Analysis of cortical cell polarity by imaging flow cytometry.

Metastasis is the main cause of cancer-related death and therapies specifically targeting metastasis are highly needed. Cortical cell polarity (CCP) is a prometastatic property of circulating tumor cells affecting their ability to exit blood vessels and form new metastases that constitute a promising point of attack to prevent metastasis. However, conventional fluorescence microscopy on single cells and manual quantification of CCP are time-consuming and unsuitable for screening regulators. In this study, we developed an imaging flow cytometry-based method for high-throughput screening of factors affecting CCP in melanoma cells. The artificial intelligence-supported analysis method we developed is highly reproducible, accurate, and orders of magnitude faster than manual quantification. Additionally, this method is flexible and can be adapted to include additional cellular parameters. In a small-scale pilot experiment using polarity-, cytoskeleton-, or membrane-affecting drugs, we demonstrate that our workflow provides a straightforward and efficient approach for screening factors affecting CCP in cells in suspension and provide insights into the specific function of these drugs in this cellular system. The method and workflow presented here will facilitate large-scale studies to reveal novel cell-intrinsic as well as systemic factors controlling CCP during metastasis.

A quantitative ex vivo study of the interactions between reconstituted high-density lipoproteins and human leukocytes.

Knowledge of the interactions between nanoparticles and immune cells is required for optimal design of nanoparticle-based drug delivery systems, either when aiming to avoid phagocytic clearance of the nanoparticles or promote an immune response by delivering therapeutic agents to specific immune cells. Several studies have suggested that reconstituted high-density lipoproteins (rHDL) are attractive drug delivery vehicles. However, detailed studies of rHDL interactions with circulating leukocytes are limited. Here, we evaluated the association of discoidal rHDL with leukocytes in human whole blood (HWB) using quantitative approaches. We found that while the rHDL of various lipid compositions associated preferentially with monocytes, the degree of association depended on the lipid composition. However, consistent with the long circulation half-life of rHDL, we show that only a minor fraction of the rHDL associated with the leukocytes. Furthermore, we used three-dimensional fluorescence microscopy and imaging flow cytometry to evaluate the possible internalization of rHDL cargo into the cells, and we show increased internalization of rHDL cargo in monocytes relative to granulocytes. The preferential rHDL association with monocytes and the internalization of rHDL cargo could possibly be mediated by the scavenger receptor class B type 1 (SR-BI), which we show is expressed to a higher extent on monocytes than on the other major leukocyte populations. Our work implies that drug-loaded rHDL can deliver its cargo to monocytes in circulation, which could lead to some off-target effects when using rHDL for systemic drug delivery, or it could pave the way for novel immunotherapeutic treatments aiming to target the monocytes.

CD46 activation induces distinct CXCL-10 response in monocytes and monocyte-derived dendritic cells.

CD46 is an important immune regulatory receptor with dual functions, however, the CD46 isoform distribution and the effect of CD46 activation on the cytokine production in monocytes and monocyte-derived dendritic cells (moDCs) is unclear. Here, we show that CD46 activation of moDCs downregulates LPS-induced CXCL-10 expression, while the expression of CXCL-10 in monocytes is unaffected. Furthermore, the differentiation of moDCs induces a switch towards dominance of CYT-2 isoforms of CD46. These data indicate that CD46 activation exerts different functions in monocytes and moDCs and this correlates with a switch in CD46 isoform expression upon differentiation of moDCs.

Pressure and stretch differentially affect proliferation of renal proximal tubular cells.

Renal obstruction is frequently found in adults and children. Mechanical stimuli, including pressure and stretch in the obstructed kidney, contribute to damage; animal models of obstruction are characterized by increased cellular proliferation. We were interested in the direct effects of pressure and stretch on renal tubular cell proliferation. Human HKC-8 or rat NRK-52E proximal tubule cells were subjected to either pressure [0, 60 or 90 mmHg] or static stretch [0 or 20%] for 24 or 48 h. Cell proliferation was measured by cell counting, cell cycle analyzed by flow cytometry, and PCNA and Skp2 expression were determined by qPCR or western blot. Blood gases were determined in an iSTAT system. Proliferation was also assessed in vivo after 24 h of ureteral obstruction. There was a significant increase in HKC-8 cell number after 48 h of exposure to either 60 or 90 mmHg pressure. Western blot and qPCR confirmed increased expression of PCNA and Skp2 in pressurized cells. Cell cycle measurements demonstrated an increase in HKC-8 in S phase. Mechanical stretching increased PCNA protein expression in HKC-8 cells after 48 h while no effect was observed on Skp2 and cell cycle measurements. Increased PCNA expression was found at 24 h after ureteral obstruction. We demonstrate direct transduction of pressure into a proliferative response in HKC-8 and NRK-52E cells, measured by cell number, PCNA and Skp2 expression and increase in cells in S phase, whereas stretch had a less robust effect on proliferation.

Uniform Combretastatin-induced Effect on Monocytes and Neutrophils in Peripheral Blood but Not in Tumors.

