Inhibition of migration of MDA-MB-231 and HCC1806.

<p>MDA-MB-231 (A) or HCC1806 (B) cells were harvested, washed and resuspended in RPMI1640-serum free media. Migration assays were performed using type I collagen (10 µg/ml) as an adhesive substrate in the lower compartment of Transwell by incubating at 37°C for 4 hours. The aptamers were added in both upper and lower chambers at the indicated concentrations. Experiments were performed in triplicate. Statistical significance was calculated by Student’s two-tailed paired <i>t</i>-test.</p

CD44 associated with EphA2 on breast cancer cells.

<p>(A) HCC38 cells were lysed with 100 mM Tris-HCl (pH 7.5) containing 1% Brij35, 0.14 M NaCl, 1 mM CaCl₂, 1 mM MnCl₂, and a protease inhibitor cocktail by scraping and pipetting. The cell lysates were precleared and immunoprecipitated with anti-CD44 antibody clone (clone 156-3C11)-, anti-EphA2- or control IgG-protein G beads for 4 hours at 4°C. The bound proteins were released by boiling at 95°C in SDS-sample buffer under reducing conditions and separated on SDS-PAGE followed by western blotting with anti-EphA2 antibody followed by HRP-secondary antibody. The proteins are visualized with enhanced chemiluminescence (ECL) reaction. (B) Cells were harvested using 5 mM EDTA in PBS and were lysed in 100 mM Tris-HCL (pH 7.5) containing 1% Brij35, 0.14 M NaCl, 1 mM CaCl₂, 1 mM MnCl₂, and a protease inhibitor cocktail. The lysates were precleared with anti-FLAG (M2) agarose beads for 4 hours by shaking at 4°C. The cleared lysates were incubated with recombinant protein CD44v10 P-FLAG-anti-FLAG (M2) antibody-agarose beads (CD44v10) or anti-FLAG (M2) antibody-agarose beads (Con.) in the presence or absence of aptamers (Apt#4 and Apt#7) by shaking at 4°C for 4 hours. The beads were washed extensively with the lysis buffer. The bound proteins were released by boiling at 95°C in SDS-sample buffer under reducing conditions and separated on SDS-PAGE followed by western blotting with anti-EphA2 followed by HRP-secondary antibody or HRP-conjugated anti-FLAG (M2) antibody. The proteins are visualized with enhanced chemiluminescence (ECL) reaction. CD44 exon v10 peptide was used as a loading control for ensuring the same amount of proteins was loaded in each lane.</p

Inhibition of migration of HCC38 cells with anti-CD44 exon v10 antibody.

<p>(A) Cells were harvested, washed, and resuspended in RPMI1640-serum free media. Migration assays were performed using type I collagen (10 µg/ml) as an adhesive substrate in the lower compartment of Transwell by incubating at 37°C for 4 hours. The antibodies were added in both upper and lower chambers at a concentration of 5 µg/ml. Experiments were performed by triplicates three times. Statistical significance was calculated by Student’s two-tailed paired <i>t</i>-test. (B) Cells were harvested, washed, resuspended in RPMI1640 containing 1 mg/ml BSA (adhesion buffer) at a concentration of 10⁵ cell/ml. Cells (100 µl/well) were incubated on plates coated with type I collagen (5 µg/ml) for 1 hour in the presence or absence of antibodies (5 µg/ml). Plates were washed, fixed followed by staining with crystal violet. After extensive washing to remove excess dye, cells were lysed with 100 µl of PBS containing 2% SDS and then the absorbance was measured at 550 nm. Experiments were performed in quadruplicates and repeated three times. The representative data were shown as mean +/− S.D. of absorbance values.</p

FACS analysis of aptamers against CD44 exon v10.

<p>HCC38 or SK-Br3 cells were harvested and resuspended in RPMI1640 containing 1 mg/ml BSA and 0.025% NaN₃ (FACS buffer). Cells were incubated with aptamers of which 5′-end was conjugated to biotin (5 µg/ml), anti-panCD44 (clone 156-3C11) (5 µg/ml), or anti-CD44 exon v10 antibody (AB2082) (5 µg/ml) for 1 hour at 4°C. After washing with the same buffer, cells were incubated with Streptavidin-FITC, FITC-goat anti-mouse IgG, or FITC-goat anti-rabbit IgG for 1 hour at 4°C. After extensive washing with FACS buffer, cells were resuspended in PBS containing 1% paraformaldehyde. Expression was measured on FACS Calibur (Becton-Dickinson, NJ USA) by collecting 10,000 events and analyzed by CellQuest.</p

Inhibition of cell migration by aptamers against CD44 exon v10.

<p>(A) HCC38 cells were harvested, washed and resuspended in RPMI1640-serum free media. Migration assays were performed using type I collagen (10 µg/ml) as an adhesive substrate in the lower compartment of Transwell by incubating at 37°C for 4 hours. The aptamers were added in both upper and lower chambers at the indicated concentrations in the text. Experiments were performed in triplicate. Statistical significance was calculated by Student’s two-tailed paired <i>t</i>-test. (B) MCF-7 cells were stably transfected with CD44E according to the methods described in Materials and Methods. MCF-7 cells transfected control vector, [MCF-7(pIRES2)], MCF-7 (CD44E), and parental MCF-7 cells (NT) were lysed and subjected to western blotting for the expression of CD44E using anti-CD44 exon v10 antibody followed by HRP-conjugated goat-anti-rabbit IgG. The signal was detected by ECL reactions. Actin was used as a loading control. (C) The migration of these cells was evaluated as described above in the presence or absence of aptamers against CD44 exon v10 at a concentration of 10 nM as described above. Experiments were performed in triplicate. Statistical significance was calculated by Student’s two-tailed paired <i>t</i>-test.</p

Protocol for isolating CD44 exon v10-specific DNA aptamers by Systematic Evolution of Ligands by Exponential Enrichment (SELEX).

