Single cell adhesion measuring apparatus (SCAMA): application to cancer cell lines of different metastatic potential and voltage-gated Na<Superscript>+</Superscript> channel expression

We have developed a simple yet effective apparatus, based upon negative pressure directed to the tip of a micro-pipette, to measure the adhesiveness of single cells. The “single cell adhesion measuring apparatus” (SCAMA) could differentiate between the adhesion of strongly versus weakly metastatic cancer cells as well as normal cells. Adhesion was quantified as “detachment negative pressure” (DNP) or “DNP relative to cell size” (DNPR) where a noticeable difference in cell size was apparent. Thus, for rat and human prostate and human breast cancer cell lines, adhesiveness (DNPR values) decreased in line with increased metastatic potential. Using the SCAMA, we investigated the effect of tetrodotoxin (TTX), a specific blocker of voltage-gated Na⁺ channels (VGSCs), on the adhesion of rat and human prostate cancer cell lines of markedly different metastatic potential. Following pretreatment with TTX (48 h with 1 μM), the adhesion values for the Mat-LyLu cells increased significantly 4.3-fold; there was no effect on the AT-2 cells. For the strongly metastatic PC-3M cells, TTX treatment caused a significant (∼30%) increase in adhesion. The adhesion of PNT2-C2 (“normal”) cells was not affected by the TTX pretreatment. The TTX-induced increase in the adhesiveness of the strongly metastatic cells was consistent with the functional VGSC expression in these cells and the proposed role of VGSC activity in metastatic cell behaviour. In conclusion, the SCAMA, which can be constructed easily and cheaply, offers a simple and effective method to characterise single-cell adhesion and its modulation.     

Preniche as missing link of the metastatic niche concept explaining organ-preferential metastasis of malignant tumors and the type of metastatic disease

Here we attempt to supplement the metastatic niche concept with a stage of “preniche” that determines the site of development of a premetastatic niche and of a subsequent metastasis. The “preniche” includes all cellular and molecular events in the site of a prospective metastasis preceding the entrance of myeloid progenitor cells. The “preniche” integrates an activation of vascular endothelium of the microcirculatory vessels of target organs in the site of a future metastasis under conditions of chronic persistent productive inflammation that can be induced by cytokines from the primary tumor and independently of it. The endothelium activation is responsible for adhesion and clustering of the recruited myeloid progenitor cells and also for the retention of cells of malignant tumors. The preniche easily arises in organs enriched with organspecific macrophages (lungs, liver, brain, etc.) where the endothelium is predisposed for intensive recruiting of myeloid progenitor cells of macrophages, especially under conditions of inflammation. Introduction of the “preniche” concept allows us to avoid difficulties associated with the development of the metastatic niche concept, especially concerning the problem of organ-preferential localization of metastases, and to make some predictions for experimental verification and potential approaches for preventing metastasizing in some oncologic patients.     

Identification of cancer stem cells-associated “side population” by flow cytometry with a violet laser

The possibility of identification of a “side population” of cancer stem cells in solid tumors by a flow cytometer equipped with a 405 nm violet laser has been investigated. Ovarian cancer (Skov-3) and colorectal cancer (Colo 320) cell lines formed the “side population” after vital staining with Hoechst 33342. Analysis of cells isolated from the tumor tissue of malignant melanoma and colorectal cancer, also revealed the “side population” characterized by increased fluorescent dye exclusion. The percentage of melanoma cells included in the “side population” was the same as that of cells co-expressing the cancer stem cells markers CD34 and CD44. However, the colon cancer “side population” was significantly smaller than the minor populations of colon cancer cells identified by either CD133 expression or exclusion of Rhodamine 123 exclusion.     

Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed “glia-like” cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that “glia-like” cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5–10 days approximately 5–10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3–6 weeks in culture. At confluence the cultures contained 2–5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95–98% GFAP-negative “glia-like” cells. This population of flat “glia-like” cells was positively stained for vimentin, fibronectin, and 20–30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat “glia-like” cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that “glia-like” cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.     

