Real-time PCR-based assay to quantify the relative amount of human and mouse tissue present in tumor xenografts

<p>Abstract</p> <p>Background</p> <p>Xenograft samples used to test anti-cancer drug efficacies and toxicities in vivo contain an unknown mix of mouse and human cells. Evaluation of drug activity can be confounded by samples containing large amounts of contaminating mouse tissue. We have developed a real-time quantitative polymerase chain reaction (qPCR) assay using TaqMan technology to quantify the amount of mouse tissue that is incorporated into human xenograft samples.</p> <p>Results</p> <p>The forward and reverse primers bind to the same DNA sequence in the human and the mouse genome. Using a set of specially designed fluorescent probes provides species specificity. The linearity and sensitivity of the assay is evaluated using serial dilutions of single species and heterogeneous DNA mixtures. We examined many xenograft samples at various in vivo passages, finding a wide variety of human:mouse DNA ratios. This variation may be influenced by tumor type, number of serial passages in vivo, and even which part of the tumor was collected and used in the assay.</p> <p>Conclusions</p> <p>This novel assay provides an accurate quantitative assessment of human and mouse content in xenograft tumors. This assay can be performed on aberrantly behaving human xenografts, samples used in bioinformatics studies, and periodically for tumor tissue frequently grown by serial passage in vivo.</p

Novel antibody reagents for characterization of drug- and tumor microenvironment-induced changes in epithelial-mesenchymal transition and cancer stem cells

<div><p>The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation−mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous <i>hHGF</i> knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.</p></div

Novel antibodies detect upregulation of EMT transcription factor expression coinciding with reduced E-cadherin expression in a NSCLC xenograft model with constitutive HGF signaling.

<p>SCID or homozygous <i>hHGF</i> knock-in mice were implanted with H596 NSCLC tumors, and tumors were harvested 33 days after implantation. FFPE tissue sections were assessed by immunofluorescence microscopy after staining with DAPI (blue) or with fluorescence-conjugated anti-rabbit antibodies following application of primary antibodies to E-cadherin (green) and EMT TFs (red), including Snail <b>(A-C)</b>, Slug <b>(D-F)</b>, and Sox9 <b>(G-I)</b>. <b>(A, D, G)</b> Representative 20X magnification images for each group (SCID or <i>hHGF</i> knock-in). White arrows indicate examples of regions rich in hHGF-secreting mouse stromal cells, which are not recognized by the anti−human E-cadherin, Sox9, Slug, or Snail antibodies. White circles indicate the regions shown at higher magnification in panels <b>(C)</b>, <b>(F)</b>, and <b>(I)</b>, respectively. <b>(B, E, H)</b> Quantitation of tumor cell nuclear area positive for Snail <b>(B)</b> and Sox9 <b>(H)</b> and tumor area positive for Slug <b>(E)</b> was performed using Definiens software. Means ± standard deviations are shown (black bars). Each point represents the EMT TF expression value for a single tumor core; points with the same symbol indicate cores harvested from the same animal. Significant differences in EMT TF expression between SCID and homozygous <i>hHGF</i> knock-in mice are indicated (*<i>P</i> < 0.05, ***<i>P</i> < 0.001, N.S.: not significant; <i>n</i> = 3–6 animals per group). <b>(C, F, I)</b> High-magnification (60X) images of the circled regions in panels <b>(A)</b>, <b>(D)</b>, and <b>(G)</b>, respectively.</p

Western blot and immunofluorescence microscopy characterization of novel anti-GSC and anti-Sox9 antibodies in target protein−overexpressing cell lines and NAMEC8 xenograft models.

<p>Data are shown for GSC 1–5 <b>(A-C)</b> and Sox9 15-4 <b>(D-F)</b>. <b>(A</b>, <b>B</b>, <b>D</b> and <b>E)</b> <i>In vitro</i> target protein detection by EMT TF antibodies was assessed by comparisons of wild-type and constitutive target protein−overexpressing cell lines. <b>(A</b> and <b>D)</b> Western blot detection of GSC and Sox9, respectively. <b>(B</b> and <b>E)</b> 20X magnification immunofluorescence images show staining with DAPI (blue) and with the antibody of interest and fluorescence-conjugated anti-rabbit secondary antibody (green). <b>(C</b> and <b>F)</b> <i>In vivo</i> target protein detection by EMT TF antibodies was assessed by immunofluorescence microscopy analysis (60X magnification) of tumor tissue from NAMEC8 xenograft models (gold, EMT TF antibody; blue, DAPI; red, vimentin).</p

NSCLC tumors from xenograft models with constitutive plasma and stromal hHGF expression exhibit enhanced mesenchymal-like character.

