<i>SOX9</i> mRNA expression is associated with poor probability of survival for SQCC patients.

<p>Univariate Kaplan-Meier survival analysis of SQCC patients, stratified by <i>SOX9</i> mRNA expression. (A) Analysis using data pooled from nine cohorts and not filtered by grade or smoking history. (B) Analysis filtering on Stage I and smoking history (three cohorts). All plotted numerical data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002581#pbio.1002581.s001" target="_blank">S1 Data</a>.</p

PI3K signaling is necessary for the squamous response to SOX2.

<p>(A) The PI3K inhibitor BKM120 inhibits growth of tracheobronchial basal cells. Basal cells were grown on plastic over 7 d with fresh media and drug added every other day. Growth was quantified by alamarBlue and is normalized to control cells treated with the DMSO vehicle, which was given a value of 100%. The means ± SEM from three replicates are shown. (B-E) Inhibition of PI3K signaling prevents SOX2-driven squamous differentiation. Tracheobronchial basal cells growing on plastic were infected with Lenti-SOX2 or empty vector, co-treated with the indicated drugs or shRNA viruses for 5 d, and then assayed for lineage marker expression by qRT-PCR. Fold inductions were first calculated by comparing marker expression between Lenti-SOX2 and control vector (non-SOX2)-transduced cells. Because the magnitudes of inductions sometimes varied between biological replicate experiments, fold-inductions were directly compared between matched pairs of Lenti-SOX2 (with DMSO or shluc) and PI3K-inhibited (chemical inhibitor or shPIK3CA) Lenti-SOX2-transduced cultures. The amount of marker induction in inhibitor-treated cultures was then plotted as a percentage of the induction response seen without inhibitor treatment, which was given a value of 100. (B) Schematic for the PI3K chemical inhibitor experiments. White cells represent undifferentiated basal cells. Yellow and purple depict squamous and mucinous-differentiating cells, respectively. Red denotes cells that have been transduced with Lenti-SOX2. (C) Summary of PI3K chemical inhibitor data. Lentivirally infected cultures were co-treated with 2.5 μM BKM120, 4 μM LY294002, or DMSO vehicle. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed <i>t</i> tests. BKM120-treated <i>p</i>-values include *<i>p</i> = 0.04, <i>**p</i> = 0.004 (<i>IVL</i>), <i>***p</i> = 0.0001 (<i>TMPRSS11B</i>), 0.0002 (<i>SPRR1A</i>), and 0.000002 (<i>SPRR3</i>). LY294002-treated <i>p</i>-values include <i>**p</i> = 0.0006 (<i>TMPRSS11B</i>), 0.001 (<i>IVL</i>), <i>***p</i> = 0.0004 (<i>SPRR1A</i>), and 0.0002 (<i>SPRR3</i>). For AKT immunoblotting, lysates were prepared at 2 hr (LY294002) and 24 hr (BKM120) post-drug addition, while for SOX2 immunoblotting, at 4 d post-drug addition. (D) Schematic for the shPIK3CA experiments. Cell colors are as described in (B). (E) Summary of shPIK3CA data. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed <i>t</i> tests. <i>*p</i> = 0.01, <i>**p</i> = 0.003 (<i>SOX2</i>), 0.002 (<i>SPRR1A</i>), <i>***p</i> = 0.0004 (<i>PIK3CA</i>), 0.0001 (<i>TMPRSS11B</i>), 0.00001 (<i>IVL</i>), 0.0002 (<i>SPRR2A</i>). All plotted numerical data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002581#pbio.1002581.s001" target="_blank">S1 Data</a>.</p

PI3K signaling is high in proliferating basal cells and in squamous differentiating epithelia.

