sj-pdf-1-jao-10.1177_03913988241234547 – Supplemental material for Thick silk fibroin vascular graft: A promising tissue-engineered scaffold material for abdominal vein grafts in middle-sized mammals

Supplemental material, sj-pdf-1-jao-10.1177_03913988241234547 for Thick silk fibroin vascular graft: A promising tissue-engineered scaffold material for abdominal vein grafts in middle-sized mammals by Kaito Fukuda, Junichi Kaneko, Sho Kiritani, Yui Sawa, Masaaki Morito, Mariko Tanaka, Tetsuo Ushiku, Chieh-Jen Cheng, Takashi Tanaka, Ryo Tanaka, Tetsuo Asakura, Yoshikuni Kawaguchi, Nobuhisa Akamatsu and Kiyoshi Hasegawa in The International Journal of Artificial Organs</p

sj-pdf-3-jao-10.1177_03913988241234547 – Supplemental material for Thick silk fibroin vascular graft: A promising tissue-engineered scaffold material for abdominal vein grafts in middle-sized mammals

Supplemental material, sj-pdf-3-jao-10.1177_03913988241234547 for Thick silk fibroin vascular graft: A promising tissue-engineered scaffold material for abdominal vein grafts in middle-sized mammals by Kaito Fukuda, Junichi Kaneko, Sho Kiritani, Yui Sawa, Masaaki Morito, Mariko Tanaka, Tetsuo Ushiku, Chieh-Jen Cheng, Takashi Tanaka, Ryo Tanaka, Tetsuo Asakura, Yoshikuni Kawaguchi, Nobuhisa Akamatsu and Kiyoshi Hasegawa in The International Journal of Artificial Organs</p

sj-pdf-2-jao-10.1177_03913988241234547 – Supplemental material for Thick silk fibroin vascular graft: A promising tissue-engineered scaffold material for abdominal vein grafts in middle-sized mammals

Supplemental material, sj-pdf-2-jao-10.1177_03913988241234547 for Thick silk fibroin vascular graft: A promising tissue-engineered scaffold material for abdominal vein grafts in middle-sized mammals by Kaito Fukuda, Junichi Kaneko, Sho Kiritani, Yui Sawa, Masaaki Morito, Mariko Tanaka, Tetsuo Ushiku, Chieh-Jen Cheng, Takashi Tanaka, Ryo Tanaka, Tetsuo Asakura, Yoshikuni Kawaguchi, Nobuhisa Akamatsu and Kiyoshi Hasegawa in The International Journal of Artificial Organs</p

Genetic and phenotypic determinants of morphologies in 3-dimensional cultures and xenografts of lung tumor cell lines

   Genetic and phenotypic determinants of morphologies in 3-dimensional cultures and xenografts of lung tumor cell lines     The images of the colonies of 40 NSCLC cell lines in 3D culture, and the images of HE, alphaSMA, and MT stains of the xenograft tumors of the 28 NSCLC cell lines </p

Tumor Content Chart-Assisted <i>HER2/</i>CEP17 Digital PCR Analysis of Gastric Cancer Biopsy Specimens

<div><p>Evaluating <i>HER2</i> gene amplification is an essential component of therapeutic decision-making for advanced or metastatic gastric cancer. A simple method that is applicable to small, formalin-fixed, paraffin-embedded biopsy specimens is desirable as an adjunct to or as a substitute for currently used HER2 immunohistochemistry and in situ hybridization protocols. In this study, we developed a microfluidics-based digital PCR method for determining <i>HER2</i> and chromosome 17 centromere (CEP17) copy numbers and estimating tumor content ratio (TCR). The <i>HER2</i>/CEP17 ratio is determined by three variables—TCR and absolute copy numbers of <i>HER2</i> and CEP17—by examining tumor cells; only the ratio of the latter two can be obtained by digital PCR using the whole specimen without purifying tumor cells. TCR was determined by semi-automatic image analysis. We developed a Tumor Content chart, which is a plane of rectangular coordinates consisting of <i>HER2</i>/CEP17 digital PCR data and TCR that delineates amplified, non-amplified, and equivocal areas. By applying this method, 44 clinical gastric cancer biopsy samples were classified as amplified (n = 13), non-amplified (n = 25), or equivocal (n = 6). By comparison, 11 samples were positive, 11 were negative, and 22 were equivocally immunohistochemistry. Thus, our novel method reduced the number of equivocal samples from 22 to 6, thereby obviating the need for confirmation by fluorescence or dual-probe in situ hybridization to < 30% of cases. Tumor content chart-assisted digital PCR analysis is also applicable to multiple sites in surgically resected tissues. These results indicate that this analysis is a useful alternative to HER2 immunohistochemistry in gastric cancers that can serve as a basis for the automated evaluation of <i>HER2</i> status.</p></div

Primer and TaqMan probe sequences for digital PCR.

