agar and Breast-Neoplasms

We aimed to build cellular aggregates of TS/A and normal fibroblasts (LX-2) or CAFs (ME-iLX-2), verifying the value of this model in the screening of anticancer drugs and demonstrating the effect of CD44 on aggregate formation. We improved soft agar culture medium to coculture CAFs (NFs) and TS/A and compared the amount and area of cellular aggregates. Eugenol was added to this model to test its value. The transcription of human CD44 was analyzed through RT-qPCR. Cellular aggregates were formed, and both the amount and area of aggregates in the TS/A-ME-iLX-2 coculture group were higher than those in other groups. The eugenol inhibited the formation of TS/A-fibroblasts aggregates. Human CD44 was highly transcripted in TS/A-ME-iLX-2 aggregates. Cocultured cellular aggregates of fibroblasts and TS/A were successfully formed in the improved soft agar culture medium, and the promotion effect of CAFs on cancer cells was further confirmed. The eugenol test showed its value in the screening of anticancer drugs. The RT-qPCR results demonstrated the important effect of CD44 on aggregate formation.

Topics: Agar; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Culture Media; Eugenol; Female; Fibroblasts; Humans; Mice

In currently, biosynthesis of copper oxide nanoparticles (CuO NPs) are most widely used numerous in biological applications such as biosensor, energy, medicine, agriculture, environmental and industrial wastewater treatment. The hierarchical CuO NPs was synthesized via green chemistry method by using of Abutilon indicum (A. indicum) leaf extract, its nontoxic, facile and low-cost approaches. Biogenic synthesized CuO NPs was characterized by using a UV-visible absorption spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and Field mission scanning electron microscopy (FE-SEM) with Energy-dispersive X-ray spectroscopy (EDX) analysis. The synthesized CuO NPs was performed antibacterial activity against human pathogenic organisms of both Gram negative (Escherichia coli and Salmonella typhi) and Gram positive (Bacillus subtilis and Staphylococcus aureus) bacteria by using agar well diffusion method. Biological synthesized CuO NPs was showed potential bactericidal activity against Gram positive bacteria of B. subtilis than compared to Gram negative bacteria of E. coli. The cytotoxic effect of A. indicum mediated synthesized CuO NPs was evaluated against to human lung A549 and breast MDA-MB-231cancer cell lines by determined using of MTT assay. In furthermore, photocatalytic dye degradation was performed that synthesized CuO NPs have effectively removed 78% of malachite green dye molecule. Our investigation results suggested that the green synthesized CuO NPs potential biological activity of antibacterial activity against Gram positive bacterial, anticancer activity was effectively against MDA-MB-231cancer cell line and good dye degradation was exhibited in malachite green. The A. indicum aqueous leaf extract mediated synthesized CuO NPs has strongly suggested promising nano-biomaterials for fabrication of biomedical applications.

Topics: Agar; Anti-Bacterial Agents; Breast Neoplasms; Copper; Escherichia coli; Female; Gram-Positive Bacteria; Humans; Lung; Malvaceae; Metal Nanoparticles; Microbial Sensitivity Tests; Oxides; Plant Extracts; Rosaniline Dyes; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

We present a new formulation for a breast tissue-mimicking phantom for combined microwave and ultrasound imaging to assist breast cancer detection. Formulations based on coconut oil, canola oil, agar and glass beads were used to mimic skin and fat tissues. First, 36 recipes were fabricated, and properties were measured to determine the relationship and possible interaction between ingredients with the ultrasound and microwave properties. Based on these results, the formulae were developed to mimic different tissues found in breast, including skin, fat, fibroglandular, and tumour tissues. All phantoms contained a base of agar and glass beads at different proportions depending on the tissue mimicked. Tumour and fibroglandular tissues were best mimicked by adding polyvinylpyrrolidone (PVP), while using coconut oil for skin and canola oil for fat produced the best results. Five final phantoms with different internal structures were fabricated and imaged using B-mode ultrasound and a microwave transmission system. Microwave permittivity maps were obtained from the microwave system and compared to ultrasound images. The structure and composition of the phantoms were all confirmed through this microwave and ultrasound imaging.

Topics: Agar; Breast; Breast Neoplasms; Coconut Oil; Female; Humans; Microwave Imaging; Microwaves; Phantoms, Imaging; Rapeseed Oil; Ultrasonography

In this work the selective uptake of native horse spleen ferritin and apoferritin loaded with MRI contrast agents has been assessed in human breast cancer cells (MCF-7 and MDA-MB-231). The higher expression of L-ferritin receptors (SCARA5) led to an enhanced uptake in MCF-7 as shown in T2 and T1 weighted MR images, respectively. The high efficiency of ferritin internalization in MCF-7 has been exploited for the simultaneous delivery of curcumin, a natural therapeutic molecule endowed with antineoplastic and anti-inflammatory action, and the MRI contrast agent Gd-HPDO3A. This theranostic system is able to treat selectively breast cancer cells over-expressing ferritin receptors. By entrapping in apoferritin both Gd-HPDO3A and curcumin, it was possible to deliver a therapeutic dose of 167 μg ml(-1) (as calculated by MRI) of this natural drug to MCF-7 cells, thus obtaining a significant reduction of cell proliferation.

Topics: Agar; Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Apoferritins; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Contrast Media; Curcumin; Drug Carriers; Female; Ferritins; Horses; Humans; Iron-Binding Proteins; Magnetic Resonance Imaging; MCF-7 Cells; Receptors, Cell Surface; Scavenger Receptors, Class A; Spleen; Temperature; Theranostic Nanomedicine

Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment.

Topics: Adenosine Triphosphate; Agar; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Chemistry, Pharmaceutical; Drug Design; Drug Screening Assays, Antitumor; Female; Fibroblasts; Flow Cytometry; High-Throughput Screening Assays; Humans; Open Reading Frames; Ovarian Neoplasms

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Topics: Agar; Antineoplastic Agents; Breast Neoplasms; Carcinogenesis; Carcinoma in Situ; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Proliferation; Disease Progression; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; Humans; Hydrolases; Neoplastic Stem Cells; Ornithine; Protein-Arginine Deiminase Type 2; Protein-Arginine Deiminases; Sepharose

Since tissues and tumors are heterogenous populations containing different cell types, their transcriptomes are blends of multiple mRNA expression profiles. Although fluorescence-activated cell sorting (FACS) allows isolation of individual cell types, RNA isolation and quantification remain problematic from rare subsets, such as tissue stem cells. Likewise, identification of transcriptional changes relevant to the tumorigenic potential of mammalian cells while they are actively growing as colonies in soft agar is also hampered by limited amounts of starting material. Here we describe a convenient method that fills the gap between single cell and whole tissue mRNA analysis, enabling mRNA quantification for individual colonies picked from soft agar. Our method involves direct lysis, reverse transcription and quantitative PCR (RT-qPCR) on 500 sorted cells or a single soft agar colony, thus allowing evaluation of up to 20 transcripts in functionally distinct subpopulations without the need for RNA isolation or amplification.

Topics: Agar; Animals; Breast; Breast Neoplasms; Cell Count; Cells, Cultured; Epithelial Cells; Female; Flow Cytometry; Gene Expression Profiling; Humans; Mice; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

To report the design and imaging methodology of a photoacoustic scanner dedicated to imaging hemoglobin distribution throughout a human breast.. The authors developed a dedicated breast photoacoustic mammography (PAM) system using a spherical detector aperture based on our previous photoacoustic tomography scanner. The system uses 512 detectors with rectilinear scanning. The scan shape is a spiral pattern whose radius varies from 24 to 96 mm, thereby allowing a field of view that accommodates a wide range of breast sizes. The authors measured the contrast-to-noise ratio (CNR) using a target comprised of 1-mm dots printed on clear plastic. Each dot absorption coefficient was approximately the same as a 1-mm thickness of whole blood at 756 nm, the output wavelength of the Alexandrite laser used by this imaging system. The target was immersed in varying depths of an 8% solution of stock Liposyn II-20%, which mimics the attenuation of breast tissue (1.1 cm(-1)). The spatial resolution was measured using a 6 μm-diameter carbon fiber embedded in agar. The breasts of four healthy female volunteers, spanning a range of breast size from a brassiere C cup to a DD cup, were imaged using a 96-mm spiral protocol.. The CNR target was clearly visualized to a depth of 53 mm. Spatial resolution, which was estimated from the full width at half-maximum of a profile across the PAM image of a carbon fiber, was 0.42 mm. In the four human volunteers, the vasculature was well visualized throughout the breast tissue, including to the chest wall.. CNR, lateral field-of-view and penetration depth of our dedicated PAM scanning system is sufficient to image breasts as large as 1335 mL, which should accommodate up to 90% of the women in the United States.

