Cecilia Magnusson

Microfluidic, Label-Free Enrichment of Prostate Cancer Cells in Blood Based on Acoustophoresis

Circulating tumor cells (CTC) are shed in peripheral blood at advanced metastatic stages of solid cancers. Surface-marker-based detection of CTC predicts recurrence and survival in colorectal, breast, and prostate cancer. However, scarcity and variation in size, morphology, expression profile, and antigen exposure impairs reliable detection and characterization of CTC. We have developed a noncontact, label-free microfluidic acoustophoresis method to separate prostate cancer cells from white blood cells (WBC) through forces generated by ultrasonic resonances in microfluidic channels. Implementation of cell prealignment in a temperature-stabilized (±0.5 °C) acoustophoresis microchannel dramatically enhanced the discriminatory capacity and enabled the separation of 5 μm microspheres from 7 μm microspheres with 99% purity. Next, we determined the feasibility of employing label-free microfluidic acoustophoresis to discriminate and divert tumor cells from WBCs using erythrocyte-lysed blood from healthy volunteers spiked with tumor cells from three prostate cancer cell-lines (DU145, PC3, LNCaP). For cells fixed with paraformaldehyde, cancer cell recovery ranged from 93.6% to 97.9% with purity ranging from 97.4% to 98.4%. There was no detectable loss of cell viability or cell proliferation subsequent to the exposure of viable tumor cells to acoustophoresis. For nonfixed, viable cells, tumor cell recovery ranged from 72.5% to 93.9% with purity ranging from 79.6% to 99.7%. These data contribute proof-in-principle that label-free microfluidic acoustophoresis can be used to enrich both viable and fixed cancer cells from WBCs with very high recovery and purity.

Acoustofluidics 8: Applications of acoustophoresis in continuous flow microsystems

This acoustofluidics tutorial focuses on continuous flow-based half wavelength resonator systems operated in the transversal mode, where the direction of the primary acoustic force acts in plane with the microchip. The transversal actuation mode facilitates integration with up- and downstream microchannel networks as well as visual control of the acoustic focusing experiment. Applications of particle enrichment in an acoustic half wavelength resonator are discussed as well as clarification of the carrier fluid from undesired particles. Binary separation of particle/vesicle/cell mixtures into two subpopulations is outlined based on the different polarities of the acoustic contrast factor. Furthermore, continuous flow separation of different particle/cell types is described where both Free Flow Acoustophoresis (FFA) and binary acoustophoresis are utilized. By capitalizing on the laminar flow regime, acoustophoresis has proven especially successful in performing bead/cell translations between different buffer systems. Likewise, the ability to controllably translate particulate matter across streamlines has opened a route to valving of cells/particles without any moving parts, where event triggered cell sorting is becoming an increasing area of activity. Recent developments now also enable measurements of fundamental cell properties such as density and compressibility by means of acoustophoresis. General aspects on working with live cells in acoustophoresis systems are discussed as well as available means to quantify the outcome of cell and particle separation experiments performed by acoustophoresis.

Microchannel Acoustophoresis does not Impact Survival or Function of Microglia, Leukocytes or Tumor Cells

Background The use of acoustic forces to manipulate particles or cells at the microfluidic scale (i.e. acoustophoresis), enables non-contact, label-free separation based on intrinsic cell properties such as size, density and compressibility. Acoustophoresis holds great promise as a cell separation technique in several research and clinical areas. However, it has been suggested that the force acting upon cells undergoing acoustophoresis may impact cell viability, proliferation or cell function via subtle phenotypic changes. If this were the case, it would suggest that the acoustophoresis method would be a less useful tool for many cell analysis applications as well as for cell therapy. Methods We investigate, for the first time, several key aspects of cellular changes following acoustophoretic processing. We used two settings of ultrasonic actuation, one that is used for cell sorting (10 Vpp operating voltage) and one that is close to the maximum of what the system can generate (20 Vpp). We used microglial cells and assessed cell viability and proliferation, as well as the inflammatory response that is indicative of more subtle changes in cellular phenotype. Furthermore, we adapted a similar methodology to monitor the response of human prostate cancer cells to acoustophoretic processing. Lastly, we analyzed the respiratory properties of human leukocytes and thrombocytes to explore if acoustophoretic processing has adverse effects. Results BV2 microglia were unaltered after acoustophoretic processing as measured by apoptosis and cell turnover assays as well as inflammatory cytokine response up to 48 h following acoustophoresis. Similarly, we found that acoustophoretic processing neither affected the cell viability of prostate cancer cells nor altered their prostate-specific antigen secretion following androgen receptor activation. Finally, human thrombocytes and leukocytes displayed unaltered mitochondrial respiratory function and integrity after acoustophoretic processing. Conclusion We conclude that microchannel acoustophoresis can be used for effective continuous flow-based cell separation without affecting cell viability, proliferation, mitochondrial respiration or inflammatory status.

