Functional Screen of Paracrine Signals in Breast Carcinoma Fibroblasts

<div><p>Stromal fibroblasts actively participate in normal mammary gland homeostasis and in breast carcinoma growth and progression by secreting paracrine factors; however, little is known about the identity of paracrine mediators in individual patients. The purpose of this study was to characterize paracrine signaling pathways between breast carcinoma cells and breast carcinoma-associated fibroblasts (CAF) or normal mammary fibroblasts (NF), respectively. CAF and NF were isolated from breast carcinoma tissue samples and adjacent normal mammary gland tissue of 28 patients. The fibroblasts were grown in 3D collagen gel co-culture with T47D human breast carcinoma cells and T47D cell growth was measured. CAF stimulated T47D cell growth to a significantly greater degree than NF. We detected a considerable inter-individual heterogeneity of paracrine interactions but identified FGF2, HB-EGF, heparanase-1 and SDF1 as factors that were consistently responsible for the activity of carcinoma-associated fibroblasts. CAF from low-grade but not high-grade carcinomas required insulin-like growth factor 1 and transforming growth factor beta 1 to stimulate carcinoma growth. Paradoxically, blocking of membrane-type 1 matrix metalloprotease stimulated T47D cell growth in co-culture with NF. The results were largely mirrored by treating the fibroblasts with siRNA oligonucleotides prior to co-culture, implicating the fibroblasts as principal production site for the secreted mediators. In summary, we identify a paracrine signaling network with inter-individual commonalities and differences. These findings have significant implications for the design of stroma-targeted therapies.</p> </div

Fluorescence-Based Assessment of Plasma-Induced Hydrophilicity in Microfluidic Devices via Nile Red Adsorption and Depletion

We present a simple method, called fluorescence-based assessment of plasma-induced hydrophilicity (FAPH), that enables spatial mapping of the local hydrophilicity of surfaces normally inaccessible by traditional contact angle measurement techniques. The method leverages the change in fluorescence of a dye, Nile Red, which is adsorbed on an oxygen plasma-treated surface, and its correlation with the contact angle of water. Using FAPH, we explored the effect of microchannel geometries on the penetration distance of oxygen plasma into a microchannel and found that entrance effects prevent uniform treatment. We showed that these variations have a significant impact on cell culture, and thus the design of cell-based microfluidic assays must consider this phenomenon to obtain repeatable and homogeneous results

Efficient Sample Preparation from Complex Biological Samples Using a Sliding Lid for Immobilized Droplet Extractions

Sample preparation is a major bottleneck in many biological processes. Paramagnetic particles (PMPs) are a ubiquitous method for isolating analytes of interest from biological samples and are used for their ability to thoroughly sample a solution and be easily collected with a magnet. There are three main methods by which PMPs are used for sample preparation: (1) removal of fluid from the analyte-bound PMPs, (2) removal of analyte-bound PMPs from the solution, and (3) removal of the substrate (with immobilized analyte-bound PMPs). In this paper, we explore the third and least studied method for PMP-based sample preparation using a platform termed Sliding Lid for Immobilized Droplet Extractions (SLIDE). SLIDE leverages principles of surface tension and patterned hydrophobicity to create a simple-to-operate platform for sample isolation (cells, DNA, RNA, protein) and preparation (cell staining) without the need for time-intensive wash steps, use of immiscible fluids, or precise pinning geometries. Compared to other standard isolation protocols using PMPs, SLIDE is able to perform rapid sample preparation with low (0.6%) carryover of contaminants from the original sample. The natural recirculation occurring within the pinned droplets of SLIDE make possible the performance of multistep cell staining protocols within the SLIDE by simply resting the lid over the various sample droplets. SLIDE demonstrates a simple easy to use platform for sample preparation on a range of complex biological samples

Functional screen of paracrine factors in co-culture of CAF with T47D cells. A.

<p>Red-green heat map representation of CAF co-culture-induced T47D cell growth in the presence or absence of neutralizing antibodies. Antibody treatment and calculation of Co-culture-induced T47D cell growth were performed as described in the legend of <b>Fig. 3</b>. Color changes from green to red as value increases. Each row depicts data for CAF from an individual patient (Pt number on left). The tumor grade is indicated on the left-hand side of Pt number. Cases are stratified into low grade (G1 and G2) and high grade (G3). Asterisk denotes neutralizing antibodies, where significant differences were detected between low-grade and high-grade cancer group. Each data point represents the mean of 3–6 replicates. <b>B.</b> Scatter plot representation of the data shown in panel “<b>A</b>”. Student t-test was applied to compare specific treatment vs. no antibody control. * P = 0.0006, ** P<0.0001.</p

Identification of paracrine signaling factors regulating T47D cell growth in co-culture with HMF. A.