<p>The CD44 exon v10 peptide was expressed as a fusion protein with FLAG tag. The purified recombinant peptide was immobilized on plates coated with anti-FLAG (M2) antibody. Library of DNA aptamers were incubated on the plates and extensively washed to remove non-bound aptamers. The isolated aptamers were then amplified and this selection process was repeated 10 times. Aptamers isolated from the last selection were amplified and ligated to pCR2.1-TOPOTA vector for sequencing.</p

Positive Association of Fibroadenomatoid Change with HER2-Negative Invasive Breast Cancer: A Co-Occurrence Study

<div><p>Background</p><p>Risk assessment of a benign breast disease/lesion (BBD) for invasive breast cancer (IBC) is typically done through a longitudinal study. For an infrequently-reported BBD, the shortage of occurrence data alone is a limiting factor to conducting such a study. Here we present an approach based on co-occurrence analysis, to help address this issue. We focus on fibroadenomatoid change (FAC), an under-studied BBD, as our preliminary analysis has suggested its previously unknown significant co-occurrence with IBC.</p><p>Methods</p><p>A cohort of 1667 female patients enrolled in the Clinical Breast Care Project was identified. A single experienced breast pathologist reviewed all pathology slides for each case and recorded all observed lesions, including FAC. Fibroadenoma (FA) was studied for comparison since FAC had been speculated to be an immature FA. FA and Fibrocystic Changes (FCC) were used for method validation since they have been comprehensively studied. Six common IBC and BBD risk/protective factors were also studied. Co-occurrence analyses were performed using logistic regression models.</p><p>Results</p><p>Common risk/protective factors were associated with FA, FCC, and IBC in ways consistent with the literature in general, and they were associated with FAC, FA, and FCC in distinct patterns. Age was associated with FAC in a bell-shape curve so that middle-aged women were more likely to have FAC. We report for the first time that FAC is positively associated with IBC with odds ratio (OR) depending on BMI (OR = 6.78, 95%CI = 3.43-13.42 at BMI<25 kg/m²; OR = 2.13, 95%CI = 1.20-3.80 at BMI>25 kg/m²). This association is only significant with HER2-negative IBC subtypes.</p><p>Conclusions</p><p>We conclude that FAC is a candidate risk factor for HER2-negative IBCs, and it is a distinct disease from FA. Co-occurrence analysis can be used for initial assessment of the risk for IBC from a BBD, which is vital to the study of infrequently-reported BBDs.</p></div

FH535 inhibited migration of MDA-MB231 and HCC38 cells to type I collagen.

<p>Cells were harvested, washed, and resuspended in RPMI-serum free media at a concentration of 5×10⁵ cells/ml. Type I collagen was used as a chemoattractant at a concentration of 3 g/ml. FH535 (0.01–1 µM) were added in both cell suspension and type I collagen solution and incubated for 4 hours at 37°C. Migrated cells were manually counted and expressed as mean +/− S.D. Experiments were repeated three times. * <i>p</i><0.001, ** <i>p</i><0.05 (by Student’s two-tailed paired <i>t</i>-test).</p

Proliferation of tumor cells in three-dimensional type I collagen gel in the presence of FH535.

<p>Cells (HCC38, MDA-MB-231, T47D, Sk-Br3, MCF-7) were cultured in type I collagen gel as described in material and methods. Cell/gel matrices were fixed and embedded in paraffin. The paraffin section was serially cut at 5 m and mounted on slide glasses. Tissue sections were stained with anti-Ki67 antibody or control IgG followed by HRP-conjugated secondary antibody as visualizing by DAB staining with counter staining by DAPI to localize cells. The results were demonstrated by the (mean +/− standard deviation) of % of positive cells for DAB from three independent experiments. Insets showed the typical staining pattern of anti-Ki67 antibody in HCC38 and MDA-MB-231 cells in the presence or absence of FH535 at a concentration of 10 µM. Magnification x200. * <i>p</i><0.001 (by Student’s two-tailed paired <i>t</i>-test). n.s: not significance (by Student’s two-tailed paired <i>t</i>-test).</p

Characteristics of IBC cases/controls in relationship to BBDs and risk/protective factors.

<p>*UN, Unknown, not used in Chi-square test.</p><p>Abbreviations: IBC = Invasive Breast Cancer; BBDs = Benign Breast Diseases; FA = Fibroadenoma; FAC = Fibroadenomatoid Change; FCC = Fibrocystic Changes; Num. = Number; Race, AA = African American, CA = Caucasian American; Current OC use = Current oral contraceptive use; BMI = Body Mass Index; HRT = Hormonal replacement therapy; Combo = Estrogen & Progesterone.</p><p>Characteristics of IBC cases/controls in relationship to BBDs and risk/protective factors.</p