BACKGROUND/AIM: Combretastatin A-4 phosphate (CA4P) is a vascular-disrupting agent which affects the level of circulating neutrophils. Since tumor-associated macrophages and neutrophils may collaborate, we compared the effect of CA4P treatment on monocytes/macrophages and neutrophils. MATERIAL AND METHODS: CDF1 mice with a C3H mammary carcinoma foot tumor were injected intraperitoneally with CA4P. Blood samples were taken and tumors excised at various time-points after treatment. Circulating monocytes and granulocytes were detected by flow cytometry and the tumor levels of these cell types was estimated immunohistochemically. RESULTS: CA4P induced similar oscillating effects on the level of circulating monocytes and of neutrophils, with an initial decrease followed by an increase and a return to control levels at 6-h and 24-h, respectively. In tumors, only the macrophage level decreased significantly after treatment. CONCLUSION: CA4P induced similar changes in the level of circulating monocytes and neutrophils, but only affected the fraction of macrophages significantly.

Treatment with a vascular disrupting agent does not increase recruitment of indium labelled human endothelial outgrowth cells in an experimental tumour model.

BACKGROUND: The effect of vascular disrupting agents in tumour therapy depends on both the immediate vascular shutdown, and on the following re-vascularization of the tumour. The aim of this study was to use a tumour model to investigate whether endothelial outgrowth cells (EOCs) influenced the short term treatment efficiency of combretastatin A-4 disodium phosphate (CA4P) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) by increasing EOC tumour recruitment. METHODS: In order to visualize the recruitment of EOCs to the tumours, umbilical cord blood derived human EOCs were labelled with 111Indium-tropolone in a dose of 0.37 MBq pr 3×106 cells and were injected intravenously into mice carrying a C3H mammary carcinoma on their right rear foot. DMXAA and CA4P in different concentrations and at different exposure times were used to create a hypoxic environment in the C3H mammary carcinoma in the mice. Three different mice strains with various degrees of functional immune system were used to study the homing capability of EOCs. RESULTS: Our data showed that approximately 4% of the total injected radioactive dose per gram of tissue was found in the tumour after treatment with CA4P and DMXAA. Regardless of the concentration and the treatment duration, CA4P did not increase EOC recruitment to the tumour in comparison to EOC recruitment in control tumours in any of the 3 mice strains studied. CONCLUSION: Our data showed that regardless of the grade of the immune system, ranging from a fully working to a fully compromised immune system, treatment with CA4P did not increase recruitment of xenotransplanted EOCs to tumour tissue.

Are endothelial outgrowth cells a potential source for future re-vascularization therapy?

Endothelial progenitor cells (EPCs) represent a heterogeneous cell population that is believed to be involved in vasculogenesis after ischemic diseases. EPCs could have a potential for future therapies with the purpose of enhancing endothelial repair. However, due to the low amount of these cells in circulation they have to be expanded in vitro before administration into recipients. The purpose of this study was to analyse and evaluate possible changes in morphology and functionality as a result of in vitro ageing of a subtype of EPCs called endothelial outgrowth cells (EOCs), since such changes might compromise the cells' ability to participate in vasculogenesis. EOCs were isolated and grown from human umbilical cord blood using two methodologies with varying degree of cell purification. The changes between the two culture setups and the changes occurring in EOCs over time were traced by flow cytometry and assays for growth, tube formation, and beta-galactosidase production. The cells showed to be indistinguishable from each other during the first weeks of culture. The cells showed a changed morphology with bigger and more granular cells and the growth rate decreased with time. The cells also showed an increased Beta-galactosidase expression and decreased tube formation ability in late passage EOCs. Our data indicates that EOCs undergo senescence during long-term expansion and therefore time for cell harvest has to be validated in order to achieve functional cells still maintaining a therapeutic potential. A possible application in large animal or humans could be local injection of EOCs into affected areas and thereby reducing the need for long-term expansion of the cells.

Treatment with the vascular disrupting agent combretastatin is associated with impaired AQP2 trafficking and increased urine output.

Combretastatin A-4 disodium phosphate (CA4P) is a vascular disrupting agent known to mediate its effects primarily on tumor blood vessels. CA4P has previously been shown to induce a significant increase in mean arterial blood pressure and in hemoglobin concentration in mice. In the present study, we examined whether this is associated with a general leakage of water into certain tissues or with changes in renal water handling. Munich-Wistar rats received either CA4P (30 mg/kg body wt) or saline intraperitoneally as a bolus injection. One hour later, hemoglobin concentration and mean blood pressure increased significantly. MRI showed no significant changes in tissue water content following CA4P administration. However, urine output and salt excretion increased 1 h after CA4P treatment, without changes in urinary and medullary osmolality. Aquaporin 2 (AQP2) mRNA levels in kidney inner medulla did not change 1 h after CA4P treatment, but semiquantitative confocal laser-scanning microscopy analysis demonstrated a decrease in phosphorylated AQP2 (pS256-AQP2) apical distribution within the collecting ducts of CA4P-treated rats compared with the characteristic apical localization in control rats. Furthermore, we demonstrated that CA4P cause disruption of microtubules and a weaker apical labeling of pS256-AQP2 in collecting duct principal cells within 1 h. In conclusion, our data indicate that water escapes from the vascular system after CA4P treatment, and it may take place primarily through a renal mechanism. The CA4P-mediated increase in urine output seems to be a local effect in the collecting ducts due to reduced AQP2 trafficking to the apical plasma membrane.