HB1.F3.eGFP cell movement and encirclement of U251.dsRed target cells.

<p>Frames are excised from larger wide-field sequences taken at 30 min intervals. <b><i>A</i></b>, Approach and contact formation by one HB1.F3.eGFP cell (filled arrow) and progression of contact formation by another (open arrow). Stages of HB1.F3.eGFP cell (filled arrow) approach and contact formation were typical: extension of a leading process making contact with the U251.dsRed cell (3.5 h) followed by translocation of the cell body (5.0–7.0 h) and then envelopment of the glioma cell (7.5 h). The other HB1.F3.eGFP cell (open arrow), initially in contact with one of two adjacent U251.dsRed cells, over time extended forward and laterally to move off the surface of both cells. A third HB1.F3.eGFP cell (lower right, not marked) appeared to contact the U251.dsRed cell of interest to the second HB1.F3.eGFP cell, begin to move over the U251.dsRed cell surface (1.0–3.0 h), stall and regress (3.5–7.0 h), and then begin to advance at the end of the sequence (7.5 h). <b><i>B</i></b>, Process of HB1.F3.eGFP cell encirclement of an U251.dsRed.dsRed target. Lamellipodia extend from the initial contact site (1.0–3.0 h) over almost the entire surface of the U251.dsRed glioma cell (6.0–12.0 h) to eventually encircle the U251.dsRed cell. Scale bars  = 15 µm.</p

Analyses of HB1.F3.eGFP::target cell contacts.

<p>Formation of contacts, and the extent of target cell encirclement, did not vary with types of cell encountered, including multiple human malignant lines, primary human glioma cell lines, primary mouse astroglia, and human dermal fibroblasts (adult and new born) and astrocytes. <b><i>A</i></b>, Proportions of total HB1.F3.eGFP cells contacting target cells. U251 cells were one of several malignant brain and breast cancer lines analyzed, and in addition served as a reference cell line for each additional group (marked by either dsRed or CMRA as indicated). No significant differences were observed (all <i>p</i>>0.05 within each group of target cells and across multiple groups; ANOVA with Bonferroni's multiple comparisons test, or <i>t</i> test, as appropriate). <b><i>B</i></b>, Proportions of HB1.F3::target cell pairs displaying each of the thee morphologies defined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051859#pone-0051859-g003" target="_blank">Figure 3</a> and illustrated in the legend (showing only the HB1.F3.eGFP cell). No significant differences were observed (all <i>p</i>>0.05) with the exception of HB1.F3 homotypic interactions (see text). Data are mean ± s.d. for 3 or more volumes within each of 2–4 independent experiments.</p

HB1.F3.eGFP cells will form contacts with, but not encircle, other HB1.F3 cells.

<p><i>Above</i>, Images of HB1.F3.eGFP::HB1.F3.eGFP and HB1.F3.eGFP::HB1.F3.CD (CMRA) contacting pairs. Cell boundaries are outlined using phalloidin_Alexa647 to show the cortical F-actin cytoskeleton; contact zones are marked by arrows. While the HB1.F3 cells showed filopodial extensions over their hydrogel-exposed surfaces, the contact zone was smooth and clearly delineated by the phalloidin_Alexa647. <i>Below</i>, Heterotypic HB1.F3.eGFP::U251.dsRed pairs were characterized by extension of actin-rich (arrows) lamellipodia from the HB1.F3.eGFP cell over the target glioma cell. These images illustrate the data presented quantitatively in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051859#pone-0051859-g005" target="_blank">Figure 5</a>.</p

Disruption of F-actin filaments with cytochalasin D and myosin II by blebbistatin and consequences for motor protein distributions and HB1.F3.eGFP interaction with target U251.dsRed cells.