<p>(A) <i>PIK3CA</i> is co-amplified with <i>SOX2</i> at 3q26-28 in lung SQCCs. <i>SOX2</i> and <i>PIK3CA</i> copy number variation data for 177 primary patient SQCCs from the TCGA. Numerical data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002581#pbio.1002581.s001" target="_blank">S1 Data</a>. (B) PI3K activity in ALI cultures of tracheobronchial basal cells. Basal cells were infected with Lenti-SOX2 or control vector and grown at ALI, as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002581#pbio.1002581.g002" target="_blank">Fig 2A</a>. Cultures were stained for phospho-Ser240/244-S6 (P-S6) or phospho-Thr308-AKT (P-AKT) at the indicated times. Dotted lines outline areas of squamous metaplasia, including upper differentiated layers that had detached during sectioning. Orange arrows mark basal cells in squamous metaplasia that are stained positive for nuclear P-AKT. Scale bars are 20 μm.</p

Precocious SOX2 expression in proliferating tracheobronchial basal cells induces hyperplastic squamous metaplasia and inhibits ciliogenesis.

<p>(A–F, I, J) For all ALI data, SOX2^Lo proliferating tracheobronchial basal cells were infected with Lenti-SOX2 or control vector and grown as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002581#pbio.1002581.g002" target="_blank">Fig 2A</a>. All ALI epithelia were analyzed after 5 wk of ALI culture. (A) PAS-D staining showing absence of mucin expression in areas of squamous metaplasia. Squamous metaplasia was quantified by scoring 6–7 cm of epithelia from multiple sections derived from three replicates. Mean ± SEM is shown. Significance was calculated by a two-tailed <i>t</i> test. *<i>p</i> = 0.03. (B) HMWCK (high molecular weight cytokeratin) staining, whose expression in upper layers marks squamous stratifying epithelia. (C) Staining for TMPRSS11B, a marker of squamous epithelia. (D, E) Lenti-SOX2 induces hyperplasia. ALI sections were stained for Ki-67 and TP63. At least eight sections were scored per replicate from three replicates. Means ± SEM are shown. Significance was calculated by two-tailed <i>t</i> tests. *<i>p</i> = 0.01, **<i>p</i> = 0.0007. (F) qRT-PCR analysis of <i>CDKN</i> expression in ALI cultures. Means ± SEM from four replicates are shown. Data are normalized to expression in vector-transduced cultures, which was assigned a value of 1. Significance was calculated by paired two-tailed <i>t</i> tests. *<i>p</i> = 0.012 (<i>CDKN1A</i>), 0.03 (<i>CDKN1C</i>). (G, H) Enforced SOX2 expression is sufficient to induce mucinous, squamous, and SQCC-like differentiation in tracheobronchial basal cells growing on plastic. Basal cells were infected with Lenti-SOX2 or control empty vector and marker expression was measured by qRT-PCR after 5 d. Data are normalized to expression in vector-transduced cultures, which was assigned a value of 1. Means ± SEM from three to five replicates are shown. Significance was calculated by paired two-tailed <i>t</i> tests. (G) *<i>p</i> = 0.02 (<i>MUC16</i>), 0.02 (<i>TMPRSS11B</i>), 0.05 (<i>SPRR3</i>), 0.02 (<i>IVL</i>), 0.04 (<i>SPRR1A</i>), 0.02 (<i>SPRR2A</i>). (H) *<i>p</i> = 0.01 (<i>ADH7</i>), 0.01 (<i>FGFR2</i>), **<i>p</i> = 0.003, ***<i>p</i> = 0.00003. (I) Lenti-SOX2 inhibits ciliogenesis. ALI cultures were stained <i>enface</i> for BTUB4 (a component of cilia), and expression of the ciliogenic <i>FOXJ1</i> transcription factor was quantified by qRT-PCR, with means ± SEM from four replicates shown. For BTUB4 expression, significance was calculated by a two-tailed <i>t</i> test. ***<i>p</i> = 0.000007. For <i>FOXJ1</i> expression, data are normalized to levels in vector-transduced cultures, which was assigned a value of 100, and significance was calculated by a paired two-tailed <i>t</i> test. ***<i>p</i> = 0.00007. (J) SOX2 IHC in lentivirally-infected ALI cultures. Scale bars are 50 μm (A, B, E) and 20 μm (C, I, J). All plotted numerical data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002581#pbio.1002581.s001" target="_blank">S1 Data</a>.</p

During mucociliary differentiation of tracheobronchial basal cells, SOX2 expression varies from low to high.

