Carina Strell

Extravasation of leukocytes in comparison to tumor cells

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.

Neutrophil granulocytes promote the migratory activity of MDA-MB-468 human breast carcinoma cells via ICAM-1

Tumor infiltrating neutrophil granulocytes do not only exhibit tumor eliminating functions but also promote tumor progression. We have recently shown that neutrophil granulocytes can serve as linking cells for the adhesion of MDA-MB-468 breast carcinoma cells to pulmonary endothelium. Neutrophil granulocytes but not MDA-MB-468 cells express beta(2)-integrins, the ligands of the intercellular adhesion molecule (ICAM)-1, whereas ICAM-1 is strongly expressed on MDA-MB-468 cells. Consequently, the herein presented study was performed to investigate if this interaction has also an influence on the migratory activity of the tumor cells and whether ICAM-1 signaling plays a role in this process, too. We found that the continuous release of interleukin-8 (IL-8) and GRO-alpha by MDA-MB-468 cells increases the migratory activity of neutrophil granulocytes and attracts these cells towards the tumor cells which enables direct cell-cell interactions. These interactions in turn increase the migratory activity of the tumor cells in an ICAM-1 clustering-dependent mechanism since transfection of the tumor cells with specific siRNA against ICAM-1 abolished the effect. Moreover, ICAM-1 cross-linking on tumor cells induces the phosphorylation of focal adhesion components such as focal adhesion kinase and paxillin via src kinase as well as the activation of the p38 MAPK pathway via Rho kinase in a time-dependent manner. Our results provide evidence that ICAM-1 is coupled to intracellular signaling pathways involved in tumor cell migration. Thus, neutrophil granulocytes can act as modulators of the metastatic capability of tumor cells by ligation of ICAM-1.

Distinct Effects of Ligand-Induced PDGFR alpha and PDGFR beta Signaling in the Human Rhabdomyosarcoma Tumor Cell and Stroma Cell Compartments

Platelet-derived growth factor receptors (PDGFR) α and β 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 PDGFRβ ligand. By immunohistochemistry, PDGF-CC, PDGF-DD, and PDGFRα were found in tumor cells, whereas PDGFRβ was primarily detected in vascular stroma. These results are concordant with the biologic processes and pathways identified by data mining. While PDGF-CC/PDGFRα signaling associated with genes involved in the reactivation of developmental programs, PDGF-DD/PDGFRβ signaling related to wound healing and leukocyte differentiation. Clinicopathologic correlations further identified associations between PDGFRβ 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-3α and GSK-3β. 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.

Surface molecules regulating rolling and adhesion to endothelium of neutrophil granulocytes and MDA-MB-468 breast carcinoma cells and their interaction

Abstract.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 β2-integrin. Furthermore, neutrophil granulocytes serve as linking cells for the interaction of the MDA-MB-468 cells with the endothelium, which are both β2-integrin negative, but express the β2-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.

Divergent effects of norepinephrine, dopamine and substance P on the activation, differentiation and effector functions of human cytotoxic T lymphocytes

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.

The inhibitory effect of norepinephrine on the migration of ES-2 ovarian carcinoma cells involves a Rap1-dependent pathway

Our previous studies have shown that norepinephrine induces the migratory activity of human PC-3 prostate, SW 480 colon and MDA-MB-468 breast carcinoma cells. In contrast to these results, we show here that human ES-2 ovarian carcinoma cells have a reduced migratory activity after norepinephrine treatment. This inhibitory effect is possibly mediated by a cAMP-dependent activation of the small GTPase Rap1 via Epac. Furthermore, a key signalling event of the promigratory effect of norepinephrine in the above mentioned carcinoma cells is the activation of phospholipase C enzymes. In ES-2 cells, this part of the signalling cascade is constitutively active.