Low expression of CysLT1R and high expression of CysLT2R mediate good prognosis in colorectal cancer

Colorectal cancer is the third most common cancer type in the Western world. In search of new treatment possibilities, the inflammation mediators, know as cysteinyl leukotrienes (CysLTs), have been shown to regulate intestinal epithelial cell survival and proliferation via the CysLT(1)R, and cell differentiation via the CysLT(2)R. These results prompted us to investigate the significance of CysLT(1)R and CysLT(2)R expression in colorectal cancer tissue for patient survival. The CysLT(1)R, CysLT(2)R, beta-catenin and Bcl-xL protein expression levels were evaluated by immunohistochemistry in a tissue microarray of 329 colorectal patients. We found that high nuclear expression of CysLT(1)R is associated with a poor prognosis, whereas high nuclear expression of CysLT(2)R is associated with a good prognosis. We also observed that patients with colorectal tumours characterised by high CysLT(1)R but low CysLT(2)R nuclear expression had the lowest survival expectancy, whereas patients with colorectal tumours characterised by low CysLT(1)R but high CysLT(2)R nuclear expression had the best survival expectancy. Interestingly, beta-catenin as a single prognostic marker did not exhibit any prognostic value. However, in patients with tumours characterised by a high CysLT(1)R nuclear expression, an elevated beta-catenin nuclear expression had a significantly prognostic value. In conclusion these data indicate that nuclear expressions of CysLTRs are potential prognostic indicators of colorectal cancer.

Acoustofluidic, Label-Free Separation and Simultaneous Concentration of Rare Tumor Cells from White Blood Cells

Enrichment of rare cells from peripheral blood has emerged as a means to enable noninvasive diagnostics and development of personalized drugs, commonly associated with a prerequisite to concentrate the enriched rare cell population prior to molecular analysis or culture. However, common concentration by centrifugation has important limitations when processing low cell numbers. Here, we report on an integrated acoustophoresis-based rare cell enrichment system combined with integrated concentration. Polystyrene 7 μm microparticles could be separated from 5 μm particles with a recovery of 99.3 ± 0.3% at a contamination of 0.1 ± 0.03%, with an overall 25.7 ± 1.7-fold concentration of the recovered 7 μm particles. At a flow rate of 100 μL/min, breast cancer cells (MCF7) spiked into red blood cell-lysed human blood were separated with an efficiency of 91.8 ± 1.0% with a contamination of 0.6 ± 0.1% from white blood cells with a 23.8 ± 1.3-fold concentration of cancer cells. The recovery of prostate cancer cells (DU145) spiked into whole blood was 84.1 ± 2.1% with 0.2 ± 0.04% contamination of white blood cells with a 9.6 ± 0.4-fold concentration of cancer cells. This simultaneous on-chip separation and concentration shows feasibility of future acoustofluidic systems for rapid label-free enrichment and molecular characterization of circulating tumor cells using peripheral venous blood in clinical practice.

