Mutant KRAS promotes malignant pleural effusion formation

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition

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

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

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 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

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

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