Topics: Acoustics; Agar; Breast; Breast Neoplasms; Carbon; Equipment Design; Female; Healthy Volunteers; Hemoglobins; Humans; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Mammography; Phantoms, Imaging; Reproducibility of Results; Signal-To-Noise Ratio; Tomography Scanners, X-Ray Computed

Cancer stem cells (CSC) are chemoresistant and implicated in tumor recurrence, metastasis and high patient mortality; thus substances impairing CSC activity, could be invaluable as novel cancer therapeutics. We previously showed that CAPE (caffeic acid phenethyl ester), a component of propolis, a honeybee product, inhibits growth of MDA-MB-231 (MDA-231) cells, mdr gene expression, NF-κB, EGFR, and VEGF. We hypothesized that CAPE also acts by interfering with CSC-mediated effects. We isolated breast CSC (bCSC) from MDA-231 cells, a model of human triple-negative breast cancer, and mouse xenografts. bCSC grow as mammospheres (MMS) and when dissociated into single cells, form MMS again, a sign of self-renewal. bCSC exhibited the characteristic CD44(+)/CD24(-/low) phenotype and generated progenitors in the presence of serum, a CSC trait responsible for regenerating tumor mass. CAPE caused dose-dependent bCSC self-renewal inhibition and progenitor formation. Clonal growth on soft agar was inhibited dose-dependently, but apoptosis was not induced as determined by Annexin-V/PI assay. Instead, bCSC were noted to significantly progress from a quiescent cell cycle state in G0/G1 (82%), S phase (12%) to a cycling state with an increase in S phase (41%) and subsequent decrease in G0/G1 (54%). Treatment of bCSC with CAPE (4.5-days) decreased CD44 levels by 95%, while another cell population containing 10-100-fold lower CD44 content concurrently increased. Results suggest that CAPE causes pronounced changes in bCSC characteristics manifested by inhibition of self renewal, progenitor formation, clonal growth in soft agar, and concurrent significant decrease in CD44 content, all signs of decreased malignancy potential.

Topics: Agar; Animals; Apoptosis; Bees; Breast Neoplasms; Caffeic Acids; Cell Cycle; Cell Differentiation; Cell Proliferation; Cell Separation; Drug Screening Assays, Antitumor; Female; Honey; Humans; Mice; Neoplastic Stem Cells; Phenotype; Phenylethyl Alcohol; Propolis; Spheroids, Cellular; Tumor Cells, Cultured

The depth of two-photon fluorescence imaging in turbid media can be significantly enhanced by the use of the here described fluorescence detection method that allows to efficiently collect scattered fluorescence photons from a wide area of the turbid sample. By using this detector we were able to perform imaging of turbid samples, simulating brain tissue, at depths up to 3 mm, where the two-photon induced fluorescence signal is too weak to be detected by means used in conventional two-photon microscopy.

Topics: Agar; Brain; Breast Neoplasms; Cell Line, Tumor; Computer Simulation; Diagnostic Imaging; Female; Gelatin; Humans; Microscopy, Fluorescence, Multiphoton; Microspheres; Models, Biological; Nephelometry and Turbidimetry; Scattering, Radiation; Silicon

An approach that facilitates rapid isolation and characterization of tumor cells with enhanced metastatic potential is highly desirable. Here, we demonstrate that plating GI-101A human breast cancer cells on hard (0.9%) agar selects for the subpopulation of metastasis-initiating cells. The agar-selected cells, designated GI-AGR, were homogeneous for CD44(+) and CD133(+) and five times more invasive than the parental GI-101A cells. Moreover, mice injected with GI-AGR cells had significantly more experimental brain metastases and shorter overall survival than did mice injected with GI-101A cells. Comparative gene expression analysis revealed that GI-AGR cells were markedly distinct from the parental cells but shared an overlapping pattern of gene expression with the GI-101A subline GI-BRN, which was generated by repeated in vivo recycling of GI-101A cells in an experimental brain metastasis model. Data mining on 216 genes shared between GI-AGR and GI-BRN breast cancer cells suggested that the molecular phenotype of these cells is consistent with that of cancer stem cells and the aggressive basal subtype of breast cancer. Collectively, these results demonstrate that analysis of cell growth in a hard agar assay is a powerful tool for selecting metastasis-initiating cells in a heterogeneous population of breast cancer cells, and that such selected cells have properties similar to those of tumor cells that are selected based on their potential to form metastases in mice.

Topics: Agar; Animals; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Cell Culture Techniques; Cell Proliferation; Female; Gene Expression; Gene Expression Profiling; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Neoplastic Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44(+)/CD24(-/low) and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR(+)/ESA(+) cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44(+)/CD24(-/low), ESA(+), CD133(+), CXCR4(+) and PROCR(+) in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.

Topics: Agar; Animals; Antigens, CD; Biomarkers, Tumor; Breast Neoplasms; CD24 Antigen; Cell Division; Cell Line, Tumor; Cell Lineage; Cell Separation; Endothelial Protein C Receptor; Epithelium; Female; Flow Cytometry; Humans; Hyaluronan Receptors; Mesoderm; Mice; Mice, SCID; Neoplastic Stem Cells; Receptors, Cell Surface; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Exogenous prolactin is mitogenic and antiapoptotic in breast cancer cells, and overexpression of autocrine prolactin cDNA in breast cancer cell lines has been shown to stimulate their growth and to protect against chemotherapy-induced apoptosis. We examined the effects of the 'pure' prolactin receptor antagonist Delta1-9-G129R-hPrl (Delta1-9) on the breast cancer cell number and clonogenicity, alone and in combination with chemotherapy.. The effects of doxorubicin, paclitaxel and Delta1-9 on the growth of breast cancer cell lines (MCF-7, T47D, MDA-MB-453, MDA-MB-468 and SK-BR-3) in monolayer culture were assessed by the sulphorhodamine B assay. Effects on clonogenicity were assessed by soft agar assay for the cell lines and by the mammosphere assay for disaggregated primary ductal carcinoma in situ samples. Dual-fluorescence immunocytochemistry was used to identify subpopulations of cells expressing the prolactin receptor and autocrine prolactin.. Delta1-9 as a single agent had no effect on the cell number in monolayer culture, but potentiated the cytotoxic effects of doxorubicin and paclitaxel. Doxorubicin accordingly induced expression of prolactin mRNA and protein in all five breast cancer cell lines tested. Delta1-9 alone inhibited the clonogenicity in soft agar of cell lines by ~90% and the mammosphere forming efficiency of six disaggregated primary ductal carcinoma in situ samples by a median of 56% (range 32% to 88%). Subpopulations of cells could be identified in the cell lines based on the prolactin receptor and prolactin expression.. Autocrine prolactin appears to act as an inducible survival factor in a clonogenic subpopulation of breast cancer cells. The rational combination of cytotoxics and Delta1-9 may therefore improve outcomes in breast cancer therapy by targeting this cell population.

Topics: Agar; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; DNA, Complementary; Doxorubicin; Humans; Immunohistochemistry; Paclitaxel; Prolactin; Receptors, Prolactin; Reverse Transcriptase Polymerase Chain Reaction; Rhodamines; RNA, Messenger

Stereotactic breast biopsy (SBB) is the gold standard for noninvasive breast cancer diagnosis. Current systems rely on one of two methods for needle insertion: a vertical-approach (from above the breast compression plate) or a lateral-approach (parallel to the compression plate). While the vertical-approach is more commonly used, it is not feasible in patients with thin breasts (less than 3 cm thickness after compression) or with superficial lesions. We present a novel design of lateral guidance device for SBB which addresses these limitations of the vertical-approach, and provides improvements over existing lateral guidance hardware. This device incorporates spherical linkages to allow two degrees of rotational freedom in the needle trajectory for increased targeting flexibility, as well as an adjustable rigid needle support to minimize needle deflection within the tissue. Needle placement error in SBB experiments is compared using both the new lateral guidance device and a commercial lateral guidance device in agar phantoms. The effect of elevation angle on needle placement accuracy using the new lateral guidance device is also assessed. Finally, a biopsy accuracy experiment is presented using a certified SBB phantom to compare the new design and the commercial lateral guidance device. In these experiments, SBB performed using the new lateral guidance device resulted in improved needle placement error and biopsy accuracy, while increasing targeting flexibility and maintaining procedural workflow.

Topics: Agar; Algorithms; Biopsy; Biopsy, Needle; Breast Neoplasms; Equipment Design; Humans; Image Interpretation, Computer-Assisted; Injections; Mammography; Needles; Phantoms, Imaging; Reproducibility of Results; Software; Surgery, Computer-Assisted

A limited number of receptor tyrosine kinases (e.g., ErbB and fibroblast growth factor receptor families) have been genetically linked to breast cancer development. Here, we investigated the contribution of the Ret receptor tyrosine kinase to breast tumor biology. Ret was expressed in primary breast tumors and cell lines. In estrogen receptor (ER)alpha-positive MCF7 and T47D lines, the ligand (glial-derived neurotrophic factor) activated signaling pathways and increased anchorage-independent proliferation in a Ret-dependent manner, showing that Ret signaling is functional in breast tumor cells. Ret expression was induced by estrogens and Ret signaling enhanced estrogen-driven proliferation, highlighting the functional interaction of Ret and ER pathways. Furthermore, Ret was detected in primary cancers, and there were higher Ret levels in ERalpha-positive tumors. In summary, we showed that Ret is a novel proliferative pathway interacting with ER signaling in vitro. Expression of Ret in primary breast tumors suggests that Ret might be a novel therapeutic target in breast cancer.

