The role of osteopontin in pulmonary metastasis

Together with the brain, liver, and bones, the lungs are frequent targets of tumor metastasis. Although osteopontin (secreted phosphoprotein 1, SPP1) has been associated with the dissemination of various bodily tumors, its role in lung-directed metastasis remains unknown. This study is designed in order to clarify the mechanism by which SPP1 induces pulmonary metastasis.To investigate this, we employed mouse models of spontaneous (after s.c. injection of a million tumor cells) and induced (after i.v. injection of half a million tumor cells) lung-targeted metastasis of three different cancer cell lines (MC38 colon and LLC lung adenocarcinomas; B16F10 melanoma) in syngeneic C57BL/6 mice competent (Spp1+/+) and deficient (Spp1-/-) in both Spp1 alleles. Tumor-derived SPP1 expression was modulated using stable anti-Spp1 shRNA and forced overexpression of intracellular (Spp1is2) and secreted (Spp1is4) Spp1 isoforms.We determined that lung epithelial and myeloid cells expressed predominantly Spp1is4, which played no role in pulmonary metastasis, since Spp1+/+ and Spp1-/- mice equally developed spontaneous and induced metastases. On the contrary, tumor cells expressed both Spp1is2 and Spp1is4, which exerted distinct effects: Spp1is2 promoted blood-borne tumor cell survival in the lungs via suppressing TRP53 expression, whereas Spp1is4 accelerated lung metastasis by enhancing tumor-derived CCL2 secretion. Consistently with the above findings, CCR2 gene-deficient mice (Ccr2-/-) were protected from lung metastasis. In addition, SPP1-competent and -incompetent tumor cells could equally colonize the lungs of both Ccr2+/+ and Ccr2-/- mice, indicating a major contribution of CCL2 to SPP1-mediated pro-metastatic effects.In conclusion, our data indicate that tumor-derived SPP1 promotes pneumotropic metastasis and present a possible therapeutic target aimed at preventing pulmonary dissemination of various bodily tumors.Ο πνεύμονας αποτελεί μια από τις κυριότερες θέσεις εγκαθίδρισης μεταστατικών όγκων μαζί με τον εγκέφαλο, το ήπαρ και τα οστά. Μέχρι σήμερα η οστεοποντίνη (secreted phosphoprotein 1, SPP1) έχει συσχετισθεί με την διασπορά ποικίλων όγκων στο σώμα, ωστόσο η δράση της στις πνευμονικές μεταστάσεις δεν έχει πλήρως αποσαφηνισθεί. Σκοπός της παρούσας μελέτης είναι η διερεύνηση και αποσαφήνιση του ρόλου της SPP1 στη μετάσταση στον πνεύμονα.Προκειμένου να απαντηθεί αυτό το ερώτημα, χρησιμοποιήσαμε μοντέλα ποντικών επαγόμενης (υποδόρια εμφύτευση) αλλά και αυτόματης (ενδοφλέβια χορήγηση) μετάστασης, τριών διαφορετικών καρκινικών σειρών (MC38 και LLC- αδενοκαρκίνωματα παχέος εντέρου και πνεύμονα αντίστοιχα, B16F10 μελάνωμα) σε φυσικού τύπου C57BL/6 ποντικούς (Spp1+/+) αλλά και σε υπόστρωμα ένδειας SPP1 του ξενιστή (Spp1-/-). Τα επίπεδα έκφρασης της πρωτεΐνης στα καρκινικά κύτταρα τροποποιήθηκαν με αποσιώπηση χρησιμοποιώντας anti-Spp1 shRNA και με επαγόμενη υπερέκφραση της ενδοκυττάριας (Spp1is2) και της εκκρινόμενης (Spp1is4) ισομορφής.Τα αποτελέσματά μας δείχνουν ότι τα πνευμονικά επιθηλιακά και μυελοειδή κύτταρα εξέφραζαν κυρίως την Spp1is4, η οποία ωστόσο δεν επηρέασε την πνευμονική αποίκηση, αφού τόσο οι Spp1+/+ όσο και οι Spp1-/- ποντικοί ανέπτυξαν παρόμοιο αριθμό αυτόματων και επαγόμενων μεταστάσεων. Αντίθετα, τα καρκινικά κύτταρα εξέφραζαν και τις δύο ισομορφές με διαφορετικές ωστόσο επιδράσεις στο φαινόμενο: η Spp1is2 προήγαγε την επιβίωση των καρκινικών κυττάρων στον πνεύμονα καταστέλλοντας την σταθεροποίηση του TRP53, ενώ η Spp1is4 επιτάχυνε την πνευμονική μετάσταση επάγοντας την έκκριση της χημειοκίνης CCL2. Παράλληλα, ποντικοί ενδεείς για το γονίδιο του CCR2 υποδοχέα (Ccr2-/-) εμφανίστηκαν μη προστατευμένοι στην πνευμονική μετάσταση καθώς καρκινικά κύτταρα επαρκή και μη σε SPP1 μπορούσαν να αποικήσουν παρόμοια πνεύμονες Ccr2+/+ και Ccr2-/- ποντικών, γεγονός που τονίζει την συμμετοχή της CCL2 στα επαγόμενα από SPP1 μεταστατικά φαινόμενα.Συμπερασματικά, τα αποτελέσματά μας υποδεικνύουν ότι η προερχόμενη από το καρκινικό κύτταρο SPP1 προάγει τις πνευμονικές μεταστάσεις και αποτελεί ενδεχομένως θεραπευτικό στόχο ώστε να αποφευχθεί η πνευμονική διασπορά ποικίλων όγκων του σώματος