<p>Primer and TaqMan probe sequences for digital PCR.</p

TC chart-assisted digital PCR analysis of clinical gastric cancer biopsy specimens.

<p>Each case was evaluated by HER2-IHC and digital PCR, and then validated by <i>HER2</i>-DISH (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154430#pone.0154430.t003" target="_blank">Table 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154430#pone.0154430.s002" target="_blank">S1 Table</a>). The results are plotted on the TC-chart. The findings of HER2-IHC are represented by symbols as follows; 3+ (positive) as rhombi; 2+ (equivocal) as triangles; 0~1+ (negative) as cross-mark. The results of <i>HER2</i>-DISH are depicted by colors as follows; positive, red; equivocal, purple; negative, blue. Multiple cases taken from the same patients (Pt) are highlighted. The vertical axis between 0.5 and 2.5 was linearly ordered and the upper area was logarithmically ordered. There were no positive cases (red rhombi) in the negative area, demonstrating that digital PCR can screen out negative cases with high specificity. On the other hand, cases plotted in the equivocal area were not always positive, as determined by CEP17 copy number. Three cases (#25, #26, and #42) showed markedly high scores and a clustering pattern in <i>HER2</i>-DISH (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154430#pone.0154430.g003" target="_blank">Fig 3C</a>).</p

Histological analysis of clinical gastric cancer biopsy specimens and estimation of TCR using Tissue Studio.

<p>(A–C) Representative cases are shown with HE staining, HER2-IHC, and <i>HER2</i>-DISH. (A) Case #37: HER2-IHC score = 0, <i>HER2</i>-DISH ratio = 1.09. (B) Case #6: HER2-IHC score = 2+, <i>HER2</i>-DISH ratio = 2.83. (C) Case #25: HER2-IHC score = 3+, <i>HER2</i>-DISH ratio = 6.56. (D) TC rate was calculated based on the ratio of nuclei counts of whole nucleated and cancerous cells, which were semi-automatically distinguished by nucleus size—with cancerous cells showing enlarged nuclei—by means of Tissue Studio image analysis software.</p

<i>HER2</i>/CEP17 ratios from <i>HER2</i>-DISH and digital PCR in H522 and SK-BR-3 cells and LCL.

<p><i>HER2</i>/CEP17 ratios from <i>HER2</i>-DISH and digital PCR in H522 and SK-BR-3 cells and LCL.</p

Equations and TC chart.

<p>(A) Mathematical representation of the correlation between <i>HER2</i>/CEP17 ratios obtained by digital PCR [<i>r</i>] and TCR [<i>x</i>] (upper). When [<i>B</i>/<i>A</i> = 2], [<i>r</i>] is represented by the right side of the equation (indicated by an arrow), and the right-hand member of the equation indicates a monotonic increase. A schematic model is shown for [<i>A</i> = 2] and [<i>B</i> = 4] (lower right). (B) TC chart demonstrated by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154430#pone.0154430.e001" target="_blank">Eq (1)</a>. The relationship can be plotted as a straight line (red) represented by [<i>r</i> = <i>x</i> + 1], which is the threshold between negative and equivocal areas. As the value of [<i>A</i>] exceeds 2.0, lines become increasingly convex and curve upwards according to this equation. Lines converge at the coordinates (0,1) and (1,2). In clinical samples, [<i>A</i>] was limited to a value between 2 and 8. The area enclosed by the straight red line [<i>r</i> = <i>x</i> + 1](<i>A</i> = 2) and the purple line curving upwards [<i>r</i> = (7<i>x</i> + 1)/(3<i>x</i> + 1)](<i>A</i> = 8) was designated as the equivocal area, and the area above the purple line was designated as the positive area.</p