Kurt S. Zänker

Cell migration strategies in 3-D extracellular matrix: Differences in morphology, cell matrix interactions, and integrin function

Cell migration in extracellular matrix is a complex process of adhesion and deadhesion events combined with cellular strategies to overcome the biophysical resistance imposed by three‐dimensionally interconnected matrix ligands. Using a 3‐D collagen matrix migration model in combination with computer‐assisted cell tracking for reconstruction of migration paths and confocal microscopy, we investigated molecular principles governing cell‐matrix interactions and migration of different cell types. Highly invasive MV3 melanoma cells and fibroblasts are large and highly polarized cells migrating at low speed (0.1–0.5 μm/min) and at high directional persistence. MV3 melanoma cells utilize adhesive migration strategies as characterized by high β1 integrin surface expression, β1 integrin clustering at interactions with matrix fibers, and β1 integrin‐mediated adhesion for force generation and migration. In contrast, T lymphocytes and dendritic cells are highly mobile cells of lower β1 integrin expression migrating at 10‐ to 40‐fold higher velocities, and directionally unpredictable path profiles. This migration occurs in the absence of focal adhesions and largely independent of β1 integrin‐mediated adhesion. Whereas cell‐matrix interactions of migrating tumor cells result in traction and reorientation of collagen fibers, partial matrix degradation, and pore formation, leukocytes form transient and short‐lived interactions with the collagen lacking structural proteolysis and matrix remodeling. In conclusion, the 3‐D extracellular matrix provides a spatially complex and biomechanically demanding substrate for cell migration, thereby differing from cell migration across planar ligands. Highly adhesive and integrin‐dependent migration strategies detected in morphologically large and slowly migrating cells may result in reorganization of the extracellular matrix, whereas leukocytes favor largely integrin‐independent, rapid, and flexible migration strategies lacking typical focal adhesions and structural matrix remodeling. Microsc. Res. Tech. 43:369–378, 1998.

Beta-Blocker Drug Therapy Reduces Secondary Cancer Formation in Breast Cancer and Improves Cancer Specific Survival

Laboratory models show that the beta-blocker, propranolol, can inhibit norepinephrine-induced breast cancer cell migration. We hypothesised that breast cancer patients receiving beta-blockers for hypertension would show reduced metastasis and improved clinical outcome. Three patient subgroups were identified from the medical records of 466 consecutive female patients (median age 57, range 28-71) with operable breast cancer and follow-up (>10 years). Two subgroups comprised 43 and 49 hypertensive patients treated with beta-blockers or other antihypertensives respectively, prior to cancer diagnosis. 374 patients formed a non-hypertensive control group. Metastasis development, disease free interval, tumour recurrence and hazards risk were statistically compared between groups. Kaplan-Meier plots were used to model survival and DM. Beta-blocker treated patients showed a significant reduction in metastasis development (p=0.026), tumour recurrence (p=0.001), and longer disease free interval (p=0.01). In addition, there was a 57% reduced risk of metastasis (Hazards ratio=0.430; 95% CI=0.200-0.926, p=0.031), and a 71% reduction in breast cancer mortality after 10 years (Hazards ratio=0.291; 95% CI=0.119-0.715, p=0.007). This proof-of-principle study showed beta-blocker therapy significantly reduces distant metastases, cancer recurrence, and cancer-specific mortality in breast cancer patients suggesting a novel role for beta-blocker therapy. A larger epidemiological study leading to randomised clinical trials is needed for breast and other cancer types including colon, prostate and ovary.

HER2-Positive Circulating Tumor Cells Indicate Poor Clinical Outcome in Stage I to III Breast Cancer Patients