Topics: Agar; Biopsy; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Estrogen Receptor alpha; Fibroblasts; Gene Expression Regulation, Neoplastic; Glial Cell Line-Derived Neurotrophic Factor; Humans; Models, Biological; Protein Binding; Proto-Oncogene Proteins c-ret; Signal Transduction; Steroids

Bis-[(p-methoxybenzyl)cyclopentadienyl] titanium dichloride, better known as Titanocene Y, is a newly synthesized transition metal-based anticancer drug. We studied the antitumor activity of Titanocene Y with concentrations of 2.1, 21 and 210 micromol/l against a freshly explanted human breast cancer, using an in-vitro soft agar cloning system. The sensitivity against Titanocene Y was highly remarkable in the breast cancer tumor in the full concentration range. Titanocene Y showed cell death induction at 2.1 micromol/l, well comparable to cisplatin, given at a concentration of 1.0 micromol/l. A further preclinical development of Titanocene Y was warranted and therefore an MCF-7 human breast cancer xenograft nonobese diabetic/severe combined immunodeficient mouse model was used. Titanocene Y was given for 21 days at 30 mg/kg/day (75% of the maximum tolerable dose of Titanocene Y), which resulted in the reduction of the tumor volume to around one-third, whereas no mouse was lost because of the surprisingly low toxicity of Titanocene Y.

Topics: Agar; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cisplatin; Female; Humans; Mice; Neoplasm Transplantation; Organ Culture Techniques; Organometallic Compounds; Xenograft Model Antitumor Assays

Recent evidence indicates that growth hormone-releasing hormone (GHRH) functions as an autocrine/paracrine growth factor for various human cancers. A splice variant (SV) of the full-length receptor for GHRH (GHRHR) is widely expressed in various primary human cancers and established cancer cell lines and appears to mediate the proliferative effects of GHRH. To investigate in greater detail the role of SV1 in tumorigenesis, we have expressed the full-length GHRHR and its SV1 in MCF-7 human breast cancer cells that do not possess either GHRHR or SV1. In accordance with previous findings, the expression of both GHRHR and SV1 restored the sensitivity to GHRH-induced stimulation of cell proliferation, with SV1 being more potent than the GHRHR. Furthermore, MCF-7 cells transfected with SV1 proliferated more quickly than the controls, even in the absence of exogenously added GHRH, suggesting the existence of intrinsic, ligand-independent activity of SV1 after its transfection. In agreement with the stimulation of cell proliferation, the levels of proliferation markers cyclin D1, cyclin E, and proliferating cell nuclear antigen were elevated in MCF-7 cells treated with GHRH, cultured in both serum-free and serum-containing media. In addition, SV1 caused a considerable stimulation of the ability of MCF-7 cells to grow in semisolid medium, an assay considered diagnostic for cell transformation. Collectively, our findings show that the expression of SV1 confers oncogenic activity and provide further evidence that GHRH operates as a growth factor in breast cancer and probably other cancers as well.

Topics: Agar; Alternative Splicing; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Neoplasms; Receptors, Neuropeptide; Receptors, Pituitary Hormone-Regulating Hormone; Transfection

Deregulated signaling by the epidermal growth factor receptor family of proteins is encountered in human malignancies including breast cancer. Cell cycle and apoptosis-regulatory protein-1 (CARP-1), a novel, perinuclear phosphoprotein, is a regulator of apoptosis signaling by epidermal growth factor receptors. CARP-1 expression is diminished in human breast cancers, and correlates inversely with human breast cancer grades which could be attributed to increased methylation. The expression of CARP-1, on the other hand, interferes with the ability of human breast cancer cells to invade through the matrigel-coated membranes, to form colonies in the soft agar, and to grow as s.c. tumors in severe combined immunodeficiency (SCID) mice. To test whether CARP-1 is a suppressor of human breast cancer growth, we generated transactivator of transcription (TAT)-tagged CARP-1 peptides. Treatment of human breast cancer cells with affinity purified, TAT-CARP-1 1-198, 197-454, and 896-1150 peptides caused inhibition of human breast cancer cell proliferation and elevated apoptosis. In contrast, TAT-tagged enhanced green fluorescent protein or CARP-1 (1-198(Y192/F)) peptide failed to inhibit cell proliferation or induce apoptosis. Apoptosis by CARP-1 peptides, with the exception of CARP-1 (1-198(Y192/F)), involves the activation of p38 stress-activated protein kinase and caspase-9. Moreover, administration of TAT-CARP-1 (1-198), but not TAT-tagged enhanced green fluorescent protein or TAT-CARP-1 (1-198(Y192/F)), inhibits growth of human breast cancer cell-derived tumor xenografts in SCID mice. We conclude that CARP-1 is a suppressor of human breast cancer growth, and its expression is diminished in tumors, in part, by methylation-dependent silencing.

Topics: Agar; Animals; Antineoplastic Agents; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Female; Gene Silencing; Green Fluorescent Proteins; Humans; In Vitro Techniques; Mice; Mice, SCID; Neoplasm Transplantation; Transcriptional Activation

Previous work has shown that the Simian Virus 40 T antigen (T antigen) cannot transform mouse embryo fibroblasts (MEFs) that do not express the type 1 insulin-like growth factor receptor (IGF-IR). We have now investigated the mechanism(s) by which the transforming activity of T antigen is affected by IGF-IR signaling. We demonstrate that transformation by T antigen of MEFs and several other cell lines requires an insulin receptor substrate-1 (IRS-1) phosphorylated on tyrosines. If IRS-1 is not expressed, or is serine phosphorylated or otherwise inactive, T antigen fails to transform cells in culture. For instance, while T antigen cannot transform 32D myeloid cells (that do not express IRS-1), its transforming activity is restored by the expression of a wild-type IRS-1, but not of an IRS-1 mutated at the PI3K binding sites. The importance of IRS-1 activation of PI3K in T-antigen transformation is supported by the finding that a constitutively activated p110 subunit of PI3K, a target of IRS-1, overcomes the inability of T antigen to transform MEFs with a serine phosphorylated IRS-1. Taken together, these results indicate that the IRS-1/PI3K signaling is one of the mechanisms regulating transformation by the SV40 T antigen. We propose that the requirement for a tyrosyl-phosphorylated IRS-1 provides a mechanism to explain the failure of T antigen to transform MEFs with deleted IGF-IR genes.

Topics: Agar; Animals; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor; Binding Sites; Blotting, Western; Breast Neoplasms; Cell Line; Cell Line, Transformed; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Fibroblasts; Gene Deletion; Insulin Receptor Substrate Proteins; Mice; Mutation; Neurons; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Pol1 Transcription Initiation Complex Proteins; Receptor, IGF Type 1; Ribosomes; RNA; RNA, Ribosomal; Serine; Signal Transduction; Time Factors; Transfection; Tyrosine

Women with BRCA1 gene mutations have an increased risk for breast and ovarian cancer (BOC). Classification of missense variants as neutral or disease causing is still a challenge and has major implications for genetic counseling. BRCA1 is organized in an N-terminal ring-finger domain and two BRCT (breast cancer C-terminus) domains, involved in protein-protein interaction. The integrity of the C-terminal, BRCT repeat region is also critical for BRCA1 tumor suppressor function. Several molecular partners of BRCA1 have so far been identified; among them, the tumor suppressor protein p53 seems to play a major role. This study was aimed at evaluating the impact of two missense mutations, namely the W1837R and the S1841N, previously identified in BOC patients and located in the BRCT domain of the BRCA1 gene, on the binding capacity of this protein to p53. Co-immunoprecipitation assays of E. coli-expressed wild-type and mutated BRCTs challenged with a HeLa cell extract revealed, for the S1841N variant a significant reduction in the binding activity to p53, while the W1837R mutant showed an inverse effect. Furthermore, a clonogenic soft agar growth assay performed on HeLa cells stably transfected with either wild-type or mutant BRCA1 showed a marked decrease of the growth in wild-type BRCA1-overexpressing cells and in BRCA1S1841N-transfected cells, while no significant changes were detected in the BRCA1W1837R-transfected cells. These results demonstrate that: i) distinct single nucleotide changes in the BRCT domain of BRCA1 affect binding of this protein to the tumor suppressor p53, and ii) the two missense mutations here described are likely to play a role in breast tumorigenesis. We suggest that in vitro/in vivo experiments testing the effects of unclassified BRCA1 gene variants should therefore be taken in to consideration and that increased surveillance should be adopted in individuals bearing these two BRCA1 missense alterations.