An increased expression of cysteinyl leukotriene 2 receptor in colorectal adenocarcinomas correlates with high differentiation

Increased levels of inflammatory mediators such as cysteinyl leukotrienes (CysLT) have been found in and around tumors. These data, along with our previous observation that the G-protein-coupled receptor CysLT(1)R, which signals survival and proliferation, is up-regulated in colon cancer, suggest an important role for CysLT(1)R in tumor development. The objective of this study was to examine the expression and function of the low-affinity CysLT2 receptor (CysLT2R) in colon cancer. We found lower expression levels of CysLT2R compared with CysLT(1)R in cancer cell lines as well as clinical tumor material. Interestingly, CysLT2R, like CysLT(1)R, was found to be one of few G-protein-coupled receptors that are located both at the plasma membrane and the nuclear membrane. No effect of CysLT2R signaling on cell proliferation was observed, nor was there a correlation between CysLT2R and different proliferation markers such as Ki-67 and cyclooxygenase-2 in the tumor material. Instead, we found that activation of this receptor in colon cancer cells led to cellular differentiation similar to the effects of butyrate treatment. In accordance with this finding, we found that reduced expression of CysLT2R in colon cancer was associated with poor prognosis. We report the novel finding that CysLT2R signaling leads to terminal differentiation of colon carcinoma cells and growth inhibition, and that its expression is relatively high in less malignant forms of colon cancer. These data suggest that the balance between these two receptors is important for tumor progression and disease outcome.

Cysteinyl leukotriene receptor expression pattern affects migration of breast cancer cells and survival of breast cancer patients

The fact that breast cancer patients with local or distal dissemination exhibit decreased survival, promotes a search for novel mechanisms to suppress such tumor progression. Here, we have determined the expression of proinflammatory cysteinyl leukotriene receptors (CysLTRs) in breast tumor tissue and their signaling effect on breast cancer cell functions related to tumor progression. Patients with breast tumors characterized by high CysLT1R and low CysLT2R expression levels exhibited increased risk of cancer‐induced death in univariate analysis for both the total patient group (hazard ratio [HR] = 2.88, 95% confidence interval [CI] = 1.11–7.41), as well as patients with large (>20 mm) tumors (HR = 5.08, 95% CI = 1.39–18.5). Multivariate analysis revealed that patients with large tumors exhibiting high CysLT1R and low CysLT2R expression levels had a significantly reduced survival, also when adjusted for established prognostic parameters (HR = 7.51, 95% CI = 1.83–30.8). In patients with large (>20 mm) tumors, elevated CysLT2R expression predicted an improved 5‐year survival (log‐rank test p = 0.04). Surprisingly, for longer time periods, this prognostic value was lost. This disappearance coincided with the termination of hormonal treatment. Tamoxifen preserved and even induced transcription of CysLT2R, but not CysLT1R, in estrogene receptor‐positive MCF‐7 breast cancer cells. This elevated CysLT2R expression decreased, even below the level of untreated cells, when tamoxifen was withdrawn. CysLT2R signaling reduced MCF‐7 cell migration, but had no effect on either proliferation or apoptosis. Our data indicate that low CysLT1R together with high CysLT2R expression levels might be useful parameters in prognostication and treatment stratification of breast cancer patients.

Concurrent Isolation of Lymphocytes and Granulocytes Using Prefocused Free Flow Acoustophoresis

Microchip-based free flow acoustophoresis (FFA) in combination with two-dimensional cell prefocusing enables concurrent multiple target outlet fractionation of leukocytes into subpopulations (lymphocytes, monocytes, and granulocytes); we report on this method here. We also observed significantly increased accuracy in size-based fractionation of microbeads as compared to previously presented FFA multiple outlet systems. Fluorescence microscopy illustrates the importance of two-dimensional prefocusing where a sample mixture of 3, 7, and 10 μm beads are separated into well-confined particle streams and collected in their respective target outlets. Flow cytometry data for lymphocytes and granulocytes, respectively, in their corresponding outlets verify concurrent isolation of leukocyte subpopulations with high purity (95.2 ± 0.6% and 98.5 ± 0.7%) and high recovery (86.5 ± 10.9% and 68.4 ± 10.6%). A relatively low purity and high recovery of monocytes (25.2% ± 5.4% and 83.1 ± 4.3%) was obtained in the third target outlet. No subpopulation bias was observed. These data demonstrate an unprecedented separation of leukocyte subpopulations at flow rates of ∼100 μL/min and ∼1 M cells/mL sample concentrations, not previously reported in acoustofluidic systems. Two-dimensional prefocusing FFA with multiple target outlets is a viable alternative to current methods for particle fractionation and cell isolation, requiring a minimum of sample preparation and lowering analysis time and cost.