<p>In co-culture with HMF, T47D cell growth is significantly reduced by neutralizing antibodies against FGF-2, Heparanase-1, or MT1-MMP. Neutralizing antibodies (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046685#pone.0046685.s010" target="_blank">Table S1</a></b> for details) were added to collagen gel and culture media. Co-cultures were fixed and stained after 3–4 days of treatment. Co-culture-induced T47D cell growth was calculated as described in Materials and Methods. <b>B.</b> Neutralizing antibodies do not affect HMF growth in co-culture. <b>C.</b> T47D cell growth was significantly decreased in co-culture with HMF transfected with siRNA targeting FGF-2, HB-EGF, Heparanase-1, MT1-MMP, SDF-1, or TGF-β1. HMF were transfected with siRNA oligonuleotides (100 nM) 3 days before co-culture with T47D cells. Co-cultures were incubated for 3 days, then fixed and stained. Data shown represent the mean of 3 independent experiments. In each experiment, 3–6 micro-channels were used as technical replicates for every group. Student’s t-test was applied to compare specific treatment vs. no antibody control. The asterisk denotes P<0.05.</p

siRNA screen of T47D cell growth in co-culture with CAF or NF and inter-subject heterogeneity of paracrine interactions. A & B

<p>. Red-green heat map representation of T47D cell growth stimulation in co-culture with CAF (<b>A</b>) or NF (<b>B</b>). SiRNA oligonuleotide transfection was performed as described in Materials and Methods to knock down expression of specific mediators. Co-culture-induced T47D cell growth was calculated as described for Fig. 3. Each data point represents the mean of 3–6 replicates. <b>C</b>. Inter-subject heterogeneity of T47D cell growth response to neutralizing antibody is highest in co-culture with NF and lowest in co-culture with CAF from high-grade tumors. Co-culture-induced T47D cell growth in the presence of antibody was normalized to the no-treatment control for each patient. The sample variance for the group of NF, CAF low-grade, or CAF high-grade were then calculated. F test was applied to compare variances between the groups. * P<0.05, CAF of low grade tumor vs. NF, # P<0.05, CAF of high grade tumor vs. CAF of low grade tumor.</p

Characterization of T47D and HMF growth in micro-channel collagen gel co-culture system. A.

<p>T47D cell growth is significantly induced in co-culture with HMF compared to mono-culture. Co-culture or mono-culture gels were fixed and stained at day 1 to 6. The cytokeratin-positive area was measured with Image J. <b>B.</b> HMF proliferate at a significantly higher rate in mono-culture than in co-culture with T47D cells.</p

Functional screen of paracrine factors in co-culture of NF and T47D cells. A.

<p>Red-green heat map representation of NF co-culture-induced T47D cell growth in the presence or absence of neutralizing antibodies. Antibody treatment and calculation of Co-culture-induced T47D cell growth were performed as described in the legend of <b>Fig. 3 and 4</b>. Color changes from green to red as value increases. Each row depicts data for NF from an individual patient (Pt number on left). The tumor grade is indicated on the left-hand side of Pt number. Cases are stratified into low grade (G1 and G2) and high grade (G3). <b>B.</b> Scatter plot representation of the data shown in panel “<b>A</b>”. Student t-test was applied to compare specific treatment vs. no antibody control. Each data point represents data of one NF sample.</p

Effect of Microculture on Cell Metabolism and Biochemistry: Do Cells Get Stressed in Microchannels?

Microfluidics is emerging as a promising platform for cell culture, enabling increased microenvironment control and potential for integrated analysis compared to conventional macroculture systems such as well plates and Petri dishes. To advance the use of microfluidic devices for cell culture, it is necessary to better understand how miniaturization affects cell behavior. In particular, microfluidic devices have significantly higher surface-area-to-volume ratios than conventional platforms, resulting in lower volumes of media per cell, which can lead to cell stress. We investigated cell stress under a variety of culture conditions using three cell lines: parental HEK (human embryonic kidney) cells and transfected HEK cells that stably express wild-type (WT) and mutant (G601S) <i>human ether-a-go-go related gene</i> (hERG) potassium channel protein. These three cell lines provide a unique model system through which to study cell-type-specific responses in microculture because mutant hERG is known to be sensitive to environmental conditions, making its expression a particularly sensitive readout through which to compare macro- and microculture. While expression of WT-hERG was similar in microchannel and well culture, the expression of mutant G601S-hERG was reduced in microchannels. Expression of the endoplasmic reticulum (ER) stress marker immunoglobulin binding protein (BiP) was upregulated in all three cell lines in microculture. Using BiP expression, glucose consumption, and lactate accumulation as readouts we developed methods for reducing ER stress including properly increasing the frequency of media replacement, reducing cell seeding density, and adjusting the serum concentration and buffering capacity of culture medium. Indeed, increasing the buffering capacity of culture medium or frequency of media replacement partially restored the expression of the G601S-hERG in microculture. This work illuminates how biochemical properties of cells differ in macro- and microculture and suggests strategies that can be used to modify cell culture protocols for future studies involving miniaturized culture platforms

Immunocytochemical staining of array-based micro-channel 3D collagen gel co-culture of T47D cells and HMF. A.

<p>Image of micro-channel device. Inset: Cartoon of single channel design. <b>B.</b> Top view of the image of single channel taken with fluorescent microscope. T47D cells were specifically labeled with anti-pancytokeratin antibody and Alexa 594-conjugated secondary antibody (red). HMF were specifically labeled with anti-vimentin antibody and Alexa 488-conjugated secondary antibody (green) (4x objective). <b>C.</b> Side view of the integrated Z-series image stack images taken with confocal microscope (20x objective).</p