Topics: Agar; Alleles; Breast Neoplasms; Cell Proliferation; Escherichia coli; Female; Genes, BRCA1; HeLa Cells; Humans; In Vitro Techniques; Models, Genetic; Mutation, Missense; Protein Binding; Protein Structure, Tertiary; Tumor Suppressor Protein p53

In this study, differential gene expression between normal human mammary epithelial cells and their malignant counterparts (eight well established breast cancer cell lines) was studied using Incyte GeneAlbum 1-6, which contains 65,873 cDNA clones representing 33,515 individual genes. 3,152 cDNAs showed a > or =3.0-fold expression level change in at least one of the human breast cancer cell lines as compared with normal human mammary epithelial cells. Integration of breast tumor gene expression data with the genes in the tumor suppressor p53 signaling pathway yielded 128 genes whose expression is altered in breast tumor cell lines and in response to p53 expression. A hierarchical cluster analysis of the 128 genes revealed that a significant portion of genes demonstrate an opposing expression pattern, i.e. p53-activated genes are down-regulated in the breast tumor lines, whereas p53-repressed genes are up-regulated. Most of these genes are involved in cell cycle regulation and/or apoptosis, consistent with the tumor suppressor function of p53. Follow-up studies on one gene, RAI3, suggested that p53 interacts with the promoter of RAI3 and repressed its expression at the onset of apoptosis. The expression of RAI3 is elevated in most tumor cell lines expressing mutant p53, whereas RAI3 mRNA is relatively repressed in the tumor cell lines expressing wild-type p53. Furthermore, ectopic expression of RAI3 in 293 cells promotes anchorage-independent growth and small interfering RNA-mediated depletion of RAI3 in AsPc-1 pancreatic tumor cells induces cell morphological change. Taken together, these data suggest a role for RAI3 in tumor growth and demonstrate the predictive power of integrative genomics.

Topics: Agar; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Cluster Analysis; DNA, Complementary; Down-Regulation; Epithelial Cells; Gene Expression Regulation, Neoplastic; Genome, Human; Genomics; HeLa Cells; Humans; Mammary Glands, Human; Models, Genetic; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Transcription, Genetic; Tumor Suppressor Protein p53; Up-Regulation

To testify the feasibility of electrical impedance scanning (EIS) imaging and to classify the factors influencing EIS imaging at the early stage of electrical impedance scanning breast imaging study.. Based on the EIS experiment workplace, a phantom to simulate the distribution of breast tissue was designed. Using NaCl solution and agar block with different conductance, three kinds of electric fields disturbances were simulated.. Different electric fields disturbance induced by different conductance distributions bring different imaging effects.. The imaging based on the theory of EIS is feasible. Initiative factors influencing EIS imaging are confirmed.

Topics: Agar; Breast Neoplasms; Electric Impedance; Feasibility Studies; Mammography; Phantoms, Imaging; Sodium Chloride

Classical NF-kappaB (p65/p50) transcription factors display dynamic induction in the mammary gland during pregnancy. To further elucidate the role of NF-kappaB factors in breast development, we generated a transgenic mouse expressing the IkappaB-alpha S32/36A superrepressor (SR) protein under control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. A transient delay in mammary ductal branching was observed in MMTV-SR-IkappaB-alpha mice early during pregnancy at day 5.5 (d5.5) and d7.5; however, development recovered by mid- to late pregnancy (d14.5). Recovery correlated with induction of nuclear cyclin D1 and RelB/p52 NF-kappaB complexes. RelB/p52 complexes induced cyclin D1 and c-myc promoter activities and failed in electrophoretic mobility shift assay to interact with IkappaB-alpha-glutathione S-transferase, indicating that their weak interaction with IkappaB-alpha can account for the observed recovery of mammary gland development. Activation of IKKalpha and NF-kappaB-inducing kinase was detected by d5.5, implicating the alternative NF-kappaB signaling pathway in RelB/p52 induction. Constitutively active IKKalpha induced p52, RelB, and cyclin D1 in untransformed mammary epithelial cells. Moreover, mouse mammary tumors induced by 7,12-dimethylbenz(a)anthracene treatment displayed increased RelB/p52 activity. Inhibition of RelB in breast cancer cells repressed cyclin D1 and c-Myc levels and growth in soft agar. These results implicate RelB/p52 complexes in mammary gland development and carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Agar; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Female; Glutathione Transferase; Humans; I-kappa B Kinase; I-kappa B Proteins; Immunoblotting; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mice; Mice, Transgenic; NF-kappa B; NF-kappa B p52 Subunit; NF-KappaB Inhibitor alpha; Phenotype; Pregnancy; Pregnancy, Animal; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-myc; RNA; Time Factors; Transcription Factor RelA; Transcription Factor RelB; Transfection; Transgenes

The expression of the rodent phosphoinositide-specific phospholipase C delta-4 (PLCdelta4) has been found to be elevated upon mitogenic stimulation and expression analysis have linked the upregulation of PLCdelta4 expression with rapid proliferation in certain rat transformed cell lines. The human homologue of PLCdelta4 has not been extensively characterized. Accordingly, we investigate the effects of overexpression of human PLCdelta4 on cell signaling and proliferation in this study.. The cDNA for human PLCdelta4 has been isolated and expressed ectopically in breast cancer MCF-7 cells. Overexpression of PLCdelta4 selectively activates protein kinase C-phi and upregulates the expression of epidermal growth factor receptors EGFR/erbB1 and HER2/erbB2, leading to constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in MCF-7 cells. MCF-7 cells stably expressing PLCdelta4 demonstrates several phenotypes of transformation, such as rapid proliferation in low serum, formation of colonies in soft agar, and capacity to form densely packed spheroids in low-attachment plates. The growth signaling responses induced by PLCdelta4 are not reversible by siRNA.. Overexpression or dysregulated expression of PLCdelta4 may initiate oncogenesis in certain tissues through upregulation of ErbB expression and activation of ERK pathway. Since the growth responses induced by PLCdelta4 are not reversible, PLCdelta4 itself is not a suitable drug target, but enzymes in pathways activated by PLCdelta4 are potential therapeutic targets for oncogenic intervention.

Topics: Agar; Breast Neoplasms; Cell Growth Processes; Cell Proliferation; Culture Media, Serum-Free; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; Genes, erbB-1; Genes, erbB-2; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; Isoenzymes; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phospholipase C delta; Protein Kinase C; RNA, Small Interfering; Spheroids, Cellular; Tumor Cells, Cultured; Type C Phospholipases; Up-Regulation

Real-time needle segmentation and tracking is very important in image-guided surgery, biopsy, and therapy. Due to its robustness to the addition of extraneous noise, the Hough Transform is one of the most powerful line-detection techniques nowadays and has been widely used in different areas. Unfortunately, its high computation needs often prevent it from being applied in real-time applications without the help of specially designed hardware. In order to solve this problem, a variety of fast implementation algorithms have been proposed. However, none of them can be performed in a real time on an affordable computer. In this paper, we describe a fast implementation of the Hough Transform based on coarse-fine search and the determination of the optimal image resolution. Compared to conventional techniques, our approach decreases the time for needle segmentation by an order of magnitude. Experiments with agar phantom and patient breast biopsy ultrasound (US) image sequences showed that our approach can segment the biopsy needle in real time (i.e., less than 33 ms) on an affordable PC computer without the help of specially designed hardware with the angular rms error of about 1 degrees and the position rms error of about 0.5 mm.

Topics: Agar; Algorithms; Biopsy, Needle; Breast; Breast Neoplasms; Humans; Models, Theoretical; Phantoms, Imaging; Time Factors

Long-term elevation of metastasis suppressor gene expression in micrometastases represents a novel therapeutic strategy for breast and other cancers. We searched for well-tolerated compounds that could elevate Nm23 metastasis suppressor expression in metastatic human breast cancer cell lines.. MDA-MB-435 and MDA-MB-231 human breast carcinoma cells were treated with dexamethasone or medroxyprogesterone acetate (MPA) in cultures containing either charcoal-stripped serum or FCS. Aspects of nm23 expression and function were determined.. Previous investigation of the nm23-H1 promoter suggested that glucocorticoids may contribute to the elevation of Nm23-H1 expression. Dexamethasone elevated Nm23-H1 and Nm23-H2 protein levels in two metastatic human breast carcinoma cell lines 2-3-fold over a 4-day time course when cultured in steroid-free culture medium, with high-dose inhibition, via a traditional transcriptional mechanism. Elevation of Nm23-H1 expression was not observed using FCS-containing culture medium, which contains endogenous levels of corticosteroids, limiting the potential in vivo use of dexamethasone. MPA was investigated as a glucocorticoid receptor agonist. MPA elevated breast carcinoma Nm23-H1 protein expression 3-fold over a 10 nM to 1 micro M dose range when cultured in steroid-free or FCS-containing medium, with a shorter time course. Elevation of Nm23-H1 expression in the presence of endogenous corticosteroids found in FCS involved a distinct, glucocorticoid receptor-dependent, posttranscriptional mechanism of action. MPA had no effect on proliferation in vitro but reduced the soft agar colonization of metastatic breast cancer cell lines by approximately 50%.. MPA represents a first generation lead agent for the elevation of Nm23-H1 metastasis suppressor expression and the inhibition of metastatic colonization.