Metadherin, p50, and p65 Expression in Epithelial Ovarian Neoplasms: An Immunohistochemical Study

NF-κB signaling promotes cancer progression in a large number of malignancies. Metadherin, a coactivator of the NF-κB transcription complex, was recently identified to regulate different signaling pathways that are closely related to cancer. We assessed the immunohistochemical expression of p50, p65, and metadherin in 30 ovarian carcinomas, 15 borderline ovarian tumours, and 31 benign ovarian cystadenomas. Ovarian carcinomas exhibited significantly higher expression of all 3 markers compared to benign ovarian tumours. Borderline ovarian tumours demonstrated significantly higher expression for all 3 markers compared to benign cystadenomas. Ovarian carcinomas demonstrated significantly higher expression of p50 and metadherin compared to borderline ovarian tumours, whereas no significant difference was noted in p65 expression between ovarian carcinomas and borderline ovarian tumours. There was a strong correlation with the expression levels of p50, p65, and metadherin, whereas no correlation was observed with either grade or stage. Strong p50, p65, and metadherin expression was associated with a high probability to distinguish ovarian carcinomas over borderline and benign ovarian tumours, as well as borderline ovarian tumours over benign ovarian neoplasms. A gradual increase in the expression of these molecules is noted when moving across the spectrum of ovarian carcinogenesis, from borderline ovarian tumours to epithelial carcinomas

Comprehensive Evaluation of Nuclear Factor-κΒ Expression Patterns in Non-Small Cell Lung Cancer.

Nuclear factor (NF)-κB signalling is required for lung adenocarcinoma development in mice, and both of its subunits RelA and RelB were independently reported to be highly expressed in human non-small cell lung cancer (NSCLC). To comprehensively examine NF-κB expression in NSCLC, we analyzed serial sections of primary tumor samples from 77 well-documented patients (36 adenocarcinomas, 40 squamous cell carcinomas and 3 large cell carcinomas) for immunoreactivity of RelA, RelB, P50, and P52/P100. Tumor and intratumoral stroma areas were discriminated based on proliferating cell nuclear antigen immunoreactivity and inflammatory infiltration was assessed in intratumoral stroma areas. NF-κB immunoreactivity was quantified by intensity, extent, and nuclear localization and was cross-examined with tumor cell proliferation, inflammatory infiltration, and clinical-pathologic data. We found that the expression of the different NF-κB subunits was not concordant, warranting our integral approach. Overall, RelA, RelB, and P50 were expressed at higher levels compared with P52/P100. However, RelA and P50 were predominantly expressed in intratumoral stroma, but RelB in tumor cells. Importantly, tumor area RelA expression was correlated with the intensity of inflammatory infiltration, whereas RelB expression was identified in proliferating tumor cells. Using multiple logistic regression, we identified that tumor RelB expression was an independent predictor of lymph node metastasis, and tumor P50 was an independent predictor of TNM6 stage IIB or higher, whereas tumor RelA was an independent predictor of inflammatory infiltration. We conclude that pathologic studies of NF-κB expression in cancer should include multiple pathway components. Utilizing such an approach, we identified intriguing associations between distinct NF-κB subunits and clinical and pathologic features of NSCLC

