhesion, biodegradability, and suitable stiffness through "click-chemistry" cross-linking. Our findings show that breast cancer or nontumorigenic breast cells showed high viability and cell proliferation for up to 7 days. MCF7 and MCF10A formed spheroids whereas MDA-MB-231 formed cell networks, with the expression of ECM and high drug resistance in all cases, evidencing that ELR hydrogels are a promising biomaterial for breast cancer modeling.

Topics: Biocompatible Materials; Breast Neoplasms; Elastin; Extracellular Matrix; Female; Humans; Hydrogels; Peptides

Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and dose-limiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these "Hydra-ELPs" toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucose-regulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by ∼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.

Topics: Animals; Breast Neoplasms; Drug Carriers; Elastin; Endoplasmic Reticulum Chaperone BiP; Female; Humans; Hydra; Peptides; Sirolimus; TOR Serine-Threonine Kinases

Elastin-like polypeptides (ELPs) undergo a characteristic phase transition in response to ambient temperature. Therefore, it has been be used as a thermosensitive vector for the delivery of chemotherapy agents since it can be used to target hyperthermic tumors. This novel strategy introduces unprecedented options for treating cancer with fewer concerns about side effects. In this study, the ELP system was further modified with an enzyme-cleavable linker in order to release drugs within tumors. This system consists of an ELP, a matrix metalloproteinase (MMP) substrate, a cell-penetrating peptide (CPP), and a 6-maleimidocaproyl amide derivative of doxorubicin (Dox). This strategy shows up to a 4-fold increase in cell penetration and results in more death in breast cancer cells compared to ELP-Dox. Even in doxorubicin-resistant cells (NCI/ADR and MES-SA/Dx5), ELP-released cell-penetrating doxorubicin demonstrated better membrane penetration, leading to at least twice the killing of resistant cells compared to ELP-Dox and free Dox. MMP-digested CPP-Dox showed better membrane penetration and induced more cancer cell death in vitro. This CPP-complexed Dox released from the ELP killed even Dox-resistant cells more efficiently than both free doxorubicin and non-cleaved ELP-CPP-Dox.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell-Penetrating Peptides; Doxorubicin; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Resistance, Neoplasm; Elastin; Female; Fluorescent Dyes; Humans; Matrix Metalloproteinase 2; Peptides; Rhodamines

Topics: Animals; Biological Products; Blood Pressure; Body Weight; Breast Neoplasms; Capillary Permeability; Disease Models, Animal; Elastin; Female; Gene Expression; Glomerular Filtration Rate; Heterografts; Humans; Hypotension; Mice; Molecular Mimicry; Neovascularization, Pathologic; Neovascularization, Physiologic; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Renal Insufficiency, Chronic; Swine; Toxicity Tests, Chronic; Vascular Endothelial Growth Factor A; X-Ray Microtomography

The tumor microenvironment plays a critical role in breast cancer progression. Here, we investigated tumor-infiltrating lymphocytes (TILs) and associations with macrophage numbers, tumor stromal elastosis, vascular invasion, and tumor detection mode. We performed a population-based retrospective study using data from The Norwegian Breast Cancer Screening Program in Vestfold County (2004-2009), including 200 screen-detected and 82 interval cancers. The number of TILs (CD45+, CD3+, CD4+, CD8+, and FOXP3+) and tumor-associated macrophages (CD163+) was counted using immunohistochemistry on tissue microarray slides. Lymphatic and blood vessel invasion (LVI and BVI) were recorded using D2-40 and CD31 staining, and the amount of elastosis (high/low) was determined on regular HE-stained slides. High numbers of all TIL subsets were associated with LVI (p ≤ 0.04 for all), and high counts of several TIL subgroups (CD8+, CD45+, and FOXP3+) were associated with BVI (p ≤ 0.04 for all). Increased levels of all TIL subsets, except CD4+, were associated with estrogen receptor-negative tumors (p < 0.001) and high tumor cell proliferation by Ki67 (p < 0.001). Furthermore, high levels of all TIL subsets were associated with high macrophage counts (p < 0.001) and low-grade stromal elastosis (p ≤ 0.02). High counts of CD3+, CD8+, and FOXP3+ TILs were associated with interval detected tumors (p ≤ 0.04 for all). Finally, in the luminal A subgroup, high levels of CD3+ and FOXP3+ TILs were associated with shorter recurrence-free survival, and high counts of FOXP3+ were linked to reduced breast cancer-specific survival. In conclusion, higher levels of different TIL subsets were associated with stromal features such as high macrophage counts (CD163+), presence of vascular invasion, absence of stromal elastosis, as well as increased tumor cell proliferation and interval detection mode. Our findings support a link between immune cells and vascular invasion in more aggressive breast cancer. Notably, presence of TIL subsets showed prognostic value within the luminal A category.

Topics: Aged; Antigens, CD; Biomarkers, Tumor; Breast Neoplasms; Disease-Free Survival; Elastin; Humans; Immunohistochemistry; Ki-67 Antigen; Lymphocyte Subsets; Lymphocytes, Tumor-Infiltrating; Macrophages; Middle Aged; Neoplasm Recurrence, Local; Neovascularization, Pathologic; Prognosis; Receptor, ErbB-2; Retrospective Studies; Tumor Microenvironment

The complexity and continuous evolution of cancer make the design of novel strategies of treatment a constant challenge in biomedicine. Moreover, most of cancer treatments are still not tumor-specific and provoke high systemic toxicity. Herein we have developed a novel selective nanodevice to eliminate tumor cells while leaving healthy ones intact. To achieve this objective, a polyplex carrier, comprising an elastin like-recombinamer covalently conjugated to an aptamer and complexed with therapeutic DNA, was tested. This carrier forms a double-lock multifunctional device due to specific binding to a tumor cell marker and the selective expression of therapeutic DNA inside human breast-cancer cells. Due to the stability provided by ELRs, the homogeneous population of polyplexes obtained showed selective toxicity against cancer cells in in vitro and in vivo assay. Inhibition of tumor progression was detected early being very significant at the end point, with a dose-dependent reduction in tumor mass. Histological studies revealed a specific reduction in tumor parenchyma and in specific tumor cell markers. These results represent an important step toward the rational development of an efficient, safe and more specialized gene-delivery device for tumor therapy.

Topics: Animals; Aptamers, Nucleotide; Breast Neoplasms; Cell Survival; Disease Progression; Elastin; Female; Gene Transfer Techniques; Genes, Transgenic, Suicide; Genetic Therapy; Genetic Vectors; Hep G2 Cells; Humans; MCF-7 Cells; Mice; Minisatellite Repeats; Mucin-1; Nanoparticles; Tumor Burden; Xenograft Model Antitumor Assays

The clinical utility of rapamycin (Rapa) is limited by solubility, bioavailability, and side effects. To overcome this, our team recently reported an elastin-like polypeptide (ELP) nanoparticle with high affinity, noncovalent drug binding, and integrin-mediated cellular uptake. Given the scarcity of pharmacology/toxicology studies of ELP-based drug carriers, this article explores safety and efficacy of ELP-Rapa. ELP-Rapa nanoparticles tested negative for hemolysis, did not interfere in plasma coagulation nor in platelet function, and did not activate the complement. Upon incubation with HepG2 cells, ELP-Rapa revealed significant cellular uptake and trafficking to acidic organelles, consistent with lysosomes. Internalized ELP-Rapa nanoparticles increased oxidative stress 4-fold compared to free drug or free ELP controls. However, mice bearing orthotopic hormone receptor positive BT-474 breast tumors, given a high dose (∼10-fold above therapeutic dose) of 1 month administration of ELP-Rapa, did not induce hepatotoxicity. On the other hand, tumor growth and mTOR signaling were suppressed without affecting body weight. Nanoparticles assembled using ELP technology appear to be a safe and efficient strategy for delivering Rapa.