Effect of hepatocyte growth factor/scatter factor and other growth factors on motility and morphology of non-tumorigenic and tumor cells

Summary Using an automated cell analyzer system, the effect of hepatocyte growth factor/scatter factor (HGF/SF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), endothelial acidic fibroblast growth factor (a-FGF), platelet derived growth factor (PDGF), and recombinant human insulinlike growth factor (IGF) on the motility and morphology of Madin-Darby canine kidney (MDCK), rat hepatomas, C₂, and H_5–6 and murine mammary carcinoma (EMT-6) cells was investigated. Treatment of MDCK cells with HGF/SF, bFGF, EGF, and a-FGF resulted in an increase in average cell velocity and in the fraction of moving cells. Cells treated with the PDGF and IGF did not show significant alterations in velocity. MDCK cells treated with each growth factor were classified into groups of “fast” and “slow” moving cells based on their average velocities, and the average morphologic features of the two groups were quantitated. Fast-moving cells had larger average area, circularity, and flatness as compared to slow-moving cells. Factors that stimulated cell movement also induced alterations in cell morphologic parameters including spreading, flatness, area, and circularity. HGF/SF also scattered and stimulated motility of C₂ and H_5–6 hepatoma cells. In contrast to MDCK cells, there was no significant difference between the morphology of the fast moving and slow moving C₂ and H_5–6 cells. These studies suggest that growth factor cytokines have specific effects on motility of normal and tumor cells.     

Growth advantage (“clonal dominance”) of metastatically competent tumor cell variants expressed under selective two- or three-dimensional tissue culture conditions

Summary In previous experiments it was shown that injection into syngeneic CBA/J mice of cell mixtures containing an excess of non-metastatic SP1 mouse mammary carcinoma cells with aras transfected metastatic variant of SP1 called C1, always resulted in the eventual dominance of the C1 subpopulation at the site of inoculation. This occurred despite the growth rates of the two cell populations being identical in vivo when grown separately. The means by which the C1 subpopulation achieved “clonal dominance” is thought to involve its responsiveness to stimulatory paracrine growth factors liberated by the non-metastatic SP1 population. The clonal dominance process, however, could not be recapitulated in conventional monolayer tissue culture conditions in which SP1 and C1 cells were grown together in high concentrations of serum, i.e. under non-limiting culture conditions. We now show that clonal dominance of C1 cells can be observed when the cell mixture is maintained in tissue culture for extended periods, or when the cells are grown under selective, limiting conditions, some of which may mimic growth conditions in vivo more accurately. These conditions were a) growth in low (limiting) serum concentrations; and b) growth as three-dimensional multicellular aggregates, i.e. as “tumor spheroids”. Under all of these conditions dominance of the C1 subpopulation always took place, but with an efficiency 6- to 40-fold less than generally observed in vivo. C1 cells were also able to form more stable (compact) spheroids compared to SP1 cells. Entrapment of the latter in mixed C1/SP1 spheroids increased the recovery of the SP1 cells suggesting some kind of “rescue” mechanism in which cells are protected from physical forces by three-dimensional structure. The relevance of these in vitro interactions for clonal dominance in primary tumors and metastasis in vivo are discussed.     

Stimulation of endothelial cell migration in culture by ladsin,a laminin-5-like cell adhesion protein

Summary Ladsin is a laminin-like cell-adhesive scatter factor with potent cell motility-stimulating ability and was purified from serum-free conditioned medium of a malignant human gastric adenocarcinoma cell line STKM-1. To test its possible role in tumor angiogenesis, we investigated its effect on primary culture of endothelial cells (human umbilical vein endothelial cells) and endothelial cell line ECV304 in this study. Cell adhesion and motility effects of ladsin were observed in both types of endothelial cells. In cell-attachment assay, ladsin interacted with integrin α3β1 that was expressed on the endothelial cell surface. In Boyden chambers, ladsin stimulated both directed and random migration of ECV304 cells. Ladsin induced repair of artificial wounds generated in ECV304 cell monolayers by stimulating cell migration. Ladsin did not affect the growth rate of ECV304 cells at a low cell density but significantly increased the saturation cell density. These results suggest that ladsin may be involved in the adhesion and migration of endothelial cells under some physiological and pathological conditions.     

Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue

Summary A method for the isolation and long-term culture of human microvessel endothelial cells from mammary adipose tissue (HuMMEC) obtained at breast reduction surgery has been developed. Pure cultures of HuMMEC were isolated by sequential digestion of the fat with collagenase and trypsin followed by specific selection of microvessel fragments withUlex europaeus agglutinin-1 coated magnetic beads (Dynabeads). The resulting cells formed contact-inhibited monolayers on gelatin and fibronectin substrates and capillary-like “tubes” on Matrigel; they also expressed von Willebrand factor, angiotensin-converting enzyme, and accumulated acetylated low density lipoprotein. Further immunofluorescence characterization revealed the presence of antigens for the endothelial cell specific monoclonal antibodies EN4 and H4-7/33. In addition, the origin of these cells was confirmed by the demonstration of the cell adhesion molecules, platelet endothelial cell adhesion molecule-1 (CD31), and endothelial leukocyte adhesion molecule-1 (ELAM-1/E-selectin) upon stimulation with tumor necrosis factor (TNF)α. HuMMEC were found to express-1 ELAM-1 at lower levels of TNFα (<10 ng/ml) than required by human umbilical vein endothelial cells. These cells should provide a useful in vitro model for studying various aspects of microvascular biology and pathology.     

Phosphorylation of focal adhesion kinase tyrosine 397 critically mediates gastrin-releasing peptide's morphogenic properties

We have proposed that gastrin-releasing peptide (GRP) and its receptor (GRP-R) are morphogens that when aberrantly re-expressed in colon cancer promote tumor cell differentiation and retard metastasis. Because circumstantial evidence suggested that these properties were mediated via focal adhesion kinase (FAK), the purpose of this study was to elucidate the role of GRP-induced activation of this enzyme on properties fundamental to metastasis including cell attachment, motility, and deformability. To do this, we studied 293 cells, a non-malignant epithelial cell line that we show expresses GRP and GRPR. To dissect out the role of FAK, 293 cells were modified to inducibly express the dominant negative enzyme FAK-related non-kinase (FRNK) under control of a Tet-On (i.e., doxycycline-sensitive) promoter. Under serum-free conditions, GRP acting in an autocrine manner caused FAK to be phosphorylated at Y397; and this could be completely inhibited either by incubating with the specific GRP-R antagonist D-Phe(6)(bombesin) methyl ester, or by upregulating FRNK using doxycycline. To measure cell attachment, we designed a cone-plate viscometer that recorded the shear stress required to detach cells from their underlying matrix. To assess motility, confluent cells were wounded and behavior assessed by time-lapse photography. To measure deformability, we recorded the ability of cells to be completely drawn into a micropipette <50% the size of the non-deformed cell. Control 293 cells adhered more avidly to their underlying matrix, rapidly remodeled wounded tissues without any increase in overall proliferation, and were less distensible than cells treated with antagonist or doxycycline. Thus, these findings suggest that expression of GRP/GRPR in cancer inhibits metastasis by enhancing cell attachment to the matrix, regulating motility in the context of remodeling, and decreasing deformability.     

A novel artificial substrate for cell culture: Effects of substrate flexibility/malleability on cell growth and morphology

Summary Gels of glyoxyl agarose (GA) are evaluated as a novel flexible substrate for cell culture with physical properties comparable to extracellular matrix (ECM) gels. We show here that cells adhere well to pure GA gels; in addition, specific interactions involving matrix receptors can be studied when individual matrix molecules are bound to the gel covalently. When cells are grown on such substrates, morphology is comparable to that observed on “natural” matrix gels (reconstituted gels of collagen type I or of Matrigel): rather than being flattened as in monolayer cultures on tissue culture plastic the cells assume a rounded morphology and tend to form tissue-like aggregates. The effects of the artificial matrix gels are discussed in the context of previous publications on cell interactions with the extracellular matrix, suggesting that in addition to specific recognition of matrix molecules the physical properties of ECM by themselves can be decisive for cell differentiation. We conclude that gels of glycoxyl agarose a) provide a useful model to mimic the physical properties of matrix gels without the presence of specific adhesion factors; b) may be useful as a general, non-specific ECM allowing cells to be cultured in vitro under conditions favorable for differentiation; and c) allow to design a variety of “synthetic” ECM models composed of a chemically defined gel matrix, which can be supplemented with covalently bound molecules to be recognized by cell surface receptors.     