Purpose: Early metastasis in node-negative breast cancer indicates that breast cancer cells obviously can bypass the lymph nodes and disseminate directly hematogenous to distant organs. For this purpose, we evaluated the prognostic value of blood-borne, HER2-positive circulating tumor cells (CTC) in the peripheral blood from 42 breast cancer patients with a median follow-up of 95 months. Experimental Design: Cells were isolated by the patented combined buoyant density gradient and immunomagnetic separation procedure and analyzed by immunocytochemistry. Results: We detected one to eight CTCs in the peripheral blood of 17 of 35 patients (48.6%) presenting no overt metastasis. As a positive control, 7 of 7 (100%) patients with metastatic disease presented positive. Healthy persons and patients (n = 32) operated for nonmalignant diseases presented negative for CTCs. The presence and frequency of HER2-positive CTCs correlated with a significantly decreased disease-free survival (P < 0.005) and overall survival (P < 0.05). Interestingly, in 12 patients with HER2-positive CTCs, the primary tumor was negative for HER2 as assessed by immunohistochemical score and fluorescence in situ hybridization. Conclusions: This study provides some evidence of a prognostic effect of HER2-positive CTCs in stage I to III breast cancer. Future studies have to determine the outcome of patients treated with HER2-targeting therapies with respect to HER2-positive CTC levels because it is not unlikely that high levels of HER2-positive CTCs reflect the activity of the tumor and may predict response to trastuzumab.

CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize β1 integrin-independent strategies for polarization, interaction with collagen fibers and locomotion

Cell migration may depend on integrin‐mediated adhesion to and deadhesion from extracellular matrix ligands. This concept, however, has not yet been confirmed for T lymphocytes migrating in three‐dimensional extracellular matrices. We investigated receptor involvement in T cell migration combining a three‐dimensional collagen matrix model with time‐lapse videomicroscopy, computer‐assisted cell tracking and confocal microscopy. In collagen lattices, the migration of CD4+ T cells (1) involved interactions with collagen fibers at the leading edge and uropod likewise, (2) occurred independently of the co‐clustering of β1, β2, or β3 integrins with F‐actin, focal adhesion kinase, and phosphotyrosine at interactions with collagen fibers, (3) was counteracted by high‐affinity β1 integrin binding induced by antibody TS2/16; however, (4) the migration could not be blocked by a combination of adhesion‐perturbing anti‐β1, ‐β2, ‐β3, and αv integrin antibodies. Integrin blocking neither affected cell polarization, interaction with fibers, β1 integrin distribution, migration velocity, path structure, nor the number of locomoting cells in spontaneously migrating or concanavalin A‐activated cells. Hence, T lymphocytes migrating in three‐dimensional collagen matrices may utilize highly transient interactions with collagen fibers of low adhesivity, thereby differing from focal adhesion‐dependent migration strategies employed by other cells.

S100 Family Members and Trypsinogens Are Predictors of Distant Metastasis and Survival in Early-Stage Non-Small Cell Lung Cancer

Distant metastasis is the predominant cause of death in early-stage non-small cell lung cancer (NSCLC). Currently, it is impossible to predict the occurrence of metastasis at early stages and thereby separate patients who could be cured by surgical resection alone from patients who would benefit from additional chemotherapy. In this study, we applied a comparative microarray approach to identify gene expression differences between early-stage NSCLC patients whose cancer ultimately did or did not metastasize during the course of their disease. Transcriptional profiling of 82 microarrays from two patient groups revealed differential expression of several gene families including known predictors of metastasis (e.g., matrix metalloproteinases). In addition, we found S100P, S100A2, trypsinogen C (TRY6), and trypsinogen IVb (PRSS3) to be overexpressed in tumors that metastasized during the course of the disease. In a third group of 42 patients, we confirmed the induction of S100 proteins and trypsinogens in metastasizing tumors and its significant correlation with survival by real-time quantitative reverse transcription-PCR. Overexpression of S100A2, S100P, or PRSS3 in NSCLC cell cultures led to increased transendothelial migration, corroborating the role of S100A2, S100P, and PRSS3 in the metastatic process. Taken together, we provide evidence that expression of S100 proteins and trypsinogens is associated with metastasis and predicts survival in early stages of NSCLC. For the first time, this implicates a role of S100 proteins and trypsinogens in the metastatic process of early-stage NSCLC.