Topics: Animals; Breast Neoplasms; Drug Carriers; Elastin; Female; Humans; Mice; Peptides; Sirolimus

Blood vessel invasion (BVI) is a prognostic indicator in various cancers. Elastic stain, which highlights blood vessel walls, is commonly used to detect BVI. In the breast, however, its diagnostic usefulness is limited because it also highlights some intraductal carcinoma components, which often mimic BVI. In this study, we aimed to improve BVI detection in breast cancer and developed a double staining: Victoria blue for elastin and immunohistochemistry for collagen IV. Collagen IV fibers were retained along the basement membranes of intraductal carcinoma components, whereas they were rearranged or lost in BVI. From these observations, we defined BVI as the presence of tumor cells inside an elastic ring with a rearrangement or loss of collagen IV fibers. Using these criteria, we found BVI in 148 cases (49%) among 304 cases of primary operable invasive breast carcinoma, and the presence of BVI correlated significantly with poor prognosis. By contrast, we detected BVI in 94 cases (31%) or 14 cases (5%) by elastic van Gieson or CD31 immunostaining among the same cases, respectively, with no statistically significant association with prognosis. Thus, elastin and collagen IV double staining facilitates the detection of BVI in breast cancer and is useful to predict prognosis.

Topics: Breast; Breast Neoplasms; Carcinoma, Ductal; Collagen; Elastin; Female; Humans; Immunohistochemistry; Neovascularization, Pathologic; Prognosis; Staining and Labeling

The ability to distinguish and grade malignant cells during surgical procedures in a fast, non-invasive and staining-free manner is of high importance in tumor management. To this extend, Third Harmonic Generation (THG), Second Harmonic Generation (SHG) and Fourier-Transform Infrared (FTIR) spectroscopy were applied to discriminate malignant from healthy cells in human breast tissue biopsies. Indeed, integration of non-linear processes into a single, unified microscopy platform offered complementary structural information within individual cells at the submicron level. Using a single laser beam, label-free THG imaging techniques provided important morphological information as to the mean nuclear and cytoplasmic area, cell volume and tissue intensity, which upon quantification could not only distinguish cancerous from benign breast tissues but also define disease severity. Simultaneously, collagen fibers that could be detected by SHG imaging showed a well structured continuity in benign tumor tissues, which were gradually disoriented along with disease severity. Combination of THG imaging with FTIR spectroscopy could provide a clearer distinction among the different grades of breast cancer, since FTIR analysis showed increased lipid concentrations in malignant tissues. Thus, the use of non-linear optical microscopy can be considered as powerful and harmless tool for tumor cell diagnostics even during real time surgery procedures.

Topics: Aged; Breast; Breast Neoplasms; Collagen; Elastin; Female; Humans; Lipid Droplets; Middle Aged; Multimodal Imaging; NAD; Neoplasm Grading; Second Harmonic Generation Microscopy; Spectroscopy, Fourier Transform Infrared

The application of rationally designed therapeutic peptides (TP) may improve outcomes in cancer treatment. These peptides hold the potential to directly target proliferative pathways and stimulate cell arrest or death pathways. Elastin-like polypeptide (ELP) is an elastin derived biopolymer that undergoes a thermally mediated phase transition. This study employs p50, a nuclear localization sequence derived peptide that inhibits the activation of NFκB and is implicated in cancer cell survival and metastasis. In order to effectively delivery p50, it is conjugated to SynB1-ELP1, a thermally responsive macromolecular carrier. By applying an external heat source, mild hyperthermic conditions (41 °C) induce aggregation and therefore can be used to specifically target ELP to solid tumors in cancer therapy. The addition of a cell penetrating peptide (CPP) to the N-terminus of the macromolecular carrier enhances the cellular uptake and directs the subcellular localization of the bioactive peptide. The novel TP, p50, inhibits proliferation and induces apoptosis of breast cancer cells by blocking the intranuclear import of NFκB. By expanding the repertoire of oncogenic targets, CPPs, and ELP carrier sizes, ELP-based polypeptides may be modulated to optimize the delivery of these novel therapies and allow for the flexibility to create individualized cancer therapies.

Topics: Amino Acid Sequence; Apoptosis; Breast Neoplasms; Cell Proliferation; Elastin; Endocytosis; Female; Humans; MCF-7 Cells; NF-kappa B; Peptides; Temperature; Tumor Necrosis Factor-alpha

The tumor microenvironment regulates tissue development and homeostasis, and its dysregulation contributes to neoplastic progression. Increased expression of type X collagen α-1 (ColXα1) in tumor-associated stroma correlates with poor pathologic response to neoadjuvant chemotherapy in estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2)-positive breast cancers. Evaluation of ColXα1 expression patterns suggests a potential connection with elastin fibers. To investigate the possible interaction between ColXα1 and elastin, we evaluated the expression of ColXα1 in relation to elastin fibers in normal breast tissue, ductal carcinoma in situ, and invasive breast carcinomas at cellular and subcellular levels. Our findings demonstrate that ColXα1 colocalizes with elastin in invasive breast cancer-associated stroma by immunohistochemistry, immunofluorescence, and electron microscopy. In 212 invasive breast carcinomas, this complex was aberrantly and selectively expressed in tumor extracellular matrix in 79% of ER+/HER2-, 80% of ER+/HER2+, 76% of ER-/HER2+, and 58% of triple negative breast cancers. In contrast, ColXα1 was generally absent, while elastin was present perivascularly in normal breast tissue. ColXα1 and elastin were coexpressed in 58% of ductal carcinoma in situ (DCIS) in periductal areas. In mass-forming DCIS with desmoplastic stroma, the complex was intensely expressed in periductal areas as well as within the tumor-associated stroma in all cases. Our data suggest that the breast carcinoma neoplastic process may involve aberrant expression of ColXα1 and elastin in the tumor microenvironment emerging early at the DCIS stage. Enrichment of these complexes in tumor-associated stroma may represent a stromal signature indicative of intrinsic differences between breast cancers. These findings shed light on investigation into the role of aberrant collagen complex expression in tumorigenesis and tumor progression which may be leveraged in therapeutic and theranostic applications.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Collagen Type X; Elastin; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Receptors, Estrogen; Tumor Microenvironment

Modification of protein-based drug carriers with tumor-targeting properties is an important area of research in the field of anticancer drug delivery. To this end, we developed nanoparticles comprised of elastin-like polypeptides (ELPs) with fused poly-aspartic acid chains (ELP-D) displaying DNA aptamers. DNA aptamers were enzymatically conjugated to the surface of the nanoparticles via genetic incorporation of Gene A* protein into the sequence of the ELP-D fusion protein. Gene A* protein, derived from bacteriophage ϕX174, can form covalent complexes with single-stranded DNA via the latter's recognition sequence. Gene A* protein-displaying nanoparticles exhibited the ability to deliver the anticancer drug paclitaxel (PTX), whilst retaining activity of the conjugated Gene A* protein. PTX-loaded protein nanoparticles displaying DNA aptamers known to bind to the MUC1 tumor marker resulted in increased cytotoxicity with MCF-7 breast cancer cells compared to PTX-loaded protein nanoparticles without the DNA aptamer modification.