Topics: Agar; Antineoplastic Agents, Hormonal; Blotting, Western; Breast Neoplasms; Cell Division; Cell Line, Tumor; Contraceptives, Oral, Synthetic; Dexamethasone; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Glucocorticoids; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Mutagenesis; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Promoter Regions, Genetic; Protein Biosynthesis; RNA Processing, Post-Transcriptional; Time Factors; Transcription, Genetic; Transfection

To elucidate the role of S-adenosylmethionine decarboxylase (SAMDC) in breast cancer biology, we have generated SAMDC overexpressing MCF-7 breast cancer cells. SAMDC overexpression did not alter in a major way growth properties of MCF-7 cells in soft agar, either under basal conditions or in response to estrogen and antiestrogen administration. SAMDC-MCF-7 cells, on the other hand, exhibited a markedly reduced invasive ability in matrigel (p=0.013). Furthermore, they were less tumorigenic in nude mice. The odds for control clones to form tumors were 3.13 (C.1.1.2-8.2, p=0.0184) higher than those for SAMDC clones. The odds ratio were identical in the absence and in the presence of estradiol. In addition, the growth rate of established tumors was slower for SAMDC than for control clones. Overall, our results are consistent with the notion that these phenotypic changes induced by SAMDC overexpression are primarily mediated by suppression of cellular putrescine (and, possibly, spermidine) levels.

Topics: Adenosylmethionine Decarboxylase; Agar; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Division; Collagen; DNA-Binding Proteins; Drug Combinations; Epidermal Growth Factor; Estradiol; Female; Fulvestrant; Humans; Laminin; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Proteoglycans; Sensitivity and Specificity; STAT3 Transcription Factor; Tamoxifen; Trans-Activators; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Stem Cell Assay

Breast cancer has a predilection for spreading to bone. The mechanism of preferential metastasis of breast cancer to bone is unknown. We hypothesize that breast cancer cells that develop bone metastases have the capacity to facilitate their colonization in bone. To examine this hypothesis, we established bone-seeking (MDA-231BO) and brain-seeking (MDA-231BR) clones of the human breast cancer cell line MDA-MB-231 by repeated sequential passages in nude mice and in vitro of metastatic cells obtained from bone and brain metastases, respectively. These clones were examined for distinguishing biological characteristics and compared with the MDA-231 parental cells (MDA-231P) in vivo and in vitro. Both the MDA-231BR and the MDA-231BO showed identical tumorigenicity to MDA-231P at the orthotopic site. MDA-231P that was inoculated into the heart developed metastases in bone, brain, ovary, and adrenal glands. On the other hand, MDA-231BO exclusively metastasized to bone with larger osteolytic lesions than MDA-231P. MDA-231BR exclusively disseminated to brain and failed to develop bone metastases. In culture, MDA-231BO produced greater amounts of parathyroid hormone-related protein (PTH-rP) than MDA-231BR and MDA-231P in the absence or presence of transforming growth factor beta (TGF-beta). Furthermore, the anchorage-independent growth of MDA- 231BO in soft agar was not inhibited by TGF-beta, whereas TGF-beta profoundly inhibited the growth of MDA-231P and MDA-231BR. Insulin-like growth factor I (IGF-I) markedly promoted the anchorage-independent growth of MDA-231BO, whereas marginal or no stimulation was observed in MDA-231BR or MDA-231P, respectively. Our data suggest that these phenotypic changes allow breast cancer cells to promote osteoclastic bone resorption, survive, and proliferate in bone, which consequently leads to the establishment of bone metastases.

Topics: Agar; Animals; Bone and Bones; Bone Neoplasms; Brain; Brain Neoplasms; Breast Neoplasms; Cell Adhesion; Cell Culture Techniques; Cell Division; Chemotaxis; Clone Cells; Female; Gene Expression; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasms, Experimental; Parathyroid Hormone-Related Protein; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Protein Biosynthesis; Signal Transduction; Transcriptional Activation; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth.

Topics: Agar; Apoptosis; Breast; Breast Neoplasms; Carrier Proteins; Cell Adhesion; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Chromones; Contact Inhibition; Enzyme Activation; Enzyme Inhibitors; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Female; Humans; Morpholines; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Signal Transduction; Simian virus 40; Tretinoin; Tumor Stem Cell Assay; Tumor Suppressor Proteins; Vitamin A

Dietary unsaturated fatty acids have been implicated in breast cancer. Since mutations in BRCA1 gene are known to predispose to breast cancers, and BRCA1 gene is known to be regulated by estradiol, the effect of linoleic acid, an omega-6-polyunsaturated fatty acid, with and without estradiol was studied for the expression of BRCA1 gene, in MCF-7 cell line, which has only one wild type allele. MCF-7 cells exposed to either linoleic acid or estradiol showed relatively greater number of colonies on soft agar, extent of proliferation and BRCA1 mRNA expression compared with controls. However, cells treated with both linoleic acid and estradiol showed significantly higher number of colonies, proliferation index and appreciably decreased expression of BRCA1 mRNA compared with controls or cells treated with linoleic acid or estradiol alone. The synergistic effects of these two agents were abrogated when indomethacin, an inhibitor of prostaglandin pathway, was added to the culture. From these observations it appears that diet rich in omega-6-polyunsaturated fatty acid like linoleic acid and endogenous estrogen may modulate BRCA1 gene expression thereby promoting breast cancer.

Topics: Agar; Alleles; Anti-Inflammatory Agents, Non-Steroidal; Breast Neoplasms; Cell Division; DNA, Complementary; Down-Regulation; Estradiol; Gene Expression Regulation, Neoplastic; Genes, BRCA1; Humans; Indomethacin; Linoleic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Several melanomas, carcinomas, glioblastomas and leukemias showed coordinated expression of HOXB7 and bFGF with exception of the SkBr3 mammary carcinoma that was negative for both. Transduction of HOXB7 gene into SkBr3 cells, induced bFGF expression, increased growth rate, independence from serum withdrawal and ability to form colonies in semisolid medium. ELISA assay showed that most of bFGF was associated to cell lysate when cells were cultured at 1% serum whereas in cells kept to 10% serum bFGF was detected both within cell lysate or secreted into cell supernatants. Antisense oligos to bFGF inhibited the growth of cells cultured in 1%, indicating that beside the possible activation of additional genes other than bFGF by HOXB7 transduction, only bFGF induction accounts for the observed results. Moreover, since inhibition of cell proliferation occurred in cells kept in 1% but not 10% serum, a bFGF intracrine loop appears operative in serum starved SkBr3/HOXB7 cells. Also, these results further indicate bFGF as target of HOXB7.

Topics: Agar; Breast Neoplasms; Cell Division; Culture Media; Culture Media, Conditioned; Culture Media, Serum-Free; Female; Fibroblast Growth Factor 2; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Growth Substances; Homeodomain Proteins; Humans; Recombinant Proteins; RNA, Messenger; Transfection; Tumor Cells, Cultured

Retinoids modulate cellular proliferation and mediate gene function through a series of nuclear receptors. The retinoic acid nuclear receptor beta (RAR beta) plays an important role in the differentiation of a number of cell types. We now demonstrate that RAR beta expression is confined to normal mammary tissue and is not expressed in either immortalized normal or malignant cell lines. Treatment of RAR beta-transfected MDA-MB-231 cells with 1 microM all-trans-retinoic acid (RA) significantly inhibited monolayer growth of the cells which express recombinant RAR beta. RAR beta-expressing MDA-MB-231 cells formed significantly smaller and fewer colonies in soft agar than the mock-transfected cells. Addition of 1 microM RA stimulated colony size and number in the RAR beta-transfected MDA-MB-231 cells. In contrast to the RAR beta-expressing cells, colony formation by the RAR alpha-expressing cells was similar to the mock-transfected controls and the addition of 1 microM RA to the RAR alpha-transfected cells inhibited colony formation. While demonstrating decreased colony formation in agar, RAR beta-expressing MDA-MB-231 cells failed to exhibit decreased growth in SCID mice. Our results show that RAR beta functions as a negative regulator of growth in breast epithelial cells. In addition, the growth of these cells is differentially regulated by RAR alpha and RAR beta which is most likely the result of the modulation of different genes.

Topics: Agar; Animals; Base Sequence; Breast Neoplasms; Cell Adhesion; Cell Division; Cloning, Molecular; Female; Humans; Mice; Mice, SCID; Molecular Sequence Data; Receptors, Retinoic Acid; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

The hormone dependency of the MCF-7 breast cancer cell line, while extensively tested in liquid culture, has not been previously evaluated under conditions of anchorage-independent growth in serum-free media. Using the soft agar clonogenic assay, we demonstrate that physiologically relevant concentrations of estradiol (E2), progesterone (Pg), and prolactin (PRL) similarly stimulated MCF-7 cell colony formation in the absence of serum. Addition of an anti-insulin-like growth factor-I (IGF-I) antibody inhibited E2- and Pg-stimulated growth, while PRL action was not affected. Similar results were obtained with an anti-IGF-I receptor antibody, except that its inhibitory effect on Pg-induced colony formation was modest and not statistically significant. Administration of either an anti-transforming growth factor-alpha (TGF-alpha) antibody or an anti-epidermal growth factor (EGF) receptor antibody similarly inhibited E2-stimulated MCF-7 cell growth in soft agar, while neither antibody influenced Pg or PRL effects. Addition of TGF-beta 1, -beta 2, -beta 3 similarly suppressed MCF-7 cell colony formation in a dose dependent manner to a degree comparable to that observed with 4-OH-tamoxifen (4-OH-T). Furthermore, the growth inhibitory effect of 4-OH-T was completely reversed by an anti-TGF-beta antibody. We conclude that IGFs and TGF-alpha are important mediators of E2-stimulated MCF-7 cell growth in soft agar. IGFs may also be playing a role in Pg action, while neither growth factor is involved in PRL-stimulated colony formation. Finally, TGF-beta appears to be an important mediator of antiestrogen-induced inhibition of tumor growth.

