Vessel size as a marker of survival in estrogen receptor positive breast cancer.

PURPOSE: Angiogenesis is crucial for tumor growth and is one of the hallmarks of cancer. In this study, we analyzed microvessel density, vessel median size, and perivascular a-SMA expression as prognostic biomarkers in breast cancer. METHODS: Dual IHC staining was performed where alpha-SMA antibodies were used together with antibodies against the endothelial cell marker CD34. Digital images of stainings were analyzed to extract quantitative data on vessel density, vessel size, and perivascular alpha-SMA status. RESULTS: The analyses in the discovery cohort (n = 108) revealed a statistically significant relationship between large vessel size and shorter disease-specific survival (p = 0.007, log-rank test; p = 0.01, HR 3.1; 95% CI 1.3-7.4, Cox-regression analyses). Subset analyses indicated that the survival association of vessel size was strengthened in ER + breast cancer. To consolidate these findings, additional analyses were performed on a validation cohort (n = 267) where an association between large vessel size and reduced survival was also detected in ER + breast cancer (p = 0.016, log-rank test; p = 0.02; HR 2.3, 95% CI 1.1-4.7, Cox-regression analyses). CONCLUSION: Alpha-SMA/CD34 dual-IHC staining revealed breast cancer heterogeneity regarding vessel size, vessel density, and perivascular a-SMA status. Large vessel size was linked to shorter survival in ER + breast cancer.

Endothelial IL-33 Expression Is Augmented by Adenoviral Activation of the DNA Damage Machinery.

IL-33, required for viral clearance by cytotoxic T cells, is generally expressed in vascular endothelial cells in healthy human tissues. We discovered that endothelial IL-33 expression was stimulated as a response to adenoviral transduction. This response was dependent on MRE11, a sensor of DNA damage that can also be activated by adenoviral DNA, and on IRF1, a transcriptional regulator of cellular responses to viral invasion and DNA damage. Accordingly, we observed that endothelial cells responded to adenoviral DNA by phosphorylation of ATM and CHK2 and that depletion or inhibition of MRE11, but not depletion of ATM, abrogated IL-33 stimulation. In conclusion, we show that adenoviral transduction stimulates IL-33 expression in endothelial cells in a manner that is dependent on the DNA-binding protein MRE11 and the antiviral factor IRF1 but not on downstream DNA damage response signaling.

Interleukin-33 drives a proinflammatory endothelial activation that selectively targets nonquiescent cells.

OBJECTIVE: Interleukin (IL)-33 is a nuclear protein that is released from stressed or damaged cells to act as an alarmin. We investigated the effects of IL-33 on endothelial cells, using the prototype IL-1 family member, IL-1ß, as a reference. METHODS AND RESULTS: Human umbilical vein endothelial cells were stimulated with IL-33 or IL-1ß, showing highly similar phosphorylation of signaling molecules, induction of adhesion molecules, and transcription profiles. However, intradermally injected IL-33 elicited significantly less proinflammatory endothelial activation when compared with IL-1ß and led us to observe that quiescent endothelial cells (ppRb(low)p27(high)) were strikingly resistant to IL-33. Accordingly, the IL-33 receptor was preferentially expressed in nonquiescent cells of low-density cultures, corresponding to selective induction of adhesion molecules and chemokines. Multiparameter phosphoflow cytometry confirmed that signaling driven by IL-33 was stronger in nonquiescent cells. Manipulation of nuclear IL-33 expression by siRNA or adenoviral transduction revealed no functional link between nuclear, endogenous IL-33, and exogenous IL-33 responsiveness. CONCLUSIONS: In contrast to other inflammatory cytokines, IL-33 selectively targets nonquiescent endothelial cells. By this novel concept, quiescent cells may remain nonresponsive to a proinflammatory stimulus that concomitantly triggers a powerful response in cells that have been released from contact inhibition.

The alarmin IL-33 is a notch target in quiescent endothelial cells.