Topics: Antineoplastic Agents; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Elastin; Female; Humans; MCF-7 Cells; Nanoparticles; Neoplasms; Paclitaxel

Topics: Actins; Adult; Breast; Breast Neoplasms; Clinical Decision-Making; Collagen; Elastin; Female; Humans; Macrophages; Mammaplasty; Mammary Arteries; Microsurgery; Time Factors; Treatment Outcome

Recombinant protein-polymer scaffolds such as elastin-like polypeptides (ELPs) offer drug-delivery opportunities including biocompatibility, monodispersity, and multifunctionality. We recently reported that the fusion of FK-506 binding protein 12 (FKBP) to an ELP nanoparticle (FSI) increases rapamycin (Rapa) solubility, suppresses tumor growth in breast cancer xenografts, and reduces side effects observed with free-drug controls. This new report significantly advances this carrier strategy by demonstrating the coassembly of two different ELP diblock copolymers containing drug-loading and tumor-targeting domains. A new ELP nanoparticle (ISR) was synthesized that includes the canonical integrin-targeting ligand (Arg-Gly-Asp, RGD). FSI and ISR mixed in a 1:1 molar ratio coassemble into bifunctional nanoparticles containing both the FKBP domain for Rapa loading and the RGD ligand for integrin binding. Coassembled nanoparticles were evaluated for bifunctionality by performing in vitro cell-binding and drug-retention assays and in vivo MDA-MB-468 breast tumor regression and tumor-accumulation studies. The bifunctional nanoparticle demonstrated superior cell target binding and similar drug retention to FSI; however, it enhanced the formulation potency, such that tumor growth was suppressed at a 3-fold lower dose compared to an untargeted FSI-Rapa control. This data suggests that ELP-mediated scaffolds are useful tools for generating multifunctional nanomedicines with potential activity in cancer.

Topics: Animals; Antibiotics, Antineoplastic; Breast; Breast Neoplasms; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Elastin; Female; Humans; Integrins; Mice; Mice, Nude; Nanoparticles; Peptides; Sirolimus

The search for new and biocompatible materials with high potential for improvement is a challenge in gene delivery applications. A cell type specific vector made of elastin-like recombinamer (ELR) and aptamers has been specifically designed for the intracellular delivery of therapeutic material for breast cancer therapy. A lysine-enriched ELR was constructed and complexed with plasmid DNA to give positively charged and stable polyplexes. Physical characterization of these polyplexes showed a particle size of around 140 nm and a zeta potential of approximately +40 mV. The incorporation of MUC1-specific aptamers into the polyplexes resulted in a slight decrease in zeta potential but increased cell transfection specificity for MCF-7 breast cancer cells with respect to a MUC1-negative tumor line. After showing the transfection ability of this aptamer-ELR vector which is facilitated mainly by macropinocytosis uptake, we demonstrated its application for suicide gene therapy using a plasmid containing the gene of the toxin PAP-S. The strategy developed in this work about using ELR as polymeric vector and aptamers as supplier of specificity to deliver therapeutic material into MUC1-positive breast cancer cells shows promising potential and continues paving the way for ELRs in the biomedical field.

Topics: Aptamers, Nucleotide; Biocompatible Materials; Breast Neoplasms; Cell Survival; Cells, Cultured; Elastin; Female; Gene Transfer Techniques; Genetic Therapy; Humans; Molecular Targeted Therapy; Mucin-1; Plasmids; Polymers

Cancer stem cell (CSC) inhibitors are a new category of investigational drugs to treat metastasis. Salinomycin (Sali) is one of most studied CSC inhibitors and has reached clinical tests. Several drug carriers have been developed to improve efficacy of Sali. However, Sali has not been shown to inhibit metastasis from orthotopic tumors, the gold standard for metastasis. To fill this gap, we developed an immune-tolerant, elastin-like polypeptide (iTEP)-based nanoparticle (iTEP-Sali-ABA NP) that released 4-(aminomethyl)benzaldehyde-modified Sali (Sali-ABA) under acidic conditions. We found that the NP increased the area under the curve (AUC) of Sali-ABA by 30-fold and the tumor accumulation by 3.4-fold. Furthermore, no metastasis was detected in any of the mice given the NP. However, all the mice died of primary tumor burdens. To overcome primary tumor growth and improve the overall survival, we applied a combination therapy consisting of the iTEP-Sali-ABA NP and iTEP NP-delivered paclitaxel. This therapy effectively retarded primary tumor growth, and most importantly, improved the overall survival. In conclusion, delivery of Sali-ABA by the NP, alone or in combination with paclitaxel, was more effective than free Sali-ABA in decreasing metastasis and increasing survival. This iTEP-Sali-ABA NP represents a novel and clinically promising therapy to combat metastasis.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzaldehydes; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Elastin; Electrophoresis, Polyacrylamide Gel; Female; Humans; Immune Tolerance; Mice, Inbred BALB C; Nanoparticles; Neoplasm Metastasis; Paclitaxel; Peptides; Pyrans; Tissue Distribution

Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.

Topics: Animals; Biocompatible Materials; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Disintegrins; Elastin; Human Umbilical Vein Endothelial Cells; Humans; Integrins; Mice, Nude; Microscopy, Confocal; Microscopy, Electron, Transmission; Nanoparticles; Peptides; Polymers; Recombinant Fusion Proteins; Xenograft Model Antitumor Assays

Salinomycin (Sali) has selective toxicity to cancer stem cells (CSCs), a subpopulation of cancer cells that have been recently linked with tumor multidrug resistance (MDR). To utilize its selective toxicity for cancer therapy, we sought to devise a nanoparticle (NP) carrier to deliver Sali to solid tumors through the enhanced permeability and retention effect and, hence, to increase its exposure to CSCs. First, hydrophobic Sali was conjugated to a hydrophilic, immune-tolerant, elastin-like polypeptide (iTEP); the amphiphilic iTEP-Sali conjugates self-assemble into NPs. Next, free Sali was encapsulated into the NPs alone or with two additives, N,N-dimethylhexylamine (DMHA) and α-tocopherol. The coencapsulation significantly improved the loading efficiency and release profile of Sali. The resulting NPs of the coencapsulation, termed as iTEP-Sali NP3s, have an in vitro release half-life of 4.1 h, four times longer than iTEP-Sali NP2s, the NPs that have encapsulated Sali only. Further, the NP3 formulation increases the plasma area under curve and the tumor accumulation of Sali by 10 and 2.4 times, respectively. Lastly, these improved pharmacokinetic and tumor accumulation profiles are consistent with a boost of CSC-elimination effect of Sali in vivo. In NP3-treated 4T1 orthotopic tumors, the mean CSC frequency is 55.62%, a significant reduction from the mean frequencies of untreated tumors, 75.00%, or free Sali-treated tumors, 64.32%. The CSC-elimination effect of the NP3 can further translate to a delay of tumor growth. Given the role of CSCs in driving tumor MDR and recurrence, it could be a promising strategy to add the NP3 to conventional cancer chemotherapies to prevent or reverse the MDR.