Establishment and characterization of a new murine mammary tumor cell line,balb/c-MC

Summary A new murine mammary tumor cell line (BALB/c-MC) was established from a spontaneous mammary tumor in a 17-mo.-old female mouse of the low mammary cancer strain BALB/cHe. The cell line was derived from a papillary adenocarcinoma. In monolayer culture the line exhibits a pavementlike arrangement of cells and forms “domes” or “hemicysts” as the cells become confluent. The cell line rapidly forms tumors when transplanted into young syngeneic BALB/cHe mice. The subcutaneous injection of 10⁶ cells resulted in the development of mammary tumors (typical papillary adenocarcinomas) in 33 of 37 (87% recipients within 2 to 3 mo. after injection. These mammary tumors also metastasize to lung [14 of 33 (42%) of recipients] during this time. The number of chromosomes in this cell line is hyperdiploid (average of 43, range 39 to 44).     

Low-Cost Motility Tracking System (LOCOMOTIS) for Time-Lapse Microscopy Applications and Cell Visualisation

Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81±0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17±0.004 (MDA-MB-231 breast cancer cells), 1.24±0.006 (SC5 mouse Sertoli cells) and 2.21±0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers.     

Cell electrophoresis — a method for cell separation and research into cell surface properties

In this paper, we discuss the application of various methods of cell electrophoresis in research into cell surface properties (analytical methods), and the separation of uniform cell subpopulations from cell mixtures (preparative methods). The emphasis is on the prospects of the development of simplified and versatile methodologies, i.e. microcapillary cell electrophoresis and horizontal cell electrophoresis under near-isopycnic conditions. New perspectives are considered on the use of analytical and preparative cell electrophoresis in research on cell differentiation, neoplastic transformation, cell-cell interactions and the biology of stem cells. Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7–11, 2007, “The Cell and Its Environment”. Publication cost was covered by the organisers of this meeting.     

The Immune Response of the Tasmanian Devil (Sarcophilus harrisii) and Devil Facial Tumour Disease

One of the most remarkable aspects of Devil Facial Tumour Disease (DFTD) is its infectious nature, and for successful transmission it must avoid detection by the devil’s immune system. For this to occur, the devil either is severely immunosuppressed or factors produced by the tumor contribute to its avoidance of immune detection. An analysis of the devil’s immune system revealed the presence of normal-looking lymphoid organs and lymphoid cells. At a functional level the lymphocytes proliferated in response to mitogen stimulation. Subcutaneous injection of a cellular antigen produced a strong antibody response, providing compelling evidence that the devil has a competent immune system. Tumor cell analysis demonstrated that the tumor expresses the genes of the major histocompatibility complex; however, there was a limited diversity. Therefore, the most likely explanation for devil-to-devil transmission of DFTD is that the tumor is not recognized by the devil as “non-self” because of the limited genetic diversity. With its consistent morphology and relatively stable genome, this tumor would provide a reasonable target for a vaccine approach, provided the immune system can be coaxed into recognizing the tumor as “non-self.”     

Synchronized oscillation of the segmentation clock gene in vertebrate development

In vertebrate somitogenesis, “segmentation clock” genes (her in zebrafish, hes in mouse, and hairy in chick) show oscillation, synchronized over nearby cells through intercellular interaction. In zebrafish, neighboring cells interact by Delta-Notch signaling to realize synchronization. Under Delta-Notch, however, a cell with a high expression of the segmentation clock gene tends to suppress its expression in adjacent cells, which might produce spatial heterogeneity instead of synchronized oscillation. Here we studied the conditions under which pre-somitic mesoderm cells show synchronized oscillation of gene expression mathematically. We adopted a model that explicitly considers the kinetics of the mRNA and proteins of the segmentation clock gene and cell–cell interaction via Delta-Notch signaling. From statistical study of a model with randomly generated parameters, we revealed how the likelihood that the system generates stable synchronized oscillation depends on the rate of each reaction in the gene–protein kinetics.     

