The multi-step process of the emigration of cells from the blood stream through the vascular endothelium into the tissue has been termed extravasation. The extravasation of leukocytes is fairly well characterized down to the molecular level, and has been reviewed in several aspects. Comparatively little is known about the extravasation of tumor cells, which is part of the hematogenic metastasis formation. Although the steps of the process are basically the same in leukocytes and tumor cells, i.e. rolling, adhesion, transmigration (diapedesis), the molecules that are involved are different. A further important difference is that leukocyte interaction with the endothelium changes the endothelial integrity only temporarily, whereas tumor cell interaction leads to an irreversible damage of the endothelial architecture. Moreover, tumor cells utilize leukocytes for their extravasation as linkers to the endothelium. Thus, metastasis formation is indirectly susceptible to localization signals that are literally specific for the immune system. We herein compare the extravasation of leukocytes and tumor cells with regard to the involved receptors and the localization signals that direct the cells to certain organs and sites of the body.

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Background A better definition of biomarkers and biological processes related to local recurrence and disease progression is highly warranted for ductal breast carcinoma in situ (DCIS). Stromal–epithelial interactions are likely of major importance for the biological, clinical, and pathological distinctions between high- and low-risk DCIS cases. Methods Stromal platelet derived growth factor receptor (PDGFR) was immunohistochemically assessed in two DCIS patient cohorts (n = 458 and n = 80). Cox proportional hazards models were used to calculate the hazard ratios of recurrence. The molecular mechanisms regulating stromal PDGFR expression were investigated in experimental in vitro co-culture systems of DCIS cells and fibroblasts and analyzed using immunoblot and quantitative real-time PCR. Knock-out of JAG1 in DCIS cells and NOTCH2 in fibroblasts was obtained through CRISPR/Cas9. Experimental data were validated by mammary fat pad injection of DCIS and DCIS-JAG1 knock-out cells (10 mice per group). All statistical tests were two-sided. Results PDGFRα(low)/PDGFRβ(high) fibroblasts were associated with increased risk for recurrence in DCIS (univariate hazard ratio = 1.59, 95% confidence interval [CI] = 1.02 to 2.46; P = .04 Wald test; multivariable hazard ratio = 1.78, 95% CI = 1.07 to 2.97; P = .03). Tissue culture and mouse model studies indicated that this fibroblast phenotype is induced by DCIS cells in a cell contact-dependent manner. Epithelial Jagged1 and fibroblast Notch2 were identified through loss-of-function studies as key juxtacrine signaling components driving the formation of the poor prognosis-associated fibroblast phenotype. Conclusions A PDGFRα(low)/PDGFRβ(high) fibroblast subset was identified as a marker for high-risk DCIS. The Jagged-1/Notch2/PDGFR stroma–epithelial pathway was described as a novel signaling mechanism regulating this poor prognosis-associated fibroblast subset. In general terms, the study highlights epithelial–stromal crosstalk in DCIS and contributes to ongoing efforts to define clinically relevant fibroblast subsets and their etiology.

Platelet-derived growth factor receptors (PDGFR) a and b have been suggested as potential targets for treatment of rhabdomyosarcoma, the most common soft tissue sarcoma in children. This study identifies biologic activities linked to PDGF signaling in rhabdomyosarcoma models and human sample collections. Analysis of gene expression profiles of 101 primary human rhabdomyosarcomas revealed elevated PDGF-C and -D expression in all subtypes, with PDGF-D as the solely overexpressed PDGFRb ligand. By immunohistochemistry, PDGF-CC, PDGF-DD, and PDGFRa were found in tumor cells, whereas PDGFRb was primarily detected in vascular stroma. These results are concordant with the biologic processes and pathways identified by data mining. While PDGF-CC/ PDGFRa signaling associated with genes involved in the reactivation of developmental programs, PDGF-DD/ PDGFRb signaling related to wound healing and leukocyte differentiation. Clinicopathologic correlations further identified associations between PDGFRb in vascular stroma and the alveolar subtype and with presence of metastases. Functional validation of our findings was carried out in molecularly distinct model systems, where therapeutic targeting reduced tumor burden in a PDGFR-dependent manner with effects on cell proliferation, vessel density, and macrophage infiltration. The PDGFR-selective inhibitor CP-673,451 regulated cell proliferation through mechanisms involving reduced phosphorylation of GSK-3a and GSK-3b. Additional tissue culture studies showed a PDGFR-dependent regulation of rhabdosphere formation/cancer cell stemness, differentiation, senescence, and apoptosis. In summary, the study shows a clinically relevant distinction in PDGF signaling in human rhabdomyosarcoma and also suggests continued exploration of the influence of stromal PDGFRs on sarcoma progression. Cancer Res; 73(7);