<p>(A) SOX2 immunohistochemistry (IHC) in normal native human tracheobronchial epithelia and <i>SOX2</i>-amplified primary patient lung SQCCs and SQCC patient-derived xenografts (PDXs). Arrows point to some basal cells. (B) qRT-PCR quantification of <i>SOX2</i> expression in normal tracheobronchial epithelial cells and SQCCs. Tracheobronchial cell suspensions and FACS-purified basal cells from these suspensions were derived from tissue without culturing. ADC = primary patient lung adenocarcinoma. All data, with the exception of “Proliferating basal cells on plastic” (green), are from individual biological replicates, which were generated from duplicate qRT-PCR technical replicates. Tracheobronchial basal cells proliferating on plastic were infected with Lenti-SOX2 or empty vector, with mean expression ± standard error of the mean (SEM) from three biological replicate experiments shown. Control empty vector did not alter <i>SOX2</i> expression relative to untransduced basal cells (not shown). LRR = log likelihood ratio quantification of <i>SOX2</i> gene copy number. Expression is normalized to normal tracheal suspension #1, which was assigned a value of 100. (C, D) SOX2 and mucociliary lineage marker expression during tracheobronchial basal cell differentiation in air-liquid-interface (ALI) cultures. (C) Immunofluorescence staining of SOX2 and lineage marker expression. White arrows point to some basal cells and green arrows mark FOXJ1+ cells. (D) qRT-PCR analysis of <i>SOX2</i> and lineage marker expression. Data are plotted relative to the time point with the maximal expression of the gene, which was given a value of 100. Means ± SEM from three replicates are shown. (E) SOX2 IHC in metaplastic areas of native human tracheobronchial epithelia. Scale bars are 20 μm. All plotted numerical data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002581#pbio.1002581.s001" target="_blank">S1 Data</a>.</p

<i>SOX9</i> is a repressed target of SOX2 and PI3K that is expressed oppositely to SOX2.

<p>(A) SOX9 protein expression is not detected in the surface epithelium of native human tracheal tissue, but is observed in submucosal glands. (B, C) Analysis of SOX9 expression during mucociliary differentiation of tracheobronchial basal cell ALI cultures. (B) qRT-PCR quantification of <i>SOX9</i> mRNA expression. Data are plotted relative to the time point with maximal expression, which was given a value of 100. Means ± SEM from three replicates are shown. Plotted numerical data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002581#pbio.1002581.s001" target="_blank">S1 Data</a>. (C) SOX9 immunostaining. Arrows show positions of some basal cells. (D) Immunostaining of SOX9 expression in rat tracheal xenografts that have reconstituted a human tracheal epithelium. Grafts were generated as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002581#pbio.1002581.g006" target="_blank">Fig 6A</a>. Arrows point to basal cells in different epithelial regions that appear less (green) or more (orange, pink) columnar-differentiated and have differing amounts of SOX9 expression. Magnified images from these areas are shown and are bordered with colors matching the arrow colors. (E) SOX9 expression is occasionally detected in TP63-expressing basal cells in non-fully mucociliary differentiated human tracheal surface epithelia. Arrows point to basal cells with high SOX9 expression. All scale bars are 20 μm.</p

Precocious SOX2 expression in proliferating tracheobronchial basal cells enhances mucinous differentiation.

<p>(A) Experimental design. SOX2^Lo tracheobronchial basal cells proliferating on plastic were infected overnight with Lenti-SOX2 or empty vector, seeded subconfluently at ALI, and examined after 5 wk. (B) Hematoxylin and eosin (H&E) staining showing ectopic induction of glandular-like areas and squamous metaplasia by Lenti-SOX2. (C) PAS-D (periodic acid Schiff-diastase) staining for mucins. Glandular differentiation was quantified by scoring 6–7 cm of epithelia from multiple sections derived from three replicates. Mean ± SEM is shown. Plotted numerical data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002581#pbio.1002581.s001" target="_blank">S1 Data</a>. (D) MUC16 and MUC5AC mucin staining. Arrows point to non-glandular cells with increased MUC16 expression relative to vector control cultures. Due to high MUC16 expression in Lenti-SOX2 cultures, the exposure time was shorter than in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002581#pbio.1002581.g001" target="_blank">Fig 1C</a>. (E) MUC16 and MUC5AC mucin staining in native human tracheobronchial tissue. Scale bars are 100 μm (B), 50 μm (C), and 20 μm (D, E). Significance was calculated using a two-tailed <i>t</i> test. *<i>p</i> = 0.006.</p

PI3K is necessary for squamous metaplasia in rat tracheal xenografts.