<p><b><i>A</i></b>, Images illustrating the sensitivities of cell morphology in Puramatrix (<i>a1, b1, c1</i>) and of the distributions of myosin II heavy chain B (by antibody immunofluorescence) (<i>a2, b2, c2</i>) and F-actin (by phalliodin_Alexa647 fluorescence) (<i>a3, b3, c3</i>). <b><i>a1</i></b>, Normal pattern of HB1.F3.eGFP::U251.dsRed cell interactions (inset shows U251.dsRed cell encirclement by a HB1.F3.eGFP cell). <b><i>a2, a3</i></b>, Normal distributions of MHC-IIB and F-actin. <b><i>b1</i></b>, Disruption of target cell encirclement after MHC-IIB inhibition by blebbistatin (50 µM; 18 h), contraction of MHC-IIB (<b><i>b2</i></b>) and formation of long actin-lined tubes emanating from both tumor cells and NSCs (<b><i>b3</i></b>). <b><i>c1</i></b>, Loss of lamellipodia and truncation of target cell encirclement (inset) after F-actin inhibition by cytochalasin D (1 µM; 18 h), and loss of filamentous structure and appearance of multiple aggregates by both MHC-IIB (<b><i>c2</i></b>) and F-actin (<b><i>c3</i></b>) and loss of spike-like filopodial structures emerging from both HB1.F3.eGFP and U251.dsRed cells (<b><i>c3</i></b>). <b><i>B</i></b>, <i>Above</i>, Proportions of HB1.F3.eGFP:: target cell pairs were not significantly affected by actin or myosin inhibition (<i>p</i>>0.05). <i>Below</i>, After actin disruption or myosin inhibition, encirclement was blocked and did not progress beyond type II (<i>p</i><0.05).</p

Stages of HB1.F3 contact formation and encirclement of U251.dsRed cells.

<p>Images are Z-axis projections of 28–44 optical sections taken at 1 µm intervals. <b><i>A</i></b>, Solitary HB1.F3.eGFP cell extending filopodia-tipped process into the hydrogel matrix. <b><i>B</i></b>, Initial HB1.F3.eGFP contact with a U251.dsRed cell (<b>Type I</b>). Note the morphology of the process contacting the target cell, which has shorter stubbier extensions resembling a holdfast. <b><i>C</i></b>, Loss of processes, apposition of the HB1.F3.eGFP cell body to the U251.dsRed cell, and the beginnings of lamellipodia extension (<b>Type II</b>). <b><i>D</i></b>, Complete HB1.F3.eGFP cell encirclement of a U251.dsRed cell with the cell body and lamellipodia covering much of the glioma cell surface (<b>Type III</b>).</p

Spatial relationships between HB1.F3.eGFP NSCs (green) and U251.dsRed glioma cells (red).

<p>Cells were co-cultured for 18 h in 3-dimensional Puramatrix hydrogel. Shown is the Z-axis projection of 34 optical sections of 900×900 µm spanning 102 µm. Expanded X-Z projections of individual contacting cell pairs confirm true contacts rather than superposition. Of a total of 157 HB1.F3.eGFP cells in this image, 38 (36.9%) were contacting U251.dsRed cells. Of the remaining solitary cells, 94 (59.9%) were roughly spherical or ellipsoid with minimal or no process extension, and 25 (15.9%) showed extended filopodia-tipped processes.</p

Cytokine Induction of VCAM-1 but Not IL13Rα2 on Glioma Cells: A Tale of Two Antibodies

<div><p>The interleukin-13 receptor alpha2 (IL13Rα2) is a cell surface receptor that is over-expressed by a subset of high-grade gliomas, but not expressed at significant levels by normal brain tissue. For both malignant and non-malignant cells, IL13Rα2 surface expression is reported to be induced by various cytokines such as IL-4 or IL-13 and tumor necrosis factor (TNF). Our group has developed a therapeutic platform to target IL13Rα2-positive brain tumors by engineering human cytotoxic T lymphocytes (CTLs) to express the IL13-zetakine chimeric antigen receptor. We therefore sought to investigate the potential of cytokine stimulation to induce IL13Rα2 cell surface expression, and thereby increase susceptibility to IL13Rα2-specific T cell killing. In the course of these experiments, we unexpectedly found that the commercially available putative IL13Rα2-specific monoclonal antibody B-D13 recognizes cytokine-induced VCAM-1 on glioblastoma. We provide evidence that the induced receptor is not IL13Rα2, because its expression does not consistently correlate with IL13Rα2 mRNA levels, it does not bind IL-13, and it is not recognized by IL13-zetakine CTL. Instead we demonstrate by immunoprecipitation experiments and mass spectrometry that the antigen recognized by the B-D13 antibody following cytokine stimulation is VCAM-1, and that VCAM-1, but not IL13Rα2, is induced on glioma cells by TNF alone or in combination with IL-13 or IL-4. Further evaluation of several commercial B-D13 antibodies revealed that B-D13 is bi-specific, recognizing both IL13Rα2 and VCAM-1. This binding is non-overlapping based on soluble receptor competition experiments, and mass spectrometry identifies two distinct heavy and light chain species, providing evidence that the B-D13 reagent is di-clonal. PE-conjugation of the B-D13 antibody appears to disrupt IL13Rα2 recognition, while maintaining VCAM-1 specificity. While this work calls into question previous studies that have used the B-D13 antibody to assess IL13Rα2 expression, it also suggests that TNF may have significant effects on glioma biology by up-regulating VCAM-1.</p></div

B-D13 reagent appears to contain two distinct monoclonal antibodies.