Topics: alpha-Tocopherol; Amines; Animals; Breast Neoplasms; Drug Carriers; Drug Delivery Systems; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Elastin; Female; Mice; Mice, Inbred BALB C; Nanomedicine; Nanoparticles; Neoplasm Transplantation; Neoplastic Stem Cells; Pyrans; Time Factors; Tissue Distribution

Mammography screen-detected breast cancers have a better prognosis than predicted from established prognostic markers. A search for additional features that are characteristic for these tumours and their prognosis is needed to reduce overtreatment, a recognized challenge in breast cancer patient management today. Here, we have investigated the occurrence and importance of tumour elastosis.. We performed a population based retrospective study of breast cancers detected in the Norwegian Breast Cancer Screening Programme in Vestfold County during 2004-2009. In total, 197 invasive screen-detected cancers and 75 interval cancers in patients aged 50-69 years were compared with regard to standard clinico-pathological parameters and tumour shape, as well as ER, PR, HER2 and Ki67 expression. In particular, the presence of elastotic material in tumours was graded on a 4-tiered scale (score 0-3).. Screen-detected cancers had a significantly higher content of stromal elastosis than interval cancers (p < 0.001). High content of elastosis (score 3) correlated strongly with stellate tumour shape, low histological grade, and ER+/HER2- status. Further, high elastosis score was significantly associated with lower Ki67 expression. In survival analyses, cases with high elastosis demonstrated increased recurrence free (p = 0.03) and disease-specific survival (p = 0.11) compared to cases with low elastosis.. There is a strong correlation between the presence of tumour elastosis, stellate tumour shape and mammography detection of breast cancers. To our knowledge, this is the first time elastosis has been studied in relation to breast cancer detection method. Presence of elastosis is associated with low tumour cell proliferation (Ki67) and a good prognosis.. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_230.

Topics: Aged; Breast Neoplasms; Disease-Free Survival; Elastic Tissue; Elastin; Female; Humans; Kaplan-Meier Estimate; Ki-67 Antigen; Mammography; Middle Aged; Neoplasm Grading; Neoplasm Recurrence, Local; Norway; Predictive Value of Tests; Retrospective Studies; Stromal Cells; Time Factors; Treatment Outcome

The fluorescence of paired human breast malignant and normal tissue samples was investigated using a novel fluorescence spectroscopic (S3-LED) ratiometer unit with no moving parts. This device can measure the emission spectra of key native organic biomolecules such as tryptophan, tyrosine, collagen and elastin within tissues by using LED (light emitting diode) excitation sources coupled to an optical fiber. With this device, the spectral profiles of 11 paired breast cancerous and normal samples from 11 patients with breast carcinoma were obtained. In each of the 11 cases, marked increases in the tryptophan levels were found in the breast carcinoma samples when compared to the normal breast tissues. In the breast cancer samples, there were also consistently higher ratios of the 340 to 440 nm and the 340 to 460 nm intensity peaks after 280 nm excitation, likely representing an increased tryptophan to NADH ratio in the breast cancer samples. This difference was seen in the spectral profiles of the breast cancer patients regardless of whether they were HER2 positive or negative or hormone receptor positive or negative, and was found regardless of menopausal status, histology, stage, or tumor grade.

Topics: Adult; Aged; Area Under Curve; Breast; Breast Neoplasms; Carcinoma; Collagen; Discriminant Analysis; Elastin; Female; Humans; Middle Aged; Optical Imaging; ROC Curve; Spectrometry, Fluorescence; Tryptophan; Tyrosine

Numerous nanocarriers of small molecules depend on either non-specific physical encapsulation or direct covalent linkage. In contrast, this manuscript explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for nanoparticulate drug delivery. To explore this approach, genetically engineered diblock copolymers were constructed from elastin-like polypeptides (ELPs) that assemble small (<100nm) nanoparticles. ELPs are protein polymers of the sequence (Val-Pro-Gly-Xaa-Gly)n, where the identity of Xaa and n determine their assembly properties. Initially, a screening assay for model drug encapsulation in ELP nanoparticles was developed, which showed that Rose Bengal and Rapa have high non-specific encapsulation in the core of ELP nanoparticles with a sequence where Xaa=Ile or Phe. While excellent at entrapping these drugs, their release was relatively fast (2.2h half-life) compared to their intended mean residence time in the human body. Having determined that Rapa can be non-specifically entrapped in the core of ELP nanoparticles, FK506 binding protein 12 (FKBP), which is the cognate protein target of Rapa, was genetically fused to the surface of these nanoparticles (FSI) to enhance their avidity towards Rapa. The fusion of FKBP to these nanoparticles slowed the terminal half-life of drug release to 57.8h. To determine if this class of drug carriers has potential applications in vivo, FSI/Rapa was administered to mice carrying a human breast cancer model (MDA-MB-468). Compared to free drug, FSI encapsulation significantly decreased gross toxicity and enhanced the anti-cancer activity. In conclusion, protein polymer nanoparticles decorated with the cognate receptor of a high potency, low solubility drug (Rapa) efficiently improved drug loading capacity and its release. This approach has applications to the delivery of Rapa and its

Topics: Amino Acid Sequence; Animals; Antibiotics, Antineoplastic; Breast; Breast Neoplasms; Cell Line, Tumor; Drug Carriers; Elastin; Female; Humans; Mice; Mice, Nude; Molecular Sequence Data; Nanoparticles; Peptides; Sirolimus; TOR Serine-Threonine Kinases

Poor aqueous solubility limits the therapeutic index of paclitaxel as an anti-cancer drug. Synthesis of soluble prodrugs of paclitaxel, or conjugation of the drug to macromolecular carriers have been reported to increase its water-solubility. Macromolecular drug carriers have an added advantage of targeting the drug to the tumor site due to the abnormal tumor blood and lymphatic vasculature. This study describes a thermally responsive macromolecular carrier, elastin-like polypeptide (ELP) for the delivery of paclitaxel. Paclitaxel was bound to ELP by conjugation with the 6-maleimidocaproyl hydrazone derivative of paclitaxel, an acid-sensitive paclitaxel prodrug, for the potential treatment of breast cancer. Focused hyperthermia above a specific transition temperature at the site of a tumor causes ELP to aggregate and accumulate, thereby increasing the local concentration of the drug cargo. The paclitaxel prodrug described here bears an acid-sensitive linker that is cleavable at the lysosomal/endosomal pH, which allows a controlled intracellular release of the drug. The ELP-delivered paclitaxel in the presence of hyperthermia inhibits MCF-7 cell proliferation by stabilizing the microtubule structures, arresting the cells at the G2/M stage, and inducing apoptosis in a manner similar to conventional paclitaxel. It also inhibits proliferation of a paclitaxel resistant MCF-7 cell line. These data provide an in vitro proof of concept for the use of ELP as a delivery vehicle of paclitaxel.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chemistry, Pharmaceutical; Delayed-Action Preparations; Dose-Response Relationship, Drug; Drug Carriers; Drug Compounding; Drug Resistance, Neoplasm; Elastin; Female; G2 Phase Cell Cycle Checkpoints; Humans; Hydrogen-Ion Concentration; Hyperthermia, Induced; Microtubules; Paclitaxel; Prodrugs; Recombinant Proteins; Temperature; Time Factors; Tubulin Modulators

Elastin-like polypeptide (ELP) is a macromolecular carrier with thermally responsive properties that can passively accumulate in solid tumors and additionally aggregate in tumor tissue when exposed to hyperthermia. In this study, ELP was conjugated to the anticancer drug doxorubicin (DOXO) and three different cell penetrating peptides (CPP) in order to inhibit tumor growth in mice compared to free doxorubicin. Fluorescence microscopy studies in MCF-7 breast carcinoma cells demonstrated that the three different CPP-ELP-DOXO conjugates delivered doxorubicin to the cell nucleus. All CPP-ELP-DOXO conjugates showed cytotoxicity with IC(50) values in the range of 12-30 μM at 42 °C, but the ELP carrier with SynB1 as the cell penetrating peptide had the lowest intrinsic cytotoxicity. Therefore, the antitumor efficacy of SynB1-ELP-DOXO was compared to doxorubicin under hyperthermic conditions. C57BL/6 female mice bearing syngeneic E0771 murine breast tumors were treated with either free doxorubicin or the SynB1-ELP-DOXO conjugate with or without focused hyperthermia on the tumor. Under hyperthermic conditions, tumor inhibition with SynB1-ELP-DOXO was 2-fold higher than under therapy with free doxorubicin at the equivalent dose, and is thus a promising lead candidate for optimizing thermally responsive drug polymer conjugates.

