Imaging surface and submembranous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages

Atomic force microscopy (AFM) has been used to image a wide variety of cells. Fixed and dried-coated, wet-fixed or living cells were investigated. The major advantage of AFM over SEM is the avoidance of vacuum and electrons, whereas imaging can be done at environmental pressure and in aqueous conditions. Evidence of the successful application of AFM in biological imaging is provided by comparing results of AFM with SEM and/or TEM. In this study, we investigated surface and submembranous structures of living and glutaraldehyde-fixed colon carcinoma cells, skin fibroblasts and liver macrophages by AFM. Special attention was paid to the correct conditions for the acquisition of images of the surface of these cells, because quality SEM examinations have already been abundantly presented.
AFM imaging of living cells revealed specific structures, such as the cytoskeleton, which were not observed by SEM. Membrane structures, such as ruffles, lamellipodia, microspikes and microvilli, could only clearly be observed after fixing the cells with 0.1% glutaraldehyde. AFM images of living cells were comparable to SEM images of fixed, dried and coated cells, but contained a number of artefacts due to tip–sample interaction. In addition, AFM imaging allowed the visualization of cytoplasmic submembranous structures without the necessity for further preparative steps, allowing us: (i) to follow cytoskeletal changes in fibroblasts under the influence of the microfilament disrupting agent latrunculin A; (ii) to study particle phagocytosis in macrophages. Therefore, in spite of the slow image acquisition of the AFM, the instrument can be used for high-resolution real-time studies of dynamic changes in submembranous structures.     

Multidisciplinary treatment of superior sulcus (Pancoast) tumors

Ten patients with a Pancoast tumour, seven with pulmonary carcinoma, three with a soft tissue tumour, were treated surgically with or without preoperative chemotherapy or external radiotherapy, and with postoperative external radiotherapy mostly in combination with brachytherapy using a flexible intraoperative template. The results were highly variable, e.g. one patient died after three months, another was still alive without tumour after 36 months. Optimal treatment requires cooperation of experienced surgeons, radiotherapists and medical oncologists.     

Survival and tumour characteristics of breast-cancer patients with germline mutations of BRCA1

BACKGROUND: Hereditary breast cancer has been associated with mutations in the BRCA1 and BRCA2 genes and has a natural history different from sporadic breast cancer. We investigated disease-free and overall survival for patients with a proven BRCA1 alteration. METHODS: We estimated disease-free and overall survival for 49 Dutch patients from 19 consecutive families with a proven specific BRCA1 mutation and one family with strong evidence for linkage to the BRCA1 gene. We compared clinical outcome and data on tumour size, histology, axillary nodal status, contralateral breast cancer, and oestrogen-receptor and progesterone-receptor status with those of 196 patients with sporadic breast cancer, matched for age and year of diagnosis. FINDINGS: Disease-free survival for BRCA1 and sporadic patients at 5 years was 49% (95% CI 33-64) and 51% (43-59), respectively (p=0.98). Overall survival at 5 years was 63% (47-76) and 69% (62-76), respectively (p=0.88). Recurrence and death rates did not differ significantly between groups. Hazard ratios for recurrence and death among BRCA1 patients were 1.00 (0.65-1.55) and 1.04 (0.63-1.71) relative to sporadic patients (p=0.88), and these did not differ significantly after adjustment for prognostic factors. Patients with BRCA1-associated breast cancer had twice as many progesterone-receptor-negative tumours (p<0.005) and development of contralateral breast cancer was four to five times as frequent as in the sporadic group (p<0.001). INTERPRETATION: We showed that disease-free and overall survival were similar for sporadic and hereditary breast cancer in the presence of different tumour characteristics, which has implications for screening prophylactic therapy, and different treatments of hereditary breast cancer.     

Chemotherapy in metastasized breast carcinoma

Metastatic breast cancer is still an incurable disease. Standard hormonal and chemotherapeutic treatment modalities yield at the best a survival advantage of 1 to 2 years. However, palliation is still the second, very important goal of treatment for metastatic disease. First-line chemotherapeutic treatment with an anthracycline-containing regimen induces a response in about half the patients. In second-line treatment docetaxel is an effective agent even in patients failing first-line therapy with an anthracycline-containing regimen. There is no effective standard third-line chemotherapy scheme.     

