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

The expression of vascular endothelial growth factor-C correlates with lymphatic microvessel density and lymph node metastasis in prostate carcinoma: An immunohistochemical study

Aim: To evaluate the expression of two different lymphatic vascular density (LVD) markers (D2-40 and LYVE-1) and a lymphangiogenic cytokine (Vascular Endothelial Growth Factor-C, [VEGF-C]) in prostate carcinoma and to investigate their relationship with the lymph node status. Settings and Design: Archival material study of 92 non-consecutive radical prostatectomy specimens. Materials and Methods: The mean LVD was assessed immunohistochemically in 24 prostate carcinoma specimens from patients with clinically localized disease, who were found to have nodal metastasis (pN1), and was compared with 68 pN0 cases. Furthermore, the mean LVD, VEGF-C expression, and lymphatic invasion were examined in relation to lymph node involvement. Results: Peritumoral (but not intratumoral) mean LVD assessed by D2-40 was higher in pN1 tumors (P = 0.015). LYVE-1 expression was limited and not associated with lymph node status. The VEGF-C expression was higher in the N1 cases and also correlated with the increased mean LVD in both the peri- and intratumoral compartments. Lymphatic invasion was strongly associated with nodal metastasis and higher VEGF-C expression. Conclusions: Our results indicate that increased peritumoral (but not intratumoral) LVD in the tumor specimen is associated with lymph node metastasis. Increased expression of VEGF-C is associated with higher LVD (in both intratumoral and peritumoral compartments) and with positive lymph node status, indicating a possible dual role in both lymphangiogenesis and lymphatic vessel invasion

A Molecular Lateral Flow Assay for SARS-CoV-2 Quantitative Detection

Since the onset of the SARS-CoV-2 pandemic, several COVID-19 detection methods, both commercially available and in the lab, have been developed using different biomolecules as analytes and different detection and sampling methods with high analytical performance. Developing novel COVID-19 detection assays is an exciting research field, as rapid accurate diagnosis is a valuable tool to control the current pandemic, and also because the acquired knowledge can be deployed for facing future infectious outbreaks. We here developed a novel gold-nanoparticle-based nucleic acid lateral flow assay for the rapid, visual, and quantitative detection of SARS-CoV-2. Our method was based on the use of a DNA internal standard (competitor) for quantification and involved RT-PCR, the hybridization of biotinylated PCR products to specific oligonucleotide probes, and detection with a dual lateral flow assay using gold nanoparticles conjugated to an anti-biotin antibody as reporters. The developed test allowed for rapid detection by the naked eye and the simultaneous quantification of SARS-CoV-2 in nasopharyngeal swabs with high specificity, detectability, and repeatability. This novel molecular strip test for COVID-19 detection represents a simple, cost-effective, and accurate rapid test that is very promising to be used as a future diagnostic tool

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

Osteopontin drives KRAS-mutant lung adenocarcinoma

Increased expression of osteopontin (SPP1) is associated with aggressive human lung adenocarcinoma, but its function remains unknown. Our aim was to determine the role of SPP1 in smoking-induced lung adenocarcinoma. We combined mouse models of tobacco carcinogen-induced lung adenocarcinoma, of deficiency of endogenous Spp1 alleles, and of adoptive pulmonary macrophage reconstitution to map the expression of SPP1 and its receptors and determine its impact during carcinogenesis. Co-expression of Spp1 and mutant KrasG12C in benign cells was employed to investigate SPP1/KRAS interactions in oncogenesis. Finally, intratracheal adenovirus encoding Cre recombinase was delivered to LSL.KRASG12D mice lacking endogenous or overexpressing transgenic Spp1 alleles. SPP1 was overexpressed in experimental and human lung adenocarcinoma and portended poor survival. In response to two different smoke carcinogens, Spp1-deficient mice developed fewer and smaller lung adenocarcinoma with decreased cellular survival and angiogenesis. Both lung epithelial- and macrophage-secreted SPP1 drove tumor-associated inflammation, while epithelial SPP1 promoted early tumorigenesis by fostering the survival of KRAS-mutated cells. Finally, loss and overexpression of Spp1 was, respectively, protective and deleterious for mice harboring KRASG12D-driven LADC. Our data support that SPP1 is functionally involved in early stages of airway epithelial carcinogenesis driven by smoking and mutant KRAS and may present an important therapeutic target

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

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