Topics: Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell-Penetrating Peptides; Doxorubicin; Drug Carriers; Elastin; Female; Humans; Hydrazones; Hydrogen-Ion Concentration; MCF-7 Cells; Mice; Mice, Inbred C57BL; Peptides; Tumor Burden

A novel approach to cancer detection in biomarkers spectral subspace (BSS) is proposed. The basis spectra of the subspace spanned by fluorescence spectra of biomarkers are obtained by the Gram-Schmidt method. A support vector machine classifier (SVM) is trained in the subspace. The spectrum of a sample tissue is projected onto and is classified in the subspace. In addition to sensitivity and specificity, the metrics of positive predictivity, Score1, maximum Score1, and accuracy (AC) are employed for performance evaluation. The proposed BSS using SVM is applied to breast cancer detection using four biomarkers: collagen, NADH, flavin, and elastin, with 340-nm excitation. It is found that the BSS SVM outperforms the approach based on multivariate curve resolution (MCR) using SVM and achieves the best performance of principal component analysis (PCA) using SVM among all combinations of PCs. The descent order of efficacy of the four biomarkers in the breast cancer detection of this experiment is collagen, NADH, elastin, and flavin. The advantage of BSS is twofold. First, all diagnostically useful information of biomarkers for cancer detection is retained while dimensionality of data is significantly reduced to obviate the curse of dimensionality. Second, the efficacy of biomarkers in cancer detection can be determined.

Topics: Biomarkers, Tumor; Breast Neoplasms; Collagen; Dinitrocresols; Elastin; Female; Humans; Multivariate Analysis; NAD; Principal Component Analysis; Sensitivity and Specificity; Signal Processing, Computer-Assisted; Spectrometry, Fluorescence; Support Vector Machine

Adherent scars on the hand often lead to a major functional impairment and an aesthetic deformity. The rate of recurrence after scar correction is usually very high. A 57-year-old woman with an adherent scar on the back of her hand and major functional impairment was successfully treated with Matriderm. Using Matriderm as an additional layer between the atrophic skin and the tendons adherency of the scar could be prevented. One year after surgery the patient is free of pain. There is normal mobility between the skin and the underlying tissue. Complete wrist flexion and extension could be achieved. To the best of our knowledge this is the first case reported of using Matriderm for the correction of a scar that was caused by the paravasal injection of cytostatic drugs.

Topics: Absorbable Implants; Antineoplastic Agents; Breast Neoplasms; Cicatrix; Collagen; Dermatologic Surgical Procedures; Drug Eruptions; Elastin; Extravasation of Diagnostic and Therapeutic Materials; Female; Hand; Hand Deformities, Acquired; Humans; Infusions, Intravenous; Middle Aged; Postoperative Complications; Reoperation; Skin; Skin, Artificial; Suture Techniques; Tissue Adhesions

Lysyl oxidase (LOX), an extracellular amine oxidase, catalyzes the cross-linking of collagen and elastin. LOX has been also shown to play an essential role in promoting the invasive and metastatic potential of breast tumor cells. However, the LOX-interacting factors in these processes are not known. In this study, we identified placental lactogen (PL), a member of the growth hormone/prolactin hormone family, as a LOX-interacting partner using yeast two-hybrid screens. PL is normally only expressed in placental syncytiotrophoblasts, but PL genes are amplified and expressed in a high percentage of invasive ductal breast carcinomas. We confirmed LOX-PL interactions using far Western and solid phase binding assays. In activity assays, PL was not a substrate or inhibitor of LOX. We further demonstrated that PL is expressed in breast tumor epithelial cells and detected LOX-PL interactions by coimmunoprecipitation in invasive breast cancer cells. In MCF-10A normal breast epithelial cells stably expressing LOX, PL, or both, LOX had no effect on cell proliferation, PL alone increased proliferation by 49%, and coexpression of LOX and PL led to a 121% increase in cell proliferation. Unlike in tumor cells, LOX did not induce a more migratory phenotype in MCF-10A cells; nor did PL. However, their coexpression resulted in a 240% increase in cell migration, suggesting that these interactions may be highly relevant to the transition of epithelial cells toward a migratory phenotype during the development and progression of breast carcinoma and a significant role for LOX-PL interactions in epithelial cell behavior.

Topics: Breast; Breast Neoplasms; Cell Division; Cell Line; Cell Line, Tumor; Cell Movement; Collagen; Elastin; Epithelial Cells; Female; Humans; Placental Lactogen; Protein-Lysine 6-Oxidase

The thermally responsive elastin-like polypeptide (ELP) has great potential as a macromolecular drug delivery vehicle due to its ability to be actively targeted to solid tumors by application of focused hyperthermia. Since, the toxicity properties of a new therapeutic delivery vehicle are crucial to its utility as an effective delivery vehicle, we evaluated the cytotoxicity of a thermally responsive Tat-ELP1 in various cell lines in response to hyperthermia. We report that Tat-ELP1 was not cytotoxic at 37 degrees C in SK-MEL-2, SKOV-3 and WI-38 cells, and only mildly toxic in the MCF-7 breast carcinoma cell line. Application of hyperthermia (42 degrees C) in combination with Tat-ELP1 resulted in cytotoxicity in all cell lines tested, and this toxicity was most prominent in the MCF-7 cell line, which was chosen to study the mechanism behind this increased toxicity. We found that Tat-ELP1 combined with hyperthermia caused membrane leakage and apoptosis, resulting in cell death, but no hemolytic effect was observed on murine erythrocytes.

Topics: Amino Acid Sequence; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Membrane Permeability; Elastin; Gene Products, tat; Humans; L-Lactate Dehydrogenase; Recombinant Fusion Proteins; Temperature

Elastin-like polypeptides are biopolymers composed of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly. Elastin-like polypeptides are soluble in aqueous solution below their transition temperature, but they hydrophobically collapse and aggregate when the temperature is raised above the transition temperature. Previous studies have suggested that the aggregation of these polypeptides in response to externally applied hyperthermia may be exploited in the use of elastin-like polypeptide for thermally targeted drug delivery. This work shows the application of elastin-like polypeptide as a delivery vehicle for a short peptide that can inhibit the transcriptional function of a specific oncogene. The coding sequence for elastin-like polypeptide was modified by the addition of the membrane translocating sequence penetratin and a peptide derived from helix 1 of the helix-loop-helix region of c-Myc (H1-S6A,F8A), known to inhibit c-Myc transcriptional function. The designed polypeptide (Pen-ELP-H1) was then expressed and purified from Escherichia coli. Cellular uptake of Pen-ELP-H1 is enhanced by both the penetratin sequence and by the hyperthermia-induced phase transition as shown by flow cytometry studies. Using immunofluorescence and reverse transcription-PCR, we show that Pen-ELP-H1 is able to disrupt the nuclear localization of c-Myc and inhibit transcriptional activation by c-Myc. Cell proliferation studies showed that Pen-ELP-H1 inhibits growth of MCF-7 cells. Furthermore, the use of hyperthermia increased the antiproliferative effect of a thermally responsive Pen-ELP-H1 approximately 2-fold compared with a nonthermally responsive control polypeptide. These studies show that genetically engineered elastin-like polypeptide carriers may provide a new way to thermally target specific oncogene inhibitors to solid tumors.