The molecular mechanisms that drive expression of the alarmin interleukin-33 (IL-33) in endothelial cells are unknown. Because nuclear IL-33 is a marker of endothelial cell quiescence (corroborated in this study by coexpression of cyclin-dependent kinase inhibitor p27(Kip1)), we hypothesized that Notch signaling might be involved in regulating IL-33 expression. Activation of Notch1 by immobilized Notch ligands was sufficient to induce nuclear IL-33 expression in cultured endothelial cells. Conversely, IL-33 expression was inhibited by the γ-secretase inhibitor DAPT or by inhibiting the function of Dll4, Jagged1, Notch1, or the canonical Notch transcription factor RBP-Jκ. Insensitivity to cycloheximide indicated that IL-33 was a direct target of Notch signaling, well in line with the identification of several conserved RBP-Jκ binding sites in the IL33 gene. The in vivo expression of Dll4 but not of Jagged1 was well correlated with expression of IL-33 in quiescent vessels, and subcutaneous injection of DAPT in healthy skin reduced IL-33 expression, indicating that Notch signaling was involved. On the other hand, loss of IL-33 during angiogenesis occurred despite sustained Dll4 and Notch1 expression, suggesting that other signals may override the IL-33-driving signal in this context. Taken together, our data demonstrate that endothelial nuclear IL-33 is induced by Notch and that Dll4 may be the dominant ligand responsible for this signaling in vivo.

Integrin α11ß1 is expressed in breast cancer stroma and associates with aggressive tumor phenotypes.

Cancer-associated fibroblasts are essential modifiers of the tumor microenvironment. The collagen-binding integrin α11ß1 has been proposed to be upregulated in a pro-tumorigenic subtype of cancer-associated fibroblasts. Here, we analyzed the expression and clinical relevance of integrin α11ß1 in a large breast cancer series using a novel antibody against the human integrin α11 chain. Several novel monoclonal antibodies against the integrin α11 subunit were tested for use on formalin-fixed paraffin-embedded tissues, and Ab 210F4B6A4 was eventually selected to investigate the immunohistochemical expression in 392 breast cancers using whole sections. mRNA data from METABRIC and co-expression patterns of integrin α11 in relation to αSMA and cytokeratin-14 were also investigated. Integrin α11 was expressed to varying degrees in spindle-shaped cells in the stroma of 99% of invasive breast carcinomas. Integrin α11 co-localized with αSMA in stromal cells, and with αSMA and cytokeratin-14 in breast myoepithelium. High stromal integrin α11 expression (66% of cases) was associated with aggressive breast cancer features such as high histologic grade, increased tumor cell proliferation, ER negativity, HER2 positivity, and triple-negative phenotype, but was not associated with breast cancer specific survival at protein or mRNA levels. In conclusion, high stromal integrin α11 expression was associated with aggressive breast cancer phenotypes.

Tumour cell expression of interleukin 6 receptor α is associated with response rates in patients treated with sunitinib for metastatic clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cell carcinoma, and anti-angiogenic treatment is currently first line therapy for metastatic ccRCC (mccRCC). Response rates and duration of response show considerable variation, and adverse events have a major influence on patient quality of life. The need for predictive biomarkers to select responders to receptor tyrosine kinase inhibitors upfront is urgent. We investigated the predictive value of immunohistochemical biomarkers associated with angiogenesis and systemic inflammation in mccRCC. Forty-six patients with metastatic or non-resectable ccRCC treated with sunitinib were included. Metastatic and/or primary tumour tissue was stained by immunohistochemistry for selected markers related to angiogenesis [vascular endothelial growth factor A (VEGF-A), VEGF receptor 2 (VEGFR2), platelet-derived growth factor receptor ß (PDGFRß), and heat shock protein 27 (HSP27)] and immune responses [Interleukin 6 receptor α (IL6Rα), interleukin-6 (IL6), and jagged1 (JAG1)]. The predictive potential of the candidate markers was assessed by correlations with response rates (RECIST). In addition, progression free survival (PFS) and overall survival (OS) were analysed. Low tumour cell expression of IL6Rα was significantly associated with improved response to sunitinib (Fisher's exact test, p = 0.03), but not with PFS or OS. Median/high expression of IL6Rα showed significant association with median/high expression of VEGF-A and HSP27. Furthermore, low expression of IL6 was significantly associated with improved PFS, but not OS or response rates. High expression of IL6 was significantly associated with high expression of JAG1, VEGF-A, VEGFR2, and PDGFRß. Loss of tumour cell expression of IL6Rα in mccRCC patients treated with sunitinib predicts improved treatment response, and might represent a candidate predictive marker.