Topics: Agar; Antibodies; Breast Neoplasms; Culture Media; Drug Interactions; Epidermal Growth Factor; Estradiol; Female; Growth Substances; Hormones; Humans; Insulin-Like Growth Factor I; Neoplasms, Hormone-Dependent; Progesterone; Prolactin; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Stem Cell Assay

We studied the effects human recombinant granulocyte-macrophage colony-stimulating factor and human recombinant interleukin-3 on the colony formation of three human solid tumor cell lines. Using a modified double-layer soft agar clonogenic assay rhGM-CSF enhanced colony formation of all cell lines tested in a dose dependent manner (up to twofold for the breast cancer cell line BT-20, up to 163% of the control for the hypernephroma cell line C 94 and up to 147% for the non-small cell lung cancer cell line CCL 185 at a concentration of 100 ng/ml). RhIL-3 stimulated colony formation of the cell lines C 94 and BT-20, whereas on the cell line CCL 185 rhIL-3 had no effect even at the highest dose level tested (100 ng/ml). Combinations of growth factors showed subadditive stimulation on two cell lines tested (BT-20, C 94). These data indicate that haematopoietic growth factors exert a growth promoting activity on certain solid tumor cells in vitro at physiological concentrations. Therefore our results suggest that the application of these factors in immuno- and myelosuppressed cancer patients after high dose chemotherapy should be seen in light of a possible co-stimulation of the malignant cells.

Topics: Agar; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Carcinoma, Renal Cell; Cell Division; Colony-Stimulating Factors; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Interleukin-3; Kidney Neoplasms; Lung Neoplasms; Neoplastic Stem Cells; Recombinant Proteins; Statistics as Topic; Tumor Cells, Cultured; Tumor Stem Cell Assay

The author succeeded in detecting microcalcifications within breast carcinomas, by ultrasound (US) imaging, at the beginning of 1982, and published the results of his clinical and experimental researches at the Third International Congress on the Ultrasonic Examination of the Breast held on June 10-12, 1983, in Tokyo, Japan. After that, more detailed clinical evidence was acquired and new experiments, using breast phantoms, were carried out. This paper introduces an experiment using phantoms which shows that US can detect and identify even 100-500 mu-sized tiny objects in an "ideal" low echoic area similar to microcalcifications within breast carcinomas. Actual clinical evidence is also presented.

Topics: Acrylic Resins; Agar; Breast; Breast Neoplasms; Calcinosis; Female; Gels; Glass; Humans; Mammography; Models, Structural; Ultrasonography; Xeroradiography

Two anthropomorphic phantoms, simulating the breast as it is compressed against the chest wall during ultrasonic imaging, are described in detail regarding structural configurations and distributions of ultrasonic properties. The external shape of each phantom is that of a rectangular parallelepiped; this simple shape is consistent with the breast in the compressed configuration and also facilitates the inclusion of a considerable variety of simulated lesions including comparisons of imaging as a function of depth. One of the phantoms was completed about 4 1/2 years prior to this writing, and images made with scanners which were state-of-the-art in late 1983 and 1984 are compared with images made with two current state-of-the-art imagers. Improvements in imager quality over that period are apparent. The more recently produced phantom contains a simulated tumor with a complex structure as well as realistic simulated calcifications of various sizes. The tumor has a necrotic core and an irregularly shaped boundary. This boundary possesses a roughness intended to give rise to diffuse reflection.

Topics: Agar; Breast; Breast Diseases; Breast Neoplasms; Equipment Design; Female; Gels; Humans; Models, Structural; Oils; Pressure; Scattering, Radiation; Ultrasonography

The clonogenicity in soft agar and the labeling index (LI) which represents the percentage of cells in the DNA synthesis phase, were studied in 59 breast cancer patients and these parameters were related to other known clinicopathological features, namely age, histological grading, estrogen and progesterone receptors and the status of axillary lymph nodes. Out of 59 tumors, 49 could be successfully cloned in soft agar and the mean plating efficiency (PE) was 0.1%. Low grade tumors were more frequently encountered in tumors which did not form colonies (P = 0.025). Cloned tumors had a higher mean LI (P = 0.05). A high PE was associated with low estrogen receptors (ER) (P = 0.03). Clonogenicity was not related to patient age, progesterone receptors (PR) or the status of axillary lymph nodes. These results suggest that a successful in vitro cloning and a high PE are associated with unfavorable prognostic factors in breast cancer.

Topics: Agar; Axilla; Breast Neoplasms; Colony-Forming Units Assay; Female; Humans; In Vitro Techniques; Lymphatic Metastasis; Neoplasm Staging; Prognosis; Receptors, Estrogen; Receptors, Progesterone; Tumor Stem Cell Assay

MCF-7 human breast cancer cells are used widely for studies of tumor biology and hormone mechanism of action. Conflicting results have often been obtained in studies reported from different laboratories. In this report several biological properties were studied in four MCF-7 cell lines obtained from different laboratories. MCF-7 (ATCC), MCF-7, MCF-7 (KO), and MCF-7 (S) demonstrated similar morphology in monolayer culture. Chromosome analysis revealed that three of the lines shared several structural chromosome alterations and marker chromosomes; however, MCF-7 (ATCC) was distinctly different with virtually no chromosomal alterations shared in common with the other lines. All four lines contained variable amounts of estrogen receptor (ER) and progesterone receptor (PgR). The growth rate of MCF-7 (ATCC) was 50% slower than that of the other lines, and, unlike the other three lines, cell proliferation was unaffected by estrogen or antiestrogen treatment despite the presence of receptors. Cloning efficiency of the four lines varied over a 10-fold range. Tumorigenicity in athymic nude mice also varied considerably among these lines. MCF-7 (ATCC) grew well in ovariectomized nude mice, while the other lines required estrogen supplementation. MCF-7 (S) and MCF-7 grew rapidly with estrogen supplementation; MCF-7 (KO) grew very slowly. Antiestrogen therapy inhibited growth of MCF-7, MCF-7 (S), and MCF-7 (KO) tumors, but it had no effect on MCF-7 (ATCC). These data demonstrate that MCF-7 lines from different laboratories may have unique biological properties, despite having a similar karyotype (MCF-7, MCF-7 (S), MCF-7 (KO]. The fundamental differences in karyotype and biological properties of the MCF-7 (ATCC), and the previously reported differences in DNA restriction fragment polymorphism analyses, demonstrate that this line is derived from an entirely different patient. Investigators should carefully document the source and identity of MCF-7 cells used in published experiments.

Topics: Agar; Animals; Breast Neoplasms; Cell Division; Cell Line; Estradiol; Humans; Interphase; Karyotyping; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen

The establishment of a spontaneously transformed tumorigenic human fibrosarcoma line derived from the ovary of a breast cancer patient (NO-15) is described. This tumor cell line has been characterized by tumor growth in nude mice. The cells form colonies without the usually recommended agar technique and are sensitive to cis-dichlorodiammineplatinum(II) (cis-DDP), daunorubicin (DR) and bleomycin (BLM), three well-known chemotherapeutic agents. The cells were incubated in the presence of drugs using three different methods. These agents proved to be noncytotoxic to monolayers treated for three hours. Conversely dose-dependent cytotoxicity was seen in suspension cultures treated for three hours. Optimal correlation between the three methods for the evaluation of cytotoxicity was seen in monolayers exposed to these agents for 24 hours. The results of three different methods to evaluate drug efficacy (number of colonies, growth rate and 3H-thymidine incorporation) were in good correlation. The incorporation of the radiolabeled tracer into cellular DNA is advantageous for its rapidity and quantitative reproducibility.