Topics: Amino Acid Sequence; Antineoplastic Agents; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cell-Penetrating Peptides; Elastin; Female; Humans; Hyperthermia, Induced; Microscopy, Confocal; Molecular Sequence Data; Peptides; Protein Engineering; Proto-Oncogene Proteins c-myc; Subcellular Fractions; Temperature; Transcription, Genetic

Galectin-3, a beta-galactoside binding protein, plays a significant role in cell to extracellular matrix interactions. Despite its extracellular expression, the precise physiological mechanisms that trigger its release from the intracellular milieu have not been characterized. The present analyses were, therefore, done to identify the extracellular matrix proteins with propensity to induce the release of intracellular galectin-3 from breast carcinoma cells. Our studies demonstrate that fetuin, a serum glycoprotein that is abundant in the fetal serum, is capable of inducing the rapid release (approximately 1 min) of intracellular galectin-3 from the cells. The mechanism by which galectin-3 is rapidly released appears to be novel and does not depend on changes in intracellular calcium levels. We also report that galectin-3-expressing breast carcinoma cells in serumless medium adhere and spread well on microtiter wells in the presence of fetuin and divalent ions in a carbohydrate-dependent manner. The data suggest that fetuin is a natural modulator of galectin-3 secretion/release and that the secreted galectin-3 modulates the activity of cell surface receptors for extracellular matrix proteins.

Topics: alpha-Fetoproteins; Antigens, Differentiation; Breast Neoplasms; Calcium; Cell Adhesion; Cell Movement; Culture Media, Serum-Free; Elastin; Galectin 3; Humans; Tumor Cells, Cultured

Galectin-3 is a beta-galactoside binding lectin whose precise physiological role is not yet defined. In the present studies, we questioned whether galectin-3 plays a role in the adhesion of breast carcinoma cells to elastin. The impetus for this analysis was the initial observation that the cellular receptor for elastin, the 67 kDa elastin/laminin protein may have galectin-like properties (Mecham et al. [1989] J. Biol. Chem. 264:16652-16657). We therefore analyzed the adhesion of breast carcinoma cells to microtiter wells coated with elastin under conditions which eliminate integrin participation in adhesion. The adhesion assay was done in the absence and presence of purified recombinant galectin-3. We hereby demonstrate that high concentrations of galectin-3 ligate breast carcinoma cells to microtiter wells coated with elastin. Galectin-3 also demonstrated a specific binding interaction with purified elastin in a dose and lactose dependent manner. Furthermore we demonstrated by immunoprecipitation that endogenous galectin-3 in breast carcinoma cells is associated with tropoelastin. Lastly, the breast carcinoma cells which expressed galectin-3 on their surface, demonstrated enhanced cellular proliferation on elastin compared to galectin-3 null expressing cells. These studies suggest that galectin-3 is capable of regulating the interactions between cells and elastin.

Topics: Antigens, Differentiation; Breast Neoplasms; Cell Adhesion; Elastin; Female; Galectin 3; Humans; Ligands; Models, Biological; Receptors, Cell Surface; Recombinant Proteins; Tumor Cells, Cultured

We studied immunohistochemically one thousand one hundred and thirty-seven cases of primary invasive breast cancers (NST) and adjacent normal mammary glands for tenascin expression, and compared their elastic content to verify if a relationship exists between tenascin expression and elastosis. Periductal, perivascular and stromal elastosis were graded on a scale from 0 to 3 (absent to massive). All carcinomas showed tenascin expression and elastosis with various histological appearances. In the adjacent breast, teanscon was distributed around the normal ducts or with extasia and uctal hyperplasia without atypia. Digestion of the sections with elastase prior to staining resulted in a loss of the specific staining reactions in all areas where elastosis was present. Tenascin staining was observed in the mesenchyme closely surrounding the neoplastic ducts and the cancer cell nests. Stromal tenascin staining appeared stronger in those carcinomas that exhibited marked desmoplastic reactions. The highly differentiated tumours contained more elastosis in their tumour tissue than the poorly differentiated ones, whereas tenascin expression was stronger in poorly differentiated tumours than well differentiated tumours. A strong staining for tenascin was observed in the elastotic cuff. Tenascin staining did not disappear afterwards with elastase. We did not find a statistically significant correlation between tenascin expression, elastosis and prognostic factors such as size of the tumour, lymph node metastasis, tumour necrosis and age. In our study tenascin proved to be an additional element in elastotic areas even though the significance of an association between elastosis and tenascin is still unknown, as is that of elastosis itself.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal, Breast; Elastic Tissue; Elastin; Humans; Immunohistochemistry; Middle Aged; Pancreatic Elastase; Tenascin

Murine breast cancer cell lines were developed to selectively invade the peritoneum while they proliferated in ascites form in the abdominal cavity. In a dominant form of invasion, tumor cells showed special affinity for elastin fibers and squeezed through narrow gaps in the elastic fiber meshwork of the stroma. Even in fixed tissue, such cells could be recognized as being in the process of invasive migration because of their dumbbell shape. This appearance was similar to that of diapedetic blood cells traversing bone marrow sinus endothelium. Three-dimensional STERECON graphics reconstruction from serial thick sections of 44 such cells was carried out. The reconstructions showed that, in mid-penetration, the cells spread extensively over the exterior surface of the elastic fiber meshwork. The cell surface contact of these forward projections was mainly with the elastic fiber outer coat of microfibrils, but small areas of the cell surface also fused directly to inner-core elastin. The morphological rearrangement of the cytoskeleton was minimal in both types of attachment areas. The location of these forward facing attachments is consistent with mechanisms for pulling the invasive cell through the gap. Lamellopodia formation and clustering of cytoplasmic organelles occurred more commonly at the forward-facing part of the cell. Morphometry of the reconstructions showed that a contraction of the whole cell occurred during the squeezing/migration process suggestive of an additional pushing process. However, our invasive cell lines showed marked differences in the degree of cell shrinkage. The process of adhesion and squeezing of tumor cells through elastin meshworks in vivo is clearly a complex phenomenon. Changes in cell surface activity appear to play a significant role in establishing the necessary 'foothold' component of invasion and, possibly, in the generation of tractive force as well.

Topics: Animals; Breast Neoplasms; Elastic Tissue; Elastin; Female; Mice; Microscopy, Electron; Models, Biological; Neoplasm Invasiveness; Organ Culture Techniques; Organelles; Peritoneum; Tumor Cells, Cultured

Using serial sections of frozen and AFA-fixed tissues from 34 breast cancers, we studied the presence of basement membrane material in the areas of elastosis. Various amounts of type IV collagen but not of laminin were demonstrated in areas of periductal elastosis. In some tumors, type IV collagen accumulated beneath the basement membrane. Periductal elastosis in areas of extensive fibrosis showed focal type IV collagen immunoreactivity, indicating remnants of ducts. Interstitial elastosis corresponded with weak type IV collagen reactivity. Each tumor showed type IV collagen immunostaining of the elastotic areas, with various degrees of intensity. Negative crossreactivity of the type IV collagen antibody with elastin was verified in skin biopsies with solar elastosis. Pre-incubation of the antibody with large amounts of elastin demonstrated an identical immunoreactivity. The specificity of the antibody was confirmed by ELISA and by Western blot analysis. To explain the periductal elastosis, we propose the following hypothesis. Excessive production of basement membrane material by the epithelial cells of the ducts leads to formation of a type IV collagen skeleton. This skeleton can act as the matrix for a secondary deposition of elastic material.