Norepinephrine Promotes the β1-Integrin–Mediated Adhesion of MDA-MB-231 Cells to Vascular Endothelium by the Induction of a GROα Release

The migratory activity of tumor cells and their ability to extravasate from the blood stream through the vascular endothelium are important steps within the metastasis cascade. We have shown previously that norepinephrine is a potent inducer of the migration of MDA-MB-468 human breast carcinoma cells and therefore investigated herein, whether the interaction of these cells as well as MDA-MB-231 and MDA-MB-435S human breast carcinoma cells with the vascular endothelium is affected by this neurotransmitter as well. By means of a flow-through assay under physiologic flow conditions, we show that norepinephrine induces an increase of the adhesion of the MDA-MB-231 cells, but not of MDA-MB-468 and MDA-MB-435S cells to human pulmonary microvascular endothelial cells (HMVEC). The adhesion of MDA-MB-231 cells was based on a norepinephrine-mediated release of GROα from HMVECs. GROα caused a β1-integrin–mediated increase of the adhesion of MDA-MB-231 cells. Most interestingly, this effect of norepinephrine, similar to the aforementioned induction of migration in MDA-MB-468 cells, was mediated by β-adrenergic receptors and therefore abrogated by β-blockers. In conclusion, norepinephrine has cell line–specific effects with regard to certain steps of the metastasis cascade, which are conjointly inhibited by clinically established β-blockers. Therefore, these results may deliver a molecular explanation for our recently published retrospective data analysis of patients with breast cancer which shows that β-blockers significantly reduce the development of metastases. Mol Cancer Res; 10(2); 197–207. ©2011 AACR.

The selective role of myosin VI in lymphoid leukemia cell migration

Several myosin isotypes are discussed to be involved in the migration of various cells ranging from tumor cells to leukocytes. We investigated the involvement of myosins II and VI in the lymphoid leukemia cells lines Jurkat, NB4, Dohh-2, and Molt-4 by a three-dimensional, collagen-based migration assay. Down-regulation of myosin VI by siRNA significantly reduced the migratory activity of all cells, whereas the pharmacological inhibition of non-muscle myosin II using blebbistatin had only marginal influence. Therefore, in contrast to differentiated leukocytes and cells from solid tumors, myosin VI plays a crucial role in the migration of leukemic cells.

Pancreatic stellate cells increase cancer cell expression of HMGA2 in a 3D co-culture model of pancreatic cancer

PDAC cells and PSCs were cultured in 3D, as monoand co-cultures, in a newly developed model. Gene expression (mRNA) for cancerand stellate cells within the co-culture model was investigated with real time PCR. This was done both for whole spheroids as well as for monoand co-cultured spheroids sorted by Fluorescence Activated Cell Sorting (FACS), in order to look at cell type specific expression. Protein expression was determined by immunohistochemistry (IHC). HMGA2 mRNA was also found to be higher specifically in the co-cultured vs mono-cultured Panc1 cells, in cells sorted by FACS (Fig.3.A). Further, TGFβ1-treatment of Panc1 mono-culture spheroids increased the expression of HMGA2 (Fig. 3.B). Finally, we also compared TGFβ1 and HMGA2 basal expression levels in a panel of normal pancreatic and PDAC cell lines (Fig.3C). We then identified a correlation between levels of TGFβ1 and HMGA2 in cell lines which were wild type for Smad4, a TGFβ1 downstream signaling molecule.

Abstract 836: Effect of voluntary running on metastasis in a mouse model of breast cancer

Introduction: Emerging evidence states that regular physical exercise provides a risk reduction for breast cancer by approximately 25%. In addition, recent studies suggest that physical activity may also have an effect on recurrence and mortality. Mouse models are suitable tools to study anti-neoplastic mechanisms. Aim: to evaluate the effect of voluntary running on tumor initiation, growth and metastasis in the Polyoma Middle T (PyMT) model of breast cancer. Methods: PyMT mice were housed with access either to wirelessly recording running wheels or locked control wheels. Running distances and tumor volumes were recorded. At 12 weeks, mice were sacrificed. Mammary glands were histologically staged and pulmonary metastases were quantified. In a follow up study, pre-trained mice were injected intravenously with tumor cells derived from the PyMT model and after an additional 10 weeks of voluntary running, pulmonary metastases were quantified. Results: PyMT mice ran significantly more than wildtype mice (6.4 vs 3.4 km/day). No significant effects of voluntary running on tumor- initiation, volume or stage were found. However, a trend for reduced metastasis was observed. After intravenous injections of tumor cells, runners had a significantly lower pulmonary metastasis frequency than non-runners. Conclusion: In this aggressive breast cancer model, an average of 6 km/day of voluntary running did not induce any effect on tumor formation or growth. However, the findings suggest that physical activity has impact on the metastatic process. Citation Format: Sara Mijwel, Helene Rundqvist, Carolin Lindholm, Carina Strell, Pernilla Roswall, Kristian Pietras, Randall S. Johnson, Arne Ostman. Effect of voluntary running on metastasis in a mouse model of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 836.