<p>SCID or homozygous <i>hHGF</i> knock-in (<i>hHGF</i>^ki/ki) mice were implanted with H596 NSCLC tumors (<i>n</i> = 5 animals per group), and tumors were harvested 33 days after implantation. <b>(A)</b> Representative EMT TF immunofluorescence microscopy images of H596 tumors from SCID or <i>hHGF</i> knock-in animals (20X magnification). FFPE tissue sections were assessed by immunofluorescence microscopy after staining with DAPI (blue) or fluorescence-conjugated primary antibodies to E-cadherin (green) and vimentin (red) or by light microscopy following H & E staining (far right). White arrow indicates a representative region rich in hHGF-secreting mouse stromal cells, which are not recognized by the anti−human E-cadherin or vimentin antibodies. White circle indicates the region shown at higher magnification in panel B. <b>(B)</b> High-magnification (60X) images of the circled region in panel A, with arrow indicating representative hHGF-secreting mouse stromal cells. <b>(C)</b> Quantitation of E-cadherin-only, vimentin-only, and E-cadherin + vimentin colocalized (E+V) staining in H596 tumors harvested from SCID or <i>hHGF</i> knock-in animals. Error bars represent standard deviation, and significant changes in the mean marker area per cell between the two groups are indicated (*<i>P</i> < 0.05; **<i>P</i> < 0.01). <b>(D)</b> Log₁₀(vimentin:E-cadherin) values for H596 tumors from SCID and <i>hHGF</i> knock-in animals reveal shifts toward a more mesenchymal-like phenotype for tumors exposed to constitutive stromal hHGF. Each point represents an individual animal; the significant difference between log₁₀(vimentin:E-cadherin) values for the two groups is indicated (**<i>P</i> < 0.01).</p

Western blot, immunofluorescence microscopy, and flow cytometry characterization of a novel anti-CD133 antibody in cell line, xenograft, and clinical tumor specimens.

<p>CD133 detection was assessed <i>in vitro</i> by comparing cell lines with known high and low endogenous CD133 expression (HT29 and U87 MG, respectively). <b>(A)</b> Western blot detection of CD133 by the CD133 47–10 antibody. <b>(B)</b> Immunofluorescence microscopy images of HT29 (top) and U87 MG (bottom) cells show staining with DAPI (blue) and CD133 47–10 and fluorescence-conjugated anti-rabbit secondary antibody (red) at 40X magnification. <b>(C)</b> Immunofluorescence microscopy images of tumor tissue from HT29 xenograft models, stained with DAPI (blue) and novel or commercially available anti-CD133 antibodies and fluorescence-conjugated anti-rabbit secondary antibody (red) at 40X magnification. W6B3C1, AC133, and 293C3 antibodies were obtained from Miltenyi Biotec and target unknown epitopes 1 (W6B3C1 and AC133) or 2 (293C3) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199361#pone.0199361.s006" target="_blank">S2 Table</a>). Identical primary antibody concentrations (10 μg/mL) were used for each experiment. <b>(D)</b> Immunohistochemical analysis of CD133 expression in human parotid tumor tissue using the CD133 47–10 antibody. Tumor tissue was combined with CD133 47–10 and incubated with either buffer (left) or the “A1” and “A2” peptides (right) corresponding to portions of the domain A epitope sequence recognized by CD133 47–10, each at a concentration 200-fold higher that of the antibody (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199361#pone.0199361.s007" target="_blank">S3 Table</a> for blocking peptide sequences). CD133 was visualized using an HRP-conjugated anti-rabbit antibody. <b>(E)</b> Flow cytometry analysis of CD133-expressing HT29 cells using novel and commercially available anti-CD133 antibodies. “Control” indicates unstained cells; ΔMFI values, indicating the difference in median fluorescence intensities between each antibody and its matched isotype control (rabbit for CD133 47–10 and mouse for all others), are shown for each antibody.</p

Western blot and immunofluorescence microscopy characterization of novel anti-Slug and anti-Snail antibodies in target protein−overexpressing cell lines and NAMEC8 xenograft models.

<p>Data are shown for Slug 9–12 <b>(A-C)</b> and Snail 41–7 <b>(D-F)</b>. <b>(A</b>, <b>B</b>, <b>D</b> and <b>E)</b> <i>In vitro</i> target protein detection by EMT TF antibodies was assessed by comparisons of wild-type and doxycycline (dox)-inducible target protein−overexpressing cell lines. <b>(A</b> and <b>D)</b> Western blot detection of Slug and Snail, respectively. <b>(B</b> and <b>E)</b> 20X magnification immunofluorescence images of cultured cells show staining with DAPI (blue), the antibody of interest and fluorescence-conjugated anti-rabbit secondary antibody (red), and fluorescence-conjugated anti–E-cadherin antibody (green). <b>(C</b> and <b>F)</b> <i>In vivo</i> target protein detection by EMT TF antibodies was assessed by immunofluorescence microscopy analysis (60X magnification) of tumor tissue from NAMEC8 xenograft models (gold, EMT TF antibody; blue, DAPI; red, vimentin).</p