Topics: Basement Membrane; Blotting, Western; Breast Neoplasms; Collagen; Elastic Tissue; Elastin; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Laminin; Staining and Labeling; Tissue Preservation

Elastosis is a prominent feature of the desmoplastic reaction in many invasive breast cancers. It is widely held that the elastic tissue is produced by fibroblastic cells of the breast stroma, but several studies have suggested that it derives from breast cancer epithelium. In studies directed to examining the mechanisms regulating desmoplasia in breast cancers, cell lines of human breast cancer derivation have been shown to synthesize immunoreactive tropoelastin in cell culture. Stromal fibroblasts, grown out from breast cancers, produced as much elastin as did nuchal ligament fibroblasts at similar passages. The human breast cancer cell lines, grown under similar conditions, produced elastin in culture at rates equivalent to 1.6-15% of those of the control fibroblastic cells. These included two estrogen receptor positive and one estrogen receptor negative cell types. Northern blot analysis of total RNA showed the presence, under high stringency conditions, of a 3.5-kilobase elastin mRNA band in both the fibroblastic cells and the cancer cell lines. In situ hybridization, with an elastin complementary RNA probe (prepared from a short segment of the translated region of human elastin mRNA), has been carried out on a selection of 21 invasive ductal breast cancers and 9 normal breast samples. It has been found that, while fibroblastic cells of the stroma and of the periductal region are responsible for elastin synthesis in most breast cancers, the malignant epithelium is a source of the elastin in the desmoplastic tissue of a significant proportion of such neoplasms. Vascular endothelium also expresses the elastin gene in some breast cancers. The elastotic elastin may have different cellular origins in different portions of a single ductal breast cancer. The results indicate that elastosis in breast cancers is very likely to be a complex process with multifactorial regulatory mechanisms. Subclassifying cancers according to the cellular source of the desmoplastic elastin, on the basis of in situ hybridization of elastin mRNA, may provide insights into the prognostic significance of elastosis in breast cancers.

Topics: Adult; Aged; Blotting, Northern; Blotting, Western; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Line; Elastin; Epithelium; Gene Expression; Humans; Middle Aged; Nucleic Acid Hybridization; RNA, Messenger; RNA, Neoplasm; Tropoelastin

Elastosis in benign and malignant breast lesions was studied by light microscopic immunohistochemistry for elastin and by electron microscopy. Upon immunohistochemical examination for elastin, elastosis, particularly in scirrhous-type ductal carcinoma, showed two characteristic staining patterns: fibrously and intensely stained elastic fibers and evenly stained elastic masses. Elastic fibers showing increased fibrous staining occurred mainly in the stromal areas, and were considered to be newly formed because they consisted of tannic acid-positive amorphous components and abundant microfibrils. Evenly stained elastic masses were observed mainly in the periductal areas and showed less intense stainability. These masses consisted of numerous fine amorphous components with plentiful microfibrils. In some regions within these masses, there were condensed accumulations of irregularly arranged small amorphous components associated with only a few microfibrils. These amorphous components had an ill-defined outline and were occasionally associated with spiralling collagen fibrils and cell debris. On the basis of these findings, the periductal evenly stained elastic masses were thought to be formed by excessive production of elastic fibers and degradation of pre-existing and newly formed elastic fibers.

Topics: Breast Neoplasms; Carcinoma; Connective Tissue Diseases; Elastic Tissue; Elastin; Humans; Immunohistochemistry; Microscopy, Electron; Reference Values; Skin Diseases

Elastosis associated with invasive ductal and lobular carcinomas of the breast was examined by tinctorial and immunohistochemical staining methods, enzyme digestion, and electron microscopy. The elastotic material exhibited the tinctorial staining properties of elastic fibres, and the ultrastructural appearances were those of elastic fibres although there was a higher proportion of microfibrils than in normal mature elastic fibres. The elastosis was immunostained by antisera to human fetal elastin, lysozyme and amyloid P component, as in other sites where elastic fibres are found. These findings indicate that immunohistochemically intact elastic fibres are present in the elastosis of breast cancer. They also demonstrate that lysozyme and amyloid P component are co-distributed with elastic fibres in elastosis of breast carcinoma, as distinct components with different susceptibilities to enzyme digestion. The cellular origin of elastosis in breast carcinoma remains uncertain.

Topics: Actin Cytoskeleton; Adult; Aged; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Elastic Tissue; Elastin; Female; Humans; Microscopy, Electron; Middle Aged; Muramidase; Serum Amyloid P-Component

Topics: Basement Membrane; Breast Neoplasms; Collagen; Elastin; Extracellular Matrix; Female; Fibronectins; Fluorescent Antibody Technique; Glycoproteins; Humans; Laminin; Serum Amyloid P-Component

The desmoplastic response to breast carcinoma is being studied. The stimulation of stromal cell proliferation by a preformed breast tumor matrix was shown. An additional mechanism for stimulating scleroprotein deposition is described here. On a per-cell basis, the synthesis of collagen and elastin was increased by 50% and 70%, respectively, in fibroblasts grown on the preformed breast tumor matrix compared to the same cells grown on plastic or on their own preformed matrix. Breast tumor cells themselves synthesized small amounts of collagen and elastin compared to fibroblasts. These levels were unchanged when breast tumor cells were grown on the preformed matrix of fibroblasts. Addition of steroid hormones to cultured cells grown on plastic or on preformed matrices in various combinations, did not change the levels of either collagen or elastin synthesis. The matrix of human breast tumor cells exerts a dual effect; it is mitogenic for fibroblasts, and also stimulates the level of collagen and elastin synthesis, events that could contribute to the formation of the desmoplastic response to human breast cancer in situ.

Topics: Breast Neoplasms; Cell Line; Chromatography, High Pressure Liquid; Collagen; Elastin; Estradiol; Extracellular Matrix; Fibroblasts; Humans; Hydroxycorticosteroids; Hydroxyproline; Progesterone; Proline; Testosterone

Elastosis, the deposition of large amounts of elastin, is characteristic of the desmoplastic reaction to human breast carcinoma. Dissolution of the elastin often occurs following treatment regimens that involve steroid hormones or their antagonists. Elastinolytic activities must be invoked to account for the loss of this elastin-cotaining stroma. We have utilized a tissue culture model to explore the molecular aspects of this phenomenon. The elastases of several human fibroblast and breast carcinoma cell lines were examined. The tumor cells had 10- to 30-fold higher elastase activity than did the fibroblasts. Three separate elastinolytic activities were observed in the tumor cell lines, and partial purification was achieved. The effect of steroid hormones on these elastases was examined. No stimulation of activity was found with any of the hormones, in any combination. However, there was marked inhibition of elastase with estradiol, progesterone, and dexamethasone of the ZR75-1 cell line. This is the estrogen receptor positive line that is estrogen responsive. The corticosteroids also inhibited the elastases of the estrogen receptor positive, non-responder cell line ZR75-30. No effect was seen on the elastases of receptor negative cells ZR75-31A with any of the steroid hormones. Stimulation of elastinolytic activities in these tumor cells must occur by some as yet unidentified pathway.