MCP-1 induces migration of adult neural stem cells

As a model for brain inflammation we previously studied transcriptional profiles of tumor necrosis factor-alpha (TNF)treated U373 astroglioma cells. In previous work we were able to demonstrate that the chemokine monocyte chemoattractant protein-1 (MCP-1, SCYA2, CCL2, MCAF) expression in U373 cells was inducible by TNF-alpha treatment. Demonstrably MCP-1 mRNA and protein expression in U373 cells was sustainable over time and at the highest level of all genes analyzed (Schwamborn et al., BMC Genomics 4, 46, 2003). In the hematopoietic system MCP-1 is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. In search of further functions in brain inflammation we tested the hypothesis that MCP-1 acts as a chemokine on neural stem cells. Here we report that MCP-1 activates the migration capacity of rat-derived neural stem cells. The migration of stem cells in a Boyden chamber analysis was elevated after stimulation with MCP-1. Time-lapse video microscopy visualized the migration of single stem cells from neurospheres in MCP-1-treated cultures, whereas untreated cultures depicted no migration at all, but showed signs of sprouting. Expression of the MCP-1 receptor CCR2 in neurosphere cultures was verified by RT-PCR and immunofluorescence microscopy. Supernatants from TNF-treated U373 cells also induced migration of neural stem cells.

Induction of cancer cell migration by epidermal growth factor is initiated by specific phosphorylation of tyrosine 1248 of c-erbB-2 receptor via EGFR

Induction of tumor cell migration is a key step in invasion and metastasis. Here we report that the epidermal growth factor (EGF)‐induced cell migration of breast cancer cells is attributed to a transient, rather than a sustained, activation of phospholipase C (PLC) ‐γ1 due to c‐erbB‐2 signaling. EGF stimulation of EGF receptor (EGFR) overexpressing cells resulted in long‐term PLC‐γ1 tyrosine phosphorylation and sustained levels of inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG) producing sinusoidal calcium oscillations. In contrast, c‐erbB‐2/EGFR expressing cells displayed baseline transient calcium oscillations after EGF treatment due to short‐term PLC‐γ1 tyrosine phosphorylation and short‐term IP3 and DAG turnover. A third cell line expressing a point‐mutated c‐erbB‐2 receptor that lacks the autophosphorylation Y1248 was generated to investigate whether the different PLC‐γ1 activation was attributed to this structure. Neither PLC‐γ1 tyrosine phosphorylation nor IP3 and DAG turnover and calcium oscillations were observed in this cell line, indicating the modulation of the PLC‐γ1 activation time course by c‐erbB‐2 signaling. Induction of cell migration was solely observable in the c‐erbB‐2‐positive cell line as proved by the mode of actin reorganization and a cell migration assay, using a 3D‐ collagen lattice. In summary, c‐erbB‐2 up‐regulation switches on the cell migration program by modulating the time course of PLC‐γ1 activation.

Adhesion molecules and chemokines: the navigation system for circulating tumor (stem) cells to metastasize in an organ-specific manner.

To date, cancer is still the second most prevalent cause of death after cardiovascular diseases in the industrialized word, whereby the primary cause of cancer is not attributed to primary tumor formation, but rather to the growth of metastases at distant organ sites. For several years it was considered that the well-known phenomenon of organ-specific spreading of tumor cells is mostly a mechanical process either directed passively due to size constraints (mechanical trapping theory) or due to a fertile environment provided by the organ in which tumor cells can proliferate (seed and soil hypothesis). Both mechanisms strongly depend on the adhesive properties of tumor cells either to endothelial cells and/or cancer cells, which are facilitated by a variety of cell adhesion molecules including carbohydrates and integrins. Within the past years it became evident that the organ-specific metastatic spreading of tumor cells does not only rely on heterotypic and homotypic adhesive interactions, but also on the interplay of chemokines and their appropriate receptors. Moreover, the identification of cancer stem cells in various tumor tissues has opened new questions. Cancer stem cells possess self-renewal, differentiation, and tumor-initiating capacities. Thus these cells are ideal candidates to be the seed of a secondary tumor. In the present review we will give a brief overview about the complex process of organ-specific metastasis formation depending on the interplay of adhesion molecules, chemokines, and the putative role of cancer stem cells in metastasis formation.