<p>Soluble receptor competition study evaluating the specificity of IL13Rα2 and VCAM-1 recognition by the B-D13-unc antibodies (Cell Sciences and Santa Cruz) using (A) PBT008 cells cultured overnight in media alone (black histogram) versus cytokine (TNF; blue histogram); or (B) IL13Rα2-expressing U251T cells (blue histogram). Cells were stained with the indicated antibody that was pre-incubated with soluble recombinant human IL13Rα-Fc (purple histograms) or VCAM-1-Fc (green histograms). Relative fluorescence index (RFI) compared to staining without the soluble competitors (i.e., the control/blue histograms) are indicated in each histogram. (C) Unconjugated B-D13 antibodies from Cell Sciences (top) and Abcam (bottom) were reduced and analyzed by LC/MS. Shown is the spectra of the deconvoluted protein masses depicting two distinct mass species for both the heavy and light chains. (D) Extracted ion chromatograms (EIC) for the two light chain species, of the Cell Sciences B-D13-unc reagent from (C).</p

The cytokine-induced cell surface antigen recognized by B-D13-PE does not bind IL-13.

<p>(A) D283 cells engineered to express IL13Rα2 (D283- IL13Rα2) (blue histograms) and D283 parental (black histograms) cells were evaluated by flow cytometry for expression of constitutive IL13Rα2 using the IL13Rα2-specific antibody AF146, and biotinylated recombinant human IL-13 (IL13-bio) followed by PE-conjugated strepavidin (SA-PE). Data are representative of 2 separate experiments. (B) U251T (grown in the absence of cytokines) were evaluated by flow cytometry for constitutive IL13Rα2 expression using AF146, and for binding to IL13-bio/SA-PE in the presence and absence of 10-fold molar excess of recombinant human IL-4 or IL-13. Black histograms represent staining with istoype control antibody or SA-PE alone. Data are representative of 2 separate experiments. (C) THP-1 and PBT008 grown in media alone (black histograms) or induced overnight with TNF and IL-4 (red histograms) were analyzed by flow cytometry for expression of constitutive IL13Rα2 (AF146), for expression of the induced antigen (B-D13-PE), and for binding to IL13-bio/SA-PE. Data are representative of 3 separate experiments. (D) IL13-zetakine⁺ CD8⁺ CTL recognize and kill U251T glioma targets expressing constitutive IL13Rα2 (AF146-positive), but not cytokine-induced PBT003 cells (B-D13-positive). Percentage specific lysis (mean ± S.D.) of triplicate wells is depicted. *, p≤0.0002 using an unpaired Student's t-test to compare U251T vs. PBT003-4 targets. #, p>0.05 using an unpaired Student's t-test to compare PBT003-4 targets with and without overnight cytokine stimulation. Data are representative of at least 2 separate experiments.</p

Differential recognition of constitutively-expressed versus cytokine-induced IL13Rα2 by commercially available anti-IL13Rα2 antibodies.

<p>Flow cytometric analysis of monocytic line THP-1 and various glioma lines with (A) B-D13-PE (Cell Sciences) or (B) AF146 (R&D Systems) reagents for media alone (black histogram) and cytokine (TNF/IL-4 or TNF/IL-13 overnight; red histogram) conditions. Isotype (iso-PE) and mouse anti-goat-FITC controls are shown as grey histograms. Percent positive and relative fluorescent index (RFI) of MFI cytokine/MFI media is reported for each histogram. (C) Flow cytometric detection of IL13Rα2 for D283 cells engineered to express IL13Rα2 (D283-IL13Rα2; blue histogram) and D283 parental (black histogram) stained with AF146 or B-D13-PE antibodies. All data are representative of more than three experiments each.</p