Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography.

Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.

Measuring cell motility using quantum dot probes.

The ability of cancer cells to migrate and metastasize is known to be directly related to tumor cell motility. Therefore, assaying the level of tumor cell motility is an excellent indicator of metastatic potential. We have developed an efficient and sensitive two-dimensional cell motility assay to image the phagokinetic uptake of colloidal CdSe/ZnS semiconductor nanocrystals (quantum dots [QDs]). As cells move across a thin, homogeneous layer of QDs, they engulf and uptake the nanocrystals and leave behind a fluorescent-free trail. By measuring the ratio of trail area to cell area we have discovered that it is possible to distinguish between noninvasive and invasive cancer cells lines. This technique has, therefore, the potential to be used as a rapid, robust, and quantitative in vitro measure of metastatic potential. Because the technique only relies on fluorescence detection, requires no significant data processing, and is used with live cells, it is both rapid and straightforward.

Quantum-dot-based cell motility assay.

Because of their favorable physical and photochemical properties, colloidal CdSe/ZnS-semiconductor nanocrystals (commonly known as quantum dots) have enormous potential for use in biological imaging. In this report, we present an assay that uses quantum dots as markers to quantify cell motility. Cells that are seeded onto a homogeneous layer of quantum dots engulf and absorb the nanocrystals and, as a consequence, leave behind a fluorescence-free trail. By subsequently determining the ratio of cell area to fluorescence-free track area, we show that it is possible to differentiate between invasive and noninvasive cancer cells. Because this assay uses simple fluorescence detection, requires no significant data processing, and can be used in live-cell studies, it has the potential to be a powerful new tool for discriminating between invasive and noninvasive cancer cell lines or for studying cell signaling events involved in migration.

Nanoimaging cells using soft X-ray tomography.

Soft X-ray microscopy is ideally suited to visualizing and quantifying biological cells. Specimens, including eukaryotic cells, are imaged intact, unstained and fully hydrated, and therefore visualized in a near-native state. The contrast in soft X-ray microscopy is generated by the differential attenuation of X-rays by the molecules in the specimen-water is relatively transmissive to this type of illumination compared to carbon and nitrogen. The attenuation of X-rays by the specimen follows the Beer-Lambert law, and therefore both linear and a quantitative measure of thickness and chemical species present at each point in the cell. In this chapter, we will describe the procedures and computational methods that lead to 50 nm (or better) tomographic reconstructions of cells using soft X-ray microscope data, and the subsequent segmentation and analysis of these volumetric reconstructions. In addition to being a high-fidelity imaging modality, soft X-ray tomography is relatively high-throughput; a complete tomographic data set can be collected in a matter of minutes. This new modality is being applied to imaging cells that range from small prokaryotes to stem cells obtained from mammalian tissues.

Actin-filled nuclear invaginations indicate degree of cell de-differentiation.

For years the existence of nuclear actin has been heavily debated, but recent data have clearly demonstrated that actin, as well as actin-binding proteins (ABPs), are located in the nucleus. We examined live EGFP-actin-expressing cells using confocal microscopy and saw the presence of structures strongly resembling actin filaments in the nuclei of MDA-MB-231 human mammary epithelial tumor cells. Many nuclei had more than one of these filamentous structures, some of which appeared to cross the entire nucleus. Extensive analysis, including fluorescence recovery after photobleaching (FRAP), showed that all EGFP-actin in the nucleus is monomeric (G-actin) rather than filamentous (F-actin) and that the apparent filaments seen in the nucleus are invaginations of cytoplasmic monomeric actin. Immunolocalization of nuclear pore complex proteins shows that similar invaginations are seen in cells that are not overexpressing EGFP-actin. To determine whether there is a correlation between increased levels of invagination in the cell nuclei and the state of de-differentiation of the cell, we examined a variety of cell types, including live Xenopus embryonic cells. Cells that were highly de-differentiated, or cancerous, had an increased incidence of invagination, while cells that were differentiated had few nuclear invaginations. The nuclei of embryonic cells that were not yet differentiated underwent multiple shape changes throughout interphase, and demonstrated numerous transient invaginations of varying sizes and shapes. Although the function of these actin-filled invaginations remains speculative, their presence correlates with cells that have increased levels of nuclear activity.

Quantum dot-based cell motility assay.

Motility and migration are measurable characteristics of cells that are classically associated with the invasive potential of cancer cells, but in vitro assays of invasiveness have been less than perfect. We previously developed an assay to monitor cell motility and migration using water-soluble CdSe/ZnS nanocrystals; cells engulf the fluorescent nanocrystals as they crawl across them and leave behind a fluorescent-free trail. We show here that semiconductor nanocrystals can also be used as a sensitive two-dimensional in vitro invasion assay. We used this assay to compare the behavior of seven different adherent human cell lines, including breast epithelial MCF 10A, breast tumor MDA-MB-231, MDA-MB-435S, MCF 7, colon tumor SW480, lung tumor NCI H1299, and bone tumor Saos-2, and observed two distinct behaviors of cancer cells that can be used to further categorize these cells. Some cancer cell lines demonstrate fibroblastic behaviors and leave long fluorescent-free trails as they migrate across the dish, whereas other cancer cells leave clear zones of varying sizes around their periphery. This assay uses fluorescence detection, requires no processing, and can be used in live cell studies. These features contribute to the increased sensitivity of this assay and make it a powerful new tool for discriminating between non-invasive and invasive cancer cell lines.