An immunological enrichment method for epithelial cells from peripheral blood

The ability of primary tumours to metastasize accounts for the majority of cancer deaths. The emergence of circulating carcinoma cells in the peripheral blood is supposed to be an indicator for cancer cell spread. We have focused on this phenomenon in order to develop a sensitive technique for enriching epithelial derived cells on the basis of a two-layer density gradient and subsequent immune magnetic cell sorting. Epithelial cells are possess a cytoskeleton containing an assembly of intermediate filaments. During carcinogenesis these filaments do not undergo modifications of antibody binding epitopes such as occur in the protein domains of surface markers. We have developed a two-layer density gradient in which the epithelial cells form a single density band. This was demonstrated by recovery experiments using [3H]thymidine-labelled epithelial cells which showed epithelial cells were enriched within this first step by a factor of 20. In a second step the MACS system was applied. Cells were stained with a performed FITC-conjugated mouse anti-human cytokeratin antibody bound to a rat anti-mouse antibody coupled to superparamagnetic particles (immune paramagnetic separation complex; IPSC) and subjected to high gradient magnetic fields. The two-step procedure was confirmed by dispersing 50 epithelial cells in 5 x 10(5), 5 x 10(6), 5 x 10(7), 5 x 10(8), 5 x 10(9) peripheral blood leucocytes. Specific binding of the preformed IPSC was demonstrated by flow cytometry, confocal laser, fluorescent and electron microscopy. The specificity of the method was further proved by dual staining with IPSC and anti-human PSA antibody of epithelial prostatic cells separated from peripheral blood in vitro. By means of this double-step separation method it was possible to isolate up to 15-20 cells out of 50 epithelial cells originally suspended into 5 x 10(7) to 5 x 10(9) human peripheral blood leucocytes. This represented an enrichment factor between 20,000 and 200,000, depending on the initial cell number. The immunologically captured epithelial cells can be used for further cytogenetic investigations such as in situ hybridization (ISH) and/or polymerase chain reaction (PCR) to detect cancer cell specific gene aberrations. This sensitive combined buoyant density immune magnetic cell separation technique is capable of detecting free carcinoma cells in the peripheral blood.

ISOLATION OF BLOOD-BORNE EPITHELIUM-DERIVED c-erbB-2 ONCOPROTEIN-POSITIVE CLUSTERED CELLS FROM THE PERIPHERAL BLOOD OF BREAST CANCER PATIENTS

Clinical studies including thousands of breast cancer patients have shown that c‐erbB‐2 is amplified and overexpressed in 20–30% of invasive human breast cancers and that it is associated with distant metastasis in specified patient subgroups. To isolate and characterize hematogeneously spreading c‐erbB‐2‐positive epithelium‐derived cells from the peripheral blood of breast cancer patients, a combined buoyant density gradient and immuno‐magnetic separation method has been used. The method utilizes a biotinylated anti‐cytokeratin monoclonal antibody (MAb) for capturing the epithelium‐derived cells. The expression of c‐erbB‐2 by the captured cells was detected using an anti‐c‐erbB‐2 rabbit antibody (21N) coupled to an anti‐rabbit gold‐labeled antibody, whereby immunoenzymatic cytokeratin staining was performed using a silver‐enhanced immunogold double staining protocol. In total, 29 of the 46 patients tested had either cytokeratin (24/29) or cytokeratin/c‐erbB‐2 (19/29) positive clustered cells in their peripheral blood. We thus report here the presence and the frequency of clone‐specifically stained clustered cells in the peripheral blood of breast cancer patients. The frequency of cytokeratin/c‐erbB‐2 double‐positive clustered cells in the peripheral blood was on average 10 times higher than that of double‐positive single cells. The numbers of cytokeratin/c‐erbB‐2 double‐positive clustered cells were positively correlated with the stage of tumors. Results of in vitro motility experiments using single and clustered cells from primary breast cancer tissue strongly support the assumption that cytokeratin/c‐erbB‐2 double‐positive clustered cells have a high potential for locomotion. We suggest that blood‐borne epithelium‐derived c‐erbB‐2‐positive clustered cells are the possible precursor cells responsible for the formation of distant metastases and bone marrow micrometastases. Int. J. Cancer 76:824–828, 1998.© 1998 Wiley‐Liss, Inc.