Topics: Breast Neoplasms; Cell Line; Cysteine Endopeptidases; Elastin; Endopeptidases; Female; Hormones; Humans; Metalloendopeptidases; Pancreatic Elastase; Serine Endopeptidases

Antibodies to alpha-elastin peptides, amyloid P component, lysozyme and plasma protease inhibitors have been used in an immunoperoxidase method to stain elastic fibres in frozen sections of human breast tissues. A loss of immunoreactivity seen in formalin-fixed, paraffin-embedded sections was reversed by a preliminary proteolysis. Differences in the tinctorial dye and immunohistochemical staining patterns following proteolysis by a variety of enzymes suggests a selective unmasking or removal of elastic fibre components and thus the presence of separate binding sites for individual antibodies and tinctorial dyes. Antibody blocking experiments and double immunoenzymatic labelling support the existence of several different epitopes within elastic fibres.

Topics: Amyloid; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Coloring Agents; Elastic Tissue; Elastin; Female; Humans; Immunoenzyme Techniques; Muramidase; Peptide Hydrolases; Protease Inhibitors; Serum Amyloid P-Component; Staining and Labeling

The content of elastic tissue has been evaluated in 171 primary breast carcinomas. Of the tumors, 35% had no or very little elastic tissue in the malignant areas, 42% presented with medium elastosis, and 22% had gross elastosis. The occurrence of elastin has been related to different prognostic factors. An increasing amount of elastin was found with increasing amounts of estrogen receptor (p = 0.0003), while there was only a slight correlation to the progesterone receptor content. Furthermore, the highly differentiated tumors contained more elastin in their tumor tissue than the poorly differentiated tumors (p = 0.003), and parous women had significantly more elastin than nonparous women (p = 0.02). The presence of elastin was not, however, of any demonstrable prognostic significance.

Topics: Breast Neoplasms; Elastin; Female; Humans; Prognosis; Receptors, Estrogen; Receptors, Progesterone

An ultrastructural study on elastosis of human breast tumors was made with special attention to the periductal elastosis and the cell responsible for elastic fiber formation. The elastosis was found prominently in scirrhous type of duct carcinoma. In the area of mild periductal elastosis, the elastic fibers with many microfibrils and a tiny central elastin were seen around the periductal fibroblasts which were characterized by attenuated cytoplasms with aggregates of microfilaments and slightly developed rough endoplasmic reticulum. With the thickening of the periductal wall, such an area was replaced by abundant mature elastic fibers with peripheral microfibrils and a few intervening ordinary fibroblasts. Therefore, it was suggested that the periductal fibroblasts which transformed into ordinary fibroblasts during the development of elastosis were primarily concerned with the elastic fiber formation. In the interlobular tissue in which both fibroblasts and myofibroblasts were present, the elastic fibers were larger than those of the periductal area and had less microfibrils in their periphery. The relationship between microfibrils and elastin during the early elastosis, maturation process of the elastic fibers, and cell modulation of the fibroblasts in the breast elastosis were discussed.

Topics: Adenofibroma; Adult; Breast; Breast Diseases; Breast Neoplasms; Carcinoma; Elastic Tissue; Elastin; Female; Fibroblasts; Histocytochemistry; Humans; Middle Aged

Topics: Breast; Breast Neoplasms; Cell Line; Cells, Cultured; Elastin; Female; Humans; Pancreatic Elastase

Indirect immunofluorescence with a purified antiserum to human foetal elastin has identified newly synthesized elastin on the membranes of neoplastic epithelial cells in human mammary carcinoma.

Topics: Amino Acids; Aorta; Breast Neoplasms; Elastin; Epithelium; Female; Fetus; Fluorescent Antibody Technique; Humans; Pregnancy

Topics: Animals; Arteries; Breast Neoplasms; Elastic Tissue; Elastin; Growth; Humans; Lipid Metabolism; Mesothelioma; Muscle, Smooth, Vascular; Swine

Duct elastosis was studied in 219 patients subjected to radical mastectomy for infiltrating carcinoma of the breast, with a 10-year follow-up. Duct elastosis is a frequent finding in infiltrating breast cancer (65% of our cases). It develops in tumors of all three grades of malignancy, but it is more frequent in tumors of low grade malignancy (76% and 74% in grades I and II, respectively, and 47% in grade III tumors). In spite of their greater incidence in low malignancy tumors, the elastotic cases have a greater metastatic ratio than the non-elastotic cases (66% vs 45%). The elastotic cases also contain a significantly greater proportion of scirrhous tumors than the non-elastotic cases (86% vs. 32%). Duct elastosis and scirrhous reaction are two processes which develop in parallel, but are not related etiologically. They seem to be correlated with more advanced stages of the neoplastic disease. The influence of duct elastosis upon the ten year survival of the patients is unfavorable. this influence is not direct, and it is particularly evident in the metastatic cases. It seems to be related to the greater duration of the neoplastic disease and to the slow clinical course of tumors of low degree of malignancy.

Topics: Breast Neoplasms; Elastin; Female; Humans; Life Expectancy; Mastectomy; Neoplasm Metastasis; Prognosis

Topics: Adolescent; Adult; Aged; Aging; Aorta; Arteriosclerosis; Breast; Breast Neoplasms; Child; Desmosine; Elastin; Female; Humans; Middle Aged; Pancreatic Elastase

Two neutral proteases have been isolated from aortas and human breast tumors. The aortic elastase-like enzyme has been further purified. The details of this purification procedure will be given and some of the properties of the purified enzyme (susceptibility to various kinds of substrates, degree of inhibition of serum inhibitors, alpha 1-antitrypsin and alpha 2-macroglobulin). This elastinolytic activity of the aorta increased with age and with the degree of atherosclerosis. Both parameters seem to act independently and in a cumulative fashion. Elastinolytic activity has been demonstrated in extracts of human breast carcinomas and is exponentially related to the age of the patient. There exists a parallel neosynthesis of elastin which increased also with the age of the patient. Some characteristics of the polymeric elastin isolated from the tumor tissue will be given. The possible role of this neutral protease present in human aortas and human breast carcinomas will be discussed.

Topics: Aging; alpha 1-Antitrypsin; alpha-Macroglobulins; Animals; Aorta; Arteriosclerosis; Breast Neoplasms; Elastin; Female; Humans; Kinetics; Pancreatic Elastase; Swine

Topics: Breast Neoplasms; Elastic Tissue; Elastin; Female; Humans

Polymeric elastin was isolated and chemically characterized from 34 human breast cancers. There exists a good correlation between the histological and biochemical determinations of elastin; the breast cancer elastin resembles the other elastins isolated from ligamentum nuchae or aorta. Meanwhile it differs by its lower proline content and by its degree of crosslinking as determined by the ratio (Des + IDes/4)Lys. An elastinolytic activity (elastase) was found in human breast cancer extracts. This activity increased with the elastin content of the tumors.

Topics: Amino Acids; Breast Neoplasms; Carcinoma; Chemical Phenomena; Chemistry; Elastin; Female; Histocytochemistry; Humans; In Vitro Techniques; Pancreatic Elastase

Topics: Adult; Animals; Aorta; Arteriosclerosis; Breast Neoplasms; Elastic Tissue; Elastin; Humans; Middle Aged; Rabbits

Topics: Adult; Aged; Amyloid; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Elastin; Female; Histocytochemistry; Humans; Hyalin; Middle Aged

Topics: Amyloid; Breast; Breast Neoplasms; Elastin; False Positive Reactions; Female; Fibroblasts; Histocytochemistry; Humans; Microscopy; Microscopy, Electron; Staining and Labeling

Topics: Age Factors; Breast; Breast Neoplasms; Carcinoma; Carcinoma, Intraductal, Noninfiltrating; Elastic Tissue; Elastin; Microscopy, Electron; Pancreatic Elastase; Staining and Labeling