The cysteinyl leukotriene 2 receptor contributes to all-trans retinoic acid-induced differentiation of colon cancer cells

BackgroundCysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators that are increased in samples from patients with inflammatory bowel diseases (IBDs). Individuals with IBDs have enhanced susceptibility to colon carcinogenesis. In colorectal cancer, the balance between the pro-mitogenic cysteinyl leukotriene 1 receptor (CysLT1R) and the differentiation-promoting cysteinyl leukotriene 2 receptor (CysLT2R) is lost. Further, our previous data indicate that patients with high CysLT1R and low CysLT2R expression have a poor prognosis. In this study, we examined whether the balance between CysLT1R and CysLT2R could be restored by treatment with the cancer chemopreventive agent all-trans retinoic acid (ATRA).MethodsTo determine the effect of ATRA on CysLT2R promoter activation, mRNA level, and protein level, we performed luciferase gene reporter assays, real-time polymerase chain reactions, and Western blots in colon cancer cell lines under various conditions.ResultsATRA treatment induces CysLT2R mRNA and protein expression without affecting CysLT1R levels. Experiments using siRNA and mutant cell lines indicate that the up-regulation is retinoic acid receptor (RAR) dependent. Interestingly, ATRA also up-regulates mRNA expression of leukotriene C4 synthase, the enzyme responsible for the production of the ligand for CysLT2R. Importantly, ATRA-induced differentiation of colorectal cancer cells as shown by increased expression of MUC-2 and production of alkaline phosphatase, both of which could be reduced by a CysLT2R-specific inhibitor.ConclusionsThis study identifies a novel mechanism of action for ATRA in colorectal cancer cell differentiation and demonstrates that retinoids can have anti-tumorigenic effects through their action on the cysteinyl leukotriene pathway.

Regulation of Cysteinyl Leukotriene Receptor 2 Expression—A Potential Anti-Tumor Mechanism

Background The cysteinyl leukotrienes receptors (CysLTRs) are implicated in many different pathological conditions, such as inflammation and cancer. We have previously shown that colon cancer patients with high CysLT1R and low CysLT2R expression demonstrate poor prognosis. Therefore, we wanted to investigate ways for the transcriptional regulation of CysLT2R, which still remains to be poorly understood. Methodology/Principal Findings We investigated the potential role of the anti-tumorigenic interferon α (IFN-α) and the mitogenic epidermal growth factor (EGF) on CysLT2R regulation using non-transformed intestinal epithelial cell lines and colon cancer cells to elucidate the effects on the CysLT2R expression and regulation. This was done using Western blot, qPCR, luciferase reporter assay and a colon cancer patient array. We found a binding site for the transcription factor IRF-7 in the putative promoter region of CysLT2R. This site was involved in the IFN-α induced activity of the CysLT2R luciferase reporter assay. In addition, IFN-α induced the activity of the differentiation marker alkaline phosphatase along with the expression of mucin-2, which protects the epithelial layer from damage. Interestingly, EGF suppressed both the expression and promoter activity of the CysLT2R. E-boxes present in the CysLT2R putative promoter region were involved in the suppressing effect. CysLT2R signaling was able to suppress cell migration that was induced by EGF signaling. Conclusions/Significance The patient array showed that aggressive tumors generally expressed less IFN-α receptor and more EGFR. Interestingly, there was a negative correlation between CysLT2R and EGFR expression. Our data strengthens the idea that there is a protective role against tumor progression for CysLT2R and that it highlights new possibilities to regulate the CysLT2R.