Beneficial impact of CCL2 and CCL12 neutralization on experimental malignant pleural effusion.

Using genetic interventions, we previously determined that C-C motif chemokine ligand 2 (CCL2) promotes malignant pleural effusion (MPE) formation in mice. Here we conducted preclinical studies aimed at assessing the specific therapeutic potential of antibody-mediated CCL2 blockade against MPE. For this, murine MPEs or skin tumors were generated in C57BL/6 mice by intrapleural or subcutaneous delivery of lung (LLC) or colon (MC38) adenocarcinoma cells. Human lung adenocarcinoma cells (A549) were used to induce MPEs in severe combined immunodeficient mice. Intraperitoneal antibodies neutralizing mouse CCL2 and/or CCL12, a murine CCL2 ortholog, were administered at 10 or 50 mg/kg every three days. We found that high doses of CCL2/12 neutralizing antibody treatment (50 mg/kg) were required to limit MPE formation by LLC cells. CCL2 and CCL12 blockade were equally potent inhibitors of MPE development by LLC cells. Combined CCL2 and CCL12 neutralization was also effective against MC38-induced MPE and prolonged the survival of mice in both syngeneic models. Mouse-specific CCL2-blockade limited A549-caused xenogeneic MPE, indicating that host-derived CCL2 also contributes to MPE precipitation in mice. The impact of CCL2/12 antagonism was associated with inhibition of immune and vascular MPE-related phenomena, such as inflammation, new blood vessel assembly and plasma extravasation into the pleural space. We conclude that CCL2 and CCL12 blockade are effective against experimental MPE induced by murine and human adenocarcinoma in mice. These results suggest that CCL2-targeted therapies may hold promise for future use against human MPE

Immunohistochemical detection of NF-κB in mouse models of NSCLC.

<p>NF-κB subunit expression was assessed by immunohistochemistry in urethane-induced mouse lung adenomas <b>(A and C)</b> and mutant <i>KRAS</i>-induced lung adenocarcinomas <b>(B and D)</b>. <b>(A, B)</b> Representative images. <b>(C, D)</b> Overall scoring of NF-κB subunit expression levels from four mice per group. Data presented as mean ± SD. ** and ***: P < 0.01, and P < 0.001 for the indicated color-coded subunit compared with normal bronchial and alveolar epithelium by two-way ANOVA followed by Bonferroni post-tests. Non-significant comparisons are not indicated.</p

Immunohistochemical detection of NF-κB subunits in NSCLC, juxta-tumoral normal lung structures and preneoplastic lesions.

<p><b>(A)</b> Representative images. Images in red frames representatively display differential NF-κB subunit expression in tumor and intratumoral stroma areas. <b>(B)</b> Overall scoring of NF-κB subunit expression levels. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, * and ***: P > 0.05, P < 0.05, and P < 0.001 for indicated comparisons by Friedman’s test followed by Dunn’s post-tests. <b>(C)</b> Co-expression matrixes of categorical NF-κB subunit expression levels. For this, NF-κB scores from (B) were categorized into low (0–4), intermediate (5–6), and high (7–18). ns: P > 0.05 by χ² tests followed by Fisher’s exact tests.</p

NF-κB subunit expression patterns in tumor versus intratumoral stroma areas.