<p>(A) Schematic for rat tracheal xenograft procedure. Human tracheobronchial basal cells were seeded into denuded rat tracheas, which were then implanted subcutaneously into immunocompromised mice. (B) Staining for Ki-67, SOX2, phospho-Ser240/244-S6 (P-S6), and phospho-Thr308-AKT (P-AKT) during the initial phase of squamous metaplasia (3 d) and later period of mucociliary differentiation (>30 d). (C) Basal cells were seeded ± 5 μM BKM120 into denuded rat tracheas, and epithelia were immunostained after 1 d. Representative images from duplicate experiments are shown. Arrows point to areas of squamous differentiation, as evidenced by involucrin (IVL) expression. Dotted line indicates underlying tracheal tissue that moved into the lumen during sectioning. Scale bars are 20 μm (B) and 50 μm (C).</p

Real-time intravital optical tracking of radiation-induced platelet thrombus formation in tumor microvascular <i>in vivo</i>.

<p><b>A.</b> FITC-Dextran (green) and APC-labeled anti-mouse CD41 antibodies (fuchsia) were used to visualize tumor microvasculature and platelets, respectively, in relation to the DsRed-Me180 tumor cells (red) before and (1 and 24 h) after a single fraction of 30 Gy. Blood flow is from top to bottom of images. <b>B.</b> Intravital fluorescence imaging (<i>inset of panel A</i>) taken pre-irradiation, 1 h and 24 h after irradiation, with serial images taken consecutively at 30 sec intervals, demonstrating transient formation of platelet thrombi. In pre-irradiation images, fast-moving blood cells, FITC-Dextran and APC-CD41 can be observed as horizontal lines (<i>orange arrows, inset of pre-irradiation images in panel B</i>) in the confocal fluorescence angiograms, as they pass out of the confocal scanning area faster than the image acquisition time (∼15 sec/channel). Compared to pre-irradiation, blood vessel dilation and blockage (<i>white arrow, B</i>), and impeded blood perfusion (<i>yellow arrow, B</i>) were seen 1 h after irradiation in some vessels when platelet thrombi form. Optical tracking of adhered FITC-Dextran-labeled RBCs (<i>red arrows, B</i>) and APC-CD41-labeled thrombotic plaques (<i>blue arrow, B</i>) formed early (1 h) after irradiation but clear after 24 h. After 24 h, early forming thrombi adhered to vessels walls were cleared in many cases. <b>C.</b> Microvascular function was compromised as early as 1 h after radiation by blockages caused mostly by larger (∼20–40 µm diameter) platelet thombi (<i>white arrow, C</i>), while microthrombi (∼2–5 µm diameter) transiently adhered to vessel walls. <b>D.</b> Typically, smaller vessels (∼12 µm diameter) were more prone at 1 and 24 h post-irradiation to thrombosis-induced occlusion (<i>blue arrows, D</i>) than larger (∼25 µm diameter) vessels where small thrombi forming at the vessel wall eventually cleared. <b>E.</b> Confocal fluorescence microscopy also revealed FITC-Dextran labeled RBCs aggregating with platelet thrombi to occlude capillaries, and in many cases adhering to microvessel walls (<i>blue arrows in inset, E</i>). <i>Scale bars: 125 µm (A), 25 µm (B), 50 µm (C), 125 µm (D), 25 µm (E) and 6 µm (inset, E).</i></p

Characterization of SOX2 and SOX9 expression in primary patient SQCCs.

<p>Characterization of SOX2 and SOX9 expression in primary patient SQCCs.</p