The role of adhesion molecules in the recruitment of hepatic natural killer cells (pit cells) in rat liver

Previous studies showed that blood large granular lymphocytes (LGL), which possess natural killer (NK) activity, develop within rat liver sinusoids into high-density (HD) and subsequently into low-density (LD) pit cells which show an increasing level and spectrum of tumor cytotoxicity. In this study, we investigated the role of adhesion molecules, such as CD2, CD11a, CD18, and CD54 in the recruitment of pit cells to the liver. Immunostaining for electron microscopy, and two color flow cytometry showed that most pit cells expressed CD2, CD11a, CD18, and CD54. After intravenous injections into rats with anti-CD2, anti-CD11a, and anti-CD18 antibodies, the number of pit cells per square millimeter in frozen sections of liver tissue decreased. Treatment of rats with zymosan increased the number of pit cells fivefold, whereas subsequent treatment with anti-adhesion-molecule antibodies resulted in approximately 60% lower number of pit cells. Anti-CD54, supposed to block CD54 expression on sinusoidal endothelial cells, also decreased the number of pit cells. The number of blood LGL was, however, not affected by these antibodies. These results indicate that blocking of CD2, CD11a, CD18, and CD54 antigens on blood LGL and/or liver endothelium decreased the number of pit cells in the liver. These adhesion molecules therefore play an important role in the recruitment of pit cells in the liver.     

The estimated accuracy of the EU reference dissection method for pig carcass classification

This experiment was designed to describe the accuracy of the EU-reference dissection method, and to describe the types of factors influencing the accuracy and assess their size. The experiment was conducted in four different European countries at two abattoirs within each country. A total of 128 carcasses was selected according to carcass weight, fat class and sex, and 8 butchers from different countries dissected the carcasses. Due to the experimental design of the experiment a variation in pig type was found between countries. The accuracy was expressed by the repeatability and reproducibility standard deviation, which were found to be 0.87 and 1.10, respectively, and by the reliability, found to be 0.87. This indicates a high accuracy, although a significant effect was found on the estimation of lean meat percentage (LMP) of butcher, and also that jointing of the carcass was of overall importance to the accuracy of the EU-reference dissection method.     

A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells

Atomic force microscopy (AFM) has been used to image a wide variety of cells and has proven to be successful in cellular imaging, by comparing results obtained by AFM with SEM or TEM. The aim of the present study was to investigate further the conditions for AFM imaging of living cells and compare the results with those obtained by SEM. We chose to image skin fibroblast and liver sinusoidal endothelial cells of two different sources, because these cells have been well described and characterized in earlier studies. AFM imaging of living cells mainly reveals submembranous structures, which could not be observed by SEM. This concerns the visualization of the overall cytoskeletal architecture and organelles, without the necessity of any preparative steps. The AFM study of living cells allows a time lapse study of dynamic changes of the actin cytoskeleton under the influence of the cytoskeleton-disturbing drug cytochalasin B in cells that can be followed individually during the process. However, softer samples, such as the fenestrated parts of living rat liver sinusoidal endothelial cells in culture could not be visualized. Apparently, these cell parts are disrupted due to tip-sample interaction in contact mode. To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was applied, resulting in images of higher quality. Still, endothelial fenestrae could not be visualized. In contrast, contact imaging of immortomouse liver sinusoidal endothelial cells, which are devoid of fenestrae, could easily be performed and revealed a detailed filamentous cytoskeleton.     

Cell cycle arrest and growth inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells

UCN-01 is a derivative of staurosporine, initially developed as a potentially selective inhibitor of the Ca(2+)- and phospholipid-dependent protein kinase C, but with the capacity to inhibit a number of tyrosine and serine/threonine kinases. UCN-01 inhibits the growth of 5 breast carcinoma cell lines with a 50% inhibitory concentration range of 30-100 nM during 6 days of continuous exposure. In MCF-7, MDA-MB453, and SK-BR-3 cells, UCN-01 is 5-fold more potent in growth inhibition than its diastereomer UCN-02, but the 2 compounds are equipotent in the inhibition of MDA-MB468 and H85787 cell growth. A differential sensitivity to a 24-h period of exposure to UCN-01 followed by drug removal and growth for 5 subsequent days was observed. The rank order for persistent inhibition of cells by UCN-01 was MCF-7, MDA-MB453 > SK-BR-3 > H85787 > MDA-MB468. MCF-7 and MDA-MB453 cells did not resume proliferation within the 5 days after brief exposure to UCN-01. In contrast, MDA-MB468 and H85787 cells showed no net growth inhibition after a 24-h pulse of UCN-01, followed by 5 more days of growth in drug-free medium. In MDA-MB468 cells, 150 nM UCN-01 retards but does not prevent cell cycle progression through S phase, but the cells are clearly blocked from exit of G1 and entry into S. Progression through S phase is completely inhibited by 600 nM UCN-01. The development of a G1 to S block by UCN-01 in MDA-MB468 cells occurs in conjunction with inhibition of [32P]orthophosphate labeling and decreased phosphotyrosine mass of discrete cellular phosphoproteins.