Topics: Adult; Agar; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Transformed; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Culture Media; Female; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Ovary; Tumor Cells, Cultured; Tumor Stem Cell Assay

The role of prolactin (PRL) in supporting the growth of human breast cancer is still unclear. The ability to grow primary breast cancer specimens in the soft agar clonogenic assay in the absence of serum gave us the opportunity to evaluate the growth-promoting effect of PRL and to compare it to that of estradiol in the same tumor samples. PRL was tested both at physiological concentrations (20 ng/ml) as well as in pharmacological amounts (200 ng/ml) comparable to circulating blood levels in hyperprolactinemic states. Estradiol was simultaneously tested in physiological amounts (10(-8)M). In 17 infiltrating ductal carcinomas, the lower dose of PRL stimulated colony formation to 126 +/- 5.2% (SE) of control, while the higher dose increased colony number to 159 +/- 10.4% of control. This latter effect was comparable to that observed with estradiol (159 +/- 8.5% of control). The effect of PRL was more pronounced in estrogen receptor-positive tumors. Nine of ten estrogen receptor-positive tumors were PRL sensitive, while three of seven estrogen receptor-negative tumors exhibited a clear response to PRL administration. PRL did not stimulate colony formation in a malignant cystosarcoma phylloides and in two benign lesions (fibroadenoma and fibrocystic disease). We conclude that, at least under the conditions of the soft agar clonogenic assay, PRL exerts a dose-dependent growth-promoting effect on human breast cancer. Such effect is comparable to that of estradiol when PRL is added in concentrations similar to circulating blood levels in hyperprolactinemic patients.

Topics: Adult; Agar; Aged; Breast Neoplasms; Cells, Cultured; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Female; Humans; Middle Aged; Prolactin; Receptors, Estrogen; Tumor Stem Cell Assay

Leukocyte migration inhibition (LMI) in patients with breast cancer was examined by using 3 M-KCl tissue extracts of breast tumors. Tumor extracts were obtained from 12 breast carcinomas of various histological types. In 20 of 65 (30.8%) leukocyte preparations from 25 patients with breast cancer migration was inhibited by breast cancer tissue extracts. In 26 leukocyte preparations from eight healthy persons and 22 preparations from eight patients with benign diseases of the breast migration was not inhibited by the breast cancer extracts. In only two of 47 (4.3%) leukocyte preparations from 12 patients with other cancers migration was inhibited by the extracts. The occurrence of LMI was the highest in leukocytes from the patients with breast cancer showing marked nuclear pleomorphism (10/19, 52.6%) or showing marked mononuclear cell infiltration (6/12, 50.0%). These results suggest that 3 M-KCl tissue extracts of breast cancer may contain tumor-associated antigens in solubilized form and that there may be a correlation between LMI and nuclear pleomorphism of cancer cells of the leukocyte donor as well as mononuclear cell infiltration in the tumor of the leukocyte donor.

Topics: Adenofibroma; Agar; Antigens, Neoplasm; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Migration Inhibition; Cell Nucleus; Cell Separation; Female; Humans; Leukocytes; Lymphocytes; Mitotic Index; Neoplasm Staging; Sepharose; Staining and Labeling; Tissue Extracts

To investigate the influence of culture conditions on the in vitro responses of tumour cells to anticancer drugs, the sensitivities observed with the soft agar methods of Hamburger & Salmon (1977) (H-S) and of Courtenay & Mills (1978) (C-M) were compared. In all cases the ID50 values were determined from dose-response curves. Six human tumour cell lines exposed to 10 different agents, and 9 patients' melanomas exposed to 5 different agents, were examined. In the studies of cell lines the H-S method gave higher sensitivity values than the C-M method in 38 out of 52 cases, whereas in 14 cases the results were the same. In the patients' tumours the H-S method gave higher sensitivity in 21 of 35 cases, equal sensitivity in 11, and lower sensitivity in 3 cases. In many instances the ID50 values obtained with the two test systems differed by factors of 10 or more, both in the case of cell lines and tumour specimens. Systematic alterations in the culture conditions indicated that the presence or absence of rat erythrocytes is the most important factor responsible for the differences observed. Also, other factors, such as supplements (in the H-S method) and the use of different serum types, appeared to influence both colony growth and chemosensitivity.

Topics: Agar; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Count; Cell Line; Cell Survival; Culture Media; Dose-Response Relationship, Drug; Erythrocytes; Humans; Melanoma; Neoplasms; Oxygen; Rats; Temperature

One hundred and thirty three specimens from mammary and ovarian adenocarcinoma and from melanoma were cultured according to an agar/agar clonogenic assay. Melanoma and ovarian cancers exhibited a 70 per cent rate of success for culture; 50 per cent of the mammary adenocarcinomas were successfully cultured. Fifty-nine ovarian cancers were cultured in order to test the in vitro effectiveness of Cisplatinum and Adriamycin. Thirty percent of cultured tumors gave rise to relevant chemograms. The chemoresistance measured in vitro was correlated to the ineffectiveness of the patient's treatment. In contrast, we were unable to predict chemosensitivity. Taking into account the technical difficulties encountered in these assays, human tumor clonogenic assays cannot at present be proposed as a routine procedure in the prediction of the effectiveness of chemotherapeutic treatments. Nevertheless, they must be developed in order to determine the spectrum of activity of new antineoplastic agents on various human tumors.

Topics: Adenocarcinoma; Agar; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cisplatin; Colony-Forming Units Assay; Cyclophosphamide; Doxorubicin; Drug Resistance; Female; Humans; Melanoma; Melphalan; Ovarian Neoplasms; Tumor Stem Cell Assay

Four established human breast tumour cell lines with different biologic properties were selected for study and requirements for their clonogenic growth in semisolid cultures were identified. The conventional conditions were modified by factors that enhanced colony formation of 3 or more of these cell lines. The modified culture conditions were then applied to the growth in agar of primary breast tumours. A 5-fold improvement in plating efficiency was observed when cultures of 105 primary tumours grown under these modified conditions were compared to those of 52 tumours grown earlier under conventional conditions, and a 4-fold improvement resulted from the addition of hormones and conditioned medidum in 26 tumours cultured simultaneously under both conditions. The biologic relevance of these clonogens recovered in vitro was substantiated by a 70% concordance of in vitro and in vivo tumour sensitivity to anticancer drugs.

Topics: Agar; Blood; Breast Neoplasms; Cell Division; Cell Line; Clone Cells; Culture Media; Female; Hormones; Humans; Neoplasms, Hormone-Dependent; Neoplastic Stem Cells

Topics: Agar; Animals; Biopsy; Breast Neoplasms; Cell Survival; Colonic Neoplasms; Colony-Forming Units Assay; Culture Techniques; Female; Humans; Male; Melanoma; Melphalan; Mice; Neoplasms; Ovarian Neoplasms; Rectal Neoplasms; Tumor Stem Cell Assay

Topics: Agar; Biopsy; Breast Neoplasms; Cell Survival; Clone Cells; Culture Techniques; Female; Humans; Lung Neoplasms; Male; Melanoma; Methylcellulose; Microscopy, Electron; Neoplasms; Pleural Effusion

An in vitro soft agar culture system was utilized to evaluate the colony growth of cells from primary breast carcinoma. A total of 53 specimens from fifty-three patients were placed in culture. Of these, 29 samples (55 per cent) formed at least 30 colonies per 500,000 cells plated. In relation to histologic type of tumor and clinical status of the disease, 4 of 4 samples from mucous carcinoma grew into colonies and, then, t-categories, i.e. histological extent of primary tumor, and colony growth showed an inverse correlation. Estrogen receptor status did not appear to influence growth of the colonies. The in vitro sensitivity studies to adriamycin showed a dose dependent increase in lethality. However, the in vitro response rate was relatively low. This assay system can be used to study the biology and clinical approaches to treatment of breast carcinoma.

Topics: Adult; Agar; Aged; Breast Neoplasms; Carcinoma; Cell Division; Cell Survival; Cells, Cultured; Colony-Forming Units Assay; Doxorubicin; Female; Humans; Middle Aged; Receptors, Estrogen; Tumor Stem Cell Assay

Human tumors were cultured by the two-layer soft-agar technique and the time course of tumor colony development was evaluated during periods of up to 6 weeks in culture. All colony counting was performed with an automated tumor colony counter (Omnicon; Bausch and Lomb, Inc, Rochester, NY, USA). This instrument provided colony counts per culture plate in six size categories from greater than 60 microns diameter colonies to greater than 149 microns diameter colonies. Six to 24 culture plates were used for each "growth curve", generally 24. Control (non-drug-treated) cultures were obtained from 117 tumors, of which 25 also provided enough cells to allow evaluation of the time course of colony development after exposure to cytostatic agents. The development of colonies in non-drug-treated plates usually demonstrated a lag phase, a logarithmic growth phase to maximum colony development and a subsequent deterioration of colonies. In spite of clumps seeded into the agar, real colony growth could be recognized by frequent colony counting of culture dishes, although the temporal patterns of growth were sometimes different if pure single-cell suspensions were compared with suspensions containing clumps from the same tumor. Drug pre-incubation caused changes in the temporal pattern of colony growth as well as in the total number of colonies. Some cultures showed drug sensitivity when evaluated at certain time points while evaluation at later time points showed only borderline drug effect or none at all. The potential utility of tumor colony growth curves in the clinical applications of tumor colony cultures is discussed.

Topics: Agar; Animals; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Small Cell; Colony-Forming Units Assay; Cystadenoma; Female; Humans; Male; Ovarian Neoplasms; Prostatic Neoplasms; Rats; Skin Neoplasms; Staining and Labeling; Time Factors; Tumor Stem Cell Assay

Relationship between histology, cloning efficiencies and estrogen receptors was studied in the group of breast cancer patient. Mechanical disaggregation gave poor cellular yields since only in 66% cells were ready for the bioassay. Comparison between clonogenic assay, histology and estrogen status of human breast cancer is of a limited value only, because of a small number of patients in our study.