Single-cell transcriptomics provides us with completely new insights into the molecular diversity of different cell types and the different states they can adopt. The technique generates inventories of cells that constitute the building blocks of multicellular organisms. However, since the method requires isolation of discrete cells, information about the original location within tissue is lost. Therefore, it is not possible to draw detailed cellular maps of tissue architecture and their positioning in relation to other cells. In order to better understand the cellular and tissue function of multicellular organisms, we need to map the cells within their physiological, morphological, and anatomical context and space. In this review, we will summarize and compare the different methods of in situ RNA analysis and the most recent developments leading to more comprehensive and highly multiplexed spatially resolved transcriptomic approaches. We will discuss their highlights and advantages as well as their limitations and challenges and give an outlook on promising future applications and directions both within basic research as well as clinical integration.

The extravasation of leukocytes and tumor cells is a multi-step process with the involvement of various adhesion molecules depending on the three steps rolling, adhesion, and diapedesis. We have developed an in vitro model, by which we investigated the rolling and adhesion of neutrophil granulocytes and MDA-MB-468 human breast carcinoma cells to lung endothelial cells under physiological flow-conditions. We found that norepinephrine had an inhibitory function on the fMLP-promoted adhesion of neutrophil granulocytes due to a down-regulation of beta2-integrin. Furthermore, neutrophil granulocytes serve as linking cells for the interaction of the MDA-MB-468 cells with the endothelium, which are both beta2-integrin negative, but express the beta2-integrin ligand ICAM-1. In addition, we show here that N-cadherin is up-regulated on the endothelial cells and on neutrophil granulocytes in response to fMLP. This up-regulation resulted in a significant increase of adherent MDA-MB-468 cells, which are also N-cadherin positive.

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BackgroundNeurotransmitters are important regulators of the immune system, with very distinct and varying effects on different leukocyte subsets. So far little is known about the impact of signals mediated by neurotransmitters on the function of CD8+ T lymphocytes. Therefore, we investigated the influence of norepinephrine, dopamine and substance P on the key tasks of CD8+ T lymphocytes: activation, migration, extravasation and cytotoxicity.ResultsThe activation of naïve CD8+ T lymphocytes by CD3/CD28 cross-linking was inhibited by norepinephrine and dopamine, which was caused by a downregulation of interleukin (IL)-2 expression via Erk1/2 and NF-κB inhibition. Furthermore, all of the investigated neurotransmitters increased the spontaneous migratory activity of naïve CD8+ T lymphocytes with dopamine being the strongest inducer. In contrast, activated CD8+ T lymphocytes showed a reduced migratory activity in the presence of norepinephrine and substance P. With regard to extravasation we found norepinephrine to induce adhesion of activated CD8+ T cells: norepinephrine increased the interleukin-8 release from endothelium, which in turn had effect on the activated CXCR1+ CD8+ T cells. At last, release of cytotoxic granules from activated cells in response to CD3 cross-linking was not influenced by any of the investigated neurotransmitters, as we have analyzed by measuring the β-hexosamidase release.ConclusionNeurotransmitters are specific modulators of CD8+ T lymphocytes not by inducing any new functions, but by fine-tuning their key tasks. The effect can be either stimulatory or suppressive depending on the activation status of the cells.