Oligomycin A induces autophagy in the IPLB-LdFB insect cell line

Functional and morphological modifications in the IPLB-LdFB insect cell line were examined following a short treatment with a reversible inhibitor of mitochondrial ATP synthase, oligomycin A, and subsequent incubation for various times in oligomycin-A-free medium. Oncosis, apoptosis and autophagy at variable percentages were observed under the various experimental conditions. Together with oncotic and apoptotic pathways that lead directly to cell death, the insect cells responded to ATP depletion with autophagy. Our results revealed that, in most cases, autophagy failed to restore cellular homeostasis, probably because of a massive sequestration of mitochondria in autophagic vacuoles. This critical event was a “point of no return” and ultimately resulted in cell necrosis. However, cells with a misshapen body and nucleus resembling “resistant forms” were observed at the end of the experiments. Our findings indicate that oligomycin-A-induced autophagy can promote cell protection or cell destruction and is an open-ended process that can lead to survival or death depending on a combination of concomitant factors.This work was supported by MIUR (Italy) grants to M.deE. and E.O. and by the Centro Grandi Attrezzature (University of Insubria, Varese, Italy).Gianluca Tettamanti and Davide Malagoli contributed equally to this work.     

Non-sufficient cell cycle control as possible clue for the resistance of human malignant glioma cells to clinically relevant treatment conditions

Summary. Objectives. Human gliomas have a catastrophic prognosis with a median survival in the range of one year even after therapeutic treatment. Relatively high resistance towards apoptotic stimuli is the characteristic feature of malignant gliomas. Since cell cycle control has been shown to be the key mechanism controlling both apoptosis and proliferation, this study focuses on DNA damage analysis and protein expression patterns of essential cell cycle regulators P53 and P21^waf1/cip1 in glioma under clinically relevant therapeutic conditions. Material and methods. U87MG cell line, characterised by wild p53-phenotype relevant for the majority of primary malignant glioblastomas, was used. Glioma cells underwent either irradiation or temozolomide treatment alone, or combined radio/chemo treatment. DNA damage was analysed by the “Comet Assay”. Expression rates of target proteins were analysed using “Western-Blot” technique. Results and conclusions. “Comet Assay” demonstrated extensive DNA damage caused by temozolomide treatment alone and in combination with irradiation, correlating well with the low survival rate observed under these treatment conditions. In contrast, irradiation alone resulted in a relatively low DNA damage, correlating well with a high survival rate and indicating a poor therapeutic efficiency of irradiation alone. Unusually low up-regulation of P53 and P21^waf1/cip1 expression patterns was produced by the hereby tested stressful conditions. A deficit in cell cycle control might be the clue to the high resistance of malignant glioma cells to established therapeutic approaches.     

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca²⁺ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell–endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell–ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell–cell contact region, and thus inhibits cell–cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca²⁺-CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell–cell contact regions might be essential for this process under hypoxic stress. W.-G. Shen and W.-X. Peng Contributed to this paper equally     

Characterization of the KG1a cell line for use in a cell migration based screening assay

High-throughput screening has become a popular method used to identify new “leads” for potentially therapeutic compounds. Further screening of these lead compounds is typically done with secondary assays which may utilize living, functioning cells as screening tools. A problem (or benefit) with these cell-based assays is that living cells are very sensitive to their environment. We have been interested in the process of stem cell migration and how it relates to the cellular therapy of bone marrow transplantation. In this study we describe a secondary, cell-based assay for screening the effects of variousin-vitro conditions on Immature Hematopoietic Cell (IHC) migration. Our results have revealed many subtle factors, such as the cell's adhesive characteristics, or the effect of a culture's growth phase, that need to be accounted for in a screening protocol. Finally, we show that exponentially growing KG1a cells (a human IHC cell line) were 10 times more motile than those in the lag or stationary phases. These data strongly suggest that KG1a cells secrete a chemokinetic factor during the exponential growth phase of a culture.