LLC metastasis growth in DUSP3^-/- mice is driven by hematopoietic cells.

<p>Tumours were established by injection (i.v.) of 10⁶ LLC-Luc+ cells to DUSP3^+/+ or DUSP3^-/- BM-transplanted irradiated DUSP3^+/+ mice. <b>(A)</b> Western blot analysis for DUSP3 expression from spleen lysates of transplanted mice (M = mouse). (<b>B</b>) Representative xenogen bioluminescence imaging results. <b>(C)</b> Quantitative imaging data (day 14). <b>(D)</b> Correlation between size of tumours (quantified as photons/second) and DUSP3 expression. <b>(E)</b> Comparison of lungs weights of DUSP3^+/+ or DUSP3^-/- BM-transplanted irradiated DUSP3^+/+ mice. <b>(F)</b> Hematoxylin-eosin staining of lung section from DUSP3^+/+ or DUSP3^-/- BM-transplanted irradiated DUSP3^+/+ mice. <b>(G)</b> Comparison of tumour areas from DUSP3^+/+ or DUSP3^-/- BM-transplanted irradiated DUSP3^+/+ mice. Student t-test was used for (<b>C</b>) and (<b>E</b>) and Mann-Whitney test was used for (<b>G</b>). *p < 0,05, **p < 0.01. 4 mice were used in each group and for each experiment. Data shown are representative of 2 different experiments.</p

Macrophages depletion reduces LLC tumour growth in DUSP3^-/- mice.

<p>Tumours were established by i.v. injection of 10⁶ LLC-Luc+ cells to clodronate-liposomes-depleted mice. <b>(A)</b> Representative xenogen imaging results and <b>(B)</b> quantification of the xenogen bioluminescence imaging data shown in A at day 14 after LLC injection. <b>(C)</b> Comparison of lung weights. <b>(D)</b> Representative hematoxylin eosin staining of lung sections from DUSP3^+/+ and DUSP3^-/- mice injected with PBS-lipsomes or clodronate-liposomes. <b>(E)</b> Comparison of tumour areas from each group. Student-t-test was used for statistical analysis. *p < 0.05, **p < 0.01 and ***p < 0.001. 3 mice were used in each group and for each experiment. Data shown are representative of 3 different experiments.</p

DUSP3 deletion does not impact experimental B16 metastasis growth.

<p>B16 tumour growths were monitored by xenogen bioluminescence imaging. Tumours were established by i.v. injection of 10⁶ B16-Luc+ cells to DUSP3^+/+ and DUSP3^-/- mice. <b>(A)</b> Representative xenogen imaging results and <b>(B)</b> quantitative xenogen bioluminescence imaging data (day 14). <b>(C)</b> Representative lung macroscopic view and <b>(D)</b> comparison of lung weights from DUSP3^+/+ and DUSP3^-/- mice. <b>(E)</b> Hematoxylin eosin staining of lung sections from each experimental group. <b>(F)</b> Comparison of tumour areas from each group. Student t-test was used for (<b>B</b>) and (<b>D</b>) and Mann-Whitney test was used for (<b>F</b>). *p < 0,05, **p < 0.01. 5 mice in each group were used for each experiment. Data shown are representative of 4 different experiments.</p

LLC metastasis growth in DUSP3^-/- mice is associated with the presence of higher monocytes and macrophages in lungs and with increased proliferation.

<p>Immune cell population phenotyping in LLC tumour-bearing lungs by flow cytometry. <b>(A)</b> Percentages of neutrophils, eosinophils, alveolar macrophages and monocytes/macrophages in DUSP3^+/+ and DUSP3^-/- lungs. <b>(B)</b> Absolute number of neutrophils, eosinophils, alveolar macrophages and monocytes/macrophages in DUSP3^+/+ and DUSP3^-/- lungs. <b>(C-E)</b> Monocytes/macrophages phenotype characterization in LLC tumours: Comparison <b>(C)</b> and percentage <b>(D)</b> of Ly6B^hi, Ly6B^int and Ly6B^low macrophages in DUSP3^+/+ and DUSP3^-/- mice. <b>(E)</b> Expression of specific macrophages markers in distinct Ly6B⁺ populations. Alveolar Ф = alveolar macrophages; Mo/MФ = monocytes/macrophages. Student-t-test was used for statistical analysis. *p < 0,05. 3 mice were used in each group and for each experiment. Data shown are representative of 4 different experiments.</p

Increased LLC tumour mass in DUSP3^-/- mice lungs is associated with increased in situ proliferation of these cells.

<p>Sections of 5-μm thickness were cut from DUSP3^-/- and DUSP3^+/+ LLC bearing lungs embedded in paraffin blocks. Immunohistochemistry for Ki67 was carried out. Revelation was performed using AEC+ Red. Negative control sections were traited similarly except that they were not stained with anti-KI67. <b>(A)</b> repesentative images from each group of mice are shown at 1x and 10x magnifications. Dark color indicate positive staining for Ki67. <b>(B)</b> Quantification of the positively stained cells from the entire tumor represented in the section using Image J software. Student-t-test was used for statistical analysis. *p < 0.05. Data shown are representative of 5 different sections scanned from 5 individual mice from each genotype.</p

DUSP3^-/- monocytes and macrophages migration is enhanced in response to LLC-conditioned medium.

<p><i>In vitro</i> migration assay of DUSP3^+/+ and DUSP3^-/- peritoneal macrophages <b>(A),</b> BMDMs <b>(B)</b> and BMMs <b>(C)</b> in presence of LLC-CM. Migration of DUSP3^+/+ or DUSP3^-/- peritoneal macrophages <b>(D)</b>, BMDMs <b>(E)</b> and BMMs <b>(F)</b> in presence of B16-CM. Migration of DUSP3^+/+ or DUSP3^-/- bone marrow sorted neutrophils in presence of LLC-CM <b>(G)</b> and B16-CM <b>(H</b>). BMDM = Bone Marrow-Differentiated Macrophages; CM = conditioned-medium. Student-t-test was used for statistical analysis. *, p < 0,05 and **, p < 0.01. For each experiment, cells were pooled from 3 mice. Data shown are representative of 3 different experiments.</p

DUSP3 deletion accelerates experimental LLC metastasis growth.

<p>LLC tumour growths were monitored by xenogen bioluminescence imaging. Tumours were established by iv injection of 10⁶ LLC-Luc+ cells to DUSP3^+/+ and DUSP3^-/- mice. <b>(A)</b> Representative xenogen imaging results. <b>(B)</b> Quantification of xenogen bioluminescence imaging data shown in A at day 14 after LLC injection. <b>(C)</b> Representative lung macroscopic view. <b>(D)</b> Comparison of lung weights from DUSP3^+/+ and DUSP3^-/- mice. <b>(E)</b> Hematoxylin eosin staining of lung sections from DUSP3^+/+ and DUSP3^-/- mice. <b>(F)</b> Comparison of tumour areas from DUSP3^+/+ and DUSP3^-/- mice. Student t-test was used for (<b>B</b>) and (<b>D</b>) and Mann-Whitney test was used for (<b>F</b>). *p < 0,05, **p < 0.01. 4 mice were used in each group and for each experiment. Data shown are representative of 5 different experiments.</p