Topics: Agar; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Clone Cells; Female; Humans; Lymph Nodes; Receptors, Estrogen

The effect of platelet lysate on the growth of human breast cancer cells was studied in the two-layer soft agar culture system. Lysate was prepared from frozen-thawed outdated human platelets. Eighteen to 446 colonies (mean: 85) grew from 50 of 57 tumors plated at a density of 5 X 10(5) cells/plate, yielding control plating efficiencies from 0.004-0.089. The viability of the cells plated ranged from 10-80%. All tumors were also plated with 200 micrograms/ml platelet lysate. Significant enhancement of colony formation (CF) (p less than 0.025) appeared in 26 of the 57 tumors tested. The number of colonies grown from these 26 tumors ranged from 36-580 (mean: 152). The extent of enhancement varied from 1.25-3.19. Significant suppression of CF (p less than 0.025) appeared in 6 of the 57 tumors. The extent of the suppression varied from 0.35-0.67. Platelet lysate induced no change in CF in the 7 tumors that failed to form colonies in control plates. There was no relationship between tumor cell viability and either the number of colonies appearing in control plates or the response of CF to platelet lysate. By contrast, there was a significant correlation (p less than 0.005) between viability and the extent of enhancement of CF by lysate. Concentrations of lysate varying from 50-400 micrograms/ml did not yield a consistent dose-related increase in CF. The results indicate that platelet lysate significantly enhances colony formation from a substantial percentage of human breast tumors.

Topics: Agar; Breast Neoplasms; Cell Division; Cells, Cultured; Female; Humans; Platelet-Derived Growth Factor; Stimulation, Chemical

A human tumor cloning system has been utilized to grow human breast carcinoma. A total of 225 specimens have been placed in culture. One hundred thirty-two were from primary chest cancer specimens and 93 were from metastatic lesions. Of these, 71% of the primary breast carcinomas and 75% of metastases formed greater than or equal to 5 colonies per 500,000 cells plated. Forty-five percent of the primary breast carcinomas and 52% of the metastases formed enough colonies (greater than or equal to 30 colonies per 500,000 cells plated) to perform meaningful in vitro drug testing. Estrogen receptor status did not influence the percentage of tumors which formed colonies in vitro. Histologic and nude mouse studies provided confirmatory evidence the colonies were composed of breast cancer cells. In 176 in vitro chemotherapeutic drug tests, the anticancer agents commonly used clinically for treatment of breast cancer, i.e., Adriamycin, 5-fluorouracil, etc., showed some in vitro activity. This activity was not as dramatic as is seen in the clinic with these conventional agents. Future work should concentrate on improving the number of colonies which form from breast cancer specimens and on prospective use of the system for screening for new agents for the treatment of human breast cancer.

Topics: Agar; Antineoplastic Agents; Breast Neoplasms; Clone Cells; Cytological Techniques; Drug Resistance; Female; Humans; Neoplasm Transplantation; Receptors, Estrogen

Single cell suspensions prepared from human breast cancer specimens by collagenase digestion were cultured in soft agar with phytohemagglutinin-stimulated human lymphocyte-conditioned medium (PHA-LCM). In 6 of 10 different tumors, PHA-LCM-dependent clonal growth was develop. After 12-14 days of incubation, two morphologic types of colony containing 20-500 cells were recognized. Both were composed of lymphocytes of T-cell nature, as judged by cell morphology in smears, cytochemical properties, capacity to form rosettes with sheep erythrocytes, and electron microscopic appearances. Contamination of the tumor cell suspensions by blood could be excluded as a source of the colony-forming lymphocytes, and the incidence of colony-forming cells correlated well with the degree of lymphocyte infiltration of the tumors. Some of the colonies in agar were expanded further in liquid culture in the continuous presence of PHA-LCM. These clones were apparently high in proliferating capacity as compared with the proliferating activity of peripheral T-cell clones obtained from normal blood. These clones were considered to be highly activated T-lymphocytes and to be stimulated to grow in vitro by the T-cell growth factor contained in PHA-LCM. The direct cloning and expansion of such activated T-lymphocytes infiltrating the tumors will be useful for studies on the functional characteristics of these cells.

Topics: Agar; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Clone Cells; Culture Media; Culture Techniques; Female; Humans; T-Lymphocytes

Topics: Agar; Breast Neoplasms; Electrons; Evaluation Studies as Topic; Humans; Lung; Mastectomy; Patient Care Planning; Radiation, Ionizing; Radiotherapy; Radiotherapy Dosage; Silicone Elastomers; Thorax; Ultrasonography

Fourteen breast cancer lines (8 human, 5 rat, and 1 mouse) have been studied in terms of their ability to form multicellular tumor spheroids (MTS) with the agar-base method. Only 8 of the lines formed MTS in contrast to a 100% efficiency in a series of 11 varied tumors reported in the initial studies with this method. We have compared the lines that do and do not form MTS in terms of a variety of characteristics (e.g., estrogen receptors, time in serial passage, growth in nude mice, etc.), and only one characteristic, the source of the original tumor cells, was predictive of MTS-forming ability. All 8 of the breast cancer lines (and the original 11 lines) that formed MTS had been obtained from solid growths (primaries or metastases), while the 6 breast cancer lines that did not form MTS were all derived from pleural effusions. Similarly, artificial selection for an ascites variant of the MTS-forming rat 13762 adenocarcinoma line produced the 13762-A line, which could no longer form MTS. These results suggest that breast cancer cells derived from pleural effusions are genetically different from the bulk of the tumor cells in solid breast cancer samples, that they are unable to grow in true solid form, and that these differences persist in spite of prolonged propagation in tissue culture.

Topics: Adaptation, Physiological; Agar; Animals; Breast Neoplasms; Cell Aggregation; Cell Line; Culture Techniques; Female; Humans; Mammary Neoplasms, Experimental; Mice; Neoplasm Metastasis; Neoplasms, Experimental; Pleural Effusion; Rats

Topics: Agar; Breast Neoplasms; Cell Differentiation; Electrophoresis; Embryo, Mammalian; Female; Humans; Isoenzymes; L-Lactate Dehydrogenase; Mitosis; Necrosis

Topics: Abdominal Neoplasms; Agar; Aged; Antigens, Neoplasm; Breast Neoplasms; Cell Division; Cells, Cultured; Clone Cells; Colonic Neoplasms; Female; Humans; Immune Sera; Immunity, Cellular; Kidney Neoplasms; Liposarcoma; Lymphocytes; Male; Melanoma; Methods; Middle Aged; Neoplasms; Prostatic Neoplasms; Rectal Neoplasms; Thyroid Neoplasms

Topics: Agar; Animals; Antineoplastic Agents; Arsenicals; Breast Neoplasms; Cell Survival; Depression, Chemical; DNA Nucleotidyltransferases; DNA, Neoplasm; Dogs; Drug Evaluation, Preclinical; Humans; In Vitro Techniques; Iodoacetates; Leukocytes; Mice; Neoplasms; Succinate Dehydrogenase; Thymidine; Tritium

Topics: Adenocarcinoma; Adult; Agar; Animals; Antineoplastic Agents; Anus Neoplasms; Breast Neoplasms; Carcinoma, Squamous Cell; DNA; Dogs; Humans; Leucine; Leukocytes; Liver; Mice; Middle Aged; Neoplasms; Neoplasms, Experimental; Parotid Neoplasms; Proteins; Radioisotopes; Rectal Neoplasms; RNA; Sulfhydryl Compounds; Thoracic Neoplasms; Thymidine; Uridine

Topics: Agar; Aged; Blood Protein Electrophoresis; Breast Neoplasms; Carcinoma, Bronchogenic; Colonic Neoplasms; Esophageal Neoplasms; Female; gamma-Globulins; Humans; Kidney Neoplasms; Lung Neoplasms; Middle Aged; Neoplasm Metastasis; Neoplasms; Stomach Neoplasms

Topics: Adult; Agar; Blood Protein Electrophoresis; Breast Neoplasms; Contraceptives, Oral; Estrogens; Female; Gels; Humans; Male; Pregnancy; Pregnancy Complications; Progesterone; Prostatic Neoplasms; Thyroid Gland; Thyroxine-Binding Proteins

Topics: Agar; Blood Protein Electrophoresis; Blood Proteins; Breast Neoplasms; Female; Humans

Topics: Agar; Breast Diseases; Breast Neoplasms; Carcinoma; Computers; Electrophoresis; Isoenzymes; L-Lactate Dehydrogenase

Topics: Adult; Agar; Aged; Anemia; Blood Proteins; Breast Neoplasms; Bronchitis; Colitis; Electrophoresis; Female; Gels; Humans; Infections; Inflammation; Male; Middle Aged; Neoplasms; Orchitis; Pneumonia; Testicular Neoplasms