<p><b>(A)</b> Representative images. <b>(B, D)</b> Scoring of NF-κB subunit expression levels in tumor (B) and stroma (D) areas. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns and ***: P > 0.05 and P < 0.001 for indicated comparisons by Friedman’s test followed by Dunn’s post-tests. <b>(C, E)</b> Co-expression matrixes of categorical NF-κB subunit expression levels in tumor (C) and stroma (E) areas. For this, NF-κB scores from (B) and (D) were categorized into low (0–4), intermediate (5–6), and high (7–18). ns: P > 0.05 and P: probability values by χ² tests followed by Fisher’s exact tests. <b>(F)</b> Co-expression matrixes of tumor versus stroma NF-κB subunit expression. ns: P > 0.05 and P: probability values by χ² tests followed by Fisher’s exact tests. <b>(G)</b> Correlation of tumor and stroma P100/P52 expression scores. Shown are data points, linear regression line with 95% confidence interval, squared Spearman’s correlation coefficient, and probability value.</p

Beneficial Impact of CCL2 and CCL12 Neutralization on Experimental Malignant Pleural Effusion

Using genetic interventions, we previously determined that C-C motif chemokine ligand 2 (CCL2) promotes malignant pleural effusion (MPE) formation in mice. Here we conducted preclinical studies aimed at assessing the specific therapeutic potential of antibody-mediated CCL2 blockade against MPE. For this, murine MPEs or skin tumors were generated in C57BL/6 mice by intrapleural or subcutaneous delivery of lung (LLC) or colon (MC38) adenocarcinoma cells. Human lung adenocarcinoma cells (A549) were used to induce MPEs in severe combined immunodeficient mice. Intraperitoneal antibodies neutralizing mouse CCL2 and/or CCL12, a murine CCL2 ortholog, were administered at 10 or 50 mg/kg every three days. We found that high doses of CCL2/12 neutralizing antibody treatment (50 mg/kg) were required to limit MPE formation by LLC cells. CCL2 and CCL12 blockade were equally potent inhibitors of MPE development by LLC cells. Combined CCL2 and CCL12 neutralization was also effective against MC38-induced MPE and prolonged the survival of mice in both syngeneic models. Mouse-specific CCL2-blockade limited A549-caused xenogeneic MPE, indicating that host-derived CCL2 also contributes to MPE precipitation in mice. The impact of CCL2/12 antagonism was associated with inhibition of immune and vascular MPE-related phenomena, such as inflammation, new blood vessel assembly and plasma extravasation into the pleural space. We conclude that CCL2 and CCL12 blockade are effective against experimental MPE induced by murine and human adenocarcinoma in mice. These results suggest that CCL2-targeted therapies may hold promise for future use against human MPE

Association of NF-κB subunit expression with tumor-related inflammation and cellular proliferation in NSCLC.

<p><b>(A)</b> Representative images of hematoxylin-stained samples showing different degrees of inflammatory infiltration of stroma areas. <b>(B)</b> NF-κB subunit expression scores of tumors with varying degrees of inflammatory infiltration. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, **, and ***: P > 0.05, P < 0.01, and P < 0.001 for indicated comparisons by Kruskal-Wallis tests followed by Dunn’s post-tests. <b>(C)</b> Representative images of PCNA-stained NSCLC subtype samples. <b>(D)</b> Nuclear co-localization of PCNA immunoreactivity with <i>Rel</i>B (arrows), but not with <i>Rel</i>A, was identified using dual immunostaining of samples of 10 patients (representative images shown).</p

Association of NF-κB expression with clinical and pathologic parameters in 77 patients with NSCLC.

<p><b>(A)</b> NF-κB expression levels subdivided by clinical and pathological parameters. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, *, and **: P > 0.05, P < 0.05, and P < 0.0501 for indicated comparisons by Wilcoxon signed rank tests or Kruskal-Wallis tests followed by Dunn’s post-tests, for two or multiple comparison groups, respectively. <b>(B)</b> Results of binary logistic regression analyses using NF-κB subunit expression scores as the input (independent variables) and dichotomized clinical and pathologic parameters as the output (dependent variables). RR, risk ratios; CI, confidence intervals; P, probability values.</p