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Metastasis is the main cause of death in cancer patients, and understanding mechanisms that control tumor cell dissemination may lead to improved therapy. Tumor cell adhesion receptors contribute to cancer spreading. We noted earlier that tumor cells can expressing the adhesion receptor integrin αvβ3 in distinct states of activation, and found that cells which metastasize from the blood stream express it in a constitutively high affinity form. Here, we analyzed steps of the metastatic cascade in vivo and asked, when and how the affinity state of integrin αvβ3 confers a critical advantage to cancer spreading. Following tumor cells by real time PCR, non-invasive bioluminescence imaging, intravital microscopy and histology allowed us to identify tumor cell extravasation from the blood stream as a rate-limiting step supported by high affinity αvβ3. Successful transendothelial migration depended on cooperation between tumor cells and platelets involving the high affinity tumor cell integrin and release of platelet granules. Thus, this study identifies the high affinity conformer of integrin αvβ3 and its interaction with platelets as critical for early steps during hematogenous metastasis and target for prevention of metastatic disease.

Cell cycle progression is dependent on the nuclear localization and transcriptional effects of activated extracellular signal-regulated kinase (ERK)1 and ERK2 mitogen-activated protein (MAP) kinases (ERK1/2). Phosphoprotein enriched in astrocytes (PEA-15) binds ERK1/2 and inhibits their nuclear localization, thus blocking cell proliferation. Here, we report that phosphorylation of PEA-15 blocks its interaction with ERK1/2 in vitro and in vivo and that phosphorylation of both Ser(104) and Ser(116) is required for this effect. Using phosphomimetic and nonphosphorylatable mutants of PEA-15, we found that PEA-15 phosphorylation abrogates its capacity to block the nuclear localization and transcriptional activities of ERK1/2; this phosphorylation therefore enables the proliferation of cells that express high levels of PEA-15. Additionally, we report that PEA-15 phosphorylation can modulate nontranscriptional activities of ERK1/2, such as the modulation of the affinity of integrin adhesion receptors. Finally, we used a novel anti-phospho-specific PEA-15 antibody to establish that PEA-15 is phosphorylated in situ in normal mammary epithelium. These results define a novel posttranslational mechanism for controlling the subcellular localization of ERK1/2 and for specifying the output of MAP kinase signaling.

Immunohistochemistry (IHC) is a useful method for visualizing antigens in tissues using target-specific antibodies in drug discovery and clinical research. Color reactions with horseradish peroxidase enzymatic activity have been used for IHC. This method has a long history and is highly reliable when applied to in vitro diagnostics. However, an IHC method with high sensitivity is required for low-expression target molecules in antibody-drug conjugates and immunotherapy. Therefore, many studies have been conducted in recent years to improve the sensitivity using highly brilliant and light-resistant phosphor-integrated dots (PIDs). In this method, streptavidin-coated PIDs are linked via biotinylated antibodies and the target molecules are visualized (PID-AbL). PID-AbL is more sensitive than IHC with chromophores. Hence, this study aimed to improve the sensitivity and quantitative results of IHC by combining PIDs with a biotinylated tyramide linker (PID-TL). PID-TL is able to detect ultralow human epidermal growth factor receptor 2-expressed cells. Moreover, PID-TL may be applied to human tissue samples as well as in vitro diagnostic kits. This new approach may help detect low-expression target molecules for clinical research. [ABSTRACT FROM AUTHOR]

The incidence of brain metastasis is rising and poses a severe clinical problem, as we lack effective therapies and knowledge of mechanisms that control metastatic growth in the brain. Here we demonstrate a crucial role for high-affinity tumor cell integrin [[alpha].sub.v][[beta].sub.3] in brain metastatic growth and recruitment of blood vessels. Although [[alpha].sub.v][[beta].sub.3] is frequently up-regulated in primary brain tumors and metastatic lesions of brain homing cancers, we show that it is the [[alpha].sub.v][[beta].sub.3] activation state that is critical for brain lesion growth. Activated, but not non-activated, tumor cell [[alpha].sub.v][[beta].sub.3] supports efficient brain metastatic growth through continuous up-regulation of vascular endothelial growth factor (VEGF) protein under normoxic conditions. In metastatic brain lesions carrying activated [[alpha].sub.v][[beta].sub.3], VEGF expression is controlled at the post-transcriptional level and involves phosphorylation and inhibition of translational respressor 4E-binding protein (4E-BP1). In contrast, tumor cells with non-activated [[alpha].sub.v][[beta].sub.3] depend on hypoxia for VEGF induction, resulting in reduced angiogenesis, tumor cell apoptosis, and inefficient intracranial growth. Importantly, the microenvironment critically influences the effects that activated tumor cell [[alpha].sub.v][[beta].sub.3] exerts on tumor cell growth. Although it strongly promoted intracranial growth, the activation state of the receptor did not influence tumor growth in the mammary fat pad as a primary site. Thus, we identified a mechanism by which metastatic cells thrive in the brain microenvironment and use the high-affinity form of an adhesion receptor to grow and secure host support for proliferation. Targeting this molecular mechanism could prove valuable for the inhibition of brain metastasis. angiogenesis | brain metastasis | integrin activation | 4E-BP1

Context.--Novel therapeutics often target complex cellular mechanisms. Increasingly, quantitative methods like digital tissue image analysis (tIA) are required to evaluate correspondingly complex biomarkers to elucidate subtle phenotypes that can inform treatment decisions with these targeted therapies. These tIA systems need a gold standard, or reference method, to establish analytical validity. Conventional, subjective histopathologic scores assigned by an experienced pathologist are the gold standard in anatomic pathology and are an attractive reference method. The pathologist's score can establish the ground truth to assess a tIA solution's analytical performance. The paradox of this validation strategy, however, is that tIA is often used to assist pathologists to score complex biomarkers because it is more objective and reproducible than manual evaluation alone by overcoming known biases in a human's visual evaluation of tissue, and because it can generate endpoints that cannot be generated by a human observer. Objective.--To discuss common visual and cognitive traps known in traditional pathology-based scoring paradigms that may impact characterization of tIA-assisted scoring accuracy, sensitivity, and specificity. Data Sources.--This manuscript reviews the current literature from the past decades available for traditional subjective pathology scoring paradigms and known cognitive and visual traps relevant to these scoring paradigms. Conclusions.--Awareness of the gold standard paradox is necessary when using traditional pathologist scores to analytically validate a tIA tool because image analysis is used specifically to overcome known sources of bias in visual assessment of tissue sections. (Arch Pathol Lab Med. 2017; 141:1267-1275; doi: 10.5858/arpa.2016-0386-RA), Anatomic pathology, as a discipline, seeks to render a precise and complete diagnosis of lesions produced by a particular disease, to the correct patient, in a timely fashion, and in [...]